构建叶酸修饰的乳腺癌靶向纳米超声造影微粒

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

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m PP/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m PPF/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m PP/PFOB或m PPF/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m PP/PFOB与m PPF/PFOB,C组在加入Cy5标记的m PP/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m PPF/PFOB纳米粒与生理盐水混悬液,C组以m PPF/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:m PPF/PFOB主要聚集在MCF-7细胞膜周围,m PP/PFOB则分布在胞浆。(4)体外超声显影结果:m PPF/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。

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. Cy5-1abled 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 Cy5-1abled 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 deteci 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.