Studies On The Surface Modification Of Mesoporous Carbon Nanoparticles And Its Biocompatibility

Nanomaterials have great application in the field of biological medicine, and carbon-based nanomaterials such as carbon nanotubes, graphene and mesoporous carbons occupy an important position. Mesoporous carbon nanomaterials are a new kind of porous carbon materials and have great potential applications. High surface area and large pore volume endow mesoporous carbons with high drug-loading capacity, and adjustable pore size and pore structure can control the release of drug molecules inside. Using mesoporous carbons in drug delivery system has great pontentiaL However, before these novel materials being safely applied in a clinical setting, it is aimed to fully understand its physical and chemical properties and adverse reactions. Their toxicity and biocompatibility needs to be carefully assessed. In the previous research, our teams have been successfully fabricated of PEGlyated oxidated mesoporous carbon nanoparticles as the carrier of antitumor drugs doxorubicin (DOX). The nanoparticle has good hydrophilicity and high drug loading efficiency. DOX releases in the system in a pH-responsive way and the drug system have good antitumor activity in vivo and in vitro. Other existing studies have also reported modification with certain molecules such as polyethylene glycol (PEG)/polyvinylpyrrolidone (PVP) which improve the materials of dispersibility and stability in the blood. It avoided the carrier of being captured of the reticuloendothelial systemand significantly prolonged the half-life. It aimed to improve the biocompatibility of carbon nanomaterials.Therefore, on the basis of previous work, here we use polyvinylpyrrolidone (PVP) and methoxyPEG2000-phospholipid conjugates to modify to the surface of mesoporous carbon nanoparticles. It is aimed to study the biocompatibility of modified and unmodified mesoporous carbon nanoparticles in the cellular level, immune system and animal level so that we can provide a theoretical basis for its application in the field of clinical medicine.The first chapter mainly introduces the synthesis of mesoporous carbon nanoparticles, surface modification and characterization. Mesoporous carbon nanoparticles were successfully synthesized by a low-concentration hydrothermal synthesis. The diameter of mesoporous carbon nanoparticles was about 90 nm, with uniform particle size, regular spherical and a good mesoscopic structure. Rely mainly on the surface of mesoporous carbon nanoparticles strong adsorption to modify. Mesoporous carbon nanoparticles were characterized by transmission electron microscopy, fourier transform infrared spectrometer, heat analysis, NMR, UV visible spectrophotometer and Malvern particle diameter. The results proved that PVP and DSPE-mPEG2000 were successfully modified to the surface of mesoporous carbon nanoparticles, which improved its dispersion and stability in aqueous solution. The results of cellular uptake with flow cytometry and fluorescence microscopy showed that the mesoporous carbon nanoparticles could uptake by normal phagocytosis and tumor cell in a short period of time (2h).The second chapter mainly studied the cytotoxicity of mesoporous carbon nanoparticles and cell oxidative stress level before and after the modification. This part was studied by CCK-8 test, cell apoptosis test and active oxygen detection kit. In the 12.5～100μg/ml concentration range, when MCN, MCN-PVP and MCN-PEG were incubated with L929 cells for 24h, MCN showed slight cytotoxicity and the mesoporous carbon nanoparticles with PVP and DSPE-mPEG2000 modification could decrease the toxicity to L929 cells; cell survival rate was increased in the different concentrations of MCN for 48h, which respect that the toxicity of MCN itself to L929 cells was low. When the modified and unmodified mesoporous carbon nanoparticles with HeLa cells were incubated with 24h or 48h, no cytotoxic effect was observed, which showed that the mesoporous carbon nanoparticles was relatively safe. When the concentration was less than 100μg/ml, the modified and unmodified mesoporous carbon would not induce the apoptosis of L929 and HeLa cells (apoptosis experiment). In the 12.5～100μg/ml concentration range, MCN could induce to product ROS in L929 and HeLa cells, which the production of ROS also increased with the increase of the concentration of nanoparticles. Modified mesoporous carbon nanoparticles could significantly reduce the amount of ROS produced by stimulating cells, the level of cellular oxidative stress and decrease stimulation of cells.In the third chapter, we have completed the preliminary evaluation of the immune toxicity of mesoporous carbon nanoparticles. MCN, MCN-PVP and MCN-PEG with the concentration of 100μg/ml could stimulate mDCs surface molecules CD11c, I-Ab, CD40 expression increase and promote the mDCs phenotype mature. At a concentration of 100μg/ml, PVP and DSPE-mPEG2000 modified mesoporous carbon nanoparticles could significantly reduce the level of TNF-a and IL-6 in the mice dendritic cells than the unmodified mesoporous carbon nanoparticles. In the concentration range of 12.5～100μg/ml, MCN, MCN-PVP and MCN-PEG could induce the apoptosis of T lymphocytes, and the more the apoptosis of T lymphocytes increased with the concentration of nanoparticles. In high concentration, T lymphocytes had higher immune toxicity and PVP and DSPE-mPEG2000 modification did not significantly reduce the apoptosis rate of T lymphocytes. Through the transmission electron microscope, we observed that the mesoporous carbon nanoparticles entered into RAW264.7 cells through endocytosis and aggregated in the cytoplasm, which as drug carriers could carry drugs through cell membranes. Modified and unmodified mesoporous carbon nanoparticles had lower the toxicity of RAW264.7 cells; possibly due to the mouse macrophage had strong phagocytosis and degradation ability.In the fourth chapter:the preliminary study of the biological toxicity effect of mesoporous carbon nanoparticles. In the concentration range of 0～100μg/ml, MCN, MCN-PVP and MCN-PEG were no hemolysis phenomenon (HR%<5%), which indicated a good biocompatibility. In the series of 7 days by tail vein injection dosage of 0.2ml/10g and concentration of lmg/ml of MCN, MCN-PVP and MCN-PEG, tissue sections of various organs showed that nanoparticles did not cause mice tissue fibrosis, degeneration and necrosis of pathological changes. But in the lung tissue sections gathered mesoporous carbon nanoparticles could be observed may due to the effects of mesoporous carbon nanoparticles in vivo hemodynamic. The mesoporous carbon nanoparticles after modified with PVP or DSPE-mPEG2000 could decrease in pulmonary residual volume, because the mesoporous carbon nanoparticles easily deposited in the vessel wall, but the modified nanoparticles improved dispersibility and improved its mobility in the blood vessels. But did not affect the function of liver and kidney of mice or activate mouse complement C3. The reason is not clear. The complement activation may occur in the later time.The above results showed that the mesoporous carbon nanoparticles were prepared and the surfaces of the nanoparticles were successfully modified with biocompatible polymer PVP and PEG. From the results of cells we can get that mesoporous carbon nanoparticle itself has relatively good biocompatibility but still have some certain stimulus to cells. Surface modification can improve the biological safety. Mesoporous carbon nanoparticles have a dose dependent on T lymphocyte toxicity, but the toxicity to T lymp hocytes could not be reduced after the modification. We can know mesoporous carbon nanoparticles with good biocompatibility before and after the modification from the tests of animals. Mesoporous carbon nanoparticles in the field of clinical medicine should be used to provide certain theoretical basis of toxicology.