Kozyreva Zhanna V, Demina Polina A, Sapach Anastasiia Yu, Terentyeva Daria A, Gusliakova Olga I, Abramova Anna M, Goryacheva Irina Yu, Trushina Daria B, Sukhorukov Gleb B, Sindeeva Olga A
Vladimir Zelman Center for Neurobiology and Brain Rehabilitation, Skolkovo Institute of Science and Technology, 30 b.1 Bolshoy Boulevard, 121205, Moscow, Russia.
Science Medical Center, Saratov State University, 83 Astrakhanskaya Str., 410012, Saratov, Russia.
Heliyon. 2024 May 5;10(10):e30680. doi: 10.1016/j.heliyon.2024.e30680. eCollection 2024 May 30.
Tracing individual cell pathways among the whole population is crucial for understanding their behavior, cell communication, migration dynamics, and fate. Optical labeling is one approach for tracing individual cells, but it typically requires genetic modification to induce the generation of photoconvertible proteins. Nevertheless, this approach has limitations and is not applicable to certain cell types. For instance, genetic modification often leads to the death of macrophages. This study aims to develop an alternative method for labeling macrophages by utilizing photoconvertible micron-sized capsules capable of easy internalization and prolonged retention within cells. Thermal treatment in a polyvinyl alcohol gel medium is employed for the scalable synthesis of capsules with a wide range of fluorescent dyes, including rhodamine 6G, pyronin B, fluorescein, acridine yellow, acridine orange, thiazine red, and previously reported rhodamine B. The fluorescence brightness, photostability, and photoconversion ability of the capsules are evaluated using confocal laser scanning microscopy. Viability, uptake, mobility, and photoconversion studies are conducted on RAW 264.7 and bone marrow-derived macrophages, serving as model cell lines. The production yield of the capsules is increased due to the use of polyvinyl alcohol gel, eliminating the need for conventional filtration steps. Capsules entrapping rhodamine B and rhodamine 6G meet all requirements for intracellular use in individual cell tracking. Mass spectrometry analysis reveals a sequence of deethylation steps that result in blue shifts in the dye spectra upon irradiation. Cellular studies on macrophages demonstrate robust uptake of the capsules. The capsules exhibit minimal cytotoxicity and have a negligible impact on cell motility. The successful photoconversion of RhB-containing capsules within cells highlights their potential as alternatives to photoconvertible proteins for individual cell labeling, with promising applications in personalized medicine.
在整个细胞群体中追踪单个细胞的路径对于理解其行为、细胞通讯、迁移动态和命运至关重要。光学标记是追踪单个细胞的一种方法,但通常需要进行基因改造以诱导光可转换蛋白的产生。然而,这种方法存在局限性,并不适用于某些细胞类型。例如,基因改造常常导致巨噬细胞死亡。本研究旨在开发一种替代方法,通过利用能够轻松内化并在细胞内长期保留的光可转换微米级胶囊来标记巨噬细胞。在聚乙烯醇凝胶介质中进行热处理,用于可扩展地合成包含多种荧光染料的胶囊,这些染料包括罗丹明6G、派洛宁B、荧光素、吖啶黄、吖啶橙、噻嗪红以及先前报道的罗丹明B。使用共聚焦激光扫描显微镜评估胶囊的荧光亮度、光稳定性和光转换能力。以RAW 264.7和骨髓来源的巨噬细胞作为模型细胞系,进行活力、摄取、迁移和光转换研究。由于使用了聚乙烯醇凝胶,胶囊的产量得以提高,无需传统的过滤步骤。包裹罗丹明B和罗丹明6G的胶囊满足单个细胞追踪中细胞内使用的所有要求。质谱分析揭示了一系列脱乙基步骤,这些步骤导致染料光谱在照射后发生蓝移。对巨噬细胞的细胞研究表明胶囊具有强大的摄取能力。这些胶囊表现出最小的细胞毒性,对细胞运动的影响可忽略不计。细胞内含有RhB的胶囊成功进行光转换,突出了它们作为光可转换蛋白用于单个细胞标记的替代品的潜力,在个性化医学中具有广阔的应用前景。
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