基于改性复合多糖的载阿霉素纳米粒子的制备及其靶向肝癌细胞的研究

目的 制备一种具有氧化还原敏感性的载阿霉素(DOX)纳米粒子,并研究其体外释放及靶向肝癌细胞的性能.方法 以1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐/N-羟基琥珀酰亚胺为催化剂,使透明质酸(HA)侧链接枝胱胺,进一步通过Schiff碱反应偶联β-环糊精(β-CD)制备β-环糊精接枝透明质酸(HACD).然后以HACD为载体材料,采用透析法制备载DOX纳米粒子(HACD/DOX),并对其载药量、包封率、粒径及分布、zeta电位等理化性质及体外释放行为进行表征;采用细胞计数试剂盒(CCK-8)检测HACD/DOX纳米粒子对肝癌细胞HepG2的毒性作用;通过流式细胞术及激光共聚焦显微镜(CLSM)研究HACD/DOX纳米粒子对HepG2细胞的靶向作用.结果 成功制备了HACD,其可携载DOX形成形态均匀的纳米粒子.DOX在纳米粒子中的载药量为(16.1±0.2)％,包封率为(64.2±0.9)％.透射电子显微镜结果显示其为球形结构;粒度分析结果表明,HACD/DOX纳米粒子的平均粒径为(203.1±2.5) nm,多分散系数为0.202,zeta电位为(-29.1±0.8)mV.该纳米粒子的体外释放行为具有明显的氧化还原敏感性.体外毒性结果显示,空白载体材料HACD对肝癌细胞无明显毒性,而HACD/DOX纳米粒子可有效杀伤肝癌细胞,48 h的半数抑制浓度(IC50)值为0.38 μg/ml.流式细胞术和CLSM结果均显示HACD/DOX纳米粒子是通过HA的介导而发挥肝癌靶向作用的.结论 制备的HACD/DOX纳米粒子具有适宜的粒径、高载药量和包封率,能在还原剂刺激下释放药物,且具有明显靶向肝癌细胞的作用,有望成为一种具有良好应用前景的靶向治疗肝癌的药物递送系统.

Preparation of doxorubicin-loaded nanoparticles based on modified popaccharide and its targeting effect on hepatocellular carcinoma cells

Objective To prepare a redox-responsive doxorubicin-loaded nanoparticle,and to study its in vitro realease behavior and targeting effect on heptoma cells.Methods Cystamine was grafted on the side chains of hyaluronic acid with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide catalyst,and then β-cyclodextrin (β-CD) was conjugated on the amine groups of the cystamine by Schiff's base reaction to prepare β-CD modified hyaluronic acid (HACD).The HACD/DOX nanoparticles were prepared by encapsulating DOX into HACD using dialysis method.The drug loading,encapsulation efficiency,particle size and distribution,zeta potential and other physical and chemical properties,as well as in vitro drug release behavior of the HACD/DOX nanoparticles were characterized.The cytotoxicity of HACD/DOX nanoparticles to HepG2 cells was studied by cell counting kit-8 (CCK-8) method.The targeting effect of HACD/DOX nanoparticles on HepG2 cells was studied using flow cytometry and confocal laser scanning microscopy (CLSM).Results HACD were successfully synthesized,which could carry DOX to form uniform homogeneous nanoparticles.The drug loading of DOX in the nanoparticles was (16.1±0.2)％ and the encapsulation efficiency was (64.2±0.9)％.The transmission electron microscope images indicated that the shape of the HACD/DOX nanoparticles was homogeneous sphere.The results of granularity analysis showed that the average size of the HACD/DOX nanoparticles was (203.1 ±2.5) nm with a narrow size distribution (PDI =0.202).The zeta potential of the HACD/DOX nanoparticles was (-29.1±0.8) mV.The in vitro release behavior of the nanoparticles exhibited obvious redox-sensitivity.The results of in vitro cytotoxicity showed that the blank carrier material HACD had no obvious toxicity to hepatoma cells,and the HACD/DOX nanoparticles could effectively kill hepatoma cells with the 0.38 μg/ml half maximal inhibitory concentration (IC50) value at 48 h.Flow cytometry and CLSM results demonstrated that the HACD/DOX nanoparticles could target hepatoma cells through the mediating effect of hyaluronic acid.Conclusions The prepared HACD/DOX nanoparticles have suitable particle size,high drug loading and encapsulation efficiency,and can release DOX under the stimulation of reducing agent.These nanoparticles have obvious targeting effect on hepatoma cells,which is expected to be applied as the drug delivery system of hepatocellular carcinoma (HCC) therapy.