Carlo Yuvienco

Long-term efficient gene delivery using polyethylenimine with modified Tat peptide.

Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (≈5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.

Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis.

The HIV-1 Tat peptide has been successfully used for intracellular gene delivery. Likewise, various lipid-based methods have shown increased endocytosis and can influence endosomal escape. This study combines the favorable properties of Tat peptide with that of lipid systems for DNA delivery. We combined the lipid FuGENE HD (FH) with the Tat peptide sequence modified with histidine and cysteine residues (mTat). mTat/FH transfection was evaluated by luciferase expression plasmid in five cell types. mTat/FH produced significant improvement in transfection efficiency of all cell lines when compared to FH or mTat. Treatment with chloroquine, associated with energy-dependent endocytosis, significantly increased transfection efficiency with mTat/FH while incubation at low temperature decreased it. The zeta potential of mTat/FH/DNA was significantly higher compared to FH, mTat, or their DNA combination in the presence of serum, and it was correlated with transfection efficiency. The particle size of the FH/DNA complex was significantly reduced by addition of mTat. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/FH transfection, but transfection was increased by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. These findings demonstrated the feasibility of using a combination of mTat with lipids, utilizing temperature-dependent and caveolae-mediated endocytosis, as a potentially attractive non-viral gene vector.

Artificial Protein Block Copolymers Blocks Comprising Two Distinct Self-Assembling Domains

Synthetic block copolymers comprising two or more compositionally distinct chains have attracted significant attention due to their ability to self-assemble into ordered microstructures. Although tremendous progress has been made in the chemical synthesis of polymers, the unsurpassed degree of control and diversity of monomers combined with advances in recombinant DNA technology permits the synthesis of unique artificial protein-derived block polymers. These include silk–elastin, elastin–elastin hybrids of varying elastin blocks, and helix–random coil–helix triblock combinations. These polymers consist of nearly similar self-assembling chains, as in the case of silk–elastin and elastin–elastin hybrids, or one self-assembling motif fused to a disordered random motif. Herein, we describe three block copolymers comprising two distinct self-assembling chains—elastin (E) and cartilage oligomeric matrix protein coiled-coil (COMPcc; C)—fused in two orientations (EC and CE) and a final construct in which an additional E block is appended (ECE; Figure 1A–C). Remarkably, the polymer structures as well as temperature and small-moleculedependent assembly rely on the block orientation and the number of blocks. Elastins consist of pentapeptide (VPGXG)n repeating units, where X is an interchangeable amino acid, that self-assemble into helical b-spirals. 7] Elastins exhibit a lower critical solution temperature (Tt) behaviour that can be tuned by varying the identity of X and the number of repeats (n). By contrast, COMPcc self-assembles into a homopentamer of parallel a-helical coiled-coils to produce a hydrophobic pore that is 7.3 nm long with a diameter of 0.2–0.6 nm. Individually, the E and C domains exhibit unique modes of self-assembly and conformation; E undergoes phase separation while C can bind small molecules. Each polymer consists of compositionally identical E and C motifs into which a short A2(TA)n spacer is incorporated at the juncture between the domains to ensure that each block is able to self-assemble as required (Figure 1A–C). A critical feature of smart biomaterials is the ability of the polymers to self-assemble as a function of environmental cues such as pH and ionic strength. Previous studies have shown that elastin and coiled-coil domains can be influenced by pH and salt conditions. The synthetic versatility of these block polymer constructs permits the exploration of how the orientation and the number of blocks influence their physicochemical properties. All block polymers have been overexpressed, purified and characterised. The molar masses of EC, CE and ECE are 22731, 22911 and 35188 Da, respectively. Although SDSPAGE analysis of the purified polymers reveals a slightly higher molecular weight for EC, CE and ECE, due to the E portion of the block polymers (Figure 1D), the exact masses were confirmed by MALDI. To determine the conformations of the block polymers, far-UV circular dichroism (CD) measurements were conducted. The homopolymer C adopted a helical structure that exhibited a transition to random configuration as the temperature was raised. In contrast, E adopted an initial b-turn that loses its structure at higher temperatures. Although nearly identical in composition, the EC and CE diblocks differed in secondary structure and exhibited distinct temperature-dependent conformational changes (Figure 2A, B). In the case of Figure 1. Amino acid sequences and structures of A) EC, B) CE and C) ECE. D) SDS-PAGE identifying protein fusions. E) Vitamin D3.

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.

Supramolecular assembly and small molecule recognition by genetically engineered protein block polymers composed of two SADs

Genetically engineered protein block polymers are an important class of biomaterials that have gained significant attention in recent years due to their potential applications in biotechnology, electronics and medicine. The majority of the protein materials have been composed of at least a single self-assembling domain (SAD), enabling the formation of supramolecular structures. Recently, we developed block polymers consisting of two distinct SADs derived from an elastin-mimetic polypeptide (E) and the alpha-helical COMPcc (C). These protein polymers, synthesized as the block sequences--EC, CE, and ECE--were assessed for overall conformation and macroscopic thermoresponsive behavior. Here, we investigate the supramolecular assembly as well as the small molecule binding and release profile of these block polymers. Our results demonstrate that the protein polymers assemble into particles as well as fully or partially networked structures in a concentration dependent manner that is distinct from the individual E and C homopolymers and the E+C non-covalent mixture. In contrast to synthetic block polymers, the structured assembly, binding and release abilities are highly dependent on the composition and orientation of the blocks. These results reveal the promise for these block polymers for therapeutic delivery and biomedical scaffolds.

Protein based therapeutic delivery agents: Contemporary developments and challenges

As unique biopolymers, proteins can be employed for therapeutic delivery. They bear important features such as bioavailability, biocompatibility, and biodegradability with low toxicity serving as a platform for delivery of various small molecule therapeutics, gene therapies, protein biologics and cells. Depending on size and characteristic of the therapeutic, a variety of natural and engineered proteins or peptides have been developed. This, coupled to recent advances in synthetic and chemical biology, has led to the creation of tailor-made protein materials for delivery. This review highlights strategies employing proteins to facilitate the delivery of therapeutic matter, addressing the challenges for small molecule, gene, protein and cell transport.

Engineered Protein Polymer-Gold Nanoparticle Hybrid Materials for Small Molecule Delivery

We have fabricated protein polymer-gold nanoparticle (P-GNP) nanocomposites that exhibit enhanced binding and delivery properties of the small hydrophobic molecule drug, curcumin, to the model breast cancer cell line, MCF-7. These hybrid biomaterials are constructed via in situ GNP templated-synthesis with genetically engineered histidine tags. The P-GNP nanocomposites exhibit enhanced small molecule loading, sustained release and increased uptake by MCF-7 cells. When compared to the proteins polymers alone, the P-GNPs demonstrate a greater than 7-fold increase in curcumin binding, a nearly 50% slower release profile and more than 2-fold increase in cellular uptake of curcumin. These results suggest that P-GNP nanocomposites serve as promising candidates for drug delivery vehicles.

Improved Stability and Half-Life of Fluorinated Phosphotriesterase Using Rosetta

Recently we demonstrated that incorporating p‐fluorophenylalanine (pFF) into phosphotriesterase dramatically improved folding, thereby leading to enhanced stability and function at elevated temperatures. To further improve the stability of the fluorinated enzyme, Rosetta was used to identify multiple potential stabilizing mutations. One such variant, pFF‐F104A, exhibited enhanced activity at elevated temperature and maintained activity over many days in solution at room temperature.

Engineered Coiled-Coil Protein for Delivery of Inverse Agonist for Osteoarthritis

Osteoarthritis (OA) results from degenerative and abnormal function of joints, with localized biochemistry playing a critical role in its onset and progression. As high levels of all- trans retinoic acid (ATRA) in synovial fluid have been identified as a contributive factor to OA, the synthesis of de novo antagonists for retinoic acid receptors (RARs) has been exploited to interrupt the mechanism of ATRA action. BMS493, a pan-RAR inverse agonist, has been reported as an effective inhibitor of ATRA signaling pathway; however, it is unstable and rapidly degrades under physiological conditions. We employed an engineered cartilage oligomeric matrix protein coiled-coil (CccS) protein for the encapsulation, protection, and delivery of BMS493. In this study, we determine the binding affinity of CccS to BMS493 and the stimulator, ATRA, via competitive binding assay, in which ATRA exhibits approximately 5-fold superior association with CccS than BMS493. Interrogation of the structure of CccS indicates that ATRA causes about 10% loss in helicity, while BMS493 did not impact the structure. Furthermore, CccS self-assembles into nanofibers when bound to BMS493 or ATRA as expected, displaying 11-15 nm in diameter. Treatment of human articular chondrocytes in vitro reveals that CccS·BMS493 demonstrates a marked improvement in efficacy in reducing the mRNA levels of matrix metalloproteinase-13 (MMP-13), one of the main proteases responsible for the degradation of the extracellular cartilage matrix compared to BMS493 alone in the presence of ATRA, interleukin-1 beta (IL-1β), or IL-1 β together with ATRA. These results support the feasibility of utilizing coiled-coil proteins as drug delivery vehicles for compounds of relatively limited bioavailability for the potential treatment of OA.