Tully J. Speaker

Aqueous-based microcapsules are detected primarily in gut-associated dendritic cells after oral inoculation of mice

We previously found that aqueous-based microencapsulation enhanced virus-specific humoral immune responses after oral inoculation of mice. However, the mechanism by which microencapsulation enhances immunogenicity remains unclear. We found that spermine-alginate microcapsules were detected primarily in gut-associated dendritic cells (i.e. CD11c/CD18+, Ia+, CD11b-, CD45R-) after oral inoculation of adult mice. Microencapsulation may enhance immunogenicity by involving antigen presenting cells which are more efficient than those recruited during natural infection.

Aqueous-based microencapsulation enhances virus-specific humoral immune responses in mice after parenteral inoculation

Vaccines are commonly administered by the parenteral route. Therefore, adjuvant strategies which include parenteral immunization may improve the efficacy of a number of current vaccines. The capacity of aqueous-based microencapsulation to enhance virus-specific IgG responses in mice inoculated intramuscularly with small quantities of antigen was evaluated. Mice were inoculated with either 10(4), 10(3), or 10(2) p.f.u. of microencapsulated rotavirus (bovine strain WC3), placebo microcapsules plus free virus, or virus alone. Mice were subsequently bled 1, 2, 4, 6, and 9 months after inoculation. Microencapsulation of rotavirus enhanced virus-specific humoral immune responses. In addition, virus-containing microcapsules composed of spermine-chondroitin sulfate induced levels of virus-specific antibodies greater than those found after inoculation with virus-containing microcapsules composed of spermine-alginate. Mechanisms by which microencapsulation may enhance virus-specific humoral immunity are discussed.

Water-Based Microsphere Delivery System for Proteins

This paper describes formulation of a model protein, horseradish peroxidase (HRP), in a water based microcapsule delivery system and demonstrates the utility of this delivery system for proteins. Aqueous solutions (1 mg/mL) of the enzyme were separately blended with aqueous solutions of the neutral sodium salt of the anionic polymer iota carrageenan (0.6 mM in repeat unit). These blends were instilled as uniform microdroplets into aqueous solutions of a series of eleven mono-, di-, or oligo-amines (as neutral hydrochloride or acetate salts). Essentially instantaneous salt exchange interaction of the sodium salt of anionic polymer with amine hydrochloride resulted in formation of microparticles of amine/polymer complex. The enzyme was captured in the resulting capsules. The particles were washed by repeated centrifugation and resuspension in water and their particle size distribution was determined. HRP in washed pelleted microspheres was analyzed for fragmentation/aggregation by SDS-PAGE and size exclusion chromatography, for unfolding by fluorescence spectroscopy, and for specific enzymatic activity, capture efficiency and release studies by absorbance spectroscopy. Dependent on amine employed, capture efficiencies ranged from 1 to 72%. Encapsulation produced no adverse effect on protein size as no molecular fragments or aggregates were visible below or above 44 kDa. The tryptophan fluorescence spectrum of the protein did not change after encapsulation indicating no conformational change in tertiary structure. There was an apparent substrate diffusion related reduction in activity of encapsulated HRP, but almost 100% of activity was recovered on lysis of the capsules. It is concluded that water based charged film encapsulation used as a drug delivery system for proteins does not alter structural conformation or specific activity of the model protein tested and provides protein release at a constant rate.

Effect of microencapsulation on immunogenicity of a bovine herpes virus glycoprotein and inactivated influenza virus in mice

We previously found that aqueous-based spermine-alginate or spermine-chondroitin sulfate microcapsules enhanced rotavirus-specific humoral immune responses after intramuscular inoculation of mice. To extend our observations with whole, infectious rotavirus to vaccine strategies which include inactivated virus and purified proteins, we determined the capacity of aqueous-based microcapsules to enhance virus-specific immune responses to bovine herpes virus type 1 glycoprotein D (BHV-1-gD) or ether-treated influenza virus. We found that spermine-alginate microcapsules decreased the quantity of BHV-1-gD necessary to induce protein-specific antibodies about 5000-fold. However, spermine-alginate microcapsules did not enhance influenza virus-specific antibody responses. Microcapsules composed of spermine-chondroitin sulfate did not enhance either BHV-1-gD or influenza virus-specific immune responses. Possible mechanisms of enhancement of virus-specific antibody responses by microencapsulation are discussed.

Development and characterization of different low methoxy pectin microcapsules by an emulsion–interface reaction technique

In the controlled release area, biodegradable microcapsules are one of the most useful devices to deliver materials in an effective, prolonged and safe manner. A new charged film microcapsular carrier system, using three different pectins, is described. The study utilized pectin microcapsules prepared by two encapsulation mechanisms of interfacial reaction explored through interaction of charged droplet–oil-anionic surfactant-calcium or oil-cationic surfactant with negatively charged pectin. A method for drug encapsulation was developed based on the type of pectin, surfactants and emulsification technique. Both types of surfactant, anionic sodium dodecyl sulphate (SDS) and cationic benzalkonium chloride (BzACl) promoted polymer film formation on the oil droplet surfaces, probably through cross-linking and electrostatic interaction, respectively. Microcapsules consisting of pectin as shell and hydrophobic oil as core were characterized. The resulting microcapsules were relatively small particles (d<3 µm), had high total particle number, specific surface area and drug encapsulation efficiency. They also demonstrated good stability with minimum particle aggregation. Correlation between physicochemical and drug release kinetic parameters were investigated with regard to the effect of pectin macromolecular structure and nature of surfactant used as a counterion in the manufacturing of microcapsules. The release rate of the encapsulated material (prednisolone) in three microcapsules can be controlled by manipulating the conformational flexibility of pectins in the presence of different counterions. As a result, biodegradable pectin microcapsules offer a novel approach for developing sustained release drug delivery systems that have potential for colonic drug delivery.

Carrageenan as an anionic polymer for aqueous microencapsulation.

All aqueous microencapsulation involving the use of the anionic polymer alginate as its amine salt with spermine has been recently studied for the encapsulation of live viruses and isolated proteins. It was of interest to study the influence of another anionic polymer, carrageenan, on the encapsulation of a single protein. Trypsin, used as a the model protein, was encapsulated in spermine carrageenan macrocapsules. It was found that trypsin retains its structure as evidenced by SDS-PAGE analysis and its functional integrity measured as specific enzymatic activity. Thus, carrageenan can serve as anionic polymer in all aqueous charged film encapsulation systems.

A quantitative luminescence assay for measuring cell uptake of aqueous-based microcapsules in vitro

We recently developed a system of microencapsulation consisting of aqueous-based polymers (e.g. alginate) and aqueous amines (e.g. spermine). We found that microencapsulation enhanced virus-specific protective immune responses. In addition, we found that microencapsulation may enhance virus-specific immune responses by selecting for antigen-presenting cells (APC) that are more efficient at processing and presenting viral antigens than those involved after natural infection. To determine the intracellular trafficking patterns and fate of microcapsules within APC, we developed a luminescence assay that permits the determination of specific quantities of proteins introduced into cells by microcapsules. We found that the time-dependent uptake of horseradish peroxidase (HRP)-labeled microcapsules was accurately detected in lysates of peritoneal exudate cells using luminol. The amplitude of HRP-catalyzed chemiluminescence in cell lysates correlated with the capture efficiency and retention kinetics of HRP in three different microcapsule preparations. HRP was most efficiently captured and retained by linking biotinylated HRP to microcapsulses chemically modified at the amine moiety with egg avidin. This preparation yielded more accurate and sensitive quantitation of HRP contained within cells than preparations capturing HRP or HRP-conjugated goat antibody into the microcapsular matrix by ionic interactions.

Determination of efficiency of attachment of biotinylated antibodies to avidin-linked, aqueous-based microcapsules by flow cytometry.

We used flow cytometry to determine the percentage of aqueous-based microcapsules bearing antibodies specific for various antigen-presenting cells (APCs) within a given population of putative APC-specific microcapsules. Flow cytometry offers a high-throughput, rapid and simple method to analyze antibody binding to noncellular, nonspherical material.

Specific attachment of aqueous-based microcapsules to macrophages, B cells, and dendritic cells in vitro

Microcapsules were previously prepared composed of aqueous anionic polymers (e.g. alginate) and aqueous amines (e.g. spermine) and it was found that the aqueous-based microcapsules enhanced rotavirus-specific immune responses after oral or parenteral immunization of mice. In these studies, one has modified the amine moiety of aqueous-based microcapsules to bind covalently to avidin and the avidin-bearing microcapsules were linked to biotinylated antibodies specific for surface markers on murine macrophages, dendritic cells, or B cells. Using fluorescence flow cytometry, it was found that antibody-coated microcapsules bound specifically to antigen-presenting cells (APC) in vitro. The availability of APC-specific microcapsules should allow for the uptake of antigens by specific APC, and further ones understanding of the relative capacities of different APC to induce antigen-specific immune responses.

Viral Microencapsulation Delays Protection after Intramuscular Inoculation of Mice with Rotavirus

Intramuscular (i.m.) immunization of mice with microencapsulated infectious rotavirus delayed protection against rotavirus challenge. Whereas unencapsulated rotavirus induced protection 6 but not 16 weeks after i.m. immunization, microencapsulated rotavirus induced protection 16 but not 6 weeks after i.m. immunization. Protection 16 weeks after immunization was associated with increased production of virus-specific immunoglobulin G (IgG) by small intestinal lamina propria (LP) lymphocytes. These findings demonstrate that i.m. immunization with microencapulated rotavirus can delay the onset of protection against challenge and the development of intestinal humoral immune responses. Simultaneous i.m. immunization with both unencapsulated and microencapsulated virus may induce prolonged protection from an intestinal pathogen, thus obviating the need for booster doses. Possible mechanisms of delayed protection induced by i.m. immunization with microencapsulated immunogens are discussed.