A bacteria-responsive drug release platform for the local treatment of bacterial vaginosis
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Bacterial vaginosis (BV) is a common vaginal anaerobic dysbiosis affecting millions of women, with a high recurrence rate probably due to the stubborn existence of biofilms and Gardnerella vaginalis (G. vaginalis) as the primary pathogen. Antimicrobial therapy has been the standard treatment for BV for several decades, which includes oral and intravaginal therapy. However, the major drawback of using antibiotics extensively is drug resistance, leading to the ineffectiveness in killing bacterial pathogens. Stimuli-responsive systems are a promising solution to address bacterial resistance because they release drugs only in the presence of specific stimulus, e.g., changes in physical micro-environment and bacterial factors, which greatly avoids overdosing. In terms of treatment method, intravaginal formulations are superior to the conventional oral route with reduced side effects such as stomach irritation and more effective drug dose. This thesis focused on the development and characterization of a bacteria-responsive drug release platform that is aimed for intravaginal therapy to cure BV. Vaginolysin (VLY), a virulence factor produced specifically by G. vaginalis, was utilized as the stimulus to trigger drug release with the aim of achieving high specificity and improving antibacterial activity. Series of liposome formulations were developed, optimized, and characterized in terms of preparation method, purification method, encapsulation efficiency, size distribution and zeta potential. We demonstrated that centrifugation was the optimal method to separate liposomes from the model free drug (calcein) while keeping liposomes stable without much aggregation. Increasing the initial calcein concentration and total amount of lipids facilitated a higher encapsulation efficiency. All liposomes had an average diameter of around 300 nm with a polydispersity index (PDI) less than 0.3 and an average zeta potential of -60 mV. Second, we optimized bacteria culture and evaluated the responsive calcein release in the presence of G. vaginalis and Lactobacillus crispatus (L. crispatus) supernatants. All liposome formulations showed sustained calcein release (cumulative release: 46.7%-51.8%) in response to G. vaginalis supernatant and very little calcein release (cumulative release: 1.1%) in the presence of L. crispatus supernatant, indicating that the responsive drug release platform had good specificity. We then constructed an Escherichia coli (E. coli)-based recombinant protein expression system to express recombinant VLY which could be used as a standard to quantify VLY produced by G. vaginalis. After that, a series of optimizations in terms of host strain, cultivation conditions, cell lysis method, purification process optimization, etc. regarding recombinant VLY expression were carried out to promote soluble protein production. Unfortunately, we did not achieve the desired results as the recombinant VLY was expressed in the form of inclusion bodies. Overall, the bacteria-responsive drug release platform has great potential, since it will be the first time to realize controlled drug release activated by a specific pathogen for BV prevention and treatment. This on-demand therapy can be potentially used by industry to provide millions of women patients with more sexual and reproductive health care.
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Chuying Feng (2022). A bacteria-responsive drug release platform for the local treatment of bacterial vaginosis. UWSpace. http://hdl.handle.net/10012/18463