中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (30): 4425-4433.doi: 10.3969/j.issn.2095-4344.2016.30.003

• 纳米生物材料 nanobiomaterials • 上一篇    下一篇

构建叶酸修饰的乳腺癌靶向纳米超声造影微粒

陈园园1，徐 枫1，杨 辉2，刘 婷1，周建桥3，蔡晨蕾4，叶园园1，刘培峰4，韩宝三1   

  1. 1上海交通大学医学院附属新华医院普外科、普外科实验室，上海市 200092；2教育部高分子合成与功能构造重点实验室，浙江大学高分子科学与工程学系，浙江省杭州市  310027；3上海交通大学医学院附属瑞金医院超声科，上海市  200025；4上海交通大学医学院附属仁济医院中心实验室，上海市 200127
  • 出版日期:2016-07-15 发布日期:2016-07-15
  • 通讯作者: 韩宝三，主任医师，上海交通大学医学院附属新华医院普外科、普外科实验室，上海市 200092
  • 作者简介:陈园园，女，1989年生，江苏省泗洪县人，汉族，上海交通大学医学院在读硕士，医师，主要从事乳腺癌的临床与基础及纳米材料的研究。
  • 基金资助:

    国家自然科学基金(81172078)，宁波市社会发展重大择优科技攻关项目(2012C5013)

Construction of folate-modified nanoparticles as ultrasound contrast agent targeting breast cancer

Chen Yuan-yuan1, Xu Feng1, Yang Hui2, Liu Ting1, Zhou Jian-qiao3, Cai Chen-lei4, Ye Yuan-yuan1, Liu Pei-feng4, Han Bao-san1   

  1. 1Department of General Surgery, General Surgery Laboratory, Affiliated Xinhua Hospital, Medical School of Shanghai Jiao Tong University, Shanghai 200092, China; 2Key Laboratory of Macromolecular Synthesis and Functionalization of Ministry of Education, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, Zhejiang Province, China; 3Department of Ultrasound, Affiliated Ruijin Hospital, Medical School of Shanghai Jiao Tong University, Shanghai 200025, China; 4Medical Science Research Center, Affiliated Renji Hospital, Medical School of Shanghai Jiao Tong University, Shanghai 200127, China
  • Online:2016-07-15 Published:2016-07-15
  • Contact: Han Bao-san, Chief physician, Department of General Surgery, General Surgery Laboratory, Affiliated Xinhua Hospital, Medical School of Shanghai Jiao Tong University, Shanghai 200092, China
  • About author:Chen Yuan-yuan, Studying for master’s degree, Physician, Department of General Surgery, General Surgery Laboratory, Affiliated Xinhua Hospital, Medical School of Shanghai Jiao Tong University, Shanghai 200092, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81172078; Major Science and Technology Project of Ningbo Social Development, No. 2012C5013

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摘要：

文章快速阅读：

文题释义：
纳米级超声对比剂：具有较强的穿透能力，能够穿越血管内皮进入组织间隙，使血管外靶组织显像成为可能，从而超越了微米级对比剂仅能发生血池内显像的局限性，推动了超声分子显像技术的发展，特别是针对肿瘤等重大疾病的早期诊断技术；使构建纳米级靶向超声对比剂成为国内外研究的热点。
超声靶向分子成像技术：是利用对比剂表面固有的化学特性或通过对对比剂表面进行特殊处理，构建特异性靶向超声对比剂，使其经静脉注入后能靶向性聚集并较长时间滞留于靶组织器官，再通过超声对比显影，产生分子水平显影，提高超声对早期病变的诊断及鉴别能力。

 

背景：研究表明，纳米级超声对比剂具有较强的穿透能力，使血管外靶组织显像成为可能，超越了微米级对比剂仅能发生血池内显像的局限性。
目的：构建叶酸修饰的乳腺癌靶向纳米超声造影微粒，评估其与细胞靶向结合的能力及体外超声成像效果。
方法：采用超声乳化-蒸发法制备对比剂聚乙二醇化乳酸羟基乙酸共聚物包裹液态氟碳的纳米粒(记为mPP/PFOB)和叶酸修饰的聚乙二醇化乳酸羟基乙酸共聚物包裹液态氟碳的纳米粒(记为mPPF/PFOB)。①生物相容性检测：取对数生长期的人皮肤成纤维细胞HFF-1、人乳腺癌MCF-7细胞，分别加入0，0.005，0.01，0.02，0.05，0.1，0.2，1 g/L的mPP/PFOB或mPPF/PFOB，培养24 h后检测细胞活力。②体外寻靶能力检测：取对数生长期的HFF-1、MCF-7细胞，均分3组干预，A、B组分别加入Cy5标记的mPP/PFOB与mPPF/PFOB，C组在加入Cy5标记的mPP/PFOB前以叶酸处理2 h，培养0.5 h后，流式细胞仪检测荧光强度；培养20 min后，共聚焦显微镜观察对比剂在细胞中的分布。③体外超声显影：实验分3组，A组为生理盐水，B组制备 mPPF/PFOB纳米粒与生理盐水混悬液，C组以 mPPF/PFOB纳米粒与生理盐水混悬液重悬MCF-7细胞沉淀，制备纳米粒与细胞混悬液，将3种液体加入乳胶手套中扎紧，采用超声诊断仪检测体外超声成像效果。
结果与结论：①生物相容性检测结果：两种纳米粒无明显的细胞毒性；②流式细胞仪检测结果：在MCF-7细胞中，B组平均荧光强度明显高于A组和C组；而在HFF-1细胞中，3组平均荧光强度无显著差别；③共聚焦显微镜观察结果：mPPF/PFOB主要聚集在MCF-7细胞膜周围，mPP/PFOB则分布在胞浆。④体外超声显影结果：mPPF/PFOB与MCF-7细胞结合后，在体外能增强超声回声；⑤结果提示：叶酸修饰的聚乙二醇化乳酸羟基乙酸共聚物包裹液态氟碳的纳米粒具有良好的生物相容性，具有与乳腺癌MCF-7细胞靶向结合能力和增强超声显像效果。

ORCID: 0000-0002-0322-5403(韩宝三)

关键词: 生物材料, 纳米材料, 纳米粒, 聚乙二醇, 聚乳酸-羟基乙酸共聚物, 叶酸受体, 超声诊断, 靶向对比剂, 乳腺癌, 国家自然科学基金

Abstract:

BACKGROUND: Studies have testified that nano-ultrasound contrast agents have a strong permeability, making it possible to image the targeted tissues outside blood vessels and overcome the limitation that micron contrast agents are only available for the blood pool imaging.
OBJECTIVE: To construct the folate-modified nanoparticles targeting breast cancer as ultrasound contrast agents, as well as to observe their ability to specifically bind to cells and imaging effect in vitro.
METHODS: Both contrast agents, pegylated lactic acid-glycolic acid copolymer wrapping liquid fluorocarbon formed nanoparticles (mPP/PFOB) and folate modified pegylated lactic acid-glycolic acid wrapping liquid fluorocarbon formed nanoparticles (mPPF/PFOB), were constructed by phacoemulsification-evaporation method. (1)Biocompatibility detection: HFF-1 and MCF-7 cells in the logarithmic phase were cultivated with various concentrations (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 1 g/L) of mPP/PFOB or mPPF/PFOB for 24 hours respectively, and then the cell viability was measured. (2)Targeting ability detection in vitro: HFF-1 and MCF-7 cells in the logarithmic phase were divided into three groups. Cy5-labled mPP/PFOB and mPPF/PFOB were added into groups A and B, respectively; the cells in group C were pretreated with folate for 2 hours, and sequentially Cy5-labled mPPF/PFOB was added into group C. Fluorescence intensity was detected by flow cytometry after 0.5 hours of culture. The distribution of contrast agents in cells was observed using confocal microscopy after 20 minutes of culture. (3)Ultrasound imaging in vitro: there were three groups: saline was as group A; the suspension of saline and mPPF/PFOB nanoparticles was prepared as group B; MCF-7 cells were resuspended with the mixture of saline and mPPF/PFOB nanoparticles to prepare the suspension of nanoparticles and cells as group C. In each group, the suspension was added into latex gloves, that were then tightened and immersed in water. Finally, the ultrasound was use to detect the ultrasound imaging effect in vitro.
RESULTS AND CONCLUSION: Neither nanoparticles were with significant cytotoxicity. The flow cytometry showed that the mean fluorescence intensity in MCF-7 cells of group B was significantly higher than that of groups A and C. But there were no significant differences in the mean fluorescence intensity in HFF-1 cells among the three groups. It was observed that mPPF/PFOB mainly gathered around the MCF-7 cell membrane, while mPP/PFOB randomly distributed in the cytoplasm. After mPPF/PFOB binding to MCF-7 cells, they could enhance ultrasound echo in vitro. These findings indicate that the targeted nanoparticles mPPF/PFOB have good biocompatibility and can specifically bind to breast cancer MCF-7 cells in vitro and enhance the imaging capability.

Key words: Nanoparticles, Ultrasonography, Contrast Media, Tissue Engineering

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