AMBITI DEI PROGETTI DI TESIAMBITI DEI PROGETTI DI TESIAMBITI DEI PROGETTI DI TESIAMBITI DEI PROGETTI DI TESI:

Nanobiotecnologie, Nanomedicina,

Nanotossicologia, proteine ricombinanti, farmaci

biotecnologici

SVOLGIMENTOSVOLGIMENTOSVOLGIMENTOSVOLGIMENTO: CRIBI, DSB

RELATORIRELATORIRELATORIRELATORI: Prof. Emanuele Papini, dr. Regina Tavano,

Prof. Alessandro Negro

DSBDipartimento Scienze Biomediche

Supervisors: Prof. E. Supervisors: Prof. E. Supervisors: Prof. E. Supervisors: Prof. E. Papini; Prof. F. Papini; Prof. F. Papini; Prof. F. Papini; Prof. F. Mancin Mancin Mancin Mancin

PROGETTO 1PROGETTO 1PROGETTO 1PROGETTO 1....

INDIVIDUAZIONE E STUDIO DI

RIVESTIMENTI MOLECOLARI INNOVATIVI

DI NANOSISTEMI “STEALTH” PER

MIGLIORARE IL LORO USO E RENDERLI

“INVISIBILI” ALLE DIFESE IMMUNITARIE

UMANE

Macrofago

NH3,

Brij 35

Surface Passivation w/ Organic Polymers

PEG

no coating

Polyoxazolines

020406080

1 001 201 401 601 80

Alp

ha-2

-M

SA

A1

Tra

nsth

yret

in

Apo

-AII

C3

[3]

Apo

-AI

Pro

per

din

[1]

Tra

nsth

yret

inV

NIg

G1

(H)

Apo

-D

IgM

(H

)H

emog

lobi

n β

Pro

thro

mbi

nA

po-C

IIA

po-A

II

Apo

-CIIIA

po-C

IA

po-E

Apo

-AI

Apo

-AIV

Clu

s

SA

A4

02 04 06 08 0

10 012 014 016 018 0

����

����

���

� P O Z -N P s

F

D

f irs t 2 0 p o lyp e p tid e s (o rd e r o f a b u n d a n cy)

Pro

thro

mbi

n

Apo

-EIg

G1

(H)

Apo

-AIV

Tra

nsth

yret

in

Apo

-AI

HS

AP

rop

erd

in [

21]

C3

[25]

IgM

(H)A

po-A

IIA

po-C

IIIC

lus

Apo

-CII

A p o -C I

mol

ecul

e nu

mbe

r/N

Ps

CF

AH

IgM

(J)

SA

A1

CD

5 an

tigen

-like

C4b

p

d o n o r:

A

� P E G -N P s

�

02 04 06 08 0

10 012 014 016 018 0

IgG

3 (H

)

CF

AH

-RP

1Ig

G2

(H)

Apo

-CII

Thy

mos

in β

-4Ig

L k

Alp

ha-1

-ant

itryp

sin

Pro

per

din

[9]

HS

AIg

M(H

)

IgL

λ1Ig

L λ2

C3

[18]

A

po-C

IIIA

po-C

IA

po-E

Apo

-AI

Clu

s

A p o -A IV

020406080

100120140160180

HS

A

Apo

-CII

cyto

plas

mat

ic a

ctin

C

FA

HS

kele

tal a

ctin

Ig

L k

IgL

λ1Ig

L λ2

CF

AH

-RP

1

Apo

-AIV

IgM

(H)

pro

per

din

[21

]A

po-A

IC

3 [3

0]A

po-A

IIA

po-C

IIA

po-C

IIIC

lus

A p o -C I

02 04 06 08 0

10 012 014 016 018 0

Fib

rinog

en α

cha

inH

emog

lobi

n β

PF

-4Ig

L k

Pro

per

din

[4]

C3

[5]

IgM

(H)

Fib

rinog

en γ

cha

inF

ibrin

ogen

α c

hain

Fib

rinog

en β

cha

inH

emog

lobi

n β

Pro

thro

mbi

nA

po-C

IIH

SA

Hem

oglo

bin

αA

po-C

IA

po-E

Apo

-AIV

Apo

-CIII

Apo

-AII

Apo

-AI

Clu

s

020406080

1 001 201 401 601 80

Pro

thro

mbi

nIg

L k

Hem

oglo

bin

α

Fib

rinog

en β

cha

inIg

M(H

)

C3

[10]

Pro

per

din

[11

]A

po-A

IVA

po-E

HS

APF

-4A

po-C

I

Apo

-AII

Apo

-CIII

Apo

-AI

Clu

s

ITI-

HC

4

0 2 0 0 0 0 4 0 0 0 0 6 0 0 0 0 8 0 0 0 00

2 5 0

5 0 0

7 5 0

1 0 0 0

F GB

AF

G

G

F

BBA

� P O Z - N P s� P E G - N P s

mac

roph

age

capt

ure

(MF

I) � n a k e d - N P s

C 3 β c h a in b a n d in te n s i t y ( d e n s ito m e t r y , A U )

A

A - G = H S d o n o r s

Fig. 6

A B C D E F G H H S (N =6)0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

n a k e d -N P s P E G -N P s P O Z -N P s

D o n o r:

mac

roph

age

capt

ure

(MF

I)

0

20 0

40 0

60 0

80 0

MF

I

**

**

*

**

*

*

donor variability

HS

(N

=6

)

PEG low/POZhigh PEGintermediate/POZhigh PEG high/POZhigh

A

….

H

NPs:

PEG POZnaked

Serum donors:

macrophage

cell capture

pro

tein

an

aly

sis

Human mouse

NPs:

PEG POZnaked

Serum from:

Macrophages from:

cell capture, effects

pro

tein

an

aly

sispig

Human pig mouse

Flow cytofluorimetry, confocal microscopy (high throuput),

Elisa, suspension array cytokines detection, cytotoxicology,

-SDS-PAGE

-Proteomic

(in situ,

shot-gun)

-Western

Blot

-Elisa assay

Inter-species variability of nanomedicine-host interactions and the underlying biochemical mechanisms

Coll. dr. S. Nardelli Istituto Zooprofilattico Sperimentale delle Venezie

Supervisors: Prof. E. Supervisors: Prof. E. Supervisors: Prof. E. Supervisors: Prof. E. Papini; Papini; Papini; Papini; Dr. R. Tavano Dr. R. Tavano Dr. R. Tavano Dr. R. Tavano

PROGETTO PROGETTO PROGETTO PROGETTO 2.2.2.2.

NANOFARMACI ANTIOSSIDANTI

Acido lipoico

NPs

Ring Opening Thiol-Disulfide Exchange Polymerization performed in solution (RODEP)

Experimental: NP synthesis

Monomer in acetone

solution (5mg/ml)

Nucleation

and

growthAqueous phase

The nanoprecipitation method combined with RODEP: into the aqueous solution of 20 mM PBS (pH=7,4) and 2 mM F127 surfactant (first beaker on the left), the monomer in acetone solution was added (second beaker). Acetone diffuses and lipoic acid derivatives precipitate into F127 micelles, inside which the RODE polymerization occurred.

RODE polymerizationRODE polymerization

PBS and F127

Pluronic® F127

30 min

Polymerization

and final

shaping

+ Octanethiol

(5 mM)120 min

+ Iodoacetamide

(5 mM)30 min

RODE polymerizationRODE polymerization

Purification

Results: fluorescent-doped NP

Rhodamine B derivative conjugated

with lipoic acid (5 mg/ml)

Monomer 1

Monomer 3

1% v/v1% v/v

1% v/v1% v/v

Supervisors: Supervisors: Supervisors: Supervisors: Prof. A. Prof. A. Prof. A. Prof. A. Negro ,ProfNegro ,ProfNegro ,ProfNegro ,Prof. e. e. e. e. Papini. Papini. Papini. Papini

PROGETTO 3PROGETTO 3PROGETTO 3PROGETTO 3....

PROTEINE RICOMBINANTI PER LE

BIOTECNOLOGIE: Biofilm-surface layer

protein A from Bacillus subtilis e tecnologie

di ingegnerizzazione proteica innovative

(A) Schematic representations of BslA structure in ribbon and cartoon form.(B–D) Photographs and schematics (inset) showing time evolution of a droplet of an aqueous solution of BslA (0.06 mg/mL)deposited onto a hydrophobized silicon wafer. Immediately after placing the drop on the silicon surface the drop has a contact angle of roughly 115°, t = 0 min. (C) t = 5 min.The contact angle decreased slightly, likely due to protein adsorption onto the hydrophobic substrate, and stabilized around roughly 90°. (D) t = 10 min. The inset schematics show BslA assembly into a flat film at the top of thedroplet as this is where flattening was observed.

Scale bar is 0.5 mm.

Published in: Gilad Kaufman; et al Langmuir 2017, 33, 13590-13597.DOI: 10.1021/acs.langmuir.7b03226Copyright © 2017 American Chemical Society

Biofilm-surface layer protein Afrom Bacillus subtilis

Optical microscopy images of arrested coalescence structures produced by water-in-oil BslA droplets.

PDMS flow-focusing microfluidic device with an aqueous solution of BslA as the outer phase and a mineral oil dispersed

phase

Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag

and SpyCatcher

SpyRing generation: the protein of interest is

genetically fused with an N-terminal SpyTag and

C-terminal SpyCatcher (red), which

spontaneously lock together.

(b)SpyRing resilience to aggregation. β-

lactamase were heated to the indicated

temperature,

c) Preservation of enzyme activity of cyclised

(red) compared to wild type β-lactamase (blue)

at room temperature, following heating to 100 °C

Cyclisation for enzyme resilience