Peptide-amphiphile nanofibers: A versatile scaffold for the preparation of self-assembling materials Jeffrey D. Hartgerink, Elia Beniash, and Samuel I. Stupp* Departments of Chemistry and Materials Science and Engineering and the Medical School, Northwestern University, Evanston, IL 60208 Edited by Jack Halpern, University of Chicago, Chicago, IL, and approved February 12, 2002 (received for review December 26, 2001) Twelve derivatives of peptide-amphiphile molecules, designed to inscribe biological signals in the self-assembled structure. In this self-assemble into nanofibers, are described. The scope of amino system, PA 4 (Fig. 1) was chosen for the self-assembling building acid selection and alkyl tail modification in the peptide-amphiphile block to combine the advantages of peptides with those of molecules are investigated, yielding nanofibers varying in mor- amphiphiles that are known to self-assemble into sheets, spheres, phology, surface chemistry, and potential bioactivity. The results rods, disks, or channels depending on the shape, charge, and demonstrate the chemically versatile nature of this supramolecular environment (25). Amphiphiles with a conical shape in which the system and its high potential for manufacturing nanomaterials. In hydrophilic head group is somewhat bulkier than its narrow addition, three different modes of self-assembly resulting in nano- hydrophobic tail have been shown to form cylindrical micelles. fibers are described, including pH control, divalent ion induction, Examples include single-chain lipids with ionic head groups such and concentration. as SDS and hexadecyltrimethylammonium bromide (26). De- pending on the composition, amphiphilic peptides have been reprogrammed noncovalent bonds, within and between mol- shown to organize peptide secondary structure and aggregation Pecules, build highly functional and dynamic structures in state (27–30). PAs synthesized previously with mono- or di-alkyl biology, which motivates our interest in self-assembly of synthetic tails were found to associate in conformations such as triple systems. Over the past few decades a substantial amount of helical structures found in collagen (31–38). Modification of this literature describing noncovalent self-assembly of nanostruc- strategy allowed us to design a cone-shaped, ionic amphiphile tures has accumulated (1–14). However, it is still difficult to that could assemble into cylindrical micelles or peptide nanofi- design supramolecular structures, particularly if we want to start bers (17). Because of the ionic nature of the head group, with designed molecules and form objects that measure between self-assembly could be induced reversibly by changing the pH of nanoscopic and macroscopic dimensions. Developing this ability the PA solution. will take us closer to the broad, bottom-up approach of self- To enhance the physical and chemical robustness of these assembly observed in biology. supramolecular fibers, an intermolecular crosslinking scheme Our laboratory has studied over the past decade self-assembly using four consecutive cysteine residues was included in our of designed molecules into macromolecular structures of two- design. Upon oxidation of the self-assembled fibers intermolec- dimensional (15, 16), one-dimensional (17, 18), and zero- ular disulfide bonds are formed that stitch the fiber into a high CHEMISTRY dimensional nature (19–22). These self-assembled objects con- molecular weight fibrous polymer. Because this crosslinking tain between 101 and 105 molecules and thus resemble synthetic scheme uses disulfide bonds this also allows the crosslinking to and biological polymers in molar mass. The interactions that lead be self-correcting and reversible as is often seen in natural to the formation of these structures include chiral dipole–dipole protein folding and disulfide bond formation (39). Because the interactions, ␲–␲ stacking, hydrogen bonds, nonspecific van der surface chemistry of the fiber can be controlled by modifying the Waals interactions, hydrophobic forces, electrostatic interac- C-terminal region of the peptide, it is also a good scaffold to tions, and repulsive steric forces. All systems studied involved nucleate crystals or adsorb molecules in a specific orientation. In combinations of these forces that counterbalance the enormous this regard our initial work on this subject demonstrated nucle- SPECIAL FEATURE translational and rotational entropic cost caused by polymolecu- ation of hydroxyapatite nanocrystals with c-axis orientation lar aggregation. In some cases the possibility of internally linking along the fiber axis, which mimics the geometrical relationship these self-assembled structures through covalent bonds has been between apatite crystals and collagen fibrils in bone (40, 41). explored (1, 17, 20, 23). A cross-linking produces actual polymers In this work we demonstrate the structural versatility of the PA whose various shapes and dimensionalities are controlled by self-assembly into nanofibers. By modifying the alkyl tail length self-assembly and are very different from the well-known and peptide amino acid composition we examine the role played ‘‘beads-on-a-chain’’ structures of traditional polymers. by different structural units in these designed molecules. Fur- In our studies of self-assembling systems we also have explored thermore we also describe here three different methods to self-organization at length scales much greater than those of the induce self-assembly, thereby further expanding its versatility. aggregates themselves, reaching into scales of microns, millime- ters, and even centimeters. We also have been interested in Materials and Methods functionalities that emerge from self-assembly at these larger- Chemicals. Except as noted below, all chemicals were purchased length scales. An interesting example was the layering and polar from Fisher or Aldrich and used as provided. Diisopropyleth- stacking of mushroom-shaped supramolecular structures each ylamine and piperidine were redistilled before use. Amino acid measuring about 5 nm. The stem-to-cap layers of these nano- derivatives, derivatized resins, and 2-(1h-benzotriazole-1-yl)- structures result in centimeter-scale films that are spontaneously 1,1,3,3-tetramethyluronium hexafluorophosphate were pur- piezoelectric (24). The search for useful systems in the micro- chased from Nova Biochem. All water used was deionized with scopic and macroscopic regime that take advantage of molecular a Millipore Milli-Q water purifier operating at a resistance of self-assembly probably will require a combination of top-down 18 M⍀. and bottom-up approaches such as ours. Very recently we have explored the self-assembly of peptide amphiphiles (PA) in water into one-dimensional cylindrical This paper was submitted directly (Track II) to the PNAS office. objects, nanometers in diameter but microns in length (17). Abbreviations: PA, peptide-amphiphile; TEM, transmission electron microscopy. These molecules are interesting because they can be used to *To whom reprint requests should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.072699999 PNAS ͉ April 16, 2002 ͉ vol. 99 ͉ no. 8 ͉ 5133–5138 Downloaded by guest on September 29, 2021 Fig. 1. Chemical structure of PA 4. Synthesis of the PAs. The PAs were prepared on a 0.25-mmol scale by using standard fluorenylmethoxycarbonyl chemistry on an Applied Biosystems 733A automated peptide synthesizer. All peptides prepared have a C-terminal carboxylic acid and were made by using prederivatized Wang resin with the exception of peptide 10. In the case of peptide 10, 1 equivalent of Wang resin was reacted with three equivalents of fluorenylmethoxycarbonyl- Ser(PO3Bnzl)-OH, four equivalents of 1-(mesitylene-2- sulfonyl)-3-nitro-1H-1,2,4-triazole, and six equivalents of 1-methylimidazole in dichloromethane. After the peptide por- Fig. 2. Time sequence of pH-controlled PA self-assembly and disassembly. tion of the molecule was prepared, the resin was removed from (Upper) From left to right molecule 6 dissolved in water at a concentration of the automated synthesizer and the N terminus was capped with 0.5% by weight at pH 8 is exposed to HCl vapor. As the acid diffused into the a fatty acid containing 6, 10, 16 or 22 carbon atoms. The solution a gel phase is formed, which self-supports upon inversion (Far Left). alkylation reaction was accomplished by using two equivalents of (Lower) The same gel is treated with NH4OH vapor, which increases the pH and the fatty acid, two equivalents 2-(1h-benzotriazole-1-yl)-1,1,3,3- disassembles the gel, returning it to a fully dissolved solution. tetramethyluronium hexafluorophosphate, and six equivalents of diisopropylethylamine in dimethylformamide. The reaction matrix-assisted laser desorption ionization–time of flight MS and was allowed to proceed for at least6hafterwhich the reaction were found to have the expected molecular weight. was monitored by ninhydrin. The alkylation reaction was re- peated until the ninhydrin test was negative. In general the Acid-Induced Self-Assembly. Samples of the PA in question were longer the fatty acid the more repetitions were required to drive prepared as above and placed in a small glass vial with an open the reaction to completion. top. This vial and a second vial filled with 12 M HCl were placed Cleavage and deprotection of the PAs containing cysteine was together in a sealed glass chamber where the HCl vapor was done with a mixture of triflouroacetic acid, water, triisopropyl