Establishment of myocardial targeted nanoparticles and preliminary evaluation of its effects on prevention and treatment of myocardial injury
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摘要: 目的 制备3-{ 4-[ 2-羟基- (1-甲基乙胺基) 丙氧基]苯基}丙酸十六醇酯 (PAC) 修饰的纳米粒,观察其对心肌细胞的靶向性及对心肌的保护作用。 方法 (1)将HL-1心肌细胞按随机数字表法分成花青素Cy3标记的非靶向小干扰RNA(Cy3-siNC)组和针对NF-κB-p65基因设计的花青素Cy3标记的小干扰RNA(Cy3-si435)组,每组3孔。Cy3-siNC组细胞转染Cy3-siNC,Cy3-si435组细胞转染Cy3-si435。转染24 h,实时荧光定量PCR检测细胞NF-κB-p65 mRNA的表达。(2)采用复乳溶剂挥发法制备PAC修饰且载有Cy3-siNC(Cy3-siNC-PAC)的纳米粒和PAC修饰且载有Cy3-si435(Cy3-si435-PAC)的纳米粒。扫描电镜观察Cy3-si435-PAC纳米粒的形态,纳米粒度及zeta电位仪检测Cy3-si435-PAC纳米粒的粒径和电势,紫外分光光度计测定Cy3-si435-PAC纳米粒的包封率、载药量,透析法测定Cy3-si435-PAC纳米粒的Cy3-si435释放量。(3)另取HL-1细胞,按随机数字表法分成4组,每组9孔。阴性对照组加入5 μL PBS;25、50、100 mg/mL Cy3-si435-PAC纳米粒组分别加入5 μL 25、50、100 mg/mL的Cy3-si435-PAC纳米粒。分别于转染6、12、24 h时,每组各取3孔,噻唑蓝法检测细胞的增殖活性。(4)另取HL-1细胞,培养24 h后加入100 μL Cy3-si435-PAC纳米粒。分别于转染0、4、8、12、24 h时各取3孔细胞,流式细胞仪检测摄取Cy3-si435-PAC纳米粒的细胞百分比。(5)另取HL-1细胞,按随机数字表法将细胞分成2组,每组3孔。Cy3-siNC-PAC组加入100 μL Cy3-siNC-PAC纳米粒,Cy3-si435-PAC组加入100 μL Cy3-si435-PAC纳米粒。转染24 h,实时荧光定量PCR检测细胞NF-κB-p65 mRNA的表达。(6)取6只雄性C57BL/6J小鼠,按随机数字表法分为2组,每组3只。Cy3-siNC-LPS组和Cy3-si435-LPS组小鼠尾静脉分别注射500 μL Cy3-siNC-PAC纳米粒和Cy3-si435-PAC纳米粒(50 mg/mL),注射后24 h,再分别腹腔注射LPS(10 mg/kg),诱导心肌损伤。伤后24 h,小动物成像仪检测纳米粒在小鼠体内的分布情况。(7)另取9只雄性C57BL/6J小鼠,按随机数字表法分为3组,每组3只。Cy3-siNC-生理盐水(NS)组和Cy3-siNC-LPS组小鼠尾静脉注射500 μL 50 mg/mL的Cy3-siNC-PAC纳米粒,Cy3-si435-LPS组小鼠注射500 μL 50 mg/mL的Cy3-si435-PAC纳米粒。注射后24 h,Cy3-siNC-NS组小鼠腹腔注射NS;Cy3-siNC-LPS组和Cy3-si435-LPS组小鼠腹腔注射10 mg/kg LPS,诱导心肌损伤。伤后24 h,取各组小鼠心肌组织,HE染色观察心肌病理变化。对数据行
t 检验、单因素方差分析。 结果 (1)Cy3-si435组细胞NF-κB-p65 mRNA表达量为0.183±0.004,明显低于Cy3-siNC组的1.003±0.092(t =15.46,P <0.01)。(2)制备的Cy3-si435-PAC纳米粒形态良好,粒径大小为146.0 nm,电势为-29.2 mV,包封率为(86.9±1.1)%,载药量为(25.4±0.9)%,Cy3-si435释放稳定。(3)转染6、12、24 h,4组细胞增殖活性总体比较,差异均无统计学意义(F 值为0.129~2.512,P 值均大于0.05)。(4)转染0、4、8、12、24 h时,摄取Cy3-si435-PAC纳米粒的细胞百分比分别为(0.79±0.06)%、(31.04±1.59)%、(51.64±2.67)%、(68.15±2.60)%、(83.68±4.67)%。(5)Cy3-si435-PAC组细胞NF-κB-p65 mRNA的表达量为0.286±0.015,明显低于Cy3-siNC-PAC组细胞的1.002±0.073(t =16.62,P <0.01)。(6)伤后24 h,Cy3-siNC-LPS组和Cy3-si435-LPS组小鼠心肌细胞都能够观察到分布均匀的纳米粒。(7)Cy3-siNC-NS组小鼠心肌纤维结构致密,无炎性细胞浸润,胞质分布均匀;Cy3-siNC-LPS组小鼠心肌纤维结构疏松,炎性细胞浸润,胞质分布散乱;Cy3-si435-LPS组小鼠心肌纤维结构较致密,未见明显炎性细胞浸润,胞质分布均匀。 结论 Cy3-si435-PAC纳米粒形态良好,粒径大小均一,电势分布正常,无细胞毒性;能够被HL-1细胞有效摄取并抑制细胞NF-κB-p65 mRNA的表达;能够有效靶向到小鼠心肌细胞并减少炎性细胞浸润,减轻LPS诱导的小鼠心肌损伤。Abstract: Objective To establish 3-{4-[2-hydroxyl-(1-methylethylamino) propoxy] phenyl} propionic acid cetylesters (PAC) modified nanoparticles, and preliminarily explore its cardiomyocyte-targeting function and protection effects on myocardium. Methods (1) HL-1 myocardial cells were divided into cyanidin-3 (Cy3) marked non-targeted small interference RNA (Cy3-siNC) group and Cy3 marked small interference RNA designed for the nuclear factor kappa B (NF-κB)-p65 gene (Cy3-si435) group according to the random number table, with 3 wells in each group. Cells in Cy3-siNC group were transfected with Cy3-siNC, while cells in Cy3-si435 group were transfected with Cy3-si435. At transfection hour 24, the mRNA expression of NF-κB-p65 of cells was determined by real-time fluorescent quantitative polymerase chain reaction. (2) Multiple emulsificating solvent evaporating method was adopted to prepare PAC modified nanoparticles carried with Cy3-siNC (Cy3-siNC-PAC) and PAC modified nanoparticles carried with Cy3-si435 (Cy3-si435-PAC). The morphology of Cy3-si435-PAC nanoparticles was observed with scanning electron microscope, and the size and potential of Cy3-si435-PAC nanoparticles were detected by nanometer particle size and zeta potential analyzer. The entrapment efficiency and drug loadings of Cy3-si435-PAC nanoparticle were determined with ultraviolet spectrophotometer. The release of Cy3-si435 of Cy3-si435-PAC nanoparticles was determined by dialysis method. (3) Another batch of HL-1 cells were divided into 4 groups according to the random number table, with 9 wells in each group. Cells in negative control group were added with 5 μL phosphate buffer. Cells in 25, 50, and 100 mg/mL Cy3-si435-PAC nanoparticles groups were added with 5 μL 25, 50, and 100 mg/mL Cy3-si435-PAC nanoparticles, respectively. At transfection hour 6, 12, and 24, proliferation activity of cells in 3 wells of each group was detected by methyl thiazolyl tetrazolium method, respectively. (4) Another batch of HL-1 cells were cultured for 24 h, and then treated with 100 μL Cy3-si435-PAC nanoparticles. At transfection hour 0, 4, 8, 12, and 24, the percentage of cells uptaking Cy3-si435-PAC nanoparticles in 3 wells were detected by flow cytometry, respectively. (5) Another batch of HL-1 cells were divided into 2 groups according to the random number table, with 3 wells in each group. Cells in Cy3-siNC-PAC group were added with 100 μL Cy3-siNC-PAC nanoparticles, while cells in Cy3-si435-PAC group were added with 100 μL Cy3-si435-PAC nanoparticles. At transfection hour 24, the mRNA expression of NF-κB-p65 of cells was determined by real-time fluorescent quantitative polymerase chain reaction. (6) Six male C57BL/6J mice were divided into 2 groups according to the random number table, with 3 mice in each group. Mice in Cy3-siNC-lipopolysaccharide (LPS) group and Cy3-si435-LPS group were respectively injected with 500 μL Cy3-siNC-PAC nanoparticles and Cy3-si435-PAC nanoparticles (50 mg/mL) in the tail vein. At injection hour 24, mice in the two groups were intraperitoneally injected with 10 mg/kg LPS to induce myocardial injury. At post injury hour 24, the distribution of nanoparticles in mice was detected with small animal imager. (7) Another 9 male C57BL/6J mice were divided into 3 groups according to the random number table, with 3 mice in each group. Mice in Cy3-siNC-normal saline (NS) group and Cy3-siNC-LPS group were injected with 500 μL 50 mg/mL Cy3-siNC-PAC nanoparticles in the tail vein, while mice in Cy3-si435-LPS group were injected with 500 μL 50 mg/mL Cy3-si435-PAC nanoparticles. At injection hour 24, mice in Cy3-siNC-NS group were intraperitoneally injected with NS, while mice in Cy3-siNC-LPS group and Cy3-si435-LPS group were injected with 10 mg/kg LPS to induce myocardial injury. At post injury hour 24, pathological changes of myocardium of mice in each group were observed with HE staining. Data were processed witht test and one-way analysis of variance. Results (1) The mRNA expression of NF-κB-p65 of cells in Cy3-si435 group was 0.183±0.004, significantly lower than 1.003±0.092 in Cy3-siNC group (t =15.46,P <0.01). (2) The form of prepared Cy3-si435-PAC nanoparticles was good, with particle size of 146.0 nm, potential of -29.2 mV, entrapment efficiency of (86.9±1.1) %, drug loadings of (25.4±0.9) %, and stable Cy3-si435 release. (3) At transfection hour 6, 12, and 24, there were no statistically significant differences in proliferation activity of cells in the 4 groups (withF values from 0.129 to 2.512,P values above 0.05). (4) At transfection hour 0, 4, 8, 12, and 24, the percentages of cells uptaking Cy3-si435-PAC nanoparticles were (0.79±0.06)%, (31.04±1.59)%, (51.64±2.67)%, (68.15±2.60)%, and (83.68±4.67)%, respectively. (5) The mRNA expression of NF-κB-p65 of cells in Cy3-si435-PAC group was 0.286±0.015, significantly lower than 1.002±0.073 in Cy3-siNC-PAC group (t =16.62,P <0.01). (6) At post injury hour 24, uniform distribution of nanoparticles could be observed in cardiomyocytes of mice in Cy3-siNC-LPS group and Cy3-si435-LPS group. (7) The structure of myocardial fibers of mice in Cy3-siNC-NS group was dense, with no inflammatory cells infiltration and uniform distribution of cytoplasm. The structure of myocardial fibers of mice in Cy3-siNC-LPS group were loose, with inflammatory cells infiltration and scattered distribution of cytoplasm. The structure of myocardial fibers of mice in Cy3-si435-LPS group was denser, with no obvious inflammatory cells infiltration and uniform distribution of cytoplasm. Conclusions Cy3-si435-PAC nanoparticles have good morphology, uniform particle size, normal potential distribution, and no cell cytotoxicity. Cy3-si435-PAC nanoparticles can be effectively uptaked by HL-1 cells and suppress NF-κB-p65 mRNA expression. They also can effectively target to mice cardiomyocytes to reduce inflammatory cells infiltration and alleviate the myocardial injury of mice induced by LPS.-
Key words:
- Sepsis /
- NF-kappa B /
- RNA, small interfering /
- Myocardial injury /
- Targeted nanoparticles
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