| In recent years,sufficient efforts have been made to develop fluorescent small-sized(diameter smaller than 10 nm)silicon nanoparticles(SiNPs)with strong fluorescence and robust photostability,providing high promise for myriad biological and biomedical applications.It is worth noting that the study of the biological effects and toxicity of nanomaterials is of essential importance for widespread applications of nanomaterials.However,there currently exists scanty information about intracellular behaviors of the small-sized SiNPs,particularly the underlying mechanism of entry into cells and intracellular delivery and fate.In this dissertation,we have conducted a systematic study on the interaction between fluorescent small-sized(diameter:4 nm)SiNPs and cells.This study helps us understand the internalization,transportion and location of SiNP of ca.4 nm diameter,together with toxicity in vitro level.The main research results are as follows:Chapter I:In this chapter,we give an introduction to the recent research progresses of nanomaterials and silicon nanoparticles,and discuss the objectives and methods of the study in this dissertation.Chapter 2:In this chapter,we systematically investigate the mechanism of the cellular uptake of these small-sized SiNPs.First,cells are incubated with SiNPs,and then the fluorescence intensity and fluorescence position are monitored at different incubation times.The results show that the cellular uptake of SiNPs is concentration-and time-dependent.Typically,the SiNPs first gather onto the cell membrane,and then gradually enter into the cytoplasm and finally positioning in the nuclear periphery,not the nucleus.Furthermore,through energy-dependent experiments and the use of several specific endocytosis inhibitors,we find that SiNPs enter into cells is energy-dependent endocytosis,predominantly by clathrin-mediated endocytosis and partially via caveolae-mediated endocytosis.This study elucidates the uptake kinetics and mechanism of SiNPs.Chapter 3:The transport and location of small-sized SiNPs are investigated systematically.The colocalization of SiNPs with various organelles(early endosomes,late endosomes,lysosomes,Golgi apparatus and endoplasmic reticulum)is analyzed by confocal laser scanning microscopy(CLSM)under different incubation times.The experimental results show that after being internalized into the cells,SiNPs firstly locate in the early endosomes,and then gradually mature into late endosomes with the prolongation of incubation time and eventually most of them end up in the lysosomes,while a small fraction is secreted out of the cell through Golgi apparatus.By further using cytoskeletal inhibitors and analyzing the location of SiNPs,we understand that the transport of SiNPs in cells is mainly mediated by microtubules.Chapter 4:The cytotoxicity of small-sized SiNPs is investigated in systematic manners.The results show that cells treated with SiNPs are in good shape,with no significant cell death,and the intracellular ATP content is not significantly affected and the cell membrane remaine intact.These results indicate that SiNPs have negligible cellular toxic and side effects.Then,several cell signal pathways related to proliferation such as NF-KkB signaling pathway,AKT signaling pathway and ERK signaling pathway are further studied.The results show that SiNPs have no significant effect on these signaling pathways.It is worthwhile to point out that,while we find that although SiNPs do not affect cell proliferation,there are some inhibitory effects on several signaling pathways,such as Hedgehog and Wnt signaling in certain specific conditions.In summary,this paper systematically studies the cell behaviors and in vitro toxicity of small-sized SiNPs,including the intracellular uptake kinetics,the endocytosis pathway,transportion and location in cell,as well as toxicity analysis.This study is helpful for understanding the biological effects of small-sized SiNPs,facilitating the development of SiNPs for biomedical applications. |
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