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.

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Objective: Sensorineural hearing loss leads to the progressive degeneration of spiral ganglion cells (SGC). Next to postoperative fibrous tissue growth, which should be suppressed to assure a close nerve-electrode interaction, the density of healthy SGC is one factor that influences the efficiency of cochlear implants (CI), the choice of treatment for affected patients. Rolipram, a phosphodiesterase-4 inhibitor, has proven neuroprotective and anti-inflammatory effects and might also reduce SGC degeneration and fibrosis, but it has to pass the cellular membrane to be biologically active. Methods: Lipidic nanocapsules (LNC) can be used as biodegradable drug carriers to increase the efficacy of conventional application methods. We examined the biological effects of rolipram and LNC's core encapsulated rolipram on SGC and dendritic cell (DC) tumor necrosis factor-α (TNF-α) production in vitro and on SGC survival in systemically-deafened guinea pigs in vivo. Results: Our results prove that rolipram does not have a beneficial effect on cultured SGC. Incorporation of rolipram in LNC increased the survival of SGC significantly. In the DC study, rolipram significantly inhibited TNF-α in a dose-dependent manner. The rolipram-loaded LNC provided a significant cytokine inhibition as well. In vivo data do not confirm the in vitro results. Conclusion: By transporting rolipram into the SGC cytoplasm, LNC enabled the neuroprotective effect of rolipram in vitro, but not in vivo. This might be due to dilution of test substances by perilymph or an inadequate release of rolipram based on differing in vivo and in vitro conditions. Nevertheless, based on in vitro results, proving a significantly increased neuronal survival when using LNC-rolipram compared to pure rolipram and pure LNC application, we believe that the combination of rolipram and LNC can potentially reduce neuronal degeneration and fibrosis after CI implantation. We conclude that rolipram is a promising drug that can be used in inner ear therapy and that LNC have potential as an inner ear drug-delivery system. Further experiments with modified conditions might reveal in vivo biological effects.

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We propose a regenerative solution in the treatment of critical limb ischaemia (CLI). Poly-lactic/glycolic acid microcarriers were prepared and coated with laminin to be sterilized through γ-irradiation of 25 kGy at low temperature. Stromal vascular fraction (SVF) cells were extracted through enzymatic digestion of adipose tissue. Streptozotocin-induced diabetic mice underwent arteriotomy and received an administration of SVF cells combined or not with biomimetic microcarriers. Functional evaluation of the ischaemic limb was then reported, and tissue reperfusion was evaluated through fluorescence molecular tomography. Microcarriers were stable and functional after γ-irradiation until at least 12 months of storage. Mice that received an injection of SVF cells in the ischaemic limb have 22% of supplementary blood supply within this limb 7 days after surgery compared with vehicle, whereas no difference was observed at Day 14. With the combined therapy, the improvement of blood flow is significantly higher compared with vehicle, of about 31% at Day 7 and of about 11% at Day 14. Injection of SVF cells induces a significant 27% decrease of necrosis compared with vehicle. This effect is more important when SVF cells were mixed with biomimetic microcarriers: -37% compared with control. Although SVF cells injection leads to a non-significant 22% proprioception recovery, the combined therapy induces a significant recovery of about 27% compared with vehicle. We show that the combination of SVF cells from adipose tissue with laminin-coated poly-lactic/glycolic acid microcarriers is efficient for critical limb ischaemia therapy in a diabetic mouse model.

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