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Fall 2008 Group Meeting

Fall 2008 Group Meeting. Jon Nickels Schmidt Group September 25, 2008. Biomaterial Modification. Affinity. Strengths of the Affinity Technique: Preserves structure and properties of PPy Specific and reversible interactions with the substrate. Equilibrium Binding Study Design.

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Fall 2008 Group Meeting

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  1. Fall 2008 Group Meeting Jon Nickels Schmidt Group September 25, 2008

  2. Biomaterial Modification Affinity • Strengths of the Affinity Technique: • Preserves structure and properties of PPy • Specific and reversible interactions with the substrate

  3. Equilibrium Binding Study Design • T59-GGGSK-Biotin used for this assay. • ELISA gives signal amplification for low surface density calculations. • This is a potential method to immobilize a biomolecule. Neutravidin-HRP T59 Peptide PPyCl

  4. Qualitative Result 0 T59-B 1 μg/ml T59-B Streptavidin-FITC T59 Peptide PPyCl

  5. Homologous Competition • Use a set T59-B input and titrate with T59. Neutravidin-HRP Neutravidin-HRP

  6. Homogeneous Competition • Performed at three detectible input concentrations • 5nM, 0.5nM, 0 nM • Fitting the data for 0.5 nM input of T59-B • Kd = IC50 – [L] • Kd = 36.03 nM • Still very strong and no reliable bottom of the curve. • Too low starting conentration.

  7. XPS Basics • Use X-ray beam to excite atoms. • We detect the emitted photon’s kinetic energy. • This correlates to binding energy via: • Ebinding = Ephoton - Ekinetic - Φ • Technique also known as ESCA.

  8. Figure 1. (top) XPS results confirming the ability to track T59GGGSC addition to a PPyCl surface. Figure 2. (bottom) XPS results of a competition style assay of T59GGGSC and standard T59. Addition of T59 competes for surface binding sites. We can compute IC50 (x) if we assume homologous competition. XPS Results • In the first figure the additional 5 amino acids more relevant to the surface concentration. • Kd = IC50 – [L] • Yields a Kd value of 10.513 µM.

  9. AFM Technique • Direct, single molecule determination of the off rate, koff. • T59-GGSSSC attached to tip. • T59-PEG8-C8H16-S-Au

  10. AFM Single Molecule Methods • Analysis • Plot most probable unbinding force versus the ln(r) • F = (kBT / xb) • ln (r xb / koff kBT) • koff allows a complete experimental picture of the binding strength. # F (pN) F (pN) Koff ln (R (pN/s))

  11. Figure 3. (left) Histograms of the unbinding forces observed using the AFM method for different loading rates, from 715 pN/s to 71,550 pN/s. Figure 4. (right) Log plot of the most probable unbinding forces determined from single molecule methods. Line shows the best fit to the data and the y-intercept corresponds to Koff. AFM Results

  12. Binding Constant Summary • Fairly weak binding pair with a rapid equilibrium.

  13. T59 Variants

  14. Cell Binding Predictions • Most studies were performed at 1 or 10 µg/ml or about .5 or 5 µM. • An example of an effective YIGSR surface concentration was 447 ± 37 fmol/cm2 by Saneinejad and Shoichet (1998).

  15. PC-12 Cell Model • PPyCl surfaces incubated with T59-Laminin Fragment solution, then washed. • PC12 Cells Seed on surfaces, cultured for 2 days. • Stained w/ phaloidin and DAPI. • T59-YIGSR showed most improved cell adhesion. • T59-IKVAV showed the most neurites generated.

  16. PC-12 Cell Model with Electrical Stimulation • Above figures show samples of stained images. From left, PBS, 10 ug/ml T59-IKVAV, 10 ug/ml T59-YIGSR. • Added IKVAV and YIGSR peptides to the surfaces to test nonspecific adsorption. • Figure at right shows a count of cells adhered to the surface.

  17. PC-12 Cell Model with Electrical Stimulation • Top figure shows the ratio of observed neurites over total cells. • The bottom figure shows neurite length.

  18. Subcutaneous Implant Method • Compare response of T59 treated PPy to untreated PPy • 2 month old male Sprauge-Dawley rats 10 μg/ml T59-B 1 μg/ml T59-B 1 cm x 1 cm PPy PLGA N=4 Control Implant Sham Surgery

  19. in vivo Results 4 x Magnification 20 x Magnificaton 10 μg/ml T59-B Control Implant

  20. in vivo Results 20 x Magnification DAPI Nuclear Stain 10 μg/ml T59-B Control Implant

  21. in vivo Results • Nuclear density profile shows no difference between treated and untreated implants. • Need to complete study for shorter time points. • Results suggest there is no negative effect of T59.

  22. Cell Binding Observation • 1 time study on spinal astrocytes. (Units Cells/Frame) • PPyCl Substrates with 1 ug/ml T59-IKVAV, 1 ug/ml T59-YIGSR or no surface treatment. • Shows Significant Binding to the IKVAV Peptide.

  23. Fin

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