Controlling the Surface Properties of Nanoparticles ... · 1. Core-Shell Nanoparticles O Si O O OEt...

NANOCYTES

Tovar_DECHEMA_9.März-2005© Tovar, Fraunhofer-Gesellschaft Institut für

GrenzflächenverfahrenstechnikUniversität Stuttgart

Institut für Zellbiologie und ImmunologieUniversität Stuttgart

Surfaces and Interfaces – Engineering at the Nanoscale, DECHEMA, 9. März 2005

Controlling the Surface Properties of Nanoparticles – Bioorganically modified Nanoparticles for Cell Signaling (NANOCYTES)

Dr. habil. Günter TovarFraunhofer-Institute for Interfacial Engineering and Biotechnology& University of Stuttgart, Institute for Interfacial Engineering

NANOCYTES




Outline

TNF-NANOCYTESFunctional Principle

Nanoparticle Synthesis and Characterisation

Bioconjugation

TNF-Nanoparticles in Biological Systems

Conclusion

NANOCYTES




Functional Principle TNF–NANOCYTES

SignalTransduktion

2

TNF-αKonversion

Enzym(TACE)

RezeptorBindung

TNF

gelöstes TNF-αMembran TNF-α NANOCYTES

?

Immobilisierung

TNF-Receptor 2

TNF-Rezeptor 1

Partikel

SignalTransduktion

1

TNFZelle

SignalTransduktion

2

TNF-αKonversion

Enzym(TACE)

RezeptorBindung

TNF

gelöstes TNF-αMembran TNF-α NANOCYTES

?

Immobilisierung

TNF-Receptor 2

TNF-Rezeptor 1

Partikel

SignalTransduktion

1

TNFZelle

Key to Control of TNF Cell-Signalling Behaviour: Surface-Bound TNF

Synthetic-Bionic Hybrid-Particles

NANOCYTES




RequirementsImmobilization as a TrimerOrientation to Bind ReceptorSuitable Ligand DensityMinimized Nonspecific Binding

TNF–NANOCYTES®

Strategy for Immobilization

Target-cellTNF-R2

TNF

R4

R4

R4R1 R2 R3+ +

NANOCYTES




Nanoparticle Preparation

1. Core-Shell Nanoparticles

O SiO

OO Et

Et

Et

Et OH-

Hydrolysis Condensation

-H20O Si

O

OO H

H

H

H

Si O HO

OO

Amorphous Silica

100 nm

Free Choice of Size. Here: 100 nm, 1 µm, 10 µmVarious fluorescence labels integrated in organic shell:green, red, violet

2. Single-Step Bioreactive Polymer NanoparticlesSurfmer-Technology

O

O

S+

R

3. Biodegradable Polymer Nanoparticles

NANOCYTES




Surface Modification of Silica Nanoparticles1st Shell Reaction: Silanization

NH2SiOOO

EtEt

Et

NH2SiOO

OH+/OH- NH2Si

OOO

HH

HHydrolyse Condensation

NH

OOH

O

SiOO

ONH2SiO

OO

O

O

O

2nd Shell Reaction: Organic Derivatization

+

Quality Control

3 4 5 6 7 8 9 10 11

-60

-50

-40

-30

-20

-10

0

10

20

30

40

Si-OH -NH2 -COOH

pH

Zeta

pote

ntia

l[m

V]

ζ-potential

e.g. by Elemental Analysis andSurface Charge Determination:

90 µmol functional groupsper 1 g particles

NANOCYTES




Shell Modification by Polymer Grafting ReactionReduction of Nonspecific Binding NSB (e.g. Pegylation)

NH

OOH

O

SiOO

O H2N- PEGEDC

-O-Me

0

20

40

60

80

-PEG

1x washing cycle 2x washing cycle

Dextra

n-NH 2

Dextra

n-COOH

-Spa

cer-N

H 2

-COOH

-NH 2

SiOH

BS

A a

dsor

bed

[%]

Low NSB of Proteins

+

Zero NSB

NANOCYTES




Shell with Low NSB and Reactive Anchor GroupsGrafting of Functional PEG

H2N

O

O

NH

O

O( )n

Boc-Amino-PEG-Amine (MW 644, n: ~11)

+ EDC-COOH

TFA-NH2

100 nm

Particle size: SEM

-2 0 2 4 6 8 10 12 14 16-6

-4

-2

0

2

4

6

8

10

12

Zeta

pote

ntia

l (m

V)

Modifier content (mg/ml)

Boc protected deprotected -NH2

Surface characterization:ζ−potential determination

NANOCYTES




RequirementsImmobilization as a TrimerOrientation to Bind ReceptorSuitable Ligand DensityMinimized Nonspecific Binding

TNF–NANOCYTES®

Strategy for Immobilization

Target-cellTNF-R2

TNF

R4

R4

R4R1 R2 R3+ +

NANOCYTES




Synthetic Nanoparticle Protein Conjugation R1 = NH2 R2——R3 = R4 = SHSulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)

HS

HS

R2 R3

NANOCYTES




TNF-Nanoparticles Bind to TNF-Receptor 2 (TNF-R2)Cell-Membrane Ligand-Receptor Study by Confocal MicroscopyLigand TNF-Nanocytes® Binding to TNF-Receptor (HeLa80-R2)

Control: unmodified particles on HeLa80-R2

Particles OverlayTNF-R2

NANOCYTES




TNF-Nanoparticles Bind to TNF-Receptor 2Cell-Membrane Ligand-Receptor Study by Confocal MicroscopyLigand TNF-Particles Binding to TNF-Receptor (HeLa80-R2)

NANOCYTES




TNF-Nanoparticles Stimulate Cell Action via TNF-Receptor 2Signal Pathway Study by Confocal MicroscopyTNF-Particles on HeLa80-R2 with Transcription Factor (NFκB-GFP)

NANOCYTES




TNF-Nanoparticles Stimulate Cell Action via TNF-Receptor 2Signal Pathway Study by Confocal MicroscopyTNF-Nanocytes® on HeLa80-R2 with Transcription Factor (NFκB-GFP)

15 min 30 min

60 min

NANOCYTES




TNF-Nanoparticles Induce Apopotosis via TNF-Receptor 2TNF-Nanocytes® Action on Target Cell (MF-R2-Fas with FADD-GFP)

NANOCYTES




1E-3 0,01 0,1 1 10 100

0,4

0,8

1,2

1,6

2,0

soluble TNF TNF modified nanoparticlesCe

ll ac

tivity

OD 50

Concentration TNF (ng/ml)

0,0

0,4

0,8

1,2

1,6

2,0

TNF-Nanoparticles Induce Apopotosis via TNF-Receptor 2Bioactivity in Cell Assay

Kym-1 cells:TNF-Receptor 1

MF-R2 cells:TNF-Receptor 2

NANOCYTES





22 min 30 min15 min 40 min

NANOCYTES




Conclusions

Synthesis of Reactive Core-Shell Nanoparticles

Reduced Non-Specific Binding of Proteins

Bioactive Conjugation of TNF to Nanoparticles

Hybrid Particles Mimick Membrane-Like TNF

TNF-Particles Recruit TNF-Receptor

TNF-Nanoparticles Stimulates Specific Cell Responses by Receptor-Binding

NANOCYTES




Acknowledgements

IGB and IGVT

Prof. Herwig BrunnerDr. Thomas SchiestelDr. Achim WeberDr. Marc HeroldDr. Carmen GruberInstitute for Cell Biologyand ImmunologyUniversity of Stuttgart

Prof. Klaus PfizenmaierProf. Peter ScheurichDr. Ingo GrunwaldDr. Susanne Bryde

Institut für Zellbiologie und Immunologie

Universität Stuttgart

Funding by BMBF, Program Nanobiotechnology

NANOCYTES





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Controlling the Surface Properties of Nanoparticles ... · 1. Core-Shell Nanoparticles O Si O O OEt...

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