Synthesis and Primary Characterization of Self-Assembled Peptide-Based Hydrogels

  • Radhika P. Nagarkar
  • Joel P. Schneider
Part of the Methods in Molecular Biology™ book series (MIMB, volume 474)


Hydrogels based on peptide self-assembly form an important class of biomaterials that find application in tissue engineering and drug delivery. It is essential to prepare peptides with high purity to achieve batch-to-batch consistency affording hydrogels with reproducible properties. Automated solid-phase peptide synthesis coupled with optimized Fmoc (9-fluorenylmethoxy-carbonyl) chemistry to obtain peptides in high yield and purity is discussed. Details of isolating a desired peptide from crude synthetic mixtures and assessment of the peptide's final purity by high-performance liquid chromatography and mass spectrometry are provided. Beyond the practical importance of synthesis and primary characterization, techniques used to investigate the properties of hydrogels are briefly discussed.

Key Words Biomaterial HPLC hydrogel peptide self-assembly solid-phase peptide synthesis 



We acknowledge the National Institutes of Health grant R01 DE016386-01. We also thank Lisa A. Haines-Butterick for optimization of the synthesizer chemistry and her helpful discussions for this chapter as well as Karthikan Rajagopal for performing the MAX3 studies.


  1. 1.
    Rajagopal K, Schneider JP. (2004) Self-assembling peptides and proteins for nano-technological applications. Curr. Opin. Struct. Biol. 14(4), 480–486.CrossRefGoogle Scholar
  2. 2.
    Whitesides GM, Mathias JP, Seto CT. (1991) Molecular self-assembly and nano-chemistry — a chemical strategy for the synthesis of nanostructures. Science 254(5036), 1312–1319.CrossRefGoogle Scholar
  3. 3.
    Carny O, Shalev DE, Gazit E. (2006) Fabrication of coaxial metal nanocables using a self-assembled peptide nanotube scaffold. Nano Lett. 6(8), 1594–1597.CrossRefGoogle Scholar
  4. 4.
    Ray S, Drew MGB, Das AK, Banerjee A. (2006) The role of terminal tyrosine residues in the formation of tripeptide nanotubes: a crystallographic insight. Tetrahedron 62(31), 7274–7283.CrossRefGoogle Scholar
  5. 5.
    Crisma M, Toniolo C, Royo S, Jimenez AI, Cativiela C. (2006) A helical, aromatic, peptide nanotube. Org. Lett. 8(26), 6091–6094.CrossRefGoogle Scholar
  6. 6.
    Leclair S, Baillargeon P, Skouta R, Gauthier D, Zhao Y, Dory YL. (2004) Micrometer-sized hexagonal tubes self-assembled by a cyclic peptide in a liquid crystal. Angew. Chem. Int. Ed. 43(3), 349–353.CrossRefGoogle Scholar
  7. 7.
    Horne WS, Stout CD, Ghadiri MR. (2003) A heterocyclic peptide nanotube. J. Am. Chem. Soc. 125(31), 9372–9376.CrossRefGoogle Scholar
  8. 8.
    Amorin M, Castedo L, Granja JR. (2005) Self-assembled peptide tubelets with 7 angstrom pores. Chemistry 11(22), 6543–6551.CrossRefGoogle Scholar
  9. 9.
    Block MAB, Hecht S. (2005) Wrapping peptide tubes: merging biological self-assembly and polymer synthesis. Angew. Chem. Int. Ed. 44(43), 6986–6989.CrossRefGoogle Scholar
  10. 10.
    Lu K, Jacob J, Thiyagarajan P, Conticello VP, Lynn DG. (2003) Exploiting amyloid fibril lamination for nanotube self-assembly. J. Am. Chem. Soc. 125(21), 6391–6393.CrossRefGoogle Scholar
  11. 11.
    Gao X Y, Matsui H. (2005) Peptide-based nanotubes and their applications in bionanotechnology. Adv. Mater. 17(17), 2037–2050.CrossRefGoogle Scholar
  12. 12.
    Woolfson DN, Ryadnov MG. (2006) Peptide-based fibrous biomaterials: some things old, new and borrowed. Curr. Opin. Chem. Biol. 10(6), 559–567.CrossRefGoogle Scholar
  13. 13.
    Aggeli A, Nyrkova IA, Bell M, et al. (2001) Hierarchical self-assembly of chiral rod-like molecules as a model for peptide beta-sheet tapes, ribbons, fibrils, and fibers. Proc. Natl. Acad. Sci. U. S. A. 98(21), 11857–11862.CrossRefGoogle Scholar
  14. 14.
    Bitton R, Schmidt J, Biesalski M, Tu R, Tirrell M, Bianco-Peled H. (2005) Self-assembly of model DNA-binding peptide amphiphiles. Langmuir 21(25), 11888–11895.CrossRefGoogle Scholar
  15. 15.
    Deechongkit S, Powers ET, You SL, Kelly JW. (2005) Controlling the morphology of cross beta-sheet assemblies by rational design. J. Am. Chem. Soc. 127(23), 8562–8570.CrossRefGoogle Scholar
  16. 16.
    Elgersma RC, Meijneke T, Posthuma G, Rijkers DTS, Liskamp RMJ. (2006) Self-assembly of amylin(20–29) amide-bond derivatives into helical ribbons and peptide nanotubes rather than fibrils. Chemistry 12(14), 3714–3725.CrossRefGoogle Scholar
  17. 17.
    Lowik D, Garcia-Hartjes J, Meijer JT, van Hest JCM. Tuning secondary structure and self-assembly of amphiphilic peptides. Langmuir 2005;21(2):524–526.CrossRefGoogle Scholar
  18. 18.
    Matsumura S, Uemura S, Mihara H. (2004) Fabrication of nanofibers with uniform morphology by self-assembly of designed peptides. Chemistry 10(11), 2789–2794.CrossRefGoogle Scholar
  19. 19.
    Zhang SG. (2003) Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 21(10), 1171–1178.CrossRefGoogle Scholar
  20. 20.
    Fairman R, Akerfeldt KS. (2005) Peptides as novel smart materials. Curr. Opin. Struct. Biol. 15(4), 453–463.CrossRefGoogle Scholar
  21. 21.
    Bonzani IC, George JH, Stevens MM. (2006) Novel materials for bone and cartilage regeneration. Curr. Opin. Chem. Biol. 10(6), 568–575.CrossRefGoogle Scholar
  22. 22.
    Mart RJ, Osborne RD, Stevens MM, Ulijn RV. (2006) Peptide-based stimuli-responsive biomaterials. Soft Matter 2(10), 822–835.CrossRefGoogle Scholar
  23. 23.
    Nowak AP, Breedveld V, Pakstis L, et al. (2002) Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles. Nature 417(6887), 424–428.CrossRefGoogle Scholar
  24. 24.
    Stendahl JC, Rao MS, Guler MO, Stupp SI. (2006) Intermolecular forces in the self-assembly of peptide amphiphile nanofibers. Adv. Funct. Mater. 16(4), 499–508.CrossRefGoogle Scholar
  25. 25.
    Schneider JP, Pochan DJ, Ozbas B, Rajagopal K, Pakstis L, Kretsinger J. (2002) Responsive hydrogels from the intramolecular folding and self-assembly of a designed peptide. J. Am. Chem. Soc. 124(50), 15030–15037.CrossRefGoogle Scholar
  26. 26.
    Caplan MR, Schwartzfarb EM, Zhang SG, Kamm RD, Lauffenburger DA. (2002) Control of self-assembling oligopeptide matrix formation through systematic variation of amino acid sequence. Biomaterials 23(1), 219–227.CrossRefGoogle Scholar
  27. 27.
    Collier JH, Messersmith PB. (2004) Self-assembling polymer-peptide conjugates: nanostructural tailoring. Adv. Mater. 16(11), 907–910.CrossRefGoogle Scholar
  28. 28.
    Ramachandran S, Trewhella J, Tseng Y, Yu YB. (2006) Coassembling peptide-based biomaterials: effects of pairing equal and unequal chain length oligopep-tides. Chem. Mater. 18(26), 6157–6162.CrossRefGoogle Scholar
  29. 29.
    Zhang SG. (2002) Emerging biological materials through molecular self-assembly. Biotechnol. Adv. 20(5–6), 321–339.CrossRefGoogle Scholar
  30. 30.
    Yang JY, Xu C Y, Wang C, Kopecek J. (2006) Refolding hydrogels self-assembled from N-(2-hydroxypropyl)methacrylamide graft copolymers by antiparallel coiled-coil formation. Biomacromolecules 7(4), 1187–1195.CrossRefGoogle Scholar
  31. 31.
    Shen W, Zhang KC, Kornfield JA, Tirrell DA. (2006) Tuning the erosion rate of artificial protein hydrogels through control of network topology. Nat. Mater. 5(2), 153–158.CrossRefGoogle Scholar
  32. 32.
    Ciani B, Hutchinson EG, Sessions RB, Woolfson DN. (2002) A designed system for assessing how sequence affects alpha to beta conformational transitions in proteins. J. Biol. Chem. 277(12), 10150–10155.CrossRefGoogle Scholar
  33. 33.
    Lee HJ, Lee J-S, Chansakul T, Yu C, Elisseeff JH, Yu SM. (2006) Collagen mimetic peptide-conjugated photopolymerizable PEG hydrogel. Biomaterials 27(30), 5268–5276.CrossRefGoogle Scholar
  34. 34.
    Kotch FW, Raines RT. (2006) Self-assembly of synthetic collagen triple helices. Proc. Natl. Acad. Sci. U. S. A. 103(9), 3028–3033.CrossRefGoogle Scholar
  35. 35.
    Yang ZM, Liang GL, Wang L, Bing X. (2006) Using a kinase/phosphatase switch to regulate a supramolecular hydrogel and forming the supramoleclar hydrogel in vivo. J. Am. Chem. Soc. 128(9), 3038–3043.CrossRefGoogle Scholar
  36. 36.
    Jun HW, Yuwono V, Paramonov SE, Hartgerink JD. (2005) Enzyme-mediated degradation of peptide-amphiphile nanofiber networks. Adv. Mater. 17(21), 2612–2617.CrossRefGoogle Scholar
  37. 37.
    Lutolf MP, Hubbell JA. (2005) Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 23(1), 47–55.CrossRefGoogle Scholar
  38. 38.
    Pochan DJ, Schneider JP, Kretsinger J, Ozbas B, Rajagopal K, Haines L. (2003) Thermally reversible hydrogels via intramolecular folding and consequent self-assembly of a de Novo designed peptide. J. Am. Chem. Soc. 125(39), 11802–11803.CrossRefGoogle Scholar
  39. 39.
    Ozbas B, Kretsinger J, Rajagopal K, Schneider JP, Pochan DJ. (2004) Salt-triggered peptide folding and consequent self-assembly into hydrogels with tunable modulus. Macromolecules 37(19), 7331–7337.CrossRefGoogle Scholar
  40. 40.
    Kretsinger JK, Haines LA, Ozbas B, Pochan DJ, Schneider JP. (2005) Cytocompatibility of self-assembled ss-hairpin peptide hydrogel surfaces. Biomaterials 26(25), 5177–5186.CrossRefGoogle Scholar
  41. 41.
    Haines LA, Rajagopal K, Ozbas B, Salick DA, Pochan DJ, Schneider JP. (2005) Light-activated hydrogel formation via the triggered folding and self-assembly of a designed peptide. J. Am. Chem. Soc. 127(48), 17025–17029.CrossRefGoogle Scholar
  42. 42.
    Rajagopal K, Ozbas B, Pochan DJ, Schneider JP. (2006) Probing the importance of lateral hydrophobic association in self-assembling peptide hydrogelators. Eur. Biophys. J. Biophys. Lett. 35(2), 162–169.CrossRefGoogle Scholar
  43. 43.
    Chan WC, White PD. (2000) Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press, New York.Google Scholar
  44. 44.
    Fields GB, Fields CG. (1991) Solvation effects in solid-phase peptide-synthesis. J. Am. Chem. Soc. 113(11), 4202–4207.CrossRefGoogle Scholar
  45. 45.
    Kates SA, Sole NA, Beyermann M, Barany G, Albericio F. (1996) Optimized preparation of deca(L-alanyl)-L-valinamide by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis on polyethylene glycol-polystyrene (PEG-PS) graft supports, with 1,8-diazobicyclo[5.4.0]-undec-7-ene (DBU) deprotection. Peptide Res. 9(3), 106–113.Google Scholar
  46. 46.
    Angell YM, Alsina J, Albericio F, Barany G. (2002) Practical protocols for step-wise solid-phase synthesis of cysteine-containing peptides. J. Peptide Res. 60(5), 292–299.CrossRefGoogle Scholar
  47. 47.
    Fasman GD. (1996) Circular Dichroism and the Conformational Analysis of Biomolecules. Plenum Press, New York.Google Scholar
  48. 48.
    Cantor CR, Schimmel PR. (1980) Biophysical Chemistry. Freeman, New York.Google Scholar
  49. 49.
    Larson RG. (1999) The Structure and Rheology of Complex Fluids. Oxford University Press, New York.Google Scholar
  50. 50.
    Williams DB, Carter CB. (1996) Transmission Electron Microscopy: A Textbook for Materials Science. Springer, New York.Google Scholar
  51. 51.
    Cohen SH, Lightbody ML. (1997) Atomic Force Microscopy/Scanning Tunneling Microscopy 2. Springer, New York.Google Scholar
  52. 52.
    Higgins JS, Benoît HC. (1997) Polymers and Neutron Scattering. Oxford University Press, New York.Google Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Radhika P. Nagarkar
    • 1
  • Joel P. Schneider
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of DelawareNewark

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