Darrin J. Pochan

Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles

Protein-based hydrogels are used for many applications, ranging from food and cosmetic thickeners to support matrices for drug delivery and tissue replacement. These materials are usually prepared using proteins extracted from natural sources, which can give rise to inconsistent properties unsuitable for medical applications. Recent developments have utilized recombinant DNA methods to prepare artificial protein hydrogels with specific association mechanisms and responsiveness to various stimuli. Here we synthesize diblock copolypeptide amphiphiles containing charged and hydrophobic segments. Dilute solutions of these copolypeptides would be expected to form micelles; instead, they form hydrogels that retain their mechanical strength up to temperatures of about 90 °C and recover rapidly after stress. The use of synthetic materials permits adjustment of copolymer chain length and composition, which we varied to study their effect on hydrogel formation and properties. We find that gelation depends not only on the amphiphilic nature of the polypeptides, but also on chain conformations—α-helix, β-strand or random coil. Indeed, shape-specific supramolecular assembly is integral to the gelation process, and provides a new class of peptide-based hydrogels with potential for applications in biotechnology.

Controlling hydrogelation kinetics by peptide design for three-dimensional encapsulation and injectable delivery of cells

A peptide-based hydrogelation strategy has been developed that allows homogenous encapsulation and subsequent delivery of C3H10t1/2 mesenchymal stem cells. Structure-based peptide design afforded MAX8, a 20-residue peptide that folds and self-assembles in response to DMEM resulting in mechanically rigid hydrogels. The folding and self-assembly kinetics of MAX8 have been tuned so that when hydrogelation is triggered in the presence of cells, the cells become homogeneously impregnated within the gel. A unique characteristic of these gel–cell constructs is that when an appropriate shear stress is applied, the hydrogel will shear-thin resulting in a low-viscosity gel. However, after the application of shear has stopped, the gel quickly resets and recovers its initial mechanical rigidity in a near quantitative fashion. This property allows gel/cell constructs to be delivered via syringe with precision to target sites. Homogenous cellular distribution and cell viability are unaffected by the shear thinning process and gel/cell constructs stay fixed at the point of introduction, suggesting that these gels may be useful for the delivery of cells to target biological sites in tissue regeneration efforts.

Rheological properties of peptide-based hydrogels for biomedical and other applications.

Peptide-based hydrogels are an important class of biomaterials finding use in food industry and potential use in tissue engineering, drug delivery and microfluidics. A primary experimental method to explore the physical properties of these hydrogels is rheology. A fundamental understanding of peptide hydrogel mechanical properties and underlying molecular mechanisms is crucial for determining whether these biomaterials are potentially suitable for biotechnological uses. In this critical review, we cover the literature containing rheological characterization of the physical properties of peptide and polypeptide-based hydrogels including hydrogel bulk mechanical properties, gelation mechanisms, and the behavior of hydrogels during and after flow (219 references).

Encapsulation of curcumin in self-assembling peptide hydrogels as injectable drug delivery vehicles.

Curcumin, a hydrophobic polyphenol, is an extract of turmeric root with antioxidant, anti-inflammatory and anti-tumorigenic properties. Its lack of water solubility and relatively low bioavailability set major limitations for its therapeutic use. In this study, a self-assembling peptide hydrogel is demonstrated to be an effective vehicle for the localized delivery of curcumin over sustained periods of time. The curcumin-hydrogel is prepared in-situ where curcumin encapsulation within the hydrogel network is accomplished concurrently with peptide self-assembly. Physical and in vitro biological studies were used to demonstrate the effectiveness of curcumin-loaded β-hairpin hydrogels as injectable agents for localized curcumin delivery. Notably, rheological characterization of the curcumin-loaded hydrogel before and after shear flow have indicated solid-like properties even at high curcumin payloads. In vitro experiments with a medulloblastoma cell line confirm that the encapsulation of the curcumin within the hydrogel does not have an adverse effect on its bioactivity. Most importantly, the rate of curcumin release and its consequent therapeutic efficacy can be conveniently modulated as a function of the concentration of the MAX8 peptide.

Self-Assembly and Hydrogelation of an Amyloid Peptide Fragment

The self-assembly of a fragment of the amyloid beta peptide that has been shown to be critical in amyloid fibrillization has been studied in aqueous solution. There are conflicting reports in the literature on the fibrillization of Abeta (16-20), i.e., KLVFF, and our results shed light on this. In dilute solution, self-assembly of NH 2-KLVFF-COOH is strongly influenced by aromatic interactions between phenylalanine units, as revealed by UV spectroscopy and circular dichroism. Fourier transform infrared (FTIR) spectroscopy reveals beta-sheet features in spectra taken for more concentrated solutions and also dried films. X-ray diffraction and cryo-transmission electron microscopy (cryo-TEM) provide further support for beta-sheet amyloid fibril formation. A comparison of cryo-TEM images with those from conventional dried and negatively stained TEM specimens highlights the pronounced effects of sample preparation on the morphology. A comparison of FTIR data for samples in solution and dried samples also highlights the strong effect of drying on the self-assembled structure. In more concentrated phosphate-buffered saline (PBS) solution, gelation of NH 2-KLVFF-COOH is observed. This is believed to be caused by screening of the electrostatic charge on the peptide, which enables beta sheets to aggregate into a fibrillar gel network. The rheology of the hydrogel is probed, and the structure is investigated by light scattering and small-angle X-ray scattering.

Macromolecular diffusion and release from self-assembled β-hairpin peptide hydrogels

Self-assembling peptide hydrogels are used to directly encapsulate and controllably release model FITC-dextran macromolecules of varying size and hydrodynamic diameters. MAX1 and MAX8 are two peptide sequences with different charge states that have been designed to intramolecularly fold and self assemble into hydrogels at physiological buffer conditions (pH 7.4, 150 mM NaCl). When self-assembly is initiated in the presence of dextran or protein probes, these macromolecules are directly encapsulated in the gel. Self-diffusion studies using fluorescence recovery after photobleaching (FRAP) and bulk release studies indicate that macromolecule mobility within, and release out of, these gels can be modulated by varying the hydrogel mesh size. The average mesh size can be modulated by simply varying the concentration of a given peptide used to construct the gel or by altering the peptide sequence. In addition, results suggest that electrostatic interactions between the macromolecules and the peptide network influence mobility and release. Depending on probe size, release half-lives can be varied from 8h to over a month.

Modular synthesis of amphiphilic Janus glycodendrimers and their self-assembly into glycodendrimersomes and other complex architectures with bioactivity to biomedically relevant lectins

The modular synthesis of 7 libraries containing 51 self-assembling amphiphilic Janus dendrimers with the monosaccharides D-mannose and D-galactose and the disaccharide D-lactose in their hydrophilic part is reported. These unprecedented sugar-containing dendrimers are named amphiphilic Janus glycodendrimers. Their self-assembly by simple injection of THF or ethanol solution into water or buffer and by hydration was analyzed by a combination of methods including dynamic light scattering, confocal microscopy, cryogenic transmission electron microscopy, Fourier transform analysis, and micropipet-aspiration experiments to assess mechanical properties. These libraries revealed a diversity of hard and soft assemblies, including unilamellar spherical, polygonal, and tubular vesicles denoted glycodendrimersomes, aggregates of Janus glycodendrimers and rodlike micelles named glycodendrimer aggregates and glycodendrimermicelles, cubosomes denoted glycodendrimercubosomes, and solid lamellae. These assemblies are stable over time in water and in buffer, exhibit narrow molecular-weight distribution, and display dimensions that are programmable by the concentration of the solution from which they are injected. This study elaborated the molecular principles leading to single-type soft glycodendrimersomes assembled from amphiphilic Janus glycodendrimers. The multivalency of glycodendrimersomes with different sizes and their ligand bioactivity were demonstrated by selective agglutination with a diversity of sugar-binding protein receptors such as the plant lectins concanavalin A and the highly toxic mistletoe Viscum album L. agglutinin, the bacterial lectin PA-IL from Pseudomonas aeruginosa, and, of special biomedical relevance, human adhesion/growth-regulatory galectin-3 and galectin-4. These results demonstrated the candidacy of glycodendrimersomes as new mimics of biological membranes with programmable glycan ligand presentations, as supramolecular lectin blockers, vaccines, and targeted delivery devices.

Helix self-assembly through the coiling of cylindrical micelles

Both single and double helical superstructures have been created through solution self-assembly of cylindrical micelles for the first time. Helical micelles were formed from the co-assembly of poly(acrylic acid)-block-poly(methyl acrylate)-block-polystyrene (PAA-b-PMA-b-PS) triblock copolymers with different multiamines. The helix pitch could be adjusted by adjusting the amount and type of multiamine added. The helical structure exhibits unprecedented regularity for a nanostructure self-assembled from solution indicating the presence of strong, synergistic forces underlying the helix formation.

The effect of protein structure on their controlled release from an injectable peptide hydrogel.

Hydrogel materials are promising vehicles for the delivery of protein therapeutics. Proteins can impart physical interactions, both steric and electrostatic in nature, that influence their release from a given gel network. Here, model proteins of varying hydrodynamic diameter and charge are directly encapsulated and their release studied from electropositive fibrillar hydrogels prepared from the self-assembling peptide, MAX8. Hydrogelation of MAX8 can be triggered in the presence of proteins for their direct encapsulation with neither effect on protein structure nor the hydrogels mechanical properties. Bulk release of the encapsulated proteins from the hydrogels was assessed for a month time period at 37 °C before and after syringe delivery of the loaded gels to determine the influence of the protein structure on release. Release of positively charged and neutral proteins was largely governed by the sterics imposed by the network. Conversely, negatively charged proteins interacted strongly with the positively charged fibrillar network, greatly restricting their release to <10% of the initial protein load. Partition and retention studies indicated that electrostatic interactions dictate the amount of protein available for release. Importantly, when protein encapsulated gels were delivered via syringe, the release profiles of the macromolecules show the similar trends as those observed for non-sheared gels. This study demonstrates that proteins can be directly encapsulated in self assembled MAX8 hydrogels, which can then be syringe delivered to a site where subsequent release is controlled by protein structure.

Injectable solid hydrogel: mechanism of shear-thinning and immediate recovery of injectable β-hairpin peptide hydrogels

β-Hairpin peptide-based hydrogels are a class of injectable hydrogel solids with significant potential use in injectable therapies. β-hairpin peptide hydrogels can be injected as preformed solids, because the solid gel can shear-thin and consequently flow under a proper shear stress but immediately recover back into a solid on removal of the stress. In this work, hydrogel behavior during and after flow was studied in order to facilitate fundamental understanding of how the gels flow during shear-thinning and how they quickly recover mechanically and morphologically relative to their original, pre-flow properties. While all studied β-hairpin hydrogels shear-thin and recover, the duration of shear and the strain rate affected both the gel stiffness immediately recovered after flow and the ultimate stiffness obtained after complete rehealing of the gel. Results of structural analysis during flow were related to bulk rheological behavior and indicated gel network fracture into large (>200 nm) hydrogel domains during flow. After cessation of flow the large hydrogel domains are immediately percolated which immediately reforms the solid hydrogel. The underlying mechanisms of the gel shear-thinning and healing processes are discussed relative to other shear-responsive networks like colloidal gels and micellar solutions.