Abstract
pH-sensitive micelles are desirable for co-drug delivery in cancer chemotherapy. Herein, a novel, very pH-sensitive and biodegradable citric acid grafted poly maleate-block-poly lactic-co-glycolic acid was synthesized and assembled as micelles via ultrasonication. The engineered homogeneous nanomicelles were used for the first time for doxorubicin and conferone combination chemotherapy in the MDA-MB-231 breast cancer cell line. The physicochemical properties of the micelles were investigated via 13CNMR, 1HNMR, FTIR, CHNS, DSC, SEM, and DLS-zeta analysis, and the in vitro degradation of the synthetic copolymer was investigated to confirm its biodegradability. The critical micelle concentration (CMC) value of the micelles was determined using pyrene as a probe and a spectrofluorometer. The drug release process was studied in acidic and neutral pH. The anti-tumoral properties of the dual drug-loaded micelles were investigated via MTT assay, cell cycle, and apoptosis experiments. The apoptosis was confirmed by Annexin-V, qRT-PCR and western blotting. The particle size (51.9 nm), zeta potential (-6.57 mV) and CMC (1.793 μg mL-1) of the co-drug loaded micelles were in the acceptable range for electrostatic stability. The uptake of the co-drug loaded micelles in the MDA-MB-231 cell line and spheroids was 97% and 36.1%, respectively. The cell cycle and apoptosis tests revealed that the cells treated with the co-drug-loaded micelles showed the highest amount of apoptosis (95.35%) in comparison to the single drug-loaded micelles and free drugs. Reverse transcription PCR (RT-PCR) showed that the expression levels of the proapoptotic genes were significantly up-regulated in the presence of the co-drug loaded micelles versus the single-drug loaded micelles and free drugs. Western blotting revealed that the co-drug-loaded micelles promoted apoptosis via the caspase-dependent pathway. Our findings confirmed that the pH-responsive biodegradable micelles containing doxorubicin and conferone are novel and effective for combination chemotherapy and offer a promising strategy for future in vivo studies.