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

Transkript

1 Surfaces and Interfaces NANCYTES Engineering at the Nanoscale, DECHEMA, 9. März 2005 Controlling the Surface Properties of Nanoparticles Bioorganically modified Nanoparticles for Cell Signaling (NANCYTES) Dr. habil. Günter Tovar Fraunhofer-Institute for Interfacial Engineering and Biotechnology & University of Stuttgart, Institute for Interfacial Engineering

2 NANCYTES utline TNF-NANCYTES Functional Principle Nanoparticle Synthesis and Characterisation Bioconjugation TNF-Nanoparticles in Biological Systems Conclusion

3 NANCYTES Functional Principle TNF NANCYTES Membran TNF-α Zelle TNF TNF- Receptor 2 TNF- Rezeptor 1 TNF-α Konversion Enzym (TACE) Rezeptor Bindung gelöstes TNF-α Immobilisierung? NANCYTES TNF Partikel Key to Control of TNF Cell-Signalling Behaviour: Surface-Bound TNF Signal Transduktion 1 Signal Transduktion 2 Synthetic-Bionic Hybrid-Particles

4 NANCYTES TNF NANCYTES Strategy for Immobilization TNF TNF-R2 Targetcell Requirements Immobilization as a Trimer rientation to Bind Receptor Suitable Ligand Density Minimized Nonspecific Binding R 1 + R 2 R 3 + R 4 R 4 R 4

5 NANCYTES Nanoparticle Preparation 1. Core-Shell Nanoparticles Et Et Si Et Et H - Hydrolysis Amorphous Silica H -H 2 0 H Si H Si H H Condensation 100 nm Free Choice of Size. Here: 100 nm, 1 µm, 10 µm Various fluorescence labels integrated in organic shell: green, red, violet 2. Single-Step Bioreactive Polymer Nanoparticles Surfmer-Technology R S + 3. Biodegradable Polymer Nanoparticles

6 NANCYTES Surface Modification of Silica Nanoparticles 1 st Shell Reaction: Silanization Et Si NH 2 H + /H - H Si NH 2 Si NH 2 Et Hydrolyse H Et H Condensation 2 nd Shell Reaction: rganic Derivatization Si NH 2 + Si N H H Quality Control e.g. by Elemental Analysis and Surface Charge Determination: 90 µmol functional groups per 1 g particles Zetapotential [mv] ζ-potential Si-H -NH 2 -CH ph

7 NANCYTES Shell Modification by Polymer Grafting Reaction Reduction of Nonspecific Binding NSB (e.g. Pegylation) Si N H + H N- PEG H 2 Low NSB of Proteins --Me EDC 80 1x washing cycle 2x washing cycle BSA adsorbed [%] SiH -NH 2 -Spacer-NH 2 Dextran-NH 2 -CH Dextran-CH -PEG Zero NSB

8 NANCYTES Shell with Low NSB and Reactive Anchor Groups Grafting of Functional PEG Boc-Amino-PEG-Amine (MW 644, n: ~11) -CH + H 2 N ( ) n NH EDC TFA -NH 2 Particle size: SEM Surface characterization: ζ potential determination Boc protected deprotected -NH 2 Zetapotential (mv) nm Modifier content (mg/ml)

9 NANCYTES TNF NANCYTES Strategy for Immobilization TNF TNF-R2 Targetcell Requirements Immobilization as a Trimer rientation to Bind Receptor Suitable Ligand Density Minimized Nonspecific Binding R 1 + R 2 R 3 + R 4 R 4 R 4

10 NANCYTES Synthetic Nanoparticle Protein Conjugation R 1 = NH 2 R 2 R 3 = R 4 = SH Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) R 2 R 3 HS HS

11 NANCYTES TNF-Nanoparticles Bind to TNF-Receptor 2 (TNF-R2) Cell-Membrane Ligand-Receptor Study by Confocal Microscopy Ligand TNF-Nanocytes Binding to TNF-Receptor (HeLa80-R2) TNF-R2 Particles verlay Control: unmodified particles on HeLa80-R2

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

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

14 NANCYTES TNF-Nanoparticles Stimulate Cell Action via TNF-Receptor 2 Signal Pathway Study by Confocal Microscopy TNF-Nanocytes on HeLa80-R2 with Transcription Factor (NFκB-GFP) 15 min 30 min 60 min

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

16 NANCYTES TNF-Nanoparticles Induce Apopotosis via TNF-Receptor 2 Bioactivity in Cell Assay 2,0 1,6 1,2 soluble TNF TNF modified nanoparticles Kym-1 cells: TNF-Receptor 1 Cell activity D 50 0,8 0,4 0,0 2,0 1,6 1,2 0,8 MF-R2 cells: TNF-Receptor 2 0,4 1E-3 0,01 0, Concentration TNF (ng/ml)

17 NANCYTES TNF-Nanoparticles Induce Apopotosis via TNF-Receptor 2 TNF-Nanocytes Action on Target Cell (MF-R2-Fas with FADD-GFP) 15 min 22 min 30 min 40 min

18 NANCYTES 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

19 Acknowledgements NANCYTES IGB and IGVT Prof. Herwig Brunner Dr. Thomas Schiestel Dr. Achim Weber Dr. Marc Herold Dr. Carmen Gruber Institute for Cell Biology and Immunology University of Stuttgart Prof. Klaus Pfizenmaier Prof. Peter Scheurich Dr. Ingo Grunwald Dr. Susanne Bryde Funding by BMBF, Program Nanobiotechnology

20 NANCYTES TNF-Nanoparticles Induce Apopotosis via TNF-Receptor 2 TNF-Nanocytes Action on Target Cell (MF-R2-Fas with FADD-GFP) 15 min 22 min 30 min 40 min