Self-assembling peptide am-phiphile nanofiber matrices

for cell entrapment*

DONGJIN DANIEL LIM

*Beniash E, Hartgerink JD, Storrie H, Stendahl JC, Stupp SI (2005) Acta Biomate-rialia 1 : 387~397

Introduction• Requirement of temporary scaffold materi-

als– 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

• 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

Materials and methods• Synthesis of peptide amphiphiles

– Producing the peptide portions– Coupling the peptide portions with

palmitic acids

• 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+

– ‘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

• FTIR Studies

– Bio-Rad FTS-40 FTIR spectrometer– Lyophilized PA gels embedded in KBr pel-

lets

• Cell Culture

– MC3T3-E1 cells were maintained in MEMα supplemented with 10% FBS and 1% anti-botics

– 10mg/ml PA solutions were sterilized un-der UV light over night after filtering with 0.25 ㎛ filter.

• 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

• TEM Studies

– Gels were prefixed with 2% glutaralde-hyde 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 cacody-

late 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 so-

lutions– Incubated twice for 10min in propylene

oxide* 2% glutaraldehyde, 2% formaldehyde, 0.1M cacodylate buf-fer, pH 7.5

– 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 ex-change

– Transferred into fresh resin and polymer-ized 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 micro-

scope at 10kV * Lecia Ultracut ultramicrotome

• Light microscopy

– A Nikon TE200 inverted microscope equipped with a Spot RT CCD camera con-trolled 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 epifluores-cence attachment on a Nikon TE200 in-verted microscope

• Analysis of celluar metabolism

– Glucose and latate concentraion were measured in the media using a YSI 2700 Select Biochemical Analyzer

• 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 buf -fer (0.1μg/mL, pH 7.5)

– Excited at 346nm, fluorescence emission at 460nm was monitored on a fluores-cence 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

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

• Complex viscosities with various ion salts

– Gels prepared with alkaline earth metals had

lower moduli than those prepared with transiton metals

‘

• Results of the ‘self-supporting gel’ test– Similar results obtained in other nega-

tively 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

• 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 interac-tions

between its side chains each other*

* Amphilic peptides assembled into ribbon-like sturctures upon addition of monovalent salts

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

nanofibers

• A gel can form in culture medium • PA molecules assemble into nanofiber with

aliphatic tails in the core

• 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

• The postion of Amide I band and no obserava-tion of 1690 cm-1 suggest that the PA mole-cule is a parallel beta-sheet

• Cell Entrapment - Examined with PA molecules 5 (Glu-Gln-Ser,

random)

• Cells remained spherical and became dead

• Cell Entrapment - Examined with PA molecules 3 (Lys-Gly-Glu,

similar charge to RGD)

• Cells in a KGE-containing PA nanofiber matrix proliferated

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