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 one's understanding of the relative capacities of different APC to induce antigen-specific immune responses.

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.

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.

Amine composition influences apparent activity of enzyme in charged film microcapsules.

It has been shown that, when captured in charged film microcapsules prepared from spermine alginate, intact viruses retain infectivity, isolated viral proteins retain immunogenicity, and trypsin retains enzymatic activity. However, it was unclear whether the greater anionic strength of hemisulfate residues such as those in carrageenan might alter protein conformation and activity unfavourably in comparison with the lesser influence of alginate carboxylates. Further, the influence of the structure of the amine used to prepare the capsules was largely unknown. To examine these questions, trypsin, used as a model protein, was encased in microcapsules prepared from iota-carrageenan and oligoamines drawn from either the homologous series spermine, spermidine, putrescine or ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine. The gross structures of encapsulated and native trypsin were compared by denaturing electrophoresis and their enzymatic activity by the method of Hummel. In all encapsulations SDS PAGE gave no evidence of alteration of protein structure. When encapsulated, the apparent activity of trypsin was reduced by about 60 to 75%, but when the capsules were lysed in hypertonic saline activity was restored. This apparent reduction in activity is attributed to the diffusional barrier imposed by the encapsulating membrane but it should be recognized that it may be the result of reversible denaturation.

Influence of dextran molecular weight on capture in and release from decylamine carboxymethylcellulose capsules.

A series of dextran molecular weight markers were encapsulated in decylamine carboxymethylcellulose microcapsules to serve as probes of capsule retentivity. The capsules were prepared by allowing microdrops of aqueous sodium carboxymethylcellulose to fall into aqueous decylamine acetate solution. Salt exchange reaction at the droplet pseudointerface resulted in self-assembling films which essentially instantaneously enclosed the droplets. Concentrations of anionic polymer were varied in the range from 1-3%. Chromophore-bearing dextrans were incorporated into these capsules by blending the dextrans with the carboxymethylcellulose prior to the encapsulation step. Four dextrans of differing (light scattering) molecular weights were used: 2 x 10(6), 6 x 10(5), 7 x 10(4), and 1.9 x 10(4) amu. The mass balance of dextran retained in the capsules, released on washing the capsules or which escaped encapsulation was determined spectrophotometrically. To measure total dextran in a population of washed capsules, the capsules were lysed in a 0.3 M solution of sodium chloride. To monitor dextran release, washed capsules were suspended in water and dextran concentration in the supernatant was measured. Encapsulation efficiency exceeded 80% for high molecular weight dextran but was lower with the smaller dextrans.

Effect of water-based microencapsulation on protection against EDIM rotavirus challenge in mice.

We determined the capacity of microcapsules formed by the combination of sodium alginate, an aqueous anionic polymer, and spermine hydrochloride, an aqueous cationic amine, to enhance protection against rotavirus challenge in mice. Adult BALB/c mice were orally inoculated with either free or microencapsulated rotavirus (simian rotavirus strain RRV) and challenged 6 or 16 weeks later with murine rotavirus strain EDIM. Virus-specific humoral immune responses were determined at the time of challenge and 4 days after challenge by intestinal fragment culture. We found that spermine-alginate microcapsules enhanced protection against challenge 16 weeks after immunization but not 6 weeks after immunization. Quantities of virus-specific immunoglobulin A produced by small intestinal lamina propria lymphocytes were correlated with the degree of protection against challenge afforded by spermine-alginate microcapsules. Possible mechanisms by which microcapsules enhance protection against rotavirus challenge are discussed.

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.

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.

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.

Retention of trypsin activity in spermine alginate microcapsules.

We have previously shown virus particles encapsulated in aqueous spermine alginate constructs retain immunogenicity and infectivity both in vitro and in vivo. However, because virions are complex structures with multiple reinforcing components, it was uncertain if isolated single proteins would retain functional integrity when similarly encapsulated. To examine this question trypsin, used as a model protein, was blended with aqueous sodium alginate and the blend was dispersed as fine droplets in aqueous spermine hydrochloride to generate self-assembling, trypsin-containing microcapsules. Trypsin was assayed spectrophotometrically for retention of enzymatic activity using N-alpha-p-tosyl-L-arginine methyl ester as substrate. Neither of the encapsulating reagents alone inhibited enzyme activity. Enzyme that escaped capture was assayed directly in the manufacturing supernatant. In mass balance studies we found that about 20-30% of activity was retained in intact capsules with the remainder resident in the aqueous manufacturing supernatant and washes. However, we found that the capsule wall appeared to inhibit enzyme activity by retarding substrate diffusion into and product diffusion out from the capsules, as evidenced by an increase in activity on lysis. Thus, it is clear that a single protein, as represented by trypsin, can retain functional integrity when encapsulated in this all aqueous system.

Orally administered microencapsulated reovirus can bypass suckled, neutralizing maternal antibody that inhibits active immunization of neonates.

Purified reovirus serotype 1, encapsulated in biodegradable aqueous microcapsules, was found to bypass maternal antibody passively transferred by suckling to neonates. Genetically identical, immunocompetent F1 scid/+ mice were generated by the reciprocal crosses of C.B17 scid/scid and normal congenic +/+ adult mice. The immunocompetent +/+ dams were either orally infected with reovirus prior to mating or not. Thus, these immunocompetent F1 pups developed either in the absence or in presence of passively transferred maternal immunity. The F1 mice were orally immunized on day 10 with either live virus, microencapsulated reovirus, or empty microcapsules plus live virus. The immune responses were assessed in the neonatal gut-associated lymphoid tissues (GALT). Examination of reovirus specific immunoglobulin A in the serum and GALT, taken on days 7, 14, and 21 postimmunization, clearly demonstrated that microencapsulated reovirus could bypass the normal effect of maternal antibodies, passively acquired by suckling, to inhibit active priming of neonates by oral route. These observations seem relevant to the development of efficacious oral vaccines that also allow passive, protective immunity via suckled maternal antibodies while permitting active oral immunization of neonates.

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.

A method to attach lectins to the surface of spermine alginate microcapsules based on the avidin biotin interaction.

To serve as model cell or tissue specific drug delivery systems spermine alginate capsules were surface modified by attachment of proteins with carbohydrate specific binding properties, i.e. lectins. In the first of a two step process avidin was covalently bound to the capsule surfaces using a hydroxy-succinimide catalysed carbodiimide reagent. Surface bound avidin was quantitated by lysing the capsules in hypertonic phosphate buffered saline (PBS) and measuring the change in absorbance at 500 nm in the interaction with 2-(4'hydroxyazobenzene) benzoic acid. A time course study of the avidin-binding reaction showed avidin surface concentrations averaged 5.4 nM/cm2 after 16h. In the second step avidin-coated capsules were incubated in aqueous solutions of biotinylated-lectin derivatives. To quantitate lectin uptake, avidin-coated capsules were lysed in PBS and titrated to the turbidance endpoint with ten different biotinylated lectins. Lectin surface concentrations ranged from 2.0 to 6.8 nm/cm2, well below the theoretical limit of 4 biotinylated ligands per molecule of avidin. Individual lectins bound to capsular surfaces retained their respective ligand specific binding properties as demonstrated by measurement of the selective saturable uptake of radiolabeled ligands when incubated with variously lectin coated capsules. The presence of porcine gastric mucin in concentrations up to 4% w/v did not inhibit binding of 14C-labelled mannose or galactose by concanavalin A-coated capsules.

Oral inoculation of mice with low doses of microencapsulated, noninfectious rotavirus induces virus-specific antibodies in gut-associated lymphoid tissue.

The capacity of an aqueous-based system of microencapsulation to enhance virus-specific humoral immune responses was evaluated in mice orally inoculated with noninfectious rotavirus (simian rotavirus strain RRV). Mice were orally inoculated with 1.75 or 0.35 microgram of inactivated RRV (iRRV) or microencapsulated iRRV. Sera, intestinal contents, and organ cultures of gut-associated lymphoid tissues (GALT) were tested for the presence of rotavirus-specific antibodies. Virus-specific IgA was produced by small intestine lamina propria lymphocytes in animals inoculated with 1.75 or 0.35 microgram of microencapsulated virus, but not in mice inoculated with unencapsulated virus. Virus-specific IgA in sera and intestinal contents were not predictive of intestinal organ culture responses. Microencapsulation may be an efficient way of inducing virus-specific immune responses in GALT after oral inoculation with small quantities of viral antigen. In addition, delayed release of virus from microcapsules may obviate the need for booster immunizations.

Enhancement by microencapsulation of rotavirus-specific intestinal immune responses in mice assessed by enzyme-linked immunospot assay and intestinal fragment culture.

The capacity of microencapsulation to enhance the humoral immune response to rotavirus in the gut-associated lymphoid tissue (GALT) of mice was determined by using a system of microencapsulation based on the ionic linkage of aqueous anionic polymers and an aqueous amine. Inoculation of mice with microencapsulated rotavirus enhanced the frequencies of virus-specific IgA-secreting cells in the lamina propria as well as the quantities of virus-specific IgA produced in GALT. In addition, an enhanced virus-specific immune response was associated with enhanced production of presumably polyclonal, non-rotavirus-specific antibodies in GALT. The mechanism by which microencapsulation enhances the humoral immune response remains to be determined.

Enhancement of rotavirus immunogenicity by microencapsulation.

It was determined whether microencapsulation of rotavirus enhanced virus-specific immunity in mice. Combinations of several water-soluble anionic polymers and amines were tested for their capacity to form microcapsules which were stable in the presence of simulated gastric acid. Using the combinations of sodium alginate and spermine hydrochloride or sodium chondroitin sulfate and spermine hydrochloride we found that microcapsules (1) captured infectious rotavirus, (2) penetrated into the persisted in gut-associated lymphoid tissue (GALT) after oral inoculation, (3) delivered rotavirus antigen to GALT at levels greater than those detected after oral inoculation with free virus, and (4) enhanced the virus-specific humoral immune response after oral or parenteral immunization.

A study of the interaction of selected drugs and plastic syringes.

It is common hospital pharmacy practice to preload syringes with selected drugs and store them ready for use. Because the several components of syringes, (such as barrels, gasket seals, etc.) may vary among manufacturers, there exists the possibility that syringe components of differing provenance might interact e.g., by sorption, with stored drugs to differing degrees. To examine possible interactions, three brands of commercially available syringes were compared to determine what influence, if any, short term storage of injectable solutions might exert on the solutions or the syringes. Four drugs; dexamethasone sodium phosphate, diazepam, diatrizoate meglumine and nitroglycerin USP were individually loaded into 3 mL syringes and stored at temperatures between -20 degrees C and + 25 degrees C for periods from 6 hours to 30 days. The syringes were examined for any gross changes. Drug solutions were analyzed after storage to determine the presence of organic leachates from the syringes and any change from original drug concentration values. No syringes showed gross physical changes after storage with drug solution nor were any drug solutions found to contain leachates on gas chromatographic-mass spectroscopic analysis. Drug concentrations were seen to change following storage, the greatest changes occurring with the highly lipophilic drugs dexamethasone and diazepam. In most instances loss of drug concentration was most rapid at room temperature. Although there were clear differences among the three brands of syringe, no overall pattern emerged which might allow the selection or rejection of one syringe over another for the extemporaneous preloading of the drugs examined.

The influence of a block copolymer on the efficiency of interfacial microencapsulation.

Both the presence of poloxamer in concentrations from 1 to 100 per cent w/v and variation in non-aqueous phase ratio in the range 0.2 to 2.0 influence the extent to which acetanilide is incorporated into piperazine arabate microcapsules compared to acetanilide encapsulation without polymer. Low poloxamer concentrations and phase ratios nearly double the amount of acetanilide encapsulated, but high concentrations or phase ratios or both decrease the efficiency of incorporation.