NanoBio&Med 2014 Abstracts Book

nov. 18-21, barcelona (spain)www.nanobiomedconf.com

organisers

abstracts book

NanoB io&Med2014

2 no v embe r 1 8 - 2 1 , 2 0 1 4 - B a r c e l o n a ( S p a i n )

Foreword 2

Organising

Committee 3

Sponsors 3

Exhibitors 4

Speakers 5

Posters 101

On behalf of the Organizing Committee, we take

great pleasure in welcoming you to Barcelona

(Spain) for the NanoBio&Med 2014 International

Conference.

The NanoBio&Med 2014, after successful editions

organized within ImagineNano in Bilbao 2011 &

2013, is going to present the most recent

international developments in the field of

Nanobiotechnology and Nanomedicine and will

provide a platform for multidisciplinary

communication, new cooperations and projects

to participants from both science and industry.

Emerging and future trends of the converging

fields of Nanotechnology, Biotechnology and

Medicine will be discussed among industry,

academia, governmental and non-governmental

institutions. NanoBio&Med 2014 will be the perfect

place to get a complete overview into the state of

the art in those fields and also to learn about the

research carried out and the latest results. The

discussion in recent advances, difficulties and

breakthroughs will be at his higher level.

This year, an industrial forum will also be

organized to promote constructive dialogue

between business and public leaders and put

specific emphasis on the technologies and

applications in the nanoBioMed sector.

We are indebted to the following Scientific

Institutions, Companies and Government

Agencies for their financial support: Universitat de

Barcelona, Institute for Bioengineering of

Catalonia (IBEC), The Centre for BioNano

Interactions (CBNI), ICEX España Exportación e

Inversiones, NanoSciences Grand Sud-Ouest,

NanoMed Spain and the health campus of the

University of Barcelona (HUBc).

In addition, thanks must be given to the staff of all

the organising institutions whose hard work has

helped planning this conference.

NanoB io&Med2014

n o v embe r 1 8 - 2 1 , 2 0 1 4 - B a r c e l o n a ( S p a i n ) 3

Antonio CORREIAAntonio CORREIAAntonio CORREIAAntonio CORREIA

President of the Phantoms Foundation

(Spain)

Dietmar Dietmar Dietmar Dietmar PUMPUMPUMPUM

Deputy Head of the Biophysics Institute –

BOKU (Austria)

Josep SAMITIERJosep SAMITIERJosep SAMITIERJosep SAMITIER

Director of the Institute for Bioengineering

of Catalonia – IBEC (Spain)

NanoB io&Med2014

4 no v embe r 1 8 - 2 1 , 2 0 1 4 - B a r c e l o n a ( S p a i n )

The Centre for BioNano Interactions (CBNI) is a

multi-disciplinary platform for Nanotoxicology

and NanoMedicine.

The Centre for Bio NanoInteractions is Ireland’s

National Platform for BioNanoInteraction

science, and draws together specialists from its

Universities, Institutes and companies. We are

one of the world’s leading Centres of knowledge

for bionanointeractions applied to the fields of

nanosafety, nanobiology and nanomedicine,

and we are pioneering many of the new

techniques and approaches in the arena. We

have strong links and co-operations with

academia, institutions, industry, and

governments world-wide.

We seek to set standards through commitment

to excellence in research and innovation,

blended with caution and attention to detail in

the science, and public dissemination.

We appreciate the multilateral responsibilities

to promote knowledge, economic development,

and above all the advancement of these in a

safe and sustainable manner. As such, the

Centre is founded on principles of integrity and

transparency in all of its activities.

More info: www.ucd.ie/cbni

IBEC is a non-profit foundation established at

the end of 2005 by the Ministries of Innovation,

Universities and Enterprises and Health of the

Generalitat de Catalunya (Autonomous

Government of Catalonia), by the University of

Barcelona (UB) and by the Technical University

of Catalonia (UPC).

The governing body of IBEC is its Board of

Trustees, composed of members of the four

founding institutions. IBEC's Board of Trustees

receives advice from the director of the institute

and from the International Scientific

Committee.

IBEC's International Scientific Committee plays

a key role in the activities of the institute,

focusing especially on the selection and

evaluation processes of the research group

leaders. The International Scientific Committee

is composed of international renowned

scientists in different bioengineering fields, as

well as prestigious professionals in key areas

within the activities of IBEC, such as research

results valorization or medical technologies

validation.

IBEC is funded by its founding institutions, by

competitive research projects, whether national

or international, and by R&D contracts with

companies.

More info: www.ibecbarcelona.eu

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Speakers list:

Alphabetical order (by surname)

Authors Page Alcaraz, Jordi (University of Barcelona, Spain) Aberrant mechanical microenvironment in lung cancer probed at the micro- and nano-scales

Oral 9 Barrán Berdón, Ana Lilia (Universidad Complutense de Madrid, Spain) Calix[4]arene TMAC4 as efficient non-viral vector in gene therapy

Oral 11 Benita, Simon (The Hebrew University of Jerusalem, Israel) Improved Oral Absorption of Exenatide using a Novel Nanoencapsulation and Microencapsulation Approach

Keynote 13 Benny, Ofra (The Hebrew University of Jerusalem, Israel) Nanomicelles for Targeting the Tumor Microenvironment

Keynote 14 Blázquez, María (INKOA SISTEMAS, SL, Spain) Selection of Nanotechnology enabled products for nano-release assessment throughout their life cyle in the

NANOSOLUTIONS project

Oral 15

Brossel, Rémy (Cell Constraint & Cancer SA, France) Action of mechanical Cues in vivo on the Growth of a subcutaneously grafted Tumor: Proof of Concept

Oral 16 Cassinelli, Nicolás (nanoScale Biomagnetics, Spain) Setting a standard on magnetic heating of nanoparticles for bioapplications

Oral 18 Cognet, Laurent (CNRS & Université de Bordeaux, France)

Single-molecule and super-resolution microscopies in biology: taking the best of fluorescent dyes, gold

nanoparticles and carbon nanotubes Keynote 19

Contant, Sheila ((IQAC) /CSIC /CIBER-BBN, Spain)

Preparation of Colloidal Dispersions of Magnetic Nanoparticles Coated with Biocompatible Polymers Oral 21

Daban, Joan-Ramon (Universitat Autònoma de Barcelona, Spain) Use of Nanomechanical Data to Validate a Supramolecular Multilayer Model That Explains the Dimensions,

Topology, and Physical Properties of Condensed Metaphase Chromosomes Oral 22

Della Pia, Eduardo Antonio (University of Copenhagen/Nano Science Center, Denmark) Conducting polymers as a versatile platform for protein nanoarrays technologies

Oral 25 Domènech, Oscar (University of Barcelona, Spain) Lipid composition modulates nanomechanics of transmembrane proteins

Oral 27 Dostalek, Jakub (Austrian Institute of Technology, Austria) Plasmonic biosensors advanced by functional hydrogels

Keynote 29 Elezgaray, Juan (Université Bordeaux, France) Localized, DNA based logical circuits as components for biodetection

Keynote 30 Eritja, Ramon (IBMB-CSIC, Spain) Development of modified siRNA for gene silencing

Keynote 31 Espejo Rodriguez, Consuelo (OEPM, Spain) How can I protect my invention? BioMed Patents

Keynote 32 Fornaguera, Cristina (Institut de Química Avançada de Catalunya (IQAC-CSIC) & CIBER-BBN, Spain) Polymeric nanoparticles, prepared from nano-emulsion templating, as novel advanced drug delivery systems

crossing the Blood-Brain Barrier Oral 33

Franzese, Giancarlo (Universitat de Barcelona, Spain) Kinetics of the protein corona assembly on nanoparticlesinetics of the protein corona assembly on nanoparticles

Keynote 35

Gierlinger, Notburga (BOKU, Austria & ETH Zurich, Switzerland) Imaging molecular structure of plant cells by Confocal Raman microscopy

Keynote 36 Gorostiza, Pau (ICREA & IBEC, Spain) Optopharmacology to regulate endogenous proteins with light

Keynote 38 Guari, Yannick (Université Montpellier II, France) Cyano-bridged coordination polymers nanoparticles as contrast agents for Biomedical Imaging

Keynote 39

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Authors Page Kanioura, Anastasia (Institute of Nuclear and Radiological Sciences & Technology, Energy

& Safety, NCSR “Demokritos", Greece) Effect of nanoscale surface roughness on the adhesion and proliferation of normal skin fibroblasts and HT1080

fibrosarcoma cells

Oral 40

Katakis, Ioanis (Universitat Rovira i Virgili, Spain) Screen printed superhydrophobic surfaces as enablers for Capillarity-Driven Biodetection Devices for Food Safety

and Clinical Analysis: towards ASSURED sensors Keynote 42

Lagunas, Anna (Institut de Bioenginyeria de Catalunya (IBEC), Spain) Large-scale dendrimer-based uneven nanopatterns of RGD towards improved architectural networks in chondrogenesis

Invited 43

Llop Roig, Jordi (CIC biomaGUNE, Spain) Following the degradation and biological fate of polymeric poly (lactic-co-glycolic acid) nanoparticles

Invited 45 Marco, M.-Pilar (IQAC-CSIC/CIBER-BBN, Spain) Biofunctional surfaces for Multiplexed Diagnostic Platforms using Site-Encoded DNA Strategies

Keynote 47 Mendoza, Ernest (Universitat Politècnica de Catalunya / Goldemar, Spain) From Basic Research to an Industrial Product: The case of Goldemar

Invited 48 Misawa, Masaki (National Institute of Advanced Industrial Science and Technology (AIST), Japan) Radiosensitizing Effect of Gold Nanoparticles under kV- and MV- X-ray Irradiations

Oral 49 Navajas, Daniel (Universitat de Barcelona / IBEC and CIBER of Respiratory Diseases, Spain) Nanomechanics of the extracellular matrix of lung and heart tissues

Keynote 51 Paez Aviles, Cristina (University of Barcelona, Spain) Cross-cutting KETs: Innovation and Industrialization challenges for Nanobiotechnology and Nanomedicine towards

Horizon 2020 Oral 53

Paoli, Roberto (Institute for Bioengineering of Catalonia (IBEC), Spain) DC studies of Layer-by-layer nanopores electrical properties tuning on Polycarbonate Membranes

Oral 56 Pardo Jimeno, Julian (University of Zaragoza, Aragon Centre for Biomedical Research (CIBA), Spain) Bona fide induction of apoptosis in transformed cells during photothermal therapy using gold nanoprisms

Oral 58 Pla-Roca, Mateu (Institute for Bioengineering of Catalonia (IBEC), Spain) Nanotechnology Platform at the Institute for Bioengineering of Catalonia: description of capabilities and examples

Keynote 59 Polo, Ester (CBNI / University College Dublin, Ireland) A microscopic molecular basis for Nanoparticle Interactions with Organisms

Keynote -

Porath, Danny (Hebrew University of Jerusalem, Israel) The Quest for Charge Transport in single Adsorbed Long DNA-Based Molecules

Keynote 60 Puntes, Victor (Institut Català de Nanociència i Nanotecnologia (ICN2) & ICREA, Spain) Nanomedicine 2.0

Keynote 62 Rigat-Brugarolas, Luis G. (Institite for Bioengineering of Catalonia (IBEC) & CIBER-BBN, Spain) Developing new tools for drug testing: introducing a microfluidic platform mimicking the spleen for future

pharmacological trials Oral 64

Rodea Palomares, Ismael (Universidad Autónoma de Madrid, Spain) PAMAM dendrimers internalizes quickly in microalgae and cyanobacteria causing toxicity and oxidative stress

Oral 66 Sáenz, Juan José (Universidad Autónoma de Madrid, Spain) Speckle fluctuations resolve the interdistance between incoherent point sources in complex media

Invited 69 Sánchez, Samuel (Max Planck Institute for Intelligent Systems, Germany) Self-powered microbots towards a “Fantastic Voyage”

Keynote 71 Scheffold, Frank (University of Fribourg, Switzerland) Scattering based bead-microrheology applied to biomaterials

Keynote 72 Schroeder, Avi (Technion - Israel Institute of Technology, Israel) Targeted drug delivery and personalized medicine

Keynote 74 Schwartz Navarro, Simo (Vall d'Hebron Hospital - CIBBIM, Spain) Personalized Cancer Nanomedicine. CLINAM 2014

Keynote 75 Serrano Núñez, Juan Manuel (Sesderma Laboratories, Spain) Liposomes: Topical and Oral Bioavailability

Oral 76 Shoseyov, Oded (The Hebrew University of Jerusalem, Israel) Nano crystalline cellulose-protein composites: Super performing biomaterials for tissue engineering and

regenerative medicine

Keynote 78

Soares, Paula (Universidade Nova de Lisboa, FCT-UNL, Portugal) Studies on thermal and magnetic properties of iron oxide nanoparticles for magnetic hyperthermia application

Oral 80 Stremersch, Stephan (Ghent University, Belgium) Hijacking nature's own communication system: evaluation of extracellular vesicles as a siRNA delivery vehicle

Oral 82 Swersky Sofer, Nava (ICA, Israel) From Science to Product, in Israel and Beyond

Keynote -


Authors Page Tatkiewicz, Witold I. (Institut de Ciència de Materials de Barcelona (ICMAB-CSIC) & CIBER-BBN, Spain) 2D Microscale Surface Engineering with Novel Protein based Nanoparticles for Cell Guidance

Oral 84 Toca Herrera, Jose Luis (BOKU / Institute for Biophysics, Austria) Atomic force microscopy, life sciences and soft matter

Keynote 86 Unciti Broceta, Juan Diego (Centre for Genomics and Oncological Research (GENYO), Spain) Multiplicity of Nanofection: a New Index to Assess Nanoparticle Cellular Uptake

Oral 87 Uriarte, Juan José (Universitat de Barcelona, Institut d’Investigacions Biomèdiques August

Pi Sunyer & CIBER de Enfermedades Respiratorias, Spain) Nanomechanics of Decellularized Lung and in Vivo Lung Elastance in a Murine Model of Marfan Syndrome

Oral 90

Veciana, Jaume (Institut de Ciència de Materials de Barcelona (CSIC)-CIBER-BBN, Spain) Supramolecular organizations as novel nanomedicines for drug delivery

Keynote 92 Vicendo, Patricia (Laboratoire des IMRCP, Université de Toulouse, France) Polymeric micelles nanovectors for photodynamic therapy applications: From the structure to the activity

Oral 93 Vila, Mercedes (University of Aveiro, Portugal) Nanographene-oxide mediated hyperthermia for cancer treatment

Invited 95 Wajs, Ewelina (Universitat Rovira i Virgili, Spain) Host-guest engineered stimuli-responsive nanocapsules

Oral 97 Yudina, Tetyana (The Catalan Institute of Nanoscience and Nanotechnology (ICN2), Spain) Nanoceria

Oral 98 Zambelli, Tomaso (ETH Zurich, Switzerland) FluidFM: combining AFM and microfluidics for single-cell perturbation in vitro

Keynote 100

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Aberrant mechanical microenvironment in

lung cancer probed at the micro- and nano-

scales

Jordi Alcaraz, Marta Puig, Marta Gabasa, Roberto Lugo, Roland Galgoczy

Unit of Biophysics, School of Medicine, University of Barcelona, Casanova 143, Barcelona, Spain [email protected]

The lung is a moderately soft organ with unique

mechanical properties that are necessary for

breathing. However, the appearance of a

desmoplastic stroma rich in activated fibroblasts/myofibroblasts and pro-fibrotic

extracellular matrix (ECM) components in lung

tumors strongly suggests that the cellular microenvironment becomes abnormally stiff in

lung cancer. To test this hypothesis, we have

analyzed the expression of fibrillar collagens (a major pro-fibrotic ECM component) in vivo by

picrosirius red staining, and have assessed the

mechanical consequences of enhanced collagen

density ex-vivo by atomic force microscopy (AFM). Likewise, we have determined the fraction

of activated fibroblasts in vivo, and the

mechanical consequences of such activation in culture at the nano- and micro-scale by AFM.

Finally, we have examined cell-ECM mechanical

interactions in fibroblasts at the nano-scale with microfabricated flat-ended-AFM tips designed to

mimic few cell-ECM adhesion sites. The biological

consequences of aberrant cell-ECM mechanical

interactions in tumors were further analyzed in terms of fibroblast accumulation, which is a

major hallmark of solid tumors in the lung and

other organs.

Normal lung parenchyma exhibited weak

collagen staining and short collagen fibers,

whereas tumor samples exhibited abundant collagen staining that was frequently organized

into long and straight fibril bundles indicative of

mechanical tension. Likewise, fibril bundles were

often organized in a parallel fashion (see dashed

squares) [1], which has been previously shown to

render stiffer tissues. To examine the mechanical

effects of increased collagen deposition, we

probed the Young´s elastic modulus (E) of

collagen gels with increasing concentration by AFM. E scaled with collagen density according c

to E ~ c2. This dependence is well captured by

current models of semiflexible semidiluted polymers in which the fibril length is larger than

the pore or mesh size [2].

In normal lung parenchyma, fibroblasts were sparsely located, and α-SMA+ cells

corresponding to smooth muscle cells were

largely restricted to the perivasculature (see black arrows). In contrast, lung tumors exhibited

a stroma rich in peritumoral α-SMA+ fibroblasts.

These features were used to assess the relative amount of tumor stroma as the percentage of α-

SMA+ area, which elicited ~25% [1]. To mimic

fibroblast activation in culture, most fibroblasts

required exogenous TGF-b1, which is a potent fibrotic cytokine commonly upregulated in the

tumor microenvironment. Treatment of

fibroblasts with TGF-b1 induced de novo

expression of α-SMA incorporated into stress

fibers in culture. Concomitantly to these

cytoskeletal alterations, TGF-b1 treatment increased the Young´s modulus more than 2-fold

with respect to untreated cells.

To investigate the critical molecular machinery involved in cell-ECM force transmission, we

microfabricated flat-ended AFM tips with a

constant cross-section area of ~1 μm2, and

coated them with an RGD peptide, which is an

adhesive domain found in many ECM

components that is specifically recognized by

N a n o B i o & M e d 2 0 1 4 1 0 n o v e m b e r 1 8 - 2 1 , 2 0 1 4 - B a r c e l o n a ( S p a i n )

ECM integrin receptors. The RGD-coated flat-

ended tip was brought to contact with the

surface of a single fibroblasts up to a moderate

force, hold for 30 s to enable the formation of focal adhesion precursors, and retracted until the

contact was lost. We found a marked increase in

both cell stiffnes and cell-ECM adhesion when

using RGD-coated tips, but not RGE or other non-integrin specific coatings. Such cell stiffening and

adhesion strengthening were abrogated upon

inhibiton of actin but not microtubule polymerization, revealing that local fibroblast

mechanoresponses requires integrin-mediated

rearrangements of the actin cytoskeleton [3].

To examine the pathological consequences of

abnormal integrin mechanosensing in

fibroblasts, we analyzed how extracellular stiffening comparable to that expected within the

tumor microenvironment altered fibroblast

behavior in an integrin-specific fashion. We found

that fibroblast density markedly increased in gels with a tumor-like rigidity compared to soft gels

with normal-like rigidity values. Remarkably, such

cell density increase was abrogated upon inhibition of beta1 integrin mechanosensing

through FAKpY397, which is the most abundant

component of fibroblast integrin receptors [1].

In summary, we have collected evidence

supporting that the tumor microenvironment is

much stiffer than the normal lung parenchyma and that activated fibroblasts are largely

responsible for such tissue hardening. Of note,

we have also obtained evidence of a positive feedback loop in which activated fibroblasts

increase tissue hardening, which in turns

stimulates fibroblast accumulation in a beta1-

integrin dependent fashion, which is a hallmark of lung cancer.

References

[1] Puig M, Lugo R, Gabasa M, Giménez A,

Velásquez A, Galgoczy R, et al. Matrix stiffening and β1 integrin drive fibroblast

accumulation in lung cancer in a subtype-

dependent fashion. Mol Cancer Res. 2014;(in press).

[2] Alcaraz J, Mori H, Ghajar CM, Brownfield D,

Galgoczy R, Bissell MJ. Collective epithelial

cell invasion overcomes mechanical barriers

of collagenous extracellular matrix by a

narrow tube-like geometry and MMP14-

dependent local softening. Integr Biol (Camb). 2011;3:1153-66.

[3] Acerbi I, Luque T, Gimenez A, Puig M, Reguart

N, Farre R, et al. Integrin-specific mechanoresponses to compression and

extension probed by cylindrical flat-ended

AFM tips in lung cells. PLoS One.

2012;7:e32261.

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Calix[4]arene TMAC4 as efficient non-viral

vector in gene therapy

A.L. Barrán-Berdón1*, Belén Yélamos2, Luis García-Rio3, E. Aicart1 and E. Junquera1 1Grupo de Química Coloidal y Supramolecular, Universidad Complutense de Madrid, Madrid, Spain

2Dpto. Bioquímica y Biología Molecular I, Universidad Complutense deMadrid, Madrid, Spain 3Centro Singular de Investigación en Química Biológica y Materiales Moleculares. Universidad de

Santiago de Compostela, Praza do Obradoiro, s/n 15782-Santiago de Compostela, Spain

[email protected]

One of the major challenges in the gene therapy

process is to find efficiently non-viral gene

carriers. Several studies have been done in order to increase the number of compounds

able to compact, protect and transport nucleics

acids into the cell. The development of several kinds of macrocycles such calixarenes open a

new way in the non-viral vector tools in gene

therapy. Calixarenes are very promising in gene delivery applications for several reasons: their

synthesis is relatively easy, they present low

toxicity levels and, possessing two clearly

distinct chemical regions, allow an efficient region-selective chemistry.[1-3]

Complexes prepared by mixing the gene vector (formed by calix[4]arene TMAC4 and the

zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-

phosphoethanolamine (DOPE), at several molar

fractions, α) with plasmid pEGFP-C3 (pDNA) or linear double-stranded calf thymus DNA

(ctDNA). A wide biophysical and biochemical

characterization was performed including zeta

potential, gel electrophoresis, SAXS, cryo- TEM,

fluorescence microscopy and cell

viability/cytotoxicity to establish a structure-biological activity relationship. The study was

performed at several compositions, , between

calixarene and DOPE, and at several effective

charge ratios, ρeff, (between the gene vector and the DNA) of the complex.

Electrochemical studies (zeta potential and gel

electrophoresis) confirm that pDNA is efficiently

compacted by the TMAC4/DOPE system.

Structural characterization by SAXS shows that

diffractograms correspond to nanoaggregates

formed by a lamellar structure at any α. Cryo-

TEM studies reveal the presence of cluster-type and finger print multilamellar structures. Finally,

the biochemical studies in vitro show that

complexes TMAC4/DOPE-pDNA present moderate transfection efficiency and good cell

viability in HEK293T cells lines. Therefore, the

reported complexes can be considered as potential DNA vectors for gene therapy in vivo.

References [1] V. Bagnacani, F. Sansone, G. Donofrio, L.

Baldini, A. Casnati, R. Ungaro, Organic

Letters, 10 (2008) 3953-3956.

[2] R. Rodik, A. Klymchenko, Y. Mely, V. Kalchenko, Journal of Inclusion Phenomena

and Macrocyclic, (2014) 1-12.

[3] R.V. Rodik, A.S. Klymchenko, N. Jain, S.I.

Miroshnichenko, L. Richert, V.I. Kalchenko, Y.

Mely, Chemistry, 17 (2011) 5526-5538.


Figures

Figure 1. a) Molecular structure

of the calix[4]arene TMAC4. b)

Plot of zeta potential () against

the complex composition (L/D)

of TMAC4/DOPE-pDNA at several

molar fractions, . c)

Fluorescence micrograph of

transfected HEK293T cells.


Improved Oral Absorption of Exenatide

using a Novel Nanoencapsulation and

Microencapsulation Approach

Liat Kochavi-Soudry, Taher Nassar and Simon Benita

The Institute for Drug Research of the School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel

Oral delivery of peptides remains challenging

and continues to be one of the most attractive

alternatives to their parenteral delivery. Despite intensive efforts invested over the last two

decades, no commercial solution has yet

emerged due to several drawbacks and hurdles associated with the poor intestinal membrane

permeability of these hydrophilic

macromolecules, instability in the gut and rapid metabolism. Therefore, the development of

sophisticated delivery systems for oral

administration of peptidic drugs still remains an

attractive scientific challenge. Exenatide is a 39-amino-acid peptide approved as an adjunctive

therapy for patients with type-2 diabetes failing

to achieve glycemic control with oral antidiabetic agents. Exenatide is injected

subcutaneously (SC) twice a day and can induce

pain and possible infections at the sites of injection that could adversely affect patient

compliance. A once a week injection of

exenatide has been developed but still suffers

from the aforementioned drawbacks. In the present research, we propose a

nano/microencapsulation process of the

hydrophilic bio macromolecule to protect and control exenatide release. This unique strategy

should facilitate the controlled release of the

exenatide-loaded nanoparticles (NPs), as opposed to the release of the dissolved drug, in

the vicinity of the mucosa in an attempt to avoid

GI acidic and proteolytic enzymes degradation

of the peptide. The first line of protection was

achieved by loading the peptide into primary

NPs. Different types of NPs were prepared;

bovine serum albumin (BSA) NPs cross-linked

with glutaraldehyde, BSA mixed with dextran

NPs cross linked with sodium trimetaphosphate and conjugation of exenatide to poly lactic-co-

glycolic acid (PLGA) NPs. The second line of

protection was achieved following encapsulation of the primary NPs within

microcapsules consisting of a blend of Eudragit

L 55-100 and hydroxypropyl methyl cellulose (HPMC) using a spray drying technique.

The primary NPs and microcapsules containing

exenatide NPs were imaged by Cryo-TEM and SEM respectively. The mean diameter of the

cross-linked NPs ranged between 50-100nm or

300-500 nm depending on the cross-linker and matrix. The PLGA NPs mean diameter was 90-

150nm. The zeta potential value was around -

45mV and the encapsulation yield was above 30-40% irrespective of the formulation. The in

vitro release kinetic profiles showed that it was

possible to reduce the burst release depending

on the type of formulation up to 20% followed by a gradual slow release over 6-8 h. The

pharmacokinetic results allowed to identify an

optimal formulation based on dextran/BSA cross-linked NPs embedded in microparticles

which elicited significant plasma levels

following oral administration in rats. The marked increase in the oral bioavailability of

such a formulation is promising, confirming that

the peptide was not markedly degraded during

the manufacturing process and the transit via

the gastro-intestinal tract.


Nanomicelles for Targeting the Tumor

Microenvironment

Ofra Benny

Institute for Drug Research, Faculty of Medicine

The Hebrew University of Jerusalem, Jerusalem, Israel

[email protected]

Tumor metastases are the principal cause of mortality in the majority of cancer patients. A

hospitable tumor microenvironment, of which

the vascular system is a significant component,

is crucial in the implantation of disseminated tumor cells. Angiogenesis, the formation of new

blood vessels, is a multifactorial process that is

critical for tumor progression and metastasis. Anti-angiogenic compounds, has been widely

investigated as a strategy to treat cancer.

However, several of these drugs are limited by poor pharmacological properties, such as low

bioavailability, undesired biodistribution and

short half-life necessitating their use in high

intravenous doses which expose the patients to adverse size-effects due to off-target activity. To

overcome these drug limitations, we developed

a formulation of self-assembled nanomicelles composed of short di-block polymers,

polyethylene glycol-polylactic acid (PEG-PLA),

for conjugating small molecule drugs. We present a case of re-formulating a broad

spectrum anti-angiogenic drug from the

fumagillin family which originally had several

clinical limitations. In the new formulation, unlike the free compound, the drug showed

high oral availability, improved tumor targeting

and reduced toxicity. Dramatic anti-cancer activity was obtained in eight different tumor

types (60-90% growth inhibition) in mice, and,

importantly, the treatment was able to prevent liver metastases due to the shift from

intravenous to oral administration. The activity

was associated with reduction of microvessel

density and increased tumor apoptosis.

Nanomicelle drug delivery system has been

shown to be an efficient approach for improving

pharmacological properties of drugs and for better targeting the tumor-microenvironment.

References [1] Benny O, Fainaru O, Adini A, Cassiola F,

Bazinet L, Adini I, Pravda E, Nahmias Y,

Koirala S, Corfas G, D'Amato RJ, Folkman J.

Nat Biotechnol. 2008 Jul;26(7):799-807. [2] Benny O, Pakneshan P. Cell Adh Migr. 2009

Apr-Jun;3(2):224-9.

Figures

Figure 1. Nanomicelles for treating tumors by targeting

tumor microenvironment (A) diagram showing the self-

assembled di-block copolymer nanomicelles. (B) AFM

image of PEG-PLA Nanomicelles. (C) tumor growth

inhibition of subcutaneous Lewis Lung Carcinoma.


Selection of nanotechnology enabled

products for nano-release assessment

throughout their life cyle in the

NANOSOLUTIONS project

María Blázquez1, I. Unzueta1, E. Fernández-Rosas2, A. Vílchez2 and S. Vázquez-Campos2 1RTD Department, INKOA SISTEMAS, SL, Ribera de Axpe 11, 48950 Erandio Bizkaia, Spain

2 LEITAT Technological Center, C/Innovació, 2, 08225 Terrassa, Spain [email protected]

The hazard evaluation of engineered nanomaterials (ENMs) needs to take into

consideration that the initially synthesized

ENMs will not remain unaltered during their life cycle. The intentional introduction of surface

modifications to ENMs is a common practice

prior to the incorporation of these ENMs in other products. Later, during the use or end of life

phases, other transformation processes may

take place, so that if ENMs are released they

may share few characteristics with the initially synthesized ENMs. Possible changes include

surface coating, irreversible embedding in

matrices, dissolution, agglomeration and aggregation, surface charge modification,

whereas factors underlying the occurrence of

these changes include aging, mechanical stress, chemical stress and/or interactions with biota in

the environment in most cases in a combined

manner.

The main goal of the present work has consisted

on the selection of nano-enabled products and

the evaluation of their life cycle to study the release of ENMs in different phases, within the

framework of the NANOSOLUTIONS FP7

European research project. This project ultimately aims at identifying and elaborating

those characteristics of ENMs that determine

their biological hazard potential by providing a

means to develop a safety classification of

ENMs.

According to the specific processes undergone by the selected applications, the life cycle of the

ENMs -beyond manufacturing stage- has been

evaluated. Thereafter, the life cycle stages that are most likely to result in the transformation of

the ENMs and/or to result in the release of ENMs

have been identified prioritizing normal use conditions (releases generated in accidental

scenarios have not been considered). The

outcome of present work has enabled the

definition of realistic laboratory scaled simulation processes to be undertaken in the

execution of the project.

Preliminary findings on nanoadditivated

commercial fabrics are also introduced.

Acknowledgements: The authors would like

to thank NANOSOLUTIONS consortium. The

project has received funding from the European

Union’s Seventh Framework Programme for research, technological development and

demonstration under grant agreement #309329


Action of mechanical Cues in vivo on the

Growth of a subcutaneously grafted Tumor:

Proof of Concept

Rémy Brossel

Cell Constraint & Cancer SA, Le mas l’Hermite, 331, chemin de la Poterie, Raphèle-les-Arles, France [email protected]

The cancerous tumor tissue and its extracellular

matrix are subject to mechanical signals. The

role of pressure in tumor transformation and

growth as well as in the appearance of metastasis is more and more understood.

Hence the effect of constraint/stress on tumor

growth has been widely explored in vitro in 3-dimension cell culture. The proof of concept

delivered by the present work shows the effect

of a constraint field in vivo on tumor growth. Nude mice were grafted subcutaneously with a

mix of ferric nanoparticles and MDA MB 231

cells. The nanoparticles with a diameter of 100

nm rapidly spread around the growing tumor. The field of constraint was applied through the

magnetized nanoparticles located around the

tumor. It was generated by the action of a magnetic field gradient on the nanoparticles

using permanent magnets located outside the

animal. A very statistically significant difference (p=0.015) was observed between the volume of

tumors with nanoparticles around and

subjected to a field of constraint for 2 hours/day

for 21 days and observed to day 59 or more, and the volume of tumor of the three control groups.

This experiment provides the first evidence of

an action of mechanical signals on the growth of tumor in vivo, in animal. These results confirm

in vivo the results previously obtained in vitro on

3-dimension tissue culture models.

References

[1] Cross SE, et al. (2008) AFM-based analysis of human

metastatic cancer cells. Nanotechnology

19(38):384003.

[2] Murphy MF, et al. (2013) Evaluation of a nonlinear

Hertzian-based model reveals prostate cancer cells

respond differently to force than normal prostate cells.

Microsc Res Tech 76(1):36-41.

[3] Remmerbach TW, et al. (2009) Oral cancer diagnosis by

mechanical phenotyping. Cancer Res 69(5):1728-1732.

[4] Fuhrmann A, et al. (2011) AFM stiffness

nanotomography of normal, metaplastic and

dysplastic human esophageal cells. Phys Biol

8(1):015007.

[5] Canetta E, et al. (2014) Discrimination of bladder cancer

cells from normal urothelial cells with high specificity

and sensitivity: combined application of atomic force

microscopy and modulated Raman spectroscopy. Acta

Biomater 10(5):2043-2055.

[6] Indra I (2012) Mechanical forces and tumor cells: insight

into the biophysical aspects of cancer progression.

Wayne State University Dissertations.

http://digitalcommons.wayne.edu/oa_dissertations/4

13:Paper 413.

[7] Xu W, et al. (2012) Cell stiffness is a biomarker of the

metastatic potential of ovarian cancer cells. PLoS One

7(10):e46609.

[8] Plodinec M, et al. (2012) The nanomechanical signature

of breast cancer. Nat Nanotechnol 7(11):757-765.

[9] Lekka M, et al. (2012) Cancer cell recognition--

mechanical phenotype. Micron 43(12):1259-1266.

[10] Ingber DE, Madri JA, & Jamieson JD (1981) Role of basal

lamina in neoplastic disorganization of tissue

architecture. Proc Natl Acad Sci U S A 78(6):3901-3905.

[11] Tse JM, et al. (2012) Mechanical compression drives

cancer cells toward invasive phenotype. Proc Natl Acad

Sci U S A 109(3):911-916.

[12] Lee GY, Kenny PA, Lee EH, & Bissell MJ (2007) Three-

dimensional culture models of normal and malignant

breast epithelial cells. Nat Methods 4(4):359-365.

[13] Jonietz E (2012) Mechanics: The forces of cancer.

Nature 491(7425):S56-57.

[14] Baish JW & Jain RK (2000) Fractals and cancer. Cancer

Res 60(14):3683-3688.


[15] Bizzarri M, et al. (2011) Fractal analysis in a systems

biology approach to cancer. Semin Cancer Biol

21(3):175-182.

[16] D'Anselmi F, et al. (2011) Metabolism and cell shape in

cancer: a fractal analysis. Int J Biochem Cell Biol

43(7):1052-1058.

[17] Stein GS, et al. (1999) Implications for interrelationships

between nuclear architecture and control of gene

expression under microgravity conditions. FASEB J 13

Suppl:S157-166.

[18] Xu R, Boudreau A, & Bissell MJ (2009) Tissue

architecture and function: dynamic reciprocity via

extra- and intra-cellular matrices. Cancer Metastasis

Rev 28(1-2):167-176.

[19] Ingber DE (2006) Cellular mechanotransduction:

putting all the pieces together again. FASEB J 20(7):811-

827.

[20] Paszek MJ, et al. (2005) Tensional homeostasis and the

malignant phenotype. Cancer Cell 8(3):241-254.

[21] Montel F, et al. (2011) Stress clamp experiments on

multicellular tumor spheroids. Phys Rev Lett

107(18):188102.

Figures

Figure 1A. Schematic representation of the experimental

setup with the animal (Magnets tumor not at scale)

Figure 1B. Close up of the West and East sides with force

vector

Tumor MDA MB 231, Perls special stain, x100-Important labeling of peri-

tumoral areas

Figure 2. Spreading of the nanoparticles around a

subcutaneous grafted tumor

Figure 3. Growth curve of the tumors in the 4 groups

Volume (mm3) Median(Q1; Q3) (Min; Max) Mean (±std) Significance (p value)

Treated (N=7) 529 (502; 840) 346; 966 646±235 Significant

(p=0.015)

Controls (N=33) 1,334 (758; 1784) 256; 2106 1,250±282 IC 95%

579 (124; 1,099)

Table 1 - Tumor volume measured on D59+tumors

West East

Iron Tumor


Setting a standard in magnetic heating of

nanoparticles for bioapplications

Nicolás Cassinelli

nanoScale biomagnetics. Calle Panamá 2, Local , 50012 Zaragoza, Spain

[email protected]

Magnetic nanoparticles (MNPs) with functionalized surfaces are bringing novel and

promising ways to treat deadly diseases such as

cancer. They have multiple applications that

range from magnetic hyperthermia, localized drug delivery and release, to tissue engineering

and new materials. MNPs are designed to

attack, with high specificity, a given tissue, challenging researchers in solving biochemical

and physiological issues. Depending on the

success in such a challenge, cancer specific hyperthermia and drug delivery protocols could

be developed. Clinical success of these

techniques has been delayed for several years in

an important part because of the lack of reliable and compliant specific instrumentation.

The Spanish company nanoScale Biomagnetics, formed in 2008 as a Spin Off coming from the

University of Zaragoza, entered the market in

2010 with a set of instruments and accessories that can be described as the first high end

resource for magnetic heating experiments with

MNPs, from material characterization to in vivo

experiments. With the common goal of developing the use of the technique, nB works

with customers and institutions from all around

the globe in the search of new standards and tools.


Single-molecule and super-resolution

microscopies in biology:

taking the best of fluorescent dyes, gold

nanoparticles and carbon nanotubes

Laurent Cognet

Laboratoire Photonique Numérique et Nanosciences (LP2N). IOGS, CNRS et Université de Bordeaux

Institut d’Optique d’Aquitaine, rue François Mitterrand. 33405 Talence cedex, France [email protected]

The optical microscopy of single molecules has recently been beneficial for many applications,

in particular in biology. It allows a sub-

wavelength localization of isolated molecules and subtle probing of their spatio-temporal

nano-environments on living cells. It also allows

designing innovative strategies to obtain super-resolved optical images i.e. with resolution

below the diffraction limit.

For many single-molecule microscopy applications, more photostable nanoprobes

than fluorescent ones are desirable. For this

aim, we developed several years ago far-field photothermal methods based on absorption

instead of luminescence. Such approaches do

not suffer from the inherent photophysical

limitations of luminescent objects and allows the ultra-sensitive detection and spectroscopy

of tiny absorbing individual nano-objects such

as gold nanoparticles down to 5 nm in cells or carbon nanotubes. In order to access confined

cellular environment (adhesion sites, synapses

etc...), I will present our current efforts to reduce the functional nano-objects sizes as well as to

use new near infrared nanoprobes.

The second part of my presentation will be dedicated to the presentation of super-

resolution microscopy methods. It is indeed

crucial to study a large ensemble of molecules

on a single cell while keeping the sub-

wavelength localization provided by single

molecule microscopy. In order to study the dynamical properties of endogenous

membrane proteins found at high densities on

living cells we developed a new single molecule super-resolution technique, named uPAINT.

Interestingly, uPAINT does not require the use of

photo-activable dyes allowing easy multi-color super-resolution imaging and single molecule

tracking. Different applications of uPAINT will be

presented, in particularly the first

demonstration of super-resolution imaging of functional receptors in interaction. This last

result was obtained combining super-resolution

microscopy and single molecule FRET.

References

[1] Super-resolution microscopy approaches for

live cell imaging. A. Godin, B. Lounis, L. Cognet

Biophys. J., 107 (2014) 1777. [2] Hyper-bright Near-Infrared Emitting

Fluorescent Organic Nanoparticles for Single

Particle Tracking. E. Genin, Z. Gao, J. Varela, J. Daniel, T. Bsaibess, I. Gosse, L. Groc, L. Cognet,

M. Blanchard-Desce Adv. Mat 26 (2014) 2258-

2261. [3] Identification and super-resolution imaging of

ligand-activated receptor dimers in live cells.

P. Winckler, L. Lartigues, G.Gianonne, F. De

Giorgi, F. Ichas, J-B. Sibarita, B. Lounis and L.

Cognet Sci. Rep., 3 (2013) 2387.


[4] A highly specific gold nanoprobe for live-cell

single-molecule imaging. C. Leduc, S. Si, , J.

Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A.

Gautreau, G. Giannone, L. Cognet, and B. Lounis Nano Lett. 13, 4, (2013) 1489-1494.

[5] Integrins β1 and β3 exhibit distinct dynamic

nanoscale organizations inside focal

adhesions. O. Rossier, V. Octeau, J.B. Sibarita, C. Leduc, B. Tessier, D. Nair, V. Gatterdam, O.

Destaing, C. Albigès-Rizo, R. Tampé, L. Cognet,

D. Choquet, B. Lounis & G. Giannone Nat. Cell Biol. 14 (2012) 1057-1067.


Preparation of Colloidal Dispersions of

Magnetic Nanoparticles Coated with

Biocompatible Polymers

S. Contant, C. Solans*

Institute for Advanced Chemistry of Catalonia (IQAC)/CSIC/CIBER BBN, Barcelona, Spain [email protected]

In recent years much interest has been

dedicated to the use of magnetic nanoparticle

dispersions for the diagnostic and treatment of

diseases [1]. The colloidal dispersions need to have several characteristics such as

biocompatibility, colloidal stability, suitable size

and narrow size distribution to be used in biomedical applications. The size of the

nanoparticles has an influence not only in the

magnetic properties and colloidal stability but also in the possibility of crossing biological

barriers such as cell junctures and membranes

[2]. To assure biocompatibility, magnetic

nanoparticles require an appropriate coating. Besides making the dispersions biocompatible,

the coating is essential because it protects

magnetic nanoparticle surface from oxidation, increases blood circulation time, provides

specificity for biological target sites and gives

steric repulsion acting as a barrier against the interaction between the particles thereby

providing colloidal stability [3, 4]. The objective

of this work was to prepare dispersions of

magnetic nanoparticles coated with biocompatible polymers showing colloidal and

physicochemical characteristics suitable for

biomedical applications. Three different polymers were used for coating: polyacrylic acid

(PAA), polyethylene glycol (PEG) and

polyethylene glycol bisamine (PEG bisamine). Iron oxide nanoparticles were used as core. The

coated nanoparticle dispersions were

characterized in terms of stability, iron content,

size and morphology of the nanoparticles and

amount of coating. The dispersions prepared

showed stability over a month, concentrations

of iron over 5mg/ml, and resulted in coated

nanoparticles with small diameters (around

100nm by DLS and 10nm by TEM) and concentrations of organics between 5 and 13%.

The results showed that the coated magnetic

nanoparticles could be suitable for further biomedical applications.

References

[1] Hilger, I., Kaiser, W. A. Nanomedicine, 7 (2012)

1443-1459. [2] Salas, G., Veintemillas-Verdaguer, S., Morales,

M. P. International Journal of Hyperthermia,

(2013) 1-9. [3] Wahajuddin, Arora, S. International Journal of

Nanomedicine, 7 (2012) 3445-3471.

[4] Reddy, L. H., Arias, J. L., Nicolas, J., Couvreur, P. Chemical Reviews, 112 (2012) 5818−5878.


Use of Nanomechanical Data to Validate a

Supramolecular Multilayer Model That

Explains the Dimensions, Topology, and

Physical Properties of Condensed Metaphase

Chromosomes

Joan-Ramon Daban Dep. de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain

[email protected]

In the cell nucleus, genomic DNA molecules are

associated with histone proteins and form long

chromatin filaments containing many nucleosomes. The three-dimensional

organization of these giant DNA molecules is

undoubtedly the most challenging topological problem of structural biology. Previous TEM and

AFM studies from our laboratory showed that,

during cell division, chromatin filaments are

folded into multilayer planar structures [1,2], in which DNA forms a two-dimensional network

with a good flexibility and mechanical strength

[3]. This discovery led to the thin-plate model in which we proposed that condensed

chromosomes are formed by many stacked

layers of chromatin oriented perpendicular to the chromosome axis [4]. More recently we

found that multilayered plates can be self-

assembled from chromatin fragments obtained

by micrococcal nuclease digestion of metaphase chromosomes [5]. This finding,

together with nanotechnology results showing

that self-assembly of different structures of biological origin can produce complex

micrometer-scale materials [6-8], suggested

that chromosomes could be self-organizing structures. This communication shows that if

chromosomes are considered as typical

supramolecular assemblies, using the

nanomechanical data obtained in other

laboratories [9,10] and basic energetic

considerations, it is possible to explain the

geometry and physical properties of condensed

chromosomes.

Metaphase chromosomes of different animal

and plant species show great differences in size,

ranging from 2 to 27 µm in length, and from 0.3 to 1.3 µm in diameter. The observed

chromosome sizes are dependent on the

amount of DNA that they contain (from 35 to

7450 Mb), but in all cases chromosomes are elongated cylinders that have relatively similar

shape proportions: the average value of the

length to diameter ratio (L/D) is 13. This study demonstrates that it is possible to explain this

morphology by considering that chromosomes

are self-organizing supramolecular structures formed by stacked layers of planar chromatin

having different nucleosome-nucleosome

interaction energies in different regions [11] (see

figure). The nucleosomes in the periphery of the chromosome are less stabilized by the attractive

interactions with other nucleosomes and this

generates a surface potential that destabilizes the structure. Chromosomes are smooth

cylinders (scheme a) because this morphology

has a lower surface energy than structures having irregular surfaces (scheme b). The

symmetry breaking produced by the different

values of the surface energies in the telomeres

and in the lateral surface (εT > εL) explains the

elongated structure of the chromosomes.


The results obtained by other authors in

nanomechanical studies of chromatin [9] and

chromosome [10] stretching have been used to

test the proposed supramolecular structure [11]. It is demonstrated quantitatively that

internucleosome interactions (εnn) between

chromatin layers (scheme c) can justify the work

required for elastic chromosome stretching. Chromosomes can be considered as hydrogels

with a lamellar liquid crystal organization. These

hydrogels have outstanding elastic properties because, in addition to the covalent bonds of

the DNA backbone, they have attractive ionic

interactions between nucleosomes that can be regenerated when the chromosome suffers a

deformation. This self-healing capacity has

been observed in nanotechnology studies of

other hydrogels stabilized by ionic interactions [12]. In the cell, this may be useful for the

maintenance of chromosome integrity during

cell division.

Finally, since early studies indicated that

chromosomes are helically coiled [13], it is possible that each chromosome is formed by a

single helicoidal plate [2,11]; the successive

turns of a helicoid (scheme d) are equivalent to

the stacked layers considered in the original thin-plate model. The flat plates seen in our

micrographs and a helicoidal plate are

topologically equivalent. They can be converted into each other without changing their mean

curvature; the plane and the helicoid are both

minimal surfaces (their mean curvature is zero). A continuous helicoidal plate has good

mechanical properties and allows a

homogenous organization of chromatin that

precludes the random entanglement of the genomic DNA molecules. Furthermore, this

chromatin organization can explain the

morphology of the chromosome bands used in cytogenetic analyses for the diagnosis of cancer

and hereditary diseases

References

[1] I. Gállego, P. Castro-Hartmann, JM. Caravaca,

S. Caño and JR. Daban, Eur. Biophys. J., 38 (2009) 503.

[2] P. Castro-Hartmann, M. Milla and JR. Daban,

Biochemistry, 49 (2010) 4043.

[3] I. Gállego, G. Oncins, X. Sisquella, X. Fernández-Busquets and JR. Daban, Biophys. J., 99 (2010)

3951.

[4] JR. Daban, Micron, 42 (2011) 733. [5] M. Milla and JR. Daban, Biophys, J., 103 (2012)

567.

[6] GM. Whitesides and B. Grzybowski, Science, 295 (2002) 2418.

[7] WJ. Chung, JW. Oh, K. Kwak, BY. Lee,… and

SW. Lee, Nature, 478 (2011) 364.

[8] T. Guibaud, E. Barry, MJ. Zakhary, M. Henglin,… and Z. Dogic, Nature, 481 (2012)

348.

[9] Y. Cui and C. Bustamante, Proc. Natl. Acad. Sci. USA., 97 (2000) 127.

[10] MG. Poirier and JF.Marko, J. Muscle Res. Cell

Motil., 23 (2002) 409. [11] JR. Daban, J. Royal Soc. Interface, 11 (2014)

20131043.

[12] JY. Sun, X. Zhao, WRK. Illeperuma, O.

Chaudhuri,… and Z. Suo, Nature, 489 (2012) 133.

[13] E. Boy de la Tour and UK. Laemmli, Cell, 55

(1998) 937.

Acknowledgements: Research supported in

part by grant BFU2010-18939 from the Ministerio de Economía y Competitividad


Figures


Conducting polymers as a versatile platform

for protein nanoarrays technologies

Eduardo Antonio Della Pia1*, N. Lloret1, J. Holm2, M. Zoonens3, J.-L. Popot3, J. Nygård2, K. L. Martinez1 1Bio-Nanotechnology Laboratory, University of Copenhagen, Copenhagen, Denmark

2Niels Bohr Institute & Nano-Science Center, Copenhagen, Denmark 3C.N.R.S./Université Paris-7 UMR 7099, Institut de Biologie Physico-Chimique Paris, France

[email protected] and [email protected]

Micro- and nano-arrays of biomolecules such as

DNA, peptides and proteins offer exciting

opportunities in both basic and applied

research (i.e. diagnostics, drug screening and drug discovery) [1]. High-density miniaturized

biochips can increase assays sensitivity and

throughput while reducing sample consumption and processing time [1, 2]. While

DNA micro-arrays are currently being realized

and are showing all their potential in genomic applications, protein arrays are still in their

infancy due to the delicate nature of proteins

and their challenging interaction with solid

substrates [1, 2]. Even though substantial advances in nano-patterning techniques have

been achieved and protein nano-patterns have

been realized using dip-pen lithography, electron beam lithography or nanografting,

examples of proteins nano-arrays are still rare

and without evidence of proteins activity and stability [1, 2].

Here we report a versatile platform for spatially

and selective functionalization of electrically contacted gold micro- and nano-structures with

biological molecules such as proteins. The

method is based on the electrochemical functionalization of the gold surfaces with

conducting polymers bearing biotin or metal

ion units [3]. We first demonstrate that biotin-binding molecules such as streptavidin or

histidine-tagged proteins can be selectively

immobilized on the polymeric film [4, 5]. We

then show that protein multiplexed nano-arrays

can be successfully prepared by sequential

polymerizations and biomolecular

immobilizations. The platform can be further

used to immobilize complex membrane

proteins stabilized in amphipathic polymers

(amphipols) [6]. In fact, by taking advantage of the high affinity between biotin and

streptavidin, we immobilize distinct membrane

proteins onto different electrodes via amphipols modified with a biotin tag (biotinylated

amphipols, Figure 1) [7]. Antibody-recognition

events indicate that the membrane proteins are stably anchored to the substrate and that the

electropolymerization is compatible with their

protein-binding activity. Finally we take

advantage of the good conductivity properties of the conducting polymers and measure the

direct electron transfer properties of a redox-

active membrane protein bound to the substrate [8].

The platform described here is a first step for fabricating functional arrays of membrane

proteins and we believe it will be a candidate of

choice to produce electronically transduced

nano-biosensors.

References

[1] Wu, Chien‐Ching, David N. Reinhoudt, Cees

Otto, Vinod Subramaniam, and Aldrik H.

Velders., "Strategies for Patterning Biomolecules with Dip‐Pen Nanolithography."

Small 7.8 (2011): 989-1002

[2] Christman, Karen L., Vanessa D. Enriquez-Rios,

and Heather D. Maynard. "Nanopatterning

proteins and peptides." Soft Matter 2.11

(2006): 928-939.

mailto:[email protected]


[3] Cosnier, Serge, and Michael

Holzinge."Electrosynthesized polymers for

biosensing." Chemical Society Reviews 40.5

(2011): 2146-2156. [4] Della Pia, Eduardo Antonio, Jeppe V. Holm,

Noemie Lloret, Christel Le Bon, Jeam-Luc

Popot, Manuela Zoonens, Jesper Nygård and

Karen Laurence Martinez. “A step closer to membrane protein multiplexed nanoarrays

using biotin-doped polypyrrole” ACS nano 8.2

(2014): 1844-1853. [5] Della Pia, Eduardo Antonio, Caroline Lindberg,

Maeva Vignes, Martinez. "A novel surface for

strong and reversible immobilization of his-tagged proteins” Manuscript in preparation.

[6] Zoonens, Manuela, and Jean-Luc Popot.

"Amphipols for each season." The Journal of

membrane biology (2014): 1-38. [7] Della Pia, Eduardo Antonio, Randi Westh

Hansen, Manuela Zoonens, and Karen L.

Martinez. "Functionalized amphipols: a versatile toolbox suitable for applications of

membrane proteins in synthetic biology." The

Journal of membrane biology (2014): 1-12. [8] Laursen, Tomas, Peter Naur, and Birger

Lindberg Møller. "Amphipol trapping of a

functional CYP system." Biotechnology and

applied biochemistry 60.1 (2013): 119-127.

Figures

Figure 1. Fluorescent microscopy image of three gold

electrodes functionalized with biotin-doped polypyrrole

film and (top and central electrode) streptavidin and

neutravidin Oregon Green (bottom electrode). By

successive polymerization and protein incubation, the

membrane protein tOmpA trapped in biotinylated

amphipols and NBD fluorescent-labelled amphipols was

immobilized on the top electrode and the membrane

protein bacteriorhodopsin trapped in biotinylated

amphipols and Alexa 647 fluorescent-labelled amphipols

was immobilized on the central electrode. The image is

obtained by overlaying fluorescence images obtained in

three different channels. Scale bar is 5 μm.


Lipid composition modulates

nanomechanics of transmembrane proteins

Domènech, Ò., Vázquez-González, M.L., de Robles, B., Montero, M.T. and Hernández-Borrell, J.

University of Barcelona, Avda. Joan XXIII s/n, Barcelona, Spain

[email protected]

There is a need to investigate the increasingly emergent problem of antibiotic resistance

because of the social impact and economic

consequences [1]. The use of new nanoscale

techniques opens new perspectives in the study of the mechanisms involved in the generation of

resistances [2]. Particularly, multidrug efflux

pumps are under the spotlight to understand the molecular and physicochemical basis of the

efflux mechanism to decrease the antibiotic

concentration inside the bacterium. We used lactose permease (Lac Y) from Escherichia coli as

a paradigm for the secondary transport proteins

that couple the energy stored in an

electrochemical ion gradient to a concentration gradient (ß-galactoside/H+ symport) to study

the effect of the lipid matrix in its nanostructure.

Firstly we characterized with the Atomic Force Microscope (AFM) the nanomechanics of the

lipids in Supported Lipid Bilayers (SLBs)

mimicking the lipid composition of bacteria (Figure 1). Secondly we incorporate the protein

to the lipid bilayers and investigate the changes produced when modifying the lipid

environment (Figure 2). We found that proteins

were segregated into liquid-crystalline phases

(L) whilst the forces needed to extend a single protein were higher when the unsaturation in

the hydrocarbon chains of the lipids decreased.

This fact could be related to the lateral pressure on the protein in the lipid bilayer evidenced

during the unfolding of a single protein when

pulling it with the AFM tip.

References

[1] Alanis, A.J., Archives of Medical Research, 36 (2005) 697.

[2] Longo, G., Alonso-Sarduy, L., Marques Rio,

L., Bizzini, A., Trampuz, A., Notz, J., Dietler, G., Kasas, S. Nature Nanotechnology, 8

(2013) 522.

Figures

Figure 1. Nanomechanics of lipids

forming SLBs mimicking E. coli

inner membrane. Comparative of

the phase diagram obtained with

DSC and AFM.


Figure 2. AFM topographic

image and height profile analysis

of a SLB composed of

POPE:POPG (3:1,mol/mol) with

LacY at a LPR (w/w) of 0.5

(Z scale = 15 nm) (A). Insert in A

presents a magnified image (470

× 280 nm, Z = 3 nm) where

domains with LacY can be

distinguished from domains

without LacY. Histograms

present the distribution of forces

of domains with LacY (red) and

domains without LacY (green) for

Fy (B) and Fadh (C). Fittings to a

Gaussian distribution are

represented in solid lines.


Plasmonic biosensors advanced by

functional hydrogels

Jakub Dostalek1, Ulrich Jonas2,3, Christian Petri1,2, Nityanand Sharma1,4 1AIT-Austrian Institute of Technology, Muthgasse 11, Vienna 1190, Austria 2University of Siegen, Adolf-Reichwein-Strasse 2, Siegen 57076, Germany

3Foundation for Research and Technology Hellas, P.O. Box 1527, 71110 Heraklion, Crete, Greece 4Nanyang Technological University, Centre for Biomimetic Sensor Science, Singapore 637553

Rapid and sensitive detection of biomarkers is

of a key interest in the field of medical

diagnostics. The paper will present current

advances in optical biosensors for the analysis of trace amounts of biomolecules that combine

plasmonic metallic nanostructures and

hydrogel materials. When post-modified with ligands for the specific capture of target analyte,

these materials can serve as a matrix for specific

capture of target analyte on the surface with good resistance to unspecific sorption of other

molecules present in complex samples. The

capture of target analyte in the hydrogel matrix

can be probed by evanescent field of guided waves. Depending on the thickness of the

hydrogel matrix (from around hundred

nanometers to several micrometers in swollen state), it can be probed by surface plasmons

with probing depth adjusted from around

hundred nanometers (regular surface plasmons) to about micrometer (long range

surface plasmons) or even above (by waveguide

modes supported by hydrogel layer itself) [1, 2].

In addition, matrices prepared from hydrogels that are responsive to external stimulus can be

advantageous for plasmonic sensors relying on

surface plasmons with highly confined field distribution as they can be collapsed after the

capture of the analyte into the plasmonic

hotspot where the maximum field strength occurs. Examples of the implementation of

hydrogel materials for the direct refractometric

detection of small molecules by using antibody

and molecularly imprinted polymer

nanoparticles will be discussed based on

spectroscopy of guided waves [3, 4]. In addition,

surface plasmon-enhanced fluorescence

spectroscopy biosensors that take advantage of

responsive hydrogel binding matrices will be

presented with the limit of detection at low femtomolar concentrations [5].

Acknowledgements: Authors gratefully acknowledge partial support from the Austrian

Science Fund (FWF) through the project

ACTIPLAS (P 244920-N20) and by the International Graduate School Bio-Nano-Tech, a

joint Ph.D. program of the University of Natural

Resources and Life Sciences Vienna (BOKU), the

Austrian Institute of Technology (AIT), and the Nanyang Technological University (NTU).

References

[1] A. Aulasevichet al, Macromolecular Rapid Communications, vol. 30, pp. 872-877, 2009.

[2] J. Dostalek et al., Plasmonics, vol. 2, pp. 97-

106, 2007.

[3] N. Sharma et al., Macromolecular Chemistry and Physics, in press, 2014.

[4] Q. Zhang et al., Talanta, vol. 104, pp. 149-154,

2013. [5] Y. Wanget al., Analytical Chemistry, vol. 81, pp.

9625-9632, 2009.


Localized, DNA based logical circuits as

components for biodetection

Juan Elezgaray

CBMN, UMR 5248, Allée Saint Hilaire, Bat. B14. 33600 Pessac, France

[email protected]

Recent advances in the field of molecular

programming [1,2] have shown that enzyme-free, DNA based circuits can be designed to

perform the basic steps of molecular detection.

Those include amplification and transduction of

non nucleic-acid inputs. The implementation of these circuits as a set of bulk reactions faces

difficulties which include leaky reactions and

intrinsically slow, diffusion-limited reaction rates. In this presentation, I will consider simple

examples of these circuits when they are

attached to platforms (DNA origamis [3]). After discussing their thermodynamic properties [4], I

will show that these platforms can be used to

precisely control the interaction between

different gates. As expected, constraining distances between gates leads to faster reaction

rates. However, it also induces side-effects that

are not detectable in the solution-phase version of this circuitry. In particular, strand

displacement without toehold needs to be

taken into account. Finally, I will present recent results showing how aptamers [5] can be

interfaced with DNA origamis to generalize the

triggering of DNA circuits by non nucleic-acid

inputss

References

[1] Zhang,D.Y. and Seelig,G. (2011) Dynamic DNA

nanotechnology using strand-displacement reactions. Nat. Chem., 3, 103–113.

[2] Li, B., Ellington, A.D. and Che, X. (2011)

Rational, modular adaptation of enzyme-free

DNA circuits to multiple detection methods,

Nucl. Ac. Res., 39, e110.

[3] P. W. K. Rothemund (2006) Folding DNA to

create nanoscale shapes and patterns, Nature, 440, 297.

[4] J.M. Arbona, J.P. Aimé and J. Elezgaray,

Cooperativity in the annealing of DNA

origamis, J. Chem. Phys. 138, 015105. [5] Durand, G., Lisi, S., Ravelet, C., dausse, E.,

Peyrin, E. and Toulmé, J-J (2014)

Riboswitches Based on Kissing Complexes for the Detection of Small Ligands, Ang. Chem.

Int. Ed. 53, 6942-6945.


Development of modified siRNA for gene

silencing

Santiago Grijalvo, Anna Aviñó, Montserrat Terrazas, Adele Alagia, Ramon Eritja

Instituto de Química Avanzada de Cataluña (IQAC), CSIC, CIBER-BBN, Barcelona, Spain

[email protected]

With the advent of RNA interference as a means

to silence gene expression, small interfering RNA (siRNA) oligonucleotides have been

recognized as powerful tools for targeting

mRNAs and eliciting their gene inhibitory

properties [1]. Small RNA duplexes are recognized by a protein complex called RISC

provoking the specific degradation of

messenger [2]. As a consequence of this discovery, siRNA oligonucleotides are now being

intensively investigated as potential therapeutic

agents for various biomedical indications [3]. siRNA are not readily taken up into tissues and

are also susceptible to degradation by

nucleases in the blood. For these reasons the

interest in the design and preparation of modified RNA derivatives that are more stable,

easier to produce at large scale and with a

higher cellular uptake it is of vital importance to improve RNAi limitations [3].

Specifically we will show the development of siRNAs carrying conformationally restricted

pseudonucleosides [4] as well as the synthesis

and properties of siRNA conjugates with

molecules that may enhance cellular uptake such as peptides, lipids and intercalating agents

[5].

References

[1] T. M. Rana. Nature Reviews Mol. Cell Biol. 2007, 8, 23-36

[2] S. M. Elbashir, J. Harborth, W. Lendeckel, A.

Yalcin, K. Weber, T. Tuschl. Nature 2001, 411,

494-498

[3] J. K. Watts, G. F. Deleavey, M. J. Damha. Drug

Discovery Today 2008, 13, 842-855 [4] M. Terrazas, S.M. Ocampo, J. C. Perales, V.

Márquez, R. Eritja. ChemBioChem 2011, 15,

1056-1065

[5] S. Grijalvo, S. M. Ocampo, J. C. Perales, R. Eritja. J. Org. Chem. 2010, 75, 6806-6813


How can I protect my invention?

BioMed Patents

Consuelo Espejo Rodríguez

Spanish Patent Office Examiner

[email protected]

Inventions are one of the most powerful

intangible values within a company, and Industrial Property can be the most effective

way of protection. The objective of this lecture is

to give an overview of what and how the

inventions (particularly the nanoinventions applied in Phama and Bio fields) can be

protected.

We will also answer some questions such as:

Do the industrial property titles expire

and are territorially limited?

What I should and I shouldn’t do before

making available any important

technical information?

What are the patent requirements in nanotechnology?

How Pharma and Bio inventions are

evaluated by the Patent Offices?

What are the exceptions to patentability in Biotechnology?

Did you know that all nanopatents are

classified in a specific technical “drawer”

and that you have free access to all of

them?

Have you heard about the Nanopharma

Technological alerts?

How to search in Espacenet, ChemBL (Chemical structure drawing) and NCBI

(Mesh)?

Is there any tips for drafting a successful

patent?


Polymeric nanoparticles, prepared from

nano-emulsion templating, as novel

advanced drug delivery systems crossing the

Blood-Brain Barrier

Cristina Fornaguera1, Aurora Dols-Pérez1, Gabriela Calderó1, M.José García-Celma2, Conxita Solans1 1Institut de Química Avançada de Catalunya (IQAC-CSIC) and CIBER de Bioingeniería, Biomateriales y

Nanomedicina (CIBER-BBN), C/ Jordi Girona 18 – 26, 08034, Barcelona, Spain 2Department of Pharmacy and Pharmaceutic Technology, University of Barcelona, Barcelona, Spain

[email protected]

Introduction

The administration of drugs to the Central

Nervous System (CNS) is a key issue for the treatment of neural diseases. The intravenous

administration, compared to the highly invasive

intracranial administration, represents a promising alternative. However, due to the

presence of the Blood-Brain Barrier (BBB), most

drugs do no reach the CNS, thus producing low

therapeutic efficiencies [1].

In this context, the need for effective drug

delivery systems to the CNS is still a challenge. Polymeric nanoparticles constitute a promising

strategy to target drugs through the BBB using

the intravenous route. Diverse types of molecules have been previously used for the

nanoparticle functionalization to target the

BBB, such as permeabilization agents to

achieve a passive targeting or monoclonal antibodies against receptors in the BBB to

achieve an active targeting [2]. However, current

approaches are not sufficiently efficient on the BBB crossing [3].

Therefore, to develop polymeric nanoparticles that efficiently cross the BBB, non-toxic,

biocompatible and biodegradable materials are

required, together with a safety method of

preparation [3]. The use of a preformed polymer

instead of the in situ polymerization and the

nano-emulsion templating technology

constitutes an interesting and versatile strategy

[4]. Nano-emulsions are fine emulsions with

droplet sizes typically between 20 – 200 nm, showing high kinetic stability against

sedimentation / creaming and a transparent to

translucent appearance [5]. Their preparation by low-energy emulsification methods

represents an alternative to high-energy

methods not only for obtaining nano-emulsions

with smaller and less polydisperse droplets, with an energy and cost efficient procedure, but

also due to the high versatility of achieving

nano-emulsions with the desired characteristics. Among low-energy methods, the

phase inversion composition (PIC) method is

advantageous for the pharmaceutical industry [5] because it can be performed under mild

conditions (e.g. mild temperatures). Once nano-

emulsions are prepared, the formation of

nanoparticles is achieved by solvent evaporation (schematic representation of

nanoparticle production methodology on

Figure 1).

Objectives

The aim of this work was to design polymeric nanoparticles from nano-emulsions templating

that efficiently cross the BBB, once

administered by the intravenous route.


Results

Poly-(lactic-co-glycolic acid) (PLGA)

nanoparticles were designed by the PIC nano-

emulsification method followed by solvent evaporation. Nano-emulsions were stabilized

with the polysorbate 80 surfactant, since

previous studies reported its ability to enhance

BBB permeability. Loperamide was incorporated into nanoparticles, prior to nano-

emulsion formation, with the aim to study the

BBB nanoparticles crossing via in vivo analgesia measurements, since the drug loperamide

hydrochloride (LOP) produces a central

analgesia, but it does not cross the BBB by itself. Nanoparticle surface was further functionalized

with the anti-transferrin receptor monoclonal

antibody (anti-TfR mAb), overexpressed in the

BBB.

Polymeric nano-emulsions were obtained in the

electrolyte solution (W) / polysorbate 80 (O) / [4wt% PLGA + 0.1wt% LOP in 20/80

ethanol/ethyl acetate] system, at 25ºC. Nano-

emulsions with 90wt % of water content, with an O/S ratio of 70/30 were chosen due to the

compromise between the low surfactant

content and sizes appropriate for the

intravenous administration (around 120 nm). Polymeric nanoparticles, formed by solvent

evaporation from template nano-emulsions,

showed hydrodynamic radii of around 100 nm and negative surface charges. Loperamide

encapsulation efficiency was found to be very

high (>99wt%) and the in vitro release profile from nanoparticles was sustained, as compared

with the drug in aqueous solution. The covalent

binding of the anti-TfR mAb to the nanoparticle

surface was successfully achieved by means of the carbodiimide reaction. A concentration step

was required to achieve therapeutic loperamide

concentrations. In vitro toxicity determinations demonstrated that nanoparticles were non-

hemolytic neither non-toxic at the in vivo

required concentrations. Central analgesia was

measured in vivo by means of the hot plate test.

The passive targeting of the BBB by non-

functionalized nanoparticles produced slight

analgesic effects, while the active BBB targeting

by the anti-TfR mAb produced a marked

analgesia (50% more than the basal level). Therefore, it could be concluded that the

formulated nanoparticles, functionalized with

the anti-TfR mAb constitute a promising

alternative to deliver drugs to the CNS by the intravenous route of administration.

References

[1] Kabanov A.V., Gendelman H.E., Progress in

Polymer Science, 32 (2007) 1054 – 1082. [2] Wohlfart S., Gelperina S., Kreuter J., J Control

Release, 161(2) (2012) 264.

[3] Tosi G., Fano R.A:, Bondioli L., Badiali L.,

Benassi R., Rivasi F., Ruozi B., Forni F., Vandelli A., Nanomedicine, 6(3) (2011) 423 – 436.

[4] Anton N., Benoit J.P., Saulnier P., J Control

Release, 128(3) (2008) 185. [5] Solans C., Solè I., Curr Opin Colloid Interf Sci,

17 (2012) 246.

Figures

Figure 1. Schematic representation of the whole process

for the nanoparticles production.


Kinetics of the protein corona assembly on

nanoparticlesinetics of

the protein corona assembly on

nanoparticles

Giancarlo Franzese

Departament de Fisica Fonamental, Universitat de Barcelona. Marti i Franques 1.Barcelona - Spain

[email protected]

Nanoparticles (NPs) in the extracellular matrix

are immediately coated by layers of biomolecules forming a "protein corona". The

protein corona gives to the NPs a "biological

identity" that regulates the NP-cell interaction. Therefore, the cell uptake of the NPs is strongly

affected by the protein corona. For this reason

learning to predict the biological identities of NPs based on a partial experimental knowledge

is essential to foresee a priori the safety

implications of a NP for human health and,

more in general, the environment.

To this goal we propose a multiscale approach

that, adopting numerical techniques from all-atoms simulations [1] to coarse-grained models

for protein-protein [2] and protein-NP

interactions [3], accounts for the effect of

interfaces on the hydration layer [4,5] in the description of proteins [6] and NPs in water [7].

The approach allows us to predict the protein

corona assembly based on a partial

experimental knowledge of the protein affinities

for NPs with a specific physico-chemical

composition and the size [8].

Specifically, we study, by numerical simulations,

the competitive adsorption of proteins on a NP suspended in blood plasma as a function of

contact time and plasma concentration. We

consider the case of silica NPs in a "simplified" blood plasma made of three competing

proteins: Human Serum Albumin, Transferin

and Fibrinogen. These proteins are of particular interest because they have a high concentration

in plasma, or because they are the most

abundant in the corona of silica NPs. Our results are compared with experiments made under

the same conditions showing that the approach

has a predictive power [4].

References

[1] M. Bernabei, G. Franzese et al. in preparation; see also T.

Kesselring, G. Franzese, S. V. Buldyrev, H. J. Herrmann, H.

E. Stanley, Nanoscale Dynamics of Phase Flipping in

Water near its Hypothesized Liquid-Liquid Critical Point,

Scientific Reports (Nature Publishing Group) 2, 474

(2012).

[2] L. Xu, S. V. Buldyrev, H. E. Stanley, G. Franzese,

Homogeneous Crystal Nucleation Near a Metastable

Fluid-Fluid Phase Transition, Physical Review Letters

109, 095702 (2012).

[3] P. Vilaseca, K.A. Dawson, G. Franzese, Understanding

and modulating the competitive surface-adsorption of

proteins, Soft Matter, 9, 6978 (2013).

[4] M. G. Mazza, K. Stokely, S. E. Pagnotta, F. Bruni, H. E.

Stanley, G. Franzese, More than one dynamic crossover

in protein hydration water, Proceedings of the National

Academy of Sciences of the USA 108, 19873 (2011).

[5] V. Bianco and G. Franzese, Critical behavior of a water

monolayer under hydrophobic confinement, Scientific

Reports (Nature Publishing Group) 4, 4440 (2014).

[6] G. Franzese, and V. Bianco, Water at Biological and

Inorganic Interfaces, Food Biophysics, 8, 153 (2013).

[7] E. G. Strekalova, M. G. Mazza, H. E. Stanley, and G.

Franzese, Large decrease of fluctuations for

supercooled water in hydrophobic nanoconfinement,

Physical Review Letters 106, 145701 (2011).

[8] O. Vilanova, J.J. Mittag, P. M. Kelly, S. Milani, K.A.

Dawson, J. Rädler, G. Franzese, Predicting the kinetics of

Protein-Nanoparticle corona formation in model

plasma, in preparation.


Imaging molecular structure of plant cells by

Confocal Raman microscopy

Notburga Gierlinger1,2, Batirtze Prats Mateu1, Barbara Stefke1, Ursula Lütz-Meindl3 1Dep. of Materials Science and Process Engineering, BOKU-University of Natural Resources and Life

Sciences, Peter Jordan Str. 82, 1190 Vienna, Austria 2Institute for Building Materials, ETH Zurich, Zurich, Switzerland

3Cell Biology Dep., Plant Physiology Division, University of Salzburg, Salzburg, Austria

[email protected]

During the last years Confocal Raman

microscopy evolved as a powerful method to

get insights into chemistry and structure of plant cells and cell walls with a spatial

resolution of around 300 nm. Two-dimensional

spectral maps can be acquired of selected areas and Raman images calculated by integrating

the intensity of characteristic spectral bands or

by using multivariate data analysis methods. This enables direct visualization of changes in

the molecular structure and analyzing the

spectra laying behind the chemical images

reveals detailed insights into cell wall chemistry and structure [1-4].

Insights have been gained into the design of plant cell walls to achieve movement in wooden

parts of trees or in roots by means of gelatinous

fibers. Plant cell walls are based on cellulose microfibrils embedded in a matrix of

hemicelluloses and lignin. The orientation of the

cellulose microfibrils (alignment with respect to

the fiber axis) on the nanolevel, the arrangement of different layers on the

microlevel, as well as the amount of lignin

determine mainly properties and functionalities. These parameters are elucidated in-situ in

context with the microstructure and reveal thus

the design of e.g. so called gelatinous fibers. Almost pure cellulose has been identified as the

main swelling core of this fibers, functionalized

by a small outer lignified layer with high

microfibril angle [4-7].

Recently the potential of method has also been

shown on the algal model system Micrasterias

denticulata. The changes in the molecular structure within the different cell organelles and

structures can be followed as well as the

changes in the outer cell wall during growth (Figure 1).

Funding: Austrian Science Fund (FWF): START Project Nr. Y-728-B1

References

[1] Gierlinger, N et al. Nature Protocols (2012)

[2] Gierlinger, N et al. Journal of Experimental Botany 62 (2) (2010) 587-595.

[3] Gierlinger, N. Frontiers in Plant Science (2014)

doi: 10.3389/fpls.2014.00306 [4] Gierlinger, N. & Schwanninger, M. Plant

Physiology 140, (2006) 1246-1254.

[5] Goswami, L et al., Plant Journal. 56-4 (2008)

531-538.


Figures

Figure 1. Raman spectroscopic image of Micrasterias

denticulata (160 x 160 µm, 0.5 µm step size, 532 nm

WITec300RA). Based on the 102 400 Raman spectra images

were calculated with the help of non-negative matrix

factorization (NMF), a method to evaluate distribution

maps of different components and demixed basis spectra.

The different colours represent the different basis spectra

(components). The blue colour represents the outer

cellulosic cell wall, which is more highlighted in the old

half of the cell (lower part of the image) due to higher

cellulose amount and crystallinity than in the newly

formed young part (upper smaller side). In the inner part

the red colour corresponds to starch and highlights the

small round pyrenoids in the older cell half, which are

embedded in the chloroplast. Proteins, pectins and fats

are coloured in green and yellow.


Optopharmacology to regulate endogenous

proteins with light

Pau Gorostiza

IBEC (Institut de Bioenginyeria de Catalunya) & ICREA. Parc Científic de Barcelona, Barcelona

[email protected]

The development of light-regulated drugs

(optopharmacology) has important applications to neuronal receptors and enables

the remote stimulation of neurons without

genetic manipulation. Controlling drug activity

with light offers the possibility of enhancing pharmacological selectivity with spatial and

temporal regulation, thus enabling highly

localized therapeutic effects and precise dosing patterns. Recent advances of the laboratory will

be presented, including the development and

characterization of the first photoswitchable allosteric modulator of a G protein-coupled

receptor.


Cyano-Bridged coordination polymers

nanoparticles as contrast agents for

Biomedical Imaging

Y. Guari, J. Larionova and J. Long

Institut Charles Gerhard, UMR 5253 CNRS-UM2-ENSCM-UM1, Université Montpellier II Place Eugène Bataillon, 34095, Montpellier Cx 5, France

From 1704, year of the discovery of the oldest

coordination polymer, Prussian blue, to now,

many cyano-bridged coordination polymers

were synthesised and extensively studied. This research field remains very active with the

development of materials featuring magnetic,

photomagnetic, sorption or catalytic properties.

Significant parts of the current research activity on these materials is devoted to the synthesis

and study of size and shape controlled cyano-

bridged coordination polymer materials at the

nanoscale [1]. These nanomaterials have the

same advantages as the corresponding bulk

materials. Among them may be mentioned the

versatility of precursors that can be assembled, the adjustable porosity and the possibility to

combine several properties within a single

nano-object [2].

In addition, the ease of synthesis of these nanoparticles under mild conditions allows

control of their size, shape and sometimes their

organization and thus control over their

properties. We will illustrate the latest

developments made in our research group on

synthetic methodologies that we developed for

the preparation of nano-objects or nano-composites of these materials and magnetic,

magneto-optic or sorption properties

associated therewith.

We will also address the potential application of cyano-bridged coordination polymers

nanoparticles in the field of medical imaging.

Keywords: Prussian blue, nanoparticles,

medical imaging.

References

[1] (a) S. P. Moulik, G. C. De, A. K. Panda, B. B.

Bhowmik, A. R. Das, Langmuir 1999, 15, 8361; (b) S. Vaucher, M. Li, S. Mann, Angew. Chem.,

Int. Ed. 2000, 39, 1793; (c) J. Larionova, Y. Guari,

C. Sangregorio, Ch. Guérin, New J. Chem. 2009, 33, 1177; (d) F. Volatron, L. Catala, E. Riviere, A.

Gloter, O. Stephan and T. Mallah, Inorg. Chem.,

2008, 47, 6584. [2] (a) G. Maurin-Pasturel, J. Long, Y. Guari, F.

Godiard, M.-G. Willinger, Ch. Guérin, J.

Larionova Angew. Chem. Int. Ed. 2014, 53,

3872; (b) M. Perrier, S. Kenouche, J. Long, T. Kalaivani, J. Larionova, C. Goze-Bac, A.

Lascialfari, M. Mariani, N. Baril, C. Guérin, B.

Donnadieu, A. Trifonov, Y. Guari Inorg. Chem., 2013, 52, 13402.

Figures

Figure 1. Biomedical imaging using cyano-bridged

coordination polymer nanoparticle.


Effect of nanoscale surface roughness on the

adhesion and proliferation of normal skin

fibroblasts and HT1080 fibrosarcoma cells

Kanioura A.1, Bourkoula A.1, Tsougeni K.2, Petrou P.1, Kletsas D.3, Tserepi A2, Gogolides E.2, Kakabakos S.1 1Institute of Nuclear and Radiological Sciences & Technology, Athens, Greece

2Institute of Nanoscience & Nanotechnology, NCSR “Demokritos”, Athens, Greece 3Institute of Biosciences and Applications, NCSR “Demokritos”, Aghia Paraskevi, Athens, Greece

[email protected]

Separation and enrichment of cancer cells from

a mixture with normal cells are important steps for cancer diagnosis. The methods used for the

separation of cancer from normal cells are

based on observation of morphological features, labeling of the cells with specific

markers or differences in physical properties

between cancer and normal cells (e.g. cell size, density, adhesion, dielectric properties) [1].

Surface nanotopography, as it has been

reported in the literature, affects cell adhesion,

proliferation and viability [2]. However, there are few reports about the potential of such

nanostructured surfaces for

separation/enrichment of cancer cells from mixtures with normal ones [3]. Here, we

investigated the effect of surface nanotexturing

on the adhesion, viability, and proliferation of normal fibroblasts and fibrosarcoma HT1080

cancer cells on thin PMMA films nanotextured

through oxygen plasma etching in comparison

to flat untreated surfaces.

Randomly nanotextured PMMA film surfaces

were prepared following a published procedure [4]. Briefly, a 25% (w/w) PMMA solution was spin

coated on Si wafers at 1500 rpm followed by

baking for 1.5 h at 150 oC. The films were treated with O2 plasma. The etching conditions were:

bias voltage: -100 Volts; electrode temperature:

15 oC; etching time: 3 min; source power: 1900

W; pressure: 0.75 Pa and oxygen flow: 100 sccm

in a Helicon Plasma reactor (MET system,

Adixen). The plasma treated surfaces (Fig. 1)

along with untreated ones were then used as

substrates to culture 10000 cells/ml normal fibroblasts or HT1080 cells for periods of 1 and 3

days. The adhered cells were fixed and stained

with phalloidin-Atto 488 (F-actin) and DAPI (nucleus) for cell counting and observation

using an epifluorescence microscope, as

described previously [5].

It was found that on the untreated surfaces after

1-day culture the number of adhered cells per

surface area was approx. 1200 and 1000 cells/cm2 for the HT1080 and the normal

fibroblasts, respectively. After 3 day culture the

HT1080 cell population increased 4 times on these surfaces and the normal fibroblasts 1.5

times. On the other hand, concerning O2 plasma

treated surfaces after 1-day culture approx. 2500 HT1080 cells and 800 normal fibroblasts had

been adhered per sq. cm. A significant finding

was that after 3-day culture the HT1080 cells

population per surface area was increased almost 4 times (9800 cells/cm2) whereas, the

number of normal fibroblasts was decreased by

20% (640 cells/cm2) compared to 1-day culture. In addition, as it is depicted in Fig. 2A and B the

morphology of normal fibroblasts on the

nanotextured surfaces was considerably affected after 3-day culture, as witnessed by the

excessive distortion of cytoskeleton, compared

to the untreated surfaces. In contrast, surface

nanotexturing did not influence the morphology

of HT1080 cells (Fig. 3A and B). The reduced cell

population of normal fibroblasts on the rough


surfaces after 3-day culture was not due to

apoptosis as it was proved through

staurosporine assay. Therefore to explain this

finding we performed focal points staining using fluorescently labeled anti-vinculin antibody. It

was found that the number of focal points per

normal fibroblast cell was reduced by 40%

whereas, that of HT1080 was increased by 30% on the nanotextured surfaces compared to the

untreated ones. Therefore the decreased

number and the distortion of the cytoskeleton of normal fibroblasts on the nanotextured

surfaces could be ascribed to the considerable

decrease of the focal points formation, which affects cell adhesion and viability. In conclusion,

taking into account that adhesion and

proliferation of normal skin fibroblasts is

inhibited on O2 plasma nanotextured PMMA surfaces in contrast to HT1080 cells, these

surfaces could be useful for the enrichment and

isolation of fibrosarcoma cells derived from tissues suspected for neoplasias and help to

improve cancer diagnosis.

References

[1] J.H. Kim, J.S. Kim, H. Choi, S.M. Lee, B.H. Jun, K.N. Yu, E. Kuk, Y.K. Kim, D.H. Jeong, M.H. Cho,

Y.S. Lee, Anal. Chem. 78 (2006) 6967-6973.

[2] K. Anselme, P. Davidson, A.M. Popa, M. Giazzon, M. Liley, L. Ploux, Acta Biomater. 6

(2010) 3824-3846.

[3] K.W. Kwon, S.S. Choi, S.H. Lee, B. Kim, S.N. Lee, M.C. Park, P. Kim, S.Y. Hwang, K.Y. Suh,

Lab on a Chip 7 (2007) 1461-1468

[4] E. Gogolides, V. Constantoudis, D.

Kontziampasis, K. Tsougeni, G. Boulousis, M. Vlachopoulou, A. Tserepi, J. Phys. D: Appl.

Phys. 44 (2011) 174021.

[5] D. Kontziampasis, A. Bourkoula, P.Petrou, A. Tserepi, S. Kakabakos, E. Gogolides,

Proceedings of SPIE 8765 (2013) 87650B.

Figures

Figure 1. SEM images of O2 plasma treated PMMA surfaces

at bias voltage 100V for 3 min.

Figure 2. Fluorescence microscope images of normal skin

fibroblasts cultured for 3 days on untreated flat PMMA

surfaces (A) or plasma treated surfaces (100 V, 3 min) (B).

Cytoskeleton (F-actin) was stained with phalloidin-Atto488

and cells nuclei with DAPI.

Figure 3. Fluorescence microscope images of HT1080

fibrosarcoma cells cultured for 3 days on untreated flat

PMMA surfaces (A) or plasma treated surfaces (100 V, 3

min) (B). Cytoskeleton and cells nuclei were stained as

described in Fig. 2.

A B

A B


Screen printed superhydrophobic surfaces

as enablers for Capillarity-Driven

Biodetection Devices for Food Safety and

Clinical Analysis: towards ASSURED sensors Ioanis Katakis

Bioengineering and Bioelectrochemistry Group, Universitat Rovira i Virgili,. Tarragona, Spain

[email protected]

Lab-on-Chip (LOC) concepts are usually realized as microsystems fabricated by microfabrication technologies of varying degrees of complexity and

operated by control equipment that commonly

require external power sources, fluid movement devices, and detection systems. Investment in purpose-built manufacturing lines, for micron or sub-micron featured microsystems and

sophisticated control apparatus is justified for high throughput analytical tasks based on limited-

volume samples.

Most analytical needs in food safety and decentralized diagnostics/theranostics are not

limited by the available sample volume and are not high throughput in nature; rather it is cost and ease

of use that eventually decide their large scale

adoption. A convenient alternative for the realization of such application-oriented LOC concepts is to

manufacture simple, basic microsystems by 3-D screen printing. Such elemental microsystems can

be operated almost autonomously: fluid movement can be achieved through capillary action, and both

fluidic control and detection by electrochemistry. Screen printing manufacturing requires a simple and relatively low cost production line and provides the

flexibility to incorporate different materials in the 3-D design accommodating both structural and actuation or detection elements. We demonstrate that basic unit operations such as dissolution,

separation, mixing, reaction, flow manipulation and

detection can be satisfactorily realized and controlled for most detection applications with low power requirements.

We applied such simple architectures in integrated devices that can detect pathogens in food and

activated sludge. In a particular product developed, Salmonella could be detected in poultry meat extracts with limit of detection of 10-20 CFUs within

15 hours of sampling. When proteins need to be

detected by immunochemical methods in lateral flow-type devices rendered by the 3-D screen printing method, we demonstrate that flow control is crucial for signal development and successful

immunorecognition. We provide such flow control with electrochemically activated stop/go printed

microvalves that modulate the hydrophilicity of the

device walls. We thus achieve successful detection

of -lactoglobulin (a potential food allergen) or HCG

(a pregnancy indicator). We therefore present a simple to manufacture, generic, low cost, and easy

to use technology platform that can tackle a variety of analytical problems.

We discuss how these technologies in combination

with nanochemical solutions can provide a possible platform towards ASSURED diagnostics

/theranostics.

Acknowledgements: This work was made possible through support by the Spanish Ministry of Science and Innovation (BIO2010-20359 MICROCAP) and by

the Catalan agency of support of University research (AGAUR grant 2010VALOR00063 SALMONELLA TRUST).


Large-scale dendrimer-based uneven

nanopatterns of RGD towards improved

architectural networks in chondrogenesis

Anna Lagunas

IBEC (Institut de Bioenginyeria de Catalunya) Parc Científic de Barcelona, Barcelona [email protected]

Cartilage damage is the main cause of joint

disorders, having a huge impact on an

increasingly ageing population. Cartilage

inability to spontaneous repair and regenerate has stimulated clinical and experimental work

towards optimal cartilage regeneration.

Transplantation of mesenchymal stem cells (MSCs), which have a vast proliferative capacity

and differentiation potential, has emerged as a

promising strategy to treat joint defects. However direct implantation of undifferentiated

MSCs without any preconditioning lead to

calcification of the implanted cells, fibrogenesis

and heterotopic tissue formation in the cartilage [1].

As in most biological systems showing multi-level organization with cross-level

interdependence, extensive cell-cell

communication networks are formed during cartilage development. In the initial stages of

chondrogenesis MSCs condensation takes

place, leading to a marked decrease of the

intercellular space, and the occurrence of a large number of cell-to-cell contacts of the gap-

junction (GJ) type. Signaling in multi-cellular

networks is strongly influenced by the system architecture: in conventional culture systems of

chondrogenic differentiation of MSCs, a hyaline-

like, zonal-distributed cartilage structure, in which nearly cylindrical cells are aligned and

connected side-by-side and end-to-end along

the proximal-distal axis of the limb, is not

sustained; instead, irregularly shaped cells

spread randomly, resulting in randomly

distributed cell junctions.

In this sense it is of crucial importance to

provide an appropriate cell environment that

allows the establishment and maintenance of

cell-to-cell interactions during the different stages of MSCs differentiation, and that while

still favoring strong cell anchorage, allows the

subsequent transplantation and release in the injured area. Taking advantage of a recently

developed dendrimer-based large-scale

nanopatterning approach [2], surfaces of poly-L-lactic acid (PLA) nanopatterned with cell

adhesive dendrimers, at different initial bulk

concentrations, were used as substrates for

chondrogenesis. Surface nanopatterning is applied to modulate cell-biomaterial

interaction in order to better mimic cartilage

architecture.

References

[1] Cui, J. H., Park, S. R., Park, K., Choi, B. H., Min, B.

H. Preconditioning of mesenchymal stem cells

with low-intensity ultrasound for cartilage formation in vivo. Tissue Eng. 2007; 13: 351-60.

[2] Lagunas, A., Castaño, A. G., Artés, J. M., Vida, Y.,

Collado, D., Pérez-Inestrosa, G., Gorostiza, P., Claros, S., Andrades, J. A., Samitier, J. Large-

scale dendrimer-based uneven nanopatterns

for the study of local RGD density effects on cell adhesion. Nano Research 2014; 7: 399-409.


Figures

Figure 1. (a) AFM image (scale bar = 250 nm) of nanopatterned dendrimers. Inset: magnified phase image of one of the

nanodomains (scale bar = 50 nm). (b) dmin probability contour plot showing regions of high local ligand density. Color scale:

dmin values in nm. (c) Fluorescent micrograph of a fibroblast after 4.5 h in culture on the nanopatterns. Inset: magnified

portion of FAs formed at the cell periphery. Scale bar = 20 μm. (d) Optical microscope image showing early hMSCs cell

condensation on nanopatterns (chondrogenesis, day 3).


Following the degradation and biological

fate of polymeric poly (lactic-co-glycolic

acid) nanoparticles

Jordi Llop, Marco Marradi, Pengfei Jiang, María Echeverría, Shan Yu, Boguslaw Szczupak, Maria

Puigivila, Vanessa Gómez-Vallejo and Sergio E. Moya. CIC biomaGUNE, Paseo Miramón 182, 20009 San Sebastián, Spain

[email protected]

Due to their small size and unique physic-

chemical properties, nanoparticles (NPs) have

been proposed as diagnostic, therapeutic or even theragnostic tools. By appropriate multi-

functionalization, NPs can be administered

systemically and directed towards specific organs or tissues, providing thus enhanced

therapeutic/diagnostic efficacy while reducing

significantly undesired side- or toxicological effects [1].

When moving to in vivo applications, the

determination of the pharmacokinetic properties and biological fate of NPs is of

paramount importance, both to assess

potential toxicological effects and to anticipate therapeutic efficacy. However, NPs are

extremely difficult to detect and quantify once

distributed in a biological system. One alternative to overcome this problem consists of

labeling the NPs with radionuclides that can

lead to their detection with ultra-high sensitivity

using in vivo imaging techniques such as Positron Emission Tomography (PET) or Single

Photon Emission Computerized Tomography

(SPECT) [2,3]. Of note, radiolabelling and subsequent imaging studies provide

information about the loci of the radionuclide,

but no information about the radiochemical integrity or the chemical stability of the NPs is

obtained.

Here, we present an unprecedented dual-

labeling strategy to assess simultaneously the

pharmacokinetic properties and biological fate

of core-shell NPs after intravenous

administration in rodents. Fe3O4/poly(lactic-co-

glycolic acid) (PLGA)/Bovine serum albumin (BSA) NPs (Figure 1) were simultaneously

labelled with 111In, which was entrapped into the

Fe3O4 crystal lattice, and 125I, which was covalently attached to the tyrosine residues of

BSA. Both isotopes emit gamma rays with

different energies (171 and 245 keV for 111In, 35.5 keV for 125I).

In a first step, biodistribution studies were

performed in mice using dissection/gamma counting. With that aim, dual-labelled NPs

(containing c.a. 111 kBq of 125I and 370 kBq of 111In) were administered to animals, which were sacrificed at different time points (5 min-48h),

the organs were harvested and the gamma

emission spectra for each organ and blood were analyzed using a multichannel analyzer.

Progressive accumulation of 125I in the thyroid

glands, the intestine and the bladder, together

with preferential accumulation of 111In in other major organs such as the lungs and the liver,

suggest a fast degradation of the NPs after

administration.

The results were confirmed by in vivo SPECT

studies, using a microSPECZT-Visio SPECT-CT system. Labelled NPs (containing c.a. 7.4 MBq of

each radionuclide) were administered via the

tail vein and static images were acquired at 1, 24

and 48 hours after administration.

Reconstruction of the images in different energy

windows was performed, and energy-resolved


images to determine the loci of both isotopes

over time were obtained. As for

dissection/counting studies, images showed

progressive accumulation of 125I in the thyroid gland, and elimination of this isotope mainly via

urine and intestine. This pattern is compatible

with a progressive dissociation of the protein

(BSA) from the NPs and subsequent detachment of 125I.

The strategy reported here, based on incorporation of two different gamma emitters

(with different emission energies) followed by

imaging studies with energy discrimination, might be applied to the determination of the

biodistribution pattern and biological fate of a

wide range of core-shell NPs.

References

[1] Janib SM, Moses AS, MacKay JA, Adv Drug

Deliv Rev 62 (2010) 1052-1063.

[2] Pérez-Campaña C, Gómez-Vallejo V, Puigivila M, Martin M, Calvo-Fernández T, Moya SE,

Ziolo RF, Reese T, Llop J. ACS Nano 7(4) (2013)

3498-3505.

[3] Frigell J, Garcia I, Gómez-Vallejo V, Llop J, Penades S. J Am Chem Soc 136(1) (2014) 449-

457.

Figures

Figure 1. TEM image of Oleic acid-coated iron oxide

nanoparticles encapsulated with PLGA and stabilized with

BSA.


Biofunctional Surfaces for Multiplexed

Diagnostic Platforms using Site‐Encoded

DNA Strategies

M.‐Pilar Marco

Nanobiotechnology for Diagnostic (Nb4D) group, IQAC‐CSIC, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER‐BBN), Barcelona, Spain

[email protected]

Advances in genomics and proteomics, point to

a future in which clinical diagnostic will be

based on molecular signatures characteristic of the health/disease status of the individuals, for

which simultaneous detection of multiple

biomarkers will be required. Moreover, future trends in medicine demand for rapid, reliable

diagnostic technologies able to assist doctors

on a more personalized and efficient medicine. In this respect, research in

micro/nanobiotechnologies may allow the

development of a new generation of improved

diagnostic devices based on novel biosensing systems. Biosensors are devices responding to

biomolecular recognition events occurring at

the surfaces of particular micro/nanostructured materials, known as transductors, which

defined physical properties are influenced by

those specific events. To achieve this goal there is the need to construct homogeneous,

organized, biocompatible and stable functional

biohybrid surfaces in which the bioreceptor and

the material behave as a single unit. However, preparation of reliable bioreceptor protein

multiplexed platforms is still a challenge due to

the molecular variability and complex nature of proteins (different hydrophobicities, acidic or

basic characters, functionality, etc.). Thus,

development of protein microarray technology has not been as straightforward as the DNA

microarray technology. Unlike nucleic acids,

which are relatively homogeneous in terms of

structural and electrostatic properties, proteins

can be extremely diverse regarding chemical

structure and biological properties. Preventing

protein denaturation and maintaining structural

conformations and biofunctionality, while

constructing these biohybrid surfaces that will act as transductors, are key issues. An

alternative to circumvent these limitations is the

use of oligonucleotide probes with well‐known sequences and their subsequent hybridization

with their complementary oligonucleotides

previously immobilized on the surface. Examples on the use of this strategy, known as

DNA‐Directed Immobilization (DDI), to develop

fluorescence site‐encoded DNA addressable

microarrays and biosensors platforms based on distinct principles will be presented.

Figures

Figure 1. DDI schematic approach of a LSPR

immunosensor chip.


From Basic Research to an Industrial

Product: The case of Goldemar

Ernest Mendoza

Goldemar Solutions, c /Baldiri Reixac 15, 08028 Barcelona, Spain

[email protected]

Over the last years there has been a massive

research effort in the field of nanotechnology. These developments are already in the market

and many more to come in the near future.

However, the process to bring an outcome from

the basic research to an industrial product is long and complicated. With this talk I will first

introduce our company and the

nanotechnology product that we have developed in the field of cleantech. Also, I would

like to explain the process that we have

followed to bring our product to the market. Not only from a technological point of view but also

from a market, investment and financial

prospective.


Radiosensitizing Effect of Gold

Nanoparticles under kV- and MV- X-ray

Irradiations

Masaki Misawa1, Shota Kuribayashi2, Masashi Hayano2, Yukiko Iwata2, Masanori Sato2, Katsuhide Fujita1 1National Institute of Advanced Industrial Science & Technology (AIST) Ibaraki 305-8564, Japan

2Komazawa University 1-23-1 Komazawa, Setagaya-ku, Tokyo 154-8525, Japan

[email protected]

Introduction:

As a possible radiosensitizer in radiotherapy, we

investigated the generation of reactive oxygen species (ROS) from dispersed gold

nanoparticles (AuNPs) with average diameters

of 5-60nm under clinical Xray irradiation[1][2]. The same AuNPs were added to the cultures of

HeLa cells and their survivability was measured.

Contribution of ROS generation to the cell survivability was discussed.

Materials and Methods: Concentrations of

AuNPs (BBI solutions) were changed at 0, 36, 72, and 144μM in 96 multi-well plates. ROS

generation was measured by a fluorescent

reagent Aminophenyl fluorescein ( APF, Sekisui Medical), which is sensitive to hydroxyl radicals

(OH•). The integrated X-ray doses were varied

from 1 to 10.0 Gy. A Mitsubishi linac (Model:EXL-15SP) was operated at 10MV with a dose rate of

1Gy/min. Survivability of HeLa cells were

measured by absorbance of WST-1 (Roche) at

440nm.

Results and discussion:

APF fluorescent intensity indicated that ROS generation for 20 – 80nm Au colloids was

greater than that of distilled water by a factor of

5-7 in a concentration-dependent way (Fig.1). Because of APF’s specific sensitivity, we

consider that OH• was a major species

generated under x-ray irradiation. Regardless of

the same mass density, ROS generation in 5nm

and 10nm colloids was suppressed. With the

addition of 75μM 5-60nm AuNPs in HeLa cell

cultures for 24hours, X-ray doses up to 10Gy

were given. The cell survivability was decreased

as the X-ray doses. Sensitizing effect was observed over the entire dose range for 5nm

AuNPs, and over the low doses up to 5Gy for

20nm and 40nm AuNPs. Sensitizing effect was not observed for 60nm AuNPs over the entire

dose range.

Conclusion:

AuNPs function as a possible x-ray sensitizer by

causing damage with a augmented effect of

OH•. Particles size can be a key factor in ROS generation and cell damage.

References

[1] Misawa M, Takahashi J., Nanomedicine 7(5)

(2011), 604-14.

[2] Takahashi J., Misawa M., Radiation Physics

and Chemistry 78(11) (2009) 889-98.


Figures

Figure 1. APF fluorescent intensity indicates ROS

generation. Over 20nm AuNPs showed enhanced ROS

generation relative to distilled water by a factor of 5-7.

Figure 2. Decrease in HeLa cell survivability as the X-ray

dose. 5-40nm AuNPs showed sensitization effect.


Nanomechanics of the extracellular matrix

of lung and heart tissues

Daniel Navajas

Institute for Bioengineering of Catalonia, School of Medicine - University of Barcelona, and CIBER of

Respiratory Diseases. Barcelona, Spain

[email protected]

Cells sense and actively respond to mechanical features of their microenvironment. Moreover,

mechanical cues have been shown to mediate

critical cell functions including proliferation,

differentiation, gene expression, contraction, and migration. Therefore, a precise definition of

the mechanical properties of the extracellular

matrix (ECM) is needed to further our understanding of the cell-microenvironment

interplay. We use atomic force microscopy

(AFM) to study nanomechanical properties of

lung and heart ECM. Thin slices (10-20 m thick)

of decellularized rat lung parenchyma and

mouse heart left ventricle are probed with a

custom-built AFM attached to an inverted optical microscope. The Young’s modulus (E) of

the ECM is computed by fitting the tip-ECM

contact model to force-indentations curves recorded on the ECM. The complex shear

modulus (G*) is measured by placing the tip at

an operating indentation of 500 nm and

superimposing small amplitude (75 nm)

multifrequency oscillations composed of sine waves (0.1-11.45 Hz). G* is computed in the

frequency domain from the complex ratio

between oscillatory force and indentation. We found that lung ECM exhibits scale-free rheology

with a storage modulus (G’, real part of G*)

increasing with frequency as a weak power law [1]. G’ values in the lung parenchyma ECM

ranged from 6 kPa in the alveolar septum to 15

kPa in the pleural membrane. The loss modulus

(G’’, imaginary part of G*) displayed a parallel frequency dependence at low frequencies, but

increased more markedly at higher frequencies.

We assessed the effect of different decellularization procedures on the local

stiffness of the acellular lung by measuring E at different sites of rat lungs subjected to four

decelularization protocols with/without

perfusion through the lung circulatory system

and using two different detergents [2]. Lung matrix stiffness revealed considerable

inhomogeneity, but conventional

decellularization procedures did not result in substantially different local stiffness. We

measured E of ECM in healthy and bleomycin-

induced fibrotic mouse lungs [3]. The local stiffness of the different sites in acellular fibrotic

lungs was very inhomogeneous and increased

according to the degree of the structural fibrotic

lesion. We also studied ECM nanomechanics of different histological regions of the left ventricle

wall of healthy and infarcted mouse hearts [4].

The ECM of the ventricular wall was 2-fold stiffer than the lung parenchyma with G’ ranging from

10 kPa in the epicardium and collagen-rich

regions of the myocardium to 30 kPa in elastin-rich regions of the myocardium. Importantly,

infarcted ECM showed a predominant collagen

composition and was 3-fold stiffer than collagen

rich regions of the healthy myocardium. ECM rheology of both lung and heart tissues was very

well characterized by a two power law model

composed of a weak power law with an exponent 0.05, accounting for a viscoelastic

solid regime dominant at physiological

frequencies, and a second power law with an exponent of 3/4, accounting for a viscoelastic

liquid regime at high frequencies. Our AFM

measurements define intrinsic mechanical

properties of the ECM at the length scale in

which cells sense and probe their

microenvironment. Regional changes in


mechanical properties of the ECM could provide

differential mechanical cues to regulate the

spatial distribution, differentiation and function

of lung and heart cells.

Acknowledgements: Funded in part by the

Spanish Ministry of Economy and

Competitiveness grant FISPI11/00089.

References

[1] ] Luque T, Melo E, Garreta E, Cortiella J,

Nichols J, Farré R, Navajas D. Local micromechanical properties of decellularized

lung scaffolds measured with atomic force

microscopy. Acta Biomater 2013, 9:6852-9.

[2] Melo E, Garreta E, Luque T, Cortiella J, Nichols J, Navajas D, Farré R. Effects of the

Decellularization Method on the Local

Stiffness of Acellular Lungs. Tissue Eng Part C 2013; 20:412-22.

[3] Melo E, Cardenes N, Garreta E, Luque T, Rojas

M, Navajas D, Farré R. Inhomogeneity of local stiffness in theextracellular matrix scaffold of

fibrotic mouse lungs. J Mech Behav Biomed

Mater 2014; 37:186–195.

[4] Andreu I, Luque T, Sancho A, Pelacho B, Iglesias-García O, Melo E, Farré R, Prósper F,

Elizalde MR, Navajas D. Heterogeneous

micromechanical properties of the extracellular matrix in healthy and infarcted

hearts. Acta Biomater 2014, 10:3235-42.


Cross-cutting KETs: Innovation and

Industrialization challenges for

Nanobiotechnology and Nanomedicine

towards Horizon 2020

Cristina Paez-Aviles1, Esteve Juanola-Feliu1, Josep Samitier1,2,3 1Dep. of Electronics, Bioelectronics and Nanobioengineering Research Group (SIC-BIO)

University of Barcelona. Barcelona, Spain. 2IBEC-Institute for Bioengineering of Catalonia, Nanosystems Engineering for Biomedical

Applications Research Group, Baldiri Reixac 10-12, 08028 Barcelona, Spain 3CIBER-BBN-Biomedical Research Networking Center in Bioengineering, Biomaterials and

Nanomedicine,María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain

[email protected]

Integration between Key Enabling Technologies

(KETs) will be essential for competitiveness and

innovation in Europe in the coming years. In this context, the new European Commission’s

initiative Horizon 2020, the biggest financial

program for Research and Innovation aims to

finance different Risk Management Projects going “from fundamental research to market

innovation”. This involves the entire innovation

chain focusing on the research and development of crosscutting KETs, which are

among the priorities of the Horizon 2020

Framework strategy. This strategy identifies the need for the EU to facilitate the industrial

deployment of KETs in order to make its

industries more innovative and globally

competitive [1].

Horizon 2020 aims to redefine the cooperation

in funding and scientific research by turning scientific breakthroughs into innovative

products and services with over 74 billion €

budget [2]. Is emphasized on three main pillars: Scientific Excellence, Society Challenges and

Industrial Leadership. This last one aims to

support SMEs in the industrial development and

application of KETs [3], which have been

selected according to the economic criteria,

economic potential, capital intensity,

technology intensity, and their value adding

enabling role: Nanotechnology, Micro and Nano

Electronics, Photonics, Advanced Materials, Biotechnology Industry, and Advanced

Manufacturing Systems.

Nanotechnology, is expected to make a rapid impact on society [4],[5]. After a long R+D

incubation period, several industrial segments

are already emerging as early adopters of nanotech-enabled products and findings

suggest that the Bio&Health market is among

the most challenging field for the coming years. Nanotechnology is also considered

multidisciplinary since it is not restricted to the

realm of advanced materials, extending also to

manufacturing processes, biotechnology, pharmacy, electronics and IT, as well as other

technologies [6]. These characteristics allow the

connection to a diversified set of industries [7], implying that nanotechnologies can be involved

directly or indirectly in the other five remaining

KETs. This strong interdisciplinary character, combined with the possibility of manipulating a

material atom by atom, opens up unknown

fields and provides an endless source of

innovation and creativity.


While each KET already has huge potential for

innovation individually, their cross-fertilization

is particularly important to offer even greater

possibilities to foster innovation and create new markets. The concept of cross-cutting KETs

refers to the integration of different key enabling

technologies in a way that creates value beyond

the sum of the individual technologies for developing innovative and competitive

products, goods and services that can

contribute to solving societal challenges. The global market volume in KETS is 646 billion

euros and substantial growth is expected of

approximately 8% of EU GDP by 2015.

At present, the emerging sector of applied

nanotechnology is addressed to the

biomedicine (nanobiotechnology and nanomedicine) [8], starting to show a promising

impact in the health sciences principally in

three main areas: Diagnostics, Therapeutics and Regenerative Medicine (Figure 1) [9], [10].

Nanomedicine is considered a long-term play in

the market [11]. In fact, the global nanotechnology market is anticipated to grow

around 19% by year during 2013-2017 [12]. The

expected market size related to radical

innovation-based nanomedicines will be 1.000 M€ in 2020 and 3.000 M€ in 2025 [13]. In this

context H2020 will spend 9.7% of the total

budget in Health, demographic change and wellbeing.

Translation of innovation and time-to-market reduction are important challenges on this

framework. Nanomedicine firms have focused

primarily on the science and less on the

commercial applications resulting difficult to bring products into the market [11]. This

remarks the existence of a gap between the

current high levels of scientific performance and the industrial competitiveness [14]. The

Commission states that bridging the so called

“Valley of Death” to upscale new KET

technology based prototypes to commercial

manufacturing, often constitutes a weak link in

the successful use of KETs potential. This is

meant to be the “European industrial

Renaissance” by covering the whole value of

chain lab-to-market as the principal aim of H2020 where market is the main starting point.

References

[1] European Commission, Brussels, 2009.

[2] D. Kalisz and M. Aluchna, Eur. Integr. Stud., vol. 6, (2012) 140–149.

[3] E. Commision, 2012.

[4] ECSIP consortium, Copenhagen, 2013. [5] M. C. Roco and W. S. Bainbridge, J.

Nanoparticle Res., vol. 7, (2005) 1–13.

[6] N. Islam and K. Miyazaki, Technovation, vol.

27, no. 11, (2007) 661–675. [7] T. Nikulainen and C. Palmberg,Technovation,

vol. 30, no. 1, (2010) 3–11.

[8] K. Miyazaki and N. Islam, vol. 30, no. 4, (2010) 229–237.

[9] European Technology Platform on

Nanomedicine, 2013. [10] European Commission, RO-cKETs - multiKETs

Pilot Lines Conference, 2014.

[11] T. Flynn and C. Wei, Nanomedicine, vol. 1, no.

1, (2005) 47–51. [12] RNCOS, 2013.

[13] European Commission, 2009.

[14] K. Debackere, R D Manag., vol. 30, no. 4, (2000) pp. 323–328..


Figures

Figure 1. Fields for cross-cutting KETs innovations in the Health and Healthcare Domain.


DC studies of Layer-by-layer nanopores

electrical properties tuning on

Polycarbonate Membranes

Roberto Paoli, Maria Bulwan, Antoni Homs-Corbera, Josep Samitier

Institute for Bioengineering of Catalonia (IBEC), c/ Baldiri Reixac, 10-12, 08028 Barcelona, Spain [email protected]

Nanoporous membranes have numerous

potential biological and medical applications

that involve sorting, sensing, isolating, and

releasing biological molecules [1]. Recent advances in nanoscience are making possible

to precisely control morphology as well as

physical and chemical properties of the pores in nanoporous materials. Different researches

showed that transport selectivity through solid-

state nanopores can be effectively modulated by changing the size [2], the charge [3–5] and

the polarity of the pores [6–8] or by using

tethered receptors that are capable of selective

molecular recognition [9]. Surface modification techniques are often used in order to achieve

those results, as they can alter both physical

and chemical properties.

We investigated how polyelectrolyte layer-by-

layer (LBL) surface modification can be used to change the characteristics of nanoporous

membranes. Studies were performed with a

custom made three-dimensional multilayer

microfluidic device able to fit membrane samples. The device allowed us to efficiently

control LBL films deposition over blank low-cost

commercially available polycarbonate track-etched (PCTE) membranes. We have

demonstrated pore diameter reduction and

deposition of the layers inside the pores through confocal and SEM images.

Posterior impedance studies served to study the

effect of the LBL charges to the net inner

nanopore surface charges. Measurements were

performed using Phosphate Buffer Saline as

conductive medium. DC results generally show

dependence between the electrical resistance

and the increasing number of layers. Adding

layers on pore surface decreases the pore mean aperture, resulting in a diminution of ions flux

and thus of electric current across the

membrane. Measurements have also demonstrated contrasted behaviors depending

on the number and parity of deposited opposite

charge layers. PCTE membranes are originally negatively charged and results evidenced higher

impedance increases for paired charges LBL

depositions. Impedance decreased when an

unpaired positive layer was added.

Following Electrical Double Layer theory we

hypothesize that charges in the buffer tend to redistribute in the solution, reorganizing near

the pores surfaces and creating an opposing

charges layer which alters local conductivity.

References

[1] S. P. Adiga, C. Jin, L. A. Curtiss, N. A. Monteiro-

Riviere, R. J. Narayan, Wiley Interdiscip. Rev.

Nanomed. Nanobiotechnol., 1, (2013), 568–81. [2] K. B. Jirage, J. C. Hulteen, C. R. Martin, Science

(80-. )., 278, (1997), 655–658.

[3] S. B. Lee, C. R. Martin, Anal. Chem., 73, (2001), 768–775.

[4] K.-Y. Chun, P. Stroeve, Langmuir, 17, (2001),

5271–5275.

[5] G. Wang, B. Zhang, J. R. Wayment, J. M. Harris,

H. S. White, J. Am. Chem. Soc., 128, (2006),

7679–7686.


[6] J. C. Hulteen, K. B. Jirage, C. R. Martin, J. Am.

Chem. Soc., 120, (1998), 6603–6604.

[7] K. B. Jirage, J. C. Hulteen, C. R. Martin, Anal.

Chem., 71, (1999), 4913–4918.

[8] E. D. Steinle, D. T. Mitchell, M. Wirtz, S. B. Lee, V.

Y. Young, C. R. Martin, Anal. Chem., 74, (2002),

2416–2422.

[9] S. B. Lee, D. T. Mitchell, L. Trofin, T. K. Nevanen, H. Söderlund, C. R. Martin, Science (80-. )., 296,

(2002), 2198–2200.

Figures

Figure 1. SEM images of polycarbonate membrane: on the left, not covered by polymers; on the right, covered by

polymers.

Figure 2. Measured resistance values comparison between different functionalizations of 200nm pore size membranes.

Resistance tends to increase with the number of deposited layer, but measurements related to an odd number of

deposited layers reveal a negative offset.


Bona fide induction of apoptosis in

transformed cells during photothermal

therapy using gold nanoprisms

Julián Pardo, M. Pérez-Hernández, P. del Pino, S. G. Mitchell, B. Pelaz, E. M Gálvez and J. M. de la Fuente

Instituto Universitario de Nanociencia de Aragon (INA), Universidad de Zaragoza, Zaragoza, Spain Fundación ARAID, Spain

Aragón Health Research Institute (IIS Aragón), Biomedical Research Centre of Aragón (CIBA), Zaragoza, Spain

Instituto de Carboquímica, CSIC, 50018 Zaragoza, Spain [email protected]

Gold nanoparticles (NPs) are promising vehicles to specifically deliver drugs to cancer cells and

in addition to their use in drug targeting, they

can be used as “heaters” during photothermal therapy of solid carcinomas using near-infrared

(NIR) laser light [1,2]. We have previously shown

that functionalization of gold nanoprisms (NPRs) with glucose selectively enhances their

cellular uptake in transformed cells [3]. During

the last years several types of NPs have been

used to kill tumoural cells, although in most cases the type of cell death (necrosis, apoptosis

, autophagy, etc.) induced has not been clearly

identified so far. Here we will present data that unequivocally show that apoptosis is really

induced in transformed cells during

photothermal therapy using gold NPRs. In addition, we will show for the first time the

molecular mechanism of apoptosis during

photothermal therapy in transformed cells

following irradiation with NIR laser light [4]. To this aim we have established conditions to

readily induce apoptosis on mouse embryonic

fibroblast (MEF) cells transformed with the SV40 virus and analyzed the mechanism of apoptosis

using MEFs from different knock out mice, which

are deficient in proteins involved in the different routes of apoptosis (Bak and Bax, Bid, caspase-

3 or caspase-9). Our results show that “hot”

NPRs activate the intrinsic mitochondrial

pathway of apoptosis mediated by Bak and Bax

through the activation of the BH3-only protein

Bid and that apoptosis and cell death is

dependent on the presence of both caspase-9 and caspase-3. Our findings demonstrate how

the functionalization and dose of NPRs, as well

as laser power density and irradiation time exposure, must be regulated to specifically

induce apoptotic cell death. Moreover the

molecular mechanism presented here may help to predict the efficacy of NP-based

photothermal therapy to treat cancer patients.

References

[1] B. Pelaz, V. Grazu, A. Ibarra, C. Magen, P. del Pino, J.M. de la Fuente, Langmuir, 2012, 28,

8965.

[2] C. Bao, N. Beziere, P. del Pino, B. Pelaz, G. Estrada, F. Tian, V. Ntziachristos, J. M. de la

Fuente and D. Cui, Small, 2012, 9, 68.

[3] M. Pérez-Hernández, P. del Pino, B. Pelaz, E. M.

Galvez, J. M. de la Fuente, J. Pardo (under review).

[4] M. Pérez-Hernández, P. del Pino, S. G. Mitchell,

B. Pelaz, E. M Gálvez, J. M. de la Fuente, Julián Pardo (under review).


Nanotechnology Platform at the Institute

for Bioengineering of Catalonia: description

of capabilities and examples

Mateu Pla-Roca

Institute for Bioengineering of Catalonia (IBEC) Baldiri Reixac 10-12 (Ed. Clúster), 08028, Barcelona, Spain [email protected]

The Nanotechnology Platform, a core facility of

the Institute for Bioengineering of Catalonia

(IBEC) is an accessible and versatile research

facility featuring 100 m2 of class 10,000 clean-room space and laboratories offering state-of-

the-art equipment for the fabrication and

characterization of microdevices and micro/nanostructures. Our mission is to

facilitate advanced research support by

providing services in the fields of micro/nanofabrication and nanotechnology for

all academic and industrial researchers. Some

of the many areas of application include lab-on-

a-chip (LOC), materials science, tissue engineering, optics and biomaterials.

IBEC’s Nanotechnology Platform offers scientific and technological support that includes the

design, characterization and development of

microdevices and micro/nanostructures so academic researchers and companies alike may

use the platform to develop their innovative

ideas.

Our experience in giving support to research

groups and practical examples will be

introduced during the presentation.

Figures

Figure 1. (A) Chemical imaging/analysis of surfaces using

TOF-SIMS (B) Fabrication of microdluidic chips and

structuration of materials at the (C) micro and (D)

nanoscale. (E) Biocompatible polymeric surface

micropatterned with a fluorescent protein.


The Quest for Charge Transport in single

Adsorbed Long DNA-Based Molecules

Danny Porath

Institute of Chemistry and Center for Nanoscience and Nanotechnology

The Hebrew University of Jerusalem, 91904 Israel

[email protected]

DNA and DNA-based polymers have been at the focus of molecular electronics owing to their

programmable structural versatility. The

variability in the measured molecules and

experimental setups, caused largely by the contact problem, has produced a wide range of

partial or seemingly contradictory results,

highlighting the challenge to transport significant current through individual DNA-

based molecules. A well-controlled experiment

that would provide clear insight into the charge transport mechanism through a single long

molecule deposited on a hard substrate has

never been accomplished. In this lecture I will

report on detailed and reproducible charge transport in G4-DNA, adsorbed on a mica

substrate. Using a novel benchmark process for

testing molecular conductance in single polymer wires, we observed currents of tens to

over 100 pA in many G4-DNA molecules over

distances ranging from tens to over 100 nm, compatible with a long-range thermal hopping

between multi-tetrad segments. With this

report, we answer a long-standing question

about the ability of individual polymers to transport significant current over long distances

when adsorbed a hard substrate, and its

mechanism. These results may re-ignite the interest in DNA-based wires and devices

towards a practical implementation of these

wires in programmable circuits

References

[1] "Direct measurement of electrical transport

through DNA molecules", Danny Porath,

Alexey Bezryadin,Simon de Vries and Cees Dekker, Nature 403, 635 (2000).

[2] "Charge Transport in DNA-based Devices",

Danny Porath, Rosa Di Felice and Gianaurelio

Cuniberti, Topics in Current Chemistry Vol. 237, pp. 183-228 Ed. Gary Shuster. Springer

Verlag, 2004.

[3] “Direct Measurement of Electrical Transport Through Single DNA Molecules of Complex

Sequence”, Hezy Cohen, Claude Nogues, Ron

Naaman and Danny Porath, PNAS 102, 11589 (2005).

[4] “Long Monomolecular G4-DNA Nanowires”,

Alexander Kotlyar, Nataly Borovok, Tatiana

Molotsky, Hezy Cohen, Errez Shapir and Danny Porath, Advanced Materials 17, 1901 (2005).

[5] “Electrical characterization of self-assembled

single- and double-stranded DNA monolayers using conductive AFM”, Hezy Cohen et al.,

Faraday Discussions 131, 367 (2006).

[6] “High-Resolution STM Imaging of Novel Poly(G)-Poly(C)DNA Molecules”, Errez Shapir,

Hezy Cohen, Natalia Borovok, Alexander B.

Kotlyar and Danny Porath, J. Phys. Chem. B

110, 4430 (2006). [7] "Polarizability of G4-DNA Observed by

Electrostatic Force Microscopy

Measurements", Hezy Cohen et al., Nano Letters 7(4), 981 (2007).

[8] “Electronic structure of single DNA molecules

resolved by transverse scanning tunneling spectroscopy”, Errez Shapir et al., Nature

Materials 7, 68 (2008).

[9] “A DNA sequence scanned”, Danny Porath,

Nature Nanotechnology 4, 476 (2009).

[10] “The Electronic Structure of G4-DNA by

Scanning Tunneling Spectroscopy”, Errez


Shapir, et.al., J. Phys. Chem. C 114, 22079

(2010).

[11] “Energy gap reduction in DNA by

complexation with metal ions”, Errez Shapir, G. Brancolini, Tatiana Molotsky, Alexander B.

Kotlyar, Rosa Di Felice, and Danny Porath,

Advanced Maerials 23, 4290 (2011).

[12] "Quasi 3D imaging of DNA-gold nanoparticle tetrahedral structures", Avigail Stern, Dvir

Rotem, Inna Popov and Danny Porath, J. Phys.

Cond. Mat. 24, 164203 (2012). [13] "Comparative electrostatic force microscopy

of tetra- and intra-molecular G4-DNA", Gideon

I. Livshits, Jamal Ghabboun, Natalia Borovok, Alexander B. Kotlyar, Danny Porath, Advanced

materials 26, 4981 (2014).

[14] "Long-range charge transport in single G4-

DNA molecules", Gideon I. Livshits et. al., Nature Nanotechnology, Advanced Online

Publication (2014).

Figures


NANOMEDICINE 2.0

Victor Puntes

Institut Català de Nanotecnologia (ICN), Campus de la UAB, 08193 Bellaterra, Spain [email protected]

Last decade has seen a flourishment in the study of the properties of inorganic

nanoparticles for medical applications.

Nanoparticles display properties that are strongly determined by both morphology and

environment and in the physico-chemical

context where they are immersed, therefore

allowing to monitor and manipulate biological states. In fact, inorganic nanoparticles behave

as "artificial atoms", since their high density of

electronic states -which controls many physical properties- can be extensively and easily tuned

by adjusting composition, size and shape and

used in biological environments. In fact, nanotechnology’s ability to shape matter on the

scale of molecules is opening the door to a new

generation of diagnostics, imaging agents, and

drugs for detecting and treating disease at its earliest stages. But perhaps more important, as

I will show, it is enabling researchers to

combine a series of advances, creating thus nanosized particles that may contain drugs

designed to kill tumours together with targeting

compounds designed to home-in on malignancies, and imaging agents designed to

light up even the earliest stage of cancers. In

fact, a description of cancer in molecular terms

seems increasingly likely to improve the ways in which human cancers are detected, classified,

monitored, and (especially) treated, and for

that, nanoparticles, which are small and therefore allows addressing molecular

structures in an unique manner, may be

especially useful for those tasks.

When almost 10 years ago, Science magazine

dedicated the special issue on nanotechnology

on cancer treatment [1], clinicians and

pharmaceutics did not consider it as a real

alternative yet. Currently, this perception may

be changing thanks to the intense research efforts and exemplary bold initiatives to develop

cancer nanotechnology [2]. As a consequence,

more than a dozen nanoparticle-based imaging agents and therapeutics are either on the

market, in clinical trials, or awaiting clinical

trials [3,4]. Similarly, the use of

superparamagnetic nanoparticles for photo-ablation (hyperthermia) of brain tumours is

already applied in the clinic [5].

References

[1] ] P.A Kiberstis et al., Celebrating a Glass Half-Full. Science 312 (2006) 1157

[2] Gallego, O., & Puntes, V. What can

nanotechnology do to fight cancer? Clin.

Transl. Oncol., 8, (2006) 788–795. [3] Avnesh S., Thakor, Sanjiv S, Gambhir.

Nanooncology: The future of cancer diagnosis

and therapy CA: A Cancer Journal for Clinicians 63 (2013) 395 ;

[4] R. Juliano Nanomedicine: is the wave

cresting? Nature Reviews Drug Discovery 12 (2013), 171

[5] http://www.magforce.de/en/home.html.


Figures

Figure 1. Normally, not all that is conceived becomes reality, however, what becomes reality, has been previously in the

imagination. The magic bullet, a way to drive drugs towards the target avoiding effects to the rest of the body. The

fantastic voyage, scientist and a submarine are miniaturized to go and do medical work inside the body. Their fight against

the immune system is epic. Both precluded developments on nanomedicine.


Developing new tools for drug testing:

introducing a microfluidic platform

mimicking the spleen for future

pharmacological trials

L.G. Rigat-Brugarolas1,2, A. Elizalde3, H.A. del Portillo3,4, A. Homs-Corbera1,2 and J. Samitier1,2,5 1Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), Spain

2Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain 3Barcelona Centre for International Health Research (CRESIB, Hospital Clínic - UB), Spain

4Institució Catalana de Recerca i Estudis Avançats (ICREA), Spain 5Department of Electronics, Barcelona University (UB), Spain

[email protected]

Constant evolution and improvements on areas such as tissue engineering, microfluidics and

nanotechnology have made it possible to

partially close the gap between conventional in vitro cell cultures and animal model-based

studies. A step forward in this field concerns

organ-on-chip technologies, capable of

reproducing the most relevant physiological features of an organ in a microfluidic device.

Research in microfluidic devices that represents organ models is still in its infancy, but offers a

tantalizing glimpse into future of drug testing

and biological hypotheses evaluation. [1]

Drug testing in animal models is time-

consuming, costly, and often does not

accurately predict the adverse effects in humans. Toward a more reliable output, several

platforms, in the interface between nanobio

and tissue engineering, have been developed in the past years [2,3] with the aim to supplement

or supplant animal studies or at least try to

prioritize them.

Nevertheless, no one developed before a

spleen-like platform for studying the

importance of this organ in differente

haematological diseases.

Similar in structure to a large lymph node, the spleen is a complex three-dimensional

branched vasculature exquisitely adapted to

perform different functions containing closed/rapid and open/slow microcirculations,

compartmentalized parenchyma and sinusoidal

structure forcing erythrocytes to squeeze

through interstitial slits (IES) before reaching venous circulation. [4]

Taking into account these features, we designed and developed a multilayered microfluidic

device of the first ever functional human

splenon-on-a-chip, mimicking the hydrodynamic behavior of the spleen's red pulp,

to evaluate and simulate its activities,

mechanics and physiological responses.

Different physiological features have been translated into engineering elements that can

be combined to integrate a biomimetic splenon

model [5] (the minimal functional unit of the spleen).

This biochip-based platform should allow a deeper understanding of the underlying

mechanisms of Plasmodium parasite infection

and contribute to vaccine development and

drug testing of malaria and other hematological

disorders. Preliminary results showed significant

statistical differences in terms of cell


deformability between old vs. fresh RBCs

(p=0.001) and non-parasitized vs. P. Yoelii

parasitized reticulocytes (p=0.006) when passing

through the 2 µm constrictions simulating the IES.

Still, additional challenges remain before these

in vitro models can be used in applications such as diagnostics, but they could be the future of

drug testing and biological platforms.

Acknowledgements: Part of this work was

financially supported by the technology transfer

program of the Fundación Botín and by the Explora Program of the Ministry of Economy and

Competitiveness of the Government of Spain.

We thank David Izquierdo and Miriam Funes for

their help in this project.

References

[1] D. Huh et al. Science (2010) 328, 1662-1668.

[2] D. Huh et al. Sci Transl Me (2012) 4, 159ra147. [3] A. Neswith et al. Lab Chip (2014) 14, 3925-3936.

[4] A.J. Bowdler, The complete spleen (2010) 2nd

edition.

[5] L.G. Rigat-Brugarolas et al. Lab Chip (2014) 14, 1715-1724.


PAMAM dendrimers internalizes quickly in

microalgae and cyanobacteria causing

toxicity and oxidative stress

Rodea-Palomares I.1,2, Gonzalo S.2, Rosal R.2 Leganés F.1 & Fernandez-Piñas F.1 1Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid. Madrid, Spain

2Departamento de Ingeniería Química, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain

[email protected]

1. Introduction

Poly(amidoamine) (PAMAM) dendrimers are

hyper-branched polymeric, nanoscale molecules with exceptional properties that

make them attractive for a variety of biomedical

and technological applications [1]. Dendrimers are considered “perfect” polymers due to their

symmetry, and are classified according to their

“generation” “G”, which accounts for the number of “layers” of polymer forming the

dendrimer. Each generation doubles molecular

weight and surface functional groups.

Furthermore they are susceptible of a variety of surface functionalizations. Despite their

promising applications, they have been found

to be toxic to mammalian cells depending on generation and surface functionalization and

their possible adverse effects for aquatic life,

and especially for microalgae are largely unknown. In the present work we chose

generation G2, G3 and G4 native –NH2 (cationic)

and NH-C-(CH2OH)3 (-OH) (anionic) surface

functionalized PAMAM dendrimers in order to study the dependency of polyamidoamine

(PAMAM) dendrimer toxicity on generation and

surface functionalization. As model organisms we chose a green microalga (Chlamydomonas

reindhartii) and a cyanobacterium (Anabaena

PCC7120). We have applied a multi-method approach to get insight into the toxic

mechanisms of action of PAMAM dendrimers on

both C. reindhartii and Anabaena sp. PCC 7120

including physicochemical characterization of

PAMAM dendrimers in culture media, and

different physiological and cell biology

techniques.

2. Materials and methods

Materials and physicochemical

characterization. Amine- and hydroxyl terminated G2, G3 and G4 PAMAM

ethylenediamine core dendrimers were used

(Sigma-Aldrich). The size distribution of nanoparticles was obtained using dynamic light

scattering (DLS). Zeta potential was measured

via electrophoretic light scattering. Growth

inhibition experiments were performed with C. reindhartii and Anabaena sp. PCC 7120

following the standard OECD TG 201. Detection

of reactive oxygen species (ROS): DCF was used as indicator of intracellular ROS formation. C4-

BODIPY was used for evaluating lipid

peroxidation. Internalization studies: PAMAM-Alexa Fluor 488 conjugates were prepared

following the standard protocol (A30006,

Molecular probes). The Alexa Flour 488 reactive

dye has a tetrafluorophenyl (TFP) ester which reacts efficiently with primary amines. Anti-

Alexa fluor 488 Rabbit IgG Fraction (A-11094,

Molecular probes), was used In order to discriminate surface bound and truly

internalized dendrimers. Fluorescence studies

were performed by flow cytometry and confocal microscopy. Ultrastructure alterations were

studied by transmission electron microscopy

(TEM).


3. Results and discussion

3.1. Toxicity of PAMAM dendrimers in

cyanobacteria and microalgae All the cationic dendrimers (native –NH2) proved

toxic to both the green alga and the

cyanobacterium. G2 and G3 Anionic dendrimers

(-OH surface functionalized) were nontoxic, however, G4-OH proved toxic for both

organisms. When toxicity is referred to mass

concentration (mg/L), cationic dendrimers showed similar toxicity, apparently irrespective

of generation (size). However, considering the

large differences in molecular weight of the tested dendrimers, concentrations expressed

on a molar basis revealed a clear relationship

between dendrimer generation and toxicity for

both organisms.

3.2. Toxicity of PAMAM dendrimers correlated

with oxidative stress Increasing evidences indicate that

nanoparticles in general can generate reactive

oxygen species (ROS) and subsequently oxidative stress which might eventually lead to

cell damage and cell death [2]. When the ability

of the tested anionic and cationic dendrimers to

elicit oxidative stress was evaluated by fluorometry, flow citometry and confocal

microscopy we found that the strong

differences in toxicity between anionic and cationic PAMAM dendrimers correlated with

alterations in the ROS metabolism in both

organisms. Figure 1 showed, as an example, ROS induction kinetics along the experimental

lapse time (0 h-72 h) of Anabaena exposed to

G2-OH and G2-NH2. Interestingly, neither DCF

fluorescence (general ROS indicator), nor Bodipy fluorescence (lipid peroxidation) co-

localized with photosynthetic structures of both

organisms even when lipid peroxidation was observed in C. reinhardtii based on flow

cytometry analysis, suggesting that the

photosynthetic machinery is neither affected

nor the origin of the observed oxidative stress

which is in disagreement with previous studies

[3, 4].

3.3. PAMAM dendrimers were internalized very fast and presented low retention times in cell

envelopes.

We made a time course of dendrimers

internalization. In the cyanobacterium, the three dendrimers were quickly taken up (80% of G2

and G3 and 100% of G4 after 10 min). In the

green alga, dendrimer uptake was slower with 100% uptake after 2 h, G4 dendrimer uptake

was slightly quicker than the other two

dendrimers. The experiments with the antiAlexa antibody showed that in both organisms the

Alexa Fluor-dendrimer conjugates were largely

internalized even at the shorter time assayed

(10 min). Interestingly, similar to the ROS results, no co-localization of Alexa fluor488 and

photosynthetic membranes was found

supporting the hypothesis that oxidative stress is neither affecting nor coming from the

photosynthetic machinery. Furthermore,

derdrimers were found to target mitochondria producing mitochondrial peroxidation. It has

been found that dendrimers are internalized in

different animal and human cell systems[5];

however, to our knowledge, this is the first time that PAMAM dendrimer internalization is

confirmed in algae and cyanobacteria.

Conclusions

Cationic (-NH2) PAMAM dendrimers presented a

generation-dependent increasing toxicity in both organisms. Anionic (-OH) PAMAM of

generation G2 and G3 were non toxic, however,

G4-OH presented a similar level of toxicity to G4-

NH2 in both organisms. Internalization of PAMAM dendrimers was observed by the first

time in microalgae and cyanobacteria.

Internalization was very fast (after 10 min of exposure) and with low retention time in cell

envelopes of both organisms. Toxicity

correlated with oxidative stress and dendrimer

internalization. However, the photosintetic


machinery seemed to be unafected, and most

probably was not involved in oxidative stress.

Acknowledgements: This study was supported by the Community of Madrid grants

S-0505/AMB/0321and S-2009/AMB/1511and by

the Spanish Ministry of Science grant CGL2010-

15675/BOS and CTM2008-04239/TECNO and CTM2008-00311/TECNO.

References

[1] Svenson, S. and D.A. Tomalia, Dendrimers in biomedical applications—reflections on the

field. Advanced Drug Delivery Reviews, 2005.

57(15): p. 2106-2129.

[2] Nel, A., et al., Toxic Potential of Materials at the Nanolevel. Science, 2006. 311(5761): p. 622-

627.

[3] Petit, A.-N., et al., Effects of a cationic PAMAM dendrimer on photosynthesis and ROS

production of Chlamydomonas reinhardtii.

Nanotoxicology, 2012. 6(3): p. 315-326. [4] Petit, A.-N., et al., Toxicity of PAMAM

dendrimers to Chlamydomonas reinhardtii.

Aquatic Toxicology, 2010. 100(2): p. 187-193.

[5] Albertazzi, L., et al., Dendrimer Internalization and Intracellular Trafficking in Living Cells.

Molecular Pharmaceutics, 2010. 7(3): p. 680-

688.

Figures

Figure 1. ROS induction kinetics (DCF fluorescence

488/528nm) in Anabaena PCC7120 exposed to increasing

concentrations of anionic (-OH) and cationic (-NH2) G2

PAMAM dendrimers along the experimental lapse time

(72h).


Speckle fluctuations resolve the

interdistance between incoherent point

sources in complex media

R. Carminati1, G. Cwilich2, L.S. Froufe-Pérez3 and J.J. Sáenz4,5 1ESPCI ParisTech, PSL Research University, CNRS,Institut Langevin, 1 rue Jussieu, Paris, France

2Department of Physics, Yeshiva University, 500 W 185th Street, New York, New York 10033, USA 3Department of Physics, University of Fribourg, Chemin du Mus ee 3, CH-1700, Fribourg, Switzerland

4Condensed Matter Physics Center (IFIMAC), Depto. de Física de la Materia Condensada and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, 28049 Madrid, Spain

5Donostia International Physics Center (DIPC),Paseo Manuel Lardizabal 4, San Sebastian, Spain

[email protected]

We propose a method to capture the interaction

between two identical sources in a scattering environment, based only on the measurement

of intensity fluctuations [1]. The principle of the

method is schematically illustrated in Fig. 1, and is based on the analysis of the intensity-

intensity correlation function and the intensity

fluctuations in the speckle pattern formed by

two identical and mutually incoherent point sources. This approach permits in principle to

monitor the relative distance between the

sources in the range 10-500 nm, with a precision that is not limited by diffraction, but by the

microstructure of the scattering medium.

A key issue affecting subwavelength imaging

methods is the optical transparency of the

media surrounding the light emitters. Taking

advantage of the transparency of cells, fluorescence microscopy uniquely provides

noninvasive imaging of the interior of cells and

allows the detection of specific cellular constituents through fluorescence tagging.

However, certain biological tissues or soft-

matter systems (such as foams or colloidal suspensions) look turbid due to intense

scattering of photons traveling through them

[2]. The image formed at a given point in the

observation plane consists in a superposition of

multiple fields, each arising from a different

scattering sequence in the medium. This gives

rise to a chaotic intensity distribution with

numerous bright and dark spots known as a speckle pattern, producing a blurred image

carrying no apparent information about the

source position [3].

Techniques to measure the distance between

individual nano-objects without actually

imaging their position exist [4], Fluorescence Resonance Energy Transfer (FRET) being the

most widespread example [5]. It relies on the

near-field energy transfer between two fluorophores (donor and acceptor) emitting at

different wavelengths. The FRET signal (e.g. the

ratio between the intensities emitted by the donor and the acceptor at different

wavelengths) depends on the donor-acceptor

distance in the range 2 ∼ 10 nm. As such, it is

not very sensitive to scattering problems. However, determining distances between two

emitters in the range of 10 to 500 nm in a

scattering medium still remains a challenging problem, not accessible either by fluorescence

microscopy or FRET techniques.

Our main goal here is to introduce a new

approach to obtain information about the

relative distance between two identical

incoherent point sources in a disordered

environment, based on the analysis of the

fluctuations of the emitted light. This is an issue


of much interest, for example, in the study of

conformational changes in biomolecules in

living tissues.

References

[1] R. Carminati, G. Cwilich, L.S. Froufe-Pérez, and

J.J. Sáenz, arXiv preprint, arXiv:1407.5222v2 [2] A. Yodh and B. Chance, Physics Today 48, 34

(1995).

[3] J.C. Dainty (ed.) Laser Speckle and Related Phenomena (Springer-Verlag, Berlin, 1975);

J.W. Goodman, Speckle Phenomena in Optics:

Theory and Applications (Roberts &

Company, Englewood, 2007).

[4] X. Michalet and S. Weiss, Proc. Nat. Acad. Sci.

USA 103, 4797 (2006).

[5] For a recent review see the Special Issue Förster Resonance Energy Transfer in

ChemPhysChem 12, Issue 3, (2011) and

references therein.

Figures

Figure 1. The intensity radiated by two incoherent point

sources in a complex medium form a speckle pattern that

fluctuates in both space and time. The speckle

fluctuations encode the relative distance between the

sources [After Ref. 1]


Self-powered microbots towards a

“Fantastic Voyage”

Samuel Sánchez

Max Planck for Intelligent Systems, Stuttgart, Germany

Self-powered micro-motors are currently

subject of a growing interest due to their visionary but also potential applications in

robotics, biosensing, nanomedicine,

microfluidics, and environmental field [1]. These

micromotors are autonomous since they do not need external sources of energy in order to

move. Instead, self-powered microrobots propel

by decomposition of the fuel where they swim.

These tiny motors swim through the water and

can clean up contaminants or can swim through blood to one day transport medicines

to a targeted part of the body -like taken from a

science fiction movie Fantastic Voyage-.

Those artificial nanomotors act collectively [2]

reacting to external stimuli like chemotactic

behaviour [3] and are capable to clean polluted water [4,5]. Future operations of autonomous

intelligent multi-functional nanomachines will

combine the sensing of hazardous chemicals using bio-inspired search strategies. With

continuous innovations we expect that man-

made nano/microscale motors will have

profound impact upon in several fields such as drug delivery, biosensing and environmental

remediation, among other visions.

References

[1] Sanchez, Soler and Katuri, Angew. Chem. Int. Ed., 2014. DOI: 10.1002/anie.201406096

[2] Solovev, A. A. et al., Nanoscale, 5, 1284 (2013)

[3] Baraban, L. et al., Angew. Chem. Int. Ed., 52,

5552. (2013), b) S. Saha, et.al., Phys. Rev. E, 89,

062316, (2014); c) Y. Hong, N. M.K. et al, Phys.

Rev. Lett., 99, 178103 (2007)

[4] Soler, Ll. et al., ACS Nano, 7, 9611 (2013), b) J.

Orozco, et al Angew.Chem., Int. Ed., 52, 13276; (2013)

[5] (a) Gao, W. and Wang, J., ACS Nano, 8, 3170

(2014). (b) Soler, L. and Sanchez, S. Nanoscale,

6, 7175 (2014).


Scattering based bead-microrheology

applied to biomaterials

Frank Scheffold

Department of Physics and Fribourg Center for Nanomaterials -

University of Fribourg, 1700 Fribourg, Switzerland

The quantitative stochastic description of

Brownian motion of spherical micro- and nanobeads in complex fluids has laid the

foundations for the invention of tracer

microrheology, a powerful, noninvasive method

that allows the measurement of mechanical properties over an extended range of

frequencies using all optical instrumentation [1-

3]. Over the last 20 years the method has been applied to a large range of materials ranging

from foodstuff, dispersions, slurries to polymer

solutions and surfactant based systems [1-4]. Since only small volumes are required the

method is particularly well suited for

applications in the field of biomaterials and

bioactive substances [5-7].

The basic idea of optical microrheology is to

study the response of small (colloidal) particles embedded in the system under study. The

particle can be added as tracer particles or can

be naturally present in the system (such as oil droplets in an emulsion or fat droplets and

protein micelles in yoghurt). The motion of the

embedded probe particles can either be

controlled actively, e.g. using optical tweezers or one can analyze the thermal motion of the

particles. Both approaches can provide

quantitative information about the viscous and viscoelastic properties of the surrounding fluid.

Scattering techniques such as diffusing wave

spectroscopy (DWS) or dynamic light scattering (DLS) are some of the most popular techniques

to probe passive (thermal) particle motion

remotely. These laser-based techniques offer

the advantage to provide an ensemble average

of the probe particle motion within about one

minute measurement time. Standard sample

preparation protocols can be used and the

samples are held in common cylindrical or rectangular glass cuvettes filled with fluid

volumes of typically 0.2-1 ml. Moreover these

methods can resolve extremely fast

displacements on the order of microseconds with a sub-nanometer resolution.

Here I will briefly review the methodology and instrumentation and then discuss applications

to biomaterials and foodstuff [5], for probing

protein interactions/aggregation and for the study three-dimensional assemblies of cell

clusters [6,7]

References

[1] T. G. Mason and D. A. Weitz, Optical

Measurements of Frequency-Dependent Linear Viscoelastic Moduli of Complex Fluids,

Phys. Rev. Lett. 74, 1250 (1995).

[2] N. Willenbacher, C. Oelschlaeger, M. Schöpferer, P. Fischer, F. Cardinaux and F.

Scheffold, Broad Bandwidth Optical and

Mechanical Rheometry of Wormlike Micelle

Solutions, Physical Review Letters 99, 68302 (2007)

[3] P. Domínguez-García, Frédéric Cardinaux,

Elena Bertseva, László Forró, Frank Scheffold, Sylvia Jeney, Accounting for inertia effects to

access the high-frequency microrheology of

viscoelastic fluids, submitted, arXiv:1408.4181 [cond-mat.soft]

[4] F. Cardinaux, H. Bissig, P. Schurtenberger and

F. Scheffold, Optical microrheology of gelling

biopolymer solutions based on diffusive wave

spectroscopy, Food Hydrocolloids 20, 325-331

(2007)


[5] C. Oelschlaeger, M. Cota Pinto Coelho, and N.

Willenbacher, Chain Flexibility and Dynamics

of Polysaccharide Hyaluronan in Entangled

Solutions: A High Frequency Rheology and Diffusing Wave Spectroscopy Study,

Biomacromolecules 14, 3689 (2013).

[6] B. Fabry, G. N. Maksym, J. P. Butler, M.

Glogauer, D. Navajas, and J. J. Fredberg, Scaling the microrheology of living cells, Phys.

Rev. Lett. 87, :148102 (2001)

[7] F. Scheffold, J. Frith and J. Cooper White, Diffusing Wave Spectroscopy of Concentrated

Mesenchymal Cell Suspensions, under

preparation.


Targeted drug delivery and personalized

medicine

Avi Schroeder

Technion – Israel Institute of Technology, Israel

[email protected]

The field of medicine is taking its first steps

towards patient-specific care. Our research is aimed at tailoring treatments to address each

person’s individualized needs and unique

disease presentation. Specifically, we are

developing nanoparticles that target disease sites, where they perform a programmed

therapeutic task. These systems utilize

molecular-machines and cellular recognition to improve efficacy and reduce side effects.

Two examples will be described: the first involves a nanoscale theranostic system for

predicting the therapeutic potency of drugs

against metastatic cancer. The system provides

patient-specific drug activity data with single-cell resolution. The system makes use of

barcoded nanoparticles to predict the

therapeutic effect different drugs will have on the tumor microenvironment.

The second system makes use of enzymes, loaded into a biodegradable chip, to perform a

programed therapeutic task – surgery with

molecular precision. Collagenase is an enzyme

that cleaves collagen, but not other tissues. This enzyme was loaded into the biodegradable chip

and placed in the periodontal pocket. Once the

collagenase releases from the chip, collagen fibers that connect between the teeth and the

underlying bone are relaxed, thereby enabling

enhanced orthodontic corrective motion and reducing pain. This new field is termed

BioSurgery.

The clinical implications of these approaches

will be discussed.


Personalized Cancer Nanomedicine.

CLINAM 2014

Simo Schwartz

Vall d'Hebron Hospital - CIBBIM. P. de la Vall d'Hebron, 119-129. 08035 Barcelona, Spain

It has been hypothesized that drug delivery by

nanoparticles may well circumvent the

resistance machinery of cancer stem cells (CSC). To be able to study efficacy of nanomedicines in

population of CSC, we first developed an in vitro

model in which CSC are tagged by a fluorescent

reporter gene under the control of a CSC specific promoter. Using this system, we

demonstrated that while bulk cancer cells die,

CSC population augments after paclitaxel (PTX) treatment. We then investigated the prospects

of different targeted and non-targeted delivery

systems loaded with PTX and functionalized with specific antibodies against cancer stem cell

populations in regular breast cancer cell lines,

as well as in our CSC models. Our data shows

that reducing tumor resistance of cancer stem cells might be related to specific active targeting

of DDS and not attributed to a general

mechanism of action of nanomedicines.


Liposomes: Topical and Oral Bioavailability

Julio Cortijo, Patricia Almudéver Folch, Juan Manuel Serrano Núñez

Sesderma Laboratories, Polígono Industrial Rafelbuñol C/Massamagrell 3, Rafelbuñol 46138 Valencia, Spain [email protected]

Liposomes are small vesicles composed of one or more lipid bilayers. The size can go from 30nm up to several microns. Liposomes can encapsulate hydrophilic solutes in the aqueous core and

lipophilic solutes in the membrane. These vesicles can be classified according to their size and number

of bilayers: Multilamellar (100-10.000nm), Small Unilamellar (less than 100nm), Large Unilamellar (100-500nm). Sesderma manufactures very uniform,

unilamellar liposome populations of between 50-

150nm.

The advantages of liposomes are that, the structure

is very similar to biological membranes and thus, are biodegradable and non toxic, they can reach the deepest layers of the skin, they provide a sustained

release of the active ingredients, they prevent the

oxidation and degradation of the ingredients and

they show higher efficiencies at lower

concentrations.

We have carried out three different experiments on topical bioavailability: liposome penetration

through skin, hair follicles and nails.

All the ingredients used to prepare the liposomes

are classified as GRAS (generally recognized as safe).

In the first one, we compared the permeation

capacity through human skin, using a Franz Diffusion Cell, of two different substances

encapsulated and not encapsulated in liposomes: fluorescein and sodium ascorbate. Aliquots were taken from the receptor chamber at different times.

The concentration of sodium ascorbate was determined by high performance liquid chromatography with ultraviolet detection (HPLC-UV) and that of fluorescein by spectrofluorimetry.

The results were as follows:

These results might be due to the nature and size of

the active ingredients, and the characteristics of the

layers of the skin. The epidermis is a stratified layer with plenty of cells, this is why liposomes can get through it easier than the ingredients in solution. Fluorescein can diffuse faster through the epidermis

than sodium ascorbate because fluorescein is more lipophilic than sodium ascorbate. The dermis has

less cells and more fibres, and has a greater aqueous volume, so the preparation that permeates faster is that of sodium ascorbate solution due to its

hydrophilic nature and small size. Finally, we can

confirm that liposomes help substances pass through the skin.

In the second case, liposome ability to go across the follicular canal was assayed with liposomal fluorescein. The skin samples were extracted from

human scalp and the equipment used was the same

as in the prior experiment: Franz Diffusion Cell.

Pictures were taken at different times with a

fluorescence microscope. We concluded that the follicular canal is an excellent penetration enhancer; a liposome reservoir is formed, facilitating its pass

through the hair follicle and into the dermis.

In the third experiment, we assessed the penetration capacity of liposomal fluorescein on one hand and a

solution of fluorescein on the other hand, through human nails. The equipment utilized was a Franz

Diffusion Cell with a coupling device for nails. Aliquots were taken from the receptor chamber at

different times and the concentrations of fluorescein were determined by spectrofluorimetry. Pictures

were also taken with a fluorescence microscope.The results showed that the maximum quantity of absorption for both formulations was obtained after

2 days in contact with the products. The concentration of fluorescein (2.96 ±1. 0.2 μg/cm²) for the liposomal formulation was 2.5 times higher than the solution (1.22 ± 0.2 μg/cm²). However, the

permeability constant is very similar for both

preparations: fluorescein solution (0.006 ± 0.002 cm²/s) and liposomal fluorescein (0.008 ± 0.001

cm²/s). We could also observe that there was an increase in the thickness of the nail treated with


liposomal fluorescein whilst there were no changes

observed in the nail treated with the solution of

fluorescein.

Ascorbic Acid Oral Pharmacokinetics in Rats

Aim: Compare the pharmacokinetics of two sodium ascorbate formulations:

- Sodium ascorbate solution (extemporaneously

prepared). - Sodium ascorbate encapsulated in liposomes.

Method:

The day prior to the administration of the formulations, 12 Wistar rats (280-310 g) were

cannulated in the jugular vein to allow blood sampling at preset times. A volume of 2.5 mL of each

fresh formulation was administered orally as single dose with intraoesophageal cannula. Six replicates were performed for each formulation. 200 μl blood

samples were taken at the following times: 0 , 15 , 30,

45 , 60, 90 , 120 minutes and 3, 4 , 5, 6 , 7, 8 , 10, 12 , 23, 26 hours . The samples were centrifuged at 2000g

for 5 min to obtain plasma which was immediately

deproteinized with icecold MPA 10%

(metaphosphoric acid). The samples were filtered through a pore diameter of 0.45μm. The analytical

method used to measure vitamin C (sodium ascorbate) was HPLC-UV with UV detection at

254nm. The mobile phase used consisted of a KH2PO4 (0.1M) solution: ACN (95:5) at a pH of 2. As the stationary phase, the column Sherisorb ODS1 5uM 25x0.4mm was used and the selected flow rate

was 1 ml / min. The injection volume used was 60 μL.

Results:

Figure 1. Plasma concentration versus time after oral

administration of 250 mg of sodium ascorbate formulated

in an extemporaneous solution (black line) or in liposomes

(green line). Mean ± SEM, n = 6.

Conclusions:

Liposomes enable a better control of the release of

the drug in plasma and maintains it for a longer period of time. A liposomal formulation of sodium

ascorbate requires a smaller dose to reach the desired plasma concentration and, therefore, the desired therapeutic effect.

References

[1] ELKEEB, R., ALIKHAN, A., ELKEEB, L., HUI, X. &

MAIBACH, H. I. 2010. Transungual drug delivery:

current status. Int J Pharm, 384, 1-8.

[2] ISHIDA, A., OTSUKA, C., TANI, H. & KAMIDATE, T.

2005. Fluorescein chemiluminescence method for

estimation of membrane permeability of liposomes. Anal Biochem, 342, 338-40.

[3] KARLSEN, A., BLOMHOFF, R. & GUNDERSEN, T. E.

2005. High-throughput analysis of vitamin C in

human plasma with the use of HPLC with monolithic column and UV-detection. J Chromatogr B Analyt Technol Biomed Life Sci, 824,

132-8.

[4] KLIGMAN, A. M. & CHRISTOPHERS, E. 1963.

Preparation of Isolated Sheets of Human Stratum

Corneum. Arch Dermatol, 88, 702-5.

[5] O'GOSHI, K. & SERUP, J. 2006. Safety of sodium fluorescein for in vivo study of skin. Skin Res Technol, 12, 155-61.

[6] SZNITOWSKA, M. & BERNER, B. 1995. Polar

pathway for percutaneous absorption. Curr Probl

Dermatol, 22, 164-70.

[7] TORRES-MOLINA, F., ARISTORENA, J. C., GARCIA-

CARBONELL, C., GRANERO, L., CHESA-JIMENEZ, J.,

PLA-DELFINA, J. & PERIS-RIBERA, J. E. 1992.

Influence of permanent cannulation of the jugular

vein on pharmacokinetics of amoxycillin and

antipyrine in the rat. Pharm Res, 9, 1587-91.


Nano crystalline cellulose-protein

composites: Super performing biomaterials

for tissue engineering and regenerative

medicine

Oded Shoseyov

The Robert H Smith Institute of Plant Science and genetics. The Faculty of Agriculture

The Hebrew University of Jerusalem, Israel

A platform technology that brings together the

toughness of cellulose nano-fibers from the plant kingdom, the remarkable elasticity and resilience

of resilin that enables flees to jump as high as 400

times their height from the insect kingdom, and the adhesion power of DOPA, the functional

molecule of mussels that enable it to bind tightly

under water to organic and inorganic matter from the marine kingdom and all that combined

with Human Recombinant Type I collagen

produced in tobacco plants;

SUPERPERFORMING BIOMATERIALS.

Resilin is a polymeric rubber-like protein

secreted by insects to specialized cuticle regions, in areas where high resilience and low

stiffness are required. Resilin binds to the cuticle

polysaccharide chitin via a chitin binding domain and is further polymerized through

oxidation of the tyrosine residues resulting in

the formation of dityrosine bridges and

assembly of a high-performance protein-carbohydrate composite material. Plant cell

walls also present durable composite structures

made of cellulose, other polysaccharides, and structural proteins. Plant cell wall composite

exhibit extraordinary strength exemplified by

their ability to carry the huge mass of some forest trees. Inspired by the remarkable

mechanical properties of insect cuticle and

plant cell walls we have developed novel

composite materials of resilin and Nano-

Crystalline Cellulose (resiline-NCC) that display

remarkable mechanical properties combining

strength and elasticity. We produced a novel

resilin protein with affinity to cellulose by genetically engineering a cellulose binding

domain into the resilin. This CBD-Resilin enable,

interfacing at the nano-level between the resilin; the elastic component of the composite, to the

cellulose, the stiff component. Furthermore,

chemical and enzymatic modifications of the composite are developed to produce DOPA-

Resiline-NCC which confers adhesive and

sealant properties to the composite.

As a central element of the extracellular matrix,

collagen is intimately involved in tissue

development, remodeling, and repair and confers high tensile strength to tissues.

Numerous medical applications, particularly,

wound healing, cell therapy, and bone reconstruction, rely on its supportive and

healing qualities. Its synthesis and assembly

require a multitude of genes and post-

translational modifications. Historically, collagen was always extracted from animal and

human cadaver sources, but bare risk of

contamination and allergenicity and was subjected to harsh purification conditions

resulting in irreversible modifications impeding

its biofunctionality. In parallel, the highly complex and stringent post-translational

processing of collagen, prerequisite of its

viability and proper functioning, sets significant

limitations on recombinant expression systems.

A tobacco plant expression platform has been

recruited to effectively express human collagen,


along with three modifying enzymes, critical to

collagen maturation. The plant extracted

recombinant human collagen type I forms

thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of

native collagen. Combining collagen at the

nano-scale with resilin to produce fibers

resulted in super-performing fibers with mechanical properties which exceed that of

natural fibers.


Studies on thermal and magnetic properties

of iron oxide nanoparticles for magnetic

hyperthermia application

Paula Soares, Isabel Ferreira and João Paulo Borges

Department of Materials Science, FCT-UNL, Campus de Caparica, 2829-516 Caparica, Portugal [email protected]

Hyperthermia is an old technique which is

recognized as a possible treatment option for

cancer [1]. Cancer is a severe disease and

currently is one of the leading causes of morbidity and mortality in the world, while

chemo- and radiotherapy present several side

effects due to their lack of specificity to the cancer type and the development of drug

resistance.

Iron oxide nanoparticles are having been

extensively investigated for several biomedical

applications such as hyperthermia and

magnetic resonance imaging for cancer

treatment. In this context, a work was

performed comparing the effect of surfactants on the stability and the heating ability of iron

oxide colloids.

Iron oxide nanoparticles were synthetized

through chemical precipitation and stabilized using two surfactants: sodium citrate and oleic

acid. The as-prepared nanoparticles were

characterized by several techniques and their

heating ability was evaluated using different

sample concentrations and field intensities.

Hysteresis loops measured at temperatures 10

and 315 K for coated iron oxide nanoparticles

are shown in Fig. 1. Comparing the effect of sodium citrate and oleic acid it is possible to

observe that oleic acid is reducing the magnetic

moments at the surface of the nanoparticles probably due to the diamagnetic contribution

of the surfactant volume. For higher

concentrations of oleic acid it seems to be an

increase in the SPA values.

Hyperthermia results (Fig. 2) show a strong

reduction on the ILP value when oleic acid is

added to the colloids, while for sodium citrate

this reduction is not so pronounced. However, ILP values are within the literature values for

commercial iron oxide nanoparticles.

These results show oleic acid has a more severe

effect on the magnetic properties and heating ability of the nanoparticles. This effect is

probably due to the surfactant viscosity and the

size of the molecule that is higher than sodium

citrate

References

[1] Soares, Paula IP; Alves, Ana MR; Pereira, Laura CJ; Coutinho, Joana T; Ferreira, Isabel MM; Novo, Carlos MM; Borges, João PMR. Journal of colloid

and interface science, 419 (2014) 46-51.

[2] IP Soares, Paula; MM Ferreira, Isabel; AGBN Igreja, Rui; MM Novo, Carlos; PMR Borges, João; Recent patents on anti-cancer drug discovery,7(1) (2012) 64-73.

[3] Soares, PIP; Dias, SJR; Novo, CMM; Ferreira, IMM; Borges, JP, Mini reviews in medicinal chemistry, 12(12) (2012) 1239-1249.

[4] Baptista, Ana; Soares, Paula; Ferreira, Isabel;

Borges, Joao Paulo; ,Nanofibers and nanoparticles in biomedical applications, Bioengineered Nanomaterials, 93, 2013,CRC Press

[5] Soares, Paula I. P., Ferreira, Isabel M.M., Borges,

João P.M.R., Topics in Anti-Cancer Research, Vol. 3;

Bentham Science Publishers, 2014, Chapter 9, 342-383.


Figures

Figure 1. Magnetization vs. applied magnetic field for Iron oxide nanoparticles coated with sodium citrate (a) and

oleic acid (b)

Figure 2. – Intrinsic loss power (ILP) vs. Surfactant concentration (left image – sodium citrate, Right image – Oleic

acid) for three field intensities with a frequency of 418.5 kHz.


Hijacking nature's own communication

system: evaluation of extracellular vesicles

as a sirna delivery vehicle

Stephan Stremersch1, K. Braeckmans1,2, R. E. Vandenbroucke3, S. De Smedt1, K. Raemdonck1 1Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical

Pharmacy, Ghent University, Ottergemsesteenweg 460, Ghent, Belgium 2Center for Nano-and Biophotonics, Ghent University, Ghent, Belgium

3Inflammation Research Center, VIB – Ghent University, Ghent, Belgium [email protected]

In order to exploit the therapeutic potential of small interfering RNA (siRNA), it is key to

overcome the various intra- and extracellular

barriers imposed by the human body. To this end, siRNA therapeutics are commonly

packaged in an appropriate nanosized drug

carrier.[1] Recently it was discovered that extracellular vesicles (EVs), i.e. lipid membrane-

sealed nanosized particles, act as nature’s own

nucleic acid delivery system.[2] EVs are secreted

by every cell type and have been shown to contain a variety of biological molecules,

including miRNA, which can be transferred to

other cells leading to phenotypic changes. For this reason, interest has surged towards

evaluating these vesicles as a new personalized

drug delivery platform for therapeutic nucleic acids, such as siRNA. Yet, to date a major

impediment in using EVs as a carrier for siRNA in

the clinic is the lack of a suitable procedure for

efficient and reproducible siRNA loading.[3]

In this work we aimed to develop and

thoroughly characterize methods for loading isolated EVs with siRNA. EVs were purified from

conditioned cell culture medium derived from a

B16F10 melanoma cell line by (density gradient) ultracentrifugation. The presence of EVs was

confirmed by means of cryo-TEM imaging,

immunoblot detection of EV-specific markers

and via their typical size and buoyant density.

In a first effort towards intrave¬sicular loading of

exogenous siRNA, we critically evaluated a

previously reported method based on electroporation of an EV/siRNA mixture with the

aim to induce transient pores in the EV

membrane, hence allowing the siRNA to migrate through the lipid bilayer. Using this approach,

siRNA encapsulation efficiencies up to 25% were

reported.[4] Importantly, duplication of these experiments in our hands under identical

experimental conditions revealed that the

afore-mentioned siRNA encapsulation was

largely due to unspecific aggregate formation, independent of the presence of extracellular

vesicles.[5] The latter aggregates resulted from

the interaction of multivalent cations, released from the metal electrodes in the electroporation

cuvettes, with hydroxyl anions present in the

electroporation buffer and were shown to co-precipitate siRNA. After blocking aggregate

formation no significant encapsulation of siRNA

could be measured. Taken together, these

results dem¬onstrate the necessity for alternative methods to load EVs with siRNA and

the importance of including the correct controls

to properly assess loading efficiencies in biological vesicles.

Next, we developed a new loading approach in which siRNA modified with a cholesterol moiety,

was used to ally siRNA to the EV lipid

membrane. The association of siRNA on the

surface of EVs was shown using different

methods based on gel electrophoresis, an

antigen-specific bead based assay and


iodixanol density gradient ultracentrifugation.

Moreover, we clearly demonstrated that this

approach required the use of chemically

stabilized siRNA, due to the presence of significant nuclease activity in the isolated EV

sample. Furthermore, we could confirm that EV

cell uptake was not affected by the siRNA

incorporation and compared the functional siRNA delivery capacity with an anionic,

fusogenic liposome in a monocyte/DC cell line

(JAWSII). Interestingly, we observed that the fusogenic liposomes clearly outperformed the

EVs in terms of siRNA delivery. Finally, we also

compared the gene silencing capacity of the cholesterol-siRNA inserted in the EV membrane

with that of the endogenously present,

intravesiclular miRNA’s. Therefore, a total

miRNA profiling of the purified EVs was done by a nCounter® miRNA expression assay. Target

mRNA’s in JAWSII cells of the most abundant

miRNA’s identified in the melanoma EVs were evaluated for specific post-transcripitional gene

suppression and compared to specific gene

silencing of the pan-leucocytic marker CD45 via the loaded cholesterol siRNA.

To conclude we can state that electroporation,

in contrast to previous reports, is not a feasible technique for loading siRNA in isolated EVs.

Instead we developed a new approach based

on a cholesterol modified siRNA to efficiently and reproducibly load EVs with exogenous

small nucleic acids (graphical abstract). Finally,

we compared the functional siRNA delivery potential between EVs and a classic, fusogenic

liposome and between exogenous siRNA and

endogenous miRNA.

References

[1] K. Raemdonck, R.E. Vandenbroucke, J. Demeester, N.N. Sanders, S.C. De Smedt, Drug

discov. today, 13 (2008) 917-931.

[2] H. Valadi, K. Ekstrom, A. Bossios, M. Sjostrand,

J.J. Lee, J.O. Lotvall, Nat. Cell Biol., 9 (2007)

654-U672.

[3] P. Vader, S.A. Kooijmans, S. Stremersch, K.

Raemdonck, Therapeutic delivery, 5 (2014)

105-107.

[4] L. Alvarez-Erviti, Y.Q. Seow, H.F. Yin, C. Betts, S. Lakhal, M.J.A. Wood, Nat. Biotech., 29 (2011)

341-U179.

[5] S.A. Kooijmans, S. Stremersch, K. Braeckmans,

S.C. De Smedt, A. Hendrix, M.J. Wood, R.M. Schiffelers, K. Raemdonck, P. Vader,

J.Control.Release, 172 (2013) 229-238.

Figures

Figure 1. EVs released by B16F10 melanoma cells were

purified via density gradient ultracentrifugation. Next,

two methods for exogenous siRNA loading were

evaluated. Electroporation appeared not a feasible

loading technique, cholesterol mediated siRNA loading

on the other hand, provided efficient and reproducible

loading. Finally, these cholesterol-siRNA loaded EVs

were evaluated for cell uptake and functional siRNA

delivery.


2D Microscale Surface Engineering with

Novel Protein based Nanoparticles for Cell

Guidance

Witold I. Tatkiewicz1,2, Joaquin Seras-Franzoso2,3,4, Elena García-Fruitós2,3, Esther Vazquez2,3,4, Nora

Ventosa1,2, Imma Ratera1,2, Antonio Villaverde2,3,4 and Jaume Veciana1,2 1Dep. of Molecular Nanoscience and Organic Materials, Institut de Ciencia de Materials de Barcelona

(CSIC), Bellaterra, 08193 Barcelona, Spain 2CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, Barcelona, Spain

3Ins. de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Barcelona, Spain 4Dep. of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain

[email protected] / [email protected] / [email protected]

The term “inclusion bodies” (IBs) was coined to

describe optically opaque moieties present in cell lumen. They have aspect of refractile

particles of up to a few hundred nanometers

and about 2 μm3 of size when observed by optical microscopy and as electron-dense

aggregates without defined organisation by

transmission electron microscopy [1].

The history of IBs turned when they were

recognized as a prospective biomaterial with desirable properties. Being a product derived

from biological synthesis, it is fully

biocompatible and preserves the functionality

of the embedded protein [2]. In a course of

investigation it was revealed that IBs size,

geometry, stiffness, wettability, z-potential, bio-adhesiveness, density/porosity etc. can be

easily fine tuned by control over basic

parameters of their production: harvesting time,

host genetic background and production conditions (e.g. temperature, pH) In addition,

their production and downstream processes are

fully scalable, cost effective and methodologically simple [3].

It is widely accepted, that cell´s responses, such

as positioning, morphological changes,

proliferation, motility and apoptosis are the result of complex chemical, topographical and

biological stimuli. Here we will show the

application of IBs as a functional biomaterial for

engineering two dimensional substrates for cell guidance. We have cultivated fibroblast cells on

supports patterned with IBs derived from green

fluorescent protein (GFP) or human basic fibroblast growth factor (FGF). Two

methodologies of pattern deposition were

applied: microcontact printing (μCP) optimized

for use with aqueous colloidal suspensions and a novel, template-free technique based on the

coffee-drop effect due to a convective self-

assembly (Figure 1) [4].

The first technique was applied in order to

deposit IBs with high resolution geometrical

patterns of various shapes and sizes. Then we

have investigated how cells react to IBs geometrical distribution. Parameters such as

orientation morphology and positioning were

thoroughly investigated based on rich statistical

data delivered by microscopy image treatment (Figure 2). The second technique has been

recently developed in order to deposit complex

and well-controlled two dimensional IB´s patterns with concentration gradients for the

study of cell motility (Figure 3). Cell movement

cultivated on such substrates was characterized and quantified based on confocal microscopy

time-lapse acquisitions [5,6].

mailto:[email protected]


In both cases a deep statistical data treatment

was preformed to characterize macroscopic

responses of cells when grown over nanoscale

profiles made with IBs concluding that cell proliferation is not only dramatically stimulated

but cell also preferentially adhere to IBs-rich

areas, align, elongate and move according to

such IBs geometrical cues. These findings prove the potential of surface patterning with

functional IBs as protein-based nanomaterials

for tissue engineering and regenerative medicine among other promising biomedical

applications

References

[1] (a) Villaverde A., Carrio M.M., Biotechnol. Lett., 25 (2003) 1385. (b) García-Fruitós E., Rodríguez-Carmona E., Diez-Gil C., Ferraz R.Mª, Vázquez E.,

Corchero J.L., Cano-Sarabia M., Ratera I., Ventosa

N., Veciana J., Villaverde A. Adv. Mater., 21 (2009) 4249. (c) Cano-Garrido, O., Rodríguez-Carmona E.,

Díez-Gil C., Vázquez E., Elizondo E., Cubarsi R.,

Seras-Franzoso J., Corchero J.L., Rinas U., Ratera I.;

et al. Acta Biomater. 9 (2013) 6134. [2] García-Fruitós E., Vazquez E., Díez-Gil C., Corchero

J.L.; Seras-Franzoso J., Ratera I., Veciana J., Villaverde A., Trends Biotechnol., 30 (2012), 65.

[3] (a) García-Fruitos E., Seras-Franzoso J., Vazquez E., Villaverde A., Nanotechnology, 21 (2010) 205101.

(b) Vazquez E., Corchero J. L., Burgueno J.F., Seras-

Franzoso J., Kosoy A., Bosser R., Mendoza R., Martínez-Láinez J.M., Rinas U., Fernandez E., Ruiz-

Avila L., García-Fruitós E., Villaverde A., Adv. Mater., 24 (2012) 1742. (c) Vazquez E., Roldán M., Diez-Gil

C., Unzueta U., Domingo-Espín J., Cedano J., Conchillo O., Ratera I., Veciana J., Daura X., Ferrer-

Miralles N., Villaverde A., Nanomedicine., 5 (2010)

259. [4] (a) Han W., Lin Z., Angew. Chem. Int. Ed., 51 (2012)

1534 (b) Hanafusa T., Mino Y., Watanabe S., Miyahara M.T., Advanced Powder Technology, 25

(2014) 811

[5] (a) Díez-Gil C., Krabbenborg S., García-Fruitós E., Vazquez E., Rodríguez-Carmona E., Ratera I., Ventosa N., Seras-Franzoso J., Cano-Garrido O.,

Ferrer-Miralles N., Villaverde A., Veciana J.,

Biomaterials, 31 (2010) 5805. (b) Seras-Franzoso J., Díez-Gil C., Vazquez E., García-Fruitós E., Cubarsi R.,

Ratera I., Veciana J., Villaverde A., Nanomedicine, 7

(2012) 79.

[6] Tatkiewicz W.I., Seras-Franzoso J., García-Fruitós E., Vazquez E., Ventosa N., Peebo K., Ratera I.,

Villaverde A., Veciana J., ACSNano, 7 (2013) 4774.

Figures

Figure 1. Schematic illustration of particle deposition.

Particles are pinning to the substrate on the edge of

meniscus, where the evaporation is more intense. Image

adapted from reference [4b].

Figure 2. IBs striped (top) and random (bottom) pattern are

compared. On the left; representative confocal microscopy

images of cells cultivated on such patterns are presented. On

the right; the overall orientation distribution of cells is

presented. It is clearly seen, that cells are guided by the

stripped pattern and they orient themselves along its

geometry, whereas no predominant orientation of cells can

be observed in the case of random pattern.

Figure 3. Example of GFP-derived IBs gradient pattern

deposited by a controlled convective self-assembly

technique. Left: fluorescence microscopy image, right: IBs

concentration calculated based on fluorescence intensity.


Atomic force microscopy, life sciences and

soft matter

José L. Toca-Herrera

Institute for Biophysics, Department of Nanobiotechnology, University of Natural Resources and Life

Sciences Vienna (BOKU), Muthgasse 11, A-1190 Vienna, Austria

In this contribution, I would like to present the

different use and development in atomic force microscopy (AFM) focusing primarily in the fields

of life sciences and soft matter. In particular, we

will see in which way the AFM is used as an

imaging machine to characterize macromolecules at different interfaces or to

follow crystallization processes. Furthermore,

the use of the AFM as a mechanical machine it will be presented. In this part, I will talk about

molecular forces, elasticity of proteins and cell

mechanics [1,2]. Finally, different possibilities to combine the scanning probe microscopy with

other microscopy techniques such as

fluorescence microscopy, RICM and STED will be

mentioned [3].

References

[1] ]. S. Moreno-Flores, R. Benitez, M. dM Vivanco

and J. L. Toca-Herrera. Stress relaxation and

creep on living cells with the atomic force microscope: a means to calculate elastic

moduli and viscosities of cell components.

Nanotechnology 21 (2010) 445101.

[2] K. A. Melzak, G. R. Lazaro, A. Hernandez-Machado, I. Pagonabarraga, J. M. Cardenas

Dıaz de Espada and J. L. Toca-Herrera. AFM

measurements and lipid rearrangements: evidence from red blood cell shape changes.

Soft Matter, 8 (2012) 3716.

[3] S. Moreno-Flores and J. L. Toca-Herrera. Hybridizing Surface Probe Microscopies:

Toward a Full Description of the Meso- And

Nanoworlds. CRC Press. 2013. Boca Raton. FL.

Figures

Figure 1. Left: force relaxation experiment after treating the

cells with cytochalasin. Note that the grey line (after actin

disruption with cytochalasin) decays faster than the black one

(control). Middle: creep compliance experiment on the same

cell before and after cytochalasin treatment. Note that the

deformation of the cell is larger (grey line) after cytochalasin

treatment. Right (above): optical image of the control MCF-7

cells. Right (below): optical image of the cells treated with

cytochalasin. Figure adapted from [2].


Multiplicity of Nanofection: a New Index to

Assess Nanoparticle Cellular Uptake

Juan D. Unciti-Broceta, Victoria Cano-Cortés, Patricia Altea Manzano, Salvatore Pernagallo, Juan J.

Díaz-Mochón, Rosario M. Sánchez-Martín.

Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional

Government (GENTO). PTS Granada,Avenida de la Ilustración, 114.18016 Granada, Spain Fac. de Farmacia, Dep. de Química Farmacéutica y Orgánica, Uni. de Granada, 18071 Granada, Spain

Nanogetic S. L. Calle Marqués de los Vélez 2, 6ºA. 18005 Granada, Spain

DestiNA Genomica S.L. Avenida de la Innovación, 1. 18100 Armilla (Granada), Spain [email protected]

Engineered nanoparticles (ENPs) for biological applications are produced from functionalized

nanoparticles (NPs) after undergoing multiple

coupling and cleaning steps, giving rise to an inevitable loss of NPs in final compositions.

Herein, we present a simple method to quantify

the number of ENPs per microliter using standard spectrophotometers and volumes of

up to one microliter. Light going through NP

suspensions is scattered via reflection,

refraction and diffraction phenomenon and the amount of the scattered light depend on the

number of NPs found in suspensions. By

measuring optical densities (OD) at 600 nm of different polystyrene NP suspensions of three

different sizes (100 nm, 200 nm and 460 nm),

linear correlations between OD600 and number of NPs were found for each NP size. These

calibration curves can then be applied to

estimate the number of ENP compositions of a

particular NP size and material (Figure 1). To exemplify the method, we introduced the

number of ENPs versus number of cells as a new

parameter to report cellular uptake assays where capacities of cells to uptake beads or NPs

(“nanofection”) need to be assessed. This

parameter allows us to introduce “multiplicity of nanofection 50” (MNF50) index, which is

defined as the number of NPs per cell needed to

“nanofect” 50% of a given cell type, as a

measure of the capacity of a cell type to uptake

certain ENPs. Three mammalian cell lines were

tested with 200 nm Cy5-PEG-NPs and, following

flow cytometry analysis, each of them presented different MNF50, being MDA MB 231 mammalian

breast cancer cell line the one with a lower

MNF50 and therefore with a higher uptaking capacity of these ENPs (Figure 2). Median of

fluorescence intensity (MFI) of Cy5 positive cells

analysis showed a linear behavior with different slopes for each cell line which is also a

parameter to assess cell capacities for NPs

uptaking (Figure 3). Furthermore, if we compare

MFI increments (ΔMFI=MFI sample/MFI untreated) same results were obtained (Figure

4). A deeper study of ΔMFI showed a surprising

data, from the closest ratio to their MNF50, the increase of the ΔMFI is doubled when the NPs

number are doubled, something which is not

observed when ratios lowers than their MNF50 are used. Importantly, this effect is the same for

the three studied cell lines. Therefore, when

MNF50 is reached, the nanofection rate is

constant and proportional to the number of nanoparticles used with cell lines presenting

similar behavior. Nowadays the efficiency of

many NPs-based delivery systems of bioactive cargoes are related to solid content (w/V) of NPs

per cell. This method allows introducing a new

parameter to analyze cellular uptake by reporting nanoparticle number versus cells

number (multiplicity of nanofection). Based on

these data we believe that the number of NPs

per cell could be reported rather than weight of

NPs per cell in any cell-based assays using NPs.

The implementation of the Multiplicity of


nanofection (MNF) will improve dramatically the

efficiency of any nanoparticle-based devices.

References

[1] V. Wagner, A. Dullaart, A. K. Bock and A. Zweck,

Nature biotechnology, 2006, 24, 1211-1217.

[2] E. Elinav and D. Peer, ACS nano, 2013, 7, 2883-2890.

[3] M. Goldsmith, L. Abramovitz and D. Peer, ACS

nano, 2014, 8, 1958-1965. [4] T. Lammers, S. Aime, W. E. Hennink, G. Storm

and F. Kiessling, Accounts of chemical

research, 2011, 44, 1029-1038. [5] Y. Namiki, T. Fuchigami, N. Tada, R.

Kawamura, S. Matsunuma, Y. Kitamoto and M.

Nakagawa, Accounts of chemical research,

2011, 44, 1080-1093. [6] J. G. Borger, J. M. Cardenas-Maestre, R.

Zamoyska and R. M. Sanchez-Martin,

Bioconjugate chemistry, 2011, 22, 1904-1908. [7] M. Bradley, L. Alexander, K. Duncan, M.

Chennaoui, A. C. Jones and R. M. Sanchez-

Martin, Bioorganic & medicinal chemistry letters, 2008, 18, 313-317.

[8] J. M. Cardenas-Maestre, A. M. Perez-Lopez, M.

Bradley and R. M. Sanchez-Martin,

Macromolecular bioscience, 2014, DOI: 10.1002/mabi.201300525.

[9] N. Gennet, L. M. Alexander, R. M. Sanchez-

Martin, J. M. Behrendt, A. J. Sutherland, J. M. Brickman, M. Bradley and M. Li, New

biotechnology, 2009, 25, 442-449.

[10] S. Kunjachan, F. Gremse, B. Theek, P. Koczera, R. Pola, M. Pechar, T. Etrych, K. Ulbrich, G.

Storm, F. Kiessling and T. Lammers, ACS nano,

2012, 7, 252-262.

[11] R. Sanchez-Martin, V. Cano-Cortes, J. A. Marchal and M. Peran, Methods in molecular

biology (Clifton, N.J.), 2013, 1058, 41-47.

[12] R. M. Sanchez-Martin, L. Alexander and M. Bradley, Annals of the New York Academy of

Sciences, 2008, 1130, 207-217.

[13] R. M. Sanchez-Martin, M. Cuttle, S. Mittoo and

M. Bradley, Angewandte Chemie

(International ed. in English), 2006, 45, 5472-

5474.

[14] S. Sengupta and A. Kulkarni, ACS nano, 2013,

7, 2878-2882. [15] A. Tsakiridis, L. M. Alexander, N. Gennet, R. M.

Sanchez-Martin, A. Livigni, M. Li, M. Bradley

and J. M. Brickman, Biomaterials, 2009, 30,

5853-5861. [16] R. M. Sanchez-Martin, M. Muzerelle, N. Chitkul,

S. E. How, S. Mittoo and M. Bradley,

Chembiochem : a European journal of chemical biology, 2005, 6, 1341-1345.

[17] J. Shi, A. R. Votruba, O. C. Farokhzad and R.

Langer, Nano letters, 2010, 10, 3223-3230. [18] R. M. Yusop, A. Unciti-Broceta, E. M.

Johansson, R. M. Sanchez-Martin and M.

Bradley, Nature chemistry, 2011, 3, 239-243.

[19] A. Unciti-Broceta, J. J. Díaz-Mochón, R. M. Sánchez-Martín and M. Bradley, Accounts of

chemical research, 2012, 45, 1140-1152.

[20] D. Schneider and K. Lüdtke-Buzug, in Magnetic Particle Imaging, eds. T. M. Buzug and J.

Borgert, Springer Berlin Heidelberg, 2012, vol.

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[22] B. D. Chithrani, A. A. Ghazani and W. C. Chan,

Nano letters, 2006, 6, 662-668. [23] B. D. Chithrani and W. C. Chan, Nano letters,

2007, 7, 1542-1550.

[24] V. V. Savchenko, A. G. Basnakian, A. A. Pasko, S. V. Ten and R. Huang, in Computer graphics,

eds. E. Rae and V. John, Academic Press Ltd.,

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[28] F. Alexis, E. Pridgen, L. K. Molnar and O. C.

Farokhzad, Molecular pharmaceutics, 2008, 5,

505-515.

Figures

Figure 1

Figure 2

Figure 3

Figure 4


Nanomechanics of Decellularized Lung and

in Vivo Lung Elastance in a Murine Model of

Marfan Syndrome

J.J. Uriarte1,3,4, P.N. Nonaka1, N. Campillo1,2,4, Y. Mendizabal5, E. Sarri5, G. Egea5, R.Farre1,3,4; D. Navajas1,2,4 1Unitat de Biofísica i Bioingeniería, Facultat de Medicina, Univ. de Barcelona, Barcelona,Spain

2Institut de Bioenginyeria de Catalunya, Barcelona, Spain 3Institut d’Investigacions Biomèdiques August Pi Sunyer, Barcelona, Spain

4CIBER de Enfermedades Respiratorias, Madrid, Spain 5Dept. Biologia Cel·lular, Immunologia i Neurociències. Fac. de Medicina, UB, Barcelona, Spain

[email protected]

Marfan syndrome (MFS) is an autosomal

dominant disorder caused by mutations in the

gene (FBN1) encoding fibrillin-1, the major component of extracellular matrix (ECM)

microfibrills. In the pathogenesis of MFS, matrix

metalloproteases and over activity of TGF-β are directly involved. The syndrome carries an

increased risk of aneurysm and dissection of the

ascending aorta, and alterations in eyes,

skeleton and lungs. Although lung mechanics in MFS could be affected by changes in elastic and

tensile strength of connective tissue, there are

no data available on the effects of this monogenetic disease in lung mechanics. The

aim of this work is to assess whether lung

scaffold stiffness and in vivo lung elastance is affected in a Marfan mouse model. Twelve 9

month-old C57BL/6 mice (6 healthy controls

and 6 Marfan mice heterozygous for an Fbn1

allele encoding a cysteine substitution, Fbn1(C1039G/+) were used. Control and Marfan mice

were intraperitoneally anesthetized (urethane,

1.5 g/kg), paralyzed (pancuronium bromide, 0.1 mg/kg) and subjected to volume-control

mechanical ventilation (100 breaths/min, 0.30

ml tidal volume). Subsequently, the chest wall was opened and a positive end-expiratory

pressure of 2 cmH2O was applied. The signals of

tracheal pressure and flow during mechanical

ventilation were recorded at the entrance of the

tracheal cannula and lung elastance was

determined by conventional linear regression.

The animals were euthanized by

exsanguination, the left lung lobule was excised

and decellularized with a conventional protocol based on freezing/thawing cycles and sodium

dodecyl sulfate detergent. Acellular lung slices

(12 micron thick) were obtained in order to measure nanomechanics (Young’s modulus) of

different regions of the lung scaffold (alveolar

septum, tunica adventitia and tunica intima)

with atomic force microscopy using pyramidal cantilevers (nominal spring constant 0.03 N/m)

at an operating indentation of 500 nm. Marfan

mice exhibited an in vivo lung elastance that was 42% lower than controls (21.7±2.7 and

37.1±2.5 cmH2O/ml, respectively; mean±SEM;

p<0.05). Remarkably, no significant differences were found in the local stiffness of the acellular

lung between Marfan mice and controls:

36.4±3.7 vs 38.4±10.0 kPa, 63.2±17.5 vs 48.2±6.8

kPa and 125.2±10.2 vs 119.8±23.7 kPa in the alveolar septum and the lung vessels tunicae

adventitia and intima, respectively. In

conclusion, these data suggest that the higher in vivo compliance observed in Marfan lungs are

not caused by a softening of the acellular lung

scaffold, as demonstrated by AFM measurements of the local nanomechanical

properties of the extracellular matrix of the lung.

These changes could be attributed to

alterations in the 3-D structure of the lung.


References

[1] Luque T, Melo E, Garreta E, Cortiella J, Nichols

J, Farré R, Navajas D. Local micromechanical properties of decellularized lung scaffolds

measured with atomic force microscopy. Acta

Biomaterialia 9 (2013) 6852–6859.

[2] Melo E, Cardenes N, Garreta E, Luque T, Rojas M, Navajas D, Farré R. Inhomogeneity of local

stiffness in the extracellular matrix scaffold of

fibrotic mouse lungs. J Mech Behav Biomed Mater 2014; 37: 186–195.

[3] Neptune ER, Frischmeyer PA, Arking DE, Myers

L, Bunton TE, Gayraud B, Ramirez F, Sakai LY, Dietz HC. Dysregulation of TGF-beta activation

contributes to pathogenesis in Marfan

syndrome. Nat Genet. 2003 Mar; 33(3):407-11.

[4] Cañadas V, Vilacosta I, Bruna I, Fuster V. Marfan Syndrome. Part 1: Pathophysiology

and diagnosis. Nat Rev Cardiol. 2010 May; 7

(5): 256-65.

Figures

Figure 1. Effective elastance (E) computed from control

(white) and Marfan mice (gray) during in vivo conventional

mechanical ventilation. Mean ± SE. Asterisk indicates p<0.05.

Figure 2. Local stiffness at different acellular lung

parenchyma of control (white) and Marfan mice (gray). Mean ±

SE. There are no significant differences.


Supramolecular organizations as novel

nanomedicines for drug delivery

Jaume Veciana

Institut de Ciència de Materials de Barcelona (CSIC) and Networking Research Center on Bioengineering,

Biomaterials and Nanomedicine (CIBER-BBN), Campus Universitari de Bellaterra, Cerdanyola, Spain

[email protected]

The objective of this lecture is to give a broad overview of how nanotechnology is impacting in

some areas of medicine and pharmacology.

This lecture will report the advantages of

nanoparticulate molecule-based organizations for drug delivery. It has been reported that

polymeric nanoparticles and nanovesicles are

efficient drug carriers that can significantly help to develop new drug delivery routes, and more

selective and efficient drugs with a higher

permeability to biological membranes and with controlled released profiles, as well as to

enhance their targeting towards particular

tissues or cells [1-2].

The potential of nanotechnology «bottom-up»

strategies, based on molecular self-assembling, is much larger than that of «top-down»

approaches for the preparation of such

nanosized supramolecular organizations. For

instance, by precipitation procedures it should

be possible to control particle size and size

distribution, morphology and particle supramolecular structure. However,

conventional methods from liquid solutions

have serious limitations and are not adequate

for producing such nanoparticulate materials at large scale with the narrow structural variability,

high reproducibility, purity and cost needed to

satisfy the high-performance requirements and regulatory demands dictated by the USA and

European medicine agencies. On the contrary,

using compressed solvent media it is possible to obtain supramolecular materials with unique

physicochemical characteristics (size, porosity,

polymorphic nature morphology, molecular

self-assembling, etc.) unachievable with

classical liquid media. The most widely used CF is CO2, which has gained considerable attention,

during the past few years as a «green substitute»

to organic solvents. Due to such properties,

during the past few years CFs based technologies are attracting increasing interest

for the preparation of nanoparticles and

nanovesicles with application in nanomedicine.

In this lecture a simple one-step and scale-up methodology for preparing multifunctional

nanovesicle-drug conjugates will be presented.

This method is readily amenable to the

integration/encapsulation of multiple bioactive

components, like peptides, proteins, enzymes,

into the vesicles in a single-step yielding

sufficient quantities for clinical research becoming, thereby, nanocarriers to be used in

nanomedicine. A couple of examples of novel

nanomedicines for solving serious diseases,

prepared by this methodology, will be

presented and their advantages discussed [3-4]

References

[1] M. E. Davis, Z. Chen, D. M. Shin, Nature Reviews-Drug Discovery 2008, 7, 771-782.

[2] J. A. Hubbell, R. Langer, Nature Materials, 2013, 12, 963-966.

[3] N. Ventosa, L. Ferrer-Tasies, E. Moreno-Calvo, M. Cano, M. Aguilella, A. Angelova, S. Lesieur, S. Ricart, J. Faraudo, J. Veciana. Langmuir, 2013, 29, 6519-6528.

[4] I. Cabrera, E. Elizondo, E. Olga; J. Corchero, M. Mergarejo, D. Pulido, A. Cordoba, E. Moreno-Calvo, U.

Unzueta, E. Vazquez, I. Abasolo, S. Schwartz, A.

Villaverde, F. Albericio, M. Royo, M. Garcia, N. Ventosa,

J. Veciana. Nano Letters, 2013, 13, 3766-3774.


Polymeric micelles nanovectors for

photodynamic therapy applications: From

the structure to the activity

P. Vicendo1, A.F. Mingotaud1, L. Gibot2 , A. Lemelle1, U. Till1,2,3, B. Moukarzel1, M.P. Rols2, C.

Chassenieux4, M. Gaucher5 and F. Violleau5 1Université de Toulouse; UPS/CNRS; IMRCP, Toulouse Cedex 9, France

2Université de Toulouse, IPBS-CNRS UMR 5089, 205 Route de Narbonne, Toulouse Cedex, France 3Technopolym, Institut de Chimie de Toulouse, 118 route de Narbonne, Toulouse Cedex 9, France.

4Université de Toulouse, Institut National Polytechnique de Toulouse – Ecole d’Ingénieurs de

Purpan, Département Sciences Agronomiques et Agroalimentaires, UPSP/DGER 115, 75 voie du

TOEC, BP 57611, F-31076 Toulouse Cedex 03, France 5LUNAM Université, Université du Maine, IMMM UMR CNRS 6283 Département PCI, Avenue Olivier

Messiaen, 72085 Le Mans Cedex 09, France

[email protected]

The recent development of light irradiation

systems has facilitated the emergence of new therapies based on light-sensitive drugs.

However, photosensitizers have a tendency to

self-associate in physiologic environment,

leading to a loss of their physical properties. Hence, nanometric formulations have been

assessed, because this limits self-association

and enables accumulation in solid tumors owing to enhanced permeability and retention

effect (EPR).

In this study, we present first a thorough

characterization of polymeric micelles based on

light scattering and Asymmetrical Flow Field

Flow Fractionation. In a second step, we examine their efficiency as photosensitizer

vectors using 2D or 3D tumor model namely

spheroids.

Polymeric micelles were formed from 4 different

amphiphilic block copolymers: poly(ethylene oxide-b- ε-caprolactone) 2000-2800,

poly(ethylene oxide-b-ε-caprolactone) 5000-

4000, poly(ethylene oxide-b-polystyrene) 3100-

2200 and poly(ethylene oxide-b-(D,L)-lactide)

2400-2000. The micelles have been

characterized by static and dynamic light

scattering, electron microscopy and

asymmetrical flow field-flow fractionation. This showed that all systems led to polymeric self-

assemblies having a size close to 20nm and a

neutral surface.

They were shown to be stable upon ageing and

dilution, even in the presence of various blood

components such as globulins or albumin, which is essential for a possible application as

vectors. Cytotoxicity and phototoxicity in the

presence of Pheophorbide a as photosensitizer were then characterized both on 2D and 3D cell

culture. PDT on spheroids enabled to

corroborate results from 2D, showing that

encapsulation of Pheophorbide yielded a strong increase of photocytotoxicity.

However, small differences for the nanovectors were highlighted: PEO-PCL 2000-2800 being the

most efficient in 2D, whereas PEO-PDLLA 2400-

2000 was the best for 3D tests. The obtained results will be discussed in relation with the

ones obtained in physical chemistry

characterizations.

Only a thorough physico-chemical

characterization coupled to in vitro experiments


may enable a critical analysis of possible

vectors. The polymeric micelles chosen in this

study were observed to yield a strong efficiency

in PDT, but the differences observed between 2D and 3D systems show that a great care

should be taken when testing such vectors.

References

[1] L. Gibot, A. Lemelle, U. Till, B. Moukarzel, A.-F.

Mingotaud, V. Pimienta, P. Saint-Aguet, M.-P. Rols, M. Gaucher, F. Violleau, C. Chassenieux,

P. Vicendo, Biomacromolecules 15(4) (2014)

1443-1455.

Figures

Figure 1. Polymers and PS used

Figure 2. Example of tumor spheroid macroscopic aspect

after PDT procedure with PEO-PS micelles loaded with

pheophorbide a.


Nanographene-oxide mediated

hyperthermia for cancer treatment

Mercedes Vila

TEMA-NRD, Mechanical Engineering Department and Aveiro Institute of Nanotechnology (AIN),

University of Aveiro, 3810-193 Aveiro, Portugal

[email protected]

One of the new trends of nanomedicine is the application of nanoparticles for targeting

tumors to achieve localized tumor cell

destruction while producing minimal side

effects on healthy cells, but their application will not be feasible without a previous

understanding of vector-cell/tissue interactions,

possible toxicity and accumulation risks. [1]

The enhanced permeability and retention effect

(EPR), provoked by the angiogenesis process of tumors, allows preferential concentration of

nanosystems on the tumor periphery, making

hyperthermia mediated by these systems a

potential efficient therapy for producing confined tumoral cell death. [2] It can induce

lethal damage to cellular components at

temperatures above 40 ºC and cancerous cells are subsequently removed by macrophages,

causing the tumor to diminish. Although

hyperthermia is a well-known concept, little is known about the type of damage, cell death

and secondary effects that these nanosystems

mediated therapies can provoke locally.

Amongst hyperthermia potential agents, nano

graphene oxide (nGO) has been proposed due

to its strong Near-Infrared (NIR 700-1100 nm range) optical absorption ability and its unique

2-dimensional aspect ratio. [3] Restricting all

dimensions at nanoscale could allow unique performing when compared to any other

nanoparticle, but it is mandatory to deeply

study the hyperthermia route and the kind of

nGO-cell interactions induced in the process.

By optimizing the nGO synthesis, it is possible to diminish the initial cell-particle interactions to

reduce possible future toxicity in healthy

cells.[3,4] Cell internalization kinetics

(specifically for targeting tumoral osteoblasts on a bone cancer model) were established for

producing a safe and efficient tumor cell

destruction avoiding damage on untreated cells as well as an evaluation of the nature of tumor

destruction that could be produced by this

hyperthermia treatment.[5,6] The type of cell damage and toxicity produced by NIR laser

irradiation was evaluated as a function of

exposure time and laser power in order to

control the temperature rise and consequent damage in the nGO containing cell culture

medium. The results showed that cell culture

temperature (after irradiating cells with internalized nGO) increases preferentially with

laser power rather than with exposure time.

Moreover, when laser power is increased, necrosis is the preferential cell death (Fig. 1).

The results suggested that controlling the type

of cell death, the threshold for producing soft or

harmful damage could be precisely controlled and so, the increase of cytokine release to the

medium, having this a direct impact on immune

system reactions. Moreover, nGO cell exposure did not stimulate proinflammatory cytokine

secretion [7] and nanoparticles incorporation by

different cell types either in the absence or in the presence of eight endocytosis inhibitors,

showed that macropynocitosis is the general

mechanism of nGO internalization, but it can

also entry through clathrin-dependent

mechanisms in hepatocytes and macrophages.

[8].


References

[1] Day ES, Morton JG, West JL. Nanoparticles for Thermal Cancer Therapy J Biomed Eng

2009;131:074001.

[2] Gonçalves G, Vila M, Portolés MT, Vallet-Regi

M, Gracio J, Marques PAAP. Nano-Graphene Oxide: a potential platform for cancer therapy

and diagnosis. Advanced Healthcare

Materials. (2013) 2(8):1072-90 [3] Vila M, Portolés MT, Marques PAAP, Feito MJ,

Matesanz MC, Ramírez-Santillán et al. Cell

uptake survey of pegylated nano graphene oxide. Nanotechnology 2012; 23:465103.

[4] Gonçalves G, Vila M, Bdikin I, de Andrés A,

Emami N, Ferreira RAS, Carlos LD, Grácio J,

Marques PAAP. Breakdown into nanoscale of graphene oxide: Confined hot spot atomic

reduction and fragmentation Nature Scientific

Reports, 2014, In press [5] Vila M.,.Matesanz M.C, Gonçalves G., Feito M.J.,

Linares J., Marques P.A.A.P., Portolés M.T.,

Vallet-Regi M. Triggering cell death by nanographene oxide mediated hyperthermia.

Nanotechnology 2014 (25) 035101

[6] Matesanz MC, Vila M, Feito MJ, Linares J,

Gonçalves G, Vallet-Regi M et al. The effects of graphene oxide nanosheets localized on f-

actin filaments on cell cycle alterations.

Biomaterials 2013; 34: 1562-9. [7] Feito MJ, Vila M, Matesanz MC, Linares J,

Gonçalves G, Marques PAAP, Vallet-Regí M,

Rojo JM, Portolés MT. In vitro evaluation of graphene oxide nanosheets on immune

function J Colloid Interface Sci. 432 (2014) 221-

228;

[8] Linares J, Matesanz MC, Vila M, Feito MJ, Gonçalves G, Vallet-Regí M, Marques PAAP,

Portolés MT. Endocytic Mechanisms of

Graphene Oxide Nanosheets in Osteoblasts, Hepatocytes and Macrophages ACS Appl.

Mater. Interfaces, 6 (2014) 13697−13706

Figures

Figure 1. Morphology evaluation by confocal microscopy of

cultured human SAOS-2 osteoblasts in the presence of GOs,

before (left) and after 7 min of 1.5 W/cm2 laser irradiation

showing necrotic cells (right).


Host-guest engineered stimuli-responsive

nanocapsules

Ewelina Wajs1, Thorbjørn Terndrup Nielsen2, Alex Fragoso1 1Nanobiotechnology & Bioanalysis Group, Universitat Rovira I Virgili, Tarragona, Spain

2 Department of Biotechnology, Aalborg University, 9000 Aalborg, Denmark

[email protected]

The supramolecular self-assembly of materials through host-guest interactions is a powerful

tool to create non-conventional materials. Thus,

biodegradable nanocapsules with redox-/or

light-responsibility were fabricated with non-covalent interactions between βCD and

ferrocene (Fc)/or αCD and azobenzene (Azo)

units. Different biocompatible polymers, dextran-βCD (βCD-Dex) and dextranferrocene

(Fc-Dex), dextran-αCD (αCD-Dex) and dextran-

azobenzene (Azo-Dex) were assembled in alternating way on gold nanoparticles of two

different sizes (100 and 400 nm). The gold

nanoparticles were removed by chemical

degradation and rhodamine B (RhB) was encapsulated inside the carriers as a model

drug. The encapsulation process of the dye

molecules was accelerated by oxidation step or by UV-light of the nanocapsules wall, thus

enabling easier and faster diffusion through the

polymer layers. Confocal laser scanning microscopy (CLSM), scanning electron

microscopy (SEM), atomic force microscopy

(AFM), RAMAN spectroscopy, UV-spectroscopy

and dynamic light scattering (DLS) measurements were employed for the

characterization of the nanocapsules.

*Financial support from Ministerio de Economía

y Competitividad, Spain (grant BIO2012-30936

to A.F.) is gratefully acknowledged.

Figures

Figure 1. Schematic representation of the formation of LbL

self-assembled nanocapsules via host-guest interactions

between complementary βCD and Fc appended dextran

polymers (the same methodology was applied for the

αCD/Azo appended dextran polymers).


Nanoceria

Tetyana Yudina, Eudald Casals, Víctor Puntes

The Catalan Institute of Nanoscience and Nanotechnology (ICN2), Edifici CIN2, 08193 Bellaterra (Barcelona), Spain

[email protected]

Cerium oxide nanoparticles (CeO2NPs or

nanoceria) is an inorganic material with many of

applications and more to come. Besides being rather a chemically inert ceramic, its flurite-like

electronic structure (Fig. 1) confers it a variety of

interesting properties, making nanoceria one of

the most interesting NPs in industry and biomedical research. What makes nanoceria

very appealing is its high capacity to buffer

electrons from an oxidant/reducing environment, which is due to its easy ability of

being oxidized and reduced, from Ce3+ to Ce4+

and vice versa [1], followed by the capture or release of oxygen, or oxygen species (as OH·).

Since redox reactions are an important class of

chemical reactions encountered in everyday processes, CeO2 NPs are widely used in a range

of industrial applications as combustion of

fuels, environmental remediation [2], water purification [3], catalysis [4] and many others.

A special interesting case is metabolism where partial reduction of oxygen produces by-

products, known as reactive oxygen species

(ROS) including superoxide anion (O2-),

hydrogen peroxide (H2O2) and the hydroxyl radical (OH·). On one hand, ROS are an

antibacterial tool in case of infection; on the

other hand, high amounts of ROS are toxic for humans and the environment. Unfortunately,

the heightened levels of ROS can damage

significantly cellular integrity, by inducing chronic inflammation, lipid peroxidation, DNA

damage, damage of oxidation sensitive

proteins, or even trigger cell death (apoptosis)

by a metabolic flux disruption. Therefore, the

oxygen storage capacity of CeO2NPs becomes

highly useful to remove them as soon as they

are generated, in situation of ROS disbalance.

That property makes nanoceria a potential

candidate as a therapeutic tool in prevention and treatment of a wide range of human

diseases with ROS disbalance, such as: cancer,

diabetes mellitus, cardiovascular disease (CDV),

age-related macular degeneration (AMD) and ophthalmology. Moreover, the overproduction

of ROS is critical in neurodegeneration,

including Alzheimer, Parkinson, Huntington, schizophrenia among others.

What is clear is that there is a strong correlation between the cellular effects of the NPs and their

engineering, including the preparation method,

morphology (size, shape, surface composition,

contaminants) and aggregation state of the nanoparticles [5].

Regarding its uses in medicine, morphology determines biodistribution and reactivity,

therefore, for the hard task of performing

precise work within the biological machinery, a fine morphological control of CeO2

nanoparticles and their aggregation state is

needed, since it drives the reactivity, colloidal

stability, interaction with proteins and pharmacokinetics of the nanoparticles within

the organism.

Up to now many of labored protocols of

nanoceria synthesis have been described, such

as high-temperature thermolysis of cerium salts, mechanochemical reactions, gas-phase

methods, non-isotermal precipitation,

supercritical synthesis methods, hydrothermal

synthesis, sol-gel, flame-spray pyrolysis and

solvothermal method, between others. Many of

them require multiple steps, as use of high


temperatures, refluxing, sonication or product

drying. Nowadays, the wet-chemical

preparations have become one of the most

widely used methods of synthesis of CeO2 nanoparticles. Even so, an obtaining of pure

and monodisperced CeO2 nanoparticles, with a

reproducible size-control is still a challenge,

since its is a highly reactive nanomaterial and tends to suffer changes in size, morphology and

aggregation state (Fig.2).

Therefore, we focused our study in an

understanding of the kinetic behavior of the

formation of nanoceria, in order to be able to control the purity, size and aggregation state of

the obtained material.

In this study we describe a preparation of CeO2 nanoparticles in aqueous phase by a kinetic

control of Ce3+ oxidation at room temperature.

We also try to give explanations of the nucleation, selective attachment and

aggregation phenomena of the nanoceria and

propose storage conditions suitable for their bio-medical or industrial purposes. The size-

dependent reactivity, scalability and bio-

compatibility are also analyzed.

References

[1] Cafun, J.D. et al., ACS Nano, 7(2013) 10726-32.

[2] Sajith, V., Sobhan C.B., Peterson G.P.,

Advances in Mechanical Engineering, Vol 2010 (2010) 1.

[3] Mellaerts, R. et al., Rsc Advances, 3(3) (2013)

900-909.

[4] Popa-Wagner, A. et al., Oxid Med Cell Longev, 2013 (2013) 963520.

[5] Dowding, J.M. et al., Acs Nano, 7(6) (2013)

4855-4868.

Figures

Figure 1. Electronic structure of CeO2NPs fluorite structure,

containing 8 coordinate Ce4+ and 4 coordinate O2–.

Figure 2. HR-TEM imaging of CeO2NPs, showing the instability

of nanoceria, reflected in phenomenas as selective

attachment (red brackets in (a) and (b)), morphological

changes of the NPs (blue brackets in (a) and (c)) and

aggregation (d).

N a n o B i o & M e d 2 0 1 4 1 0 0 n o v e m b e r 1 8 - 2 1 , 2 0 1 4 - B a r c e l o n a ( S p a i n )

FluidFM: combining AFM and microfluidics

for single-cell perturbation in vitro

Tomaso Zambelli

Laboratory of Biosensors and Bioelectronics, ETH Zurich, Switzerland

Glass micropipettes are the typical instrument

for intracellular injection, patch clamping or extracellular deposition of liquids into viable

cells. The micro pipette is thereby slowly

approached to the cell by using micro

manipulators and visual control through an optical microscope. During this process,

however, the cell is often mechanically injured

which leads to cell death and failure of the experiment. To overcome these challenges and

limitations of this conventional method we

developed the FluidFM technology, an evolution of standard AFM microscopy combining

nanofluidics via cantilevers with integrated

microfluidic channel [1]. The channel ends at a

well-defined aperture at the apex of the AFM tip while the other extremity is connected to a

reservoir. The instrument can therefore be

regarded as a multifunctional micropipette with force feedback working in liquid environment.

We are focussing on three applications for single-cell biology [2]: i) cytosolic and

intranuclear injection, ii) cell adhesion, and iii)

single virus deposition on cell surfaces.

At the same time we are using the FluidFM as

lithography tool in liquid [3].

References

[1] A. Meister et al, Nano Lett (2009) 9:2501 [2] O. Guillaume-Gentil et al, Trends Biotech

(2014) 32:381

[3] R.R. Grüter et al, Nanoscale (2013) 5:1097.

Figures

Figure 1. a) Scheme of the FluidFM. b) Two fluorescent viruses

ejected from a microchanneled cantilever. c) A yeast attached

by underpressure at the aperture of a microchanneled

cantilever.

N a n o B i o & M e d 2 0 1 4 n o v e m b e r 1 8 - 2 1 , 2 0 1 4 - B a r c e l o n a ( S p a i n ) 1 0 1

Poster list:

Alphabetical order (by surname)

Authors Country Poster title

Almendral-Parra, María Jesús Spain

Biomolecule-Quantum Dot systems for biological applications:

Size-controlled aqueous synthesis of CdS Quantum Dots in

homogeneous phase with BSA as capping ligand. Sara Sánchez Paradinas and Víctor Barba

Vicente

Ariza-Sáenz, Martha Spain

Optimization and characterization of poly (lactic-co-glycolic

acid) nanoparticles loaded with an HIV-1 inhibitor synthetic

peptide. Vega E., Espina M., Gómara M.J, Egea M.A,

Haro I., Garcia M.L.

Aviñó, Anna

Spain Nucleic Acids for biosensing applications. César Sánchez, Mar Oroval, Laura Carrascosa,

Ramón Martínez-Máñez, Laura Lechuga and

Ramon Eritja

Blanco Perez, Jordi Spain

Silver nanoparticles downregulates p53 activation and induce

desacetilation of histone 3 in epithelial human lung cancer

epithelial cells. Daisy Lafuente, Domènec Sánchez, José Luis

Domingo, Mercedes Gómez

Busquets, M. Antònia Spain Magnetoliposomes for magnetic resonance imaging.

Joan Estelrich, Josep Queralt, Montserrat

Gallardo

Cabellos, Joan

Spain In vitro and in vivo evaluation of TiO2 oral absorption. Gemma Janer, Ezequiel Mas del Molino,

Elisabet Fernández-Rosas, Socorro Vásquez- Campos

Corredor, Miriam Spain

Synthesis of modified dendrimers and conjugation with

selected apoptosis inhibitors. Ignacio Alfonso, Dietmar Appelhans, Angel

Messeguer

Deville, Sarah

Belgium

Monitoring the intracellular dynamics of polystyrene

nanoparticles in lung epithelial cells monitored by image

(cross-) correlation spectroscopy and single particle tracking.

Rozhin Penjweini, Nick Smisdom, Kristof

Notelaers, Inge Nelissen, Jef Hooyberghs, Marcel Ameloot

Estelrich, Joan Spain

Interaction of targeted of magnetoliposomes with Hela

epithelial carcinoma and 3T3 fibroblasts cell lines. M. Carmen Moran, M. Antònia Busquets

Fàbregas, Anna

Spain Thermal stability of a cationic solid lipid nanoparticle (cSLN)

formulation as a possible biocompatibility indicator. Montserrat Miñarro, Josep Ramon Ticó, Encarna García-Montoya, Pilar Pérez-Lozano,

Josep Mª Suñé-Negre

Feiner-Gracia, Natàlia Spain PLGA nanoparticles as advanced imaging nanosystems.

C. Fornaguera, A. Dols-Perez, M.J. García-

Celma, C. Solans

Giorello, Antonella

Spain

Continuous synthesis of silver nanoparticles using green

chemicals and microreactors and its evaluation as bactericidal

agents.

Santiago Ibarlín, Esteban Gioria, José Luis

Hueso, Victor Sebastián, Manuel Arruebo,

Laura Gutierrez and Jesús Santamaría

N a n o B i o & M e d 2 0 1 4 1 0 2 n o v e m b e r 1 8 - 2 1 , 2 0 1 4 - B a r c e l o n a ( S p a i n )


Gómara, María José Spain

Biodegradable polymeric nanoparticles modified with cell

penetrating peptides as an effective ocular drug delivery

system. Aimee Vasconcelos, Estefanía Vega, Yolanda

Pérez, María Luisa García, Isabel Haro

Grijalvo, Santiago Spain

Cationic vesicles based on non-ionic surfactant and synthetic

aminolipids mediate delivery of antisense oligonucleotides into

mammalian cells. Adele Alagia, Gustavo Puras, Jon Zárate, Jose Luis Pedraz and Ramon Eritja

Herance, José Raúl

Spain Gold Nanoparticles Supported on Nanoparticulate Ceria as a

Powerful Agent against Intracellular Oxidative Stress. María Gamón, Cristina Menchón, Roberto

Martín, Nadezda Apostolova, Milagros Rocha,

Victor Manuel Victor, Mercedes Alvaro,

Hermengildo García

Khanmohammadi Mohammadreza Iran

Application of magnetic chitosan nano particles for anti-

Alzheimer drug delivery systems. Hamideh Elmizadeh

Llanas, Hector Spain

The interferences of nanomaterials with hemoglobin a

handicap to study hemocompatibility. Sordé A. , Mitjans M. , Vinardell M.P.

López Martínez, Montse Spain

Nanoscale conductance imaging of electronic materials and

redox proteins in aqueous solution. J. M. Artés, I. Díez-Perez, F. Sanz and P.

Gorostiza

Lotfallah, Ahmed H. Spain

Gemini Amphiphilic Pseudopeptides for Encapsulation and

Release of Hydrophobic Molecules. Ignacio Alfonso,M. Isabel Burguete and

Santiago V. Luis

Marcos, Mercedes Spain

ph-responsive “onion nanospheres” coming from ionic liquid

crystal pamam dendrimers. Silvia Hernández-Ainsa, Joaquín Barberá, José Luis Serrano, Teresa Sierra

Nelissen, Inge Belgium

Modulation of dendritic cell sensitization by combined exposure

to allergens and nanoparticles. Birgit Baré, Sarah Deville, An Jacobs, Nathalie

Lambrechts, Peter Hoet

Ochoa-Zapater, María Amparo Spain

Toxicity assays of nebulized gold nanoparticles with potential

applications in the development of nanopesticides. J. Querol-Donat, F.M. Romero, A. Ribera, G.

Gallello, A. Torreblanca, M.D. Garcerá

Oró Bozzini, Denise

Spain Cerium oxide nanoparticles reduce portal hypertension and

show antiinflammatory properties in CCl4-treated rats. G. Fernández-Varo, V. Reichenbach, T. Yudina,

E. Casals, G. Casals,B. González de la Presa, V.

Puntes, W. Jiménez

Paez-Aviles, Cristina Spain

Bridging Research and Industrial Production towards H2020:

Future challenges for Nanomedicine with a multi-KET approach. Esteve Juanola-Feliu, Josep Samitier

Raesch, Simon S. Germany Accessing the Nanoparticle Corona in Pulmonary Surfactant.

Stefan Tenzer, Wiebke Storck, Christian Ruge,

Ulrich F. Schaefer, Claus-Michael Lehr

Ratera, Imma

Spain 2D Microscale Engineering of Novel Protein based Nanoparticles

for Cell Guidance. Witold I. Tatkiewicz, Joaquin Seras-Franzoso,

Elena García-Fruitós, Esther Vazquez, Nora Ventosa, Antonio Villaverde and Jaume

Veciana

Rubio Lorente, Laura

Spain Long-term exposures to low doses of cobalt nanoparticles

induce cell-transformation enhanced by oxidative damage. Balasubramanyam Annangi, Jordi Bach,

Gerard Vales, Laura Rubio, Ricardo Marcos, Alba Hernández

N a n o B i o & M e d 2 0 1 4 n o v e m b e r 1 8 - 2 1 , 2 0 1 4 - B a r c e l o n a ( S p a i n ) 1 0 3


Saenz del Burgo, Laura Spain

Graphene oxide application in cell microencapsulation for

bioartificial organ development. Jesús Ciriza, Gorka Orive, Rosa María

Hernández, Jose Luis Pedraz

Sánchez, Elena

Spain Tramadol Hydrochloride Released from Lipid Nanoparticles:

Studies on Modelling Kinetics.

Helen Alvarado, Prapaporn Boonme, Guadalupe Abrego, Tatiana Andreani, Monica

Vazzana, Joana F. Fangueiro, Catarina Faggio,

Carla Silva, Sajan José, Antonello Santini,

María Luisa Garcia, Ana C. Calpena, Amélia M.

Silva, Eliana B. Souto

van de Winckel, Eveline Spain

Polycationic Silicon Phthalocyanines as Photosensitizers for

Photodynamic Therapy and Photodynamic Inactivation of

Microorganisms. Andrés de la Escosura, Tomás Torres

Biomolecule-Quantum Dot systems for biological applications: Size-controlled

aqueous synthesis of CdS Quantum Dots in homogeneous phase with BSA as capping

ligand.

María Jesús Almendral Parra1, Sara Sánchez Paradinas

2, Víctor Barba Vicente

1.

1Departamento de Química Analítica, Nutrición y Bromatología. Facultad de Ciencias Químicas.

Universidad de Salamanca. Plaza de la Merced, s/n. 37008, Salamanca. Telefon: +34 923294483. e-mail:

[email protected]. 2Institut für Physikalische Chemie und Elektrochemie .Leibniz Universität Hannover. Schneiderberg

39.30167 Hannover. Raum: 2 07. Telefon: +49 511 762 16076. e-mail: [email protected]

hannover.de.

The literature contains few works reporting on the in situ generation of a reagent for

the obtaining of nanocrystals with quantum characteristics. Yang and Xiang1 have

described the aqueous synthesis of nanocrystals of CdS using CdSO4 and Na2S2O3 as

precursors in the presence of thioglycerol as a dispersant. However, in that

experimental work the authors failed to study the changes in size of the nanocrystals

as a function of different conditions and, additionally, the dispersive behaviour of

thioglycerol is small, since colloidal solutions of CdS are obtained and these have low

stability. The same precursors and the same dispersant were used by Unni et al. for the

synthesis of nanocrystals of CdS spiked with Zn2+

or Cu2+

, with the above drawback of

the low capacity of the dispersant to stabilize the solutions in which the nanocrystals

are formed.

Serum albumins have been used as a model protein for many and diverse biophysical,

biochemical and physicochemical studies. Due to the high homology between bovine

serum albumin (BSA) and Human Serum Albumin (HSA) it is possible to investigate

systems aiming at future applications in medicine and biology.

In this work, we reported the bioconjugation of CdS Quantum dots directly with bovine

serum albumin (BSA) as capping ligand via an aqueous route. In an earlier work2 by our

team, we performed the synthesis of CdS nanocrystals in aqueous medium starting out

from the precursors CdCl2 and Na2S and using mercaptoacetic acid as the dispersant.

We performed an in-depth study of several variables that affect size, surface state,

fluorescence and stability of the aqueous solutions containing the CdS nanocrystals.

In the present work we describe a similar set of experiments, but with a fundamental

difference in that the S2-

ion was generated in situ from the precursor thioacetamide

CH3C(S)NH2, which was slowly hydrolyzed in basic aqueous solution. Cd(ClO4)2.6H2O

was used as the precursor of Cd2+

and bovine serum albumin(BSA) was employed as

capping ligand.

We study the variables affecting the hydrolysis rate of CH3C(S)NH2 (pH, temperature).

For these variables we studied the evolution of the size of the nanoparticles (NPs) with

the time, the surface characteristics governing their fluorescence properties and their

stability. We compared the above characteristics of the NPs of CdS obtained with both

methods, deducing the advantages conferred by synthesis in homogeneous phase.

1 Y. J. Yang, J. W. Xiang. Template-free synthesis of CuS nanorods with a simple aqueous reaction at ambient conditions. Appl. Phys.

A: Mater Sci. Proc. 81 (2005) 1351-1353 2 M. J. Almendral Parra, A. Alonso Mateos, S. Sánchez Paradinas, J. F. Boyero Benito, E. Rodríguez Fernández and J. J. Criado

Talavera. Procedures for controlling the size, structure and optical properties of CdS Quantum Dots during synthesis in aqueous

solution. Journal of Fliorescence 22 (2012) 59-69.

OPTIMIZATION AND CHARACTERIZATION OF POLY (LACTIC-CO-GLYCOLIC ACID)

NANOPARTICLES LOADED WITH AN HIV-1 INHIBITOR SYNTHETIC PEPTIDE

Ariza-Sáenz M1,2

, Vega E1, Espina M

1, GómaraM.J

2, Egea M.A

1,

Haro I2, Garcia, ML

1

1. Department of Physical Chemistry, Institute of Nanoscience and Nanotechnology, Faculty of Pharmacy, University of Barcelona, Avda Joan XXIII s/n 08028 Barcelona, Spain. 2. Unit of Synthesis and Biomedical Applications of Peptides, IQAC-CSIC, Jordi Girona 18-26 08034

Barcelona, Spain [email protected]

Introduction: It has been reported previously that patients coinfected with HIV and GB virus C (GBV-C) have a prolonged survival. Recently, it has been shown that the GBV-C envelope glycoproteins are enabling to interfere with HIV-1 fusion and entry (1). We report herein the synthesis of a peptide inhibitor of HIV-1 derived form the envelope E1 protein of GBV-C virus and the preparation, optimization and in vitro characterization of poly (lactic-co-glycolic acid) PLGA-Nanoparticles (NPs) encapsulating this peptide sequence. Methods: The peptide was synthesized by the Fmoc solid phase peptide synthesis method (2), purified by High Performance Liquid Chromatography (HPLC) and characterized by mass spectrometry (MALDI-Tof). Peptide-loaded PLGA NPs were prepared by a double emulsion/solvent evaporation technique (3). After selecting the factors that influenced physicochemical properties of the peptide-NPs, a three factors, five-level central composite rotatable design 2

3 + star was applied to optimize the formulation. The factors

selected were the volume of the inner aqueous phase, the concentration of PLGA and the concentration of peptide. The main interactions and effect of these independent variables were studied on particle size, polydispersity index (PDI), and entrapment efficiency (EE). The selected standard conditions were set as 0.8% (w/v) of PLGA; 0.025% (w/v) of peptide; 2.5% (w/v) of Poly (vinyl alcohol) PVA; 0.5 mL of inner aqueous phase; 30 seconds of primary emulsion and 90 seconds of secondary emulsion. The mean size (Z) and PDI were determined by photon correlation spectroscopy and the EE was assessed determining the non-entrapped peptide by HPLC. The destabilization process of the formulation selected was evaluated using a Turbiscan Lab Expert ®. Results: The experimental responses of a total of 16 formulations resulted in a mean size nanoparticle diameter ranging from 209 to 471 nm, with polydispersity index from 0.06 to 0.4 and EE of peptide values ranging from 13% to 84%. The formulation with 0.86 % (w/v) of PLGA; 0.025% (w/v) of peptide; 2.5% (w/v) of PVA; 0.5 mL of the inner aqueous phase, was found suitable for obtaining a high entrapment efficiency (84%) with an adequate average size of 262 nm and unimodal size distribution. The figure 1 shows the three-dimensional response surface of diagram corresponding to effects of peptide and polymer on the entrapment efficiency of peptide-NPs. As illustrated, the highest E.E was obtained with peptide concentration around of 0.025 %(w/v) and polymer concentration around 0.86 %(w/v), thus being these parameters considered relevant in the encapsulation process. Turbiscan data showed that the Peptide-NPs formulation selected has a good stability during more than 72 hours (Figure 2). Conclusion: The results obtained suggested

Besides, the factorial design is a valuable tool to provide screening trials useful to select an optimized formulation with a minimum number of experiments.

References

1. Mohr, EL, Stapleton, JT. GB virus type C interactions with HIV: the role of envelope glycoproteins. J. Virol. Hepatol. 2009; 16: 757-768

2. Fmoc solid phase peptide synthesis. A practical approach. W.C. Chan and P.D. Whire Eds. Oxford University Press Inc. New York, 2000.

3. Zhang JX, Zhu KJ. An improvement of double emulsion technique for preparing bovine serum albumin-loaded PLGA microspheres. J Microencapsul. 2004; 21 (7):775-85.

Figure 1. Surface response diagram of entrapment efficiency.

(a) (b)

Figure 2. Turbiscan profiles for a Peptide-NPs sample. (a). Transmission level (%) versus high cell (mm); (b) Backscattering (%) versus high cell (mm).

Nucleic acids for biosensing applications

Anna Aviñó, César Sánchez*, Mar Oroval#, Laura Carrascosa*, Ramón Martínez-Máñez#, Laura Lechuga*, and Ramon Eritja

Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Institute for

Advanced Chemistry of Catalonia (IQAC), CSIC, Jordi Girona 18-26 08034 Barcelona, Spain # Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Unidad Mixta Universidad

Politécnica de Valencia - Universidad de Valencia, Spain. *Nanobiosensors and Bioanalytical Applications Group CIBER-BBN and Research Center on

Nanoscience and Nanotechnology (CIN2) CSIC, Spain [email protected]

Abstract DNA biosensors are small devices which intimately couple biological recognition element interacting with the target analyte with a physycal transducer that translates the biorecognition event into a useful electrical signal. Common transducing elements are optical, electrochemical or mass-sensitive. DNA biosensors, based on nucleic acid recognition processes, are being developed towards the goal of rapid, simple and inexpensive testing of genetic or infectious deseases. In one hand, we present a biosensing approach for the label-free detection of nucleic acid sequences with special emphasis on targeting RNA sequences with secondary structures or microRNAs that are involved in several deseases. The approach is based on selecting 8-aminopurine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. On the other hand, the design of stimuli-responsive nanoscopic gated systems involving biomolecules has recently attracted great attention. Capped materials have been mainly used in drug delivery applications. However in sensing are less common. Among different biomolecules that could act as caps, nucleic acids aptamers are especially attractive for the design of gated nanosensors for sensing applications. Specifically, we have prepared an aptamer-gated delivery system (S1-TBA) for the fluorogenic detection of thrombin. The sensing mechanism arises from the high affinity between an aptamer (TBA) and it -thrombin). References [1] Aviñó A.Frieden, M Morales J.C García de la Torre B. Ramón Güimil García1, Azorín F. Gelpí,J.L. Orozco,M González C. and Ramon Eritja, Nucleic Acids Res. 30 ( 2002), , 2609-2619 [2] Laura G. Carrascosa S. Gómez-Montes, A. Aviñó, A. Nadal, M. Pla, R. Eritja and L. M. Lechuga, Nucl. Acids Res. 2 (2012), 11. [3] Oroval, M. Climent, E Coll, C. Eritja R. Aviñó A. Marcos M.D. Sancenón F. Matínez-Máñez R. and Amorós P, Chem. Commun. 49 (2013), 5480 -582A Figures

DETECTION OF RNA SEQUENCES USING PARALLEL TAIL CLAMPS AS BIORECEPTORS TO FORM STABLE TRIPLEX STRUCTURES IN A SPR BIOSENSOR

DETECTION OF THROMBIN USING AN APTAMER GATED DELIVERY SYSTEM

Silver nanoparticles downregulate p53 activation and induce desacetylation of histone 3 in human lung cancer epithelial cells

Jordi Blanco, Daisy Lafuente, Domènec Sánchez, José Luis Domingo, Mercedes Gómez

Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Universitat Rovira i Virgili, 43201 Reus, Spain.

[email protected]

Abstract

Nanomaterials have been widely used in recent years in aerospace engineering, nanoelectronics,

environmental remediation, medical health care, and consumer products. Silver nanoparticles (AgNPs)

are one of the most commonly used nanomaterials, because possess potent antibacterial and antifungal

characteristics. AgNPs have been used extensively as an antimicrobial agent in cosmetics, textiles and

the food industry, as well as a disinfectant for medical devices and for coating home appliance [1]. The

emerging number of consumer products containing AgNPs and increasing enviromental concentration,

have led to concerns, because nanoparticles may pose a risk for humans and the environment. The

main ways by which people may be exposed to AgNP are by inhalation, dermal contact, and oral

ingestion. The absorbed AgNPs can pass through the respiratory or gastrointestinal tracts and stored in

many organs such as lung, liver, spleen, kidney and the central nervous system. There is growing

evidence that AgNPs are highly toxic in terms of cytotoxicity, genotoxicity, and oxidative stress [3].

The present study evaluated the cytotoxic effects of AgNPs (20 nm of diameter coated with 0.3% of

PVP) in A549 cells. A549 cells were exposed to 0, 25, 50, 100 and 200 µg/mL of AgNPs along 72

hours. AgNPs caused cell death in a dose- and time- dependent manner (Figure 1). Cell death induced

at high doses was positively correlated with a down regulation of the expression and phosphorylation of

p53 protein and acetylation of histone 3 (H3, Figure 2). Contrarily, the expression of total H3 protein was

overexpressed at high doses.

The desacetylation of H3 at high doses of AgNPs suggests that epigenetic changes could be

happening into the chromatin. These sugestion are reinforced by the morphologic changes observed in

A549 at high doses. In the same way, downregulation of the expression of p53 could be also due to a

desacetylation of lysine residues which lead to its proteosomal degradation. The down regulation of p53

could lead to a deregulation of cell cycle and could induce arrest in S phase and thereby increase of the

expression of histones proteins. The knowledge of the mechanisms by which AgNPs induce these

changes could help to better understanding how nanoparticles could induce cancer cells death.

References

[1] Navarro E, Baun A, Behra R, Hartmann NB, Filser J, Miao AJ, Quigg A, Santschi PH, Sigg L: Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 2008, 17:372-386.

[2] Chen X, Schluesener HJ: Nanosilver: a nanoproduct in medical application. ToxicolLett 2008, 176:1-12.

[3] Kim S, Choi JE, Choi J, Chung KH, Park K, Yi J, Ryu DY: Oxidative stressdependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol In Vitro 2009, 23:1076-1084.

Figures

Figure 1.

Figure2.

Magnetoliposomes for magnetic resonance imaging Joan Estelrich

1,3, Josep Queralt

2, Montserrat Gallardo

1, M. Antònia Busquets

1,3

1Departament de Fisicoquímica.

2 Departament de Fisiologia. Facultat de Farmàcia. UB. Avda Joan

XXIII, s/n 08028 Barcelona 3Institut de Nanociència i Nanotecnologia (IN2UB)

[email protected]

Abstract The development of superparamagnetic iron oxide nanoparticles (SPIONS) has opened a new and attractive approach towards the molecular imaging of living subjects because they overcome not only many of the current limitations in diagnosis but also in the treatment and management of human diseases [1]. The combination of imaging modalities based on the use of SPIONS such as magnetic resonance imaging (MRI), optical imaging (OI) or positron emission tomography (CT) have been developed to visualize pathological situations. Among these techniques, MRI is one of the most powerful, and non-invasive modality of diagnosis due to its high soft tissue contrast, spatial resolution, and penetration depth. MRI images result from the spatial identification of hydrogen nuclei. The contrast in the images comes from local differences in spin relaxation kinetics along the longitudinal T1 (spin-lattice) and transverse T2 (spin-spin) relaxation times [2]. Contrast agents alter the signal intensity by selectively shortening the hydrogen relaxation times of the tissues and are used to improve the sensitivity and specificity of MRI. In particular, SPIONS have emerged as T2 contrast agents because their enhancement of the negative contrast, thus showing darker images of the regions of interest. This contrast is strongly related to the SPIONS coating being the most widely used dextran and its derivatives. However, these coatings have raised several controversies mainly because of their weak physisorption on the nanoparticle surface and toxicology associated to the coating [3]. To overcome these drawbacks, the SPIONS can be incorporated into lipid vesicles thus obtaining magnetoliposomes (MLs). MLs have special interest because of their low cytotoxicity, enhanced versatility and target biodistribution. To gain understanding on the magnetic relaxation processes involved in contrast generation by MLs, we seek an analysis of the impact that coating has on the relaxivity of MLs. With this purpose, we have prepared magnetite coated with polyethylene glycol (PEG) [4] and then, the resulting ferrofluid has been encapsulated into liposomes of different lipid composition in order to analyze the influence of size, physical state of the phospholipid bilayer and medium of dispersion of MLs in the MRI parameters [5]. Thus, dimyristoyl phosphatidylcholine (DMPC), dipalmytoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC) and dioleoyl phosphatidylcholine (DOPC) were chosen to study the effect of chain length and membrane physical state on T1 and T2 relaxations. In addition, ferrofluid was incorporated into DMPC:Cholesterol (CHOL) at different molar rations. Finally, the influence of size on MRI relaxivities was analyzed with non-extruded (multilamellar liposomes, MLVs) and extruded MLs (LUVs). Samples were prepared at iron concentrations ranging from 0.2 to 1.2 mM in agar (5%) fantoms as to mimic human tissues, in 8 x 6 cm wells plate (Figure 1). MRI experiments were conducted on a 7.0 T BioSpec 70/30 horizontal animal scanner (Bruker BioSpin, Ettlingen, Germany), equipped with a 12 cm inner diameter actively shielded gradient system (400 mT/m) and a receiver/transceiver coil covering the whole mouse volume. The sample was placed in a Plexiglas holder. Tripilot scans were used for accurate positioning of the sample in the isocenter of the magnet. T1 relaxometry maps were acquired by using RARE (rapid acquisition with rapid enhancement) sequence applying 9 repetition times = 354.172, 354.172, 500, 700, 1000, 1400, 2000, 3000 and 6000 ms, echo time = 10 ms, RARE factor = 1 average, number of slices = 6 for vertical view, field of view = 69.9 x 60 mm, matrix size = 128 x 128 pixels, resulting in a spatial resolution of 0.546 × 0.469 mm in 1 mm slice thickness. For the T2 relaxometry maps, MSME (multi-slice multi-echo) sequence was used with a repetition time = 4764.346 ms with 16 echo times corresponding to 14.11, 28.21, 42.32, 56.43, 70.54, 84.64, 98.75, 112.86, 126.97, 141.07, 155.18, 169.29, 183.4, 197.5, 211.61 and 225.72 ms, 1 average, number of slices = 6 for vertical view, field of view = 69.9 x 60 mm, matrix size = 448 x 384 pixels, resulting in a spatial resolution of 0.156 x 0.156 mm in 1 mm slice thickness. Data were processed using the Paravision 5.1 software (Bruker, BioSpin, Ettlingen, Germany).

In contrast to the T2 relaxation, T1 relaxation depends on a fast proton exchange between the bulk water phase with slow T1 relaxation, and protons at the surface of magnetic particle aggregates where T1

relaxation is fast. Since residence time of protons is roughly proportional to the square of the particle size, this can explain the lower values of the MLs compared with the values described in the literature [6].

DMPC

[Fe] 0,08 mM

DMPC [Fe]

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0,20mM

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0,20mM

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0,08 mM

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0,14 mM

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0,20mM

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0,08 mM

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0,08 mM

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0,14 mM

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0,20mM

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0,20mM

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0,08 mM

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0,08 mM

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0,08 mM

DMPC/chol90:10 [Fe]

0,08 mM

DMPC/chol95:5 [Fe]

0,08 mM

DMPC/chol95:5 [Fe]

0,08 mM

Figure 1. Top: MRI Maps of T1 (left) and T2 (right). Below: samples of the above maps. MLVs. multilamellar magnetoliposomes with the corresponding mM iron concentration ([Fe]). DMPC: dimyristoyl phosphatidylcholine; DPPC: dipalmytoyl phosphatidylcholine; DSPC: distearoyl phosphatidylcholine, DOPC: dioleoyl phosphatidylcholine and CHOL: cholesterol. Control samples of ferrofluid (FF) were also analyzed as control (Figure not shown).

0.00 0.02 0.04 0.06 0.08 0.102.0

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s

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s

Figure 2. T1 and T2 relaxation times of ferrofluid (blue), large unilamellar (red) and multilamellar (black) magnetoliposomes of DMPC

Acknowledgements. The authors are grateful for the financial support given by the Spanish Ministerio de Economía y Competitividad (MINECO) to the project MAT2012-36270-C04-03.

References [1] C. Corrot, P. Robert, JM Idee, M. Port, Adv. Drug Deliv. Rev. 58 (2006) 1471-1504. [2] H. Fattahi, S. Laurent, F. Liu, N. Arsalani, L. Vander Elst, R.N. Muller, Nanomedicine 6 (2011) 529-544. [3] S. Mornet, J. Portier, E. Doguet. J. Magn. Magn. Matter 293 (205) 127-134.

[4] S.García-Jimeno, J. Estelrich, Colloids Surf. A, 420 (2013) 74-81.

[5] R. Sabaté, R. Barnadas-Rodríguez, J. Callejas-Fernández, R. Hidalgo-Alvarez, J. Estelrich, Int. J. Pharm. 347 (2008) 156-162. [6] M. Hoedenius, C. Würth, J. Jayapaul, J.E. Wong, T. Lammers, J. Gätjens, S. Arns, N. Mertens, I. Slabu, G. Ivanova, J. Bornemann, M. De Cuyper, U. Resch-Genger, F. Kiessling. Contrast Media Mol. Imaging 7 (2011) 59-67.

In vitro and in vivo evaluation of TiO2 oral absorption

Joan Cabellos, Gemma Janer, Ezequiel Mas del Molino, Elisabet Fernández-Rosas, Socorro Vásquez-Campos

LEITAT, Innovació 2, Terrassa, SPAIN [email protected]

Abstract The Caco-2 monolayer permeation test is a widely used in vitro test to predict oral absorption of organic compounds, particularly by the pharmaceutical industry (Artursson et al. 2001). A few reports also exist on the use of this test system for NMs (e.g., Al-Jubory and Handy 2012; Antunes et al. 2013; Jin et al. 2013). But the predictive value of the data obtained in such test is still unclear. The main processes that determine uptake for chemicals are diffusion and active transport through membrane transporters (Grassi 2007), and the Caco-2 monolayer permeation test is a good model for both processes. However, these are not the main processes governing the oral uptake of particles. Three main pathways for absorption of particles across intestinal barriers have been described. First, paracellular transport can take place for small molecules that can pass the tight junctions and the pore-diameter which is reported to be around 0.6–5 nm (Ruenraroengsak et al. 2010). Second, transcytosis may appear across enterocytes, but mostly across M-cells located in the Peyer’s patches (Powell et al. 2010). An third, particles (nano and micro sized) can be transported across degrading enterocytes (Hillyer and Albrecht 2001; Volkheimer 1993). The three mechanisms that have been described for NMs oral absorption could theoretically occur in the Caco-2 model, particularly if this could be modified to incorporate M-cells.

In the present report, we have used a single type of TiO2 NPs (spherical, 18 ± 8 nm diameter, surface area was 89.8 m2/g) in different in vitro and in vivo systems in order to better understand how results from these studies can be extrapolated to other cell types or levels of organization. In particular, we describe the results of five studies: an in vitro cell uptake using A549 cells, an in vitro permeation test using differentiated Caco-2 cells, an in vitro permeation test using a coculture of differentiated Caco-2 cells and M-cells, and two in vivo oral absorption tests in rats (with and without fasting conditions prior to administration). To overcome the analytical challenges associated to the tracking of unlabelled TiO2

NPs, both inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) were used.

TiO2 NPs were present in form of agglomerates of different sizes within cytoplasmic vesicles of A549 cells after 72 hours exposure. In none of the cells evaluated, particles were found freely in the cytoplasm or in the nucleus. This efficient cell internalization and the fact that NPs were found as aggregates in cytoplasmic vesicles is consistent with previous reports in a variety of human cell lines.

A very small proportion of the NPs was able to cross the differentiated Caco-2 cell membrane (below or close to the detection limit, i.e., 0.1 ppm or 0.4% of the applied concentration). In an attempt to increase the biological relevance of this permeation model, we introduced Raji cells to induce the differentiation of Caco-2 cells into M-cells. The Caco-2/M-cell coculture model was established and characterized through measuring TEER values during the differentiation process, and performing morphological (histological sections and scanning electron microscopy; Figure 1) and immunostaining (ZO1 and ocludin tight junction proteins). All data confirmed that a proportion of Caco-2 cells consistently differentiated into M-cells. In such system, a very low permeation rate for TiO2 NP was recorded, although qualitatively higher (higher frequency of values above the detection limit) than that for Caco-2 cell monocultures.

The readily internalization in A549 cells and in most cell lines contrasts with an extremely low absorption by Caco-2 cells in our study and the low uptake by these cells in the study by Fisichella et al. (2012). The fact that we used the same TiO2 NPs for in vitro studies with both A549 and Caco-2 cells indicate that these differences are related to the cell line properties and not to the TiO2 NPs used. The apparent contradiction is probably related to the cell membrane morphology of the latter. Differentiated Caco-2 cells are polarised cells with microvilli in their apical side (where exposure takes place).

To evaluate in vivo absorption Sprague-Dawley male rats were administered the vehicle or the TiO2

NPs by oral gavage. Two independent experiments were performed, one in which administration of the NPs was performed in fasting conditions and one without food restrictions. Administered rats were weighted and observed to assess clinical signs of toxicity on the day of administration and on the following day, before sacrifice. At termination, spleen, liver, small and large intestines, and mesenteric lymph nodes were removed. The intestines were carefully washed with phosphate buffer to remove their

content. Peyer’s patches were excised and separated from the rest of the small intestine. The caecum was separated from the rest of the large intestine. One of the Peyer’s patches and a piece of smooth small intestine were immediately preserved in buffered glutaraldehyde-paraformaldehyde for later TEM analyses. The remaining samples were kept at -20°C until acid digestion and analysis by ICP-MS.

There was no detectable increase in titanium levels in any of the tissues evaluated 24 hours after the administration of 100 mg/kg TiO2 NPs, regardless of the food restriction conditions (only some tissues under fasting conditions: Peyer’s patches, smooth small intestine, and mesenteric lymph nodes). Smooth sections and Peyer’s patch sections of the small intestine of the animals that received TiO2 NPs without food restrictions were examined by TEM. No TiO2 NPs were observed in the smooth sections. In contrast, we did observe at least one cell containing considerable amounts of TiO2 NPs aggregates in a Peyer’s patch section (Figure 2). In this cell, the TiO2 NPs were not surrounded by membranes, but they were freely distributed in the cytoplasm. We did not observe NPs inside mitochondria or the nucleus.

The low bioavailability of TiO2 NPs in this report contrasts with the relatively high oral bioavailability study reported by Jani et al. (1994). We had hypothesized that these differences could be due to the fact that Jani et al. (1994) administered the particles after several hours of fasting (Janer et al., 2014), but the experiment that we conducted in fasting conditions does not support such hypothesis.

In summary, we showed that A549 cells readily uptake the TiO2 NPs used in this study. The results were consistent with most literature reports for TiO2 NPs and other type of NMs, suggesting a limited modulating effect of the physicochemical properties of NMs on cell uptake. However, such rapid uptake contrasted with a very low oral absorption in the in vitro and in vivo studies that we performed. The results of this study support that M-cells play an important role in the absorption of nanoparticles, and suggest that the Caco-2/M-cell coculture model is a more relevant model for the prediction of oral absorption of nanoparticles than the Caco-2 monoculture model.

References Al-Jubory AR, Handy RD, Nanotoxicology, 7 (2013) 1282-301. Antunes F, Andrade F, Araujo F, Ferreira D, Sarmento B, Eur J Pharm Biopharm 83 (2013) 427-35. Artursson P, Palm K, Luthman K, Adv Drug Deliv Rev 46 (2001) 27-43. Grassi M GG, Lapasin R, Colombo I, A Physical And Mathematical Approach. CRC Press. (2007). Powell JJ, Faria N, Thomas-McKay E, Pele LC, J Autoimmun 34 (2010) J226-33. Jin X, Zhang ZH, Li SL, Sun E, Tan XB, Song J, Jia XB, Fitoterapia 84 (2013) 64-71. Ruenraroengsak P, Cook JM, Florence AT, J Control Release 141 (2010) 265-76. Hillyer JF, Albrecht RM, J Pharm Sci 90 (2001) 1927-36. Volkheimer G. Pathologe 14 (1993) 247-52. Fisichella M, Berenguer F, Steinmetz G, Auffan M, Rose J, Prat O, Part Fibre Toxicol 9 (2012) 18. Jani PU, McCarthy DE, Florence AT, Int. J. Pharmaceutics 105 (1994) 157-168.Janer G, Mas del Molino E, Fernández-Rosas E, Fernández A, Vázquez-Campos S. Toxicol Lett. 228(2014) 103-10.

Figures

M

Figure 1. SEM images of Caco-2/Raji cocultures. Caco-2 cells show dense microvilli and contrast with larger M-cells with only rudimentary microvilli (M).

2 m

Figure 2. TEM micrograph showing the presence of TiO2 nanoparticles in a cell from a Peyer’s patch section. The arrows point to some of the NP aggregates.

Synthesis of modified dendrimers and conjugation with selected apoptosis inhibitors

Miriam Corredor1, Ignacio Alfonso1, Dietmar Appelhans2, Angel Messeguer1

1 Dep. Chemical Biology and Molecular Modelling, IQAC-CSIC C/ Jordi-Girona, 18-26 08034 Barcelona (Spain)

2 Dep. Bioactive and Responsive Polymers, Leibniz-Institut für Polymerforschung Dresden Hohe Stra e, 6 D-01069 Dresden (Germany)

[email protected]

Apoptosis is a biological process relevant to different human diseases stated that is regulated

through protein-protein interactions and complex formation.[1] One point of regulation is the formation of

a multiprotein complex known as apoptosome.[2] In our group, it has been previously reported a

peptidomimetic compound bearing a 3-substituted-piperazine-2,5-dione moiety and a seven-membered

ring moiety as potent apoptotic inhibitors.[3] We reduced the conformational freedom of the exocyclic

tertiary amide bond of the diketopiperazine by an isosteric substitution of a triazole. For one of the

proposed structures a -lactam compound was isolated, that resulted to be the most potent inhibitor.[4]

At this point, we wanted to conjugate our potential drugs with a polymer that could offer a more

specific intracellular transport and release to reach the molecular target.

Dendritic polymers are widely used as multifunctional materials with specific properties for potential

biomedical and pharmaceutical applications. These multifunctional macromolecules have been used as

carrier systems of drugs in the study of bio-interaction against different bio-active molecules and

systems.[5] The most important drawback of these types of dendrimers is their toxicity due to the

positive charge on their surface. Thus, a high generation of poly(propylene imine) dendrimers with

densely organized oligosaccharide shells in which each peripheral amino group is modified by two

chemically coupled oligosaccharide units has been reported.[6] This attachment resulted in much lower

cytotoxicity towards different cell lines.[7]

In this work, 5th generation PPI dendrimers modified with maltose units were synthesized and

coupled with previously mentioned modified small molecules which have shown activity as potential

apoptosis inhibitors.

Figure 1: Coupling of a small molecule with a dense-shell glycodendrimer bearing an amino-terminal group.

References

[1] Mondragón, L.; Orzáez, M.; Sanclimens, G.; Moure A.; Armiñán, A.; Sepúlveda, P.; Messeguer, A.; Vicent, M. J.; Pérez-Payá. J. Med. Chem. 51 (2008) 521-529. [2] Martin, A. G.; Nguyen, J.; Wells, J. A.; Fearnhead, H. O. Biochem. Biophys. Res. Comm. 319 (2004) 944-950. [3] Moure, A.; Sanclimens, G.; Bujons, J.; Masip, I.; Alvarez-Larena, A.; Pérez-Payá, E.; Alfonso, I.; Messeguer, A. Chem. Eur. J. 17 (2011) 7927-7939. [4] Corredor, M.; Bujons, J.; Orzáez, M.; Sancho, M.; Pérez-Payá, E.; Alfonso, I.; Messeguer, A. Eur. J. Med. Chem. 63 (2013) 892-896. [5] Boas, U.; Heegaard, P.M; Chem. Soc. Rev. 33 (2004) 43-63. [6] Klajnert, B.; Appelhans, D.; Komber, H.; Morgner, N.; Schwarz, S.; Richter, S.; Brutschy, B.; Ionov, M.; Tonkikh, A. D.; Bryszewska, M.; Voit, B. Chem. Eur. J. 14 (2008) 7030-7041. [7] Arima, H.; Chihara, Y.; Arizono, M.; Yamashita, S.; Wada, K.; Hirayama, F.; Uekama, K. J. Control Release 116(2006) 64-74.

NHS/ EDC

TEA / DMSON

N

N

N

N

N

N

N N

N

NN

NH2NH2NH2

NH2NH2

NH2NH2

N

N

N

N

NH2NH2

NH2NH2

N

N

NH2

NH2NH2

NH2

NH2

N N

N

N

N

NH2NH2NH2

NH2

NN

NH2

NH2

NH2NH2

NN N

NNH2NH2

NH2

NN

NH2

NH2NH2NH2NH2

N

N

N

N

N

N

N

NN

N

NN

H2NH2N

H2N

H2NH2N NH2 NH2

N

N

N

N

H2NH2N

H2NH2N

N

N

H2NH2N

H2NH2N

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NN

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N

N

H2NH2NH2N

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H2NH2N

H2NH2N

NNN

NH2N

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NN

H2NH2NH2NH2N H2N

G5-PPI

NH2

NH2

NH2

NH2

NH2

NH2 NH2

NH2

NH2

NH2

+

Monitoring the intracellular dynamics of polystyrene nanoparticles in lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking

Sarah Deville

i, ii, Rozhin Penjweini

i, Nick Smisdom

i, ii, Kristof Notelaers

i, Inge Nelissen

ii, Jef

Hooyberghsii, iii

, Marcel Amelooti

i Biomedical Research Unit, Hasselt University, Diepenbeek, Belgium. ii Flemish Institute For Technological Research (VITO), Mol, Belgium

iii Theoretical Physics, Hasselt University, Diepenbeek, Belgium

[email protected]

Abstract Interactions of nanoparticles (NPs) with living cells and resulting biological responses are largely dependent on NP uptake processes, intracellular transport and their complex behaviours. In order to demonstrate the applicability of image (cross-) correlation spectroscopy based techniques for monitoring the intracellular dynamics of NPs, 100 nm fluorescently stained carboxylated polystyrene (PS) NPs were used to expose in vitro cultured human lung epithelial A549 cells, thus mimicking NP exposure in the lungs. Transport direction, transport velocity and diffusion of PS NPs were determined to acquire more insights in the intracellular transport following NP uptake. To complement these ensemble average techniques, PS NP motions were also analysed by single particle tracking. Hereby, distinct clusters are registered and tracked frame by frame allowing access to individual PS NP dynamics. Potential dynamic interactions of NPs with the nucleus, mitochondria, early endosomes, late endosomes and lysosomes were also explored. PS NPs directed motions were shown to be highly dependent on the microtubule-assisted transport and were strongly associated with the endolysosomal compartment. Image (cross-) correlation analyses were shown to be a powerful tool for determining the kinetic behaviour of NPs inside the cell. References [1] Penjweini, R.; Smisdom, N.; Deville, S.; Ameloot, M., Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1843 (5) (2014), 855-865.

Interaction of targeted of magnetoliposomes with Hela epithelial carcinoma and 3T3 fibroblasts cell lines.

Joan Estelrich1,3

, M. Carmen Moran2,3

, M. Antònia Busquets1,3

1Departament de Fisicoquímica.

2 Departament de Fisiologia. Facultat de Farmàcia. UB. Avda Joan

XXIII, s/n 08028 Barcelona 3Institut de Nanociència i Nanotecnologia (IN2UB)

[email protected]

Abstract

In the last years, the development of iron oxide magnetic nanoparticles (IONPs) has significantly increased with respect to other nanosized particles because of their attractive properties as theranostic agents [1]. These systems combine both, therapeutic and diagnostic properties. In order to improve their versatility and biodisponibility, IONPs can be incorporated into liposomes, resulting in a new kind of nanoscale system, known as magnetoliposomes (MLs) [2]. MLs, which are biodegradable and highly versatile especially in composition, have opened great expectations for the development of personalized medicine [3]. Any biomedical use of MLs entails thorough understanding of their toxicology, establishment of principles and test procedures to ensure safe manufacture and usage, and comprehensive information about their safety and potential hazard [4]. In this way, we have designed MLs appropriate for theranostic applications. However, previously to any biomedical application, the lack of inherent toxicity must be checked. To this end, the following study has been performed according to the following steps: i) synthesis of IONPs; ii) incorporation of IONPs into liposomes of different lipid composition and, iii) analysis of the cytotoxicity and internalization of MLs in cell models. IONPs coated with polyethylene-glycol (PEG) were synthesized by the coprecipitation method according to the procedure described elsewhere [5]. As far as the lipid composition is concerned, three different lipid mixtures have been prepared, namely, a) bare liposomes: dimyristoylphosphatidylcholine (DMPC)/cholesterol (CHOL): 8:2; b) bare liposomes with PEG (DMPC/CHOL/PEG: 8:2:0.3) and; c) functionalized MLs or bare liposomes with the cyclic RGD peptide (DMPC/CHOL/PEG/RGDc: 8:2:0.3:0.03). For internalization studies, MLs were decorated with the fluorescent label 0.05% Rhodamine-B. The model cells chosen for the study were 3T3 fibroblasts and Hela epithelial carcinoma cell lines. Both cells are rich in integrin membrane proteins but they are different concerning which kind of ligand is recognized. In this way, 3T3 is rich in collagen-receptor integrins, whereas HeLa in RGD-receptor integrins. Therefore, the rationale of MLs compositions was the selective targeting of the functionalized MLs towards HeLa cells. Thus, bare and PEG-MLs are considered control MLs with no affinity for the above mentioned cells. Results obtained by confocal microscopy and flow cytometry were concordant with the possibility of the formation of the so called protein corona around the MLs [6]. Potser referenciar les figures 1 I 2 al text.

Figure 1. Flow cytometry of control (black); bare MLs of DMPC/CHOL/Rho-PE (80:20:0.05) (red) and, functionalized MLs DMPC/CHOL/PEG/RGDc/Rho-PE (80:20:3: 0.3:0.05) incubated 4h with 3T3 cells (left) or Hela cells (right).

100 101 102 103 104

FL 4 Log

0

77

154

231

309

Counts

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FL 4 Log

0

27

54

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108

Counts

Figure 2. Laser confocal microscopy images of bare (left) and functionalized (right) MLs upon incubation for 4h with 3T3 cells (top) and Hela cells (bottom). Membrane cell was labeled with Alexa; the nucleus with DAPI and magnetoliposomes with Rhodamine B.

References

[1] D. Ho, X.L. Sun, S.H. Sun, Accounts of Chemical Research 44 (2011) 875-882. [2] N. Crawley, M. Thompson, Analytical Chemistry 86 (1) (2014) 130-160 [3] H. Fattahi, S. Laurent, F. Liu, N. Arsalani, L. Vander Elst, R.N. Muller, Nanomedicine 6 (2011) 529-544. [4] Arora, S.; Rajwade, J.M., Paknikar, K.M. Toxicol. Applied Pharmacol. 258 (2012) 151-165

[5] S.García-Jimeno, J. Estelrich, Colloids Surf. A, 420 (2013) 74-81.

[6] M.P. Monopoli, Aberg, C. A. Salvati, K. Dawson, Nature Nanotech. 7 (2012) 779-786

Acknowledgements. MAB and JE are grateful for the financial support to the project MAT2012-36270-C04-03 and, MCM to the project MAT2012-38047-C02-01 given by the Spanish Ministerio de Economía y Competitividad (MINECO). MCM acknowledges the support of the MICINN (Ramon y Cajal contract RyC 2009-04683).

Thermal stability of a cationic solid lipid nanoparticle (cSLN) formulation as

a possible biocompatibility indicator

Anna Fàbregas, Montserrat Miñarro, Josep Ramon Ticó, Encarna García-Montoya, Pilar Pérez-

Lozano, Josep Mª Suñé-Negre

Drug Development Service (SDM). Dept. Pharmacy and Pharmaceutical Technology. Faculty of Pharmacy (Universitat de Barcelona), Avda. Joan XXIII, s/n 08028, Barcelona, Spain

[email protected]

Abstract

Cationic solid lipid nanoparticles have become a non-viral delivery system for nucleic acid transfection

and further genomic regulation and delivery of encapsulated drugs [1].

A formulation of cationic solid lipid nanoparticles intended for gene delivery [2] has been analyzed in

terms of thermal stability at different temperatures. The aim is to determine in a short-term study the

influence of temperature on particle size and surface potential, in order to assess what is the best

temperature that contributes to maintain cSLN without or low aggregation and proper surface potential

[3]. Short-term thermal storage study can serve as well for an approach to behavior at physiological

temperature when the study is carried out at 37 ºC.

The cSLN formulation consists of stearic acid, octadecylamine and surfactant Poloxamer 188 [2].

Thermal behaviour is studied at 4 ºC, 25 ºC and 37 ºC.

The cSLN are synthesized using the hot microemulsification method [4]. Then, the nanoparticles are

distributed in vials and stored at the temperatures mentioned above. The particle size determinations

are carried out in a Mastersizer 2000 laser diffractometre (Malvern Instruments, UK) and Z potential

values are determined on a Zetasizer Nano-Z (Malvern Instruments, UK). Both measures are performed

daily during a week.

The results are represented graphically (figure 1), and show the evolution of this formulation at the

different temperatures in terms of particle size (given as surface weighted mean in nm) and surface

charge (given as Z potential in mV). Mean value and standard deviation (table 1) show that at 37 ºC,

these nanoparticles suffer the lowest variation both in particle size and Z potential.

Thus, cSLN formulation presents a thermal behavior which results in a stable state at 37 °C in

comparison to 25 ºC and 4 ºC, with particle size and Z potential showing slightly changes, then

indicating that at this temperature the formulation is still able during a week for acid nucleic binding.

Additionally, while 37 ºC corresponds to physiological temperature at which cSLN would be

administered, it may be taken into consideration as a possible indicator of biocompatibility, although the

influence of other variables such as thermal behavior after nucleic acid binding should be taken into

account in further studies.

It can be concluded that regarding low tendency to aggregation or modification of surface potential in

the first days after its synthesis when stored at 37 ºC, these cSLN may represent a proper non-viral

delivery system following nucleic acid binding intended for immediate and short-term administration.

References [1] Ekambaran P et al., Scientific & Chemical Communications, 2 2012 80-102.

[2] Fàbregas et al., International Journal of Pharmaceutics, 1-2 2014 270-279.

[3] Vauthier C et al., European Journal of Pharmaceutics and Biopharmaceutics, 2 2008 466-475.

[4] Mehnert W et al. Advanced Drug Delivery Reviews, 64 2012 83-101.

Table 1. Values of particle size and surface potential at different temperatures during 7 days.

Figure 1. Graphical representation of changes on particle size and surface potential as a function of time.

>

>

>

Z Potential (mV)

Days 4 ºC 25 ºC 37 ºC

1 27.7 35.8 32.5

2 29.9 35.6 30.1

3 27.1 24.0 31.6

4 34.9 39.2 34.2

5 38.6 37.0 35.2

6 34.2 43.7 40.2

7 29.9 31.6 27.3

Mean 31.7 35.3 33.0

SD 4.2 6.2 4.1

Surface weighted mean (nm)

Days 4 ºC 25 ºC 37 ºC

1 269 217 236

2 124 339 119

3 126 55194 113

4 237 40709 132

5 206 126742 121

6 65070 58070 122

7 86870 75887 282

Mean 21843 51023 161

SD 37507 44112 68

PLGA nanoparticles as advanced imaging nanosystems

N. Feiner-Gracia, C. Fornaguera, A. Dols-Perez, M.J. García-Celma, C.Solans

1 QCI group, Institut de Química Avançada de Catalunya (IQAC-CSIC), C/ Jordi Girona 18-26,

08034, Barcelona, Spain 2 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)

3 Departament de Farmàcia i Tecnologia Farmacèutica, Universitat de Barcelona (UB), Av/ Joan

XXIII S/N, 08028, Barcelona, Spain

[email protected] Abstract

Introduction

Polymeric nanoparticles (NP) are of increasing interest in the biomedical field. They represent a

promising strategy for in vivo diagnosis as medical imaging nanosystems [1]. Due to the

possibility of functionalizing nanoparticle surface, these systems can be vectorized to the tissue

of interest. In addition, if they include a fluorescent dye nanoparticle tracking can be monitored.

Biocompatible, biodegradable and safety materials are required for the preparation of

nanoparticles intended for biomedical applications. Therefore, poly(lactic-co-glycolic acid)

(PLGA) polymer is appropriate to prepare polymeric nanoparticles using nano-emulsion

templating, which is a simple, well-known and versatile method. Nano-emulsions are colloidal

systems with droplet size in the range of 20-200 nm. The phase inversion composition method

(PIC), a low energy emulsification method, is a suitable methodology to prepare nano-

emulsions for pharmaceutical applications as the process can be performed at mild temperature

[2]. Following, polymeric nanoparticles can be easily obtained from polymeric O/W nano-

emulsions by solvent evaporation, if the oil component (internal phase) of nano-emulsions

consist in a preformed polymer dissolved in a volatile organic solvent. The fluorescent dye can

be solubilized in the oil phase prior to nano-emulsion formation to enhance high loading

efficiency.

Objectives

The aim of this work was to obtain biomedical imaging systems appropriate for intravenous

administration.

Results

O/W polymeric nano-emulsions were prepared in a system PBS/ polysorbate80 surfactant/ [4%

PLGA and 0.1% fluorescent dye in an organic solvent]. The organic solvent consisted in ethyl

acetate or 80/20 ethyl acetate/ethanol. The fluorescent dyes selected were Coumarin 6 (C6)

and Rhodamine 6G due to their non-toxic character, appropriate to be used in the biomedical

field and also due to their solubility characteristics in the oil phase of the selected system. Nano-

emulsions were prepared by the PIC method, at 25ºC. Nanoparticles (Figure 1) were obtained

from nano-emulsion templating. Both (nano-emulsions and nanoparticles) were characterized

using Zeta Potential (surface charge), dynamic light scattering (DLS, hydrodynamic size) and

visual aspect (stability). Nanoparticles and their template nano-emulsions showed

hydrodynamic radii below 100 nm and negative surface charges. Nanoparticles sizes were

lower than those of their template nano-emulsions. The stability of the nanoparticles allows their

use as medical imaging systems. Moreover, the encapsulation efficiency achieved was nearly

complete, for both fluorescent dyes, attributed to the nanoparticle preparation method. The

fluorescent release was studied for nanoparticle dispersions and for an aqueous and a micellar

solutions, for comparative purposes. The Rhodamine 6G release from nanoparticles was slower

than that from the aqueous solution (Figure 2), which is of great interest due to the fact that

nanoparticles will reach the target tissue before the fluorescent dye begins to be released.

Conclusion

The formulated polymeric nanoparticles are promising as fluorescent delivery systems for

biomedical applications.

References

[1] S. Mura, P. Couvrer, Advanced Drug Delivery Reviews, 64 (2012) 1394-1416 [2] G. Calderó, MJ García-Celma, C. Solans, J of Colloid and Interface Science, 535 (2) (2010) 406-411 Figures

Figure 1. Visual appearance of nanoparticle dispersions. From left to right: free-NP, C6-NP, Rho 6G-NP

Figure 2. Release of Rhodamine 6G as a function of time for nanoparticles, micellar solution

and aqueous solution

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Aqueous solution

Continuous synthesis of silver nanoparticles using green chemicals and microreactors and its evaluation as bactericidal agents

Santiago Ibarlín3, Esteban Gioria

3, Antonella Giorello

1,3, José Luis Hueso

1,2, Victor Sebastián

1,2,

Manuel Arruebo1,2

, Laura Gutierrez3 and Jesús Santamaría

1,2

1Instituto de Nanociencia de Aragón (INA), University of Zaragoza, Spain

2Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain

3Instituto de Investigaciones en Catálisis y Petroquímica (INCAPE), UNL – CONICET, Santiago del

Estero 2829, Santa Fe, 3000, Argentina

[email protected]

Silver nanoparticles have been extensively studied in medicine and microbiology mainly because of their bactericidal properties [1]. There are many methods for the synthesis of these nanoparticles but only a few of them are really reproducible and use protocols and reactants that are environmentally friendly. In this work, the synthesis of silver nanoparticles using green reactants is described. Moreover, a continuous production method is proposed based on the use of microreactors [2]. Green chemicals such as glucose and starch are used as reducer and stabilizing agents, respectively. Different synthesis parameters such as reactant ratios and temperatures are thoughtfully evaluated and optimized to maximize the production of silver nanoparticles. The synthetized materials are fully characterized by TEM, UV.Vis and XPS. Likewise, the bactericidal activity of selected nanoparticles has been evaluated against Escherichia coli.

References

[1] Knetsch, M.L.W. and L.H. Koole. Polymers. New Strategies in the Development of Antimicrobial Coatings: The Example of Increasing Usage of Silver and Silver Nanoparticles, 2011) p.340-366.

[2] Sebastian, V., M. Arruebo, and J. Santamaria. Small. Reaction Engineering Strategies for the Production of Inorganic Nanomaterials, (2014) p. 835-853.

Collection flask

Thermostatic bath

Microreactor

Syringe pump

Glucose + Starch

Aging hose

AgNO3

Collection flask

Thermostatic bath

Microreactor

Syringe pump

Glucose + Starch

Aging hose

AgNO3

Figure 1. Scheme of continuous synthesis device.

Figure 2. TEM micrographs of AgNp synthesized with Ag / Gluc = 1: 5 at 40°C.a: Flow, b: Batch.

Figure 3. Results of the exposure of different concentrations of the selected particles (1.25, 5 y 15 ppm) against E.coli (10

6 UFC/ml). Positive control (+: culture medium with bacterias) and negative control (-

B19 and –F19: culture medium with nanoparticles) were also made.

Biodegradable polymeric nanoparticles modified with cell penetrating peptides as an effective ocular drug delivery system

María José Gómara1, Aimee Vasconcelos1, Estefanía Vega2, Yolanda Pérez3,

María Luisa García2, Isabel Haro1

1. Unit of Synthesis and Biomedical Applications of Peptides, IQAC-CSIC, Jordi Girona, 18-26 08034 Barcelona, Spain. 2. Department of Physical Chemistry, Institute of Nanoscience and Nanotechnology,

Faculty of Pharmacy, University of Barcelona, Avda Joan XXIII s/n 08028 Barcelona, Spain. 3. NMR Unit, IQAC-CSIC, Barcelona, Spain.

[email protected]

Abstract

The bioavailability of ophthalmic drugs in aqueous solutions is usually low due to their rapid elimination after mucosal instillation; a consequence of reflex blinking and tear drainage, as well as of the presence of the corneal barrier. In fact, only 5% of the applied dose reaches intraocular tissues after corneal penetration [1]. Research into biomaterials has therefore included the use of biodegradable polymeric nanoparticles (NPs) in ocular drug delivery; one of the most promising applications of NPs, as they offer a controlled release profile of a drug which is entrapped in the polymeric matrix [2,3]. These are advantages that suggest that the required therapeutic effects could easily be achieved [4]. Over the years, the potential of a variety of synthetic biodegradable polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their copolymer, poly(lactic-co-glycolic acid) (PLGA), for the production of NPs has been extensively explored due to their biocompatibility, biodegradability and mechanical strength [5]. On the other hand, flurbiprofen (FB) is a non-steroidal anti-inflammatory drug which has been introduced into ocular therapy recently not only for the management of inflammatory diseases that affect ocular structures, but also for use during eye surgery. FB has previously been formulated in PLGA NPs by Vega et al. [6], who achieved good stability and appropriate physicochemical properties for ocular administration, without causing ocular irritancy at any level. Recently, a novel cell penetrating peptide (CPP) for ocular delivery was reported (peptide for ocular delivery; POD) that is capable of transporting both small and large molecules across the plasma membrane [7]. The main aim of this work is to improve the corneal epithelium penetration of NPs composed of PLGA-PEG by means of conjugating POD, the final objective being to achieve a longer sustained release of FB which has been used as an example of NSAID drug. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEG-peptide copolymer followed by NP formulation. The physicochemical and biopharmaceutical properties of the resulting NPs (morphology, in vitro release, cell viability and ocular tolerance) were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eye after topical instillation of sodium arachidonate. PLGA-PEG-POD-NPs exhibited suitable entrapment efficiency and sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (HET-CAM) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD-NPs are promising vehicles for ocular drug delivery.

References

[1] Zhang W, Prausnitz MR, Edwards A. J Control Release, 99 (2004) 241-258.[2] Pignatello R, Bucolo C, Spedalieri G, Maltese A, Puglisi G. Biomaterials, 23 (2002) 3247-3255. [3] Pignatello R, Bucolo C, Ferrara P, Maltese A, Puleo A, Puglisi G. Eur J Pharm Sci, 16 (2002) 43-46. [4] Dillen K, Weyenberg W, Vandervoort J, Ludwig A. Eur J Pharm Biopharm, 58 (2004) 539-549. [5] Deshpande AA, Heller J, Gurny R. Crit Rev Ther Drug Carrier Syst, 15 (1998) 381-420. [6] Vega E, Egea MA, Valls O, Espina M, García ML. J Pharm Sci. 95 (2006) 2393-2405. [7] Johnson LN, Cashman SM, Kumar-Singh R. Mol Ther, 16 (2007) 107-114.

Figure

0 30 60 90 120

150

180

210

0

5

10

15

20

25

SA Ocufen ® PLGA-PEG-NPs PLGA-PEG-POD-NPs

* **

*** ******

******

***

***$$

******

***$$

******

***

***

***

$$

$

Time (min)

Ocu

lar

inflam

ati

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sco

re

Comparison of anti-inflammatory efficacy of PLGA-PEG-NPs, PLGA-PEG-POD-NPs and Ocufen® in the prevention of ocular inflammation induced by SA in the rabbit eye. Values expressed as mean ±SD. *P<0.05, **P<0.01 and ***P<0.001 significantly lower than the inflammatory effect induce by AS. ($P<0.05,$$P<0.01 and $$$P<0.001 significantly lower than anti-inflammatory efficacy of Ocufen®.

NPs SA

Cationic vesicles based on non-ionic surfactant and synthetic aminolipids mediate delivery of antisense oligonucleotides into mammalian cells

Santiago Grijalvo,

1 Adele Alagia,

1 Gustavo Puras,

2 Jon Zárate,

2 Jose Luis Pedraz,

2 and Ramon Eritja

1*

1Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Department of Chemical and Biomolecular

Nanotechnology and Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain. 2NanoBioCel group, University of the Basque Country (EHU-UPV), Vitoria and Networking Research

Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) [email protected]

Abstract A formulation based on a synthetic aminolipid containing a double-tailed with two saturated alkyl chains along with a non-ionic surfactant polysorbate-80 has been used to form lipoplexes with an antisense oligonucleotide capable of inhibiting the expression of Renilla luciferase mRNA [1]. The resultant lipolexes were characterized in terms of morphology, zeta potential, average size, stability and electrophoretic shift assay. The lipoplexes did not show any cytotoxicity in cell culture up to 150 mM concentration. The gene inhibition studies demonstrated that synthetic cationic vesicles based on non-ionic surfactant and the appropriate aminolipid play an important role in enhancing cellular uptake of antisense oligonucleotides obtaining promising results and efficiencies comparable to commercially available cationic lipids in cultured mammalian cells (Figure 1). Based on these results, this amino lipid moiety could be considered as starting point for the synthesis of novel cationic lipids to obtain potential non-viral carriers for antisense and RNA interference therapies. References [1] Santiago Grijalvo, Adele Alagia, Gustavo Puras, Jon Zárate, Jose Luis Pedraz and Ramon Eritja, Colloids and Surfaces B: Biointerfaces 119, (2014) 30-37. Figure 1

Gold Nanoparticles Supported on Nanoparticulate Ceria as a Powerful Agent against Intracellular Oxidative Stress

José Raúl Herance,1,2

María Gamón,1 Cristina Menchón,

2 Roberto Martín,

3 Nadezda Apostolova,

1

Milagros Rocha,1 Victor Manuel Victor,

1 Mercedes Alvaro,

3 Hermengildo García.

3

1 Foundation for the Promotion of Healthcare and Biomedical Research in the Valencian Community

(FISABIO)/ University Hospital Doctor Peset (Service of Endocrinology), Juan de Garay 19-21, 46017, Valencia, Spain.

2 Institut d'Alta Tecnologia-PRBB/ CIBER-BBN/CRC-Centre d'Imatge Molecular, Dr.

Aiguader 88, 08003, Barcelona, Spain. 3 Instituto de Tecnología Química CSIC-UPV/Departamento de

Química, Universidad Politécnica de Valencia, Av. De los Naranjos s/n, 46022 Valencia.

[email protected]

Abstract

Ceria-supported gold nanoparticles are prepared exhibiting peroxidase activity and acting as radical

traps. Au/CeO2 shows a remarkable biocompatibility as demonstrated by measuring cellular viability,

proliferation, and lack of apoptosis for two human cell lines (Hep3B and HeLa). The antioxidant activity

of Au/CeO2 against reactive oxygen species (ROS) is demonstrated by studying the cellular behavior of

Hep3B and HeLa in a model of cellular oxidative stress. It is determined that Au/CeO2 exhibits higher

antioxidant activity than glutathione, the main cytosolic antioxidant compound, and its CeO2 carrier.

Overall the result presented here shows the potential of implementing well-established nanoparticulated

gold catalysts with remarkable biocompatibility in cellular biology.

References

[1] Menchón C, Martín R, Apostolova N, Victor VM, Alvaro M, Herance JR, García H., Small, 8 (2012) 1895.

Figures

Fig 1. Effect of Au/CeO2 (20 µg/ml), CeO2 (20 µg/ml) and glutathione (100 µM) on Rotenone-induced ROS production. Bar charts showing DCFH fluorescence in Hep3B cells.

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Fig 2. Effect of Au/CeO2 and the carrier CeO2 on cellular proliferation and viability in Hep3B and HeLa cells (a). Cell count over 3 days by static cytometry (data represented as mean ± S.E.M, n= 3). (b) MTT assay of exponentially growing cells after 24 h of culture (data represented as mean ± S.E.M, n= 5-6) were analyzed by Student´s t-test, significance vs control * p<0.05.

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Fig 3. Assessment of apoptosis in He3B cells after 24 h incubation with the nanoparticles or the positive apoptotic control, staurosporine (STS). (a) Representative histograms (Bivariate Annexin V/PI analysis) of untreated control, the carrier CeO2 and 1µM STS-treated cells. The table shows the % of each sub-population for all the conditions studied. (b) Summary histogram of AnnexinV and PI fluorescence data and (c) nuclear morphology changes (mean Hoechst fluorescence and nuclear area). Data (mean ± S.E.M, n=4) was analyzed by Student´s t-test significance vs control * p<0.05 and *** p<0.001.

a ba b

a b

c

a b

c

Application of magnetic chitosan nano particles for anti-Alzheimer drug delivery systems

Mohammadreza Khanmohammadi, Hamideh Elmizadeh

Chemistry Department, Faculty of Science, IKIU, Qazvin, Iran

[email protected]

Abstract

Nanoparticles have become an important area of research in the field of drug delivery because they

have the ability to deliver a wide range of drugs to different areas of the body at appropriate times [1].

Polymers used to form nanoparticles can be two types, hydrophobic and hydrophilic. Nanoparticles

based on hydrophilic polymers such as chitosan are appropriate candidates for drug delivery systems

[2 4]. Chitosan nanoparticles and magnetic chitosan nanoparticles can be applied as delivery systems

for the anti-Alzheimer drug tacrine. Investigation was carried out to elucidate the influence of process

parameters on the mean particle size of chitosan nanoparticles produced by spontaneous

emulsification. The method was optimized using design of experiments (DOE) by employing a 3-factor,

3-level Box Behnken statistical design. This statistical design is used in order to achieve the minimum

size and suitable morphology of nanoparticles. Also, magnetic chitosan nanoparticles were synthesized

according to optimal method. The designed nanoparticles have average particle size from 33.64 to

74.87nm, which were determined by field emission scanning electron microscopy (FE-SEM). Drug

loading in the nanoparticles as drug delivery systems has been done according to the presented optimal

method and appropriate capacity of drug loading was shown by ultraviolet spectrophotometry. Chitosan

and magnetic chitosan nanoparticles as drug delivery systems were characterized by Diffuse

Reflectance Fourier Transform Mid Infrared spectroscopy (DR-FTMIR).

References

[1] M.L. Hans, A.M. Lowman, Biodegradable nanoparticles for drug delivery and targeting, Curr. Opin.

Solid State Mater. Sci. 6 (2002) 319 327..

[2] E. Lee, J. Lee, I.H. Lee, M. Yu, H. Kim, S.U. Chae, S. Jon, Conjugated chitosan as a novel platform

for oral delivery of paclitaxel, J. Med. Chem. 51 (2008) 6442 6449.

[3] A. Trapani, J. Sitterberg, U. Bakowsky, T. Kissel, The potential of glycol chitosan nanoparticles as

carrier for low water soluble drugs, Int. J. Pharm. 375 (2009) 97 106.

[4] Y. Zhang, M. Huo, J. Zhou, D. Yu, Y. Wu, Potential of amphiphilically modified low molecular weight

chitosan as a novel carrier for hydrophobic anticancer drug: synthesis, characterization, micellization

and cytotoxicity evaluation, Carbohydr. Polym. 77 (2009) 231 238.

FT-IR Spectra chitosan nanoparticles (1) and magnetic chitosan nanoparticles (2)

The interferences of nanomaterials with hemoglobin a handicap to study hemocompatibility

Llanas H, Sordé A, Mitjans M, Vinardell MP

Departament de Fisiologia, Facultat de Farmàcia, Av. Joan XXIII s/n, 08028 Barcelona, Spain [email protected]

Abstract The interactions of nanomaterials with membrane cells are an important research area because such interactions are critical in many applications such as biomedical imaging, drug delivery, disease diagnostics and DNA/protein stricter probing [1]. More and more nanomaterials are designed for biological applications, and this raises new concerns about the safety of nanotechnology [2,3]. Nanotechnology-derived devices and drug carriers are emerging as alternatives to conventional small-molecule drugs, and in vitro evaluation of their biocompatibility with blood components is a necessary part of early preclinical development. Special attention should be paid to the interaction of nanomaterials (NMs) with erythrocytes and for this reason the haemolysis assay is recommended as a reliable test for material biocompatibility [4]. The method used was the hemolysis assay as described in previous papers [5] and adapted to the study of NMs. Briefly, red blood cells obtained by centrifugation from fresh blood were incubated at room temperature for 1, 3 and 24 hours with different concentrations of the different nanomaterials studied. At the end of the incubation period, tubes were centrifuged and the amount of hemoglobin on the supernatant has been determined by spectroscopy at 540 nm to determine the percentage of hemolysis induced by the chemicals, compared to red blood cells totally hemolysed. We have used red blood cells from human, rat and rabbit. One of the possible limitations of the hemolysis assay is the absorption of the NMs at 540 nm and this should be discarded [6,7]. Another the possible interference of the nanomaterials with the endpoint of the hemoglobin determination is the adsorption of the hemoglobin by the nanomaterial and/or the protein denaturation. In order to study these possible interferences we have exposed the hemoglobin obtained from erythrocytes by hypotonic haemolysis to the nanomaterials under study. The haemoglobin spectrum was recorded with an UV/visible spectrophotometer. We have studied different nanomaterials such as nano aluminum oxide as nanopowder (13 and 50 nm) and nanowires, zinc oxide nanopowder (50 and 100nm) (Sigma-Aldrich) and a commercial preparation of hydroxyapatite (nanoXIM.CarePaste®, supplied by Fluidinova). This is a highly dispersed hydroxyapatite aqueous paste specially designed to be incorporated in high performance Oral Care products, with special highlight in toothpastes and mouthwashes aiming enamel remineralization and reduction of teeth sensitivity. The hemolysis phenomena is usually concentration-dependent (higher test concentration induces higher hemolysis). In some cases this is not observed, a decrease in the hemolysis is observed when the concentrations of the test substances increase. This can be observed in the hemolysis induced by Al2O3 nanowire after 3 hours incubation (Figure 1). This effect could be done by the adsorption of the hemoglobin by the nanowire and this could be demonstrated by the spectrum of hemoglobin treated with the nanowire (Figure 2). Clearly, we can observe that the spectrum is not modified, then there is no denaturation and the decrease in absorbance could be attributed to the adsorption phenomena. Similarly, we have observed this phenomenon with a commercial hydroxyapatite preparation (nanoXIM) (Figures 3). In figure 4 we can observe the decrease in the supernatant color and increase in pellet color with increasing concentrations of the nanomaterial. In the case of nano zinc oxide we can observe a significant color change and the hemoglobin spectrum shows an alteration due to the protein denaturation induced by the nanoparticle at higher concentration. This effect is not observed with lower concentrations.

References [1] Verma A, Stellaci F, Small 6 (2010) 12. [2] Nel E, Madler L, Velegol D, Xia T, Hoek EMV, Somasundaran P, Klaessig F, Castranova V, Thompson M, Nat. Mater. 8 ( 2009) 543. [3] Ai J, Biazar E, Jafarpour M, Montazeri M, Majdi A, Aminifard S, Zafari M, Akbari HR, Rad HG. Int J Nanomedicine. 6 (2011) 1117. [4] Lu S, Duffin R, Poland C, Daly P, Murphy F, Drost E, Macnee W, Stone V, Donaldson K. Environmental Health Perspectives 117 (2009) 241. [5] Nogueira DR, Mitjans M, Infante MR, Vinardell MP. Acta Biomaterialia 7 (2011) 2846. [6] Dobrovolskaia M.A., Clogston J.D, Neun B.W., Hall J.B., Patri A.K., MacNeil S. Nanoletters 8 (2008) 2180. [7] Neun BW, Dobrovolskaia MA.Methods Mol Biol. 697 (2011) 215. Figures

Figure 2: Rabbit hemoglobin spectrum and effect of

Al2O3 nanowire at 60 and 80 mg/ml after 3 hours incubation time

Figure 1: Hemolysis induced by nanowire of Al2O3 after 3 hours incubation

Figure 3: Human hemoglobin spectrum and effect of nanoXIM after 24 hours incubation time (31 to 1.5 mg/mL)

Figure 4: Human erythrocytes treated with increasing concentration of nanoXIM. The supernatant shows decrease in color with concentration and the pellet shows the adsorption of hemoglobin

Figure 5: Human hemoglobin spectrum and effect of nano ZnO 100 nm after 24 hours incubation time at 37ºC. Spectrum alteration after treatment with 2 mg/mL

Nanoscale conductance imaging of electronic materials and redox proteins in aqueous solution

M. López-Martínez

1,2,3, J. M. Artés

1, I. Díez-Perez

1,3, F. Sanz

1,2,3 and P. Gorostiza

1,2,4,*

1Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 15-21, 08028 Barcelona, Spain

2 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)

3 Physical Chemistry Department, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain

4 Institució Catalana de Recerca i Estudis Avançats (ICREA)

(*) [email protected] Electron Transfer (ET) plays essential roles in chemistry and biology, as it is involved in electrochemical reactions and in crucial biological processes such as cell respiration and photosynthesis. ET takes place between redox proteins and in protein complexes, and it displays an outstanding efficiency and environmental adaptability. Although the fundamental aspects of ET processes are well understood, more experimental methods are needed to determine electronic pathways, especially in complex systems like organic and inorganic nanostructures as well as in biomolecules like nucleic acids and proteins. Understanding how ET works is important not only for fundamental reasons, but also for the potential technological applications of these redox-active nanoscale systems.

Electrochemical Scanning Tunneling Microscopy (ECSTM) is an excellent tool to study electronic materials and redox molecules including proteins

1. It offers atomic or single molecule resolution and

allows working in aqueous solution, in nearly physiological conditions in the case of proteins, and under full electrochemical control. Beyond imaging, ECSTM allows performing current-voltage and current-distance tunneling spectroscopy. We have adapted this spectroscopy mode of ECSTM to include a sinusoidal voltage modulation to the STM tip, and current measurement by means of a lock-in amplifier, which renders a signal that is proportional to the differential conductance dI/dV of the studied surface

2.

We have used this setup to record for the first time spatially resolved, differential conductance images under potentiostatic control (differential electrochemical conductance (DECC) imaging). We have validated and optimized the technique using an iron electrode, whose reversible oxidation in borate buffer is well characterized

3 (Figure 1).

We have applied DECC imaging to gold Au <111> surfaces coated with P. Aeruginosa Azurin, a redox metalloprotein with a copper center involved in the respiratory chain of denitrifying bacteria. Azurin can be immobilized on single crystal Au <111> surfaces via a dithiol covalent bond, and it has become a model system to study biological ET processes

4. DECC imaging provides simultaneously the surface

topography and local conductance with a resolution of a few nanometers, and reveals regions with different conductance within the protein. The characterization of conduction pathways in redox proteins at the nanoscale would enable important advances in biochemistry and would cause a high impact in the field of nanotechnology

5. This method can be used to study more complex biosystems, like multi-

center redox proteins and protein redox complexes, and lead to a deeper understanding of their electronic properties and ET pathways. References 1 Friis, E. P. et al. An approach to long-range electron transfer mechanisms in metalloproteins: In situ scanning

tunneling microscopy with submolecular resolution. Proceedings of the National Academy of Sciences of the United

States of America 96, 1379-1384, doi:10.1073/pnas.96.4.1379 (1999).

2 Robinson, R. S. & Widrig, C. A. Differential conductance tunneling spectroscopy in electrolytic solution. Langmuir

8, 2311-2316, doi:10.1021/la00045a039 (1992).

3 Diez-Perez, I., Guell, A. G., Sanz, F. & Gorostiza, P. Conductance maps by electrochemical tunneling spectroscopy

to fingerprint the electrode electronic structure. Analytical Chemistry 78, 7325-7329, doi:10.1021/ac0603330 (2006).

4 Artes, J. M., Diez-Perez, I., Sanz, F. & Gorostiza, P. Direct Measurement of Electron Transfer Distance Decay

Constants of Single Redox Proteins by Electrochemical Tunneling Spectroscopy. Acs Nano 5, 2060-2066,

doi:10.1021/nn103236e (2011).

5 Artes, J. M., Diez-Perez, I. & Gorostiza, P. Transistor-like Behavior of Single Metalloprotein Junctions. Nano letters

12, 2679-2684 (2012).

Figures

ph-

Silvia Hernández-Ainsa1,3

, Joaquín Barberá1, Mercedes Marcos

1, José Luis Serrano

2 , Teresa

Sierra1

1. Instituto de Ciencia de Materiales de Aragón (ICMA), Universidad de Zaragoza CSIC, Pedro

Cerbuna 12, 50009, Zaragoza, Spain. 2. Instituto Universitario de Nanociencia (INA),

Universidad de Zaragoza, Pedro Cerbuna 12, 50009, Zaragoza, Spain. 3. Current address:

Biological and Soft Systems, Cavendish Laboratory, JJ Thomson Avenue, Cambridge, CB3

0HE. Liquid crystal order allows the control of the supramolecular arrangement, thus providing a powerful tool to obtain ordered structures capable of executing a function. For instance, many lyotropic liquid crystals have been studied as bioinspired synthetic materials mimicking cellular membranes. In this way, supramolecular self-assembly in water constitutes an active topic of research because the possibility to produce a variety of nanoobjets with different shapes. These structures have opened a wide range of potential applications in fields as different as Material Science or Biomedicine. Among the great variety of molecules investigated in this context, dendrimers posses some specific characteristics that make them of great relevance to address the desired self-assembly process. Nearly all amphiphilic dendrimers for guest encapsulation have been obtained by means of covalent bonds, which is laborious and require several purification steps. The preparation of dendrimers by non-covalent self-assembly processes, such as the ionic interaction, is a very convenient synthetic method due to its simplicity, (it is made in one-step) and its versatility because variation of the functional groups can easily be carried out. Some of these ionic dendrimers have shown liquid crystalline properties. With the aim to obtain amphiphilic molecules a series of ionic amphiphilic dendrimers constituted by the grafting of poly(amidoamine) (PAMAM) of different generations (G=0-4) with linear carboxylic acids bearing hydrophobic chains has been prepared. Almost all of the compounds present liquid crystalline behaviour as shown by differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and X-ray diffractometry (XRD) studies. Smectic A mesomorphism has been found for most of the compounds and a rectangular columnar mesophase is displayed for the highest generation compound at low temperature. Interestingly these amphiphilic dendrimers are also capable to self-assemble in water depending on their hydrophobic/hydrophilic balance forming some nanoobjects. In most of the cases these nanoobjects resemble nanospheres whose morphology has been studied by means of transmission electronic microscopy (TEM). The stability of these nanospheres is disrupted in acid or basic media and their amphiphilic nature makes them suitable for trapping both hydrophobic -carotene) and hydrophilic (Rhodamine B) molecules. These features make these easy to synthesize systems promising and versatile candidates as molecular nanocarriers for a number of biomedical and technological applications.

50 nm a b c d a), b), c) Representative TEM images of nanospheres formed by self-assembly in water of the ionic dendrimer derived from PAMAM and myristic acid., d) POM textures of G4(C14) at 59ºC in the first cooling process.

Gemini Amphiphilic Pseudopeptides for Encapsulation and Release of

Hydrophobic Molecules

Ahmed H.Lotfallah,a Ignacio Alfonso,

b M. Isabel Burguete

a and Santiago V. Luis

a

a Departamento de Química Inorgánica y Orgánica, Universiat Jaume I, Avenida Sos Baynat

s/n, Castellón, Spain. b

Departamento de Química Biológica y Modelización Molecular, Instituto

de Química Avanzada de Cataluña (IQAC-CSIC), Jordi Girona 18-26, Barcelona, Spain.

E-mail: [email protected]

Abstract:

Gemini amphiphilic pseudopeptides (GAPs) are non-biogenic peptide like molecules [1], able to

self-assemble into well-ordered nanostructures through the cooperative action of polar (H-

bonding and dipole-dipole) and non-polar (van der Waals) interactions [2-4]. In acidic medium,

GAPs are able to form vesicles which have been studied in the solid state (SEM, TEM and

AFM; Fig. 1) and in the liquid state (optical fluorescent microscope, Fig. 3). This vesicular

morphology is attributed to the hydrophobic interactions which play a major role in the stability

of the folding state [3]. In addition, GAPs provide o/w emulsion that remains stable for months

and also shows good stability toward the acidic pH and centrifugation effect Fig. 3&2. The

capability of this system to encapsulate hydrophobic molecules such as dimethylanthracene

(DMA) and dansyldiethyl amine (DEA) was evaluated by fluorescence spectroscopy and

microscopy respectively. The results showed that the DMA fluorescence was highly enhanced

after 24 hours and I1/I3 fluorescence intensity ratio increased by almost 0.6 Fig. 4. Additionally,

DEA was efficiently incorporated into the inner hydrophobic core of GAPs vesicles rendering

green colored balls Fig 2. Ultimately, such system can be enzymatically disassembled resulting

in the destruction of vesicles and release of its contents Fig. 5 [5]. Therefore, the GAPs here

considered are promising system for drug delivery.

References:

[1] Fundamentals of Protein Structure and Function" Engelbert Buxbaum, Springer, 1 edition,

2007, ISBN:0387263527.

[2] (a) S. Cavalli, F. Albericio, A. Kros, Chem. Soc. Rev., 2010, 39, 241.

(b) R. J.Brea, C.Reiriz, J. R. Granja, Chem. Soc. Rev., 2010, 39, 1448.

(c) I. W. Hamley, Soft Matter, 2011, 7, 4122.

[3] J. Rubio, I. Alfonso, M. I. Burguete, S V. Luis, Soft Matter, 2011, 7, 10737.

[4] J. Rubio, I. Alfonso, M. I. Burguete, S V. Luis, Chem. Commun., 2012, 48, 2210.

[5] S. Bai, C. Pappas, S. Debnath, P. J. M. Frederix, J. Leckie, S. Fleming, R. V. Ulijin

2014, 8, 7005.

Figures:

Fig. 1 Micrographs of GAPs model grown from 1:1 MeOH : H2O + HCl captured by (a) SEM, (b)

AFM and (c) TEM techniques.

Fig. 2 Fluorescent microscope images of o/w GAPs encapsulated DEA emulsion; Long term

stability (a) after 1 week, (b) after 1 month, (c) after 3 months.

Fig. 3 Optical microscope images of o/w GAPs emulsion after centrifugation at 3000 rpm for 30

minutes; Mechanical stability test (a) 5 min, (b) 15 min, (c) 30 min.

Fig. 4 Fluorescence spectroscopy of Dimethyl anthracene; (a) free DMA, (b) encapsulated

DMA.

Fig. 5 Optical microscope images of o/w GAPs emulsion; (a) without thermolysin, (b,c) after

adding 1.5 mg/mL thermolysin.

Modulation of dendritic cell sensitization by combined exposure to allergens and nanoparticles

Inge Nelissena, Birgit Baré

a, Sarah Deville

a,b, An Jacobs

a, Nathalie Lambrechts

a, Peter Hoet

c

a Flemish Institute for Technological Research (VITO), Applied Bio&molecular Systems, Mol, Belgium

b Hasselt University, Biomedical Research Institute (BIOMED), Diepenbeek, Belgium

c Catholic University Leuven (KULeuven), Lung Toxicology Unit, Leuven, Belgium

[email protected]

Abstract The adjuvant activity of air pollution particles in allergic airway sensitization is well known, but a similar role of manufactured nanoparticles in allergic sensitization has not been clarified. The goal of our study was to assess the possible alteration of an allergen-induced sensitization response by gold nanoparticles (NPs) through in vitro studies. Immature myeloid dendritic cells (CD34-DC), differentiated from human cord blood-derived CD34

+

progenitor cells, were incubated in the presence of subtoxic concentrations of two sensitizing compounds and citrate-stabilized 50-nm gold NPs (4.4 µg/ml) for 24 hours, either as separate inducers or as a mixture. The chemical sensitizer nickel sulphate (NiSO4, 160 and 430 µg/ml) and a whole Der p protein allergen mixture (20, 100 and 200 µg/ml) were used as model allergens. Activation and maturation of CD34-DC were studied as indicators of a sensitization response by measuring cell surface expression of the antigen-presenting HLA-DR receptor, the co-stimulatory molecules CD80, CD86 and CD83, and the integrin CD11c using flow cytometry. Exposure of CD34-DC to NPs induced significant upregulation of the three co-stimulatory molecules as compared to dispersant treated cells. Der p alone did not stimulate any of the studied cell surface markers, but when co-incubated with the NPs it was observed to significantly inhibit NP-induced CD34-DC activation in a dose-dependent way. Sole exposure to NiSO4 significantly upregulated CD86 and CD83, while downregulating CD80 expression in CD34-DC. When NiSO4 and gold NPs were combined during co-exposure, we observed a cell activation pattern and levels similar to those induced by NiSO4 alone, and thus significantly lower than an additive effect of both inducers (Figure). These results indicate that gold NPs interfere with the allergens in the CD34-DC culture, resulting in decreased sensitizing effects. This may either be mediated via a physico-chemical or immune regulatory mechanism. Further investigation will enhance our insight in the possible impact that nanoparticles may pose to our health. Figure

Mixture effect of Au-NPs and NiSO4 on a sensitization response in CD34-DCs. Cells were co-exposed to Au-NPs (4.4 µg/ml) and NiSO4 (430 µg/ml) for 24 hours, harvested, and analysed for surface marker expression. Mean log2 SI ± standard deviation (N=5) are shown (log2 SI of solvent control (SC) = 0). Significantly altered expression compared to respective SC is indicated with * (p<0.05) and ** (p<0.005) only for conditions with |SI|>1.5.

Toxicity assays of nebulized gold nanoparticles with potential applications in the development of nanopesticides

M.A. Ochoa-Zapater

a*, J. Querol-Donat

a, F.M. Romero

b, A. Ribera

b, G. Gallello

c, A. Torreblanca

a, M.D.

Garceráa.

a Departamento de Biología Funcional y Antropología Física, Universitat de València, Dr. Moliner 50,

46100 Burjassot, Valencia, Spain. b Instituto de Ciencia Molecular, Universitat de València, Catedrático José

Beltrán, 2. 46980, Paterna, Valencia, Spain. c Departamento de Química Analítica, Universitat de València, Dr. Moliner 50, 46100 Burjassot,

Valencia, Spain. *[email protected]

Abstract In recent years, nanotechnology applications in agriculture had led to the development of a wide range of plant protection products described as nanopesticides: these products include polimer based formulations [1], formulations containing inorganic nanoparticles [2] and nanoemulsions [3]. The main reasons for the development of these products are the growing need for alternative pesticides to prevent damage on non-target organisms and delay the development of resistances [4]. Moreover, some of these alternative pesticides can benefit from these nanoformulations, which can provide delivery systems for active ingredients with reduced solubility, as well as increase stability and protect them from premature degradation [5]. The toxicity of nebulized gold nanoparticles (AuNPs), which could be functionalized for the formulation of nanopesticides, was tested in two laboratory reared insect species, the german cockroach Blattella germanica, considered an important urban pest with serious implications in public health [6], and the milkweed bug Oncopeltus fasciatus. AuNPs were synthetized following the methodology described by Bastús et al. [7] and characterized by UV-Vis and Transmission Electron Microscopy (TEM). Adult insects (15 females and 15 males, aged 1-6 days) were exposed to 1mL and 2mL of AuNPs in sodium citrate with the aid of a nebulizer based system (figure 1), with times of total exposure ranged between 15 to 90 minutes (table 1). Mortality rates were monitored 24, 48, 72 and 96 hours post-treatment, and enzymatic activities related to oxidative stress and insecticide resistance [8], such as glutathione S-transferases (GSTs) and esterases (p-NPA), were measured in exposed insects frozen immediately after nebulization and insects frozen at 96h post-treatment. Also, a comparison between the obtained activity rates and results from our previous studies in tarsal contact toxicity bioassays were made for the two insect species. Finally, in order to study the persistence of nanoparticles in treated insects, inductively coupled spectroscopy (ICP-OES) was performed in insects frozen at times 0 and 96 hours after AuNPs exposure. References [1] Adak T, Kumar J, Shakil NA, Walia S, J. Environ. Sci. Health B, 47 (3) (2012) 217 25. [2] Song M-R, Cui S-M, Gao F, Liu Y-R, Fan C-L, Lei T-Q, Liu D-C, J. Pestic. Sci., 37 (3) (2012) 258 60. [3] Kumar RSS, Shiny PJ, Anjali CH, Jerobin J, Goshen KM, Magdassi S, Mukherjee A, Chandrasekaran N, Environ. Sci. Pollut. Res., 20 (4) (2013) 2593 602. [4] Bhattacharyya A, Bhaumik A, Rani PU, Mandal S, Epidi T, Afr. J. Biotechnol., 9 (2010) 3489 3493. [5] Nguyen HM, Hwang IC, Park JW, Park HJ, J. Microencapsul., 29 (6) (2012) 596 604. [6] Roberts J, Br. Med. J., 312 (1996) 1630. [7] Bastús NG, Comenge J, Puntes V, Langmuir, 27 (2001) 11098-11105. [8] Hemingway J, Ranson H, Annu. Rev. Entomol., 45 (2000) 371-391.

Figures Table 1. Total exposure time to nebulized AuNPs

Treatment Volume (mL) Duty (%)a

Exposure time (h:m:s)

Blattella germanica Oncopeltus fasciatus

Mean SD Mean SD

AuNP 1 100 0:15:15 0:00:05 0:14:53 0:00:17

50 0:16:59 0:00:13 0:16:19 0:00:08

5 0:46:01 0:01:51 0:46:15 0:00:31

AuNP 2 100 0:16:50 0:00:06 0:16:55 0:00:34

50 0:20:26 0:00:03 0:20:02 0:00:03

5 1:14:40 0:02:15 1:18:27 0:02:15 a % of nebulized solution per cycle (1 cycle equals 6 seconds)

Figure 1. Adult cockroaches being exposed to nebulized AuNPs in the nebulization chamber

CERIUM OXIDE NANOPARTICLES REDUCE PORTAL HYPERTENSION AND SHOW ANTIINFLAMMATORY PROPERTIES IN CCl4-TREATED RATS

D. Oró1, G. Fernández-Varo1,3, V. Reichenbach1, T. Yudina2 , E. Casals2, G. Casals1,B. González de la Presa1, V. Puntes2, W. Jiménez1,3.

1Biochemistry and Molecular Genetics Service, Hospital Clínic de Barcelona, IDIBAPS, Centro de Investigación Biomédica en Red de Enfermedades

Hepáticas y Digestivas (CIBERehd), 2Institut Català de Nanotecnologia (ICN), Bellaterra, Spain, 3Department of Physiological Sciences I, University of

Barcelona, Barcelona, Spain.

[email protected]

Background and Aims. During the last few years nanoparticles (NPs) have emerged as a new

technology allowing enhanced levels of precision in treating disease. Cerium oxide (CeO2) NPs

have proven to behave as free radical scavenger and/or antiinflammatory agents. However,

whether CeO2NPs are of therapeutic value in liver disease is not known. We assessed the

organ distribution, subcellular localization, metabolic fate and systemic and hepatic effects of

the iv administration of CeO2NPs to CCl4-treated rats. The aim of the study was to determine

whether CeO2NPs display hepatoprotective properties in experimental liver disease. Methods.

Organ and subcellular distribution of NPs was assessed using magnetic resonance imaging

(MRI) and transmission electron microscopy (TEM), respectively. The metabolic fate of

CeO2NPs was investigated by measuring daily urinary and fecal excretion of Ce (ICP-MS). The

systemic and hepatic effects of NPs were assessed in CCl4-treated rats receiving CeO2NPs (0.1

mg/kg, n=10) or vehicle (n=15) twice weekly for two weeks and CCl4 treatment was continued

for 8 additional weeks. Thereafter, mean arterial pressure (MAP) and portal pressure (PP) were

assessed and serum samples obtained to measure standard hepatic and renal function tests.

Liver samples were also obtained to evaluate mRNA expression of genes related to

-smooth muscle actin -SMA)

expression and hepatic apoptosis. Results. More than 90% of the NPs were located in the liver

and spleen 30 min after administration. The remaining targeted lungs and kidneys. No NPs

were located in the brain. CeO2NPs were internalized by parenchymal cells and found in either,

peroxisomes or free in the cytoplasm. Most NPs were excreted by the urine. CeO2NPs

ameliorated systemic inflammatory biomarkers (LDH: 879±229 vs 392±67, ALT: 1287±419 vs

304±40 U/L; p<0.05) and improved PP (9.9±0.4 vs 8.2±0.4 mm Hg, p<0.05) without affecting

MAP. A marked reduction in mRNA abundance of i 53±11.1 vs

18.3±4.7, 61.6±10 vs 31.2±5.4; p<0.05), iNOS: (537±158 vs 94±35, p<0.05) and ET-1

(14.2±2.7 vs 6.3±1.9, p<0.05), infiltration of macrophages (29.5±0.8 vs 25.7±0.7 cells/field) and

protein expression of caspase-3 (21±2.9 vs 7.1±2.7 DAU, p<0.05) -SMA (7.1±0.3 vs

5.8±0.3 %, p<0.01) was observed in the liver of rats receiving CeO2NPs. Conclusions.

CeO2NPs administration to CCl4-treated rats protects against chronic liver injury by markedly

attenuating the intensity of the inflammatory response and reducing portal hypertension, thereby

suggesting that CeO2NPs may be of therapeutic value in chronic liver disease.

! "!

Bridging Research and Industrial Production towards H2020: Future challenges for Nanomedicine with a multi-KET approach

Cristina Paez-Aviles1, Esteve Juanola-Feliu

1, Josep Samitier

1,2,3

1Department of Electronics, Bioelectronics and Nanobioengineering Research Group (SIC-BIO),

University of Barcelona, Martí i Franquès 1, Planta 2, 08028 Barcelona, Spain. [email protected] for Bioengineering of Catalonia, Nanosystems Engineering for Biomedical Applications

Research Group, Baldiri Reixac 10-12, 08028 Barcelona, Spain 3CIBER-BBN-Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine,

María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain

Abstract

It is stated that pilot production builds the bridge between research and industrial production since this activity is among technology and commercialization. However, pilot scalability is considered a bottleneck in the way to commercialization, even more in the Health domain where scalability is more complex. In this context, the new European Commission’s initiative Horizon 2020, the biggest financial program for Research and Innovation, plans to finance different Risk Management Projects going “from fundamental research to market innovation” involving the entire innovation chain. H2020 is particularly focused on the research and development of Key Enabling Technologies (KETs), which are among the priorities of the framework strategy, thatidentifies the need for the EU to facilitate the industrial deployment of KETs in order to make its industries more innovative and globally competitive [1]. Six KETs have been selected according to an economic criteria, economic potential, capital intensity, technology intensity, and their value adding enabling role: Nanotechnology, Micro and Nano Electronics, Photonics, Advanced Materials, Biotechnology Industry, and Advanced Manufacturing Systems[2].

While each KET already has huge potential for innovation individually, their cross-fertilization is particularly important to offer even greater possibilities to foster innovation and create new markets. Integration between KETs will be essential for create jobs in industry, improve competitiveness and innovation, and at the same time address today’s burning societal challenges in Europe in the coming years. The concept of cross-cutting KETs refers to the integration of different key enabling technologies in a way that creates value beyond the sum of the individual technologies for developing innovative and competitive products, goods and services oriented to solve societal needs. The global market volume in KETS is €646 billion andsubstantial growth is expected of approximately 8% of EU GDP by 2015. In this context, Horizon 2020 will invest €5.96 billion in the industry sector for the development of the KETs and about 1/3 of this budget will be assigned to projects integrating different KETs [3].

Most high tech pilot production problems are inherently multi-KETs. The scale up of nanomedicines for clinical testing is severely hindered by a lack of knowledge about how and where to manufacture such entities according to Good Manufacturing Practice (GMP) and taking into account the medical regulatory requirements. The Commission states that bridging the so called “Valley of Death” to upscale new KET technology based prototypes to commercial manufacturing, often constitutes a weak link in the successful use of KETs potential[4].

Translation of innovation and time-to-market reduction are important challenges on H2020. After a long R+D incubation period, several industrial segments are already emerging as early adopters of nanotech-enabled products and findings suggest that the Bio&Health market is among the most challenging field for the coming years. As a major application of Nanotechnology, the field of Nanomedicine fits naturally amongst the Key Enabling Technologies defined by the European Commission. It is considered multidisciplinary since it is not restricted to the realm of advanced materials, extending also to manufacturing processes, biotechnology, pharmacy, electronics and IT, as well as other technologies [5]. These characteristics allow the connection to a diversified set of industries [6]. Inherent interactions exist between these sectors and could be mutually beneficial in terms of research innovation(Fig. 1). For example, the use of quantum dots and shape-shifting nanomaterials for medical applications could greatly benefit from the latest progress in photonics, and nanomedicine

! #!

sensors from biotechnology and biological pores. Additionally, new medical therapies enabled by Nanotechnology and Advanced Materials, can contribute to personalised health care. This strong interdisciplinary character, combined with the possibility of manipulating a material atom by atom, opens up unknown fields and provides an endless source of innovation and creativityin the healthcare domain.

Fig. 1: A Multi-KET approach of Nanomedicine: common R&D topics with the KETs [7]

References

[1] European Commission, Brussels, 2009.[2] B. Aschhoff, D. Crass, K. Cremers, C. Grimpe, F. Brandes, F. Diaz-lopez, R. K.

Woolthuis, M. Mayer, and C. Montalvo, 2010[3] E. Commision, 2013.[4] M. De Heide, M. Butter, D. Kappen, A. Thielmann, A. Braun, M. Meister, D. Holden, F.

Livese, E. O. Sullivan, C. Hartmann, M. Zaldua, N. Olivieri, L. Turno, M. Deschryvere, J. Lehenkari, P. Ypma, P. Mcnally, and M. De Vries, 1–58, 2013

[5] N. Islam and K. Miyazaki, Technovation, vol. 27, no. 11, (2007) 661–675.[6] T. Nikulainen and C. Palmberg,Technovation, vol. 30, no. 1, (2010) 3–11.[7] European Technology Platform on Nanomedicine, 2013.

Accessing the Nanoparticle Corona in Pulmonary Surfactant

Simon S. Raesch1,2

, Stefan Tenzer3, Wiebke Storck

3, Christian Ruge

1, Ulrich F. Schaefer

1, Claus-

Michael Lehr1,2

1Biopharmaceutics and Pharmaceutical Technology, Saarland University, 66123 Saarbruecken,

Germany2Department of Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland, 66123

Saarbruecken, Germany3Institute for Immunology, University Medical Center of Mainz, 55101 Mainz, Germany

[email protected]

Abstract

Nanoparticles (NP) that come in contact with a biological fluid are opsonized by biomolecules such as proteins, which build a “corona”.This time-dependent layer of adherent biomolecules typifies the actual biological identity of the NP. Considering the many different NP with varying surface modifications which are produced worldwide, differences in resulting corona seem plausible and were identified in the coronas on NP in plasma [2, 3].An attractive pharmaceutical target for various nanoparticles is the lung, as the air-blood barrier is a less than 2 µm thin layer with an enormous alveolar surface area larger than 100m

2. The vast amount of

potentially polluted air, which passes through the lung, makes an effective maintenance system essential. In the alveolar region cells are only covered by a thin pulmonary surfactant (PS) layer and clearance is mainly carried out by alveolar macrophages. PS, secreted by type II alveolar cells, allows gas diffusion and its surface tension lowering effect is thus essential for stability of the alveoli during abreathing cycle. The surfactant layer consists of approximately 90% lipids (mainly phospholipids, especially DPPC) and 10% proteins with about half of them being surfactant specific proteins (SP). Before NP are either taken up by the alveolar cells or ingested by macrophages, they are coated by the PS building a lipid-protein-“corona”.So far, it remains to be elucidated whether the fate of inhaled NP depends on the coating obtained from the surfactant layer, though there is evidence for the influence of the SP on macrophage uptake [1]. Although understanding the surfactant-NP interaction is fundamental for the fate of NP in the lung, there is, so far, no reproducible method for the analysis of the NP-corona.

The unique composition, structure, and properties of the lipid-rich PS require different and more advanced analytical methods for the assessment of the NP-corona in the deep lung.Hence, we used a native pulmonary surfactant preparation, isolated from porcine lungs, for the development of a method to access the lipid-protein-corona. Centrifugation, magnetic separation and density centrifugation were compared with three magnetic model particles (Phosphatidylcholine-, PEG-coated, and plain PLGA). Magnetic separation of NP was found to be superior to the other commontechniques.SDS-PAGE showed the impact of hydrophobicity on the PS corona and was verified by advanced label-free proteomics.

References

[1] Ruge, C. A. et al., PLoS ONE, 7(7) (2012) e40775[2] Monopoli, M. P. et al., J. Am. Chem. Soc., 133(8) (2011) 2525–34[3] Tenzer, S. et al., Nat. Nanotechnol., 8(Oct) (2013) 772–781

Graphical Abstract

2D Microscale Engineering of Novel Protein based Nanoparticles for Cell Guidance

Witold I. Tatkiewicz,

1,2 Joaquin Seras-Franzoso,

2,3,4, Elena García-Fruitós,

2,3 Esther Vazquez,

2,3,4 Nora

Ventosa,1,2

Imma Ratera,1,2

Antonio Villaverde,2,3,4

and Jaume Veciana1,2

1Department of Molecular Nanoscience and Organic Materials, Institut de Ciencia de Materials de

Barcelona (CSIC), Bellaterra, 08193 Barcelona, Spain, 2CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193 Barcelona,

Spain, 3Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Bellaterra, 08193

Barcelona, Spain, 4Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193

Barcelona, Spain,

[email protected], [email protected], [email protected]

Abstract ies (IBs) was coined to describe optically opaque moieties present in cell

lumen. They have aspect of refractile particles of up to a few hundred nanometers and about 2 3 of

size when observed by optical microscopy and as electron-dense aggregates without defined organisation by transmission electron microscopy.[1]

The history of IBs turned when they were recognized as a prospective biomaterial with desirable properties. Being a product derived from biological synthesis, it is fully biocompatible and preserves the functionality of the embedded protein [2]. In a course of investigation it was revealed that IBs size, geometry, stiffness, wettability, z-potential, bio-adhesiveness, density/porosity etc. can be easily fine tuned by control over basic parameters of their production: harvesting time, host genetic background and production conditions (e.g. temperature, pH) In addition, their production and downstream processes are fully scalable, cost effective and methodologically simple.[3]

It is widely accepted, that cell´s responses, such as positioning, morphological changes, proliferation, mottility and apoptosis are the result of complex chemical, topographical and biological stimuli. Here we will show the application of IBs, as a functional biomaterial for engineering two dimensional substrates for cell guidance. We have cultivated fibroblast cells on supports patterned with IBs derived from green fluorescent protein (GFP) or human basic fibroblast growth factor (FGF). Two methodologies of pattern deposition were applied: microcontact printing ( CP) optimized for use with aqueous colloidal suspensions and a novel, template-free technique based on the coffee-drop effect due to a convective self-assembly (Figure 1).[4]

The first technique was applied in order to deposit IBs with high resolution geometrical patterns of various shapes and sizes. Then we have investigated how cells react to IBs geometrical distribution. Parameters such as orientation morphology and positioning were thoroughly investigated based on rich statistical data delivered by microscopy image treatment (Figure 2)The second technique has been recently developed in order to deposit complex and well-controlled two dimensional IB´s patterns with concentration gradients for the study of cell motility (Figure 3). Cell movement cultivated on such substrates was characterized and quantified based on confocal microscopy time-lapse acquisitions.[5,6]

In both cases a deep statistical data treatment was preformed to characterize macroscopic responses of cells when grown over nanoscale profiles made with IBs concluding that cell proliferation is not only dramatically stimulated but cell also preferentially adhere to IBs-rich areas, align, elongate and move according to such IBs geometrical cues.. These findings prove the potential of surface patterning with functional IBs as protein-based nanomaterials for tissue engineering and regenerative medicine among other promising biomedical applications.

References

[1] (a) Villaverde A., Carrio M.M., Biotechnol. Lett., 2003, 25, 1385 (b) E. García-Fruitós, E. Rodríguez-Carmona, C. Diez-Gil, R. Mª Ferraz, E. Vázquez, J. L. Corchero, M. Cano-Sarabia, I. Ratera, N. Ventosa, and J. Veciana, A. Villaverde Adv. Mater., 2009, 21, 4249 (c) Cano-Garrido, O.; Rodríguez-Carmona, E.; Díez-Gil, C.; Vázquez, E.; Elizondo, E.; Cubarsi, R.; Seras-Franzoso, J.; Corchero, J. L.; Rinas, U.; Ratera, I.; et al.. Acta Biomater. 2013, 9, 6134.

[2] García-Fruitós E., Vazquez E., Díez-Gil C., Corchero J.L.; Seras-Franzoso J., Ratera I., Veciana J., Villaverde A., Trends Biotechnol., 2012, 30, 65

[3] (a) García-Fruitos E., Seras-Franzoso J., Vazquez E., Villaverde A., Nanotechnology, 2010, 21, 205101 (b) Vazquez E., Corchero J. L., Burgueno J.F., Seras-Franzoso J., Kosoy A., Bosser R., Mendoza R., Martínez-Láinez J.M., Rinas U., Fernandez E., Ruiz-Avila L., García-Fruitós E., Villaverde A., Adv. Mater., 2012, 24, 1742 (c) E. Vazquez , M. Roldán , C. Diez-Gil , U. Unzueta, J. Domingo-Espín, J. Cedano, O. Conchillo, I. Ratera, J. Veciana , X. Daura, N. Ferrer-Miralles, A. Villaverde, Nanomedicine., 2010, 5, 2, 259

[4] (a) Han W., Lin Z., Angew. Chem. Int. Ed., 2012, 51, 1534 (b) Hanafusa T.,Mino Y., Watanabe S., Miyahara M.T., Advanced Powder Technology 2014, 25, 811

[5] (a) C. Díez-Gil, S. Krabbenborg, E. García-Fruitós, E. Vazquez, E. Rodríguez-Carmona, I. Ratera, N. Ventosa, J. Seras-Franzoso, O. Cano-Garrido, N. Ferrer-Miralles, A. Villaverde, J. Veciana, Biomaterials, 2010, 31, 5805 (b) J. Seras-Franzoso, C. Díez-Gil, E. Vazquez , E. García-Fruitós , R. Cubarsi, I. Ratera, J. Veciana, A. Villaverde, Nanomedicine, 2012, 7(1):79-93

[6] W. I. Tatkiewicz, J. Seras-Franzoso, E García-Fruitós, E. Vazquez, N. Ventosa, K. Peebo, I. Ratera, A. Villaverde, J.Veciana, ACSNano, 2013, 7(6), 4774

Figures

Figure 1. Schematic illustration of particle deposition. Particles are pinning to the substrate on the edge of meniscus, where the evaporation is more intense. Image adapted from reference [4b].

Figure 2. IBs striped (top) and random (bottom) pattern are compared. On the left; representative confocal microscopy images of cells cultivated on such patterns are presented. On the right; the overall orientation distribution of cells is presented. It is clearly seen, that cells are guided by the stripped pattern and they orient themselves along its geometry, whereas no predominant orientation of cells can be observed in the case of random pattern.

Figure 3. Example of GFP-derived IBs gradient pattern deposited by a controlled convective self-assembly technique. Left: fluorescence microscopy image, right: IBs concentration calculated based on fluorescence intensity.

Long-term exposures to low doses of cobalt nanoparticles induce cell-transformation enhanced by oxidative damage.

Laura Rubio: Balasubramanyam Annangi, Jordi Bach, Gerard Vales, Laura Rubio, Ricardo Marcos,

Alba Hernández.

Universitat Autònoma de Barcelona, Campus Bellaterra Facultat de Biociències, Departament de Genètica i Microbiologia, Barcelona, Spain

[email protected]

Abstract A great effort is being done by the scientists to increase our knowledge on the role of nanoparticles-

associated genotoxic and carcinogenic effects [1, 2]. Although important research activity took place in

this area for >10 years, most of the findings concerning to the genotoxic and cell transforming potential

of NPs are limited to short-term in vitro studies [3, 4]. Comprehensively, acute or short-term studies use

high environmentally irrelevant single doses to study the adverse effects of engineered NPs, which

could not be enough to draw plausible conclusions about the potential human health risk of NPs

exposure [5]. Until now, only a few in vitro long-term exposure studies have been carried out with NPs

[6,7,8,9]. Keeping this in mind, it seems necessary to increase the amount of in vitro investigations

focusing on long-term or chronic exposures at sub-toxic doses.

Despite the usefulness of cobalt nanoparticles (CoNPs) in various fields [10], they are also potentially

harmful to humans. Some studies have found that in vitro acute exposure of CoNPs induce oxidative

stress, DNA damage, morphological transformation and inflammatory responses in different cell types,

among other kind of effects [3, 11, 12, 13, 14,15]. However, there is no conclusive information available

on the in vitro carcinogenic potential of CoNPs under chronic settings so far. In vitro cell transformation

assays have been proposed as alternatives to long term animal studies. In fact, OECD has specific

[16] with accumulated

evidence that the cellular and molecular processes involved in vitro cell transformation are similar to

those of in vivo carcinogenesis [17].

In this study, we have evaluated the cell transforming ability of cobalt nanoparticles (CoNPs) after long-

term exposures (12 weeks) to sub-toxic doses (0.05 and 0.1 µg/mL). To get further information on

whether CoNPs-induced oxidative DNA damage is relevant for CoNPs carcinogenesis, the cell lines

selected for the study were the wild-type mouse embryonic fibroblast (MEF Ogg1+/+

) and its isogenic

Ogg1 knockout partner (MEF Ogg1 ), unable to properly eliminate the 8-OH-dG lesions from DNA. Our

initial short-term exposure experiments demonstrate that low doses of CoNPs are able to induce

reactive oxygen species (ROS) and that MEF Ogg1 cells are more sensitive to CoNPs-induced acute

toxicity and oxidative DNA damage. On the other hand, long-term exposures of MEF cells to sub-toxic

doses of CoNPs were able to induce cell transformation, as indicated by the observed morphological

cell changes, significant increases in the secretion of metalloproteinases (MMPs) and anchorage-

independent cell growth ability, all cancer-like phenotypic hallmarks. Interestingly, such changes were

significantly dependent on the cell line used, the Ogg1 cells being particularly sensitive. Altogether,

the data presented here confirms the potential carcinogenic risk of CoNPs and points out the relevance

of ROS and Ogg1 genetic background on CoNPs-associated effects.

References

[1] Arora S, Rajwade JM, Paknikar KM. Toxicol Appl Pharmacol. 258(2012):151 65.

[2] Becker H, Herzberg F, Schulte A, Kolossa-Gehring M. Int J Hyg Environ Health 214(2011):231 8.

[3] Colognato R, Bonelli A, Ponti J, Farina M, Bergamaschi E, Sabbioni E, et al. Mutagenesis 23(2008):377 82.

[4] Ponti J, Broggi F, Mariani V, De Marzi L, Colognato R, Marmorato P, et al. Nanotoxicology 7(2013):221 33.

[5] Hristozov DR, Gottardo S, Critto A, Marcomini A. Nanotoxicology 6(2012):880 98.

[6] Hackenberg S, Scherzed A, Technau A, Froelich K, Hagen R, Kleinsasser N. J Biomed Nanotechnol 9(2013):86 95.

[7] Huang S, Chueh PJ, Lin YW, Shih TS, Chuang SM. Toxicol Appl Pharmacol 241(2009):182 94.

[8] Jacobsen NR, Saber AT, White P, Møller P, Pojana G, Vogel U, et al. Environ Mol Mutagen 48(2007):451 61.

[9] Kocbek P, Teskac K, Kreft ME, Kristl J. Small 6(2010):1908 17.

[10] Horev-Azaria L, Kirkpatrick CJ, Korenstein R, Marche PN, Maimon O, Ponti J, et al. Toxicol Sci 122(2011):489 501.

[11] Magaye R, Zhao J, Bowman L, Ding M. Exp Ther Med 4(2012):551 61.

[12] Papageorgiou I, Brown C, Schins R, Singh S, Newson R, Davis S, et al. Biomaterials 28(2007): 2946 58.

[13] Papis E, Gornati R, Prati M, Ponti J, Sabbioni E, Bernardini G. 2007. Toxicol Lett 170(2007):18592.

[14] Petrarca C, Perrone A, Verna N, Verginelli F, Ponti J, Sabbioni E, et al. Int J Immunopathol Pharmacol 19(2006):11 14.

[15] Ponti J, Broggi F, Mariani V, De Marzi L, Colognato R, Marmorato P, et al. Nanotoxicology 7(2013):221 33.

[16] Vasseur P, Lasne C. Mutat Res 744(2012):8 11.

[17] Combes R, Balls M, Curren R, Fischbach M, Fusenig N, Kirkland D, et al. ATLA 27(1989):745 67.

Graphene oxide application in cell microencapsulation for bioartificial organ development

Laura Saenz del Burgo*, Jesús Ciriza*, Gorka Orive, Rosa María Hernández, Jose Luis Pedraz

NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain

The Biomedical Research Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)

* Both authors contributed equally to the development of the present work [email protected]

Abstract Cell microencapsulation represents a great promise for the development of new long-term drug delivery systems. However, several challenges need to be overcome before it can be translated extensively into the clinic. For instance, the long term cell survival inside the microcapsules. On this regard, graphene oxide has shown to promote the proliferation of different cell types both in two and three dimension cultures. Therefore, we planned to combine the use of graphene oxide together with the cell microencapsulation technology and analyze the biocompatibility of this chemical compound with cells within alginate-poly-L-lysine (APA) microcapsules. We have been able to produce 200 µm-diameter APA microcapsules with increasing concentrations of graphene oxide in their inside and prove that the physical chemical parameters of the traditional microcapsules were no modified. Moreover, microcapsules containing graphene oxide enhanced the viability of the encapsulated cells, providing another step for the future pre-clinical application of graphene oxide in combination with cell microencapsulation. References [1] Orive, G.; Santos, E.; Pedraz, J. L.; Hernandez, R. M., Adv Drug Deliv Rev, 67-68 (2013) 3-14. [2] Basta, G.; Montanucci, P.; Luca, G.; Boselli, C.; Noya, G.; Barbaro, B.; Qi, M.; Kinzer, K. P.; Oberholzer, J.; Calafiore, R., Diabetes Care, 34(11) (2011) 2406-9. [3] Goenka, S.; Sant, V.; Sant, S., J Control Release, 173 (2013) 75-88. [4] Lee, W. C.; Lim, C. H.; Shi, H.; Tang, L. A.; Wang, Y.; Lim, C. T.; Loh, K. P., ACS Nano, 5(9) (2011) 7334-41. [5] Li, N.; Zhang, Q.; Gao, S.; Song, Q.; Huang, R.; Wang, L.; Liu, L.; Dai, J.; Tang, M.; Cheng, G., Sci Rep, 3 (2013) 1604. [6] Ruiz, O. N.; Fernando, K. A.; Wang, B.; Brown, N. A.; Luo, P. G.; McNamara, N. D.; Vangsness, M.; Sun, Y. P.; Bunker, C. E., ACS Nano 5(11) (2011) 8100-7. Figures

Figure 1.- Microscopy images of bright field (A) and fluorescence after calcein ethidium staining (B) from microcapsules containing graphene oxide [1) without oxide graphene, 2) 10 µg/ml, 3) 25 µg/ml, 4) 50 µg/ml and 5) 100 µg/ml] and C2C12 myoblasts 4 days after encapsulation. Scale bar 100 µm.

Figure 2.- Viability of encapsulated C2C12 myoblasts in alginate microcapsules containing different concentrations of graphene oxide [0-100 µg/ml]. A) Metabolic activity measured in the cell counting kit 8 (CCK8) assay and B) Membrane integrity measured by the lactate dehydrogenase activity (LDH) assay, both expressed as the ratio between day 8 and 1 after microencapsulation.

Tramadol Hydrochloride Released from Lipid Nanoparticles:

Studies on Modelling Kinetics

Elena Sánchezª, Helen Alvaradoa,b*

, Prapaporn Boonmec, Guadalupe Abrego

a,b,

Tatiana Andreanid,e

, Monica Vazzanaf, Joana F. Fangueiro

b, Catarina Faggio

f, Carla

Silvag, Sajan José

h, Antonello Santini

i, María Luisa Garcia

a, Ana C. Calpena

b

Amélia M. Silvad,e

, Eliana B. Soutoj*

a Department of Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain

b Department of Biopharmacy and Pharmacology, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain

cFaculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand dDepartment of Biology and Environment, University of Trás-os Montes e Alto Douro, Portugal; eCentre for Research and Technology of Agro-Environmental and Biological Sciences, Portugal;

fDepartment of Biological and Environmental Sciences, University of Messina, Italy; gCenter for Nanotechnology and Smart Materials, Portugal;

hDepartment of Pharmaceutical Sciences, Mahatma Gandhi University, India; iDepartment of Pharmacy, University of Napoli, Italy;

jDepartment of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra

*E-mail address corresponding author: [email protected]; [email protected]

Keywords: tramadol hydrochloride, HPLC, lipid nanoparticles, kinetic model

A reverse-phase (RP) high performance liquid chromatography (HPLC) method was

developed according to the International Conference on Harmonisation (ICH) guidelines,

for the determination of tramadol hydrochloride (THC) from lipid nanoparticles (LNs).

THC is an opioid analgesic drug, mainly acting on the central nervous system (CNS) and

structurally related to codeine and morphine, but clinically 10-fold less potent than codeine

and 6000-fold less than morphine. It was developed in 1970s and is currently in use for the

management, treatment and relief of moderate to severe pain conditions. A method for the

preparation of THC standard and N1,N1-dimethylsulfanilamide (used as the internal

standard) has been described. HPLC analysis was performed on a 250x4 mm

chromatographic column with LiChrospher 60 RP-selectB 5-

acetonitrile:0.01 M phosphate buffer, pH 2.8 (3:7, v/v) as mobile phase. Fluorescence

detection was done at 296 nm (THC) and at 344 nm (N1,N1-dimethylsulfanilamide). THC-

loaded LNs dispersions were produced by hot high pressure homogenization technique,

using Compritol®

888ATO as solid lipid, stabilized with 3% (w/w) Phospholipon®

80H

and 1% (w/w) Tyloxapol®

as surfactants. Particles ranging between 79.4±0.3 and 144.6±14

nm in size were obtained, with a mean zeta potential of -10.2±1.2 mV. Four kinetic models

(i.e., zero order, Higuchi, Baker-Lonsdale and Korsmeyer Peppas) were selected to fit the

data to describe the THC release profile from LNs. The in vitro release profile of THC

from LNs was compared with that from the commercial oral suspension (Tramal®

), in pH

6.8 phosphate buffer. Commercial THC suspension depicted a 100% release in the first

hour; whereas for LNs, a biphasic sustained release profile was observed. According to the

obtained R2 values, Korsmeyer-Peppas model was reported as the best fit modelling kinetic

profile for THC release from LNs. The recorded n = 0.63 value typical for anomalous non-

Fickian transport is in agreement with the biphasic mechanism of drug release from LNs.

Polycationic Silicon Phthalocyanines as Photosensitizers for Photodynamic Therapy and Photodynamic Inactivation of Microorganisms

Eveline van de Winckel, Andrés de la Escosura, Tomás Torres

Departamento de Química Orgánica, Universidad Autónoma de Madrid, Cantoblanco,

28049 Madrid, España [email protected]

Abstract Photodynamic therapy (PDT) is a form of therapy that uses light-sensitive compounds, which upon selective exposure to light become toxic to targeted malignant and other diseased cells

[1]. Since its

incidental discovery in 1900, photodynamic therapy (PDT) and all related aspects, ranging from its mechanism of action, the different photosensitizers that can be employed and its clinical applications have been studied in great detail. In general, it is well-known that three components are required for PDT to occur; a photosensitizer, oxygen and a light source. In the presence of oxygen, irradiation of the photosensitizer of choice can lead to the generation of singlet oxygen, which is a powerful, indiscriminate oxidant that reacts with a variety of biological molecules. Singlet oxygen is indeed the main reactive oxygen species (ROS) in PDT, responsible for the destruction of tumor cells, bacteria, viruses, etc.

[2]. Following the absorption

of light, the photosensitizer is transformed from its ground singlet state (S0) into an electronically excited triplet state (T1) via a short-lived excited singlet state (S1). The excited triplet can undergo two kinds of reactions as shown by the Jablonski diagram depicted in Fig 1. Firstly, it can participate in an electron-transfer process with a biological substrate to form radicals and radical ions that, after interaction with oxygen, can produce oxygenated products (type I reaction). Alternatively, it can undergo a photochemical process known as a type II reaction, which results in the conversion of stable triplet oxygen (

3O2) into the short-lived but highly reactive singlet oxygen (

1O2) species, the

putative cytotoxic agent. Phthalocyanines are an important class of non-natural organic pigments that have received considerable attention in the field of PDT

[3].Our focus will be centered on the design of novel silicon

phthalocyanines with different substitution patterns in their axial positions (Fig 2) as new photosensitizers for their use in photodynamic therapy. For different and multipurpose reasons, it has been chosen to incorporate a series of polyamine ligands on one face of the phthalocyanine core, while on the other face a series of hydrophobic ligands are incorporated. In this way, the obtained photosensitizer molecules will be amphiphilic, which is a desirable characteristic to facilitate their possibility to cross cell membranes and improve their cell uptake. Another reason to incorporate various polyamine chains in one of the ligands is the fact that under physiological pH these polyamine chains will be protonated. This is useful to target Gram negative bacteria, which, in contrast to Gram positive bacteria that only possess an inner cell membrane and peptidoglycan layer, also possess an outer negatively charged cell membrane. For this reason, for the photosensitizer to be able to cross this membrane it is required to be positively charged. Furthermore, polyamines are naturally occurring compounds that play multifunctional roles in a number of cell processes including cell proliferation and differentiation. Rapidly dividing cells such as tumor cells require large amounts of polyamines to sustain the rapid cell division. Part of these materials can be biosynthesized internally, while the majority is imported from exogenous sources through active and specific polyamine transporters (PAT). These features have led to the use of polyamines as potent vectors for the selective delivery of chemotherapeutic and DNA-targeted drugs into cancer cells

[4].

In summary, we have designed and are currently preparing a library of ligands to incorporate in the silicon (IV) phthalocyanines, making use of different substitution patterns in their axial positions. An overview of the series of ligands to incorporate can be seen in Fig 2. The resulting amphiphilic phthalocyanines are expected to be non-aggregated in aqueous media, because of their axial substituents, and are expected to have an enhanced photoinduced singlet oxygen generation in the pH range from 5 to 7.

References [1] T. Hasan et al. Photochem. Photobiol. Sci., 3 (2004) 436 450 [2] A. Almeida et al., Bioorg. & Med. Chem., 21 (2013) 4311-4318 [3] a) J.R. Castón et al., Chem. Sci., 5 (2014) 575-581 b) J.L.M. Cornelissen et al., J. Am. Chem. Soc., 133(18) 2011 6878-6881 [4] a) D.K.P. Ng et al., J. Med. Chem., 54 (2011) 320-330 , b) D.K.P. Ng et al., Chem. Comm., 49 (2013) 4274-4276 Figures

Fig 1. Jablonsky diagram that illustrates the photophysical and photochemical processes occurring when a photosensitizer (PS) is irradiated for PDT purposes.

Fig 2. General structure of novel silicon phthalocyanine photosensitizers with different substitution patterns in their axial positions, and a representation of several of the ligands to be incorporated (top: series of polycationic ligands, bottom: series of

hydrophobic ligands)

Calle Alfonso Gomez 17

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