Ernesto Soto

Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation

Gene silencing by double-stranded RNA, denoted RNA interference, represents a new paradigm for rational drug design. However, the transformative therapeutic potential of short interfering RNA (siRNA) has been stymied by a key obstacle—safe delivery to specified target cells in vivo. Macrophages are particularly attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Here we report the engineering of β1,3-d-glucan-encapsulated siRNA particles (GeRPs) as efficient oral delivery vehicles that potently silence genes in mouse macrophages in vitro and in vivo. Oral gavage of mice with GeRPs containing as little as 20 μg kg-1 siRNA directed against tumour necrosis factor α (Tnf-α) depleted its messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum Tnf-α levels. Screening with GeRPs for inflammation genes revealed that the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) is a previously unknown mediator of cytokine expression. Importantly, silencing Map4k4 in macrophages in vivo protected mice from lipopolysaccharide-induced lethality by inhibiting Tnf-α and interleukin-1β production. This technology defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease.

Glucan particles for selective delivery of siRNA to phagocytic cells in mice.

Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi (RNA interference) therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex 5 component glucan-based encapsulation system for siRNA (small interfering RNA) delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA-Endo-Porter complexes in glucan shells of 2-4 μm diameter in the absence of other components. The resulting glucan particles containing fluorescently labelled siRNA were readily internalized by macrophages, but not other cell types, and released the labelled siRNA into the macrophage cytoplasm. Intraperitoneal administration of such glucan particles containing siRNA-Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These results from the present study indicate that specific targeting to phagocytes is mediated by the glucan, whereas Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyse escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.

Glucan Particles for Macrophage Targeted Delivery of Nanoparticles

Glucan particles (GPs) are hollow, porous 2–4 μm microspheres derived from the cell walls of Bakers yeast (Saccharomyces cerevisiae). The 1,3-β-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing β-glucan receptors. GPs have been used for macrophage-targeted delivery of soluble payloads (DNA, siRNA, protein, and small molecules) encapsulated inside the hollow GPs via core polyplex and layer-by-layer (LbL) synthetic strategies. In this communication, we report the incorporation of nanoparticles as cores inside GPs (GP-NP) or electrostatically bound to the surface of chemically derivatized GPs (NP-GP). GP nanoparticle formulations benefit from the drug encapsulation properties of NPs and the macrophage-targeting properties of GPs. GP nanoparticle formulations were synthesized using fluorescent anionic polystyrene nanoparticles allowing visualization and quantitation of NP binding and encapsulation. Mesoporous silica nanoparticles (MSNs) containing the chemotherapeutic doxorubicin (Dox) were bound to cationic GPs. Dox-MSN-GPs efficiently delivered Dox into GP phagocytic cells resulting in enhanced Dox-mediated growth arrest.

Beta-Glucan Particles as Vaccine Adjuvant Carriers.

Glucan particles (GPs) are spherical hollow particles derived from Saccharomyces cerevisiae cell walls and mainly consist of β-1, 3-D-glucans. The inner hollow cavity of glucan particles can be loaded with different compounds, including protein antigens, and delivered to macrophages and dendritic cells. Moreover, the GP delivery system possesses β-glucans intrinsic immunostimulatory properties. Therefore, GPs serve as both an antigen-presenting cell-targeted delivery system and an adjuvant.Here, we describe the production of GPs from S. cerevisiae using hot alkaline and solvent extraction and characterization of these particles for morphology, particle density, and hydrodynamic volume. A detailed protocol for loading and entrapping a model antigen, ovalbumin (OVA), into these particles using yeast RNA is presented. Similar methods are used to load pathogen-specific antigens (peptides, proteins, soluble extracts) which then can be tested in in vivo vaccination models.

Targeted Delivery of Glucan Particle Encapsulated Gallium Nanoparticles Inhibits HIV Growth in Human Macrophages

Glucan particles (GPs) are hollow, porous 3–5 μm microspheres derived from the cell walls of Bakers yeast (Saccharomyces cerevisiae). The 1,3-β-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing β-glucan receptors. GPs have been used for macrophage-targeted delivery of a wide range of payloads (DNA, siRNA, protein, small molecules, and nanoparticles) encapsulated inside the hollow GPs or bound to the surface of chemically derivatized GPs. Gallium nanoparticles have been proposed as an inhibitory agent against HIV infection. Here, macrophage targeting of gallium using GPs provides for more efficient delivery of gallium and inhibition of HIV infection in macrophages compared to free gallium nanoparticles.

Preparation and characterization of yeast cell wall beta-glucan encapsulated humic acid nanoparticles as an enhanced aflatoxin B1 binder

This study aimed to assess the effect of encapsulating humic acid inside yeast cell walls (YCW) to detoxify AFB1 in in vitro gastrointestinal models. Glucan Mannan Lipid Particles (GMLPs) from Saccharomyces cerevisiae cell walls showed the highest AFB1 adsorption in simulated gastric fluid (SGF) after 10 min, and in simulated intestinal fluid (SIF) after 1 h. GMLPs are hollow 3-4 micron porous microspheres that provide an efficient system for the synthesis and encapsulation of AFB1-absorbing nanoparticles (NPs). Humic acid nanoparticles (HA-NPs) were synthesized within the GMLP cavity by complexation with ferric chloride. Encapsulating HA-NPs in GMLPs increased HA-NP stability in SIF. The hybrid GMLP HA-NP formulation synergistically enhanced AFB1 binding compared to individual GMLP and HA components in SGF and in SIF. Cytotoxicity on a murine macrophage cell line demonstrated that GMLP HA-NP-AFB1 complexes were stable in both SGF and SIF, detoxified AFB1 and are suitable for in vivo testing.