中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (34): 5468-5472.doi: 10.3969/j.issn.2095-4344.1968

• 药物控释材料 drug delivery materials • 上一篇    下一篇

构建载基因多功能高分子微泡靶向乳腺癌细胞

林  敏1，2，魏巍丽1，何以敉1，陈志奎1
  

  1. 1福建医科大学附属协和医院超声科，福建省福州市  350001；2福建医科大学附属龙岩第一医院，福建省龙岩市  364000
  • 收稿日期:2019-06-26 出版日期:2019-12-08 发布日期:2019-12-08
  • 通讯作者: 陈志奎，博士，副主任医师，副教授，福建医科大学附属协和医院超声科，福建省福州市 350001
  • 作者简介:林敏，女，1987年生，福建省龙岩市人，汉族，硕士，现就职于福建医科大学附属龙岩第一医院，主治医师，主要从事超声造影研究。
  • 基金资助:

    福建省自然科学基金面上项目(2014J01416)，项目负责人：陈志奎

Construction of a gene-loaded multifunctional polymer microbubble targeting breast cancer cells

Lin Min1,2, Wei Weili1, He Yimi1, Chen Zhikui1
  

  1. 1Department of Ultrasound, Affiliated Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China; 2Longyan First Hospital of Fujian Medical University, Longyan 364000, Fujian Province, China
  • Received:2019-06-26 Online:2019-12-08 Published:2019-12-08
  • Contact: Chen Zhikui, MD, Associate chief physician, Associate professor, Department of Ultrasound, Affiliated Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China
  • About author:Lin Min, Master, Attending physician, Department of Ultrasound, Affiliated Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China; Longyan First Hospital of Fujian Medical University, Longyan 364000, Fujian Province, China
  • Supported by:

    The Natural Science Foundation of Fujian Province (General Program), No. 2014J01416 (to CZK)

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摘要：

文章快速阅读：

 

文题释义：
高分子微泡：以高分子聚合物材料作为外壳、含氟碳惰性气体为内核，采用乳化或机械振荡等方法制备的微米级超声对比剂，其稳定性好、体内存留时间较长且抗压性能高，采用超声靶向微泡破裂后产生的生物效应，可阻断局部血液循环、提高细胞通透性、促进基因转染。
阳离子聚合物：为含有大量阳离子的聚合物，其通过阳离子骨架静电结合浓缩 DNA，通过导向配体与细胞受体结合，促进细胞吞噬聚合物/DNA复合体，在细胞内涵体中形成吸收质子的“海绵”，使内涵体崩解，DNA在细胞核内释放，进而提高基因转染效率。
 
 
背景：高分子微泡的稳定性好，具有更长的体内存留时间。
目的：构建载基因靶向高分子微泡，评估其体外靶向结合受体的能力。
方法：以高分子聚合物单甲氧基聚乙二醇左旋聚乳酸共聚物、聚乳酸-羟基乙酸-聚乙二醇-COOH及胆固醇3β-N-(二甲基-氨基-乙基)氨基甲酸叔丁酯盐酸盐作为微泡外壳材料，全氟戊烷气体为内核，单乳化法制备高分子微泡，利用电荷的静电作用将质粒DNA连接到微泡上，碳二亚胺法靶向修饰微泡，形成载基因靶向高分子微泡，显微镜观察微泡形貌、大小、粒径及分布，荧光显微镜及流式细胞术鉴定微泡与质粒DNA及抗体的连接率。观察人类表皮生长因子受体2(+)乳腺癌细胞生长至融合度约80%时，分2组干预：一组加入高分子微泡孵育1 h；另一组预先加入曲妥珠单抗孵育1 h，再加入高分子微泡孵育1 h，显微镜下观察微泡体外靶向结合人类表皮生长因子受体2(+)乳腺癌细胞的能力。
结果与结论：①载基因靶向高分子微泡呈圆形，分散良好，平均粒径(3.0±1.5) μm，大小分布较均匀，浓度为8.8×1010 L-1；②荧光显微镜下可见载基因靶向高分子微泡表面曲妥珠单抗-IgG-FITC发出绿色荧光、质粒DNA-PI发出红色荧光，流式细胞术测得高分子微泡同时连接质粒DNA与曲妥珠单抗的比率为96.28%；③载基因靶向高分子微泡与乳腺癌细胞孵育1 h后，显微镜下观察可见细胞表面结合了大量微泡，而曲妥珠单抗阻断后，高分子微泡与细胞结合量显著减少；④结果表明单乳化法制备的高分子微泡，可有效携带质粒DNA及抗体药物，并且具有良好的靶向功能。

关键词: 超声微泡, 高分子聚合物, 单乳化法, 乳腺癌, 人表皮生长因子受体, 基因治疗, 靶向治疗, 流式细胞术

Abstract:

BACKGROUND: Polymer microbubbles have good stability and long retention time.
OBJECTIVE: To construct gene-loaded polymer microbubbles and to assess their ability to target receptors in vitro.
METHODS: The microbubbles were prepared with polymers (mPEG-PLLA, PLGA-PEG-COOH and DC-Chol) as shell and PFP as core by emulsion method, then modified with herceptin and cationic polymer coated plasmid DNA to form the gene-loaded targeting polymer microbubbles. The morphology, size, and distribution were determined by optical microscopy. The rate of the polymer microbubbles conjugating with both plasmid DNA and trastuzumab-IgG-FITC was determined by fluorescence microscopy and flow cytometry. When human epidermal growth factor receptor  2 (+) breast cancer cells grew to about 80% confluency, they were divided into two groups: one group was incubated with polymer microbubbles for 1 hour; the other group was pre-incubated with trastuzumab for 1 hour, and then with polymer microbubbles for another 1 hour. The ability of the microbubbles binding to human epidermal growth factor receptor 2 (+) breast cancer cells in vitro was detected under a microscope.
RESULTS AND CONCLUSION: The gene-targeted polymer microbubbles were round and well dispersed, with an average particle size of (3.0±1.5) μm and they had uniform size distribution with a concentration of 8.8×1010/L. Under the fluorescence microscope, trastuzumab-IgG-FITC on the surface of gene-loaded breast cancer cells-targeting polymer microbubbles emitted green fluorescence, and plasmid DNA-PI emitted red fluorescence. Flow cytometry showed that the rate of polymer microbubbles both conjugating with plasmid DNA and trastuzumab was 96.28%. After the gene-loaded polymer microbubbles and breast cancer cells were co-incubated and for 1 hour, microscopy showed that a large number of polymer microbubbles were bound on the cell surface, and after blocking with trastuzumab, the amount of polymer microbubbles bounding to breast cancer cells was significantly reduced. The results showed that the polymer microbubbles prepared by the single emulsion method can effectively carry plasmid DNA and antibody drugs, and have good targeting function.

Key words: microbubbles, polymer, single emulsion method, breast cancer, human epidermal growth factor receptor, gene therapy, targeted therapy, flow cytometry

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