(M0930-10-57) Targeted Liposomes for Selective Delivery of Cisplatin to Ovarian Cancer

Purpose: Cisplatin (CPT) is a first-line chemotherapeutic agent for the treatment of ovarian cancer patients. However, CPT has severe adverse effects. The purpose of this study is to develop targeted liposomes that target the xCT transporter which is upregulated in ovarian cancer. Targeted liposomes encapsulating CPT, target ovarian cancer and selectively deliver CPT only to tumor, thereby avoiding the adverse effects of CPT in the normal tissues. We evaluated the stability, the cytotoxicity, the cellular uptake of targeted liposomes in ovarian cancer cell lines and their anticancer effect in a mouse model of orthotopic ovarian cancer.

Methods: CPT was encapsulated in liposomes by the remote loading method. The stability of liposomes was evaluated by measuring the drug-leakage from liposomes and the growth of liposome size stored at 4°C for a 30-day period. The drug-leakage from the targeted liposomes was evaluated by placing the CPT loaded liposomes into a dialysis tubing, then removing aliquots and analyzing twice weekly for CPT content. The liposome size was evaluated using Dynamic Light Scattering (Nano Brook Omni, Brookhaven Instruments). The cellular uptake of targeted liposomes (encapsulating the fluorescent dye sulforhodamine B, SRB) was evaluated in drug-resistant (A2870cis, OVCAR3, OVCAR4, IGROV1, and NCI/ADR-RES) and drug sensitive (A2870 and OVCAR8) ovarian cancer cell lines were evaluated using flow cytometry (BD Accuri C6). The cytotoxicity of CPT in targeted liposomes was evaluated in the various ovarian cell lines using the MTS assay (Novus Biologicals). The anticancer effect of CPT encapsulated in targeted liposomes was evaluated in female mice (NCI Athymic NCr-nu/nu, purchased from Charles River) injected orthotopically with 1x10⁶ A2780-cis cells. Two weeks later the mice were injected IV with 3.5 mg/kg/week of cisplatin encapsulated in targeted liposomes, nontargeted and free cisplatin and the survival of mice was recorded.

Results: The targeted liposomes over the 30-day period, show that 6.8% of the total loaded CPT leaked out of the liposomes. During that period the growth of the liposomes was < 5% for the liposomes stored at 4°C. The cellular uptake of ovarian cancer cell lines, A2870, A2870cis, OVCAR3, OVCAR4, OVCAR8, and NCI/ADR-Res the targeted SRB loaded liposomes showed significantly more fluorescence intensity when compared to free SRB and non-targeted liposomes (p < 0.05). In the cell line IGROV1, there is a similar trend seen, where there was a higher fluorescence intensity compared to free SRB and non-targeted. The fluorescence intensity was found to be higher compared to non-targeted liposomes (p < 0.0001) but not statistically significant higher compared to free SRB. For cytotoxicity, a similar trend is seen in cell lines A2870, A2870cis, IGROV1, OVCAR3, OVCAR4, OVCAR8, and NCI/ADR-res, where the cisplatin loaded targeted liposomes had the lowest IC₅₀ compared to free drug and non-targeted liposomes. Non-targeted liposomes had the highest IC₅₀ values compared to free drug and targeted liposomes. In cell lines A2870, A2870cis, IGROV1, OVCAR3, OVCAR4, OVCAR8, and NCI/ADR-res the IC₅₀ values show a 2.4, 5.3, 4.1, 3.3, 1.7, 1.6, and 2.5-fold lower values compared to free cisplatin. Mice that received cisplatin in targeted liposomes show higher survival rates compared to mice received cisplatin in nontargeted liposomes and free cisplatin.

Conclusion: Targeted liposomes encapsulating CPT are stable in terms of size and drug leakage, show enhanced in-vitro cellular uptake and anticancer activity in resistant and sensitive ovarian cancer cell lines. Furthermore, cisplatin in targeted liposomes significantly increased the survival of mice with orthotopic ovarian tumors.

Acknowledgements: We thank the NCI for their support, award R41CA247009.