Visualizing the anti-cancer effects of chrysin nanoparticles by flow cytometry, microscopy and Fourier transform infrared spectroscopy.

Nano formulations of anti-cancer natural products have revolutionized anti-cancer therapy and its challenges such as short half-life, low therapeutic index, non-specificity, efficiency, and bioavailability. Chrysin (Chr), a dihydroxyflavone isolated from a marine fungus Chaetomium globosum, is a known anti-cancer natural product. However, its free use in clinical scenarios is disadvantaged due to poor water solubility, rapid metabolism, and non-specific toxicity. In this study, Chr-nanoparticles (NPs) were prepared by the emulsion-diffusion evaporation technique. The optimized formulation of Chr-NPs was characterized for zeta size, potential, polydispersity index, transmission electron microscopy (TEM), and Fourier Transform Infrared Spectroscopy (FTIR). Chr-NPs had an average zeta size of 217.93 +/- 3.45 nm, and a zeta potential of -15.63 +/- 3.9 mV. As seen under the TEM, Chr-NPs measured an average size of 98.55 +/- 4.01 nm. The NPs demonstrated an initial burst followed by a sustained release behaviour. The anti-cancer effects of Chr-NPs were investigated in human cervical cancer cells (HeLa) and human embryonic kidney (HEK) cells. MTT and propidium iodide live/dead flow cytometry assays demonstrated an improved IC50 value over the free drug treatment. An interplay of apoptosis and autophagy cell death mechanisms in Chr-NP treated HeLa cells was observed by Acridine Orange and MDC staining, visualized by confocal microscopy. Chr-NPs induced structural changes in the HeLa cell surface proteins and lipids were studied by FTIR spectroscopy. Natural products are the largest source of drug discovery; this study can pave the way to improve their chemistry sustainably and explore the cell death mechanisms.