| Nephroblastoma,also known as Wilm's tumor(WT),is the most common primary renal malignancy in children.The combined treatment of surgery,chemotherapy and radiotherapy is effective for most of nephroblastoma,resulting in the 5-year survival rate of them exceeding 85%.Carboplatin,as a second-generation platinum antitumor drug,has less nephrotoxicity than cisplatin and is mainly used in the treatment of children with recurrent or initially failed nephroblastoma.However,due to the low uptake of carboplatin in tumor tissue,most of the drugs are excreted in urine in a short time,which affects its anti-tumor effect.In recent years,in order to reduce the side effects of platinum-based antitumor drugs and to improve their anti-tumor effects,researches on platinum-based drug nanocarriers have been increasing.As the only alkaline polysaccharide in nature,chitosan has good biocompatibility and degradability.Its polymer nanoparticles act as a drug transport carrier,which has the functions of targeting,sustained release,increasing drug absorption and improving drug efficacy.Therefore,in order to enhance the therapeutic effect of carboplatin on nephroblastoma,we prepared chitosan nanoparticles loaded with carboplatin using chitosan as raw material,and then examined its physicochemical properties.SK-NEP-1 cell model was used to evaluate the inhibitory effect of carboplatin-chitosan nanoparticles on nephroblastoma in vitro.Objectives:1.To prepare carboplatin-chitosan nanoparticles,to investigate their general properties,such as the morphology,the diameter distributions,the loading capacity,the encapsulation efficiency,the kinetics of drug release process in vitro.2.To evaluate the effects of the inhibitory effect of carboplatin-chitosan nanoparticles on nephroblastoma human nephroblastoma cell line SK-NEP-1 in vitro.Methods:1.The carboplatin-chitosan nanoparticles were prepared by ionic gelation method,and their morphology was observed by transmission electron microscope and atomic force microscope.The particle size distribution was determined by laser particle size analyzer.The encapsulation efficiency and loading capacity of carboplatin-chitosan nanoparticles were determined by high performance liquid chromatography(HPLC),and the release kinetics of carboplatin-chitosan nanoparticles were studied by high performance liquid chromatography(HPLC)with PBS buffer as the release medium in vitro.2.Different concentrations of carboplatin and carboplatin-chitosan nanoparticles were added into the cell culture medium to incubate SK-NEP-1 cells until the set time.The morphological changes of each group were observed by optical microscope.The effects of carboplatin-chitosan nanoparticles on the survival rate of SK-NEP-1 cells was detected by trypan blue dye exclusion and MTT assays.The cell colony formation assay was used to detect the effects of carboplatin-chitosan nanoparticles on the colony-forming ability of SK-NEP-1 cells.The effect of carboplatin-chitosan nanoparticles on apoptosis of SK-NEP-1 cells was detected by Hoechst staining and TUN EL assay.Results:1.The prepared carboplatin-chitosan nanoparticles were spherical or spheroidal under a transmission electron microscope,with uniform size and clear boundaries between each other.Atomic force microscopy imaging further confirmed that the nanoparticles were circular andappeared flat on the surface of mica.The average particle size of carboplatin-chitosan nanoparticles measured by laser particle size analyzer is about 160 nm(160±20.5 nm),and the distribution is relatively concentrated.2.The amount of carboplatin added can affect the encapsulation efficiency of chitosan nanoparticles.When the amount of carboplatin is 3%of the amount of chitosan nanoparticles,the encapsulation efficiency is 92.7%.When the amount of carboplatin is 25%of chitosan nanoparticles,the encapsulation efficiency decreases rapidly to 44.2%.here was no significant difference in the encapsulation efficiency of carboplatin-chitosan nanoparticles when carboplatin content was 5%,8%and 15%(P>0.05);there was significant difference in the encapsulation efficiency of carboplatin-chitosan nanoparticles when carboplatin content was 15%and 25%(P<0.01).3.The release of carboplatin from chitosan nanoparticles can be divided into three phases:rapid release,uniform release,and slow release.The rapid release phase was that the release amount of the nanoparticle drug can reach 51.2%within 24 hours after administration;the uniform release phase was the 2-4 days after administration;after the fourth day,the drug release entered the slow release phase for at least 2 days.4.The morphology of cells was observed under optical microscope.SK-NEP-1 cells in the untreated control group maintained their typical morphology and evenly spreaded over the entire coverslips.SK-NEP-1 cells treated with free carboplatin were irregular in morphology and decreased in number.Over 80%of SK-NEP-1 cells treated with carboplatin-chitosan nanoparticles were not adherent,and the rest crinkled to circle.5.The results of trypan blue exclusion assay showed that carboplatin and carboplatin-chitosan nanoparticles had significant inhibitory effects on SK-NEP-1 cells,and the effect of carboplatin-chitosan nanoparticles was greater than that of the free carboplatin(P<0.05).At the concentration of 10 ?g/mL,the growth of cells in the carboplatin-chitosan nanoparticles group decreased by about 80%after 24 hours of treatment,while that in the free carboplatin group decreased by only about 40%.6.The MTT assay showed that the carboplatin-chitosan nanoparticles had strong dose-dependent cytotoxicity in SK-NEP-1 cells,and the effect was significantly greater than that of the free carboplatin.At the concentration of 1 ?g/mL,the cell viability of the free carboplatin group and the carboplatin-chitosan nanoparticle group remained at 72%and 56%respectively;at 10 ?g/mL,the cell viability of the carboplatin group and the carboplatin-chitosan nanoparticle group remained at 34%and 18%respectively.7.The colony formation assay showed that the colony formation of SK-NEP-1 cells in the drug-free control group was not inhibited,while the coloning abilities of SK-NEP-1 cells treated with carboplatin and carboplatin-chitosan nanoparticles were inhibited significantly,and the latter was more effective.Specifically,after 14 days of treatment,70%of SK-NEP-1 cells in the carboplatin-chitosan nanoparticles group were inhibited in colony formation,whereas only 35%of the ability of SK-NEP-1 cells in the carboplatin group were affected.8.Hoechst staining and TUNEL assay showed that SK-NEP-1 cells treated with carboplatin-chitosan nanoparticles showed more apoptotic characteristics than untreated control group.Specifically,the fluorescence intensity of the control cells was weak,while the cells treated with carboplatin and carboplatin-chitosan nanoparticles showed a markedly enhanced fluorescence intensity marking apoptosis.Quantitative data further showed that the apoptosis rate of cells in the carboplatin-chitosan nanoparticles group was nearly 30%,only 15%in the free carboplatin group and 2%in the control group.Conclusions:1.The carboplatin-chitosan nanoparticles prepared by the ionic gel method were uniform in size and shape,and had drug loading and releasing properties.2.The carboplatin-chitosan nanoparticles had stronger inhibitory effect on SK-NEP-1 cells than free carboplatin in vitro. |
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