Biophysical, bioinformatical, cellular, and molecular investigations on the effects of graphene oxide nanosheets on the hemoglobin structure and lymphocyte cell cytotoxicity

Mohammad Mahdi Nejadi Babadaei,1,* Mina Feli Moghaddam,1,* Sara Solhvand,1 Ehsan Alizadehmollayaghoob,1 Farnoosh Attar,2 Elham Rajabbeigi,3 Keivan Akhtari,4 Soyar Sari,1 Mojtaba Falahati5 1Department of Cellular and Molecular Biology, Faculty of Advance Science and Technology, Pharmaceutical Sciences Branch, Islamic Azad University (IAUPS), Tehran, Iran; 2Department of Biology, Faculty of Food Industry and Agriculture, Standard Research Institute (SRI), Karaj, Iran; 3Department of Biochemistry, Faculty of Advance Science and Technology, Medical Sciences Branch, Islamic Azad University, Tehran, Iran; 4Department of Physics, University of Kurdistan, Sanandaj, Iran; 5Department of Nanotechnology, Faculty of Advance Science and Technology, Pharmaceutical Sciences Branch, Islamic Azad University (IAUPS), Tehran, Iran *These authors contributed equally to this work Background: Implementations of nanoparticles have been receiving great interest in medicine and technology due to their unique characteristics. However, their toxic impacts on the biological system are not well explored.Aim: This study aims to investigate the influence of fabricated nano graphene oxide (NGO) sheets on the secondary and quaternary structural alterations of human hemoglobin (Hb) and cytotoxicity against lymphocyte cells.Materials and methods: Different spectroscopic methods, such as extrinsic and synchronous fluorescence spectroscopy and far circular dichroism (CD) spectroscopy, molecular docking investigation, cellular assays (trypan blue exclusion, cellular uptake, ROS, cell cycle, and apoptosis), and molecular assay (fold changes in anti/proapoptotic genes [B-cell lymphoma-2 {BCL2}/BAX] expression levels) were used in this study.Results: Transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and zeta potential investigations revealed the nano-sized nature of NGOs with good colloidal stability. Extrinsic fluorescence spectroscopy by using 8-anilinonaphthalene-1-sulfonic acid and synchronous fluorescence spectroscopy showed that NGOs can unfold the quaternary structure of Hb in the vicinity of Tyr residues. The CD investigation demonstrated that the α-helicity of Hb experienced substantial alteration upon interaction with increasing concentrations of NGOs. The molecular docking study showed that NGOs interacted with polar residues of Hb. Cellular and molecular assays revealed that NGOs lead to ROS formation, cell cycle arrest, and apoptosis through the BAX and BCL2 pathway.Conclusion: These data reveal that NGOs can induce some protein structural changes and stimulate cytotoxicity against normal cell targets. Therefore, their applications in healthy systems should be limited. Keywords: nano graphene oxide, human hemoglobin, lymphocyte cell, fluorescence spectroscopy, circular dichroism spectroscopy, docking, cytotoxicity, ROS, cell cycle, flow cytometr