Effect Of Physicochemical Properties Of Nanomaterials On Cellular Uptake, Endocytosis Mechanism And Biosecurity

As fluorescent probes and drug carriers, nanomaterials have great potential for cancer diagnosis and therapy. In recent years, the physicochemical properties of nanomaterials(such as size, shape, surface charge, surface modification and surface topology, etc.) have been systematically investigated to demonstrate their influence on cellular uptake, endocytosis mechanism, biodistribution and biosecurity. However, the studies such as the shape effect of drug nanocrystals against anticancer activities and systematic toxicities in vivo, the effect of surface roughness on cellular uptake and internalization mechanism of dye nanoparticles, and the biocompatibility of organic dye nanoparticles, are insufficient. Therefore, in this thesis we focused on the optimization of the physicochemical properties of nanomaterials and achieved some new results as follows:1. We prepared HCPT drug nanocrystals in two shapes(nanoparticles and nanorods) with similar hydrodynamic sizes, and then studied the shape effect of nanodrugs on the antitumor efficacy and systematic toxicity in vivo. The results showed that NRs were internalized by cells more easily and observed to exhibit much higher antitumor efficacy than that of NPs. However, systemic toxicology studies suggested that the NRs possessed obviously higher toxicities than that of NPs. The MTD for HCPT NPs and free HCPT is 8 mg/kg, while that of NRs is only 5 mg/kg. Both blood analysis and histology examinations demonstrated an acute liver and kidney injury for free HCPT and long-term liver toxicity for HCPT NRs treated groups, while slight toxicity was found in HCPT NPs treated groups. These results suggested that the balance between anticancer activities and systematic toxicity should be taken into account in future design of drug nanocrystals.2. We prepared an organic dye nanoparticles(NPAPF NPs) and changed surface charges and surface roughness by coating a layer of silica on the surface of NPAPF NPs(NPAPF NPs@SiO2), then investigated the effects of surface properties on cellular uptake, internalization mechanism and the intracellular fate of these two dye nanoparticles. It was found that although the particles with positive surface charge can be more effectively internalized by cells through electrostatic interactions with the negatively charged cell plasma membrane, surface roughness played a predominant role on the cellular uptake efficacy. Meanwhile, we discovered that combined pathways of clathrin and caveolae-mediated endocytosis were the main pathway for internalization of nude NPAPF NPs, while the clathrin- and caveolae-independent endocytosis most probably played a crucial role on the uptake of rough NPs due to the change of surface roughness. In vitro co-localization with lysosome and excretion rate of nanoparticles studies showed that rough NPs could bypass the endolysosomal compartments and stay in cells for much longer time. All the above results show that rough surface enhance the cellular uptake and regulate the intercellular fate of dye nanoparticles, which has significant implication for long-term, real-time tracking in living cells.3. We used C18PMH-PEG to modify the as-prepared NPAPF NPs to obtain the NPAPF NPs-PEG and then investigated the effect of surface modification on biosecurity in vitro. Due to the existencen of PEG, the cellular uptake of NPs-PEG was remarkablely decreased by the human normal liver cells(HL-7702), while no obvious influence was found in liver cancer cells(HepG2). The results of MTT test and cell apoptosis experiment showed that the NPAPF NPs affected the cellular activities when the concentration is increased up to 40 μM, while the NPs-PEG displayed only slight toxicity with concentrations up to 100 μM. The level of reactive oxygen speciess(ROS) showed that NPs may bring a higher toxicity in both HepG2 and HL-7702 cells at high concentration compared to NPs-PEG. We also found that both NPs and NPs-PEG had no ability to escape the lysosome and therefore they were more easily to be excreted by cells. Our research shows that the modification on the surface of nanomaterials would partly reduce the toxicity in vitro, and contribute to the design on nanomaterial for bioapplication.