Shikonin-loaded antibody-armed nanoparticles for targeted therapy of ovarian cancer

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin ( SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells ( OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and -lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment ( TME), we aimed to engineer SHK as polymeric nanoparticles ( NPs) with targeting moiety toward tumor -microvasculature. To this end, using single/double emulsion solvent evaporation/-diffusion technique with sonication, we formulated biodegradable NPs of poly( lactic-co-glycolic acid) ( PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol ( PEG) and tumor endothelial marker 1 ( TEM1)/ endosialin-targeting antibody ( Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was similar to 80%-90%, which was reduced to similar to 50%- 60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loadedAb-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.