Micro et Nanomédecines Biomimétiques-MINT, INSERM U 1066, Angers F-49933, France.
J Control Release. 2013 Sep 28;170(3):334-42. doi: 10.1016/j.jconrel.2013.06.014. Epub 2013 Jun 19.
Numerous studies on nanocarriers use fluorescent dye labeling to investigate their biodistribution or cellular trafficking. However, when the fluorescence dye is not grafted to the nanocarrier, the question of the stability of the labeling arises. How can it be validated that the fluorescence observed during an experiment corresponds to the nanocarriers, and not to the free dye released from the nanocarriers? Studying the integrity of the labeling is challenging. Therefore, an innovative approach to confirm the labeling stability was developed, based on the transfer of a fluorescent dye from its hosting nanocarrier to a lipophilic compartment. Lipid nanocapsules (LNC) and triglyceride oil were used as models. The protocol involved mixing of LNC suspension and oil, and then separation by centrifugation. The quality of the separation was controlled by light scattering, using the derived count rate tool. Dye transfer from loaded LNCs to the lipophilic compartment or from a lipophilic compartment containing dye to non-loaded LNC was investigated by varying the nature of the dye and the oil, the oil volume and the LNC dilution. Tensiometry was used to define the dye location in the nanocarrier. Results showed that when dyes such as Nile Red and Coumarin-6 are located in oily core, the transfer occurred in a partition-dependent manner. In contrast, when the dye was entrapped in the surfactant shell of LNCs such as lipophilic indocarbocyanines (i.e. DiO, DiI and DiD), no transfer was observed. Dye diffusion was also observed in cell culture, with Nile Red inside lipid bodies of HEI-OC1 cells, without uptake of LNCs. In contrast, DiO-loaded LNCs had to be internalized to observe fluorescence inside the cells, providing a further confirmation of the absence of transfer in this case, and the stability of fluorescence labeling of the LNCs.
许多关于纳米载体的研究都使用荧光染料标记来研究它们的生物分布或细胞内转运。然而，当荧光染料未嫁接到纳米载体上时，就会出现标记稳定性的问题。如何验证在实验中观察到的荧光与纳米载体有关，而不是与从纳米载体中释放的游离染料有关？研究标记的完整性具有挑战性。因此，基于荧光染料从其宿主纳米载体转移到亲脂性隔室的方法，开发了一种确认标记稳定性的创新方法。脂质纳米胶囊（LNC）和甘油三酯油被用作模型。该方案涉及 LNC 悬浮液和油的混合，然后通过离心分离。通过使用衍生的计数率工具进行光散射来控制分离的质量。通过改变染料和油的性质、油的体积和 LNC 的稀释度，研究了负载 LNC 中的染料向亲脂性隔室或含有染料的亲脂性隔室向未负载 LNC 的染料转移。张力计用于定义纳米载体中染料的位置。结果表明，当 Nile Red 和 Coumarin-6 等染料位于油性核心时，转移以分区依赖的方式发生。相比之下，当染料被包封在 LNC 的表面活性剂壳中，如亲脂性吲哚氰（即 DiO、DiI 和 DiD）时，观察不到转移。在细胞培养中也观察到了染料扩散，Nile Red 在内质体中，而没有 LNC 的摄取。相比之下，只有当 DiO 负载的 LNC 被内化时，才能观察到细胞内的荧光，这进一步证实了在这种情况下没有转移，并且 LNC 的荧光标记稳定性。
