Haresh T. More

Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing

Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC). Addition of a cationic engineered supercharged coiled-coil protein (CSP) in a 10:1:1 ratio of DNC:CSP:siRNA produced a stable lipoproteoplex (LPP) nanoparticle, with optimal siRNA complexation, minimal cytotoxicity, and increased transfection efficacy. In a humanized murine diabetic wound healing model, our optimized LPP formulation successfully delivered siRNA targeted against Keap1, key repressor of Nrf2 which is a central regulator of redox mechanisms. Application of LPP complexing siKeap1 restored Nrf2 antioxidant function, accelerated diabetic tissue regeneration, and augmented reduction-oxidation homeostasis in the wound environment. Our topical LPP delivery system can readily be translated into clinical use for the treatment of diabetic wounds and can be extended to other cutaneous diseases with genetic components.

Modulating Supramolecular Assemblies and Mechanical Properties of Engineered Protein Materials by Fluorinated Amino Acids

Here we describe the biosynthesis and characterization of fluorinated protein block polymers comprised of the two self-assembling domains (SADs): elastin (E) and the coiled-coil region of cartilage oligomeric matrix proteins (C). Fluorination is achieved by residue-specific incorporation of p-fluorophenylalanine (pFF) to create pFF-EC, pFF-CE, and pFF-ECE. Global fluorination results in downstream effects on the temperature-dependent secondary structure, supramolecular assembly, and bulk mechanical properties. The impact of fluorination on material properties also differs depending on the orientation of the block configurations as well as the number of domains in the fusion. These studies suggest that integration of fluorinated amino acids within protein materials can be employed to tune the material properties, especially mechanical integrity.

Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex

A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP·DNA(8:1)·FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat·FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP·DNA(8:1)·FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery.

Influence of fluorination on protein-engineered coiled-coil fibers.

We describe the design and characterization of fluorinated coiled-coil proteins able to assemble into robust nano- and microfibers. Fluorination is achieved biosynthetically by residue-specific incorporation of 5,5,5-trifluoroleucine (TFL). The fluorinated proteins C+TFL and Q+TFL are highly α-helical as confirmed via circular dichroism (CD) and more resistant to thermal denaturation compared to their nonfluorinated counterparts, C and Q. The fluorinated proteins demonstrate enhanced fiber assembly at pH 8.0 with higher order structure in contrast to nonfluorinated proteins, which are unable to form fibers under the same conditions. Ionic strength dependent fiber assembly is observed for fluorinated as well as wild-type proteins in which the fluorinated proteins exhibited more stable, thicker fibers. The fluorinated and nonfluorinated proteins reveal metal ion-dependent small molecule recognition and supramolecular assemblies. In the presence of Zn (II), enhanced thermal stability and fiber assembly is observed for the fluorinated proteins and their nonfluorinated counterparts. Whereas Ni (II) promotes aggregation with no fiber assembly, the stabilization of α-helix by Zn (II) results in enhanced binding to curcumin by the fluorinated proteins. Surprisingly, the nonfluorinated proteins exhibit multiple-fold increase in curcumin binding in the presence of Zn (II). In the context of the growing number of protein-based fiber assemblies, these fluorinated coiled-coil proteins introduce a new paradigm in the development of highly stable, robust self-assembling fibers under more physiologically relevant pH conditions that promotes the binding and release of small molecules in response to external cues.

Efficient Dual siRNA and Drug Delivery Using Engineered Lipoproteoplexes

An engineered supercharged coiled-coil protein (CSP) and the cationic transfection reagent Lipofectamine 2000 are combined to form a lipoproteoplex for the purpose of dual delivery of siRNA and doxorubicin. CSP, bearing an external positive charge and axial hydrophobic pore, demonstrates the ability to condense siRNA and encapsulate the small-molecule chemotherapeutic, doxorubicin. The lipoproteoplex demonstrates improved doxorubicin loading relative to Lipofectamine 2000. Furthermore, it induces effective transfection of GAPDH (60% knockdown) in MCF-7 breast cancer cells with efficiencies comparing favorably to Lipofectamine 2000. When the lipoproteoplex is loaded with doxorubicin, the improved doxorubicin loading (∼40 μg Dox/mg CSP) results in a substantial decrease in MCF-7 cell viability.