Targeted core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin

Abstract

Core-crosslinked thermosensitive and biodegradable polymeric micelles were actively targeted to EGFR-overexpressing cancer cells by conjugating an anti-EGFR nanobody on the surface of the micelles. A methacrylated doxorubicin derivative containing an acid sensitive hydrazone spacer was encapsulated and subsequently covalently attached during core-crosslinking of the micelles. Encapsulation efficiency was 60% and doxorubicin (DOX) was completely released after 24 h at pH 5, while hardly any DOX was released at pH 7.4. DOXloaded micelles showed toxicity similar to free doxorubicin towards ovarian carcinoma cells. Introduction: Conventional anticancer treatments include the use of cytotoxic compounds that kill tumor cells. However, these compounds also inhibit the growth and viability of various healthy cells like bone marrow and gastrointestinal tract cells, causing severe systemic side effects. For such cytotoxic agents, efficient drug delivery systems (DDS) are needed, that specifically deliver these compounds in tumor cells. For intravenous administration of hydrophobic drugs, polymeric micelles are an interesting type of DDS. Thermosensitive and biodegradable polymeric micelles composed of mPEG-pHPMAmlactate have been used in the encapsulation of several anticancer drugs, as well as an MRI contrast agent [1-4]. However, even though these micelles (when core-crosslinked) display prolonged circulation and enhanced passive tumor accumulation due to the EPR effect, the loaded drug is rapidly diffused out after injection in the blood stream. So in this study doxorubicin, a cytotoxic drug, was selected for covalent attachment to the core of these micelles, using a methacrylated derivative of doxorubicin Figure Presented This prodrug could be co-crosslinked in the hydrophobic core of the micelles composed of methacrylated block copolymers (Fig. 1a) by means of KPS and TEMED polymerization. Importantly, this doxorubicin derivative contains a biodegradable hydrazone linker, which is stable at physiological pH, but hydrolyzes in acidic environment [5]. Thus it was hypothesized that doxorubicin wouwas less thanld be retained in the micelles during the circulation, but would be released at the low endosomal pH of the tumor cells, upon arrival to the tumor tissue through the EPR effect and/or active targeting and subsequent tumor cell internalization by endocytosis. Figure Presented. Experimental methods: For active targeting, the polymer was modified with PDP (pyridyldithio propionate) on the PEG block. This way, PDP surface modified micelles could be formed, on which a SATA modified anti-EGFR nanobodywas conjugated via disulfide bonding by mixing and reacting at room temperature overnight. For doxorubicin covalent entrapment, 20 mg/ml of polymer was dissolved in ammonium acetate buffer, and KPS (90 μl, 30 mg/ml in buffer), TEMED (50 μl,120 mg/ml in buffer) and DOX-MA (30 mg/ml in methanol) were subsequently added, followed by rapid heating at 50 °C, to result inmicelle formation. The DOX loading and release were determined by HPLC. Finally to obtain information about the efficacy of the formulation, the cell viability of ovarian carcinoma cells wasmonitored using aWST assay, after 72 h incubation at 37 °C with free DOX, DOX-MA, and DOX loaded micelles. Results and discussion To ensure tumor cell recognition and uptake of the corecrosslinked micelles, an anti-EGFR EGa1 nanobody was attached on the surface of fluorescently labeled empty micelles using disulfide bonds, and the cell association was studied in cancer cells overexpressing EGFR. The conjugation was successful; cell association experiments in EGFR expressing cancer cells (A431) showed increased association of the nanobody micelles compared to empty micelles, which also increased with concentration (Fig. 2). It was shown that around 60% of the methacrylated doxorubicin added was covalently attached to the core of the micelles (without nanobodies on the surface), while the free drug that remained was less than 5%. The micelles released the entire drug payload within approximately 24 h incubation at pH 5 (where hydrolysis of the hydrazone bond occurs), while release at pH 7.4 was less than 10% Figure Presented Finally, in vitro cytotoxicity experiments in tumor cells showed no toxicity for empty micelles (data not shown), while DOX-loaded micelles showed similar toxicity as free DOX (Fig. 4), indicating possible cell internalization and release in the acidic lysosomes. Figure Presented. Conclusion: A novel delivery system was developed, which can covalently entrap doxorubicin through a biodegradable linker that releases the drug preferentially in an acidic environment, and which can be targeted via the conjugation of an anti-EGFR nanobody on the micellar surface.