125:583

1. 125:583 Biointerfacial Characterization of Nanoparticles and Nanoscale Biointerfaces II P. Moghe

2. Outline • Measuring surface charge/zeta potentials on nanoparticles (Anthony) • Characterization of biofunctionalization of nanoparticles (Moghe) • Characterization of cell adhesive responses to biofunctional nanoscale interfaces (Moghe)

3. How to characterize biofunctionalization of nanoparticles? • Use of fluorometry • Use ELISA for biospecific signal • Use DLS if the biocomplexation leads to change in size. • Electron Microscopy; AFM for morphological changes • Modeling

4. Example: Characterization of Biofunctionalization • Example from Sharma et al., In Review, Biomaterials (2005). • Albumin nanoparticles (ANP) were derivatized with fibronectin fragments and then adsorbed on TCPS. • Albumin-specific ELISA was used to quantify the amount of backbone NPs, while FNf-specific ELISA was used to quantify the biofunctionalization.

5. Ligand Loading on NanoparticlesExample: Fibronectin fragment derivatized to albumin nanocarriers (ANC)

6. Exposure of bioactive sites on nanoparticle-derivatized ligands

8. Particle Sizing Following PEGylation

9. Morphology of biofunctionalized nanoparticles

10. Ligand Concentration

13. How to characterize the density and spatial organization of nanoscale ligands?

14. Model System for Clustered Presentation of Ligands

16. Biofunctionalized Comb Polymers

17. AFM measurement of ligand clustering

18. AFM of Nanospheres for Clustered Biointerfaces

19. Characterization of Cell Adhesive Reponses to Nanoscale Clustered Ligands

20. Hypothesis: Presentation of an integrin ligand in a clustered format may enhance the efficiency of clustering of ligand- bound integrins in comparison to those elicited by equivalent levels of the ligand presented in a sparse manner. Alteration in the ligand presentation format may be a basis to alter cell adhesion strength (& motility)

21. Characterization of RGD star polymers

22. Cell culture and ligand system • WT NR6 cells, a 3T3-derived murine fibroblast cell line lacking endogenous EGF receptor, transfected with a wild type human EGFR (Chen et al., J Cell Biol, 124, 547) These cells express avb3 and a5b1 integrins, which bind to RGD adhesion ligands. • Cells cultured in MEM-a medium with FBS, P-S,etc. Studies for adhesion & migration excluded FBS and included Hepes buffer. • YGRGD ligand was presented via polyethylene oxide (PEO) tethers against an inert substrate of radiation-crosslinked PEO hydrogel on PEO-silane treated glass coverslips.

23. Ligand and Substrate System • YGRGD peptide was attached to the PEG hydrogel modified coverslips using star PEO tethers, in order to vary the average surface density, and the local spatial distribution (50 nm scale) of RGD peptide. Star PEO has many PEO arms. 1-n RGD peptides were linked to each start, blocking each unreacted star arm, and diluting RGD-modified stars with blank stars. These were then grafted to the surface. • Stars with an average of 1, 5 or 9 ligands per star were achieved. (verified using 125I-YGRGD?) • Next, RGD conjugated stars and unconjugated stars (in correct proportion, f) were added to PEO hydrogel substrates. Unreacted chain ends were blocked with Tris-HCl. By varying f, average ligand densities (1000 (L) - 200,000 (H) molecules/cm2) were obtained. Average cluster-cluster distance = 6-300 nm.

24. Cell Adhesion Assays • Silanized glass coverslips were glued (!) to 35 mm dishes (m) or 24 well plates (a), and incubated with 0.2 ml FN solution/cm2 for 2 h. Substrates were then blocked with BSA for 1 h. FN molecular densities were calculated (assuming each FN offers 1 integrin binding site?) • A centrifugal cell detachment assay was performed. Cells were incubated for 12 h (serum-free) in coverslip glued 24 well plates, and medium w/ or w/o EGF was added. Media was filled, wells sealed, and plates spun at 800g for 10 minutes.

25. • RGD ligand presentation determines cell-substrate adhesion strength.