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

125:583

Biointerfacial Characterization of Nanoparticles and

Nanoscale Biointerfaces II

P. Moghe

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)

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

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.

Ligand Loading on Nanoparticles

Example: Fibronectin fragment derivatized to albumin

nanocarriers (ANC)

Exposure of bioactive sites on nanoparticle-derivatized ligands

Particle Sizing Following PEGylation

Morphology of biofunctionalized

nanoparticles

Ligand Concentration

How to characterize the density and

spatial organization of nanoscale ligands?

Model System for Clustered Presentation of Ligands

Biofunctionalized Comb Polymers

AFM measurement of ligand clustering

AFM of Nanospheres for Clustered Biointerfaces

Characterization of Cell Adhesive Reponses to Nanoscale Clustered

Ligands

Hypothesis: Presentation of an integrin ligand in a clusteredformat may enhance the efficiency of clustering of ligand-bound integrins in comparison to those elicited byequivalent levels of the ligand presented in a sparse manner.

Alteration in the ligand presentation format may be a basisto alter cell adhesion strength (& motility)

Characterization of RGD star polymers

Cell culture and ligand system

• WT NR6 cells, a 3T3-derived murine fibroblast cell linelacking endogenous EGF receptor, transfected with awild type human EGFR (Chen et al., J Cell Biol, 124, 547)These cells express avb3 and a5b1 integrins, which bindto RGD adhesion ligands.

• Cells cultured in MEM-a medium with FBS, P-S,etc.Studies for adhesion & migration excluded FBS and includedHepes buffer.

• YGRGD ligand was presented via polyethylene oxide (PEO)tethers against an inert substrate of radiation-crosslinked PEOhydrogel on PEO-silane treated glass coverslips.

Ligand and Substrate System

• YGRGD peptide was attached to the PEG hydrogel modifiedcoverslips using star PEO tethers, in order to vary the averagesurface density, and the local spatial distribution (50 nm scale)of RGD peptide. Star PEO has many PEO arms. 1-n RGDpeptides were linked to each start, blocking each unreactedstar 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 correctproportion, ) were added to PEO hydrogel substrates. Unreactedchain ends were blocked with Tris-HCl. By varying , averageligand densities (1000 (L) - 200,000 (H) molecules/cm2) wereobtained. Average cluster-cluster distance = 6-300 nm.

Cell Adhesion Assays

• Silanized glass coverslips were glued (!) to35 mm dishes (m) or 24 well plates (a), andincubated with 0.2 ml FN solution/cm2 for 2 h.Substrates were then blocked with BSA for 1 h.FN molecular densities were calculated (assumingeach FN offers 1 integrin binding site?) • A centrifugal cell detachment assay was performed.

Cells were incubated for 12 h (serum-free) in coverslipglued 24 well plates, and medium w/ or w/o EGF was added. Media was filled, wells sealed, and platesspun at 800g for 10 minutes.

• RGD ligand presentation determines cell-substrate adhesion strength.