载阿霉素聚合物纳米粒的制备、表征与体内外性能研究

Preparation, characterization, in vitro and in vivo evaluation of doxorubicin-loaded polymeric nanoparticles

目的 以两亲性三嵌段共聚物聚己内酯-聚乙二醇-聚己内酯（PCL-b-PEG-b-PCL）为载体材料,制备包载抗肿瘤药物阿霉素（DOX）的聚合物纳米粒,并对其进行体内外性能研究.方法 以PCL-b-PEG-b-PCL作为载体材料,通过薄膜水化超声分散法制备出载DOX的聚合物纳米粒,并对其形态、粒径及其分布、载药量及包封率等理化性能进行表征.采用MTS法研究载DOX聚合物纳米粒对EMT6乳腺癌细胞的细胞毒性,激光扫描共聚焦显微镜（CLSM）观察EMT6细胞对纳米粒的细胞吞噬,离体脏器荧光成像研究纳米粒在荷EMT6乳腺癌小鼠的组织分布.结果 通过薄膜水化超声分散法成功制备出载DOX聚合物纳米粒,透射电镜和扫描电镜结果表明,该纳米粒呈球形,大小均匀,具有明显的核壳结构.粒度分析表明,载DOX聚合物纳米粒的平均粒径为130.8 nm,且粒径分布较窄（多分散系数为0.200）.DOX在聚合物纳米粒中的包封率和载药量分别为（86.71±2.05）％和（8.71±0.57）％.细胞毒性研究发现,空白纳米粒对EMT6细胞无毒性,而载入DOX后,DOX-NPs的细胞毒性具有时间和剂量依赖性;在DOX质量浓度较高（20 μg/ml和40μg/ml）和孵育时间较长（72 h）时,载DOX聚合物纳米粒与游离DOX的细胞毒性相当,差异无统计学意义（P＞0.05）.CLSM观察发现,EMT6乳腺癌细胞与载DOX聚合物纳米粒共同孵育后,DOX的荧光在细胞质和细胞核中均有分布,但与游离DOX共同孵育后,DOX的红色荧光主要出现在细胞核中.离体脏器荧光成像研究表明,分别对荷EMT6乳腺癌小鼠尾静脉注射载DOX聚合物纳米粒及游离DOX后,载DOX聚合物纳米粒可通过增强渗透和滞留效应（EPR）在肿瘤部位有效聚集.结论 载DOX聚合物纳米粒具有适合静脉注射的粒径、高载药量和包封率及良好的被动靶向特性,是一种在肿瘤治疗中具有潜在应用前景的纳米药物递送系统.

Objective To prepare and evaluate the polymeric nanoparticles based on PCL-PEG-PCL amphiphilic triblock copolymers for doxorubicin delivery against breast cancer.Methods PCL-PEG-PCL amphiphilic triblock copolymers were used to encapsulate doxorubicin by thin-film hydration and an ultrasonic dispersion method.The prepared DOX-loaded polymeric nanoparticles were characterized in terms of morphology,particle size and size distribution,drug loading content and encapsulation efficiency.In vitro cytotoxicity against EMT6 cell line was assessed by MTS assay.Confocal laser scanning microscopy （CLSM） was used to evaluate the cellular uptake of DOX-loaded polymeric nanoparticles.Ex vivo DOX fluorescence imaging of the isolated major organs and tumors in EMT6 tumor-bearing mice was observed.Results The DOX-loaded polymeric nanoparticles were successfully prepared by thin-film hydration and ultrasonic dispersion method.Transmission electron microscope and scanning electron microscope verified that the DOX-loaded polymeric nanoparticles were homogeneous spherical shapes with apparent core-shell morphology.The average particle size of the DOX-loaded polymeric nanoparticles was 130.8 nm with a narrow size distribution （polydispersity index was 0.200）.DOX was entrapped in the polymeric nanoparticles with encapsulation efficiency and loading content of （86.71±2.05）% and （8.72±0.57）%,respectively.In vitro cytotoxicity assay against EMT6 cells demonstrated that the blank nanoparticles exhibited no cytotoxicity,while the cytotoxic effect of DOX-loaded polymeric nanoparticles gradually approached that of free DOX when increasing the concentration and the incubation time.CLSM results showed that the DOX fluorescence was distributed both in the cytoplasm and nucleus for DOX-loaded polymeric nanoparticles treated cells,while the red fluorescence was observed mostly in the nucleus for free DOX treated cells.Furthermore,ex vivo DOX fluorescence imaging revealed that DOX-loaded polymeric nanoparticles had highly efficient targeting and accumulation at the implanted site of an EMT6 xenograft tumor in vivo through enhanced permeability and retention effect （EPR）.Conclusions The prepared DOX-loaded polymeric nanoparticles showed satisfactory size and narrow size distribution,high encapsulation efficiency and drug loading content,efficient passive targeting via EPR.These results suggest that the DOX-loaded polymeric nanoparticles might have the potential to be developed as a nano-drug delivery system for cancer chemotherapy.