Carbon Quantum Dots Induced Apoptosis Of Human Osteosarcoma 143B Cell Line And The Underlying Mechanisms

Objectives:Osteosarcoma is the most common primary malignant tumor in both children and adolescents and accounts for about 35%of malignant bone tumors.In recent years,with the development of limb-salvaging surgery,neoadjuvant chemotherapy,immunotherapy,gene therapy,molecular targeted therapy,and other comprehensive treatments,the 5-year survival rate of osteosarcoma patients has increased to more than 80%.However,such treatments have several deficiencies,such as cancer recurrence,incision infection,high expense,and sideeffects.Carbon-based nanomaterials have been studied for biomedical applications in recent years.Compared with other nanomaterials,carbon nanomaterials have attracted attention due to their unique physicochemical and biological effects,such as low density,good chemical stability,low price,low toxicity,and strong cell penetration.New forms of carbon nanostructures have been applied for drug delivery,antibacterial activity,clinical detection,antitumor activity,and bioengineering.However,the biocompatibility and bioavailability of nanocarbon materials require further study.To date,few studies have demonstrated that carbon-based nanomaterials present different cellular effects.Here,we describe novel carbonbased nanomaterials,named Carbon quantum dots(CQDs),and their potential in cancer therapy.We investigated the effects of colloidal CQDs on tumor cells in vitro to evaluate the CQDs'cytotoxicity and a xenograft model was used to detect antitumor effects in vivo.Methods:CCK-8,NR and LDH release assay were used to detect the cell morphology and cytotoxicity of human osteosarcoma 143B cell line cultured with high(276?g/mL),medium(138?g/mL)and low(69?g/mL)CQDS for 24h,48h and 72h.Cell apoptosis was detected by Annexin V-FITC/PI double dyeing flow assay after 24h,48h and 72h co-culture of CQDS and 143B cell line.Fluorescent probes DCFH-DA and JC-10 were used to detect the changes of ROS and MMP in CQDS cells cultured with 143B cell line for 12h,24h and 48h.The expression of apoptotic proteins(Bax,Bcl-2,Cytochrome C,caspase-3,Cleaved caspase-3,PARP1,and Cleaved PARP1)in 143B osteosarcoma cells cultured with carbon quantum dots for 72h was determined by Western-blot and immunofluorescence(IF).Use health 4 to 6 weeks of SPF male NU/NU Nude mice subcutaneously 143 b cell suspension tumor-burdened Nude mice model,the experimental group and control group two groups,experimental group stomach every day CQDs sol 0.5 ml,control the stomach filling amount of PBS solution,generally observed a tumor-burdened Nude mice every day,every week to measure changes in tumor size,the anatomy of the tumor to be put to death in the two groups after 4 weeks Nude mice compared to the tumor size and weight;SPF grade male BALB/c mice aged 4-6 weeks were selected to verify the biosafety of CQDS.The experiment was divided into two groups:4-week group and 12-week group,Within each group is divided into 3 groups:control group,low concentration CQDs set of 69 mu(g/ml)and high concentration of CQDs 276 mu(g/ml)group,low concentration and high concentration of mice CQDs by stomach 0.5 ml amount of different concentrations of CQDs sol,control group mice stomach 0.5 ml PBS solution,generally observe mice every day,every 2 weeks weigh weight in mice,4 weeks and 12 weeks of mice executed corresponding detection of blood routine and blood biochemistry,anatomy of mice heart,liver,spleen,lung,kidney,brain,HE staining were used to detect stomach CQDs mice after the histological changes of the main organ;Immunohistochemical method(IHC)was used to detect the expression of lymphocytes(CD3+,CD4+)in macrophages(F4/80)in spleen and liver,the main immune organ of mice.Results:CCK-8,NR and LDH release assay showed that high concentration(276?g/mL)of CQDS had obvious cytotoxicity on human osteosarcoma 143B cells(P<0.05).Annexin VFITC/PI double staining flow assay showed that high concentration of CQDS(276?g/mL)could significantly induce apoptosis of 143B cells in osteosarcoma(P<0.05).Fluorescent probe DCFH-DA detection results showed that high concentration of CQDS(276?g/mL)could significantly increase the expression of ROS in 143B cells(P<0.05).JC-10 fluorescent probe results showed that CQDS(276?g/mL)could significantly reduce the level of mitochondrial membrane potential of 143B cells(P<0.05).Western-blot and immunofluorescence(IF)results showed that high concentration of CQD(276?g/mL)induced apoptosis of 143B cells by activating mitochondrial apoptosis signaling pathway,increasing the expression of apoptotic proteins Bax,Cytochrome C,Caspase-3,Cleaved Caspase-3,and Cleaved Parp1(P<0.05),and reducing the expression of apoptotic protective proteins Bcl-2 and Parpl(P<0.05).In NU/NU Nude mice right thigh lateral skin injection 143 b cell suspension,after 1 month to tumor growth to 100 mm3 successfully established a tumor-burdened Nude mice model of stomach high concentrations of CQDs 276 mu(g/ml)4 weeks after visible experimental tumor-burdened Nude mice tumor size significantly less than the control group,the tumor inhibition rate of 38.9%volume weight(P<0.05),tumor inhibition rate was 30.1%(P<0.05);The body weight and blood routine indexes of normal BALB/c mice fed with high concentration(276?g/mL)and low concentration(69?g/mL)CQDS were not significantly different from those of the control group(P>0.05),but the ALT,AST and Cr values of CQDS group were lower than those of the control group.Whether CQDS has protective effect on liver and kidney function remains to be further studied.He staining tissues of heart,liver,spleen,lung,kidney and brain showed no local inflammation,hyperplasia or necrosis.The expressions of macrophages(F4/80)and lymphocytes(CD3+,CD4+)in spleen of mice were increased(P<0.05).Conclusions:High concentration of CQD(276?g/mL)had obvious cytotoxicity on human osteosarcoma 143B cells and induced apoptosis by activating mitochondrial apoptosis signaling pathway.Gastric administration of high concentration of CQDS in tumor-bearing nude mice not only showed anti-tumor effect,but also showed good biosafety to normal mice.In conclusion,CQDS not only has anti-tumor effect,but also has high biological safety and biocompatibility.As a new carbon nanomaterial,CQDS has great potential in cancer treatment.