Joshua A. Gustafson

Silk-elastinlike protein polymers for matrix-mediated cancer gene therapy.

Silk-elastinlike protein polymers (SELPs) are recombinant polymers designed from silk fibroin and mammalian elastin amino acid repeats. These are versatile materials that have been examined as controlled release systems for intratumoral gene delivery. SELP hydrogels comprise monodisperse and tunable polymers that have the capability to control and localize the release and expression of plasmid DNA and viruses. This article reviews recent developments in the synthesis and characterization of SELP hydrogels and their use for matrix-mediated gene delivery.

Silk-elastinlike recombinant polymers for gene therapy of head and neck cancer: from molecular definition to controlled gene expression

Silk-elastinlike protein polymers (SELPs) are block copolymers of silk-like and elastin-like tandem repeats. With appropriate sequence and composition SELPs can be liquid at room temperature and form hydrogels at body temperature. The influence of polymer structure and concentration on biodegradation in vitro and controlled delivery of adenoviruses carrying reporter genes to head and neck tumors in vivo was evaluated using hydrogels made from three SELP analogs. SELP-815K, with eight silk and fifteen elastin units and a lysine (K) modified elastin, was compared to SELP-415K and SELP-47K. Hydrogels with higher silk content and concentration degraded at a slower rate compared to other analogs. Intratumoral injection of adenoviruses with SELPs enhanced gene expression in tumor tissue up to 10 fold compared to viral injection without polymer. Viruses delivered with SELP-815K at 4 wt.% polymer concentration showed the highest level of gene expression. Tumor to liver ratio of expression was up to 55 fold higher for SELP-mediated systems. This study demonstrates the influence of exquisite control over polymer structure using recombinant techniques on spatial and temporal control over adenoviral delivery and establishes the utility of SELP matrices as biodegradable systems for gene therapy of head and neck cancer.

Fabrication of highly uniform nanoparticles from recombinant silk-elastin-like protein polymers for therapeutic agent delivery.

Here we generate silk-elastin-like protein (SELP) polymeric nanoparticles and demonstrate precise control over their dimensions using an electrospray differential mobility analyzer (ES-DMA). Electrospray produces droplets encompassing several polymer strands. Evaporation ensues, leading polymer strands to accumulate at the droplet interface, forming a hollow nanoparticle. The resulting nanoparticle size distributions, which govern particle yield, depend on buffer concentration to the -1/3 power, polymer concentration to the 1/3 power, and ratio of silk-to-elastin blocks. Three recombinantly tuned ratios of 8:16, 4:8, and 4:16, respectively named SELP-815K, SELP-47K, and SELP-415K, are employed, with the latter ratio resulting in a thinner shell and larger diameter for the nanoparticles than the former. The DMA narrows the size distribution by electrostatically classifying the aerosolized nanoparticles. These highly uniform nanoparticles have variations of 1.2 and 1.4 nm for 24.0 and 36.0 nm particles, respectively. Transmission electron microscopy reveals the nanoparticles to be faceted, as a buckling instability releases compression energy arising from evaporation after the shell has formed by bending it. A thermodynamic equilibrium exists between compression and bending energies, where the facet length is half the particle diameter, in agreement with experiments. Rod-like particles also formed from polymer-stabilized filaments when the viscous length exceeds the jet radius at higher solution viscosities. The unusual uniformity in composition and dimension indicates the potential of these nanoparticles to deliver bioactive and imaging agents.

Gold nanorod mediated plasmonic photothermal therapy: A tool to enhance macromolecular delivery

Plasmonic photothermal therapy (PPTT) with gold nanostructures has been used to generate significant heat within tumors to ablate vasculature. Here we report the use of gold nanorod (GNR) mediated PPTT to induce moderate hyperthermia as a tool to enhance the delivery of macromolecules. GNRs were injected intravenously in a mouse sarcoma (S-180) tumor model. After 24h Evans blue dye (EBD) was injected and the right tumor was radiated with a laser diode for 10 min. EBD content in the right and left tumors were extracted in formamide, measured spectrophotometrically and expressed as a thermal enhancement ratio (TER). Enhanced delivery of EBD was observed (up to 1.8-fold) when tumor temperatures reached 43°C or 46°C. No statistical difference was observed between tumors at these two temperatures, though significant hemorrhage was observed in tumors and surrounding areas receiving the higher thermal dose (46°C). These results indicate that tumor directed PPTT may be used to induce moderate hyperthermia and therefore selectively increase the delivery of macromolecules with therapeutic anticancer drugs.

Silk-elastinlike protein polymers improve the efficacy of adenovirus thymidine kinase enzyme prodrug therapy of head and neck tumors

Adenoviral‐directed enzyme prodrug therapy is a promising approach for head and neck cancer gene therapy. The challenges faced by this approach, however, comprise transient gene expression and dissemination of viruses to distant organs.

Synthesis and characterization of a matrix-metalloproteinase responsive silk-elastinlike protein polymer

Silk-elastinlike protein polymers (SELPs) are recombinant polymers consisting of tandem repeats of silk (GAGAGS) and elastin (GVGVP) units. By modification of the length and composition of these repeats, the properties of SELP hydrogels can be controlled for specific applications including nucleic acid and virus delivery and tissue engineering. Here, the structure of SELPs is further modified to include a sequence that is sensitive to matrix-metalloproteinases (MMPs). MMPs are a ubiquitous family of extracellular matrix-modifying enzymes that are commonly associated with numerous vital processes. Increased levels of MMPs are found at high levels locally in many types of solid tumors. By modifying the SELP backbone with MMP-sensitive peptide sequences, a hydrogel that is degradable by MMPs was produced. The MMP-sensitivity of the polymer was examined by incubation with MMP-2 and MMP-9, which yielded complete cleavage of all full-length polymers by 36 hours and 48 hours, respectively, with no observable effect on unmodified SELP. Hydrogel sensitivity was tested by exposure to MMP-2 or MMP-9 for 2 weeks, during which samples were taken to analyze protein loss from the hydrogel and release of 100 nm fluorescent beads. Following the incubation period, hydrogels were tested in mechanical compression to examine the loss of hydrogel stiffness due to degradation. It was found that MMP-2 and MMP-9 caused 63% and 44% increased protein loss and 65% and 95% increased release from MMP-sensitive hydrogels, while the compressive modulus decreased by 41% and 29%. These results suggest the potential of MMP-responsive SELPs for localized delivery of bioactive agents where MMPs are overexpressed.

Silk-Elastin-like Hydrogel Improves the Safety of Adenovirus-Mediated Gene-Directed Enzyme−Prodrug Therapy

Recombinant silk-elastin-like protein polymers (SELPs) are well-known for their highly tunable properties on both the molecular and macroscopic hydrogel levels. One specific structure of these polymers, SELP-815K, has been investigated as an injectable controlled delivery system for the treatment of head and neck cancer via a gene-directed enzyme prodrug therapy (GDEPT) approach. Due to its pore size and gelation properties in vivo, SELP restricts the distribution and controls the release of therapeutic viruses for up to one month. It has been shown that SELP-mediated delivery significantly improves therapeutic outcome of the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system in xenograft models of human head and neck cancer. However little is known about potential benefits of this approach with regard to toxicity in the presence of a fully intact immune system. The studies presented here were designed to assess the change in toxicity of the SELP-mediated viral delivery compared to free viral injection in a non-tumor-bearing immune competent mouse model. Toxicity was assessed at 1, 2, 4, and 12 weeks via body weight monitoring, complete blood count (CBC), and blood chemistry. It was found that in the acute and subacute phases (weeks 1-4) there is significant toxicity in groups combining the virus and the prodrug, and matrix-mediated gene delivery with SELP demonstrates a reduction in toxicity from the 2 week time point through the 4 week time point. At the end of the subchronic phase (12 weeks), signs of toxicity had subsided in both groups. Based on these results, recombinant SELPs offer a significant reduction in toxicity of virus-mediated GDEPT treatment compared to free virus injection in the acute and subacute phases.

Genetically engineered nanocarriers for drug delivery

Cytotoxicity, low water solubility, rapid clearance from circulation, and off-target side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or non-polymeric. This review summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins.

A Hybrid Protein–Polymer Nanoworm Potentiates Apoptosis Better than a Monoclonal Antibody

B-cell lymphomas continue to occur with a high incidence. The chimeric antibody known as Rituximab (Rituxan) has become a vital therapy for these patients. Rituximab induces cell death via binding and clustering of the CD20 receptor by Fcγ expressing effector cells. Because of the limited mobility of effector cells, it may be advantageous to cluster CD20 directly using multivalent nanostructures. To explore this strategy, this manuscript introduces a nanoparticle that assembles from a fusion between a single chain antibody and a soluble protein polymer. These hybrid proteins express in Escherichia coli and do not require bioconjugation between the antibody and a substrate. Surprisingly a fusion between an anti-CD20 single chain antibody and a soluble protein polymer assemble worm-like nanostructures, which were characterized using light scattering and cryogenic transmission electron microscopy. These nanoworms competitively bind CD20 on two B-cell lymphoma cell lines, exhibit concentration-dependent induction of apoptosis, and induce apoptosis better than Rituximab alone. Similar activity was observed in vivo using a non-Hodgkin lymphoma xenograft model. In comparison to Rituximab, systemic nanoworms significantly slowed tumor growth. These findings suggest that hybrid nanoworms targeted at CD20 may be useful treatments for B-cell related malignancies. Because of the ubiquity of antibody therapeutics, related nanoworms may have uses against other molecular targets.

Comparison of silk-elastinlike protein polymer hydrogel and poloxamer in matrix-mediated gene delivery.

The silk-elastinlike protein polymer, SELP 815K, and poloxomer 407, a commercially available synthetic copolymer, were evaluated to compare their relative performance in matrix-mediated viral gene delivery. Using a xenogenic mouse tumor model of human head and neck squamous cell carcinoma, the efficacy of viral gene-directed enzyme prodrug therapy with these polymers was characterized by viral gene expression in the tumor tissue, tumor size reduction, and survivability with treatment. Viral injection in SELP 815K produced a greater level and more prolonged extent of gene expression in the tumor, a statistically greater tumor size reduction, a longer time until tumor rebound, and a significantly increased survivability, as compared to injection of virus alone or in Poloxamer 407. Safety of treatment with these polymers was evaluated in a non-tumor bearing immunocompetent mouse model. Compared to virus injected alone or in Poloxamer 407, virus injected in SELP 815K had fewer and less severe indications of toxicity related to treatment as assessed by blood analysis, body weight, and histopathology of distant organs and the injection sites. Similar to virus alone or in Poloxamer 407, virus injected in SELP 815K elicited a mild injection site inflammatory response characterized primarily by a mononuclear leukocyte infiltrate and the formation of granulation tissue. Virus injected in SELP 815K resulted in fewer animals with elevated white blood cell counts and a less pronounced local toxicity reaction than was observed with virus in Poloxamer 407. In contrast to virus injected alone or in Poloxamer 407, which were not retained in the injection site tissues beyond week 1, SELP 815K was retained at the injection sites and by the end of the study (week 12), displayed limited absorption, and mild encapsulation. These results demonstrate the benefits of SELP 815K for matrix-mediated gene delivery over the injection of free virus and the injection of virus in Poloxamer 407. Virus in SELP 815K had greater efficacy of tumor suppression, promoted greater levels and greater duration of viral gene expression, and displayed reduced levels of injection site toxicity. Combining these performance and safety benefits with the degree of control with which they can be designed, synthesized and formulated, SELPs continue to show promise for their application in viral gene delivery.