Intracellular Trafficking And Cellular Uptake Mechanism Of Ascorbic Acid Carbon Dots In MC3T3 Cells

Background:Oral and maxillofacial bone defects caused by many factors such as trauma,infection and tumor are becoming more and more common in clinic,which seriously affect their diet,beauty and mental health.There are various disadvantages of traditional therapies: allogeneic bone suppression causes immune rejection,autologous bone suppression causes loss of bone tissue in other parts of the body,and scaffold materials fail to have ideal biocompatibility.Therefore,the development of a new and effective method to repair the bone tissue is quite necessary.Transgenic therapies are currently the hot topics in the treatment of bone defects,but pure gene is of instability and easy to be degraded,so we need gene vectors to protect the genes.The transfection of viral vectors is efficient,but they often cause immune responses and biological toxicity cannot be ignored.Non-viral vectors,especially nano-particles with smaller particle size and larger specific surface area,are easy to be modified.As a result,they are more and more used in gene therapy.As a new type of nanoparticle,carbon dots have the advantages of low cost,convenient manufacture and excellent photoluminescence.They can be used in biosensing and bioimaging and have excellent performance in gene therapy.Therefore,it is of great significance to study the transmembrane transport and intracellular distribution of carbon dots and to explore their biocompatibility,which help us to use carbon dots as a non-viral gene carrier.Methods:Microwave method was used to prepare ascorbic acid-PEI composite carbon dots.MTT assay was used to detect the effect of carbon dots on cell proliferation.Annexin V-PITC/PI double staining method was used to detect the effect of carbon dots on cell apoptosis by flow cytometry.At the same time,cell cycle,intracellular ATP levels,and intracellular ROS levels were measured to evaluate the biocompatibility of carbon dots.We use endocytosis inhibitors and flow cytometry to clarify how MC3T3 cells uptake carbon dots.What is more,we verified the result by atomic force microscopy and transmission electron microscopy.Every kind of cell organelle was labeled with a dye,and the distribution of carbon dots on the subcellular structure was clearly defined by detecting the green fluorescence of the carbon dots and red fluorescence of different kinds of organelle.We verified the result by the use of transmission electron microscopy.Results:The carbon dots could be excited to emit green fluorescence under blue fluorescence.After entering the cells,they were mainly distributed to the cytoplasm and could show the outline of the cells.When the cells were treated with 40 mg·L-1 carbon dots for 24 h,the cell proliferation rate was still above 80%.Carbon dots had little effect on the early apoptosis rate of cells(P>0.05).Carbon dots reduced the proportion of cells in G0 and G1 phases(P<0.05)and increased the proportion of cells in S phase(P<0.05).Carbon dots could increase ROS levels in cells,but not significantly(P>0.05).Carbon dots reduced the ATP level of cells(P<0.05).When chlorpromazine is used to inhibit clathrin-mediated endocytosis,the cellular uptake of carbon dots was reduced;When nocodazole or 4°C incubation was used to inhibit the energy-dependent endocytic pathway,the cellular uptake rate of carbon dots decreased significantly(P<0.05),but there were still carbon dots that could be taken up by the cells.When the carbon dots were co-incubated with the cells for 15 min,the undulation of the cell surface was larger than that of the control group,and many depressions with a diameter of about 100 nm were generated,but when the carbon dots were co-incubated with the cells for 3 h,the cell membrane surface became flattened.Transmission electron microscopy results showed that the carbon dots entered the cell with the formation of invagination on cell membrane.The green fluorescence of the carbon dots overlapped well with the red fluorescence of the labeled lysosomes and mitochondria,but only partially overlapped with the red fluorescence of the endoplasmic reticulum,while the red fluorescence of the labeled Golgi complex was not very obvious.Transmission electron microscopy results showed that carbon dots could be found in the endoplasmic reticulum,mitochondria,and lysosomes,but no obvious Golgi apparatus was observed.In addition,carbon dots could also be found in the nucleus.Conclusion: 1.The carbon dots have excitation dependence,they can emit green fluorescence under blue fluorescence excitation and emit red fluorescence under green fluorescence excitation,but they mainly emit green fluorescence.The carbon dots have good microscopic cell imaging capabilities and can enter the cells,mainly dispute in the cytoplasm.They can show the outline of the cells.Carbon dots have low cytotoxicity and good biocompatibility.2.Carbon dots can be endocytosed into cells by clathrin-mediated endocytosis and can also enter cells through non-energy-dependent pathways.Carbon dots can accumulate before they enter the cell,which can accompany the invagination of the cell membrane in the corresponding region.3.The endocytosis of carbon dots is mainly distributed to the endomembrane system of cells and distributed to lysosomes,mitochondria and endoplasmic reticulum.They can be transported in the vesicles.In addition,carbon dots can enter the cell nucleus.