Sigot Valeria
Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077, Götingen, Germany,
Methods Mol Biol. 2014;1199:113-27. doi: 10.1007/978-1-4939-1280-3_9.
Lipid nanoparticles composed of mixtures of PEGylated-lipids; cationic and neutral lipids prepared by detergent dialysis can encapsulate biological active molecules and show considerable potential as systemic therapeutic agents. Addition of biotinylated lipids to this formulation allows surface modification of these particles with a suitable ligand or probe conjugated to streptavidin for specific cell targeting. Monitoring long circulating particles and cellular uptake requires stable and bright fluorescent probes. Quantum dots (QDs) constitute a relatively new class of fluorescent probes that overcome the limitations of organic fluorophores in biological imaging applications. Here, a protocol for the encapsulation of QD655 (red) in biotinylated lipid particles (BLPs) prepared by a detergent dialysis technique is presented followed by characterization of the loaded liposomal vehicles. Then, a protocol for BLPs surface modification via biotin-streptavidin linkage with preformed complexes of ligand-QD525 (green) for specific cell targeting of the nanoparticle is detailed. Conditions for cell binding and uptake of two colors QD labeled BLPs as well as basic microscopic settings for confocal live cell imaging are described.
由聚乙二醇化脂质、阳离子脂质和中性脂质混合物组成的脂质纳米颗粒，通过去污剂透析法制备，可封装生物活性分子，并显示出作为全身治疗剂的巨大潜力。向该制剂中添加生物素化脂质，可通过与链霉亲和素偶联的合适配体或探针，对这些颗粒进行表面修饰，以实现特异性细胞靶向。监测长循环颗粒和细胞摄取需要稳定且明亮的荧光探针。量子点（QDs）是一类相对较新的荧光探针，克服了有机荧光团在生物成像应用中的局限性。本文介绍了一种通过去污剂透析技术将QD655（红色）封装在生物素化脂质颗粒（BLPs）中的方案，随后对负载的脂质体载体进行表征。然后，详细介绍了通过生物素 - 链霉亲和素连接对BLPs进行表面修饰的方案，该连接使用配体 - QD525（绿色）的预形成复合物，用于纳米颗粒的特异性细胞靶向。描述了双色QD标记的BLPs细胞结合和摄取的条件以及共聚焦活细胞成像的基本显微镜设置。
