Oleanolic acid-conjugated poly (D, l-lactide)-based micelles for effective delivery of doxorubicin and combination chemotherapy in oral cancer

Abstract

Here, an amphiphilic polymer, oleanolic acid conjugated methoxy-poly (ethylene glycol)-poly (D, l-lactide) (mPEG-PLA-OA) was synthesized, which self-assembled in aqueous environment and loaded the poorly soluble anticancer agent, doxorubicin. Various physico-chemical characterization studies on polymer and DOX-loaded polymeric micelles (DOX-mPEG-PLA-OA) at various drug to polymer (1:5/10/15) (DTP) ratios were performed, which included characterizations via UV, IR, NMR spectroscopy and gel permeation chromatography for polymers, and dynamic light scattering, DSC, XRD techniques, determination of critical micelles concentration, percent drug loading, encapsulation efficiency, drug release, kinetic stability and haemolysis studies for micelles. Further, DOX-mPEG-PLA-OA were evaluated for their cellular uptake, cytotoxicity, ability to induce apoptosis, DNA fragmentation, nuclear damage, ROS generation, and mitochondrial membrane depolarization in oral squamous cell carcinoma (FaDu HTB-43) and murine melanoma (B16F10) in monolayer and in FaDu HTB-43 3D cell culture systems. DOX-mPEG-PLA-OA at DTP ratios. 1:5/10/15 showed physico-chemical parameters in the following range: particle sizes. ~210.3–225.1 nm, DOX encapsulation efficiency. 29–35% and DOX-loading. 2.2–5%. DOX-mPEG-PLA-OA micelles (1:15) were stable as proven by kinetic stability study, and showed sustained release of encapsulated DOX for over 60 h. The cell viability study revealed that DOX-mPEG-PLA-OA micelles (1:15) demonstrated highest time- and dose dependent cytotoxicity compared to free DOX and micelles of other DTP ratios. Following treatment, the DOX-mPEG-PLA-OA micelles (1:5/10/15) induced highest cellular apoptosis in the range of 28.17–48.1, compared to free DOX (9.5 ± 2.52), and mPEG-PLA-OA micelles (25.4 ± 2.91), DOX-mPEG-PLA-OA micelles (1:15) being the strongest apoptosis inducer (48.1 ± 2.68) as determined by flow cytometry-mediated annexin V assay. DOX-mPEG-PLA-OA micelles (1:15) induced significantly higher DNA fragmentation, nuclear condensation, ROS generation, and mitochondrial membrane permeabilization compared to free Dox in monolayer FaDu HTB-43. DOX-mPEG-PLA-OA micelles (1:15) showed reasonably higher penetration and stronger growth inhibition in FaDu HTB-43 spheroids compared to free DOX and micelles of other DTP ratios. In-vivo pharmacokinetic studies showed that the DOX-mPEG-PLA-OA micelles demonstrated enhanced blood circulation time with decreased rate of clearance than free Dox (t1/2. 33.5 ± 2.5 h vs. 1.77 ± 0.32 h, and Cmax. 59.7 ± 2.2 vs. 2.3 ± 0.1, respectively). Immunohistochemistry revealed no significant toxicity in major organs of DOX-mPEG-PLA-OA micelles treated rats. The series of studies indicated that the OA-conjugated mPEG-PLA micelles served as an efficient nanocarriers for DOX, demonstrated DOX, and OA combined superior anticancer effect, sustained DOX level in blood circulation for up to 96 h, and no sign of major organs toxicity. Therefore, this newly developed DOX, OA combined micelles displayed its potential as promising alternative to DOX-based conventional therapy, and warrants further investigation for clinical application, especially for the treatment of oral cancer.