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中华医学超声杂志（电子版）

中华医学超声杂志（电子版） ›› 2020, Vol. 17 ›› Issue (04) : 363 -369. doi: 10.3877/cma.j.issn.1672-6448.2020.04.013

所属专题： 文献；

基础研究

IR780-VPN靶向纳米粒的制备及其对肾母细胞瘤的抑制作用
徐晓川1, 唐毅1,†(), 陈镜宇1, 计晓娟1, 杨春江1, 朱丽容1   
  1. 1. 400014　重庆医科大学附属儿童医院超声科　儿童发育疾病研究教育部重点实验室　国家儿童健康与疾病临床医学研究中心（重庆）　儿童发育重大疾病国家国际科技合作基地　儿科学重庆市重点实验室
  • 收稿日期:2020-01-03 出版日期:2020-04-01
  • 通信作者: 唐毅
  • 基金资助:
    重庆市社会事业与民生保障科技创新专项(cstc2017shmsA130082); 中国博士后基金特别资助项目(2018T110949)

Preparation of IR780 and vincristine-loaded poly (lactic-co-glycolic acid) nanoparticles and antinep-hroblastoma cells proliferation in vitro

Xiaochuan Xu1, Yi Tang1,†(), Jingyu Chen1, Xiaojuan Ji1, Chunjiang Yang1, Lirong Zhu1   

  1. 1. Department of Ultrasound, Ministry of Education Key Laboratory of Child Development and Disorders; National Clinical Research Center for Child Health and Disorders (Chongqing); China International Science and Technology Cooperation Base of Child Development and Critical Disorders; Chongqing Key Laboratory of Pediatrics; Children′s Hospital of Chongqing Medical University, Chongqing 400014, China
  • Received:2020-01-03 Published:2020-04-01
  • Corresponding author: Yi Tang
  • About author:
    Corresponding author: Tang Yi, Email:
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引用本文：

徐晓川, 唐毅, 陈镜宇, 计晓娟, 杨春江, 朱丽容. IR780-VPN靶向纳米粒的制备及其对肾母细胞瘤的抑制作用[J]. 中华医学超声杂志（电子版）, 2020, 17(04): 363-369.

Xiaochuan Xu, Yi Tang, Jingyu Chen, Xiaojuan Ji, Chunjiang Yang, Lirong Zhu. Preparation of IR780 and vincristine-loaded poly (lactic-co-glycolic acid) nanoparticles and antinep-hroblastoma cells proliferation in vitro[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2020, 17(04): 363-369.

目的

制备同时携带IR780碘化物和硫酸长春新碱（VCR）的聚乳酸-羟基乙酸（PLGA）靶向纳米粒（IR780-VPN），并观察其在体外对肾母细胞瘤（SK-NEP1）的靶向作用及抗肾母细胞瘤增殖效率。

方法

制备IR780-VPN，检测其基本理化特性，培养SK-NEP1细胞作为体外肿瘤细胞模型，研究IR780-VPN的体外靶向性，观察以下6组的体外抗SK-NEP1细胞增殖效率：A组，PBS（对照组）；B组，游离VCR；C组，空白纳米粒（PN）；D组，载VCR纳米粒（VPN）；E组，载IR780纳米粒（IR780-PN）；F组，IR780-VPN。

结果

制备出的IR780-VPN平均粒径为（290.82±3.22）nm，粒径的分散指数（PDI）为0.024，平均Zeta电位为（1.16±0.87）mV。用细胞膜绿色荧光探针染色后于倒置荧光显微镜下观察，IR780-VPN大小均匀、形态规则，无聚集、粘连；透射电镜下观察IR780-VPN为球形或类球形，表面光滑，粒径分布均匀。其平均包封率为72.80%，平均载药量为2.80%，平均连靶率为91.30%。体外寻靶实验中可见SK-NEP1细胞表面有大量的IR780-VPN聚集。体外抗SK-NEP1细胞增殖实验证实，在相同药物质量浓度条件下，IR780-VPN组较VCR、VPN组的体外抗肿瘤细胞增殖效率高（P均<0.001），且游离VCR、VPN、IR780-VPN对SK-NEP1细胞增殖的抑制率具有浓度依赖性，药物质量浓度越大，对细胞的抑制作用越强（F=13254.105、54354.510、1370.059，P均<0.001）；而随着药物质量浓度增加，PN、IR780-PN组的细胞增殖抑制率差异均无统计学意义（P均>0.05），PBS（对照组）对SK-NEP1细胞增殖的抑制率为（2.83±0.32）%。

结论

本实验成功制备了性质稳定的靶向IR780-VPN，对肾母细胞瘤细胞有良好的的靶向性，并提高了体外抗肾母细胞瘤增殖效率，为体内的肿瘤分子靶向研究提供了实验基础。

关键词: 靶向纳米粒, 超声造影剂, 肾母细胞瘤, 细胞增殖
Objective

To prepare IR780 and vincristine (VCR)-loaded poly(lactic-co-glycolic acid)(PLGA) nanoparticles (IR780-VPN) and verify their specific targeting ability and anti-proliferative efficiency in vitro.

Methods

IR780-VPN were prepared to detect their basic physical and chemical characteristics. SK-NEP1 cells were used as a tumor cell model to verify the specific targeting ability and the anti-proliferative efficiency of IR780-VPN. SK-NEP1 cells were divided into six groups: group A: PBS; group B: free VCR; group C: PLGA nanoparticles; group D: VCR-PLGA nanoparticles; group E: IR780-PLGA nanoparticles; group F: IR780-VPN.

Results

The average particle size of IR780-VPN was (290.82±3.22) nm, the particle dispersion index (PDI) was 0.024, and the average zeta potential of IR780-VPN was (1.16±0.87) mV. IR780-VPN showed stable physical and chemical properties, and they were spherical, uniform in size, and regular in shape, without aggregation oradhesion under a microscope. The morphology of IR780-VPN was spherical or quasi-spherical with a smooth surface, and the particle size distribution was uniform. The average encapsulation efficiency of IR780-VCR-PLGA nanoparticles was about 72.80%, the average loading efficiency was 2.80%, and the specific targeting efficiency was 91.30%. Many IR780-VPN were observed to target tumor cells in vitro. The cytotoxicity was the highest in group F when compared with group B and group D at the same concentration (P<0.05), and cytotoxicity was concentration-dependent in group B, group D, and group F: the higher the drug concentration, the stronger the cytotoxicity (F=13254.105, 54354.510, and 1370.059, P<0.05). There was no significant difference in group C and group E with the increase of drug concentration (P>0.05). The rate of reduced cell proliferation in group A was (2.83±0.32)%.

Conclusion

IR780-VPN with stable properties have been successfully prepared, which can specifically target tumor cells and have improved anti-proliferative efficiency in vitro.

Key words: Targeted nanoparticles, Ultrasound contrast agents, Wilms tumor, Cell proliferation
图1 携带IR780碘化物和硫酸长春新碱的聚乳酸-羟基乙酸纳米粒（IR780-VPN）粒径图
图2 携带IR780碘化物和硫酸长春新碱的聚乳酸-羟基乙酸纳米粒（IR780-VPN）的物理形态。图a为倒置荧光显微镜下观（DiO染色×100），可见IR780-VPN大小均匀、形态规则，无聚集；图b为透射电子显微镜下观（×20000），可见IR780-VPN表面光滑，为球形或者类球形
图3 硫酸长春新碱（VCR） PBS标准曲线及携带IR780碘化物和VCR的聚乳酸-羟基乙酸纳米粒（IR780-VPN）体外释放曲线。图a为VCR PBS标准曲线及回归方程式；图b为将1、2、4、8、12、24、41、150 h的累积释放液测得的吸光度，带入上述标准曲线回归方程计算累积释放率，绘制出IR780-VPN释药曲线图
图4 携带IR780碘化物和硫酸长春新碱的聚乳酸-羟基乙酸纳米粒（IR780-VPN）与未包载IR780的VPN激光共聚焦显微镜下观（蓝色为经DAPI染色的细胞核；红色为经DiI染色的IR780-VPN ×400）。图a为未包载IR780的VPN与SK-NEP1细胞的结合情况，可见细胞周围罕有VPN聚集；图b为IR780-VPN与SK-NEP1细胞的结合情况，可见细胞周围大量IR780-VPN聚集
表1 不同药物浓度的PN和IR780-PN溶液对SK-NEP1细胞增殖的抑制率（±s）
药物浓度（mg/ml） 抑制率（%）
PN IR780-PN
2.5 2.96±0.88 2.98±0.71
5.0 2.97±0.99 2.95±0.42
10.0 2.99±0.89 2.96±0.06
20.0 2.92±0.30 2.96±0.31
40.0 2.91±0.06 2.98±0.31
F值 0.007 0.003
P值 >0.05 >0.05
表2 不同药物浓度的VCR、VPN和IR780-VPN溶液对SK-NEP1细胞增殖的抑制率（±s）
药物浓度（mg/ml） 抑制率（%） F值 P值
VCR VPN IR780-VPN
2.5 21.90±0.36 28.01±0.16 32.79±1.29a 146.931 <0.001
5.0 35.99±0.19 43.68±0.25 51.52±1.39a - <0.001
10.0 50.48±0.52 62.85±0.23 70.96±0.57a 342.782 <0.001
20.0 71.28±0.20 84.80±0.20 86.40±0.28a 4059.570 <0.001
40.0 73.73±0.30 86.03±0.05 88.07±0.14a 4932.526 <0.001
F值 13254.105 54354.510 1370.059    
P值 <0.001 <0.001 <0.001    
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