File S1 - A Novel Liposome-Based Nanocarrier Loaded with an LPS-dsRNA Cocktail for Fish Innate Immune System Stimulation

Supporting information Table and Figures S1–S7. Table S1. Rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) specific primers for PCR and Q-PCR. Figure S1. Evaluation of toxicity of cationic liposomes without encapsulated immunostimulants (NL1,n and NL2,n). Viability of ZFL cell line was assessed with the MTT assay (A) or LDH assay (B) after a dose response (0.1 µg/ml-10 mg/ml) with the two liposomal formulations (NL1,n and NL2,n). Viability of HepG2 cell line was determined with the MTT assay (C) and with the LDH assay (D) after a dose response (0.1 µg/ml-10 mg/ml) with the two liposomal formulations (NL1,n and NL2,n). Non-treated cells were used as 100% viability control (dotted line) in the MTT assays and non-treated cells were used as control of the basal death (dotted line) in the LDH assays. 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. Figure S2. Cytotoxicity of NLc formulation in ZFL cells by LDH assay. (A) Viability of ZFL after 24 h incubation with the 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 control treatment with liposomes without encapsulated immunostimulants (NL2,n, 1 mg/ml liposome) and the blue bar is the non-encapsulated LPS control (50 µg/ml). (B) Viability of ZFL after 24 h incubation with 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 control treatment with empty liposomes (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 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 control treatment with empty liposomes (NL2,n, 1.5 mg/ml liposome), the blue bar is the non-encapsulated LPS (25 µg/ml) and the red bar represents the non-encapsulated poly (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<0.05; ***, p<0.001. Figure S3. In vitro cytotoxicity of NLc formulation in trout macrophages. (A) The cytotoxicity of NLc was assessed by the LDH assay. Viability of the trout macrophage primary cell culture after 24 h incubation with NLc encapsulating both poly (I:C) and LPS (green bars) at Dose 1 = 0.75 mg/ml liposome with 25 µg/ml poly (I:C) and 12.5 µg/ml LPS and Dose 2 = 0.375 mg/ml liposome with 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS. The white bar is the control treatment with non-encapsulating liposomes (NL2,n, 0.75 mg/ml liposome) and the grey bar is the non-encapsulated poly (I:C) and LPS control (25 µg/ml and 12.5 µg/ml, respectively). Basal dead cells of the non-treated cells were used as control (dotted line). Data represent means ± SD of 3 independent experiments. Differences were analyzed using One-way ANOVA followed by Tukey's post test **, p<0.01. Figure S4. In vitro cytotoxicity of endocytosis inhibitors. (A) Viability of ZFL cells after 1 h exposure (16 h in the case of the chloroquine) to different endocytosis inhibitors, assessed by the MTT assay. (B) Viability of trout macrophages after 1 h exposure to different endocytosis inhibitors, assessed by the MTT assay. Non-treated cells were used as a 100% viability control (Control bar). Figure S5. Time-course of NLc uptake in vitro. (A) Flow cytometry time course of NLc uptake (grey bars, liposomes at 750 µg/ml containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS) by ZFL cells. To study the metabolization of NLc, ZFL cells were also pretreated for 1 h with chloroquine at 100 µM (red bars). Then, liposomes were added (750 µg/ml liposome containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS), and left to incubate in the constant presence of chloroquine. (B) Flow cytometry time course of NLc uptake (grey bars, liposomes at 750 µg/ml containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS) by trout macrophages. Cells not exposed to NLc were used as controls (white bars). Data represent means ± SD of triplicates of three independent experiments. Figure S6. In vivo NLc toxicity assay controls. Survival of zebrafish embryos was recorded every 24 h at 120 h post fertilization (hpf) (A) and 72 h post hatching (hph) (B) after exposure to non-encapsulated LPS (black, 25 µg/ml and 100 µg/ml), non-encapsulated poly (I:C) (green, 50 µg/ml) and non-encapsulated LPS (25 µg/ml) and poly (I:C) (50 µg/ml) in combination (orange). Non-treated embryos (blue) were used as controls. Survival curves were analyzed using the log rank test (n = 24 individual). Figure S7. Analysis of gene expression in zebrafish larvae after time-course exposure to liposome preparation. NL2,n = liposomes without encapsulated immunostimulants (1.5 mg/ml), NLc = liposomes (1.5 mg/ml) with 50 µg/ml poly (I:C) and 25 µg/ml LPS and LPS+poly (I:C) = stimulation control (50 µg/ml poly (I:C), 25 µg/ml LPS). Non-treated embryos were used as control (Ctrl). Elongation factor (EF1) was the reference gene and TLR3, GIG2, TNFα and iNOS mRNA abundance was analyzed by conventional PCR (right panel). Representative images of three independent experiments are shown.
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辅助信息：表S1及图S1~S7。
表S1：用于聚合酶链式反应（PCR, Polymerase Chain Reaction）与定量聚合酶链式反应（Q-PCR, Quantitative PCR）的虹鳟（*Oncorhynchus mykiss*）及斑马鱼（*Danio rerio*）特异性引物。
图S1：未包被免疫刺激剂的阳离子脂质体（NL1,n与NL2,n）的毒性评价。针对两种脂质体制剂（NL1,n与NL2,n）设置浓度梯度（0.1 μg/ml~10 mg/ml），分别采用MTT法（MTT assay）与乳酸脱氢酶释放法（LDH assay）检测ZFL细胞系的存活率；针对HepG2细胞系，采用MTT法（对应面板C）与LDH法（对应面板D）检测其存活率。MTT实验中以未处理细胞作为100%存活率对照（虚线），LDH实验中以未处理细胞作为基础死亡对照（虚线）。数据以三次独立实验的平均值±标准差（SD）表示，差异分析采用单因素方差分析（One-way ANOVA）结合Tukey事后检验（Tukey's post test），标记：*，p<0.05；**，p<0.01；***，p<0.001。
图S2：NLc制剂在ZFL细胞中的细胞毒性（LDH法检测）。
(A) ZFL细胞与脂质体包被脂多糖（LPS, Lipopolysaccharide，NL2,LPS，绿色柱）孵育24 h后的存活率，设置三个剂量组：剂量1=1 mg/ml脂质体+50 μg/ml LPS，剂量2=0.5 mg/ml脂质体+25 μg/ml LPS，剂量3=0.20 mg/ml脂质体+10 μg/ml LPS。白色柱为未包被免疫刺激剂的脂质体对照组（NL2,n，1 mg/ml脂质体），蓝色柱为未包被LPS对照组（50 μg/ml）。
(B) ZFL细胞与脂质体包被聚肌胞苷酸（poly(I:C)，NL2,poly(I:C)，绿色柱）孵育24 h后的存活率，设置三个剂量组：剂量1=1.5 mg/ml脂质体+50 μg/ml poly(I:C)，剂量2=0.75 mg/ml脂质体+25 μg/ml poly(I:C)，剂量3=0.375 mg/ml脂质体+10 μg/ml poly(I:C)。白色柱为空白脂质体对照组（NL2,n，1.5 mg/ml脂质体），红色柱为未包被poly(I:C)对照组（50 μg/ml）。
(C) ZFL细胞与脂质体包裹LPS-聚肌胞苷酸复合物（NLc，绿色柱）孵育24 h后的存活率，设置三个剂量组：剂量1=1.5 mg/ml脂质体+50 μg/ml poly(I:C)+25 μg/ml LPS，剂量2=0.75 mg/ml脂质体+25 μg/ml poly(I:C)+12.5 μg/ml LPS，剂量3=0.375 mg/ml脂质体+12.5 μg/ml poly(I:C)+6.25 μg/ml LPS。白色柱为空白脂质体对照组（NL2,n，1.5 mg/ml脂质体），蓝色柱为未包被LPS对照组（25 μg/ml），红色柱为未包被poly(I:C)对照组（50 μg/ml）。未处理细胞作为100%存活率对照（虚线）。数据以三次独立实验的平均值±标准差表示，差异分析采用单因素方差分析结合Tukey事后检验，标记：*，p<0.05；***，p<0.001。
图S3：NLc制剂在虹鳟巨噬细胞中的体外细胞毒性。(A) 采用LDH法检测NLc的细胞毒性：将虹鳟巨噬原代细胞与同时包被poly(I:C)与LPS的NLc孵育24 h，设置两个剂量组：剂量1=0.75 mg/ml脂质体+25 μg/ml poly(I:C)+12.5 μg/ml LPS，剂量2=0.375 mg/ml脂质体+12.5 μg/ml poly(I:C)+6.25 μg/ml LPS。白色柱为未包被脂质体对照组（NL2,n，0.75 mg/ml脂质体），灰色柱为未包被poly(I:C)与LPS对照组（分别为25 μg/ml与12.5 μg/ml）。以未处理细胞的基础死亡数作为对照（虚线）。数据以三次独立实验的平均值±标准差表示，差异分析采用单因素方差分析结合Tukey事后检验，**，p<0.01。
图S4：内吞抑制剂的体外细胞毒性。(A) 采用MTT法检测ZFL细胞经不同内吞抑制剂处理1 h（氯喹处理时长为16 h）后的存活率。(B) 采用MTT法检测虹鳟巨噬细胞经不同内吞抑制剂处理1 h后的存活率。以未处理细胞作为100%存活率对照（对照组柱）。
图S5：NLc体外摄取的时间进程。(A) 流式细胞术（Flow Cytometry）检测ZFL细胞对NLc的摄取时间进程（灰色柱：750 μg/ml脂质体，含25 μg/ml poly(I:C)与12.5 μg/ml LPS）。为研究NLc的代谢过程，ZFL细胞预先用100 μM氯喹处理1 h（红色柱），随后加入NLc（750 μg/ml脂质体，含25 μg/ml poly(I:C)与12.5 μg/ml LPS），并在孵育过程中持续存在氯喹。(B) 流式细胞术检测虹鳟巨噬细胞对NLc的摄取时间进程（灰色柱：750 μg/ml脂质体，含25 μg/ml poly(I:C)与12.5 μg/ml LPS）。未暴露于NLc的细胞作为对照（白色柱）。数据以三次独立实验的三次重复的平均值±标准差表示。
图S6：体内NLc毒性检测对照。将斑马鱼胚胎暴露于未包被LPS（黑色组：25 μg/ml与100 μg/ml）、未包被聚肌胞苷酸（绿色组：50 μg/ml）以及未包被LPS与poly(I:C)联合组（橙色组：25 μg/ml LPS+50 μg/ml poly(I:C)），分别在受精后120 h（hpf，A）与孵化后72 h（hph，B）每24 h记录胚胎存活率。以未处理胚胎（蓝色组）作为对照组。生存曲线采用对数秩检验（log rank test）分析（n=24个个体）。
图S7：斑马鱼幼鱼经脂质体制剂暴露后的基因表达分析。设置三组处理：NL2,n=未包被免疫刺激剂的脂质体（1.5 mg/ml），NLc=包被50 μg/ml poly(I:C)与25 μg/ml LPS的脂质体（1.5 mg/ml），LPS+poly(I:C)=未包被的联合刺激对照组（50 μg/ml poly(I:C)+25 μg/ml LPS）。以未处理胚胎作为对照（Ctrl）。以延伸因子1（EF1）作为内参基因，采用常规PCR检测Toll样受体3（TLR3）、GIG2、肿瘤坏死因子α（TNFα）与诱导型一氧化氮合酶（iNOS）的mRNA丰度（右侧面板）。展示三次独立实验的代表性图像。
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