Poster T2 - Cationic Nanoparticles Loaded with Cisplatin for Local Delivery in Cervical Cancer Enhance Uptake and Anti-tumor Effects, and Reduce Leaking to Circulation

Introduction: Cervical cancer is one of the leading causes of mortality among women worldwide, and it is usually treated with a combination of chemotherapy, such as cisplatin, and radiation therapy. However, conventional therapies have yielded unsatisfactory outcomes, partially, due to inadequate delivery cisplatin to the cervix tissue, resulting from dose-limiting side effects, when delivered systemically (Federico et al., 2021). We have previously shown that localized delivery of cisplatin using a PEG-based biodegradable implant dramatically enhanced the cisplatin levels in the cervical tumors and reduced its levels in other normal organs (Federico et al., 2021); however, the released cisplatin was also shown to leak to the blood stream. The overall goal of this study is to develop a nanoparticle-based cisplatin formulation to enhance its uptake to the tumor cells in the cervix and prevent its leaking to the blood, and other normal organs.

Materials and

Methods: Formulation: Two different liposomes were prepared: one with high positive charge (cationic) and no PEGylation, to enhance cell adhesion and uptake, and reduce leakiness; and another with no positive charge and PEGylated, mimicking clinically used liposomal formulation of cisplatin, Lipoplatin, both of which were prepared using thin-film hydration method. Briefly, lipids and DiD (red fluorescence dye), were dissolved in chloroform, and the chloroform was evaporated to form a thin film of lipid. Then, the films were hydrated with a solution of cisplatin, and extruded through 400 nm, 200 nm, and 100 nm membranes. Characterization: Liposomes were subjected to DLS analysis to determine size, zeta-potential, and PDI. In addition, the encapsulation efficiency of cisplatin in the liposomes was determined by measuring the non-encapsulated drug in the supernatant using ICP-MS. Cellular Uptake: Siha cell lines were cultured in 96-well plates with increasing concentrations of the DiD-labeled liposomes for 2h, 4h, 12h or 24h. Cells were washed, and the uptake of the liposomes was determined using fluorescence plate reader. Cytotoxicity Assay: Siha cell lines were cultured in 96-well plates with increasing concentrations of free, or liposomal cisplatin for 48h, and their viability was determined using MTT assay. Leakiness of liposomal vs free cisplatin: Endothelial cells were plated in the upper chamber of a transwell migration assay chamber to confluence, then free or liposomal cisplatin was applied to the upper chamber, and its concentration in the lower chamber was determined at 2h, 4h, and 24h using ICP-MS.

Results, Conclusions, and Discussions: We have developed two liposomal formulations of cisplatin: the first is cationic with zeta-potential of +39.2 mV, a size of 165 nm, and PDI of 0.04; and the PEGylated liposomes with a negative zeta-potential, a size of 136 nm, and PDI of 0.01, mimicking the clinically used Lipoplatin. Both liposomal formulations showed cisplatin encapsulation efficiency of about 50%. The cationic liposomes showed a dose-dependent and time dependent uptake to cervical cancer cells, that peaked at a 23-fold of untreated cells. While the PEGylated liposomes showed a maximum of 3-fold uptake. The cationic liposomal cisplatin induces more cytotoxicity in cervical cancer cells than the free drug and the PEGylated formulation, in which at 10 µM the cationic liposome induced killing of over 70% of the cells, while the free drug and the PEGylated liposomal cisplatin induced 60% and 27%, respectively. Moreover, while free cisplatin showed significant leakage across endothelial cells, both liposomal formulations prevented the leakage.
We have developed a cationic non-PEGylated liposomal formulation of cisplatin that dramatically enhanced the cellular uptake of cisplatin and prevented its leaking across endothelium. The rationale behind this formulation is that we wanted the maximum cell adhesion and uptake (due to the high cationic charge), but at the same time, we did need to stealth it with PEG, since it was not designed for systemic delivery, but rather for local delivery in the cervix. Compared to the systemic cisplatin formulation (non-cationic and PEGylated, mimicking Lipoplatin), the cationic formulation introduced significantly enhanced cellular uptake and toxicity in cervical cancer cells, and similar prevention of leakiness through the endothelium.
To conclude, we developed a cationic formulation of cisplatin with high cellular uptake and toxicity for local delivery in cervical cancer. These were incorporated in our previously described (Federico et al., 2021) PEG-based biodegradable local delivery system for cervical cancer, and now being tested in animals for efficacy and biodistribution, as a basis for future clinical studies.

Acknowledgements (Optional): This work is supported by a grant from the Cancer Prevention & Research of Texas (CPRIT # RR220088).