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

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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. Introduction. Requirement of temporary scaffold materials More Biocompatible - PowerPoint PPT Presentation

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

Self-assembling peptide amphiphile nanofiber matrices for cell entrapment*


*Beniash E, Hartgerink JD, Storrie H, Stendahl JC, Stupp SI (2005) Acta Biomaterialia 1 : 387~397IntroductionRequirement of temporary scaffold materialsMore BiocompatibleMore closer to real extracellular matricesMore friendly with cells

Biomimetic Strategies

A chance to design artificial extracellular matricesPeptide-based self-assembling fibrous networksPeptide 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 moleculespH change - unstableElectrostatic attractionMetal ions

The purpose of this study isDescribing how to self-assemble the PA with metal-ions at physiological pHShowing its application in cell entrapment

Materials and methodsSynthesis of peptide amphiphilesProducing the peptide portionsCoupling the peptide portions with palmitic acids

Formation of PA gelsInducing 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 MEM 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 300120 L of 2 wt.% solution of PA molecule 3 60 L of 60mM aq. ion solutions stirring with the pipette tip measured at 25C Tested with KCl, MgCl2, CaCl2, BaCl2, ZnBr2, CuCl2 , and GdCl3

FTIR Studies

Bio-Rad FTS-40 FTIR spectrometerLyophilized PA gels embedded in KBr pelletsCell Culture

MC3T3-E1 cells were maintained in MEM supplemented with 10% FBS and 1% antibotics10mg/ml PA solutions were sterilized under UV light over night after filtering with 0.25 filter.Cell Entrapment

100 L of PA solution was placed in each chamber of an eight well multi-chamber slideCell suspension (with CaCl2) was added at a density of 20,000 cells/mLAfter mixing, the slides were incubated for 30 min to get mature fibrillar matrixAdding 0.5mL of cell mediaMedia are exchanged every forth day

TEM Studies

Gels were prefixed with 2% glutaraldehyde in medium without FBS or antibiotics for 1hr at 4CFixed in modified karnovsky fixative* for 5h at RTLet the samples stay for 12h at 4CWashing with 0.1M cacodylate buffer twice for 30minPost-fixed with 1% OsO4 in 0.1M cacodylate buffer for 30min at RTRinsed in 0.1M cacodylate buffer for 10min and twice with DI water for 10minDehydrated with serise diluted alcohol solutionsIncubated twice for 10min in propylene oxide

* 2% glutaraldehyde, 2% formaldehyde, 0.1M cacodylate buffer, pH 7.5Transferred 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 vialsTransferred into pure spipon and left in closed vials for 24h, with one resin exchangeTransferred into fresh resin and polymerized at 40, 50, and 70 C for 24h eachCut using a diamond knife* (Diatome) Contrasted with 1% lead citrate and 2% uranyl acetateExamined on a JEOL 100C elctron microscope at 10kV* Lecia Ultracut ultramicrotomeLight 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 37C, rinsed and imaged using an epifluorescence attachment on a Nikon TE200 inverted 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 60C for 16hThe digested sample (5L) were reacted with 195L Hoechst 33258 dye in TNE buffer (0.1g/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

Results and DiscussionOscillatory rheology of PA molecule 3G and G were insensitive to 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 testSimilar results obtained in other negatively charged gels (except PA molecule 7)

In the presence of KClNegatively 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 NaClCaCl2 induced PA molecule 1 and 2 gelsStable in a broad pH-range (4~11)Stable in a high temperatureStable in a large volume of water* (at least 14 days)* at a 2:1 ratio of metal ions to PAMinimum 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 saltsFibers are 5 ~ 6 nm in diameterUranyl 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 bondsAmide I region (1600~1700 cm-1) corresponds to the C=O stretch weakly coupled with C-N stretch and N-H bendingAmide II region (1500~1600 cm-1) represents C-N stretch strongly coupled with N-H bending

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

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

Cells remained spherical and became deadCell Entrapment - Examined with PA molecules 3 (Lys-Gly-Glu, similar charge to RGD)

Cells in a KGE-containing PA nanofiber matrix proliferated

ConclusionsPeptide 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