Solid lipid nanoparticles for the delivery of anti-microbial oligonucleotides.

Novel alternatives to antibiotics are urgently needed for the successful treatment of antimicrobial resistant (AMR) infections. Experimental antibacterial oligonucleotide therapeutics, such as transcription factor decoys (TFD), are a promising approach to circumvent AMR. However, the therapeutic potential of TFD is contingent upon the development of carriers that afford efficient DNA protection against nucleases and delivery of DNA to the target infection site. As a carrier for TFD, here we present three prototypes of anionic solid lipid nanoparticles that were coated with either the cationic bolaamphiphile 12-bis-tetrahydroacridinium or with protamine. Both compounds switched particles zeta potential to positive values, showing efficient complexation with TFD and demonstrable protection from deoxyribonuclease. The effective delivery of TFD into bacteria was confirmed by confocal microscopy while SLN-bacteria interactions were studied by flow cytometry. Antibacterial efficacy was confirmed using a model TFD targeting the Fur iron uptake pathway in E. coli under microaerobic conditions. Biocompatibility of TFD-SLN was assessed using in vitro epithelial cell and in vivo Xenopus laevis embryo models. Taken together these results indicate that TFD-SLN complex can offer preferential accumulation of TFD in bacteria and represent a promising class of carriers for this experimental approach to tackling the worldwide AMR crisis.

Cationic liposomal vectors incorporating a bolaamphiphile for oligonucleotide antimicrobials.

Antibacterial resistance has become a serious crisis for world health over the last few decades, so that new therapeutic approaches are strongly needed to face the threat of resistant infections. Transcription factor decoys (TFD) are a promising new class of antimicrobial oligonucleotides with proven in vivo activity when combined with a bolaamphiphilic cationic molecule, 12-bis-THA. These two molecular species form stable nanoplexes which, however, present very scarce colloidal stability in physiological media, which poses the challenge of drug formulation and delivery. In this work, we reformulated the 12-bis-THA/TFD nanoplexes in a liposomal carrier, which retains the ability to protect the oligonucleotide therapeutic from degradation and deliver it across the bacterial cell wall. We performed a physical-chemical study to investigate how the incorporation of 12-bis-THA and TFD affects the structure of POPC- and POPC/DOPE liposomes. Analysis was performed using dynamic light scattering (DLS), ζ-potential measurements, small-angle x-ray scattering (SAXS), and steady-state fluorescence spectroscopy to better understand the structure of the liposomal formulations containing the 12-bis-THA/TFD complexes. Oligonucleotide delivery to model Escherichia coli bacteria was assessed by means of confocal scanning laser microscopy (CLSM), evidencing the requirement of a fusogenic helper lipid for transfection. Preliminary biological assessments suggested the necessity of further development by modulation of 12-bis-THA concentration in order to optimize its therapeutic index, i.e. the ratio of antibacterial activity to the observed cytotoxicity. In summary, POPC/DOPE/12-bis-THA liposomes appear as promising formulations for TFD delivery.

Nanodelivery Systems as New Tools for Immunostimulant or Vaccine Administration: Targeting the Fish Immune System.

Fish disease treatments have progressed significantly over the last few years and have moved from the massive use of antibiotics to the development of vaccines mainly based on inactivated bacteria. Today, the incorporation of immunostimulants and antigens into nanomaterials provide us with new tools to enhance the performance of immunostimulation. Nanoparticles are dispersions or solid particles designed with specific physical properties (size, surface charge, or loading capacity), which allow controlled delivery and therefore improved targeting and stimulation of the immune system. The use of these nanodelivery platforms in fish is in the initial steps of development. Here we review the advances in the application of nanoparticles to fish disease prevention including: the type of biomaterial, the type of immunostimulant or vaccine loaded into the nanoparticles, and how they target the fish immune system.

Decoding the genetic and functional diversity of the DSF quorum-sensing system in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia uses the Diffusible Signal Factor (DSF) quorum sensing (QS) system to mediate intra- and inter-specific signaling and regulate virulence-related processes. The components of this system are encoded by the rpf cluster, with genes rpfF and rpfC encoding for the DSF synthase RpfF and sensor RpfC, respectively. Recently, we have shown that there exist two variants of the rpf cluster (rpf-1 and rpf-2), distinguishing two groups of S. maltophilia strains. Surprisingly, only rpf-1 strains produce detectable DSF, correlating with their ability to control biofilm formation, swarming motility and virulence. The evolutive advantage of acquiring two different rpf clusters, the phylogenetic time point and mechanism of this acquisition and the conditions that activate DSF production in rpf-2 strains, are however not known. Examination of this cluster in various species suggests that its variability originated most probably by genetic exchange between rhizosphere bacteria. We propose that rpf-2 variant strains make use of a strategy recently termed as "social cheating." Analysis of cellular and extracellular fatty acids (FAs) of strains E77 (rpf-1) and M30 (rpf-2) suggests that their RpfFs have also a thioesterase activity that facilitates the release of unspecific FAs to the medium in addition to DSF. Production of DSF in rpf-1 strains appears in fact to be modulated by some of these extracellular FAs in addition to other factors such as temperature and nutrients, while in rpf-2 strains DSF biosynthesis is derepressed only upon detection of DSF itself, suggesting that they require cohabitation with DSF-producer bacteria to activate their DSF regulatory machinery. Finally, we show that the mixed rpf-1/rpf-2 population presents synergism in DSF production and virulence capacity in an in vivo infection model. Recovery and quantification of DSF from co-infected animals correlates with the observed mortality rate.

Zebrafish liver (ZFL) cells are able to mount an anti-viral response after stimulation with Poly (I:C).

The zebrafish (Danio rerio) is a widely used model species for biomedical research and is also starting to be a model for aquaculture research. The ZFL cell line, established from zebrafish liver, has been mostly used in toxicological and ecotoxicological studies. However, no studies have previously characterised this cell line in regard to its immunological response. The aim of this work was to study the gene expression response of the ZFL cell line after incubation with different prototypical immune stimuli, such as lipopolysaccharide (LPS), peptidoglycan (PGN), zymosan, and with a special focus on the dsRNA Poly (I:C). Using PCR, microarrays, and confocal microscopy we have explored the response of the ZFL cells against Poly (I:C). This study shows that the ZFL is able to uptake very efficiently the Poly (I:C) and mount a strong anti-viral response. We can conclude that ZFL could be used not only in toxicological studies, but also in studying anti-viral responses in zebrafish.

Synthesis, culture medium stability, and in vitro and in vivo zebrafish embryo toxicity of metal-organic framework nanoparticles.

Metal-organic frameworks (MOFs) are among the most attractive porous materials available today. They have garnered much attention for their potential utility in many different areas such as gas storage, separation, catalysis, and biomedicine. However, very little is known about the possible health or environmental risks of these materials. Here, the results of toxicity studies on sixteen representative uncoated MOF nanoparticles (nanoMOFs), which were assessed for cytotoxicity to HepG2 and MCF7 cells in vitro, and for toxicity to zebrafish embryos in vivo, are reported. Interestingly, there is a strong correlation between their in vitro toxicity and their in vivo toxicity. NanoMOFs were ranked according to their respective in vivo toxicity (in terms of the amount and severity of phenotypic changes observed in the treated zebrafish embryos), which varied widely. Altogether these results show different levels of toxicity of these materials; however, leaching of solubilized metal ions plays a main role.

Amyloid formation by human carboxypeptidase D transthyretin-like domain under physiological conditions.

Protein aggregation is linked to a growing list of diseases, but it is also an intrinsic property of polypeptides, because the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including the M14B subfamily carboxypeptidases. We show here that the monomeric transthyretin-like domain of human carboxypeptidase D aggregates under close to physiological conditions into amyloid structures, with the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic ß-strands in the native state. These structural elements should serve for functional/structural purposes, because they have not been purged out by evolution, but at the same time they put proteins like carboxypeptidase D at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.

Targeting and stimulation of the zebrafish (Danio rerio) innate immune system with LPS/dsRNA-loaded nanoliposomes.

Herein we report the use of immunostimulant-loaded nanoliposomes (called NLcliposomes) as a strategy to protect fish against bacterial and/or viral infections. This work entailed developing a method for in vivo tracking of the liposomes administered to adult zebrafish that enables evaluation of their in vivo dynamics and characterisation of their tissue distribution. The NLc liposomes, which co-encapsulate poly(I:C) and LPS, accumulate in immune tissues and in immunologically relevant cells such as macrophages, as has been assessed in trout primary cell cultures. They protect zebrafish against otherwise lethal bacterial (Pseudomonas aeruginosa PAO1) and viral (Spring Viraemia of Carp Virus) infections regardless of whether they are administered by injection or by immersion, as demonstrated in a series of in vivo infection experiments with adult zebrafish. Importantly, protection was not achieved in fish that had been treated with empty liposomes or with a mixture of the free immunostimulants. Our findings indicate that stimulation of the innate immune system with co-encapsulated immunostimulants in nano-liposomes is a promising strategy to simultaneously improve the levels of protection against bacterial and viral infections in fish.

Two different rpf clusters distributed among a population of Stenotrophomonas maltophilia clinical strains display differential diffusible signal factor production and virulence regulation.

The quorum-sensing (QS) system present in the emerging nosocomial pathogen Stenotrophomonas maltophilia is based on the signaling molecule diffusible signal factor (DSF). Production and detection of DSF are governed by the rpf cluster, which encodes the synthase RpfF and the sensor RpfC, among other components. Despite a well-studied system, little is known about its implication in virulence regulation in S. maltophilia. Here, we have analyzed the rpfF gene from 82 S. maltophilia clinical isolates. Although rpfF was found to be present in all of the strains, it showed substantial variation, with two populations (rpfF-1 and rpfF-2) clearly distinguishable by the N-terminal region of the protein. Analysis of rpfC in seven complete genome sequences revealed a corresponding variability in the N-terminal transmembrane domain of its product, suggesting that each RpfF variant has an associated RpfC variant. We show that only RpfC-RpfF-1 variant strains display detectable DSF production. Heterologous rpfF complementation of ΔrpfF mutants of a representative strain of each variant suggests that RpfF-2 is, however, functional and that the observed DSF-deficient phenotype of RpfC-RpfF-2 variant strains is due to permanent repression of RpfF-2 by RpfC-2. This is corroborated by the ΔrpfC mutant of the RpfC-RpfF-2 representative strain. In line with this observations, deletion of rpfF from the RpfC-RpfF-1 strain leads to an increase in biofilm formation, a decrease in swarming motility, and relative attenuation in the Caenorhabditis elegans and zebrafish infection models, whereas deletion of the same gene from the representative RpfC-RpfF-2 strain has no significant effect on these virulence-related phenotypes.

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.