Implantable antineoplastic-loaded antibody functionalized nanomicelles for human ovarian carcinoma cell targeting by molecular and in vivo investigations

Abstract

Epithelial ovarian cancer (EOC) is the most insidious, fatal gynaecological malignancy that accounts for millions of deaths in female population. Globally, the five-year survival period is between 15–20% for patience with clinical late stage ovarian malignancy in spite of surgery and platinum treatment. This study aimed to design and develop a novel drug delivery system employing antibody-ligand functionalized antineoplastic-loaded nanomicelles encapsulated with Chitosan-Poly(vinylpyrrolidone)-Poly (N-isopropylacrylamide) (C-P-N) hydrogel to form an in situ forming Implant (ISFI) which is responsive to temperature (body temperature 370C), pH (peritoneal fluid pH ~6.6) for cancer cell-targeting following intraperitoneal implantation to increase the residence time of the nanomicelles at tumor sites over a period exceeding one month, enhancing tumor uptake of drugs and prevent recurrence and chemo-resistance. An engineered-fabricated nanomicelle system (MTX)NM’s was formed by a novel thermal ring opening co-polymerization of hydrophobic L-Aspartic acid-N-carboxyanhydride onto the backbone of hydrophilic PNIPAAm-NH2 to form amphiphilic poly (N-isopropylacrylamide)-block-poly (aspartic acid) (PNIPAAm-b-PAsp) copolymer. PNIPAAm-b-PAsp copolymer exhibited competency in forming amphiphilic nanomicelles broadening areas of its nano-application in implantable drug delivery. Utilizing (PNIPAAm-b-PAsp) micelles, variables for an experimental design were obtained. A Face-Centred Central Composite experimental design approach generated thirteen formulations thoroughly screened in terms of variables (Amount of copolymer (mg) and homogenizer speed (rpm)) affecting responses (size (nm), drug entrapment efficiency (%) and mean dissolution time). Nanomicelles with sizes ranging from 51.67 to 76.45 nm, a yield/recovery of 46.8–89.8 mg and polydispersity index (PDI ≤ 0.5) were obtained. Drug encapsulation efficacy (DEE) was initially (65.3 ±0.5%) and was ultimately optimized to 80.6±0.3%. Optimal nanomicelle formulation was surface-functionalized with anti-MUC 16 (antibody) for the targeted delivery of methotrexate to human ovarian carcinoma (NIH:OVCAR-5) cells that expressed MUC 16 as a preferential form of intraperitoneal ovarian cancer chemotherapy. Furthermore, cross-linked interpenetrating C-P-N hydrogel was synthesized for the preparation of an in situ forming implant (ISFI) for ovarian carcinoma treatment. ISFI was fabricated by encapsulating a nanomicelle comprising of anti-MUC 16 (antibody) functionalized methotrexate (MTX)-loaded PNIPAAm-b-PAsp nanomicelles (AF(MTX)NM’s) within C-P-N hydrogel. Ex vivo endocytotic internalization via confocal fluorescent microscopy and intracellular imaging studies in (NIH:OVCAR-5) cells showed positive cellular uptake in both optimal (MTX)NM’s and (AF(MTX)NM’s) with exemplary results for (AF(MTX)NM’s) due to improved intracellular delivery. Chemotherapeutic efficacy of various treatment protocols including ISFI were invivo tested on the optimal Athymic nude mouse model that was intraperitoneally and subcutaneously induced with human ovarian carcinoma cells (NIH:OVCAR-5) and tumors with associated severe ascites grew within 10 days of inoculation. Results demonstrated tumor regression including reduction in mouse weight and tumor size, as well as a significant (p<0,05) reduction in mucin 16 levels in serum and ascitic fluid and improved survival of mice after treatment with the experimental anti-MUC16/CA125 antibody-bound nanotherapeutic implant drug delivery system (p<0,05). Low quantities of drug were found in the plasma but elevated levels were observed in the peritoneal cavity. In addition, the drug was present in the surrounding tissue in high concentration even after 10 days. Based on the results of this study, the antibody-bound nanotherapeutic implant drug delivery system should be considered a potentially important immuno-chemotherapeutic agent that can be employed in human clinical trials of advanced, and/or recurring, metastatic epithelial ovarian cancer (EOC). The development of this novel implantable drug delivery system may circumvent the treatment flaws experienced with conventional systemic therapies, effectively manage recurrent disease and ultimately prolong disease-free intervals in ovarian cancer patients.