Exploring the synergistic effects of metformin and doxorubicin loaded chitosan nanoparticles for A549 lung cancer therapy.

In recent years the efforts for devising effective methods to confront cancer have grasped significant attention. Since conventional methods have failed to fulfill the favorable therapeutic outcome, efforts have been made to develop novel methods. Currently, incorporating drugs in nanocarriers are considered as an effective method. Herein, we prepared chitosan nanoparticles (Cs NPs) loaded with doxorubicin (DOX) and metformin (Met) and evaluated their physicochemical and anticancer in vitro properties. The drug loaded Cs NPs were prepared using the ionic gelation method. The size and surface charge of the Cs NPs were evaluated with DLS. Further characterization was made with SEM imaging. Drug loading was confirmed with FTIR analysis and assessed with calculating the UV absorbance of the unloaded drug. Cs NPs stability was also examined with DLS and XRD results. Drug release was investigated ultimately using semi-permeable membrane. Initially, the biosafety of the blank Cs NPs was assessed on A549 and NIH-3 cells with MTT assay. Flowcytometry was used to test the NPs uptake by A549 cells. Similarly, the cytotoxic effect of Met and DOX Cs NPs was explored. The results were analyzed with CompuSyn software and the highest synergistic dose combination (Fa50) was obtained. Using Fa50 concentrations, apoptosis, scratch, 3D cultures, anti-angiogenesis, and western blot assays were performed. The size of DOX-Cs NPs and Met-Cs NPs were 51.20 nm and 48.20 nm, and zeta potentials were 23.26 mV and 25.05 mV, respectively. The EE were 59.03% for DOX and 86.39% for Met. Drug release investigation demonstrated a pH-independent release for DOX, achieving 80% release, while Met exhibited a pH-dependent release, with 70% release. Synergistic CI value of 0.91 was achieved in the toxicity assay and nanoparticulation lowered the IC of both drugs. An increase in early (35%) and late (25%) apoptosis with the combination therapy compared to controls (15% and 10%) was achieved. 3D spheroid models demonstrated that the treatment of A549 spheroids with DOX-Met-CS NPs inhibited tumor growth by 60% compared to untreated controls. Western blot analyses revealed downregulation of oncogenes ERBB2 and PI3K in response to the combination treatment. In conclusion, it was found that the combination of DOX/Met-Cs NPs significantly inhibits A549 cells growth, highlighting the proposed approach as an efficient treatment strategy against lung cancer disease.