One Step Purification-Vaccine Delivery System.

Glucan particles (GPs) are hollow, porous 3-5 µm microspheres derived from the cell walls of Baker's yeast (Saccharomyces cerevisiae). Their 1,3-ß-glucan outer shell allows for receptor-mediated uptake by macrophages and other phagocytic innate immune cells expressing ß-glucan receptors. GPs have been used for the targeted delivery of a wide range of payloads, including vaccines and nanoparticles, encapsulated inside the hollow cavity of GPs. In this paper, we describe the methods to prepare GP-encapsulated nickel nanoparticles (GP-Ni) for the binding of histidine (His)-tagged proteins. His-tagged Cda2 cryptococcal antigens were used as payloads to demonstrate the efficacy of this new GP vaccine encapsulation approach. The GP-Ni-Cda2 vaccine was shown to be comparable to our previous approach utilizing mouse serum albumin (MSA) and yeast RNA trapping of Cda2 in GPs in a mouse infection model. This novel GP-Ni approach allows for the one-step binding of His-tagged vaccine antigens and encapsulation in an effective delivery vehicle to target vaccines to antigen-presenting cells (APCs), antigen discovery, and vaccine development.

An efficient (nano) silica - In glucan particles protein encapsulation approach for improved thermal stability.

Glucan particles (GPs) are hollow, porous microspheres derived from Saccharomyces cerevisiae (Baker's yeast). The hollow cavity of GPs allows for efficient encapsulation of different types of macromolecules and small molecules. The ß-1,3-D-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing ß-glucan receptors and uptake of particles containing encapsulated proteins elicit protective innate and acquired immune responses against a wide range of pathogens. A limitation of the previously reported GP protein delivery technology is limited protection from thermal degradation. Here we present results of an efficient protein encapsulation approach using tetraethylorthosilicate (TEOS) to lock protein payloads in a thermostable silica cage formed in situ inside the hollow cavity of GPs. The methods for this improved, efficient GP protein ensilication approach were developed and optimized using bovine serum albumin (BSA) as model protein. The improved method involved controlling the rate of TEOS polymerization, such that the soluble TEOS-protein solution was able to be absorbed into the GP hollow cavity before the protein-silica cage polymerized and becomes too large to transverse across the GP wall. This improved method provided for >90% GP encapsulation efficiency, increased thermal stabilization of GP ensilicated BSA, and was shown to be applicable for encapsulation of proteins of different molecular weights and isoelectric points. To demonstrate the retention of bioactivity of this improved method of protein delivery, we evaluated the in vivo immunogenicity of two GP ensilicated vaccine formulations using (1) ovalbumin as a model antigen and (2) a protective antigenic protein from the fungal pathogen Cryptococcus neoformans. The results show that the GP ensilicated vaccines have a similar high immunogenicity as our current GP protein/hydrocolloid vaccines, as evidenced by robust antigen-specific IgG responses to the GP ensilicated OVA vaccine. Further, a GP ensilicated C. neoformans Cda2 vaccine protected vaccinated mice from a lethal pulmonary infection of C. neoformans.

Yeast Particles for Encapsulation of Terpenes and Essential Oils.

Terpenes and essential oils are materials of great commercial use due to their broad spectra of antibacterial, antifungal, membrane permeation enhancement and antioxidant biological properties, as well as for their use as flavors and fragrances. Yeast particles (YPs) are 3-5 µm hollow and porous microspheres, a byproduct of some food-grade yeast (Saccharomyces cerevisiae) extract manufacturing processes, that have been used for the encapsulation of terpenes and essential oils with high payload loading capacity (up to 500% weight) and efficiency, providing stability and sustained-release properties. This review focuses on encapsulation approaches for the preparation of YP-terpene and essential oil materials that have a wide range of potential agricultural, food and pharmaceutical applications.

Efficient and Scalable Process to Produce Novel and Highly Bioactive Purified Cytosolic Crystals from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins. IMPORTANCE Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.

Yeast Particles Hyper-Loaded with Terpenes for Biocide Applications.

Yeast particles (YPs) are 3−5 µm hollow and porous microspheres, a byproduct of some food grade yeast (Saccharomyces cerevisiae) extract manufacturing processes. Terpenes can be efficiently encapsulated inside YPs by passive diffusion through the porous cell walls. As previously published, this YP terpene encapsulation approach has been successfully implemented (1) to develop and commercialize fungicide and nematicide products for agricultural applications, (2) to co-load high potency agrochemical actives dissolved in terpenes or suitable solvents, and (3) to identify YP terpenes with broad-acting anthelmintic activity for potential pharmaceutical applications. These first-generation YP terpene materials were developed with a <2:1 terpene: YP weight ratio. Here we report methods to increase the terpene loading capacity in YPs up to 5:1 terpene: YP weight ratio. Hyper-loaded YP terpenes extend the kinetics of payload release up to three-fold compared to the commercialized YP terpene formulations. Hyper-loaded YP-terpene compositions were further optimized to achieve high terpene storage encapsulation stability from −20 °C to 54 °C. The development of hyper-loaded YP terpenes has a wide range of potential agricultural and pharmaceutical applications with terpenes and other compatible active substances that could benefit from a delivery system with a high payload loading capacity combined with increased payload stability and sustained release properties.

Yeast Particle Encapsulation of Scaffolded Terpene Compounds for Controlled Terpene Release.

Terpenes are naturally occurring compounds produced by plants that are of great commercial interest in the food, agricultural, cosmetic, and pharmaceutical industries due to their broad spectra of antibacterial, antifungal, anthelmintic, membrane permeation enhancement, and antioxidant biological activities. Applications of terpenes are often limited by their volatility and the need for surfactants or alcohols to produce stable, soluble (non-precipitated) products. Yeast particles (YPs) are hollow, porous microspheres that have been used for the encapsulation of terpenes (YP terpenes) by passive diffusion of terpenes through the porous YP cell walls. We here report the development of a second generation YP encapsulated terpene technology that incorporates the stimuli-responsive control of terpene release using biodegradable pro-terpene compounds (YP pro-terpenes). YP terpenes and YP pro-terpenes were both produced, in which high levels of carvacrol, eugenol, thymol and geraniol were encapsulated. The YP pro-terpenes show higher encapsulation stability than YP terpenes due to pro-terpenes being non-volatile solids at room temperature and stable in suspensions at neutral pH. YP pro-terpenes and YP terpenes were evaluated for biological activity in antibacterial, antifungal and anthelmintic assays. The YP pro-terpenes retained the full biological activity of the parent terpene compound.

Anthelmintic Activity of Yeast Particle-Encapsulated Terpenes.

Soil-transmitted nematodes (STN) infect 1-2 billion of the poorest people worldwide. Only benzimidazoles are currently used in mass drug administration, with many instances of reduced activity. Terpenes are a class of compounds with anthelmintic activity. Thymol, a natural monoterpene phenol, was used to help eradicate hookworms in the U.S. South circa 1910. However, the use of terpenes as anthelmintics was discontinued because of adverse side effects associated with high doses and premature stomach absorption. Furthermore, the dose-response activity of specific terpenes against STNs has been understudied. Here we used hollow, porous yeast particles (YPs) to efficiently encapsulate (>95%) high levels of terpenes (52% w/w) and evaluated their anthelmintic activity on hookworms (Ancylostoma ceylanicum), a rodent parasite (Nippostrongylus brasiliensis), and whipworm (Trichuris muris). We identified YP-terpenes that were effective against all three parasites. Further, YP-terpenes overcame albendazole-resistant Caenorhabditis elegans. These results demonstrate that terpenes are broad-acting anthelmintics. Terpenes are predicted to be extremely difficult for parasites to resist, and YP encapsulation provides water-suspendable terpene materials without surfactants and sustained terpene release that could lead to the development of formulations for oral delivery that overcome fast absorption in the stomach, thus reducing dosage and toxic side effects.

Polydopamine Coating of Glucan Particles Increases Uptake into Peyer's Patches.

Glucan particles (GPs) are hollow, porous 3-4 µm microspheres derived from the cell walls of Baker's yeast (Saccharomyces cerevisiae). The ß-1,3-D glucan outer shell of GPs provides for receptor-mediated uptake by phagocytic cells expressing ß-glucan receptors. GPs have been used for efficient encapsulation of different types of payloads (DNA, siRNA, proteins, antigens, small molecules), and these payloads have been delivered in vivo by a variety of routes including oral delivery. It is known that GPs are transported across the intestinal epithelium by Peyer's patch M-cells and accumulate in a subset of CD11c+Langerin-positive dendritic cells (DC) in the subepithelial dome (SED). An increase in GP uptake in the intestinal epithelium is needed to improve our efforts to develop GPs for oral delivery of therapeutics and vaccines. In this Article, we report that polydopamine coating of GPs (PDA-GPs) increases transepithelial uptake. Synthesis of PDA-GPs was optimized to allow for encapsulation of payloads inside the hollow cavity of GPs. PDA-GPs and GP controls were orally administered to mice, and PDA-GPs showed a 42% increased uptake in SED phagocytes. PDA-GP uptake by SED phagocytes in control and M-cell-depleted mice demonstrated both M-cell-dependent and -independent mechanisms. In future studies, we will evaluate PDA-GPs for oral vaccine delivery and the use of PDA-functional groups for secondary surface derivatization to generate particles with ligands targeting other intestinal epithelium cell-surface receptors.

Beta-Glucan Particles as Vaccine Adjuvant Carriers.

Glucan particles (GPs) are spherical hollow particles derived from Saccharomyces cerevisiae cell walls and mainly consist of ß-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 ß-glucan's 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 Baker's yeast (Saccharomyces cerevisiae). The 1,3-ß-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing ß-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.

Glucan particles for macrophage targeted delivery of nanoparticles.

Glucan particles (GPs) are hollow, porous 2-4 µm microspheres derived from the cell walls of Baker's yeast (Saccharomyces cerevisiae). The 1,3-ß-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing ß-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.

Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery.

Nonviral gene delivery technologies have been developed using layer-by-layer self-assembly of nanomaterials held together by electrostatic interactions in order to provide nanoparticulate materials that protect and deliver DNA to cells. Here we report a new DNA delivery technology based on the in situ layer-by-layer synthesis of DNA nanoparticles caged within hollow yeast cell wall particles (YCWP). YCWP provide protection and facilitate oral and systemic receptor-targeted delivery of DNA payloads to phagocytic cells. The nanoparticles inside YCWP consist of a core of tRNA/polyethylenimine (PEI) followed by a DNA layer that is finally coated with a protective outer layer of PEI. Using fluorescein and rhodamine labeling of tRNA, PEI, and DNA, the layer-by-layer formation of the nanoparticles was visualized by fluorescent microscopy and quantitated by fluorescence spectroscopy and flow cytometry. Optimal conditions (tRNA:YCWP, PEI:YCWP ratios and DNA load levels) to synthesize YCWP encapsulated nanoparticles were determined from these results. The high in vitro transfection efficiency of this encapsulated DNA delivery technology was demonstrated by the transfection of NIH3T3-D1 cells with YCWP-tRNA/PEI/gWizGFP/PEI formulations containing low amounts of the plasmid gWizGFP per particle to maximally express green fluorescent protein (GFP).

Photocurrent generation in noncovalently assembled multilayered thin films.

Multilayered photocurrent generating thin films were fabricated by templated noncovalent assembly via stepwise assembly of molecular components. Each of films I-IV contained an underlying self-assembled monolayer (SAM) consisting of an alkanethiol linked covalently to a 2,6-dicarboxypyridine ligand that served as a binding site for attaching additional molecular components. The SAM subsequently was functionalized by sequential deposition of Cu(II), Co(II), or Fe(III) ions followed by a variety of substituted 2,6-dicarboxypyridine ligands as a means to incorporate one or more layers of pyrene chromophores into the film. The films were characterized by contact angle measurements, ellipsometry, grazing incidence IR, cyclic voltammetry, and impedance spectroscopy after deposition of each layer, confirming the formation of ordered, stable layers. Following incorporation into a three-electrode system, photoexcitation resulted in the generation of a cathodic photocurrent in the presence of methyl viologen and an anodic photocurrent in the presence of triethanolamine. Using this strategy, systems were fabricated that produced up to 89 nA/cm(2) of reproducible photocurrent.

Microscale correlation between surface chemistry, texture, and the adhesive strength of Staphylococcus epidermidis.

Staphylococcus epidermidis is among the most commonly isolated microbes from medical implant infections, particularly in the colonization of blood-contacting devices. We explored the relationships between surface wettability and root-mean-square roughness (Rq) on microbial adhesive strength to a substrate. Molecular-level interactions between S. epidermidis and a variety of chemically and texturally distinct model substrata were characterized using a cellular probe and atomic force microscopy (AFM). Substrata included gold, aliphatic and aromatic self-assembled monolayers, and polymeric and proteinaceous materials. Substrate hydrophobicity, described in terms of the water contact angle, was an insufficient parameter to explain the adhesive force of the bacterium for any of the surfaces. Correlations between adhesion forces and Rq showed weak relationships for most surfaces. We used an alternate methodology to characterize the texture of the surface that is based on a fractal tiling algorithm applied to images of each surface. The relative area as a function of the scale of observation was calculated. The discrete bonding model (DBM) was applied, which describes the area available for bonding interactions over the full range of observational scales contained in the measured substrate texture. Weak negative correlations were obtained between the adhesion forces and the area available for interaction, suggesting that increased roughness decreases bacterial adhesion when nano- to micrometer scales are considered. We suggest that modification of the DBM is needed in order to include discontinuous bonding. The adhesive strength is still related to the area available for bonding on a particular scale, but on some very fine scales, the bacteria may not be able to conform to the valleys or pits of the substrate. Therefore, the bonding between the bacterium and substrate becomes discontinuous, occurring only on the tops of ridges or asperities.

Surface-based lithium ion sensor: An electrode derivatized with a self-assembled monolayer.

Self-assembled monolayers (SAMs) of 21-(16-mercaptohexadecan-1-oyl)-4,7,13,16-tetraoxa-1,10,21-triazabicyclo[8.8.5]tricosane-19,23-dione were prepared on gold. Characterization of the SAMs was carried out by sessile drop contact angle, ellipsometry, grazing angle FT-IR spectroscopy, and electrochemical techniques. The cation recognition properties of the SAM were studied by cyclic voltammetry and impedance spectroscopy. The films show moderate selectivity for detection of Li+ ions in solution over K+ and Na+, with selectivity values calculated to be log K(Li+,Na+) approximately -1.30 and log K(Li+,K+) approximately -0.92. To the best of our knowledge, this is the first demonstration of a lithium sensor fabricated using self-assembled monolayer technology.