Osinski Victoria, Klibanov Alexander L, McNamara Coleen A
Robert M. Berne Cardiovascular Research Center, University of Virginia; Department of Pathology, University of Virginia;
Robert M. Berne Cardiovascular Research Center, University of Virginia; Department of Medicine, Division of Cardiovascular Medicine, University of Virginia.
J Vis Exp. 2020 Jul 30(161). doi: 10.3791/61585.
There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.
使用脂质体在体内递送化合物，尤其是用于靶向治疗方法，正受到越来越多的关注。根据脂质体制剂的不同，脂质体可能会被体内不同的细胞类型优先摄取。由于不同疾病的进展具有组织和细胞类型特异性，这可能会影响治疗颗粒的疗效。在本方案中，我们介绍了一种使用二硬脂酰磷脂酰胆碱（DSPC）、胆固醇和聚乙二醇-2000二硬脂酰基磷脂酰乙醇胺（PEG-2000 DSPE）以及脂质染料DiD作为荧光标记来合成和荧光标记脂质体的方法。本方案还介绍了一种在体内递送脂质体并使用流式细胞术评估脂质体细胞特异性摄取的方法。这种方法可用于确定摄取脂质体的细胞类型，并量化脂质体在不同细胞类型和组织中的摄取分布及比例。虽然本方案未提及，但其他检测方法，如免疫荧光和在细胞仪上进行单细胞荧光成像，将加强所得到的任何发现或结论，因为它们能够评估细胞内染色情况。方案可能还需要根据感兴趣的组织进行调整。
