In Vitro And In Vivo Study Of CuInS₂/ZnS-PEG

As a new type of nano-material,quantum dots are widely used in the fields of optics,informatics,biomedicine,et al.Due to their excellent and stable optical properties,low photobleaching,long life,and difficulty in being masked by the tissue itself fluorescence.The quantum dots which originally used in biomedicine are containing cadmium,such as cadmium telluride and cadmium selenide.Since these quantum dots contain cadmium,people are beginning to worry about the toxicity risks of cadmium exposure in vivo.As a newly developed non-cadmium quantum dot,CuInS₂/ZnS has more stability and more biocompatibility than traditional cadmium-containing quantum dots,and has wider application potential in biomedicine field.In order to study the biosafety of CuInS₂/ZnS quantum dots,we investigated the effects of CuInS₂/ZnS on cells and animals from both in vivo and in vitro aspects.1.In vitro toxicity study of CuInS₂/ZnS quantum dots in U87 and PC12 cell lines.In this experiment,human glioma cell line U87 and rat adrenal medulla pheochromocytoma cell line PC12 were selected as in vitro study models.Microscopy and flow cytometry were used to detect the uptake of quantum dots by these two cells.The effects of quantum dots on the survival of U87 and PC12 cells were detected by MTT assay and LDH assay.The gene expression profiles of U87 and PC12 cells were detected by extracting total RNA from U87 and PC12 cells.The results showed that red fluorescence was observed in the U87 and PC12 at a concentration of 12.5?g/mL,which was mainly located in the cytoplasm.As the concentration of CuInS₂/ZnS quantum dots was increased,the red fluorescent signal was gradually increased.The results of flow cytometry showed thatthe average uptake rate of CuInS₂/ZnS of U87 cells were?11.15±1.26?%,?95.53±0.57?%,?97.37±1.53?%,and?97.50±0.72?%when the concentration of CuInS₂/ZnS quantum dots was 12.5?g/mL,25?g/mL,50?g/mL and 100?g/mL,respectively,the average uptake rate of the CuInS₂/ZnS of PC12 cells were?59.27±3.62?%,?98±0.25?%,?98.4±0.95?%,and?97.77±0.29?%,espectively.The results of MTT showed that the survival of U87 cells in the 50?g/mL and 100?g/m L quantum dot groups increased?P<0.05?when the CuInS₂/ZnS were co-cultured with the cells for 24 h,but after 48 h and 72 h of continuous co-culture,these four concentrations of CuInS₂/ZnS did not cause any significant difference in U87 cell activity.The results of LDH method showed that the LDH released by U87 cells in the quantum dot group increased?P<0.05?when the CuInS₂/ZnS were co-cultured with the cells for 24 h.However,there was no significant difference in the amount of LDH between the the experimental group and the control group after 48 h and 72 h.The results of MTT assay for PC12 cell activity showed that the four concentrations of CuInS₂/ZnS quantum dots at three time points did not cause any significant difference.The LDH method showed that the release rate of LDH increased in the 12.5?g/mL group,the 25?g/mL group,and the 100?g/mL group at 24 h?P<0.05?.However,at 48 h and 72 h,the LDH release rates of the four concentrations were not statistically significant compared with the control group.The results of gene microarray showed that there were 220 genes with significant difference in up-regulation in U87 cells treated with quantum dots,1515 genes with significant difference in down-regulation.In the PC12 cells treated with quantum dots,there were 940 genes with significant difference in up-regulation,2241 genes with significant difference in down-regulation.2.In vivo toxicity study of CuInS₂/ZnS quantum dots in BALB/c miceMale BALB/c at 7 weeks old was selected as an animal model,and high concentration?25 mg/kg?and low concentration?2.5 mg/kg?CuInS₂/ZnS quantum dots were injected through the tail vein,respectively.After the CuInS₂/ZnS quantum dots were injected,the mice were sacrificed on days 1,3,7,14,30,60 and 90.Body weight,hematology,blood biochemistry,organ histology and biodistribution were examined at different time points.The results showed that the quantum dot group mice and the normal group mice had no abnormalities in body weight.The red fluorescence of the quantum dots was observed in both the spleen and the liver of the quantum dot treatment group for 90 days.The results of ICP-MS showed that CuInS₂/ZnS quantum dots mainly remained in the liver,spleen and lung tissues of mice.After the first day of injection,the indium contents of the liver,spleen and lung of the mice were 4.350±1.699?g/g,8.322±2.094?g/g and 4.607±2.329?g/g,respectively.Over time,indium gradually decreases in the liver,spleen,and lungs.Blood routine results showed that the majority of indicators did not show significant changes on 90days,except for platelet distribution width?PDW?.Cardiac function indicators showed that creatine kinase?CK?of the 2.5 mg/kg quantum dot group was significantly higher than the control group on days 7 after the injection of quantum dots,and then returned to normal levels.Lactate dehydrogenation Enzyme?LDH?of the two quantum dot groups were significantly higher than the control group on the 7th day,and returned to normal on days 14,significantly lower than the control group on days 30 and lasted for days 60,and returned to normal at days 90.?-hydroxybutyrate dehydrogenase??-HBDH?of the 25 mg/kg quantum dot group was significantly lower than the control group On the day 30,lasted for days 60,and returned to normal on days 90.Creatine kinase isoenzyme?CK-MB?of the two groups of quantum dots were significantly higher than the control group on days 30 and remained unchanged.The indicators of liver function showed that the triglyceride?TG?was significantly higher than the control group on day 1 and the days 7 of the two doses of quantum dots,and then returned to normal.Low-density lipoprotein?LDL-C?was increased on days 7 and then returned to normal.High-density lipoprotein?HDL-C?and Alkaline phosphatase?ALP?was significantly lower than the control group days 90 after injection of 25 mg/kg quantum dots?P<0.01?.Total cholesterol?TC?,glucose?GLU?,aspartate aminotransferase?AST?,albumin?ALB?,and globulin?GLB?showed no significant changes.The indicators of renal function showed that the uric acid?UA?on the first day after the injection of quantum dots of the 25 mg/kg quantum dot group was significantly higher than the control group,and then returned to normal levels,increased 60 days again and continued to 90 days.On the days 14,urea?URER?of the 25 mg/kg quantum dot group was significantly higher than the control group,and then increased,and decreased significantly in90 days.There was no significant change in total protein?TP?and creatinine?CR?.In summary,CuInS₂/ZnS quantum dots have less effect and low toxicity in in vitro and in vivo.