Self-assembling peptide amphiphile nanofiber matrices for cell entrapment* DONGJIN DANIEL LIM

1. Self-assembling peptide amphiphile nanofiber matrices for cell entrapment* DONGJIN DANIEL LIM *Beniash E, Hartgerink JD, Storrie H, Stendahl JC, Stupp SI (2005) Acta Biomaterialia 1 : 387~397

2. Introduction • Requirement of temporary scaffold materials • More Biocompatible • More closer to real extracellular matrices • More friendly with cells • Biomimetic Strategies • A chance to design artificial extracellular matrices • Peptide-based self-assembling fibrous networks • Peptide amphiphile (PA) molecules • √ To form self-supporting gels • √ To recreate the nanoscale structure of bone • √ To promote selective differentiation of neural • progenitor cells into neurons

3. Ways to self-assemble PA molecules • pH change - unstable • Electrostatic attraction • Metal ions • The purpose of this study is • Describing how to self-assemble the PA with metal-ions at physiological pH • Showing its application in cell entrapment

4. Materials and methods • Synthesis of peptide amphiphiles • Producing the peptide portions • Coupling the peptide portions with palmitic acids

5. Formation of PA gels • Inducing gels • (with NaCl, KCl, MgCl2, CaCl2, BaCl2, ZnBr2, Cu(ClO4)2, and GdCl3) • √ 10mM aq. PA solutions at pH 7.5 • √ The final metal ion concentrations • 20mM for polyvalent ions • 200mM for monovalent ions • √ In case of PA1 and PA2 • up to 6M of KCl and NaCl • 5 – 50 mM GdCl3 and CaCl2 • √ with MEMα and DMEM • or PBS and HBSS without Ca2+, Mg2+

6. ‘self-supporting gel’ test • ‘when remaining at the bottom of the vial after • inverting the vial’ • Oscillating rheometry • Paar Physica Modular Compact Rheometer 300 • 120 μL of 2 wt.% solution of PA molecule 3 • 60 μL of 60mM aq. ion solutions • stirring with the pipette tip • measured at 25°C • Tested with KCl, MgCl2, CaCl2, BaCl2, ZnBr2, CuCl2 • , and GdCl3

7. FTIR Studies • Bio-Rad FTS-40 FTIR spectrometer • Lyophilized PA gels embedded in KBr pellets • Cell Culture • MC3T3-E1 cells were maintained in MEMα supplemented with 10% FBS and 1% antibotics • 10mg/ml PA solutions were sterilized under UVlight over night after filtering with 0.25㎛ filter.

8. Cell Entrapment • 100 μL of PA solution was placed in each chamber • of an eight well multi-chamber slide • Cell suspension (with CaCl2) was added at a density of 20,000 cells/mL • After mixing, the slides were incubated for 30 min to get mature fibrillar matrix • Adding 0.5mL of cell media • Media are exchanged every forth day

9. TEM Studies • Gels were prefixed with 2% glutaraldehyde in medium without FBS or antibiotics for 1hr at 4°C • Fixed in modified karnovsky fixative* for 5h at RT • Let the samples stay for 12h at 4°C • Washing with 0.1M cacodylate buffer twice for 30min • Post-fixed with 1% OsO4 in 0.1M cacodylate buffer for 30min at RT • Rinsed in 0.1M cacodylate buffer for 10min and twice with DI water for 10min • Dehydrated with serise diluted alcohol solutions • Incubated twice for 10min in propylene oxide * 2% glutaraldehyde, 2% formaldehyde, 0.1M cacodylate buffer, pH 7.5

10. Transferred to a 1:1 mixture of propylene oxide and spipon 812 embedding resin (SPI) • Left in closed vials for 12h followed by 8h in open vials • Transferred into pure spipon and left in closed vials for 24h, with one resin exchange • Transferred into fresh resin and polymerized at 40, 50, and 70 °C for 24h each • Cut using a diamond knife* (Diatome) • Contrasted with 1% lead citrate and 2% uranyl acetate • Examined on a JEOL 100C elctron microscope at 10kV * Lecia Ultracut ultramicrotome

11. Light microscopy • A Nikon TE200 inverted microscope equipped with a Spot RT CCD camera controlled with Metamorph digital analysis software (200X and 400X) • Viability assays were performed with LIVE/DEAD reagent for 15min at 37°C, rinsed and imaged using an epifluorescence attachment on a Nikon TE200 inverted microscope

12. Analysis of celluar metabolism • Glucose and latate concentraion were measured in the media using a YSI 2700 Select Biochemical Analyzer

13. Cell Proliferation Assay • Digested in papain(0.125 mg/mL) with 0.1M cysteine in PBE buffer (pH 6.5) at 60°C for 16h • The digested sample (5μL) were reacted with 195μL Hoechst 33258 dye in TNE buffer (0.1μg/mL, pH 7.5) • Excited at 346nm, fluorescence emission at 460nm was monitored on a fluorescence plate reader • (using Costar opaque white clear bottom 96-well plates) • Total DNAs were determined with a known DNA content of calf thymus DNA, and the number of cells was estimated aiding of 7.7 ng of DNA per cell

14. Results and Discussion • Oscillatory rheology of PA molecule 3 • G’ and G’’ were insensitive to ω • G’ constantly greater than G’’ • (with polyvalent metal ions) • Gels have elastic character * Gels of PA molecule 3 were prepared with 20mM MgCl2

15. Complex viscosities with various ion salts • Gels prepared with alkaline earth metals had • lower moduli than those prepared with transiton metals • ‘

16. Results of the ‘self-supporting gel’ test • Similar results obtained in other negatively charged gels (except PA molecule 7) • In the presence of KCl • Negatively charged gels did not form gels • (at a molar ratio of 1:20) • PA molecule 1 and 2 did not form gels even in the presence of 6M KCl or NaCl • CaCl2 induced PA molecule 1 and 2 gels • Stable in a broad pH-range (4~11) • Stable in a high temperature • Stable in a large volume of water* (at least 14 days) * at a 2:1 ratio of metal ions to PA

17. Minimum concentrations of polyvalent ions were equal to the molarity of PA molecules • IKVAV-containing molecules were shown to form self-supporting gels at 200mM KCl • - IKVAV enhances the hydrophobic interactions • between its side chains each other* * Amphilic peptides assembled into ribbon-like sturctures upon addition of monovalent salts

18. Fibers are 5 ~ 6 nm in diameter • Uranyl acetate stains only peripheral parts of nanofibers

19. A gel can form in culture medium • PA molecules assemble into nanofiber with aliphatic tails in the core

20. Amide A is the designation for the band in the region near 3300 cm-1 of –NH stretch. • Its frequency would be affected by hydrogen bonds • Amide I region (1600~1700 cm-1) corresponds to the C=O stretch weakly coupled with C-N stretch and N-H bending • Amide II region (1500~1600 cm-1) represents C-N stretch strongly coupled with N-H bending

21. The postion of Amide I band and no obseravation of 1690 cm-1 suggest that the PA molecule is a parallel beta-sheet

22. Cell Entrapment • - Examined with PA molecules 5 (Glu-Gln-Ser, random) • Cells remained spherical and became dead

23. Cell Entrapment • - Examined with PA molecules 3 (Lys-Gly-Glu, similar charge to RGD) • Cells in a KGE-containing PA nanofiber matrix proliferated

27. Conclusions • Peptide amphiphile molecules can assemble into nanofibrillar networks at physiological pH • Cells entrapped in the networks can survive and proliferate with using the nanofiber as a nutrient