Nanostrutture di carbonio come vettori per farmaci antitumorali

Carbon nanotechnology has evolved into a truly interdisciplinary field, bridging material science with medicine. Fullerenes (C60) play a major role in this field and are currently explored for biomedical applications such as radiopharmaceuticals and contrast agents, gene delivery and as carriers for chemotherapeutics. Five fullerene derivatives (F1, F2, F3, F4 and F5), functionalized by 1,3-dipolar cycloaddition of azomethine ylides to the C60 cage, were studied in vitro for their toxicity in a number of cell types and with different assays. Cell cytotoxicity on human mammary carcinoma cell line (MCF7), evaluated with the MTT and NRU tests and further confirmed by a flow cytometry approach with DiOC6 and PI probes, showed that derivative F2 was free of necrotic or apoptotic effects even after a long lasting cell exposure. F3 (differing from F2 for an additional positive charge obtained by quaternarization of the pyrrolidinic nitrogen by introducing a methyl group) was more toxic compared to the other compounds in all cellular models employed (HCT116, MCF7, MCF7/ADR, HT-29, H460, B16F10 and MDAMB231). Its IC50 is 20 à,µM after 72 hr of incubation by MTT test, and cell accumulation in the G1 phase and arrest in G0 phase (30%) was also observed. The mechanism of cellular uptake (studied with a fluorescein-bearing derivative of F2, hereafter called derivative F2-FITC), and the intracellular distribution were analyzed on MCF7 cell line. The studies of F2 biological effects have shown that this compound is able to enter the treated cells, probably by passive diffusion, to distribute within the cell cytoplasm, without getting into the nucleoplasm or into organelles such as lysosomes and mitochondria; these processes were evaluated by flow cytometry and confirmed by confocal microscopy. Experiments were performed on a multidrug resistant human mammary carcinoma cell line MCF7/ADR, a sub-line resistant to Doxorubicin because of the overexpression of the P-glycoprotein (P-gp) extrusion pump. The choice of these cells was based on our objective to conjugate the anticancer drug Doxorubicin to the fullerene vector in order to overcome adriamycin resistance. The F2 cellular uptake on MCF7/ADR and its maintenance of a constant concentration into cells seem to be insensitive to the presence of P-gp over-expression. These data suggest the possibility for derivative F2 to be used as carrier for anticancer drugs. The experiments on conjugated F2-DOX were made above all to verify if the activity of the drug linked to the fullerene remained the same as the free drug. Furthermore we studied the cellular uptake of F2-DOX in both MCF7 and MCF/ADR lines. Cytotoxicity tests have shown that F2-DOX has an irrelevant activity compared to free drug because Doxorubicin cannot get into the nucleus to perform its activity as it remains linked to F2. Nevertheless, the internalization of F2-DOX is higher in MCF7/ADR compared to the free drug. Fluorescent microscopy technique suggested that the F2-DOX inactivity might be associated with the stability of the bond between the carrier and the drug, which is not released and so is localized in the cytosol, as we have also observed for F2. Since many studies showed contradictory results and the molecular and cellular mechanisms of the cytotoxicity of this class of nanomaterials are not yet fully understood we performed a whole genome expression analysis, by high throughput RNA sequencing, using Illumina technologies. All together, the RNA-seq expression data confirmed the experimental evidence collected with previous in vitro studies showing that F3 is definitely the derivative causing more alterations on MCF7 cells on both the molecular and the cellular levels. However, also F2 is capable of affecting the same molecular pathways, although to a much lower intensity, since neither cytotoxic effects nor cell cycle arrest could be documented. The most important, and somewhat unexpected, result of our analysis was certainly the lack of molecular evidence concerning the activation of the main cellular pathways leading to cell death and often linked to fullerene toxicity in literature. In fact apoptosis, autophagy and ROS damage does not seem to be included among the most relevant molecular effects of F3, which on the contrary are mainly linked to the arrest of the transcription and protein synthesis machineries, leading to the entry in the G0 cell cycle phase. The RNA-seq analysis was able to identify several additional effects of fullerenes which had not been previously described, offering a complete overview of the gene expression alterations induced by these compounds on a whole-transcriptome level. Therefore the combination of large scale molecular analysis and the main viability assays might represent a valuable tool for a better understanding of the toxicity of fullerenes and other nanomaterials.