In the United States, over 100,000 women are diagnosed with a gynecologic malignancy every year, with ovarian cancer being the most lethal. One of the hallmark characteristics of ovarian cancer is the development of resistance to chemotherapeutics. While the exact mechanisms of chemoresistance are poorly understood, it is known that changes at the cellular and molecular level make chemoresistance challenging to treat. Improved therapeutic options are needed to target these changes at the molecular level. Using a precision medicine approach, such as gene therapy, genes can be specifically exploited to resensitize tumors to therapeutics. This review highlights traditional and novel gene targets that can be used to develop new and improved targeted therapies, from drug efflux proteins to ovarian cancer stem cells. The review also addresses the clinical relevance and landscape of the discussed gene targets.

2.2. Poly(styrene-alt-maleic anhydride)-b-poly(styrene) Nanoparticles as a Platform to Study cCBP-Mediated Targeting of MV411 Cells Scheme 1. Synthesis of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) based diblock copolymers via one-step RAFT polymerization.

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Introduction: With a 5-year survival rate of 47%, ovarian cancer is the 5th leading cause of death amongst women worldwide. Over 75% of patients experience recurrence after initial treatment, indicating a need for improved treatment options. Drug resistance is a major barrier hindering the success of current treatment methods. Our study analyzes the characteristics of a stimuli-sensitive liposomal delivery system for combatting drug resistance. Our delivery system will deliver bioactive siRNAs targeting genes related to drug resistance, cell proliferation, and apoptosis. In this study, we investigate the characteristics of the liposomes to determine particle size, surface charge, and ability to encapsulate/bind both siRNAs. We also begin to investigate the delivery potential of the pH-sensitive liposomal formulation in vitro using ovarian cancer cell lines. Methods: Empty and siRNA loaded cationic, pH-sensitive liposomes (CHEMS-LPs) were synthesized by the thin-film hydration method. Liposome size, zeta potential, and polydispersity index (PDI) were measured by dynamic light scattering (DLS). To measure siRNA encapsulation efficiency, fluorescently labeled siRNA was loaded into CHEMS-LPs and subjected to centrifugation to pellet the LPs. Fluorescence spectroscopy was used to detect siRNA in the supernatant. The toxicity of unloaded CHEMS-LPs was determined by an MTS assay using OVCAR3 (drug-sensitive) and OVCAR3-T40 (drug-resistant) human ovarian cancer cells. Results: The size and zeta potential of blank and siRNA-loaded CHEMS-LPs were 97.88 ± 2.39 nm and 29.0 ± 2.00 mV, and 80.78 ± 0.77​ nm and 13.1 ±1.66​ mV, respectively. The positively charged zeta potential confirms the cationic nature of our liposomes. The PDI demonstrated that the liposomes were unimodal and monodisperse with PDI values of less than 0.300 for each formulation. In addition, siRNA was successfully bound to CHEMS-LPs through electrostatic interaction with the cationic lipid layer, resulting in an encapsulation efficiency of 99.6% Conclusion: CHEMS-LPs are pH-sensitive, cationic, monodisperse liposomes able to encapsulate siRNAs in order to mediate delivery into ovarian cancer cells. Their stable structure, positive charge, and low cytotoxicity is promising for future studies, including delivery of bioactive siRNAs to stimulate downregulation of target genes related to drug resistance. Acknowledgements: This work was supported in part by the National Science Foundation EPSCoR Program under Award # OIA-1655740. We would like to thank George Duran from Stanford University for donating the OVCAR3-T40 cell line. Citation Format: Kharimat Lora Alatise, Samantha Gardner, Angela Alexander-Bryant. pH-sensitive liposome for siRNA delivery to treat drug-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 281.

Introduction: Ovarian cancer is the fifth leading cause of cancer mortality in women, with nearly 75% of women who respond to initial platinum-based chemotherapy experiencing relapse due to drug resistance [1,2]. Liposomes are a promising solution to overcoming drug resistance due to their biocompatibility and capacity to encapsulate hydrophilic and hydrophobic drugs as well as complex small interfering RNA (siRNA), which have the potential to downregulate the expression of genes related to drug resistance [3]. We aim to synthesize and characterize a cationic liposomal system to deliver siRNA and paclitaxel (PTX) to ovarian cancer cells. Methods: Cholesterol (CHOL) liposomes were synthesized by the thin-film hydration method. Dynamic light scattering (DLS) was used to determine the size, polydispersity index (PDI), and zeta potential of the liposomes. Uptake of liposomes into OVCAR3 and OVCAR3-T40, a wild-type and a paclitaxel-resistant human adenocarcinoma cell line, was examined using fluorescence microscopy. The cytotoxicity of unloaded CHOL liposomes was evaluated through MTS assay on OVCAR3 and OVCAR3-T40 cells. Results: PTX- and siRNA-loaded CHOL liposomes had an average diameter of 114.9 ± 10.35 nm and a zeta potential of 27.6 ± 1.79 mV. Blank CHOL liposomes had an average diameter of 123.0 ± 2.49 nm and zeta potential of 32.3 ± 2.16 mV. All formulations of liposomes were cationic and formed monodisperse nanoparticles. The encapsulation efficiency of siRNA and PTX was 99.8% and 80.4% respectively. Coumarin 6, a hydrophobic model drug, was loaded into liposomes to verify cellular uptake through fluorescent imaging. Results demonstrated that the liposomal system was efficiently delivered intracellularly. Blank liposomes were used to determine the toxicity of the delivery system. The unloaded liposomes were not cytotoxic to both the wild-type and drug-resistant cell lines at concentrations up to 75 µg/mL, and therefore, cytotoxicity of drug-loaded liposomes can be attributed to paclitaxel, siRNA, or combination treatment. Conclusions: Liposomes were successfully formed with a monodisperse size, exhibited effective drug and siRNA loading, and were internalized into OVCAR3 and OVCAR3-T40 cells. Future work includes investigating the efficacy of the liposomal system in mediating gene silencing. Acknowledgements: This work was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740 and Clemson Creative Inquiry. References: [1] Torre, L. A., CA Cancer J Clin. 2018;68(4):284-296, [2], Norouzi-Barough L., J Cell Physiol.2018;233(6):4546-4562, [3] Farra R., Pharmaceutics. 2019;11(10):547. Citation Format: Samantha Gardner, Kharimat L. Alatise, Emily Miller, Emily Grant, Angela Alexander-Bryant. Cationic cholesterol liposomes for combination therapy to treat drug-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 306.