A new tool to ensure the fluorescent dye labeling stability of nanocarriers: A real challenge for fluorescence imaging

Abstract

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.

Labeling nanoparticles: Dye leakage and altered cellular uptake

“…We have previously shown that Nile Red (NR) leaks out of poly(butyl cyanoacrylate) (PBCA)-NPs upon cell contact (24), which is in line with various studies showing NR release from NPs (18,19,25,26). The hydrophobic analogue NR668 was found to leak much less than NR from nanoemulsions (NEs) (18), and to be suitable for tracing the PBCA-NPs, as it was not released from the NPs until they were endocytosed and degraded (12).…”

“…NR has repeatedly been shown to leak out of various NPs (18,19,25,26), and we concluded in a previous study that NR was taken up by cells through contact-mediated transfer within minutes after addition of PBCA-NPs (24). The retention of a dye in a NP is largely governed by hydrophobic and electrostatic interaction between the dye and NP.…”

“…This emphasizes the importance of choosing a fluorescent dye that is compatible with the NP-platform into which it is incorporated. Various procedures have been developed to evaluate NP-labeling stability (18,20,23,(25)(26)(27)(28)(29). However, the majority of these assays do not include cells or serum, which could strongly affect dye release as these serve as acceptor compartments for released dye in vivo (18,24,(29)(30)(31)(32)(33).…”

Impact of interfacial cholesterol-anchored polyethylene glycol on sterol-rich non-phospholipid liposomes

“…The LUV size and zeta potential were not affected by the presence of DiD, confirming that the fluorophore, at that concentration, did not affect the assembly. 34 A LUV aliquot of 400 µL was intravenously delivered (caudal veins) to each rat. At various time points, from 5 min to 24 h following the injection, blood samples (about 400 µL) were collected.…”

“…The pharmaco-kinetics was examined using DiD, a fluorescent lipophilic probe that labels the LUV walls and has, in general, limited transfer from labeled to unlabeled membranes. 34 AV-400 spectrometer operating at 400 MHz for 1 H. The diffusion measurements were carried out at 25 o C using a Bruker diffusion probe (Diff60) equipped with a 1 H/ 2 H 5-mm coil. This probe includes one gradient coil, along the z axis, capable of delivering magnetic field gradients up to 2900 G/cm.…”