Quantifying Release from Lipid Nanocarriers by Fluorescence Correlation Spectroscopy.

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.