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乳腺癌细胞来源的细胞膜纳米囊泡制备及靶向特性
Preparation and Tumor Targeting Analysis of Cell Membrane Nanovesicles Derived from Breast Cancer Cells
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摘要:目的 制备乳腺癌细胞来源的细胞膜纳米囊泡（NVs），探讨其基本特性、肿瘤细胞内吞，在荷瘤小鼠模型中的体内分布，探究其肿瘤靶向特性。方法 体外培养乳腺癌4T1细胞，通过超速离心法提取细胞膜，利用脂质体挤出仪制备细胞膜纳米囊泡。应用动态光散射方法检测囊泡粒径分布，利用透射电子显微镜研究囊泡的形貌特征。通过检测囊泡在磷酸盐缓冲液中的粒径变化分析囊泡的稳定性。应用CCK8法检测不同质量浓度（5、10、20、50和100 mg·L−1）囊泡处理后树突细胞的存活率。荧光显微镜成像检测囊泡在乳腺癌细胞中的内吞。建立皮下乳腺癌荷瘤小鼠模型，将Cy5.5标记的囊泡经尾静脉注射后通过小动物活体成像系统分析其体内分布。结果 制备了乳腺癌细胞4T1来源的囊泡，平均粒径为123.2 nm，在透射电镜下呈现为空心的圆球形结构。囊泡在磷酸盐缓冲液中孵育7 d后粒径无明显变化。CCK8结果显示不同囊泡浓度处理后树突细胞存活率均大于90%，荧光显微镜成像显示囊泡能被乳腺癌细胞摄取到细胞内，体内成像结果显示乳腺癌细胞来源的囊泡具有较正常细胞囊泡更高的肿瘤组织蓄积。结论 成功制备出4T1细胞来源的NVs，该NVs能被乳腺癌细胞摄取，并展现良好的肿瘤靶向作用。Abstract:Objective To prepare cell membrane nanovesicles (NVs) derived from breast cancer cells, to explore their basic characteristics, tumor cell endocytosis, and in vivo distribution in a tumor-bearing mouse model, and to investigate their tumor targeting properties.Methods 4T1 breast cancer cells were cultured in vitro. The cell membrane of 4T1 cells was isolated through ultracentrifugation and NVs were formulated with a liposome extruder. The size distribution of NVs was determined by way of dynamic light scattering, and the morphology properties of the NVs were examined with transmission electron microscope. The stability of NVs was analyzed by measuring the diameter changes of NVs submerged in phosphate-buffered saline (PBS). The biocompatibility of NVs was investigated by measuring the viability of dendritic cells treated with NVs at different concentrations (5, 10, 20, 50, and 100 mg·L−1) by CCK-8 assay. Fluorescence microscopy was used to analyze the cellular uptake of NVs by breast cancer cells. A mice model of breast cancer model was established with mice bearing subcutaneous xenograft of 4T1 cells. The mice were treated with Cy5.5-labeled NVs injected via the tail vein and the in vivo distribution of NVs was analyzed with an imaging system for small live animals.Results The results showed that NVs derived from 4T1 breast cancer cells were successfully prepared. The NVs had a mean diameter of 123.2 nm and exhibited a hollow spherical structure under transmission electron microscope. No obvious change in the size of the NVs was observed after 7 days of incubation in PBS solution. CCK-8 assay results showed that the viability of dendritic cells treated with NVs at different concentrations was always higher than 90%. Fluorescence microscopic imaging showed that NVs could be efficiently internalized into breast cancer cells. in vivo biodistribution analysis revealed that breast cancer cell-derived NVs showed higher distribution in tumor tissue than the NVs prepared with normal cells did.Conclusion We successfully prepared cell membrane NVs derived from 4T1 breast cancer cells. These NVs had efficient cellular uptake by breast cancer cells and sound tumor targeting properties.
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