Two complementary strategies can be used in the fabrication of molecular biomaterials. In the 'top-down' approach, biomaterials are generated by stripping down a complex entity into its component parts (for example, paring a virus particle down to its capsid to form a viral cage). This contrasts with the 'bottom-up' approach, in which materials are assembled molecule by molecule (and in some cases even atom by atom) to produce novel supramolecular architectures. The latter approach is likely to become an integral part of nanomaterials manufacture and requires a deep understanding of individual molecular building blocks and their structures, assembly properties and dynamic behaviors. Two key elements in molecular fabrication are chemical complementarity and structural compatibility, both of which confer the weak and noncovalent interactions that bind building blocks together during self-assembly. Using natural processes as a guide, substantial advances have been achieved at the interface of nanomaterials and biology, including the fabrication of nanofiber materials for three-dimensional cell culture and tissue engineering, the assembly of peptide or protein nanotubes and helical ribbons, the creation of living microlenses, the synthesis of metal nanowires on DNA templates, the fabrication of peptide, protein and lipid scaffolds, the assembly of electronic materials by bacterial phage selection, and the use of radiofrequency to regulate molecular behaviors.

A 16-residue peptide [(Ala-Glu-Ala-Glu-AlaLys-Ala-Lys)2J has a characteristic 13-sheet circular dichroism spectrum in water. Upon the addition of salt, the peptide spontaneously assembles to form a macroscopic membrane. The membrane does not dissolve in heat or in acidic or alkaline solutions, nor does it dissolve upon addition of guanidine hydrochloride, SDS/urea, or a variety of proteolytic enzymes.Scanning EM reveals a network of interwoven framents ""10-20 nm in diameter. An important component of the stability is probably due to formation of complementary ionic bonds between glutamic and lysine side chains. This phenomenon may be a model for studying the insoluble peptides found in certain neurological disorders. It may also have implications for biomaterials and origin-of-life research.Peptides of alternating hydrophilic and hydrophobic amino acid residues have a tendency to adopt a (3-sheet structure. The complete sequence of (Ala-Glu-Ala-Glu-Ala-Lys-AlaLys)2 (EAK16) was originally found in a region of alternating hydrophobic and hydrophilic residues in zuotin, a yeast protein that was initially identified for its ability to bind preferentially to left-handed Z-DNA (1). Previous studies with alternating amphiphilic-peptide polymers-e.g., poly-(Val-Lys), poly(Glu-Ala), poly(Tyr-Glu), poly(Lys-Phe), poly(Lys-Leu)-and oligopeptides [(Val-Glu-Val-Orn)j_3]-Val (2-7) have shown that these polymers can adopt (3-sheet structures and can aggregate, depending upon pH, salt, and time. However, self-complementary EAK16 is distinctive in that it forms an insoluble macroscopic membrane. MATERIALS AND METHODSPeptides. The Glu-Ala-Lys peptides were synthesized by a peptide synthesizer (Applied Biosystems), purified by reverse-phase HPLC, and eluted by a linear gradient of 5-80% acetonitrile/0.1% trifluoacetic acid. The peptide stock solutions were dissolved in water (1-5 mg/ml) or in 23% acetonitrile (10 mg/ml). The concentrations of the peptides were determined by dissolving dried peptide in water (wt/vol) and centrifuging the solution. A portion of the solution was then analyzed by hydrolysis with internal controls. The sequence of the peptides was confirmed by microsequencing. The composition of the peptides was confwrmed by hydrolytic analysis. Ala-Glu-Ala-Lys-Ala-Glu-Ala-Glu-Ala-Lys-AlaLys (EAK12) and EAK16 are acetylated and aminated at the N-and C-terminal ends, respectively. Blocking of both N and C termini of EAK16 appears nonessential for membrane formation.CD Measurement. CD spectra were gathered on an Aviv model 6ODS spectropolarimeter with 6OHDS software for data processing. Because EAK16 contains both positively and negatively charged residues, the peptide itself can serve as a buffer. CD samples were prepared by diluting stock peptide solution (1-5 mg/ml) in water.Membrane Preparations. The membranes were prepared as follows: 5-10 ,4 of the stock solution of EAK16 peptide (1-5 mg/ml) was added to 0.5-1.0 ml of phosphate-buffered saline (150 mM NaCl/10 mM sodium phosphate, pH 7.4) with 0.00001% Con...

A new type of self-assembling peptide (sapeptide) scaffolds that serve as substrates for neurite outgrowth and synapse formation is described. These peptide-based scaffolds are amenable to molecular design by using chemical or biotechnological syntheses. They can be tailored to a variety of applications. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary ␤-sheet oligopeptides under physiological conditions, producing a hydrogel material. The scaffolds can support neuronal cell attachment and differentiation as well as extensive neurite outgrowth. Furthermore, they are permissive substrates for functional synapse formation between the attached neurons. That primary rat neurons form active synapses on such scaffold surfaces in situ suggests these scaffolds could be useful for tissue engineering applications. The buoyant sapeptide scaffolds with attached cells in culture can be transported readily from one environment to another. Furthermore, these peptides did not elicit a measurable immune response or tissue inflammation when introduced into animals. These biological materials created through molecular design and self assembly may be developed as a biologically compatible scaffold for tissue repair and tissue engineering.biological materials ͉ cell attachment ͉ molecular material design ͉ primary neurons ͉ PC12 cells

Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.three-dimensional cell culture ͉ biological scaffold ͉ regenerative medicine

Several surfactant-like peptides undergo self-assembly to form nanotubes and nanovesicles having an average diameter of 30 -50 nm with a helical twist. The peptide monomer contains 7-8 residues and has a hydrophilic head composed of aspartic acid and a tail of hydrophobic amino acids such as alanine, valine, or leucine. The length of each peptide is Ϸ2 nm, similar to that of biological phospholipids. Dynamic light-scattering studies showed structures with very discrete sizes. The distribution becomes broader over time, indicating a very dynamic process of assembly and disassembly. Visualization with transmission electron microscopy of quickfreeze͞deep-etch sample preparation revealed a network of openended nanotubes and some vesicles, with the latter being able to ''fuse'' and ''bud'' out of the former. The structures showed some tail sequence preference. Many three-way junctions that may act as links between the nanotubes have been observed also. Studies of peptide surfactant molecules have significant implications in the design of nonlipid biological surfactants and the understanding of the complexity and dynamics of the self-assembly processes.amino acids ͉ charged and hydrophobic residues ͉ nonlipid surfactants ͉ simplicity to complexity ͉ prebiotic enclosures M olecular self-assembly recently has attracted considerable attention for its use in the design and fabrication of nanostructures leading to the development of advanced materials (1, 2). The self-assembly of biomolecular building blocks plays an increasingly important role in the discovery of new materials and scaffolds (3, 4), with a wide range of applications in nanotechnology and medical technologies such as regenerative medicine and drug delivery systems (5, 6). Recently, Hartgerink et al. (7) reported the design of a chimeric material consisting of a hydrophobic alkyl tail and a hydrophilic peptide containing phosphorylated serine with an RGD motif that facilitates directional alignment of mineralization of hydroxyapatite.We previously described a class of ionic self-complementary peptide that spontaneously self-assemble to form interwoven nanofibers in the presence of monovalent cations (8 -10). These nanofibers further form a hydrogel consisting of greater than 99.5% water. The constituent of the hydrogel scaffold is made of peptides with alternating hydrophilic and hydrophobic amino acids. Such a sequence has a tendency to form an unusually stable ␤-sheet structure in water (8 -10). When the peptides form a ␤-sheet, they exhibit two surfaces, a hydrophilic surface consisting of charged ionic side chains and a hydrophobic surface with hydrophobic side chains. As a result, the self-assembly of these peptides is facilitated by electrostatic interactions on one side and the hydrophobic interaction on the other, in addition to the conventional ␤-sheet hydrogen bond along the backbones. The self-assembling peptide scaffolds have been demonstrated to serve as substrate for tissuecell attachment, extensive neurite outgrowth, and formation of active n...

Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with β-Tubulin+, GFAP+ and Nestin+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology.

Nanofiber structures of some peptides and proteins as biological materials have been studied extensively, but their molecular mechanism of self-assembly and reassembly still remains unclear. We report here the reassembly of an ionic self-complementary peptide RADARADARADARADA (RADA16-I) that forms a well defined nanofiber scaffold. The 16-residue peptide forms stable ␤-sheet structure and undergoes molecular self-assembly into nanofibers and eventually a scaffold hydrogel consisting of >99.5% water. In this study, the nanofiber scaffold was sonicated into smaller fragments. Circular dichroism, atomic force microscopy, and rheology were used to follow the kinetics of the reassembly. These sonicated fragments not only quickly reassemble into nanofibers that were indistinguishable from the original material, but their reassembly also correlated with the rheological analyses showing an increase of scaffold rigidity as a function of nanofiber length. The disassembly and reassembly processes were repeated four times and, each time, the reassembly reached the original length. We proposed a plausible sliding diffusion model to interpret the reassembly involving complementary nanofiber cohesive ends. This reassembly process is important for fabrication of new scaffolds for 3D cell culture, tissue repair, and regenerative medicine.atomic force microscopy ͉ circular dichroism ͉ dynamic behaviors ͉ ionic self-complementary peptides ͉ nanofiber hydrogels M olecular design, development, and fabrication of biological materials are a prerequisite for the advancement of medical technologies. These include scaffolds for fostering tissue regeneration, tissue engineering in regenerative medicine, and controlled drug release (1-7). Synthetic polymers and biodegradable biomaterials have had a significant impact in medicine over the last two decades (8-10). However, the continuous discovery and design of materials of biological origins are of great interest to multiple and diverse scientific and medical communities. The fabrication of materials at the molecular scale from ''the bottom up,'' one molecule at a time through synthesis and one unit at a time through self-assembly, has many advantages (11,12). This approach is not only flexible and simple, but these materials can be tailor-made, thus facilitating the incorporation of many biochemically and medically desired features.We previously reported the discovery and development of a class of self-assembling peptide scaffold materials to culture cells in three dimensions (13-17). These short, 8-to 16-residue (Ϸ2.5-5 nm in length) peptides are chemically synthesized and form extremely stable ␤-sheet structures in water (13, 14). They not only self-assemble to form stable nanofibers, but also form higher-order nanofiber scaffolds, namely, hydrogels with extremely high water content [Ͼ99.5 (wt͞vol)% water] (15-17). The gelation process is accelerated either by changing to neutral pH or adding physiological concentrations of salt solutions (13-15, 18-21). However, although it has high wat...

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