Study Of Endocytosis And Exocytosis Of Titanium Dioxide Nanoparticles And Its Mechanism In Neural Stem Cells

Nano titanium dioxide （nano-TiO₂） is massived produced and widely used indiverse fields because of its inherent physical and chemical advantages. Along withthe quickly increased production of nanoparticles（NPs）, people begin to worry aboutthe unknown safety of NPs. For example, in biomedical fields, NPs used as the drugdelivery carrier may target desirably to the pathological tissues and cells, realizingsafe and effective drug treatments. It is important to know the fate of NPs in vivo,especially the most basic process of their interaction with cells, namely cellularendocytosis and exocytosis. Neural stem cells （NSCs） are ancestor of all kinds ofnerve cells, and have the potential ability of both proliferation and differentiation. Sofar, there is no systematic study on the biological toxicity, environmental effects andthe interaction of biological tissue of nano-TiO₂. Given all that, we employed threetypes of nano-TiO₂in different shapes and sizes and NSCs as cell model to measurequantitatively the endocytosis and exocytosis of nano-TiO₂.Because the physical and chemical properties of nanoparticles have directlyinfuence on their biological effects, the sufficient characterizations for the purity, size,morphology, crystal structure and surface potential of three kinds of nano-TiO₂werefirstly carried out. The cytotoxicity of nano-TiO₂is investigated using CCK-8andreactive oxygen species （ROS） assay. Cytotoxicity assay shows that nano-TiO₂insidecells are nontoxic at the low concentration. A time-dependent relationship is observed,while a dose-dependent relationship is seen only at the concentration higher than150mg/L. The size and morphology of nano-TiO₂have no influence on the cytotocixity.The study of endocytosis and exocytosis of nano-TiO₂: The uptake amountincrease with the incubation time prolonging and reach saturation after48h.nano-TiO₂in smaller particle size endocytose faster and enter cells more easily.Tubular nanoparticles are more difficult to enter the cell than spherical particles. Theendocytosis rate of nano-TiO₂is heavily dependent on the size and particularly on theshape. Notably, NTs possess much longer half-life than NP1and NP2. To reach the maximum endocytosis of nano-TiO₂, the endocytosis amount after co-incubtion for48h was selected as a starting point to study the exocytosis. A multi-step cellularsubculture method was adopted and a primary dynamic tracking of exocytosis processwas achieved by using confocol. Nanoparticles around the nucleus disperse graduallyand exocytosis from the cells finally. The ICP-AES analysis results show theremarkable decrease of Ti content within the cells with increasing incubation time.The exocytosis ratio of NP1, NP2and NTs reached to35.0%,34.6%and41.7%respectively after24h incubation at the concentration of50mg/L. But when theconcentration increased to100mg/L, the2-fold endocytosis amount does not induce2-fold exocytosis amount. For three types of nano-TiO₂, the exocytosis ratiodecreased with increasing incubation concentration. Especially in the case of NTs, theexocytosis ratio decreased from41.7%to26.7%. Our results show that the exocytosisratio of nano-TiO₂heavily depends on the original concentration, endocytosis amountand shapes, but it is less influenced with size.The mechanism study of endocytosis and exocytosis: For three types of NPs,both the endocytosis and exocytosis amount of NPs are remarkably reduced by4°Cincubation compared with the normal incubation at37°C.These results show that bothendocytosis and exocytosis of nano-TiO₂are energy-dependent processes, andnanoparticles enter into cells via a receptor mediated pathway. Further, we study theinfluence of serum on the endocytosis and exocytosis of nano-TiO₂. Our results showthat both the endocytosis and exocytosis of nano-TiO₂are inhibited when the mediumwas depleted of serum.24.9%,18.6%and30.4%endocytosis was inhibited for NP1,NP2and NTs, while23.0%,19.5%and58.2%in exocytosis, respectively. In TEMimage, there is a layer of gelatinous substance on the surface of nano-TiO₂in culturemedium. The nanoparticle-protein complexes can reduce the aggregates of NPs andincrease the amount of endocytosis and exocytosis. Cell morphology changes slightlyin the process of exocytosis which may due to the effect of nano-TiO₂on microtuluesin NSCs. Microtubules plays the role of intracellular material transport tracks, thedestruction of the microtubule will restain the intracellular material trasport, whichalso may be caused a slightly increase of ROS.