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

Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ~125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health

Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells

Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement – a stage between initial cellular internalization and final gene silencing of siRNA delivery systems

Is early antithrombotic therapy necessary after tissue mitral valve replacement?

Patients with prosthetic heart valves have a higher risk of developing valve thrombosis and arterial thromboembolism. Antithrombotic therapy during the early postoperative period after biologic mitral valve replacement (MVR) is controversial. Hence, a retrospective study was conducted to investigate the efficacy of different antithrombotic therapies in patients after MVR with bioprostheses. METHODS: Between January 2000 and January 2006, a total of 99 patients presenting with preoperative sinus rhythm underwent isolated bioprosthetic MVR. Of these patients, 59 (58%) received a bovine pericardial xenograft, and 40 (42%) a porcine bioprosthesis. The postoperative antithrombotic therapy was prescribed according to the surgeon's preference. RESULTS: Fifty-one (51%) patients received acetylsalicylic acid (ASA group, 100 mg/day), 12 (13%) did not receive any specific antithrombotic therapy (NT group), and 36 (36%) received a vitamin K antagonist (VKA group, INR 2-3). The primary endpoints were the rate of cerebral ischemic events, bleeding events, and survival. The mean follow up was 23 months (range: 3-68 months). There were five early deaths (5%), and eight late deaths (8%). There were five episodes of cerebral ischemic events; these included three patients (8.3%) in the VKA group, one patient (2.0%) in ASA group, and one patient (8.3%) in the NT group (p = 0.351). Of these episodes, two occurred between 24 h and three months after surgery. Only one (2.8%) episode of major bleeding occurred (in the VKA group), due to poor anticoagulation management. CONCLUSION: Each of the antithrombotic therapies evaluated appeared to be safe. There was no evidence to suggest that any specific antithrombotic therapy would be superior in preventing valve thrombosis in patients undergoing bioprosthetic MVR

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

Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ~125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health

Analysis of gene expression in ZFL cell culture (A) and trout macrophage primary cell culture (B) after 16 h exposure to liposomes.

<p>NL_2,n = liposomes without immunostimulants (750 µg/ml), NL_c Dose 1 = liposomes (750 µg/ml) containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS, NL_c 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 (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001) and values with letters (_a,_b) are statistically significant relative to NL_c Dose 1 (_a, <i>p</i><0.001, _b, <i>p</i><0.05). Differences were analyzed using One-way ANOVA and Tukey's post test. (<b>C</b>) TNFα secretion from trout macrophages stimulated with liposomes for 16 h was assessed by Western blot. NL_c Dose 2 = 375 µg/ml liposomes, 12.5 µg/ml poly (I:C), 6.25 µg/ml LPS, NL_2,n = empty liposomes (375 µg/ml) and LPS = stimulation control (6.25 µg/ml). A representative Western Blot is shown.</p

Characterization of liposomal formulations.

<p>(<b>A</b>) 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>B</b>) Confocal fluorescence image of a single liposome tagged on its lipid bilayer with Marina Blue-DHPE (blue) and its corresponding fluorescence intensity profile. (<b>C</b>) Confocal fluorescence image of a single Marina Blue-labeled liposome containing AlexaFluor594-labeled LPS (red) and their corresponding fluorescence intensity profiles. (<b>D</b>) Confocal fluorescence image of a single Marina Blue-labeled liposome containing fluorescein-labeled poly (I:C) and their corresponding fluorescence intensity profiles. (<b>E</b>) Schematic representation of the liposomal IS-cocktail (NL_c) showing the presence of both encapsulated LPS (red) and poly (I:C) (green) in the lipidic bilayer of liposomes. (<b>F</b>) 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.</p

Endocytosis of NL_c formulation by ZFL cells.

<p>(<b>A</b>) Flow cytometry time-course comparison of the membrane-bound (dark grey bar) versus the endocyted liposomes (light grey bar) after incubation with NL_c (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>B</b>) Effect of chemical inhibitors on the endocytosis of the NL_c (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 (NL_c 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. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001. (<b>C</b>) Confocal microscopy images of fluorescent liposomes (NL_c) endocyted by ZFL cells. Cells were incubated for 30 min, 1.5 h and 16 h with NL_c 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).</p