Confocal analysis of LNC600-WS12 uptake in zebrafish. LNC600-WS12 is labeled with DiI (red), and the results are shown for experiments performed in which 1 µM (A), 100 nM (B) or 10 nM (C) was used to treat zebrafish embryos for 6 days before the embryos were fixed. Confocal analysis showing the internalization of DiI-labeled LNC600-WS12 in the zebrafish. Zooms show the presence of LNC600-WS12 into the yolk sacs of zebrafish after treatment with 1µM and 100nM of LNC600-WS12.

Plasma membrane penetration and dynamic localization of lipid nanocapsules. (A) Electron micrographs of PC3-M8 cells treated with 100 nM of empty LNC600 (upper panels) or 100 nM of LNC600-WS12 (lower panels) for different times (5 s, 30 s, 5 min and 30 min). Lipid Nanoparticles appeared as electron-dense spots located at the plasma membrane and are indicated by an arrow. (B) Bar graphs show the quantification of the data for empty LNC600 (Bi) or LNC600-WS12 (Bii) as µm of mobile or rigid plasma membrane at different timepoints after treatment with 100 nM of empty LNC600 or LNC600-WS12. (C) Bar plot showing the quantification of empty LNC600 or LNC600-WS12 in the rigid (Ci) and mobile (Cii) fractions of plasma membranes obtained from PC3-M8 cells at different timepoints after treatment with 100 nM of empty LNC600 or LNC600-WS12. *P < 0.05 (ANOVA, Tukey’s multiple comparisons test).

Confocal visualization of DiI/WS12-containing lipid nanocapsules (LNC600-WS12-DiI) and concurrent LNC-induced [Ca^2±]_i responses in PC3 cell expressing stably TRPM8. (A) Plot (right) compares the dynamics of relative changes in Fluo-4 fluorescence reporting WS12-induced changes in intracellular Ca²⁺ concentration, [Ca²⁺]_i (ΔF/F0, green trace), DiI fluorescence reporting re-distribution of LNC content within the cell (ΔF/F0, red trace) and DiI fluorescence reporting aggregation of LNCs on the cell surface (ΔR/Ro, violet trace). The traces show mean ± S.E.M. signals from 4 cells (image, left). (B) Plots of Fluo-4 (green traces) and intracellular DiI (red traces) responses in 4 cells (numbered in A, left) during the period of interest (POI, brown bar in A, right) highlight the delay (Δt1) between internalization of LNC content and elevation of [Ca²⁺]_i, respectively. (C) The same as B but the delay (Δt2) between the elevation of [Ca²⁺]_i (green traces) and aggregation of LNCs on the cell surface (violet traces) is highlighted. The galleries below the plots show every 8^th (B) and 100^th (C) image captured during the periods corresponding to the plots. (D) Plot displays mean ± S.E.M. values of Δt1 and Δt2 measured in 40 cells upon 17 independent experiments. Note that the LNC is internalized before [Ca²⁺]_i response initiation, while aggregation of LNCs on the cell surface became prominent >3 min later. (E,F) Visualization of the 3-dimensional (3-D) distributions of DiI and Fluo-4 fluorescence 20 min after stimulation of the cell with LNCs. The results are presented by galleries showing (E) every 2^nd x-y image obtained during Z-sectioning protocol (Z-stack) and (F) rotations of the reconstructed 3-D image around Y axis with 30° step (3-D rotations). Note LNC aggregates (red) on the cell surface.

In vitro and in vivo effects of lipid nanocapsules on PC3-M8 cell viability and migration. (A) Cell viability assays performed with different TRPM8 agonists (WS12, empty LNC600, LNC600-WS12 or icilin) in PC3 and PC3-M8 cells and evaluated after 24 h (Ai) or 72 h (Aii) of incubation. MTS reagent was used to determine cell viability percentages (mean ± SEM; normalized to the control (CTRL) condition). (B) Transwell migration assays showing the effects of TRPM8 agonists on PC3 (Bi) or PC3-M8 (Bii) cells. The results are represented as a percentage of migrated cells normalized to the results observed under the CTRL condition (N = 9, mean ± SEM; *P < 0.05; **P < 0.01, ANOVA, Tukey’s multiple comparisons test for control and pairwise t-test comparison against 10 nM WS12 encapsulated or not condition, ^##P < 0.01). (C) In vivo migration assays performed in zebrafish embryos. (Ci) Graph bar representing PC3 and PC3-M8 cell migration along the zebrafish tail following the application of 100 nM of empty LNC600, 100 nM of free WS12 or 100 nM of LNC600-WS12. For each condition, PC3 and PC3-M8 cells were counted and the results normalized to the total number of migrating cells (N = 15, mean ± SEM; *P < 0.05, ANOVA, Tukey’s multiple comparisons test). (Cii) Representative confocal images of Tg[Fli1-eGFP] zebrafish embryos (vasculature shown in green) injected with DiD-labeled PC3 (yellow) and DiI-labeled PC3-M8 (red) cells. Cell migration was quantified in two segments of the ventral part of the fish (1^st and 2^nd segments).