维替泊芬PLGA纳米粒的制备及其对小鼠肝细胞癌的作用研究

Preparation of verteporfin PLGA nanoparticles and the effect on hepatocellular cancer in mice

目的制备一种维替泊芬聚乳酸-羟基乙酸共聚物(PLGA)纳米粒(PLGA-VP NPs)并对其对小鼠肝细胞癌细胞Hepa 1-6的抑制作用和体内安全性进行验证。方法用乳化蒸发法制备PLGA-VP NPs,用动态光散射(DLS)检测PLGA-VP NPs的粒径和电位。将Hepa 1-6细胞分为对照组(不做任何处理)、空载PLGA NPs组(加入空载PLGA NPs)和PLGA-VP NPs组(加入PLGA-VP NPs)。用阿尔玛蓝法检测细胞增殖情况,用流式细胞术检测细胞对PLGA-VP NPs的摄取情况及细胞凋亡率,用蛋白质印迹法检测细胞程序性死亡-配体1(PD-L1)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达水平。将C57小鼠分为正常组(尾静脉注射磷酸缓冲盐溶液100μL)和PLGA-VP NPs组(尾静脉注射2μg·μL^(-1)PLGA-VP NPs 100μL)。用苏木精-伊红(HE)染色法观察小鼠各主要器官。结果PLGA-VP NPs的粒径约为(124.00±1.80)nm,电位约为(-38.00±1.40)mV。PLGA-VP NPs质量浓度为0、0.5、1.0、2.0、2.5、3.0、4.0、5.0 mg·mL^(-1)时,细胞存活率分别为(100.00±2.02)%、(87.33±3.74)%、(55.86±9.71)%、(30.32±6.30)%、(26.03±2.60)%、(23.81±2.09)%、(23.63±1.29)%和(19.75±3.32)%,PLGA-VP NPs对Hepa 1-6的半抑制浓度为0.87 mg·mL^(-1)。0、2、4、8、12、24 h时Hepa 1-6对PLGA-VP NPs的摄取率分别为0、(3.27±0.10)%、(7.32±1.36)%、(27.03±1.11)%、(44.97±1.43)%和(68.37±0.94)%。对照组、空载PLGA NPs组、PLGA-VP NPs组的细胞凋亡率分别为(9.26±1.42)%、(11.08±1.16)%和(27.27±1.12)%,PD-L1蛋白相对表达水平分别为1.00±0.01、1.00±0.08和0.21±0.06,Bcl-2蛋白相对表达水平分别为1.20±0.02、0.43±0.01和0.54±0.07,Bax蛋白相对表达水平分别为0.35±0.02、1.09±0.07和1.30±0.06。PLGA-VP NPs组的上述指标与对照组及空载PLGA NPs组比较,在统计学上差异均有统计学意义(均P<0.05)。HE染色结果显示,与正常组比较,PLGA-VP NPs组小鼠主要器官未见明显异常。结论成功合成了PLGA-VP NPs,该纳米粒对Hepa 1-6肝癌细胞有抑制作用,可降低PD-L1蛋白的表达,且体内安全性良好。

Objective To prepare a verteporfin-loaded poly(lacticco-glycolic acid)nanoparticle(PLGA-VP NPs)and to verify its inhibitory effect on hepatocellular carcinoma cell line Hepa 1-6 and its in vivosafety.Methods PLGA-VP NPs were prepared by emulsion evaporation method,and the particle size and potential of PLGA-VP NPs were detected by dynamic light scattering(DLS).Mouse hepatocellular carcinoma cell line Hepa 1-6 was culturedin vitroand divided into control group(without any treatment),PLGA NPs group(adding PLGA NPs)and PLGA-VP NPs group(adding PLGA-VP NPs).The cell proliferation was detected by the Alamar Blue;the uptake of PLGA-VP NPs and apoptosis rate of cells were detected by flow cytometry;the expression levels of programmed death-ligand 1(PD-L1)protein,B-cell lymphoma-2(Bcl-2)protein and Bcl-2 associated X protein(Bax)were detected by Western blotting.C57 mice were divided into control group(phosphate buffered saline 100μL)and PLGA-VP NPs group(tail vein injection of 2μg·μL^(-1)PLGA-VP NPs 100μL).The main organs of mice were observed by hematoxylin-eosin(HE)staining.Results The particle size of PLGA-VP NPs was about(124.00±1.80)nm,and the potential was about(-38.00±1.40)mV.When the concentrations of PLGA-VP NPs were 0,0.5,1,2,2.5,3,4,5 mg·mL^(-1),the cell survival rates were(100.00±2.02)%,(87.33±3.74)%,(55.86±9.71)%,(30.32±6.30)%,(26.03±2.60)%,(23.81±2.09)%,(23.63±1.29)%and(19.75±3.32)%;the half maximal inhibitory concentration of PLGA-VP NPs on Hepa 1-6 was 0.87 mg·mL^(-1).The uptake rates of Hepa 1-6 to PLGA-VP NPs at 0,2,4,8,12 and 24 h were 0,(3.27±0.10)%,(7.32±1.36)%,(27.03±1.11)%,(44.97±1.43)%and(68.37±0.94)%,respectively.The apoptosis rates of the control group,the PLGA NPs group and the PLGA-VP NPs group were(9.26±1.42)%,(11.08±1.16)%and(27.27±1.12)%;the relative expression levels of PD-L1 protein were 1.00±0.01,1.00±0.08 and0.21±0.06;the relative expression levels of Bcl-2 protein were 1.20±0.02,0.43±0.01 and 0.54±0.07;the relative expression levels of Bax protein were 0.35±0.02,1.09±0.07 and 1.30±0.06,respectively.The above indexes show statistically significant differences in between the PLGA-VP NPs group and the control group,PLGA NPs group(allP<0.05).After HE staining,comparing to the normal group,there was no obvious abnormality in the main organs of the mice in the PLGA-VP NPs group.Conclusions PLGA-VP NPs were successfully synthesized,and the nanoparticles have inhibitory effects on Hepa 1-6 hepatocellular carcinoma cells,can reduce the expression of PD-L1 protein,and have goodin vivosafety.