The use of fluorescent nanocrystals (NCs) as probes for bioimaging applications has emerged as an advantageous alternative to conventional organic fluorescent dyes. Therefore their toxicological evaluation and intracellular delivery is currently a primary field of research. In this work, hydrophobic and highly fluorescent CdSe@ZnS NCs were encapsulated into the lipid bilayer of liposomes by the micelle-to-vesicle transition (MVT) method. The obtained aqueous NC−Liposome suspensions preserved the spectroscopic characteristics of the native NCs. A systematic study of the in vitro toxicological effect on HeLa cells of these red emitting NC-Liposomes was then carried out and compared to that of empty liposomes. By using liposomes of different phospholipid composition, we evaluated the effect of the lipid carrier on the cytotoxicity towards HeLa cells. Surprisingly, cells proliferation and death study along with the MTT test on HeLa cells treated with NC-Liposomes have shown that the toxic effects of NCs, at concentrations up to 20 nM, are negligible compared to those of the lipid carrier, especially when this is constituted by cationic phospholipid DOTAP. In particular, obtained data suggest that DOTAP has a dose- and time-dependent toxic effect on HeLa cells. On the contrary, the addition of PEG to the liposomes does not alter significantly the viability of the cells. In addition, the ability of NC-Liposomes to penetrate the HeLa cells was assessed by fluorescence and confocal microscopy investigation. Captured images show that NC-Liposomes are internalized into cells through the endocytic pathway, enter early endosomes and reach lysosomes in 1 h. Interestingly, red emitting NCs co-localized with endosomes and were positioned at the limiting membrane of the organelles. The overall results suggest that the fluorescent system as a whole, NCs and its carrier, should be considered for the development of fully safe biological applications of CdSe@ZnS NCs, and provide essential indications to define the optimal experimental conditions to use the proposed system as optical probe for future in vivo experiments

中文翻译：

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嵌入脂质体中的发光 CdSe@ZnS 纳米晶体：HeLa 细胞的细胞毒性研究


使用荧光纳米晶体（NC）作为生物成像应用的探针已成为传统有机荧光染料的有利替代品。因此，它们的毒理学评估和细胞内递送是目前的主要研究领域。在这项工作中，通过胶束到囊泡转变（MVT）方法将疏水性和高荧光的CdSe@ZnS NCs封装到脂质体的脂质双层中。获得的水性NC-脂质体悬浮液保留了天然NC的光谱特征。然后对这些发红光的 NC 脂质体对 HeLa 细胞的体外毒理学作用进行了系统研究，并与空脂质体进行了比较。通过使用不同磷脂组成的脂质体，我们评估了脂质载体对HeLa细胞的细胞毒性的影响。令人惊讶的是，细胞增殖和死亡研究以及用 NC 脂质体处理的 HeLa 细胞的 MTT 测试表明，与脂质载体相比，浓度高达 20 nM 的 NC 的毒性作用可以忽略不计，特别是当这是由阳离子磷脂DOTAP构成。特别是，获得的数据表明 DOTAP 对 HeLa 细胞具有剂量和时间依赖性毒性作用。相反，向脂质体中添加 PEG 不会显着改变细胞的活力。此外，还通过荧光和共聚焦显微镜研究评估了 NC 脂质体穿透 HeLa 细胞的能力。捕获的图像显示，NC-脂质体通过内吞途径内化到细胞中，进入早期内体并在 1 小时内到达溶酶体。有趣的是，发红光的 NC 与内体共定位，并位于细胞器的限制膜上。总体结果表明，荧光系统作为一个整体，NCs及其载体，应该被考虑用于CdSe@ZnS NCs完全安全的生物应用的开发，并提供必要的指示来定义使用所提出的系统的最佳实验条件用于未来体内实验的光学探针