Nanotube Functionalization and Therapeutic Applications

Nanotube Functionalization and Therapeutic Applications

by Alberto Bianco

Immunologie et Chimie Thérapeutiques, CNRS, Strasbourg, France

NanoSOFT (Roscoff), 21-25 May 2007

Immunologie et Chimie Thérapeutiques

Carbone amorphe Graphite Diamant

Fullerène Nanotube de carbone

The Allotropic Forms of Carbon

Zigzag direction

Armchair direction

STRIP OF A GRAPHENE SHEET ROLLED INTO A TUBE

Carbon Nanotube Folding

Movies from: http://www.photon.t.u-tokyo.ac.jp/~maruyama/nanotube.html

Types of Carbon Nanotubes

Single-walled carbon nanotube (SWNT) presents only one graphene layer

Multi-walled carbon nanotube (MWNT) presents several graphitic concentric layers

MWNTSWNT

10 µmHUMAN HAIR

MWNT

0.1 µm

250 nm200 nm

Ø = 1.4 - 100 nmL = 1 - several µm

Ø = 0.4 - 2 nmL = 20 - 1000 nm

BundlesØ = 10 - 30 nmL = 1 - 50 µm

Organic Functionalization of Carbon Nanotubes

CO2H

HNO3/H2SO4

HO2CCO2H

HO2C

CO2H

CO2H

Functionalization using the oxidation/cut method

CO2H

HO2CCO2H

HO2C

CO2H

CO2H

CO2-R

R-O2CCO2-R

R-O2C

CO2-R

CO2-RR-X

Organic Functionalization of Carbon Nanotubes

Functionalization using the oxidation/cut method

Functionalization using the addition/cycloaddition approach

R-X

R R

R

R

RR

101 103 104102 10510-1 107 108106

Nanometers

Water

Glucose

Antibody

Virus

Bacteria

Tennis ball

O

H

HO

H

HO

H

OH

OHHH

HO

Nanoscale devices are of the same scale of biologically important molecules

The Right Size of Nano-objects

Cancer cell

Nanodevices:• Nanoparticles• Dendrimers• Quantum Dots• Carbon nanotubes

- Carbon nanotubes as substrates for neuronal and cell growth

- Carbon nanotubes for tissue engineering and implants

- Carbon nanotubes as biosensors for sugars, antigens, proteins, antibodies

- Carbon nanotubes as substrates for engineered cell membrane surfaces

- Carbon nanotube cell uptake

- Carbon nanotubes for the delivery of therapeutic molecules

Biomedical Applications of Carbon Nanotubes

Since carbon nanotubes are metallic or semi-conductors, can they help to connect neurons which do not communicate because damaged?

Courtesy of M. Prato and L. Ballerini

Neuronal network Carbon nanotube network

Substrates for Neuronal and Cell Growth


Lovat V et al. Nano Lett. 2005, 5, 1107

MWNT remain adherent to the glass surface in the condition of cell culture

Neonatal hyppocampal neurons grow on the dispersed MWNT, with dendrites and axons extended across the tubes

Neurites travel in close contact with the nanotubes

Spontaneous postsynaptic current (PSC) shows the formation of functional synapses and it is indicative of neuronal network efficiency: neurons grown on the CNT display a 6-fold increase on the frequency of spontaneous PSC

Growing neuronal circuits on CNT grids promote an increase in the network operation

NCH3

CH2(CH2)4CH3

350 °CCH3(CH2)5CHO

CH3NHCH2COOH130 °C, DMF


Electric events are not related to an increase of surviving neurons in the presence of CNT

Cell morphology (A-D, G, H) does not present differences when the cell are cultured on glass or on CNT

Control

CNT

Density of neurons (E) and the number of neurite/cell (F) are equal when the cells are cultured on glass or on CNT

Carbon Nanotubes for Tissue Engineering

MacDonald R et al. J. Biomed. Mater. Res. 74A 2005, 489

Collagen CNT

CNT incorporated into collagen fibrils

Mechanical properties of carbon nanotubes

- Composite materials

- Reinforced fibers

- Collagen matrix with embedded CNT

Why collagen (Type I)?

- Important physical and biochemical functions

- Promise as matrix for regenerative medicine

Applications as scaffold for

- Tissue engineering

- Biosensor components

- Useful medical devices (to provide electrical signal for cardiac muscle)

Gelation and formation of a cell-seeded tissue construct

Analysis of the morphology of the cells embedded within collagen-CNT gels after 3 days : no cell morphology alteration and cell viability was above 85% along 7 days

(Red: nucleus; green actin cytoskeleton)

SEM analysis of collagen-CNT matrices (0.4 wt% SWNT) : presence of fine fibers (arrowed) distinct from the typical banded fibers of collagen. CNT strongly interact with collagen forming intersections and blends.

Carbon Nanotubes for Tissue Engineering

Carbon nanotubes as biosensors for sugars, antigens, proteins, antibodies

Electronic Sensors for the Detection of Antibodies

Chen RJ et al. PNAS 2003, 100, 4984

Carbon nanotubes as a platform for:

- Studying of surface/protein or protein/protein binding

- Electrical sensing of specific biomolecules

- Detecting clinically important species (i.e. antibodies)

Non-specific binding (NSB) of proteins

Proteins simply adsorb onto carbon nanotubes via hydrophobic interactions

Verification via AFM

Demonstration of irreversibility using quartz crystal microbalance (QCM) (a decrease of resonance frequency indicates mass uptake by CNT surface; level of detection down to 10 nM)

Similarly, conductance changes (FET) allow to detect protein absorption with a level of detection down to 100 pM

Approach to avoid non-specific binding (NSB) of proteins

Proteins do NOT adsorb onto carbon nanotubes coated with polyethylene (PEO) chains

Verification via AFM

Carbon nanotubes coated with PEO chains form stable water suspensions

No change detected with QCM

No change on conductance

Electronic Sensors for the Detection of Antibodies

Electronic Sensors for the Detection of AntibodiesSpecific bindingbetween biotin-PEOcoated CNT and SA (SA: streptavidin)

QCM detection Electricdetection

Specific binding betweenU1A-PEO coated CNT and10E3 (antigen/antibody)

QCM detection Electricdetection

Observation of Carbon Nanotubes in a Biological Environment

Cherukuri P et al. JACS 2004, 126, 15638

The detection of pristine CNT in biological systems exploits the near-infrared (NIR) intrinsic fluorescence of the tubes and the absence of endogenous fluorescence from the tissues in this region

Viability and proliferation of mouse macrophages are not affected under incubation with CNT (normal adhesion and growth, morphology and confluence)

Detection of CNT fluorescence in the NIR region after cellular uptake

The tubes into the cells present a significant broadening and red-shifting of the spectral peaks in comparison to CNT suspended in water using a surfactant (Pluronic F108)

Displacement of the surfactant coating the CNT by proteins

Mechanism of internalization depends on the temperature and consists on the typical macrophage phagocytosis (active ingestion of 1 nanotube of ~1 µm per second per cell)

After the incubation with the cell NIR light emission is localized into intracellular regions probably corresponding to small phagosomes

CNT can be used as fluorophores for the selective detection, imaging and biodistribution studies

CNT are photostable like Qdots (absence of photobleaching) and less cytotoxic

Carbon nanotubes as substrates for engineered cell membrane surfaces

Biomimetic Cell Surface Engineering

Chen X et al. Angew. Chem. Int. Ed. 2004, 43, 6112

Biomimetic approach can be used to bridge nanomaterials and biological systems

Surface modification of carbon nanotubes using glycosylated polymers designed to mimic natural cell surface glycans (mucins)

Integration of carbon nanotubes into physiological conditions

Improvement of biocompatibilty

Receptor-mediated cell-cell recognition studies


Chen X et al. JACS 2006, 128, 6292

Mucins, which are present at the cell surface, serve a dual role:

1) Molecular recognition

2) Resistance to biofouling (biofouling is the undesirable accumulation of microorganisms, plants and animals on artificial surfaces)

Do CNT coated with α-GalNAc interact with the receptor at the cell surface?

HPA is a hexavalent lectin capable of cross-linking cells and glycoproteins

Strategy to probe for biological process

Monitoring the variation in a cell’s local environment by following the changes in the electrical, mechanical and optical properties of CNT


Interaction between CNT coated with α-GalNAc and the cell receptors

Specificity of the interaction between CNT coated with mucin mimetics and the cell receptors

Cytotoxic effect of the complexes on the cell growth

Biomimetic Bacterial Surface Engineering

Gu L et al. Chem Commun 2005, 874

O

OH

H

H

HO

H

HOHH

O

OH

NH

O= = D-galactose-binding protein

Gal-SWNT E. coliE. coli

Functionalization of CNT with galactose and interaction with galactose-binding protein at the bacterial surface

Capture of pathogenic E. coli cells by sugar-modified carbon nanotubes

Interaction of Pathogens with Antibody-CarbonNanotube Conjugates

Elkin T. et al. ChemBioChem 2005, 6, 640

The development of immuno-nanotubes is based on the conjugation of oxidized CNT to bovine serum albumin (BSA) and direct adsorption of an specific antibody (anti E. coli Ab)

Capture of pathogenic E. coli cells by antibody-modified carbon nanotubes Green E. coli Red Ab-CNT Yellow

overlapping

Immuno-CNT-E. Coli interactions are specific and through the antibody bound to the nanotubesand the antigens on the cell surface

Potential development of CNT devices for rapid and ultrasensitive detection of pathogens

Control

Carbon nanotube cell uptake

Do Carbon Nanotubes Penetrate into the Cells?

OO

NH

N

OO

NH

N

OO

NH

N

Functionalized carbon nanotubes penetrate into the cells following two mechanisms:

i) via an energy-independent mechanism (passive insertion and diffusion through lipid bilayerof the cell membrane – nanoneedle penetration)(Pantarotto D et al. Chem. Commun. 2004, 16; Angew Chem. Int Ed. 2004, 43, 5242; Cai D et al. Nat. Meth. 2005, 2, 449)

ii) via an energy-dependent mechanism (endocytosis, phagocytosis)

(Kam NWS et al. JACS 2004, 126, 6850; JACS 2005, 127, 6021; Cherukuri P et al. JACS 2004, 126, 15638)

OO

HN

N

NO

O

O

S

H-Lys(FITC)-(αs384-394)-Cys-OH

Cell Uptake of Carbon Nanotubes via Energy-independent Mechanism

OO

N

HN S

HN

CO2H

O

O

OH

Pantarotto D et al. Chem. Commun. 2004, 16

Fibroblast cytoplasm localization Fibroblast nulcear localization

Capacity of Functionalized Carbon Nanotubes to Penetrate Different Cell Types

MOD-K

E. coli C. neo-formansS. cere-visiae

OOHN

O

NH

HN

HO2C

OO OH

S

NO O NH3

+Cl-

N

O

OHO

O

OH OH

OH

OH OH

OHOH

O

OHO

H2NOH

O

OO

OONH

O

NH

HN

HO2C

OO OH

S

HN

N

O

O

HN NH

CO2H

O OHO

S

NO O NH3

+Cl-

NO O

HN

S

HN

HO2C

O

O

OH

Jurkat

MacrophagesLymphocytes BLymphocytes T

Kostarelos K et al. Nature Nanotech. 2007, 2, 108

Cells Incubated at 4°C Cells treated with DNPCells treated with NaN3

Functionalized carbon nanotubes penetrate into the cells at 4°C

Functionalized carbon nanotubes penetrate into the cells in the presence of the inhibitors of endocytosis NaN3 and DNP (2,4-dinitrophenol)

Cell Uptake of Carbon Nanotubes via Energy-independent Mechanism

Pantarotto D et al. Angew. Chem. Int. Ed. 2004, 43, 5242

Direct Visualization of Carbon Nanotubes into the Cells

OO

NH3+Cl-

N

Nanoneedle Cell Penetration of Carbon Nanotubes

CELL MEDIUM

CYTOPLASM

OO

NH3+Cl-

Nchromatin

internalised MWNT

nuclear membrane

Golgi’s complex

Functionalized carbon nanotubes penetrate like nanoneedles piercing the cell membrane without inducing cell death

Cell

Cell

CNT

F

B - Effect of a static magnetic field

Carbon Nanotubes Spearing

Cai D et al. Nature Methods 2005, 2, 449

A - Effect of a rotating magnetic field

Cell

Cells on substate

CNT

CNT

A rotating magnetic field drives the carbon nanotubes (containing Ni particles) to spear the cells

A static field pulls the carbon nanotubesinto the cells

The spearing action induces the nanopenetration of the cell membrane 1 µm

500 nmControl

Kam NWS et al. JACS 2004, 126, 6850

COOH

NHHN

O OHO

S

ONH

NHHN

O

O

Alexa Fluor Streptavidin

NH

O

NH

O

NH

O

OH

O

Carbon nanotubes (100-1000 nm) conjugated to FITC (a) or fluorescent streptavidin (60 kD) via biotin (b)

The protein-CNT non covalent complexes are uptaken by the cells and they are localized into the endosomes (c)

The protein-CNT do not penetrate at 4 °C (d)

The free protein does not penetrate

Penetration is time dependent (after 4 h the fluorescence reaches the maximum) and dose dependent

The penetration is independent of the cell type and it is via endocytosis leading to the accumulation into the cytoplasm

Cell Uptake of Carbon Nanotubes via Energy-dependent Mechanism



Proteins spontaneously adsorb onto oxidized carbon nanotubes via non specific binding

Non covalent protein-CNT conjugates are transported via endocytosis into different cell lines

After release from the endosomes, the proteins express their biological functions as demonstrated for example by induction of apoptosis (programmed cell death) using cytochrome-c (Cyt-c)

Complexes between CNT and different proteins

CNT BSA

spA Cyt-c


CNT affect the transportation of protein cargos into the cells

Proteins are internalized by CNT because they do not intrinsically penetrate into the cells

Protein-CNT are localized into the cytoplasmatic endosomes

No fluorescence is detected into the nucleus

Endocytosis is inhibited at 4°C, where little uptake is observed in comparison to 37°C

Intracellular internalization of protein-CNT

Proteins do not penetrate into the cells

4°C37°C

Carbon nanotubes for the delivery of therapeutic molecules

Functionalized Carbon Nanotubes as New Vectorsfor the Delivery of Therapeutic Molecules

• Functionalization of carbon nanotubes with nucleic acids and proteins• Application on bio-macromolecule delivery

• Functionalization of carbon nanotubes with bioactive peptides• Application on vaccine delivery

• Functionalization of carbon nanotubes with small bioactive molecules• Application on drug delivery

• Formation of supramolecular complexes based on charge interactions• Application on gene delivery

Carbon Nanotube Transporters: DNA

Kam NWS et al. PNAS 2005, 102, 11600

Development of carbon nanotubes for:

- DNA delivery

- Cancer therapy

The biological system are transparent in the near-infrared (NIR) region (700-1100 nm) while carbon nanotubes have a strong optical absorbance in the same spectral region

When DNA functionalized carbon nanotubes penetrate into the cells, NIR laser pulses trigger endosomal rupture and subsequent translocation of DNA into the nucleus

When carbon nanotubes are functionalized with molecules targeting cancer cells a continuous NIR irradiation causes the death of those cells that internalized the nanotubes

Carbon nanotubes (~150 nm) functionalized with a 15-mer ssDNA labeled with Cy3 green dye

DNA-CNT are internalized by the cells and accumulate into the cytoplasm

The penetration mechanism is energy-dependent because at at 4°C the cellular uptake is minimum

Carbon Nanotube Transporters: DNA

Irradiation by applying repeated, short (few secondes) laser NIR pulses provokes the release of DNA from the endosomes and DNA nuclear translocation (yellow color indicates co-localization into the nucleus)

Increase of fluorescence is an indicative of DNA unwrapping and release due to endosomal break

Continuous NIR pulses (808 nm) increase the temperature of the solution eventually triggering cell death

Carbon Nanotube Transporters: Proteins


Cytochrome-c (Cyt-c) is a 12 kD protein which induces or activates apoptosis (programmed cell death) when injected into the cells

Cyt-c-CNT complexes are uptaken via endocytosis

The treatment of the cells with chloroquine induces the rupture of the endosomes and the release of the protein into the cytoplasm

Cell apoptosis occurs after Cyt-c migration into the cytoplasm

Carbon nanotubes are able to deliver active proteins which eventually display their biological function

Punctuated fluorescence indicates vesicle localization

Diffused and uniform fluorescence indicates vesicle release by action of chloroquine

Carbon Nanotube for Anticancer Therapy

Irradiation by applying repeated, short laser NIR pulses changes the morphology of the cell that have uptake the CNT-DNA complexes

Cells not incubated with the CNT-DNA complexes are undamaged by NIR pulses

Prolonged irradiation (2 minutes) induces extensive death of cells with CNT-DNA complexes

Cell death is accompanied by CNT aggregation, even visible to the naked eye

Kam NWS et al. PNAS 2005, 102, 11600

Carbon Nanotube for Anticancer Therapy

Cell death provoked by NIR irradiation is exploited for cancer therapy

Carbon nanotubes are functionalized with specific ligands that recognize and target cancer cells

Folate receptors (FR) are markers of cancer cells

Irradiation of overexpressing FR cells = cell dead

Irradiation of normal cells = cell alive

Cancer cells internalize the fluorescente tubes functionalized with folic acid

Normal cells shows minimal internalization because of PEG effect (inertness or blocking of non specific binding)

Liu Z et al. Nature Nanotechology 2007, 2, 47

Carbon nanotubes are functionalized with specific ligands which are recognized by tumor cells

Carbon Nanotubes for Anticancer Therapy

Delivery of peptide-based synthetic vaccines

0.45 nm1.34 nm

1.45 nm

1.36

nm

Molecular model of a helical peptide-CNT conjugate

Carbon Nanotubes as Suitable Scaffold for the Presentation of Antigens to the Immune System

Development of vaccines based on synthetic peptides

- Peptide antigens are poorly immunogenic

- Conjugation to protein carriers (BSA) are necessary to improve the Abs production

- The carrier presents the peptide to the immune system in the correct conformation

- Protein carrier are intrinsically immunogenic

- Generated Abs present low specificity

- Inert carriers system are ideal

Peptide Antigens Conjugated to Carbon Nanotubes for Synthetic Vaccine Delivery

O O

HN

N

7

N

O

O

O

SN

1

NH2

O

O

4) Ac-Cys-GSGVRGDFGSLAPRVARQL

1) Boc-Lys(Boc)-OH, DIC/HOBt in DMF, 3h

2) TFA, 2h

(Ac-Cys-FMDV) in H2O, 9h

Ac-Cys-FMDV

N O N

OO

O

O

O

3) in DMF, 3h

ONH

HN N

O

O

OAc-Cys-FMDV

S

O O

HN

N

6

NO

O

O

ON

O

O

N

O

O

O

S

2) Ac-Cys-GSGVRGDFGSLAPRVARQL (5)

1) (4) in DMF, 3h

(Ac-Cys-FMDV) in H2O, 6h

Ac-Cys-FMDV

Foot-and-Mouth Disease Virus (FMDV)Pantarotto et al. J. Am. Chem. Soc. 2003, 125, 6160 Pantarotto et al. Chem. Biol. 2003, 2003, 10, 961

Immunological Characterization of Peptide-Carbon Nanotubes

Antigenicity is the capacity of a peptide antigen to be specifically recognized by an antibody

The peptide antigen should adopt the correct conformation when bound to the carrier system

The conformation should be the same as in the case of the native structure present in the protein, which contains the peptide epitope

Immunogenicity is the capacity of a peptide antigen to elicit an immune response

Molecular model of the complex between a helical peptide-CNT conjugate and an antibody fragment (Fab: Fragment antigen binding)

Antigenic Characterization of Peptide-Carbon Nanotubes

Carbon nanotubes are a good multi-presentation system since they are able to present the peptide antigen with the correct conformation for the antibody recognition

Carbon nanotubes do not perturb the secondary structure of the peptide

Surface plasmon resonance (SPR) analysis

0

40

80

0 200 400 600Time (s)

RU

120

Bis FMDV-NT 7

Mono FMDV-NT 6

FMDV-peptide

Ac-NT

Immunogenic Characterization of Peptide-Carbon Nanotube Conjugates

FMDV(141-159)-BSA

Control-peptide-BSA

CNT

Coating antigens

- High antibody (Ab) responses

- Bis-conjugate generates higher levels of Abs than the mono-conjugate

- Carbon nanotubes devoid of the peptide are non immunogenic (no antibody production)

0

1

2

3

4

5

6

Freepeptide

Monoconjugate 6

Bisconjugate 7

Log1

0 an

tibod

ytit

er

FMDV(141-159)-BSA Control-peptide-BSA CNT

Delivery of nucleic acids

Delivery of Nucleic Acids by Carbon Nanotubes

Single-stranded DNA sequences are able to wrap around carbon nanotubes

- DNA assisted dispersion and and separation of carbon nanotubes

- Sequence dependent DNA carbon nanotube sorting

Zheng et al. Nat. Mater. 2003, 2, 338

Carbon nanotubes for the delivery of DNA, plasmid DNA, RNA

- Gene transfer applications

- Genetic vaccination

Complexes Based on Positive-negative Charge Interactions

1 base = 1 negative charge Positive charged carbon nanotube

⊕

⊕

⊕ ⊕

⊕

ΘΘΘ

ΘΘ

ΘΘ

Θ ΘΘ

ΘΘ

Θ

ΘΘΘΘ

ΘΘ

Θ Θ

ΘΘ

ΘΘ

++

Cationic macromolecules, such as peptides, dendrimers and liposomes in general achieve effective delivery of DNA leading to pronounced toxic effects at cellular level

Pantarotto et al. Angew. Chem. Int. Ed. 2004, 43, 5242Singh et al. J. Am. Chem. Soc. 2005, 127, 4388

N

NH3+ Cl-

O

O

10 nm 50 nm

III

Potential of Carbon Nanotubes on Gene Delivery

Plasmid DNA condenses on the surface of the cationic carbon nanotubes forming supercoiled and globular-like structures

Gel Electrophoresis of f-CNT:DNA Complexes

- A shift in the gel is indicative of the formation of the complex and of its stability

- A strong decrease in the fluorescence intensity and an increase in the upward shift of the free DNA bands is correlated to an increase of the +/- charge ratio

- Cationic single-walled CNT are not able to fully condense DNA

- Cationic multi-walled CNT are most efficient in condensing DNA

A. SWNT-NH3+

1 2 3 4

OCSC

B. SWNT-Lys-NH3+

1 2 3 4

OCSC

C. MWNT-NH3+

1 2 3 4

OCSC

1:1 2:1 10:1Control 1:1 2:1 10:1Control 1:1 2:1 10:1Control+/-

Delivery and Expression of Plasmid DNA by f-CNT

0.0E+00

2.0E+04

4.0E+04

6.0E+04

8.0E+04

1.0E+05

1.2E+05

Naïve DNAonly

SWNT:DNA1:1

SWNT:DNA2:1

SWNT:DNA4:1

SWNT:DNA6:1

SWNT:DNA8:1

SWNT:DNA10:1

B-g

alEx

pres

sion

(RLU

/wel

l)

- The charge ratio between NH3+ at the SWNT surface and the phosphates of the DNA backbone is a

determinant factor of the resulting levels of gene expression

- SWNT:DNA +/- charge ratios between 2:1 and 6:1 offer 5 to 10 times higher levels of gene expression compared to DNA alone

- This first generation of functionalized carbon nanotubes is less effective for transfection in vitrothan lipid:DNA system

Gao L. et al. ChemBioChem 2006, 7, 239

Characterization and Expression of Plasmid DNA by f-CNT MWNTox-NH3

+ MWNTox-NH3+-DNA

Formation of the complex detected by gel electrophoresis shift

Cationic carbon nanotubes display a reduced cellular toxicity in comparison to other transfection systems

Cationic carbon nanotubes are able to transfect the cells although less efficiently than lipofectamine

The level of transfection is dependent on the +/-charge ratio

Liu Y. et al. Angew. Chem. Int. Ed. 2005, 44, 4782

Delivery of Plasmid DNA Using Carbon Nanotubes

Alternative approach for the solublization and the preparation of cationic carbon nanotubes: conjugation of CNT to polyethylene imine (PEI)

A

B

Delivery of Plasmid DNA Using Carbon Nanotubes

PEI functionalized carbon nanotubes are as effective for transfection in vitro as PEI alone

PEI functionalized carbon nanotubes are uptaken via endocytosis and display a reduced cellular toxicity in comparison to PEI

Delivery of therapeutic agents to cells

Zhu Y et al. JACS 2005, 127, 9875

Carbon Nanotubes for Boron Neutron Capture TherapyAn ideal therapy for cancer would be one whereby all tumor cells are selectively destroyed without damaging normal tissues

Most of the cancer cells should be destroyed, either by the treatment itself or with the help from the body's immune system, otherwise the danger exists that the tumor may reestablish itself

The promise of a new experimental cancer therapy with some indication of its potential efficacy has led to work on an approach called boron neutron capture therapy (BNCT)

BNCT is a binary radiation therapy modality that brings together two components that when kept separate have only minor effects on cells. The first component is a stable isotope of boron (boron-10) that can be concentrated in tumor cells by attaching it to tumor seeking compounds. The second is a beam of low-energy neutrons. Boron-10 in or adjacent to the tumor cells disintegrates after capturing a neutron and the high energy heavy charged particles produced destroy only the cells in close proximity to it, primarily cancer cells, leaving adjacent normal cells largely unaffected

Development of water soluble carborane-CNT conjugates able to target cancer cellsHNH

Me

HNH

Me

EtO

EtO

Na+

Na+

N

N

Me

Me

Me

N3NaOH

EtOH

CNT

o-DCB

Mammalian carcinoma cells (EMT6) are transplanted into the right flank of mice

Maximum boron concentration is achieved on the tumor cells after 30 h in comparison to blood and tissues (lung, liver, spleen)

The amount is slightly lower than the desired level for effective BNCT [30 µm(boron)/g(tissue)]

The low concentration on the different organs supports the preferential uptake by tumor cells

The efficient accumulation is guaranteed by the nanotube delivery capacity since the free carboranederivatives generally show no adsorption or retention on tumor cells

The mechanism of targeting of carborane-CNT is unknown

A passive accumulation of macromolecular drugs in tumor cells is favored by an increased vascular permeability and a decrease in lymphatic drainage system in cancer cells

Saline solution DMSO

Carbon Nanotubes for Boron Neutron Capture Therapy

Carbon Nanotubes for Drug Delivery

The conjugation of a drug to carbon nanotubes might have several advantages:

i) Increase of the solubility of the molecule

ii) Decrease of the aggregation phenomena

iii) Improvement of the efficacy owing to the internalization capacity of CNT

iv) Modulation of the drug activity against different types of cells (mammalian, bacterial, fungal)

v) Reduction of the amount of drug administered

Wu W et al. Angew. Chem. Int. Ed. 2005, 44, 6258

Delivery of Antibiotics by Carbon Nanotubes

Amphotericin B (AmB) is a potent antifungal agent for the treatment of chronic fungal infections

AmB is highly toxic for mammalian cells (likely because of the formation of aggregates which reduce the solubility in water)

New approach to carbon nanotube functionalization: preparation of nanotubes carrying one or more therapeutic agent with:

i) Recognition capacity

ii) Optical signal for imaging

iii) Specific targeting

OONH

O

NH

HN

HO2C

OO OH

S

N

O

O OH

O

OHOH

OH

OHOH

OHHO

O

O OH

NH2OH

O

O O

HN

FITC

AmB

0

10

20

30

40

50

60

MWNT

4 (1 µ

g/ml)

% A

popt

otic

and/

or d

ead

cells

MWNT

4 (2 µ

g/ml)

MWNT

4 (5 µ

g/ml)

MWNT

4 (10 µ

g/ml)

MWNT

4 (20 µ

g/ml)

Contro

l

MWNT

4 (40 µ

g/ml)

AmB(1

0 µg/m

l)

N

O

O OH

O

OHOH

OH

OHOH

OHHO

O

O OH

NH2OH

O

O O

OONH

O

NH

HN

HO2C

OO OH

S

HN

The conjugation of AmB to carbon nanotubes clearly reduces the toxic affect of the antifungal agent on mammalian cells (Jurkat cells)

Effect of AmB Conjugated to Carbon Nanotubes

Antifungal Activity of AmB Conjugated to Carbon Nanotubes

*The MIC corresponds to the lowest concentration of compound that inhibited visible growth of the organism. Results given are mean values of two independent determinations performed in duplicate. C.i.: clinical isolate. In this table, the MIC values for MWNT-AmB and SWNT-AmB refer to the concentration of AmB in the conjugates (approximately one third by weight).

Minimum inhibitory concentration (MIC)* in µg/ml

C. parapsilosis ATCC90118

C. famata (c.i.) C. albicans (c.i.)

C. neoformans ATCC90112

S. cerevisiae (c.i)

AmB 20 20 > 80 5 2.5 SWNT-NH3

+ > 80 > 80 > 80 > 80 > 80 MWNT-AmB 1.6 0.8 6.4 0.8 0.8 SWNT-AmB 1.6 1.6 13.8 0.8 1.6

AmB-CNT preserve a high antifungal activity

When equal amount of free and conjugated AmB are administered the CNT conjugates are more potent against certain strains

The reduced mammalian cell cytotoxicity and the increased antimycotic activity can be explained with:

i) Rapid internalization of AmB into the cytoplasm by the CNT reduces the possibility of disruption of the cell membrane

ii) Prevention of aggregation

iii) Increased solubility of the drug

iv) Binding to CNT and the presence of multiple copies of AMB per CNT favor the interaction of the drug with the fungal membrane

An appropriate conjugation increases the effectiveness of a drug (i.e. AmB) decreasing its cytotoxicity

Health and Environment Risks of Nanomaterials

Report: 253 Pages

CARBON NANOTUBES: 157 times

Report: 64 Pages

CARBON NANOTUBES: 17 times

Reports on the Health and Environment Impact of Nanomaterials

Advantages of Functionalized Carbon Nanotubes

• Control of the functionalization

• Lack of immunogenicity

• Reduced toxicity

• No apparent tissue/organ accumulation

Factors to be Carefully Addressed

• Quality of the starting material (carbon nanotubes)

• Control of the preparations

• Long-term toxicity

Conclusions and Perspectives

Documents

Nanotube Functionalization and Therapeutic Applications