Paclitaxel-Loaded Polymersomes for Enhanced Intraperitoneal Chemotherapy

Abstract

Peritoneal carcinomatosis is present in more than 60% of gastric cancer, 40% of ovarian cancer, and 35% of colon cancer patients. It is the second most common cause of cancer-related mortality, with a median survival of 1 to 3 months. Cytoreductive surgery combined with intraperitoneal chemotherapy is the current clinical treatment, but achieving curative drug accumulation and penetration in peritoneal carcinomatosis lesions remains an unresolved challenge. Here, we used flexible and pH-sensitive polymersomes for payload delivery to peritoneal gastric (MKN-45P) and colon (CT26) carcinoma in mice. Polymersomes were loaded with paclitaxel and in vitro drug release was studied as a function of pH and time. Paclitaxel-loaded polymersomes remained stable in aqueous solution at neutral pH for up to 4 months. In cell viability assay on cultured cancer cell lines (MKN-45P, SKOV3, CT26), paclitaxel-loaded polymersomes were more toxic than free drug or albumin-bound paclitaxel (Abraxane). Intraperitoneally administered fluorescent polymersomes accumulated in malignant lesions, and immunofluorescence revealed an intense signal inside tumors with no detectable signal in control organs. A dual targeting of tumors was observed: direct (circulation-independent) penetration, and systemic, blood vessel-associated accumulation. Finally, we evaluated preclinical antitumor efficacy of paclitaxel-polymersomes in the treatment of MKN-45P disseminated gastric carcinoma using a total dose of 7 mg/kg. Experimental therapy with paclitaxel-polymersomes improved the therapeutic index of drug over free paclitaxel and Abraxane, as evaluated by intraperitoneal tumor burden and number of metastatic nodules. Our findings underline the potential utility of the polymersome platform for delivery of drugs and imaging agents to peritoneal carcinomatosis lesions. Mol Cancer Ther; 15(4); 670-9. ©2016 AACR.

Figure 1

Characterization of empty polymersomes (PS) and PTX-loaded polymersomes (PS-PTX). A: TEM images of freshly prepared empty and PTX-polymersomes. Polymersome samples were deposited on copper grids and stained with phosphotungstic acid at pH 7.4. B: DLS determination of hydrodynamic diameter and polydispersity index (PDI) values of empty and PTX-polymersomes after incubation for indicated time and temperature.

Figure 2

In vitro assessment of cytotoxicity of free and encapsulated PTX to MKN-45P, CT26, and SKOV-3 cells. Cytotoxicity measured by MTT assay of the indicated formulations at 0.5 μM of PTX after 24 hours of incubation. PS, polymersomes; PS-PTX, PTX-loaded polymersomes. Statistical analysis was performed by ANOVA. n=4; error bars indicate +SEM; *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 3

Intraperitoneally administered fluorescent polymersomes home to peritoneal tumors in vivo. Mice bearing disseminated MKN-45P (A) or CT26 (B) tumors were injected with polymersomes labeled with fluorescein and the mice were perfused after 24 hours. C: Florescence intensity of tumors and different organs from ex vivo imaging quantified with Image J. Results are normalized for tissue area. Panel D: Confocal micrographs of peritoneal tumor sections after 24 hours of FAM-polymersomes injection in MKN-45P and CT26 bearing mice. Blue, DAPI; green, FAM; red, CD31. The white arrow indicates the co-localization of green and red fluorescence and the pink arrow indicates only the green fluorescence. Representative fields from multiple sections of three independent mice are shown. Tu, tumor; Ki, kidney; He, heart; Li, liver; Lu, lung; Sp, spleen. N = 4 for MKN-45 tumor model and n =3 for CT26 tumor model. Error bars indicate + SEM. Scale bars = 20μm, for panel H, 50μm for panels A, C, E, F, G, and 100 μm for panel D.

Figure 4

Experimental therapy of MKN-45P tumor-bearing mice. Mice bearing disseminated peritoneal tumors induced with MKN-45P-Luc cells were injected with indicated formulations (1 mg PTX/kg) every other day. A: tumor growth monitored by measurement of luciferase activity. Days after MKN-45P-Luc cells injection are plotted on the x-axis. On the y-axes are the photons recovered from region of interest. The arrows indicate treatment injections. B: Quantification of metastatic nodules after the treatment. n =5 in each group. PS, polymersomes; PS-PTX, PTX-loaded polymersomes. Statistical analyses, ANOVA; error bars, mean + SEM, *** p < 0.001, ** p < 0.01.