Bouchaala Redouane, Richert Ludovic, Anton Nicolas, Vandamme Thierry F, Djabi Smail, Mély Yves, Klymchenko Andrey S
CNRS UMR 7021, Laboratoire de Bioimagerie et Pathologies, University of Strasbourg, 67401 Illkirch Cedex, France.
Laboratory of Photonic Systems and Nonlinear Optics, Institute of Optics and Fine Mechanics, University of Setif 1, 19000 Setif, Algeria.
ACS Omega. 2018 Oct 31;3(10):14333-14340. doi: 10.1021/acsomega.8b01488. Epub 2018 Oct 29.
Understanding the release of drugs and contrast agents from nanocarriers is fundamental in the development of new effective nanomedicines. However, the commonly used method based on dialysis frequently fails to quantify the release of molecules poorly soluble in water, and it is not well-suited for in situ measurements in biological media. Here, we have developed a new methodology for quantifying the release of fluorescent molecules from lipid nanocarriers (LNCs) using fluorescence correlation spectroscopy (FCS). LNCs based on nanoemulsion droplets, encapsulating the hydrophobic Nile red derivative NR668 as a model cargo, were used. Our studies revealed that the standard deviation of fluorescence fluctuations in FCS measurements depends linearly on the dye loading in the nanocarriers, and it is insensitive to the presence of less-bright molecular emissive species in solution. In sharp contrast, classical FCS parameters, such as the number and the brightness of emissive species, are strongly influenced by the fluorescence of molecular species in solution. Therefore, we propose to use the standard deviation of fluorescence fluctuations for the quantitative analysis of dye release from nanocarriers, which is unaffected by the "parasite" fluorescence of the released dyes or the auto-fluorescence of the medium. Using this method, we found that LNCs remain intact in water, whereas in serum medium, they release their content in a temperature-dependent manner. At 37 °C, the release was relatively slow reaching 50% only after 6 h of incubation. The results are corroborated by qualitative observations based on Förster resonance energy transfer between two different encapsulated dyes. The developed method is simple because it is only based on the standard deviation of fluorescence fluctuations and, in principle, can be applied to nanocarriers of different types.
了解纳米载体中药物和造影剂的释放情况是开发新型有效纳米药物的基础。然而，基于透析的常用方法常常无法对水溶性差的分子释放进行定量，且不太适合在生物介质中进行原位测量。在此，我们开发了一种新方法，利用荧光相关光谱法（FCS）对脂质纳米载体（LNCs）中荧光分子的释放进行定量。使用了基于纳米乳液滴的LNCs，其包裹了疏水性尼罗红衍生物NR668作为模型载药。我们的研究表明，FCS测量中荧光波动的标准偏差与纳米载体中的染料负载量呈线性关系，并且对溶液中亮度较低的分子发射物种的存在不敏感。与之形成鲜明对比的是，经典的FCS参数，如发射物种的数量和亮度，会受到溶液中分子物种荧光的强烈影响。因此，我们建议使用荧光波动的标准偏差对纳米载体中染料的释放进行定量分析，该方法不受释放染料的“寄生”荧光或介质自发荧光的影响。使用这种方法，我们发现LNCs在水中保持完整，而在血清介质中，它们以温度依赖的方式释放其内容物。在37℃时，释放相对缓慢，仅在孵育6小时后达到50%。基于两种不同包裹染料之间的Förster共振能量转移的定性观察证实了这些结果。所开发的方法很简单，因为它仅基于荧光波动的标准偏差，并且原则上可应用于不同类型的纳米载体。
