Biocompatibility of Carbon Nanoparticles in HeLa Cells is Dictated by Synthesis and Sterilization Procedures

Due to their unique chemical and physical properties, carbon-based NanoMaterials (C-NMs) are largely exploited in biomedicine, i.e., cell and tissue imaging, drug delivery and tissue engineering scaffold, even if reports regarding their toxicity are still conflicting. In fact, biological effects strictly depend on the dynamic physicochemical characteristics of C-NMs, which in turn are strongly influenced by the procedures of their synthesis, and nanometrological techniques, e.g., Electron Microscopy (EM)-based analysis, are becoming the main tool for researchers to characterize nanoproducts. The aim of the present work is the study of the influence of synthesis and sterilization protocols on the size, shape, stability and biocompatibility of carbon NanoParticles (C-NPs). C-NPs were synthesized by using graphite as bulk material through an electrochemical method applying a constant voltage of 30 V and different times of synthesis. The C-NPs solution was sterilized by adopting different sterilization protocols during and/or after the synthesis. Size, shape and stability were studied by TEM and spectroscopy, while biocompatibility was tested in HeLa cells. Synthesis and sterilization procedures did not influence size, shape and stability of C-NPs, but interfered with C-NPs biocompatibility. In fact, irrespective of time of electrolysis process, the NPs show spherical shape with an average diameter of 7 nm. UV-visible spectra show typical peak of carbonaceous materials that falls at 236 nm without aggregation and sedimentation. However, when NPs obtained at 90 min of synthesis were twice autoclaved the peak shifted to 257 nm. HeLa cells were incubated with different C-NPs solutions administered at different concentrations, ranging from 8×105 to 1.6×107 C-NPs/cell, for different times (4, 24 and 48h). Cell viability was C-NPs concentration- and time of culture-dependent; interestingly, also the time of electrolysis process used during particles synthesis and procedures adopted to sterilize C-NPs solutions largely influenced cells response.