Annona muricata L. leaves extract loaded on chitosan nanoparticles combined with methotrexate induce Cytotoxicity in A549 cancer cell line

Abstract Annona muricata Linn (AM) and methotrexate (MTX)-loaded chitosan (Ch) as a natural cationic, bio-degradable and biocompatible polymer were investigated for their genotoxic and cytotoxic effects in their normal and nano form. The AM-ChNP and MTX-ChNP were analyzed and characterized according to fourier transform infrared spectroscopy (FTIR), particle size, Zeta potential, transmission electron microscope (TEM) and encapsulation efficiency. The chitosan concentration (3:1) was found to be an important parameter for optimizing the dispersion of AM-ChNPs and of MTX-ChNPs nanocomposites over long time. The efficiency was found to be 87.3% and size of 100–200 nm. The stabilty of the nanocomposites offers a good sign in cancer cell line treatment. Seven groups were prepared using the A549 lung cell line: control group, A. muricata leaf extract (AM), Methotrexate (MTX), AM-ChNPs, MTX-ChNPs, AM + MTX, and AM-ChNPs + MTX-ChNPs. 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide ( MTT) assay for testing cell cytotoxicity; gene expression of Caspase- 3; Bcl- 2; TNF- α; and Cyclin A genes. Cell cycle progress and the percentages of apoptosis and necrosis additionally were assessed. The combination of AM-ChNPs and MTX-ChNPs revealed clear action against A549 cancer cell line. The IC50 values for AM-ChNPs + MTX-ChNPs nano-group compared to AM + MTX group increased by 1.7 folds. Moreover, the apoptotic Caspase-3 gene expression was significantly upregulated by 8 folds. However, antiapoptotic Bcl-2 gene expression was unsignificantly downregulated by 0.16 folds. The inflammatory TNF-α gene expression was significantly upregulated by 6 folds, and the Cyclin A gene expression was downregulated by 0.29 folds, in the combination group of AM-ChNPs + MTX-ChNPs. An increase was discovered through flow cytometric analysis in the G2/M phase cell population driven into apoptosis. This study highlights on the combined effects on genotoxicity and cytotoxicity for both AM-ChNPs and MTX-ChNPs and their free forms; as well as their apoptogenic activities.