Novel pH-sensitive and biodegradable micelles for the combined delivery of doxorubicin and conferone to induce apoptosis in MDA-MB-231 breast cancer cell line.

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--poly lactic--glycolic acid was synthesized and assembled as micelles 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 CNMR, HNMR, FTIR, CHNS, DSC, SEM, and DLS-zeta analysis, and the 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 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) 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 the single-drug loaded micelles and free drugs. Western blotting revealed that the co-drug-loaded micelles promoted apoptosis 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 studies.