Endocytosis,Translocation And Exocytosis Of Quantum Dots In Cells

Quantum dots(QDs)are excellent nanomaterials with unique optical properties,including narrow emission band,size-dependent luminescence,high quantum yield and good photostability.They have been widely used in biomedicine fields,such as biosensor,imaging,drug delivery and cancer treatment.In addition,they also are used in color converter,laser,and various photoelectric devices.Both the biological applications and safety assessments require a deep understanding of the interaction of QDs and biological systems,in which the behavior and fate of QDs in cells are important and complex aspects.Although a lot of studies on the interaction between cells and QDs have been carried out,including endocytosis,translocation,and exocytosis,there are still some problems to be solved.For example,the research on the whole process of QDs from entering to leaving cells is very limited,making it difficult to compare the data from different studies;the research on the exocytois of QDs is still in its infancy;some factors affecting the interaction between QDs and cells are not well studied,such as cell division.Based on the above background,we selected human cervical cell HeLa,mouse macrophage Raw 264.7,human lung cancer cell A549,human normal lung cell BEAS-2B,human epidermoid carcinoma cell A431,and human breast cancer cell MDA-MB-468 as cell models,to systematically and comprehensively study the cellular uptake,translocation and exocytosis of typical QDs,core-shell CdSe/ZnS QDs and carbon quantum dots(CDs),in cells,with considering the influence of conditions such as cell division,by using confocal laser scanning microscopy and flow cytometry.The main contents and results are summarized as follows:(1)The endocytosis,translocation and exocytosis of amino functionalized CdSe/ZnS quantum dots(QDs-MEA)in HeLa cells.We found that QDs-MEA were internalized by HeLa cells with a time-,dose-,and serum-dependent manner.The cellular uptake was inhibited dramatically by serum,but eventually peaked after 4-6 h incubation with/without serum.All three endocytic pathways contributed to the uptake of MEA-QDs in both serum and serum-free medium.In the medium with serum,the primary endocytosis pathway was clathrin-mediated,and actin-and microtubuledependent,however,the caveolae-mediated endocytosis and macropinocytosis were more important in the serum-free medium.MEA-QDs contacted the cell membrane,and then were wrapped into vesicles formed by depressed cell membrane and transported into cytoplasm.MEA-QDs mainly distributed in endosomes/lysosomes,and a small amount in mitochondria,endoplasmic reticulum,and Golgi apparatus.The translocation of QDs-MEA from other organelles to Golgi apparatus was observed.The exocytosis of QDs-MEA was faster than their endocytosis,reaching the maximum in about one hour after cultured in fresh culture medium,with around 40% of the internalized QDs-MEA was discharged.The exocytosis process was energy-and actindependent,and the lysosome exocytosis and endoplasmic reticulum/Golgi pathway were the main routes.(2)The effect of the surface charge on the endocytosis,distribution and exocytosis of QDs in Raw264.7 cells.The positive-charged QDs-MEA and negative-charged carboxyl functionalized CdSe/ZnS QDs(QDs-MPA)were studied.The results showed that Raw264.7 cells internalized both of the QDs,and the serum inhibited the cellular uptake of the QDs.But the endocytosis of QDs-MPA was always higher than that of QDs-MEA in culture medium with or without serum.In the presence of serum,both of QDs entered cells mainly in an energy-dependent manner.MEA-QDs could be internalized via combined pathways of clathrin/caveolae-mediated endocytosis,while QDs-MPA via clathrin-mediated mainly and supplemented with caveolae-mediated endocytosis and macropinocytosis.QDs-MEA was mainly distributed in lysosomes,while QDs-MPA was mainly distributed in mitochondria and lysosomes.The exocytosis of both of the QDs from Raw264.7 cells was very difficult,and the discharge of positive-charged QDs-MEA was more difficult than that of negative-charged QDsMPA.(3)The endocytosis and exocytosis of two typical QDs(QDs-MEA and polyethylenimine modified carbon quantum dots CDs-PEI)in different cell models(HeLa cells,A549 cells,A431 cells,MDA-MB-468 cells,and BEAS-2B cells).The results indicated that the celluar uptake of QDs was affected by the properties and concentration of QDs,culture time,and cell types.The higher the concentration of QDs,the higher the celluar uptake of QDs.Except that the celluar uptake of CDs-PEI in MDA-MB-468 cells increased continuously over time within 24 h,the celluar uptake of QDs peaked at 4-6 h,and then remained constant or slightly decreased.The order of the uptake ability from high to low was MDA-MB-468,A549/BEAS-2B,A431,and HeLa.The exocytosis of QDs depended upon the properties of QDs,exocytosis time,and cell types.The exocytosis of QDs was not influenced by the intracellular amount of QDs,except QDs-MEA in MDA-MB-468 cells.The exocytosis rate of QDs in normal cells was slower and weaker than that in cancer cells.The ability of cells to excrete CDs-PEI was higher than that to excrete QDs-MEA.This may be related to the size of the QDs.In addition,the serum did not affect the exocytosis rate and ability in the first 6 h.and then the exocyosis slowed down in the medium with serum.The exocytosis pathway of QDs was greatly influenced by the cell type and properties of QDs,The main exocytosis pathway of QDs-MEA involved lysosomal pathway,and the exocytosis depended on actin.For CDs-PEI,the Golgi apparatus were vital regulatory stations,and lysosomal pathway contributed little were tubulin dependent,except CDsPEI left MDA-MB-468 cells mainly via lysosomal pathway,not Golgi apparatus.The exocytosis of CDs-PEI in cells was microtubule dependent.