Tuning Cell Autophagy By Diversifying Multi-Walled Carbon Nanotube’s Surface Chemistry

Materials in the nanometer range often possess unique physical, optical, electronic, and biological properties compared with larger particles. The unique and advanced properties of nanomaterials have led to a rapid increase in their application. These applications include aerospace and airplanes, energy, architecture, chemicals and coatings, catalysts, environmental protection, computer memory, biomedicine and consumer products. This will increase the possibility for the NMs to enter human body. So it is necessary and urgent to evaluate the biosafety issue of NMs in human body.Autophagy, meaning self-eating, is an evolutionarily conserved catabolic process of the cells to degrade its cellular materials through the lysosome machinery. It exists in all the eukaryotic cells including yeast and mammalian cells, and is the other degradation machinery in addition to the ubiquitin-proteasome machinery. In normal circumstances, autophagy is kept to a basal level to deal with the metabolic stress, damaged organelles as well as the mis-folded or aggregated proteins in order to maintain the cell homeostasis. In time of starvation or energy deficiency, autophagy will be tuned to a higher level to provide cells with necessary nutrients or energy to survive the stress conditions. However, if the stress is constant, cells may die through an autophagy-dependent programmed cell death. Autophagy has been reported to play vital and complex roles in many diseases, including cancer, neurodegeneration, type Ⅱ diabetes, and atherosclerosis. Considering its complexity, proper regulation of autophagy turns out to be a practical strategy for maximum therapeutic outcomes.There is a growing body of literatures on the autophagy induction by nanomaterials ever since the first witness of autophagosome formation induced by quantum dots. Because of the janus role of autophagy in life, the relationship between nanomaterials and autophagy also has two sides. On one hand, nanomaterials turn out to be a good supplement to the tradational small molecule autophagy inducers. On the other hand, nanomaterials may impair the lysosome function leading to the cytotoxicity associated with the blockade of the autophagy flux. So modifying nanomaterials to tune their autophagy inducing level is very necessary and promising.To develop safe nanomaterials and nanomedicinal agents that regulate cell autophagy, we explored the possibility of controlling autophagy induction by systematically modifying the surface chemistry of MWCNTs. By analyzing autophagosome formation and autophagy-associated biomarkers, we have classified the autophagy inducing ability of MWCNTs into four categories. Seven MWCNT variants including MWCNT-COOH have the highest autophagy induction ability. While another seven MWCNTs variants lost this ability. The other67MWCNT variants belong to two categories with low and medium autophagy inducing ability. These findings demonstrate that pharmaceutical autophagy modulators and biocompatible nanomaterials can be developed through surface modifications. Our computational efforts have identified surface ligands (pharmacophores) that may switch autophagy on and off, providing a powerful method for the rational design of autophagy modulators and biocompatible nanoparticles. Studies on the molecular machinery of autophagy induction is the leading edge in the field of autophagy research. We checked the mTOR signaling activation by the seven MWCNTs variants with the highest level by western blot and found that they also deviated the autophagy induction signaling. We then chose mTOR dependent MWCNT-COOH and mTOR independent MWCNT41, which had comparable autophagy inducing ability, to further investigate their autophagy induction mechanisms. TEM showed that both MWCNT variants induced autophagosome formation in cells and western blot against LC3B indicated that their induction of autophagy is dose dependent. We then ruled out the possibility that autophagy was induced by the ligands dissociated from the nanotubes by western blot. Annexin V/7-AAD assay and western blot against cleaved caspase3indicated that both MWCNT variants did not induce apoptosis. Furthermore, cells incubated with fluorescent FITC-BSA tagged MWCNTs exhibited similar cellular uptake amount at various time points through flow cytometry analysis. And both MWCNT variants did not alter the intracellular calcium concentration at both low and high concentrations. TEM analysis found that both MWCNT variants had similar subcellular localizations. After ruling out the above major possibilites that may cause different signalings, we conducted an autophagy specific PCR array expreriment and revealed that the two MWCNT variants altered the mRNA expression of IGF1and IFNA2to different levels. Combining the our previous studies on MWCNTs and the current knowledge on autophagy, we deduced that the differences on the autophagy induction signaling was caused by different cell receptor binding preferences after modification. MWCNT-COOH induced autophagy through IGFlR/mTOR/p70S6K pathway, while MWCNT41induced autophagy by altering the expression of IFNA2through binding to IFNA2R.Differences in surface chemistry allow MWCNTs to trigger autophagy through different signaling pathways, demonstrating the flexibility and specificity of autophagy modulation by nanoparticles as a result of well-defined interactions with specific molecular signaling pathways.