盐酸阿霉素和P53蛋白的复合载药纳米粒在大鼠体内分布和对肝癌Hepa1-6细胞抑制作用

Distribution of adriamycin hydrochloride and P53 protein-loaded compound nanoparticles in rats and their inhibitory effect on Hepa1-6 cells

目的考察负载盐酸阿霉素和P53蛋白的复合载药纳米粒在大鼠体内的分布及对肝癌Hepa1-6细胞的抑制作用。方法①方法学考察:用高效液相分别测定盐酸阿霉素、空白载药纳米粒和载药纳米粒的保留时间。取SD大鼠空白血浆和各个组织匀浆匀浆后加入盐酸阿霉素标准品,分别配置成5种不同质量浓度(20,40,80,160,320μg·mL-1)的溶液,制备盐酸阿霉素在心、肝、脾、肺、肾和血浆中的线性回归方程。取SD大鼠空白血浆和各个组织匀浆,加入低、中、高3个质量浓度(1,10,50μg·mL-1)的对照品溶液100μL,取匀浆液200μL,离心取上清液,再进行复溶,测量日内精密度与日间精密度。②体内分布实验:按照体重将SD大鼠随机分为3组:盐酸阿霉素组(AH)、载盐酸阿霉素纳米粒(AHR)和空白组,每组10只。AH组大鼠尾静脉注射阿霉素4 mg·kg^-1,AHR组给予载药复合纳米粒阿霉素9 mg·kg^-1,空白组给予0.9%NaCl。用高效液相色谱法测定药物在大鼠体内心、肝、脾、肺和肾组织的分布情况。③细胞抑制率实验:将制备好的载P53蛋白的盐酸阿霉素纳米粒(P53-AHR)和AHR作用于Hepa1-6细胞,用酶标仪测定细胞抑制情况。结果①盐酸阿霉素保留时间为5.02 min,载药纳米粒保留时间为5.14 min。在0.10~250.00μg·mL^-1,相关系数均为0.99。②盐酸阿霉素在不同组织的回收率均大于90%,日间精密度均小于9%,日内精密度均小于5%;1 h时,肾AH组为(12.50±1.27)μg·mL^-1,AHR组为(27.30±3.27)μg·mL^-1,肝AH组为(14.70±1.06)μg·mL^-1,AHR组为(26.60±3.21)μg·mL^-1,脾AH组为(14.60±2.09)μg·mL-1,AHR组为(19.70±3.65)μg·mL-1。③当盐酸阿霉素浓度为0.30μg·mL^-1时,P53-AHR组的细胞抑制率为(81.62±4.98)%,AHR组的细胞抑制率为(63.02±12.16)%。结论碳酸钙复合载药纳米粒具有肝、脾的靶向性和趋向性,同时能够高效的杀死肝癌细胞且具有协同作用。

Objective To investigate the distribution of doxorubicin hydrochloride and P53 protein loaded composite drug-loaded nanoparticles in rats and their inhibitory effect on Hepa 1-6 cells.Methods①The retention time of adriamycin hydrochloride,blank loaded nanoparticles and loaded nanoparticles were determined by HPLC.The blank plasma and tissue homogenates of SD rats were added with doxorubicin hydrochloride standard.Five different concentrations(20,40,80,160,320μg·mL^-1)of doxorubicin hydrochloride were prepared.The linear regression equations of doxorubicin hydrochloride in heart,liver,spleen,lung,kidney and plasma were prepared.Take the blank plasma and tissue homogenate of SD rats,add low,medium and high concentration(1,10,50μg·mL^-1)control solution 100μL,take homogenized serum 200μL,centrifugate the supernatant,and then redissolve it,and measure the daily precision and precision.②In vivo distribution experiment:SD rats were randomly divided into three groups:adriamycin hydrochloride group(AH),adriamycin loaded nanoparticles(AHR)and blank group,10 rats in each group.The dose of adriamycin was 4 mg·kg^-1 in the tail vein of rats in the AH group,9 mg·kg^-1 in the AHR group and 0.9%NaCl in the blank group.The distribution of drugs in the heart,liver,spleen,lung and kidney of rats was determined by HPLC.③Cell inhibition rate experiment:the prepared adriamycin hydrochloride nanoparticles carrying P53 protein(P53-AHR)and AHR were applied to Hepa1-6 cells.The cell inhibition was determined by enzyme labeling.Results①The retention time of adriamycin hydrochloride was 5.02 min,the retention time of cloaded nanoparticles was 5.14 min.In the range of 0.10-250.00μg·mL^-1,the correlation coefficients are all 0.99.②The recovery of doxorubicin hydrochloride in different tissues was more than 90%,the daytime precision was less than 9%,and the intraday precision was less than 5%.At one hour,the recovery of doxorubicin hydrochloride in kidney AH group was(12.50±1.27)μg·mL^-1,AHR group was(27.30±3.27)μg·mL^-1,liver AH group was(14.70±1.06)μg·mL^-1,AHR group was(26.60±3.21)μg·mL^-1,spleen AH group was(14.60±2.09)μg·mL^-1,AHR group was(19.70±3.65)μg·mL^-1.③When the concentration of doxorubicin hydrochloride was 0.30μg·mL^-1,the inhibition rate of P53-AHR group was(81.62±4.98)%,and that of AHR group was(63.02±12.16)%.Conclusion Calcium carbonate composite drug-loaded nanoparticles have the characteristics of liver and spleen targeting and tropism,and can kill hepatocellular carcinoma cells efficiently with synergistic effect.