Infrared spectroscopy and flow cytometry studies on the apoptotic effect of nano-chrysin in HeLa cells

Mapping the structural changes in membrane lipids, proteins, polysaccharides and nucleic acids has opened new channels for understanding the mode of action of anticancer natural products. Earlier, we synthesized chrysin nanoparticles (NChr) with good bioavailability, and characterized its size, surface charge, entrapment efficiency, and drug release pattern using PLGA polymer. NChr induced concentration dependent cytotoxicity in HeLa cells with an IC₅₀ of 61.54 ± 1.2 µM in comparison with free chrysin with IC₅₀ of 86.51 ± 2.9 µM. Since nanoparticles interact dynamically with cell membranes, organelles, proteins and DNA, it is necessary to understand the interplay of nanodrug induced macromolecular changes in cancer cells. In this work, we obtained signatures of NChr-induced biochemical changes in HeLa cells by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy technique coupled with flow cytometry. NChr induced cell membrane disruption, G1 phase cell cycle arrest, and increased externalization of phosphatidylserine leading to apoptosis indicating the biochemical perturbations in membrane lipids and DNA of HeLa cells in comparison with untreated cells. The 1300-1000 cm^-1 spectral region indicated NChr interaction with the ribose sugar backbone and DNA denaturation. Spectral range 1800-1400 cm^-1 indicated a concentration dependent decrease in α helical and β sheet structures which may lead to protein degradation during apoptosis. The spectral range 3000-2800 cm^-1 indicated the lipid peroxidation in response to NChr treatment. This is the first study describing the bio-macromolecular changes induced by a nano encapsulated drug and can pave the way to investigate unconventional modes of action for bioactive formulations.