Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems.

Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

Therapeutic nanocarriers comprising extracellular matrix-inspired peptides and polysaccharides.

INTRODUCTION: The extracellular matrix (ECM) is vital for cell and tissue development. Given its importance, extensive work has been conducted to develop biomaterials and drug delivery vehicles that capture features of ECM structure and function. AREAS COVERED: This review highlights recent developments of ECM-inspired nanocarriers and their exploration for drug and gene delivery applications. Nanocarriers that are inspired by or created from primary components of the ECM (e.g. elastin, collagen, hyaluronic acid (HA), or combinations of these) are explicitly covered. An update on current clinical trials employing elastin-like proteins is also included. EXPERT OPINION: Novel ECM-inspired nanoscale structures and conjugates continue to be of great interest in the materials science and bioengineering communities. Hyaluronic acid nanocarrier systems in particular are widely employed due to the functional activity of HA in mediating a large number of disease states. In contrast, collagen-like peptide nanocarriers are an emerging drug delivery platform with potential relevance to a myriad of ECM-related diseases, making their continued study most pertinent. Elastin-like peptide nanocarriers have a well-established tolerability and efficacy track record in preclinical analyses that has motivated their recent advancement into the clinical arena.

Manipulation of the dually thermoresponsive behavior of peptide-based vesicles through modification of collagen-like peptide domains.

Materials that respond to temporally defined exogenous cues continue to be an active pursuit of research toward on-demand nanoparticle drug delivery applications, and using one or more exogenous temperature stimuli could significantly expand the application of nanoparticle-based drug delivery formulations under both hyperthermal and hypothermal conditions. Previously we have reported the development of a biocompatible and thermoresponsive elastin-b-collagen-like polypeptide (ELP-CLP) conjugate that is capable of self-assembling into vesicles and encapsulating small molecule therapeutics that can be delivered at different rates via a single temperature stimulus. Herein we report the evaluation of multiple ELP-CLP conjugates, demonstrating that the inverse transition temperature (T t) of the ELP-CLPs can be manipulated by modifying the melting temperature (T m) of the CLP domain, and that the overall hydrophilicity of the ELP-CLP conjugate also may alter the T t. Based on these design parameters, we demonstrate that the ELP-CLP sequence (VPGFG)6-(GPO)7GG can self-assemble into stable vesicles at 25°C and dissociate at elevated temperatures by means of the unfolding of the CLP domain above its T m. We also demonstrate here for the first time the ability of this ELP-CLP vesicle to dissociate via a hypothermic temperature stimulus by means of exploiting the inverse transition temperature (T t) phenomena found in ELPs. The development of design rules for manipulating the thermal properties of these bioconjugates will enable future modifications to either the ELP or CLP sequences to more finely tune the transitions of the conjugates for specific biomedical applications.

Automated High-Throughput Synthesis of Protein-Loaded Polyanhydride Nanoparticle Libraries.

The development of high-throughput techniques and combinatorial libraries can facilitate rapid synthesis and screening of biomaterial-based nanocarriers for drug and vaccine delivery. This study describes a high-throughput method using an automated robot for synthesizing polyanhydride nanoparticles encapsulating proteins. Polyanhydrides are a class of safe and biodegradable polymers that have been widely used as drug and vaccine delivery vehicles. The robot contains a multiplexed homogenizer and has the capacity to handle parallel streams of monomer or polymer solutions to synthesize polymers and/or nanoparticles. Copolymer libraries were synthesized using the monomers sebacic acid, 1,6-bis( p-carboxyphenoxy)hexane, and 1,8-bis( p-carboxyphenoxy)-3,6-dioxactane and compared to conventionally synthesized copolymers. Nanoparticle libraries of varying copolymer compositions encapsulating the model antigen ovalbumin were synthesized using flash nanoprecipitation. The amount of the surfactant Span 80 was varied to test its effect on protein encapsulation efficiency as well as antigen release kinetics. It was observed that, although the amount of surfactant did not significantly affect protein release rate, its presence enhanced protein encapsulation efficiency. Protein burst and release kinetics from conventionally and combinatorially synthesized nanoparticles were similar even though particles synthesized using the high-throughput technique were smaller. Finally, it was demonstrated that the high-throughput method could be adapted to functionalize the surface of particle libraries to aid in the design and screening of targeted drug and vaccine delivery systems. These results suggest that the new high-throughput method is a viable alternative to conventional methods for synthesizing and screening protein and vaccine delivery vehicles.

Thermoresponsive Elastin-b-Collagen-Like Peptide Bioconjugate Nanovesicles for Targeted Drug Delivery to Collagen-Containing Matrices.

Over the past few decades, (poly)peptide block copolymers have been widely employed in generating well-defined nanostructures as vehicles for targeted drug delivery applications. We previously reported the assembly of thermoresponsive nanoscale vesicles from an elastin-b-collagen-like peptide (ELP-CLP). The vesicles were observed to dissociate at elevated temperatures, despite the LCST-like behavior of the tethered ELP domain, which is suggested to be triggered by the unfolding of the CLP domain. Here, the potential of using the vesicles as drug delivery vehicles for targeting collagen-containing matrices is evaluated. The sustained release of an encapsulated model drug was achieved over a period of 3 weeks, following which complete release could be triggered via heating. The ELP-CLP vesicles show strong retention on a collagen substrate, presumably through collagen triple helix interactions. Cell viability and proliferation studies using fibroblasts and chondrocytes suggest that the vesicles are highly cytocompatible. Additionally, essentially no activation of a macrophage-like cell line is observed, suggesting that the vesicles do not initiate an inflammatory response. Endowed with thermally controlled delivery, the ability to bind collagen, and excellent cytocompatibility, these ELP-CLP nanovesicles are suggested to have significant potential in the controlled delivery of drugs to collagen-containing matrices and tissues.