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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 shown that nanometer-scale ultrasound contrast agents have strong penetration capabilities, making imaging of extravascular target tissues possible, beyond the limitations of micron-scale contrast agents that can only occur within the blood pool. Objective: To construct folic acid-modified breast cancer-targeted nano-enhanced ultrasound microparticles, evaluate its ability to combine with cell-targeting and in vitro ultrasound imaging. METHODS: A phacoemulsification-evaporation method was used to prepare contrast-coated PEGylated lactic acid glycolic acid copolymer nanoparticles encapsulated with liquid fluorocarbon (marked m PP/PFOB) and folic acid-modified PEGylated glycolic acid glycol copolymer coated liquid. Fluorocarbon nanoparticles (marked as m PPF/PFOB). (1) Biocompatibility test: Human dermal fibroblast HFF-1 and human breast cancer MCF-7 cells in logarithmic growth phase were added, and 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 1 g were added respectively. /L m PP/PFOB or m PPF/PFOB, cell viability was measured 24 h after culture. (2) Detection of in vitro homing ability: HFF-1 and MCF-7 cells in logarithmic growth phase were divided equally into 3 groups. Group A and B were added with Cy5-labeled m PP/PFOB and m PPF/PFOB, respectively. The cells were treated with folic acid for 2 h before adding the Cy5-labeled m PP/PFOB, and cultured for 0.5 h. The fluorescence intensity was measured by flow cytometry. After 20 min culture, the distribution of the contrast agent in the cells was observed by confocal microscopy. (3) Ultrasound in vitro: The experiment was divided into 3 groups. Group A was normal saline. In group B, m PPF/PFOB nanoparticles and physiological saline suspension were prepared. In group C, m PPF/PFOB nanoparticles and physiological saline suspension were used. The suspension of MCF-7 cells was prepared, and the nanoparticles and cell suspension were prepared. The three liquids were added into the latex gloves and tightened. Ultrasound diagnostic imaging instrument was used to detect the effect of ultrasound imaging in vitro. RESULTS AND CONCLUSIONS: (1) Biocompatibility test results: There was no obvious cytotoxicity of both nanoparticles; (2) Flow cytometry results: In MCF-7 cells, the mean fluorescence intensity of group B was significantly higher than that of A. In group C and group C, in the HFF-1 cells, there was no significant difference in the mean fluorescence intensity between the three groups; (3) Confocal microscopy observations: m PPF/PFOB mainly clustered around the MCF-7 cell membrane, and m PP/PFOB was distributed. In the cytoplasm. (4) In vitro ultrasound imaging results: m PPF/PFOB combined with MCF-7 cells can enhance ultrasound echo in vitro; (5) results suggest that folic acid-modified PEGylated glycolic acid copolymers entrap liquid fluorocarbons Nanoparticles have good biocompatibility and have the ability to target and bind to breast cancer MCF-7 cells and enhance ultrasound imaging.