Protein-filled polyelectrolyte microcapsules in the design of enzymic microdiagnostics

Encapsulation of enzymes (lactate dehydrogenase and urease) in polyelectrolyte shells was assessed with a view to designing enzymic microdiagnostics for low-molecular compounds in native biological fluids. Polyelectrolyte microcapsules were prepared with two polyanions [poly(styrenesulfonate) PSS and dextran sulfate DS] and two polycations [poly(allylamine) PAA and poly(diallyldimethylammonium) PDADMA]; calcium carbonate microspherulites with embedded enzymes served as “cores.” It was demonstrated that the main problem in making such a biosensor is to select a pair of oppositely charged polyelectrolytes that would be optimal for enzyme functioning. The best pairs were PAA/DS and PAA/PSS for lactate dehydrogenase, and PSS/PAA and PSS/PDADMA for urease. We designed and prepared enzyme-containing microcapsules differing in polyelectrolyte composition and number of layers, and investigated their properties.     

Polyelectrolyte-coated microcapsules and their potential applications to biotechnology

Polyelectrolyte-coated microcapsules can be prepared by adsorption of polyions onto microcapsule surfaces in aqueous solutions under appropriate pH and ionic conditions. The resulting polyelectrolyte-coated microcapsules provide a promising tool for studying pH-induced configurational changes in polyions adsorbed onto hydrophobic membranes (capsule walls). An interesting application of polyelectrolyte-coated microcapsules is the pH-sensitive on/off control of microencapsulated enzyme reactions through alterations in the substrate permeability of the capsule wall by pH-conditioned configurational changes in the adsorbed polyion layer. This paper presents an overview of pH-induced conformational changes of polyelectrolytes in solutions, preparation of polyelectrolyte-coated microcapsules with an immobilized enzyme, and on/off control of the respective enzyme reactions by pH adjustment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules

We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (p<0.05). The APA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.     

Novel dry powder preparations of whole inactivated influenza virus for nasal vaccination

The purpose of these studies was to enhance mucosal and systemic antibody production in response to increased local residence time of a whole inactivated influenza virus administered as a dry powder nasal vaccine formulation. Spray-freeze-drying (SFD) particles suitable for nasal delivery were characterized for physico-chemical properties and stability. Mucoadhesive compounds (MA) were characterized for their effects on nasal residence time of vaccine powders in rats compared with published in vitro data and elicited immune responses. SFD particles (D(50) = 26.9 microm) were spherical with a specific surface area of 1.25 m(2)/g. Thermal analysis indicated SFD powders were amorphous and demonstrated improved stability with respect to liquid formulations under various storage conditions. In vitro physico-chemical studies and in vivo scintigraphic imaging experiments indicated sodium alginate (SA) and carboxymethylcellulose-high molecular weight (CMC-HMW) powder formulations most significantly increased residence time in Brown Norway rats. Intramuscular delivery provided equivalent serum antibody titers to intranasal (IN) powder without MA, in the presence of CMC-HMW, SA, and hydroxypropyl methylcellulose (HPMC-HMW) after initial dosing and all formulations except IN powder with chitosan after boosting. IN liquid provided equivalent serum antibody titers to all IN powders after the initial vaccination and significantly greater serum antibody titers than IN powder with chitosan after boosting. Trends were consistent between residence time studies and immune response; however, no statistically significant differences between powder and liquid formulations were observed. It was concluded that enhanced serum and mucosal antibody responses were elicited by a dry powder nasal vaccine, specifically, administered in the presence of sodium alginate.     

Preparation and in vitro evaluation of polystyrene-coated diltiazem-resin complex by oil-in-water emulsion solvent evaporation method

The purpose of this study was to examine the suitability of polystyrene-coated (PS-coated) microcapsules of drug-resin complex for achieving prolonged release of diltiazem-HCl, a highly water-soluble drug, in simulated gastric and intestinal fluid. The drug was bound to Indion 254, a cation-exchange resin, and the resulting resinate was microencapsulated with PS using an oil-in-water emulsion-solvent evaporation method. The effect of various formulation parameters on the characteristics of the microcapsules was studied. Mean diameter and encapsulation efficiency of the microcapsules rose with an increase in the concentration of emulsion stabilizer and the coat/core ratio, while the same characteristics tended to decrease with an increase in the volume of the organic disperse phase. The desorption of drug from the uncoated resinate was quite rapid and independent of the pH of the dissolution media. On the other hand, the drug release from the microcapsules was prolonged for different periods of time depending on the formulation parameters and was also found to be independent of the pH of the dissolution media. Both the encapsulation efficiency and the retardation of drug release were found to be dependent on the uniformity of coating, which in turn was influenced by the formulation parameters. Kinetic studies revealed that the desorption of drug from the resinate obeyed the typical particle diffusion process, whereas the drug release from the microencapsulated resinate followed the diffusion-controlled model in accordance with the Higuchi equation. PS appeared to be a suitable polymer to provide prolonged release of diltiazem independent of the pH of the dissolution media.     

Formulation of a dry powder influenza vaccine for nasal delivery

The purpose of this research was to prepare a dry powder vaccine formulation containing whole inactivated influenza virus (VIIV) and a mucoadhesive compound suitable for nasal delivery. Powders containing WIIV and either lactose or trehalose were produced by lyophilization. A micro-ball mill was used to reduce the lyophilized cake to sizes suitable for nasal delivery. Chitosan flakes were reduced in size using a cryo-milling technique. Milled powders were sieved between 45 and 125 μm aggregate sizes and characterized for particle size and distribution, morphology, and flow properties. Powders were blended in the micro-ball mill without the ball. Lyophilization followed by milling produced irregularly shaped, polydisperse particles with a median primary particle diameter of ≈21 μm and a yield of ≈37% of particles in the 45 to 125 μm particle size range. Flow properties of lactose and trehalose powders after lyophilization followed by milling and sieving were similar. Cryo-milling produced a small yield of particles in the desired size range (<10%). Lyophilization followed by milling and sieving produced particles suitable for nasal delivery with different physicochemical properties as a function of processing conditions and components of the formulation. Further optimization of particle size and morphology is required for these powders to be suitable for clinical evaluation. Published: March 10, 2006     

Polyelectrolyte microcapsules as the systems for delivery of biologically active substances

Based on the method of the layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes, sodium alginate (Alg) and poly-L-lysine (PLL), novel biodegradable microcapsules have been prepared for delivery of biological active substances (BAS). Porous spherical CaCO₃ microparticles were used as templates. The template cores were coated with several layers of oppositely charged polyelectrolytes forming shell on the core surface. The core-shell microparticles were converted into hollow microcapsules by means of core dissolution with EDTA. Mild conditions for microcapsules preparation allow to perform incorporation of various biomolecules maintaining their bioactivity. Biocompatibility and biodegradability of the polyelectrolytes give a possibility to use the microcapsules as the target delivery systems. Chymotrypsin entrapped into the microcapsules was used as a model enzyme. The immobilized enzyme retained about 86% of the activity compared to a native chymotrypsin. The resultant microcapsules were stable in acidic medium and could be easily decomposed by trypsin treatment in slightly alkaline medium. Chymotrypsin was shown to be active after its release from the microcapsules decomposed by the trypsin treatment. Thus, the microcapsules prepared by the LbL technique can be used for the development of new type of BAS delivery systems in humans and animals.     

Entrapment of herbal extracts into biodegradable microcapsules

The microcapsules with entrapped herbal water-soluble extracts of plantain Plantago major and calendula Calendula officinalis L. (PCE) were prepared by layer-by-layer (LbL)-adsorption of carrageenan and oligochitosan onto CaCO₃ microparticles with their subsequent dissolving after the treatment of EDTA. Entrapment of PCE was performed by using adsorption and co-precipitation techniques. The co-precipitation provided better entrapment of PCE into the carbonate matrix compared to adsorption. In vitro release kinetics (AGJ) was studied using artificial gastric juice. Using the model of acetate ulcer in rats it has been demonstrated that PCE released from the microcapsules accelerates gastric tissue repair.     

Electroantennogram measurements of atmospheric pheromone concentration after aerial and ground application of gypsy moth mating disruptants

Abstract:  A portable electroantennogram (EAG) sensor was used to measure relative atmospheric pheromone concentration in forest plots treated with aerial and ground applications of gypsy moth, Lymantria dispar (L.) (Lep., Lymantriidae), mating-disruption formulations. Five treatments (Disrupt II flakes with sticker, Disrupt II flakes without sticker, Disrupt II flakes in a sticker slurry, microcapsules and hand-applied Luretape), all applied at 75 g active ingredient per hectare and an untreated control were evaluated. Gypsy moth male catch in pheromone-baited traps and fertilization of deployed females were suppressed in all treatments, and no females deployed in treated plots produced more than 5% fertile eggs. Relative pheromone concentrations were significantly higher in the two treatments in which flakes were aerially applied with sticker and in the microcapsule treatment. Pheromone concentration measurements in the flakes without sticker and hand-applied treatments were not significantly different from those in the control. Mating success was negatively correlated with relative pheromone concentration. The ability of the EAG to detect differences in pheromone concentration that are correlated with mating success suggests that this could be a useful method for predicting the effectiveness of mating-disruption treatments.