Biointerfacial Characterization of Nanoparticles and Nanoscale Biointerfaces II

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125583

• Biointerfacial Characterization of Nanoparticles
  and Nanoscale Biointerfaces II
• P. Moghe

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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)

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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

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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.

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Ligand Loading on NanoparticlesExample
Fibronectin fragment derivatized to albumin
nanocarriers (ANC)
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Exposure of bioactive sites on nanoparticle-deriva
tized ligands
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Particle Sizing Following PEGylation
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Morphology of biofunctionalized nanoparticles
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Ligand Concentration
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How to characterize the density and spatial
organization of nanoscale ligands?
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Model System for Clustered Presentation of Ligands
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Biofunctionalized Comb Polymers
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AFM measurement of ligand clustering
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AFM of Nanospheres for Clustered Biointerfaces
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Characterization of Cell Adhesive Reponses to
Nanoscale Clustered Ligands
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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)
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Characterization of RGD star polymers
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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.
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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.
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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.
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RGD ligand presentation determines
cell-substrate adhesion strength.
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