Poster CC4 - Bacterial extracellular vesicles for vaginal drug delivery

Introduction: There is a critical need for the rapid translation of innovative therapeutic approaches for gynecologic and obstetric health. Vaginal drug delivery is optimal for drug delivery to the female reproductive tract, as it has the potential to increase therapeutic concentration in target tissues while reducing off-target side effects. Vaginally administered therapies avoid the harsh gastrointestinal environment and hepatic first pass that challenge the efficacy of orally and systemically administered drugs. For maximal efficacy, vaginally administered nanoparticles must avoid steric and adhesive interactions with cervicovaginal mucus (CVM). The CVM barrier is a complex mixture of mucin and other proteins, cells, and bacteria that traps and eliminates foreign pathogens and particles, preventing infection or effective drug delivery. Rationally engineered nanoparticles are useful for vaginal drug delivery as they can target specific female reproductive tract cells, control the release of diverse cargoes, and bypass biological barriers. However, nanoparticle production is associated with limited scalability, high cost, and prolonged optimization timelines. Recent work highlights bacterial extracellular vesicles (bEVs) as potential therapeutic vehicles that can overcome some of the challenges for nanoparticles due to the low cost of cell cultures, adaptability to biomanufacturing infrastructure, and the tunability of therapeutic properties. Our work describes bEVs as a novel platform for vaginal drug delivery applications and shows that they can be tailored through genetic engineering.

Materials and

Methods: Here, we investigate the use of bEVs from the probiotic bacterium E. coli Nissle 1917 as vaginal drug delivery carriers. We genetically modified E. coli Nissle 1917 to display fluorescent proteins on the inner membrane using various signal peptides. We can modulate the amount of protein displayed using promoters with varied strength. By displaying proteins on the inner membrane, we were able to load bEVs with these model proteins. bEVs were isolated via ultracentrifugation. The size, surface charge, and protein content of bEVs were quantified using nanoparticle tracking analysis (NTA, Particle Metrix ZetaView) and bicinchoninic acid (BCA) assay. The morphology of particles was assessed using transmission electron microscopy (TEM). To determine the ability of bEVs to move through human CVM, we used established multiple particle tracking protocols and an in-house MATLAB program to quantify the average mean squared displacement of particles. To determine compatibility with the local vaginal environment, we exposed vaginal epithelial cells to bEVs and measured cell viability via CCK-8 and inflammatory response via ELISA. We also assessed the compatibility of bEVs with vaginal bacteria by counting colony forming units (CFUs) after live cultures of vaginal microbes were exposed to bEVs from genetically modified Nissle.

Results, Conclusions, and Discussions: We showed that tailored genetic modification can be designed for tracking purposes and to improve the therapeutic potential of a bEV platform. We identified bEVs isolated from cultures of E. coli Nissle 1917 using NTA and TEM. bEVs were produced at a concentration of 7.1 x 108 ± 3.8 x 108 particles/mL. Particles had an average size of 192.7 ± 10.9 nm and an average surface charge of -48.3 ± 5.6 mV. TEM images revealed circular membrane bound particles with a size range 100 – 300 nm. Using fluorescent NTA, we confirmed that bEVs were loaded with fluorescent protein. Our work highlights that the level of protein display needs to be finely tuned with promoter strength, as too much protein production can be toxic to the cell. Consistent with previous work, we found that bEVs were more mobile in CVM samples with a polymicrobial microbiome. Given that a polymicrobial microenvironment increases susceptibility to sexually transmitted infections, pelvic inflammatory disease, infertility, and preterm birth, among other indications, we pose that increased bEV mobility in these samples increases the potential use for bEV therapeutics for gynecologic and obstetric indications. We demonstrate the use of genetic engineering to reduce the cost of therapeutic manufacturing and show how the bEV platform can be leveraged to expedite the formulation of new nanomedicines for gynecologic and obstetric indications.

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