Table 1.
Composition and characterization of non-loaded liposomal formulations.
Figure 1.
Characterization of liposomal formulations.
(A) Representative Cryo-TEM image of DLPC/Chol/Cholesteryl/PEG600-Chol (5∶3.5∶1∶0.5) liposomes extruded through a 200 nm pore size membrane. (B) Confocal fluorescence image of a single liposome tagged on its lipid bilayer with Marina Blue-DHPE (blue) and its corresponding fluorescence intensity profile. (C) Confocal fluorescence image of a single Marina Blue-labeled liposome containing AlexaFluor594-labeled LPS (red) and their corresponding fluorescence intensity profiles. (D) Confocal fluorescence image of a single Marina Blue-labeled liposome containing fluorescein-labeled poly (I:C) and their corresponding fluorescence intensity profiles. (E) Schematic representation of the liposomal IS-cocktail (NLc) showing the presence of both encapsulated LPS (red) and poly (I:C) (green) in the lipidic bilayer of liposomes. (F) Confocal fluorescence image of a single liposome containing both fluorescein-labeled poly (I:C) (green) and AlexaFluor594-labeled LPS (red) and their corresponding fluorescence intensity profiles.
Table 2.
Efficiencies for the encapsulation of LPS and poly (I:C).
Figure 2.
Cytotoxicity of NL2, LPS, NL2, poly (I:C), and NLc formulations in ZFL cells by MTT-based assay.
(A) Viability of ZFL after 24 h incubation with liposome-encapsulated LPS (NL2, LPS, green bars) at Dose 1 = 1 mg/ml liposome with 50 µg/ml LPS, Dose 2 = 0.5 mg/ml liposome with 25 µg/ml LPS and Dose 3 = 0.20 mg/ml liposome with 10 µg/ml LPS. The white bar is the empty liposome control (NL2,n, 1 mg/ml liposome) and the blue bar is the free LPS control (50 µg/ml). (B) Viability of ZFL after 24 h incubation the liposome-encapsulated poly (I:C) (NL2, poly (I:C), green bars) at Dose 1 = 1.5 mg/ml liposome with 50 µg/ml poly (I:C), Dose 2 = 0.75 mg/ml liposome with 25 µg/ml poly (I:C) and Dose 3 = 0.375 mg/ml liposome with 10 µg/ml poly (I:C). The white bar is the empty liposome control treatment (NL2,n, 1.5 mg/ml liposome) and the red bar is the non-encapsulated poly (I:C) control (50 µg/ml). (C) Viability of ZFL cells after 24 h incubation with liposomal LPS-poly (I:C) cocktail (NLc, green bars) at Dose 1 = 1.5 mg/ml liposome with 50 µg/ml poly (I:C) and 25 µg/ml LPS, Dose 2 = 0.75 mg/ml liposome with 25 µg/ml poly (I:C) and 12.5 µg/ml LPS and Dose 3 = 0.375 mg/ml liposome with 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS. The white bar is the empty liposome control treatment (NL2,n, 1.5 mg/ml liposome), the blue bar indicates the free LPS (25 µg/ml) and the red bar is the free (I:C) control (50 µg/ml). Non-treated cells were used as 100% viability control (dotted line). Data represent means ± SD of three independent experiments. Differences were analyzed using One-way ANOVA followed by Tukey's post test. **, p<0.01; ***, p<0.001.
Figure 3.
Endocytosis of NLc formulation by ZFL cells.
(A) Flow cytometry time-course comparison of the membrane-bound (dark grey bar) versus the endocyted liposomes (light grey bar) after incubation with NLc (750 µg/ml liposome, 25 µg/ml poly (I:C) and 12.5 µg/ml LPS) at the indicated times. Data represent means ± SD of three independent experiments. (B) Effect of chemical inhibitors on the endocytosis of the NLc (750 µg/ml liposome, 25 µg/ml poly (I:C) and 12.5 µg/ml LPS). Inhibitors were used at the following concentrations: MβCD at 5 mM, EIPA at 50 µM, sucrose at 300 mM and W at 100 nM. The uptake of cells without inhibitors (NLc bar) was used as 100% uptake control and non-treated cells were used as control (control bar). Data represent means ± SD of three independent experiments. Differences were analyzed using One-way ANOVA followed by Tukey's post test. *, p<0.05; **, p<0.01; ***, p<0.001. (C) Confocal microscopy images of fluorescent liposomes (NLc) endocyted by ZFL cells. Cells were incubated for 30 min, 1.5 h and 16 h with NLc containing DHPE-Fluorescein (green) at a 0.05 molar ratio. Cell membranes were stained with CellMask (red) and the nucleus was stained with Hoechst (blue).
Figure 4.
Endocytosis of NLc formulation by trout macrophages.
(A) Flow cytometry time-course comparison of the membrane-bound (dark grey bar) versus the endocyted liposomes (light grey bar) after incubation with 750 µg/ml liposome-encapsulated 25 µg/ml poly (I:C) and 12.5 µg/ml LPS at the indicated times. Data represent means ± SD of three independent experiments. (B) Effect of chemical inhibitors on the endocytosis of NLc (750 µg/ml liposome-encapsulated 25 µg/ml poly (I:C) and 12.5 µg/ml LPS) macrophages uptake. Inhibitors were used at the following concentrations: MβCD at 5 mM, EIPA at 50 µM, sucrose at 150 mM and W at 100 nM. The uptake of cells not treated with inhibitors (NLc bar) was used as 100% uptake control and non-treated cells were used as control (control bar). Data represent means ± SD of 3 independent experiments. Differences were analyzed using One-way ANOVA followed by Newman-Keuls post-test. *, p<0.05; **, p<0.01. (C) Confocal microscopy images of fluorescent liposomes (NLc) endocyted by macrophages. Cells incubated 30 min, 1 h and 16 h with NLc containing DHPE-Fluorescein (green) at a 0.05 molar ratio. Cell membranes were stained with CellMask (red) and nucleus with Hoechst (blue).
Figure 5.
Analysis of gene expression in ZFL cell culture (A) and trout macrophage primary cell culture (B) after 16 h exposure to liposomes.
NL2,n = liposomes without immunostimulants (750 µg/ml), NLc Dose 1 = liposomes (750 µg/ml) containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS, NLc Dose 2 = liposomes (375 µg/ml) containing 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS, and LPS+poly (I:C) = stimulation control (25 µg/ml poly (I:C), 12.5 µg/ml LPS). Elongation factor (EF1) was used as reference gene for ZFL cells and 18S for trout macrophages. IFN (φ for ZFL and α for macrophages), GIG2, CCL4, IL-6 and TNFα abundance was analyzed by Q-PCR (left panel) and conventional PCR (right panel). Data represent means ± SD of 3 independent experiments. Values with asterisk are statistically significant relative to the control (*, p<0.05; **, p<0.01; ***, p<0.001) and values with letters (a,b) are statistically significant relative to NLc Dose 1 (a, p<0.001, b, p<0.05). Differences were analyzed using One-way ANOVA and Tukey's post test. (C) TNFα secretion from trout macrophages stimulated with liposomes for 16 h was assessed by Western blot. NLc Dose 2 = 375 µg/ml liposomes, 12.5 µg/ml poly (I:C), 6.25 µg/ml LPS, NL2,n = empty liposomes (375 µg/ml) and LPS = stimulation control (6.25 µg/ml). A representative Western Blot is shown.
Figure 6.
In vivo NLc formulation toxicities.
Survival of zebrafish embryos was recorded every 24(hpf) (A) and 72 h post-hatching (hph) (B) after exposure to four concentrations of liposomal IS cocktail (red, NLc Dose 1 = 750 µg/ml liposomes, 25 µg/ml poly (I:C) and 12.5 µg/ml LPS; NLc Dose 2 = 1.5 mg/ml liposomes, 50 µg/ml poly (I:C) and 25 µg/ml LPS; NLc Dose 3 = 3 mg/ml liposomes, 100 µg/ml poly (I:C) and 50 µg/ml LPS; NLc Dose 4 = 6 mg/ml liposomes, 200 µg/ml poly (I:C) and 100 µg/ml LPS). Liposomes without encapsulated immunostimulants (grey, NL2,n Dose 2 = 1.5 mg/ml, NL2,n Dose 4 = 6 mg/ml) and non-treated embryos (blue) were used as controls. Non-encapsulated LPS (black, 25 µg/ml and 100 µg/ml) was used as mortality control. Differences were analyzed using log rank test. *, p<0.05; **, p<0.01; ***, p<0.001.