Preparation and stability of pegylated poly(S-alkyl-L-homocysteine) coacervate core micelles in aqueous media.

We report development and preparation of synthetic polypeptide based, coacervate core polyelectrolyte complex micelles, PCMs, in aqueous media, which were characterized and evaluated for the encapsulation and in vitro release of a model single-stranded RNA, polyadenylic acid, poly(A). Cationic, α-helical polypeptides pegylated at their N-termini, PEG113-b-5bn and PEG113-b-5cn, were designed to form coacervate core PCMs upon mixing with multivalent anions in aqueous media. Sodium tripolyphosphate (TPP) and poly(A) were used as model multivalent anions that allowed optimization of polypeptide composition and chain length for formation of stable, nanoscale PCMs. PEG113-b-5c27 was selected for preparation of PCMs that were characterized under different environmental conditions using dynamic light scattering, atomic force microscopy and cryoelectron microscopy. The PCMs were found to efficiently encapsulate poly(A), were stable at physiologically relevant pH and solution ionic strength, and were able to release poly(A) in the presence of excess polyvalent anions. These PCMs were found to be a promising model system for further development of polypeptide based therapeutic delivery vehicles.

Poly(dehydroalanine): Synthesis, Properties, and Functional Diversification of a Fluorescent Polypeptide.

Via the design of a new, soluble poly(S-alkyl-l-cysteine) precursor, a route was developed for the successful preparation of long-chain poly(dehydroalanine), ADH, as well as the incorporation of dehydroalanine residues and ADH segments into copolypeptides. Based on experimental and computational data, ADH was found to adopt a previously unobserved "hybrid coil" structure, which combines the elements of 25-helical and 310-helical conformations. Analysis of the spectroscopic properties of ADH revealed that it possesses a strong inherent blue fluorescence, which may be amenable for use in imaging applications. ADH also contains reactive electrophilic groups that allowed its efficient modification to functionalized polypeptides after reactions under mild conditions with thiol and amine nucleophiles. The combined structural, spectroscopic, and reactivity properties of ADH make it a unique reactive and fluorescent polypeptide component for utilization in self-assembled biomaterials.

Active Controlled and Tunable Coacervation Using Side-Chain Functional α-Helical Homopolypeptides.

We report the development of new side-chain amino acid-functionalized α-helical homopolypeptides that reversibly form coacervate phases in aqueous media. The designed multifunctional nature of the side-chains was found to provide a means to actively control coacervation via mild, biomimetic redox chemistry as well as allow response to physiologically relevant environmental changes in pH, temperature, and counterions. These homopolypeptides were found to possess properties that mimic many of those observed in natural coacervate forming intrinsically disordered proteins. Despite ordered α-helical conformations that are thought to disfavor coacervation, molecular dynamics simulations of a polypeptide model revealed a high degree of side-chain conformational disorder and hydration around the ordered backbone, which may explain the ability of these polypeptides to form coacervates. Overall, the modular design, uniform nature, and ordered chain conformations of these polypeptides were found to provide a well-defined platform for deconvolution of molecular elements that influence biopolymer coacervation and tuning of coacervate properties for downstream applications.

Modification of Poly(5,6-epoxy-l-norleucine) Gives Functional Polypeptides with Alternative Side-Chain Linkages.

The preparation and characterization of a new epoxide containing polypeptide, poly(5,6-epoxy-l-norleucine), via postpolymerization modification of poly(l-homoallylglycine) is described. Addition of thiols to the epoxide groups in poly(5,6-epoxy-l-norleucine) was studied as a means to prepare side-chain functional polypeptides. The solution properties of the derivatized polypeptides were studied in water and compared to similar thioether containing functional polypeptides prepared via different routes. Subtle differences in side-chain linkage chemistry were found to influence polypeptide solubility, chain conformation in solution, and thermoresponsive behavior. Poly(5,6-epoxy-l-norleucine) was found to be useful as a readily prepared intermediate that can be reacted with thiols to give a variety of functional polypeptides.