Ronit Bitton

A self-assembly pathway to aligned monodomain gels

Aggregates of charged amphiphilic molecules have been found to access a structure at elevated temperature that templates alignment of supramolecular fibrils over macroscopic scales. The thermal pathway leads to a lamellar plaque structure with fibrous texture that breaks upon cooling into large arrays of aligned nanoscale fibres and forms a strongly birefringent liquid. By manually dragging this liquid crystal from a pipette onto salty media, it is possible to extend this alignment over centimetres in noodle-shaped viscoelastic strings. Using this approach, the solution of supramolecular filaments can be mixed with cells at physiological temperatures to form monodomain gels of aligned cells and filaments. The nature of the self-assembly process and its biocompatibility would allow formation of cellular wires in situ that have any length and customized peptide compositions for use in biological applications.

A bioactive self-assembled membrane to promote angiogenesis.

We report here on a bioactive hierarchically structured membrane formed by self-assembly. The membrane is formed with hyaluronic acid and peptide amphiphiles with binding affinity for heparin, and its hierarchical structure contains both an amorphous zone and a layer of fibrils oriented perpendicular to the membrane plane. The design of bioactivity is based on the potential ability to bind and slowly release heparin-binding growth factors. Human mesenchymal stem cells (hMSCs) seeded on these membranes attached and remained viable. Basic fibroblast growth factor (FGF2) and vascular endothelial growth factor (VEGF) were incorporated within the membrane structure prior to self-assembly and released into media over a prolonged period of time (14 days). Using the chicken chorioallantoic membrane (CAM) assay, we also found that these membranes induced a significant and rapid enhancement of angiogenesis relative to controls.

Nanostructure-templated control of drug release from peptide amphiphile nanofiber gels

High aspect ratio peptide nanofibers have potential as biodegradable vehicles for drug delivery. We report here the synthesis of four self-assembling peptide amphiphiles (PAs) containing a lysine ε-amine-derivatized hydrazide that was systematically placed at different positions along the backbone of the peptide sequence C(16)V(2)A(2)E(2) (where C(16) = palmitic acid). Hydrazones were formed from each hydrazide by condensation with the solvatochromic dye 6-propionyl-2-dimethylaminonaphthalene (Prodan), which is typically used to probe cell membranes. All four compounds were found to self-assemble into nanofibers, and Prodan release was measured from filamentous gels prepared by screening PA charges with divalent cations. Near zero-order release kinetics were observed for all nanofibers, but release half-lives differed depending on the position of the fluorophore in the PA sequence. Dye release kinetics were rationalized through the use of cryogenic transmission electron microscopy, small-angle X-ray scattering, fluorescence spectroscopy, fluorescence anisotropy, circular dichroism, and partition coefficient calculations. Relative release rates were found to correlate directly with fluorophore mobility, which varied inversely with packing density, degree of order in the hydrophobic PA core, and the β-sheet character of the peptide.

Electrostatic control of bioactivity.

The power of independence: When exhibited on the surface of self-assembling peptide-amphiphile nanofibers, the hydrophobic laminin-derived IKVAV epitope induced nanofiber bundling through interdigitation with neighboring fibers and thus decreased the bioactivity of the resulting materials. The inclusion of charged amino acids in the peptide amphiphiles disrupted the tendency to bundle and led to significantly enhanced neurite outgrowth.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein–peptide system

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

The role of nanoscale architecture in supramolecular templating of biomimetic hydroxyapatite mineralization

Understanding and mimicking the hierarchical structure of mineralized tissue is a challenge in the field of biomineralization and is important for the development of scaffolds to guide bone regeneration. Bone is a remarkable tissue with an organic matrix comprised of aligned collagen bundles embedded with nanometer-sized inorganic hydroxyapatite (HAP) crystals that exhibit orientation on the macroscale. Hybrid organic-inorganic structures mimic the composition of mineralized tissue for functional bone scaffolds, but the relationship between morphology of the organic matrix and orientation of mineral is poorly understood. Herein the mineralization of supramolecular peptide amphiphile templates, that are designed to vary in nanoscale morphology by altering the amino acid sequence, is reported. It is found that 1D cylindrical nanostructures direct the growth of oriented HAP crystals, while flatter nanostructures fail to guide the orientation found in biological systems. The geometric constraints associated with the morphology of the nanostructures may effectively control HAP nucleation and growth. Additionally, the mineralization of macroscopically aligned bundles of the nanoscale assemblies to create hierarchically ordered scaffolds is explored. Again, it is found that only aligned gel templates of cylindrical nanostructures lead to hierarchical control over hydroxyapatite orientation across multiple length scales as found in bone.

Physical properties of hierarchically ordered self-assembled planar and spherical membranes

The mechanical properties and water permeability of hierarchical self-assembling membranes and sacs formed from oppositely charged high molecular weight hyaluronic acid (HA) and small molecule peptide amphiphiles (PAs) were studied. Techniques to make reproducible 2D planar membranes and 3D spherical sacs from these materials were developed while membrane inflation and osmotic swelling were used to quantify the mechanical properties and water permeability of these structures. It was found that incubation time and concentration of HA used had an effect on the area modulus and water permeability of the membranes. These factors also affected the kinetics of membrane growth as evidenced in SEM micrographs, which showed differences in the structure. Area modulus of membranes changed from about 6 N m−1 for the lower weight percent HA system at the shortest incubation time of 3 minutes, up to 12 N m−1 for the higher weight percent HA system at the longest incubation time of 60 minutes. Water permeability decreased with incubation time, but the lower weight percent HA system showed a lower water permeability when compared to the higher weight percent HA system at the same incubation time. This type of characterization and understanding of the structure–property relationships in self-assembling systems are necessary steps in both using these structures for specific applications and applying this knowledge to design new and better materials in the future.

Novel Biomimetic Adhesives Based on Algae Glue

Inspired by the remarkable adhesive capabilities to wet surfaces of the secretes of the brown alga Fucus serratus, novel glues have been designed and characterized. Formulations composed of phloroglucinol, alginate, and calcium ions are capable of adhering to a variety of surfaces. Rheological data show that the presence of phloroglucinol lowers the amount of Ca(2+) ions required for sol-gel transition, which indicates interactions between the alginate and the phloroglucinol. SAXS data support this claim. The phloroglucinol adhesive binds porcine tissues together with an adhesive strength of 17-25 kPa, which indicates appropriate mechanical properties for application as a soft tissue adhesive.

Cooperative DNA binding and assembly by a bZip peptide-amphiphile

The bipartite basic zipper (bZip) GCN4 peptide, containing a leucine zipper and a basic binding region, is a well-studied transcription factor that can be rationally adapted to control dimerization or assembly. We have covalently appended alkyl tails to the C-terminus (leucine zipper terminus) of a bZip sequence, yielding mono- and dialkyl bZip peptide-amphiphiles that allowed us to investigate how molecular design can control the formation of secondary structure and self-assembled structure. We demonstrate that these peptide-amphiphiles exhibit four qualities that are representative of their modular construction. First, circular dichroism confirms that self-assembly of peptide-amphiphiles above the critical micelle concentration (CMC) results in an enhanced α-helical secondary structure as peptide head groups are confined to the assembled interface with high local concentrations. Second, the binding of the peptide-amphiphiles to DNA yields a further increase in secondary structure, where the helicity of the basic binding region is stabilized by forming native-like contacts, an “induced fit mechanism”. Third, competitive fluorescence binding assays show peptide-amphiphiles bind cooperatively to DNA well below the CMC, where DNA templates monomeric binding and hydrophobic forces promote cooperativity, but the ability of the peptide to recognize a specific DNA sequence is not retained. And fourth, SANS results demonstrate the assembly of large lamellar aggregates as peptide-amphiphiles complex with DNA, supporting a structural hypothesis in which peptide-amphiphiles bind to the DNA in a native-like ‘standing’ orientation. These designed synthetic molecular architectures are capable of hierarchical assembly making them useful as functional building blocks that may be applied to a variety of systems, including gene delivery and artificial transcription factors.

Phloroglucinol-based biomimetic adhesives for medical applications.

An adhesive that functions well under moist conditions could facilitate many surgical procedures. In recent studies we designed novel biomimetic glues which mimic the adhesion mechanism of algae, renowned for their remarkable adherence to wet surfaces. Here we extend our previous studies and propose biomimetic formulations, composed of alginate gel and native phloroglucinol, that do not induce cell cytotoxicity. Characterization of the adherence to tissues showed that adhesion was directly related to the mechanical strength of the cross-linked alginate. Therefore the adhesion strength can be altered by changing the source of the calcium cross-linker, the alginate G-content or the molecular weight of the alginate. The adhesion strength was comparable to that of Tisseel, a commercial tissue adhesive.