借助活细胞代谢及解毒途径的微囊泡原位荧光标记新策略及应用

Fluorescence labeling technology of cell-derived microvesicles (MVs) is of important significance on in vivo tracking of MVs’ biological behaviors and functions. The fluo-rescence-based labeling for imaging is the main visualization technique for MVs at present, relying on chemical conjugation or encapsulation of fluorescent dyes/nanomaterials, which exposed obviously laborious features and low efficiencies. Moreover, those exogenous labeling materials loaded by cells could influence the formation and natural properties of MVs, hindering the study of MVs’ original beha-viors and functions. Considering the limitations of current labeling approaches, it is an urgent need to develop a moderate, reliable and biofriendly strategy for more efficient MVs labeling. Herein, we present an efficient, biofriendly and automated strategy for designer self-cell-implemented labeling of microvesicles in situ with the intracellular-synthesized quantum dots (QDs). By elaborately spatial-temporal coupling intracellular metabolism and detoxification pathways, the living mammalian cells could be successfully transformed into fluorescent cells based on intracellular biosyn-thesized QDs. Therefore MVs could be straightforwardly labeled by loading of intra-cellular biosynthesized QDs and automatically secreted outward by direct budding of plasma membrane. The whole labeling process is convenient and straightforward, which needs just one step without any additional sophisticated operations. Further-more, taking advantages of the natural drug loading capability and targeting properties of MVs, the fluorescent MVs can be engineered to load therapeutic drug for simultaneous in vivo bioimaging and tumor-targeted therapy. The project herein via coupling biochemical reaction and cell inherent properties would provide a new insight to the observation and research of MVs’ biological behaviors and functions, as well as to the use of live organisms for a wide spectrum of bioapplication.

细胞源性微囊泡的荧光标记技术对于研究其在细胞之间作为信息传输载体的生物学行为及功能具有重要意义。目前，其可视化标记技术主要包括化学偶联和直接包覆染料或纳米材料法，存在着费时费力﹑标记效率低、生物功能难以保持等科学问题。本项目旨在以发展温和、高效、生物友好的标记技术为研究目的，创新性地耦合哺乳动物细胞的代谢及解毒途径，建立一种微囊泡的新型自动化的原位荧光自主标记技术。通过按照设计意图耦合胞内不同途径实现荧光量子点的生物合成，进而利用微囊泡出芽形成机制包裹胞内量子点，实现对微囊泡的荧光标记。整个标记过程无需对微囊泡及供体细胞进行任何化学修饰或基因操控。在此基础上，利用微囊泡的天然靶向传输性，探索其负载药物后在肿瘤成像与治疗方面的应用。本项目的研究工作将为借助活细胞生命过程的生物标记及其相关应用提供新方法和新思考。

细胞源性微囊泡几乎可以从所有种类的哺乳动物细胞分泌到细胞外空间，并在细胞间通讯和细胞间生物分子的运输中起关键作用。然而，由于现有标记方法的局限性，微囊泡生物行为的可视化和监测面临着巨大的挑战。本项目创新性地耦合哺乳动物细胞的代谢、解毒途径以及细胞出芽形成微囊泡的机制，建立了一种微囊泡新型自动化的原位荧光自主标记技术。通过精心设计，在时间和空间上耦合细胞的代谢和解毒途径以及细胞出芽产生微囊泡的过程，使得细胞按照设计思路完成胞内荧光量子点合成并直接原位标记细胞源性微囊泡，实现了量子点活细胞合成与原位标记的融合。通过向人乳腺癌细胞MCF-7的培养基中适时适量依次加入Na2SeO3和CdCl2，MCF-7细胞摄取外加的高价态无机硒化合物，通过胞内硒的还原代谢途径将高价态无机硒化合物还原为低价态的有机硒，可作为CdSe量子点合成的硒前体；同时，外加的镉离子激活细胞解毒途径，通过解毒过程生成的镉配合物，可作为CdSe合成的镉前体。当镉配合物在预订时间和细胞质中低价的有机硒化合物相遇反应，在胞内即可合成出发光CdSe量子点，从而点亮细胞发荧光。该方法可以让高达97.4%的细胞转化为发荧光的细胞，进而利用细胞出芽形成其微囊泡的机制，高效温和地实现了量子点对细胞源性微囊泡的标记，其标记效率高达89.9%。这种标记新策略具有普适性，已扩展至MDA-MB-231及MDCK细胞，其荧光细胞转化率分别为86.7%和95.2%，微囊泡的荧光量子点标记效率分别达到83.6%和94.8%，方法温和高效。整个标记过程无需对微囊泡及供体细胞进行任何化学修饰或基因操作，仅巧妙地利用和调控胞内错综复杂而不能自然交会的生化反应途径，如耦合胞内代谢、还原、解毒途径以及微囊泡形成过程，即可直接收集到标记好的荧光微囊泡。