Timothy J. Deming

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.

Biomimetic synthesis of ordered silica structures mediated by block copolypeptides

In biological systems such as diatoms and sponges, the formation of solid silica structures with precisely controlled morphologies is directed by proteins and polysaccharides and occurs in water at neutral pH and ambient temperature. Laboratory methods, in contrast, have to rely on extreme pH conditions and/or surfactants to induce the condensation of silica precursors into specific morphologies or patterned structures. This contrast in processing conditions and the growing demand for benign synthesis methods that minimize adverse environmental effects have spurred much interest in biomimetic approaches in materials science. The recent demonstration that silicatein—a protein found in the silica spicules of the sponge Tethya aurantia—can hydrolyse and condense the precursor molecule tetraethoxysilane to form silica structures with controlled shapes at ambient conditions seems particularly promising in this context. Here we describe synthetic cysteine-lysine block copolypeptides that mimic the properties of silicatein: the copolypeptides self-assemble into structured aggregates that hydrolyse tetraethoxysilane while simultaneously directing the formation of ordered silica morphologies. We find that oxidation of the cysteine sulphydryl groups, which is known to affect the assembly of the block copolypeptide, allows us to produce different structures: hard silica spheres and well-defined columns of amorphous silica are produced using the fully reduced and the oxidized forms of the copolymer, respectively.

Astrocyte scar formation aids central nervous system axon regeneration

Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration.

Mussel byssus and biomolecular materials

Mussel adhesive proteins are remarkable materials that display an extraordinary capability to adhere to substrates underwater. Recent investigations from groups with quite diverse areas of expertise have made substantial progress in the identification of the genes and proteins that are involved in adhesive formation. These discoveries have led to the development of recombinant proteins and synthetic polypeptides that are able to reproduce the properties of mussel adhesives for applications in medicine and biotechnology.

Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions

Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using “nunchuck” CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay (“negative Gaussian”) membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.

Nanoscale double emulsions stabilized by single-component block copolypeptides

Water-in-oil-in-water emulsions are examples of double emulsions, in which dispersions of small water droplets within larger oil droplets are themselves dispersed in a continuous aqueous phase. Emulsions occur in many forms of processing and are used extensively by the foods, cosmetics and coatings industries. Because of their compartmentalized internal structure, double emulsions can provide advantages over simple oil-in-water emulsions for encapsulation, such as the ability to carry both polar and non-polar cargos, and improved control over release of therapeutic molecules. The preparation of double emulsions typically requires mixtures of surfactants for stability; the formation of double nanoemulsions, where both inner and outer droplets are under 100 nm, has not yet been achieved. Here we show that water-in-oil-in-water double emulsions can be prepared in a simple process and stabilized over many months using single-component, synthetic amphiphilic diblock copolypeptide surfactants. These surfactants even stabilize droplets subjected to extreme flow, leading to direct, mass production of robust double nanoemulsions that are amenable to nanostructured encapsulation applications in foods, cosmetics and drug delivery.

Living polymerization of α‐amino acid‐N‐carboxyanhydrides

This article reviews recent developments in the polymerization of α-amino acid- N-carboxyanhydrides (NCAs) to form polypeptides. Traditional methods used to polymerize these monomers are described, and limitations in the utility of these systems for the preparation of polypeptides with controlled molecular weights and narrow molecular weight distributions are discussed. The development of transition-metal-based initiators, which activate the monomers to form covalent active species, permits the formation of polypeptides via the living polymerization of NCAs. In these systems, polymer molecular weights are controlled by monomer-to-initiator stoichiometry, polydispersities are low, and block copolypeptides can be prepared. The scope and limitations of these initiators and their key features and mode of operation are described in detail in this highlight.

Methodologies for preparation of synthetic block copolypeptides: materials with future promise in drug delivery

This article summarizes recent developments in the synthesis of copolypeptides, focusing on the realization of living polymerizations that allow preparation of materials with excellent attributes for drug delivery. Traditional methods used to polymerize alpha-amino acid-N-carboxyanhydrides (NCAs) are described, and limitations in the utility of these systems for the preparation of polypeptides are discussed. A system utilizing transition metal catalysis for polypeptide synthesis is described that allows preparation of materials that begin to rival biologically produced counterparts in terms of complexity and purity. Overall, new developments in NCA and beta-lactam polymerizations hold tremendous promise now that block copolypeptides of controlled dimensions (molecular weight, sequence, composition, and molecular weight distribution) can be prepared. Such well-defined polymers should greatly assist in the development of new drug carriers with increased function.

Polypeptide hydrogels via a unique assembly mechanism

There is a long history of mans use of materials derived from peptides and proteins. These natural materials possess sophisticated mechanisms of nanoscale self assembly, which have inspired the design of many synthetic and biosynthetic amino-acid based materials. These materials are attractive since they can have exceptional properties, environmental responsive behavior, biological activity, and can be metabolized. With all of their complexity, peptides and proteins rely primarily on two fundamental modes of self assembly: association of β-strands and the coiling of helices. In this context, a class of recently synthesized and characterized polypeptide materials are reviewed here, which were found to self-assemble by a fundamentally different process. This new mode of assembly was found to give rise to polypeptide hydrogels with a unique combination of properties ( heat stability and injectability) making them attractive for applications in foods, personal care products, and medicine.

Glycopolypeptides via Living Polymerization of Glycosylated-l-lysine N-Carboxyanhydrides

The preparation of new glycosylated-L-lysine-N-carboxyanhydride (glyco-K NCA) monomers is described. These monomers employ C-linked sugars and amide linkages to lysine for improved stability without sacrificing biochemical properties. Three glyco-K NCAs were synthesized, purified, and found to undergo living polymerization using transition metal initiation. These are the first living polymerizations of glycosylated NCAs and were used to prepare well-defined, high molecular weight glycopolypeptides and block and statistical glycocopolypeptides. This methodology solves many long-standing problems in the direct synthesis of glycopolypeptides from N-carboxyanhydrides relating to monomer synthesis, purification, and polymerization and gives polypeptides with 100% glycosylation. These long chain glycopolypeptides have potential to be good mimics of natural high molecular weight glycoproteins.