Kim A. Woodrow

Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA

Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protected against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternate approach, using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA in the vaginal mucosa.

Mucosal Vaccine Design and Delivery

Mucosal surfaces are a major portal of entry for many human pathogens that are the cause of infectious diseases worldwide. Vaccines capable of eliciting mucosal immune responses can fortify defenses at mucosal front lines and protect against infection. However, most licensed vaccines are administered parenterally and fail to elicit protective mucosal immunity. Immunization by mucosal routes may be more effective at inducing protective immunity against mucosal pathogens at their sites of entry. Recent advances in our understanding of mucosal immunity and identification of correlates of protective immunity against specific mucosal pathogens have renewed interest in the development of mucosal vaccines. Efforts have focused on efficient delivery of vaccine antigens to mucosal sites that facilitate uptake by local antigen-presenting cells to generate protective mucosal immune responses. Discovery of safe and effective mucosal adjuvants are also being sought to enhance the magnitude and quality of the protective immune response.

Current strategies for sustaining drug release from electrospun nanofibers

Electrospun drug-eluting fibers are emerging as a novel dosage form for multipurpose prevention against sexually transmitted infections, including HIV, and unintended pregnancy. Previous work from our lab and others show the versatility of this platform to deliver large doses of physico-chemically diverse agents. However, there is still an unmet need to develop practical fiber formulations for water-soluble small molecule drugs needed at high dosing due to intrinsic low potency or desire for sustained prevention. To date, most sustained release fibers have been restricted to the delivery of biologics or hydrophobic small molecules at low drug loading of typically <1 wt.%, which is often impractical for most clinical applications. For hydrophilic small molecule drugs, their high aqueous solubility and poor partitioning and incompatibility with insoluble polymers make long-term release even more challenging. Here we investigate several existing strategies to sustain release of hydrophilic small molecule drugs that are highly-loaded in electrospun fibers. In particular, we investigate what is known about the design constraints required to realize multi-day release from fibers fabricated from uniaxial and coaxial electrospinning.

Drug-Eluting Fibers for HIV-1 Inhibition and Contraception

Multipurpose prevention technologies (MPTs) that simultaneously prevent sexually transmitted infections (STIs) and unintended pregnancy are a global health priority. Combining chemical and physical barriers offers the greatest potential to design effective MPTs, but integrating both functional modalities into a single device has been challenging. Here we show that drug-eluting fiber meshes designed for topical drug delivery can function as a combination chemical and physical barrier MPT. Using FDA-approved polymers, we fabricated nanofiber meshes with tunable fiber size and controlled degradation kinetics that facilitate simultaneous release of multiple agents against HIV-1, HSV-2, and sperm. We observed that drug-loaded meshes inhibited HIV-1 infection in vitro and physically obstructed sperm penetration. Furthermore, we report on a previously unknown activity of glycerol monolaurate (GML) to potently inhibit sperm motility and viability. The application of drug-eluting nanofibers for HIV-1 prevention and sperm inhibition may serve as an innovative platform technology for drug delivery to the lower female reproductive tract.

Cell-Free Protein Synthesis With Prokaryotic Combined Transcription-Translation

Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells. One model system is prokaryotic cell-free protein synthesis. This technology offers an attractive and convenient approach to produce properly folded recombinant DNA (rDNA) proteins on a laboratory scale, screen PCR fragment libraries in a high-throughput format, express pharmaceutical proteins, incorporate labeled or unnatural amino acids into proteins, and activate microbial physiology to allow for investigation of biological systems. We describe the preparation of materials necessary for the expression, quantification, and purification of rDNA proteins from active Escherichia coli extracts.

Increasing PCR Fragment Stability and Protein Yields in a Cell-Free System with Genetically Modified Escherichia coli Extracts

Escherichia coli cell-free protein synthesis is a highly productive system that can be applied to high throughput expression from polymerase chain reaction (PCR) products in 96-well plates for proteomic studies as well as protein evolution. However, linear DNA instability appears to be a major limitation of the system. We modified the genome of the E. coli strain A19 by removing the endA gene encoding the endonuclease I and replacing the recCBD operon (in which recD encodes the exonuclease V) by the λ phage recombination system. Using the cell extract from this new strain increased the stability of PCR products amplified from a plasmid containing the cat gene. This resulted in CAT (chloramphenicol acetyltransferase) production from PCR products comparable to that from plasmids (500–600 µg/ml) in a batch reaction. We show that cell-free protein synthesis reactions using PCR products amplified from genomic DNA and extended with the T7 promoter and the T7 terminator give the same high yields of proteins (550 µg/ml) in 96-well plates. With this system, it was possible to rapidly express a range of cytoplasmic and periplasmic proteins.

Drug Synergy of Tenofovir and Nanoparticle-Based Antiretrovirals for HIV Prophylaxis

Background The use of drug combinations has revolutionized the treatment of HIV but there is no equivalent combination product that exists for prevention, particularly for topical HIV prevention. Strategies to combine chemically incompatible agents may facilitate the discovery of unique drug-drug activities, particularly unexplored combination drug synergy. We fabricated two types of nanoparticles, each loaded with a single antiretroviral (ARV) that acts on a specific step of the viral replication cycle. Here we show unique combination drug activities mediated by our polymeric delivery systems when combined with free tenofovir (TFV). Methodology/Principal Findings Biodegradable poly(lactide-co-glycolide) nanoparticles loaded with efavirenz (NP-EFV) or saquinavir (NP-SQV) were individually prepared by emulsion or nanoprecipitation techniques. Nanoparticles had reproducible size (d ∼200 nm) and zeta potential (-25 mV). The drug loading of the nanoparticles was approximately 7% (w/w). NP-EFV and NP-SQV were nontoxic to TZM-bl cells and ectocervical explants. Both NP-EFV and NP-SQV exhibited potent protection against HIV-1 BaL infection in vitro. The HIV inhibitory effect of nanoparticle formulated ARVs showed up to a 50-fold reduction in the 50% inhibitory concentration (IC50) compared to free drug. To quantify the activity arising from delivery of drug combinations, we calculated combination indices (CI) according to the median-effect principle. NP-EFV combined with free TFV demonstrated strong synergistic effects (CI50 = 0.07) at a 1∶50 ratio of IC50 values and additive effects (CI50 = 1.05) at a 1∶1 ratio of IC50 values. TFV combined with NP-SQV at a 1∶1 ratio of IC50 values also showed strong synergy (CI50 = 0.07). Conclusions ARVs with different physicochemical properties can be encapsulated individually into nanoparticles to potently inhibit HIV. Our findings demonstrate for the first time that combining TFV with either NP-EFV or NP-SQV results in pronounced combination drug effects, and emphasize the potential of nanoparticles for the realization of unique drug-drug activities.

Manufacturing scale-up of electrospun poly(vinyl alcohol) fibers containing tenofovir for vaginal drug delivery.

Electrospun fibers containing antiretroviral drugs have recently been investigated as a new dosage form for topical microbicides against HIV-1. However, little work has been done to evaluate the scalability of the fiber platform for pharmaceutical production of medical fabrics. Scalability and cost-effectiveness are essential criteria in developing fibers as a practical platform for use as a microbicide and for translation to clinical use. To address this critical gap in the development of fiber-based vaginal dosage forms, we assessed the scale-up potential of drug-eluting fibers delivering tenofovir (TFV), a nucleotide reverse transcriptase inhibitor and lead compound for topical HIV-1 chemoprophylaxis. Here we describe the process of free-surface electrospinning to scale up production of TFV fibers, and evaluate key attributes of the finished products such as fiber morphology, drug crystallinity, and drug loading and release kinetics. Poly(vinyl alcohol) (PVA) containing up to 60 wt% TFV was successfully electrospun into fibers using a nozzle-free production-scale electrospinning instrument. Actual TFV loading in fibers increased with increasing weight percent TFV in solution, and encapsulation efficiency was improved by maintaining TFV solubility and preventing drug sedimentation during batch processing. These results define important solution and processing parameters for scale-up production of TFV drug-eluting fibers by wire electrospinning, which may have significant implications for pharmaceutical manufacturing of fiber-based medical fabrics for clinical use.

Electrospun Solid Dispersions of Maraviroc for Rapid Intravaginal Preexposure Prophylaxis of HIV

ABSTRACT The development of topical anti-human immunodeficiency virus (HIV) microbicides may provide women with strategies to protect themselves against sexual HIV transmission. Pericoital drug delivery systems intended for use immediately before sex, such as microbicide gels, must deliver high drug doses for maximal effectiveness. The goal of achieving a high antiretroviral dose is complicated by the need to simultaneously retain the dose and quickly release drug compounds into the tissue. For drugs with limited solubility in vaginal gels, increasing the gel volume to increase the dose can result in leakage. While solid dosage forms like films and tablets increase retention, they often require more than 15 min to fully dissolve, potentially increasing the risk of inducing epithelial abrasions during sex. Here, we demonstrate that water-soluble electrospun fibers, with their high surface area-to-volume ratio and ability to disperse antiretrovirals, can serve as an alternative solid dosage form for microbicides requiring both high drug loading and rapid hydration. We formulated maraviroc at up to 28 wt% into electrospun solid dispersions made from either polyvinylpyrrolidone or poly(ethylene oxide) nanofibers or microfibers and investigated the role of drug loading, distribution, and crystallinity in determining drug release rates into aqueous media. We show here that water-soluble electrospun materials can rapidly release maraviroc upon contact with moisture and that drug delivery is faster (less than 6 min under sink conditions) when maraviroc is electrospun in polyvinylpyrrolidone fibers containing an excipient wetting agent. These materials offer an alternative dosage form to current pericoital microbicides.

Delivery of multipurpose prevention drug combinations from electrospun nanofibers using composite microarchitectures

Background Electrospun drug-eluting fabrics have enormous potential for the delivery of physicochemically diverse drugs in combination by controlling the underlying material chemistry and fabric microarchitecture. However, the rationale for formulating drugs at high drug loading in the same or separate fibers is unknown but has important implications for product development and clinical applications. Methods Using a production-scale free-surface electrospinning instrument, we produced electrospun nanofibers with different microscale geometries for the co-delivery of tenofovir (TFV) and levonorgestrel (LNG) – two lead drug candidates for multipurpose prevention of HIV acquisition and unintended pregnancy. We investigated the in vitro drug release of TFV and LNG combinations from composites that deliver the two drugs from the same fiber (combined fibers) or from separate fibers in a stacked or interwoven architecture. For stacked composites, we also examined the role that fabric thickness has on drug-release kinetics. We also measured the cytotoxicity and antiviral activity of the drugs delivered alone and in combination. Results Herein, we report on the solution and processing parameters for the free-surface electrospinning of medical fabrics with controlled microarchitecture and high drug loading (up to 20 wt%). We observed that in vitro release of the highly water-soluble TFV, but not the water-insoluble LNG, was affected by composite microarchitecture, fabric thickness, and drug content. Finally, we showed that the drug-loaded nanofibers are noncytotoxic and that the antiviral activity of TFV is preserved through the electrospinning process and when combined with LNG. Conclusion Electrospun fabrics with high drug loading create multicomponent systems that benefit from the independent control of the nanofibrous microarchitecture. Our findings are significant because they will inform the design and production of composite electrospun fabrics for the co-delivery of physicochemically diverse drugs that may be useful for multipurpose prevention.