We have developed mucus-penetrating nanoparticles (MPP) suitable for sustained delivery of small-molecule microbicides (Lai et al, Proceedings of the National Academy of Sciences 2007;104(5):1482-7). Conventional particles (CP) are mucoadhesive and stick to the outer layers of mucus that are shed most rapidly out of the vagina. By densely coating MPP with low molecular weight polyethylene glycol (PEG) we found that unexpectedly large MPP 100-500 nm in diameter can be engineered to rapidly penetrate human cervicovaginal (CV) mucus and thereby reach the unstirred layer of mucus adhering to the epithelial surface. These MPP will likely significantly increase vaginal residence time and improve epithelial microbicide distribution. The aim of this R21/R33 project is to develop MPP for the sustained delivery of small-molecule microbicides to increase their protective efficacy, acceptability, and user reliability. 'User failure'is the primary failure mode of barrier methods, and microbicides are likely to be used more reliably if applied daily on a coitally-dissociated basis. Another failure mode well-documented in animal models is inadequate microbicide distribution - the infectious inoculum reaches surfaces unprotected by the microbicide. MPP can provide a once-a-day, coitally-dissociated method that is likely to achieve complete and essentially uniform epithelial distribution. MPP will not likely provide the month-long delivery of a vaginal ring, but MPP have advantages that are not immediately apparent: 1) The vaginal epithelium is highly permeable to small water-soluble molecules - thus uniform epithelial distribution can best be achieved by uniform sustained delivery of small water soluble microbicides directly to the entire epithelial surface, not just to the vicinity of a vaginal ring. 2) Uterine peristalsis exposes the upper reproductive tract to vaginally deposited pathogens, and reliable protection of the upper tract is more likely to be achieved by MPP that can transport, and then locally deliver, small water-soluble molecules to the epithelia surfaces of the upper tract. In the R21 phase we propose to develop acyclovir-loaded MPP to evaluate in our mouse HSV models for efficacy, duration, vaginal distribution, and toxicity. The MPP will be composed biodegradable copolymers that we have shown are capable of sustained delivery of a wide range of bioactive molecules. The key milestone for R21 will be to develop acyclovir-MPP that provide at least one day of protection in the mouse. In the R33 phase, we will use the knowledge gained from the R21 phase to speed the development of an anti- HIV-MPP for sustained release of the best anti-HIV microbicide candidate then available (Fall, 2010), with tenofovir being a likely choice. The R33 anti-HIV-MPP will be optimized for drug delivery based on R21 results, and be tested for toxicity in mouse models and for efficacy in the Hu-BLT-SCID mouse/HIV model by Dr. Victor Garcia at UT Southwestern. World-wide, there is a great need for methods women can use to protect against AIDS and other sexually transmitted diseases. Several small-molecule vaginal microbicides are being developed that block HIV from infecting and/or replicating in target cells. The aim of this project is to enhance the protective efficacy of these small-molecule microbicides by developing mucus-penetrating nanoparticles that will improve coverage of susceptible tissues to increase reliability of protection, and to increase duration of protection so that the microbicides can be applied regularly, on a daily basis, and not require coitally-related applications.