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The COVID-19 pandemic has accelerated pre-clinical and clinical development of microneedle-based drug delivery technology. However the regulatory science of this emerging dosage form is immature and explicit regulatory guidance is limited. A group of international stakeholders has formed to identify and address key issues for the regulatory science of future products that combine a microneedle device and active pharmaceutical ingredient (in solid or semi-solid state) in a single entity that is designed for application to the skin. Guided by the principles of Quality by Design (QbD) and informed by consultation with wider stakeholders, this 'White Paper' describes fundamental elements of the work in an effort to harmonise understanding, stimulate discussion and guide innovation. The paper discusses classification of the dosage form (combination/medicinal product), the regulatory nomenclature that is likely to be adopted and the technical vocabulary that best describes its form and function. More than twenty potential critical quality attributes (CQAs) are identified for the dosage form, and a prioritisation exercise identifies those CQAs that are most pertinent to the dosage form and that will likely require bespoke test methods (delivered dose, puncture performance) or major adaptions to established compendial test methods (dissolution). Hopefully the work will provide a platform for the development of dosage form specific guidance (from regulatory authorities and/or international pharmacopoeias), that expedites clinical translation of safe and effective microneedle-based products., Competing Interests: Declaration of competing interest Prausnitz is an inventor of patents, founder/shareholder of companies, and consultant to companies developing MAP technologies. This conflict of interest is managed by the Georgia Institute of Technology. Coulman and Birchall are inventors of patents. This conflict of interest is managed by Cardiff University. Ryan Donnelly is an inventor of patents that have been licensed to companies developing microneedle-based products and is a paid advisor to companies developing microneedle-based products. The resulting potential conflict of interest has been disclosed and is managed by Queen's University Belfast. The companies had no role in the design of the manuscript, in the collection, analyses or interpretation of the various studies reviewed, in the writing of the manuscript or in the decision to publish., (Copyright © 2024 The Author. Published by Elsevier B.V. All rights reserved.)

Purpose: Small interfering RNAs (siRNAs) specifically and potently inhibit target gene expression. Pachyonychia congenita (PC) is a skin disorder caused by mutations in genes encoding keratin (K) 6a/b, K16, and K17, resulting in faulty intermediate filaments. A siRNA targeting a single nucleotide, PC-relevant mutation inhibits K6a expression and has been evaluated in the clinic with encouraging results., Procedures: To better understand the pathophysiology of PC, and develop a model system to study siRNA delivery and visualize efficacy in skin, wild type (WT) and mutant K6a complementary DNAs (cDNAs) were fused to either enhanced green fluorescent protein or tandem tomato fluorescent protein cDNA to allow covisualization of mutant and WT K6a expression in mouse footpad skin using a dual fluorescence in vivo confocal imaging system equipped with 488 and 532 nm lasers., Results: Expression of mutant K6a/reporter resulted in visualization of keratin aggregates, while expression of WT K6a/reporter led to incorporation into filaments. Addition of mutant K6a-specific siRNA resulted in inhibition of mutant, but not WT, K6a/reporter expression., Conclusions: Intravital imaging offers subcellular resolution for tracking functional activity of siRNA in real time and enables detailed analyses of therapeutic effects in individual mice to facilitate development of nucleic acid-based therapeutics for skin disorders.

Background: Pachyonychia congenita (PC) is a skin disorder resulting from mutations in keratin (K) proteins including K6a, K6b, K16, and K17. One of the major symptoms is painful plantar keratoderma. The pathogenic sequelae resulting from the keratin mutations remain unclear., Objective: To better understand PC pathogenesis., Methods: RNA profiling was performed on biopsies taken from PC-involved and uninvolved plantar skin of seven genotyped PC patients (two K6a, one K6b, three K16, and one K17) as well as from control volunteers. Protein profiling was generated from tape-stripping samples., Results: A comparison of PC-involved skin biopsies to adjacent uninvolved plantar skin identified 112 differentially-expressed mRNAs common to patient groups harboring K6 (i.e., both K6a and K6b) and K16 mutations. Among these mRNAs, 25 encode structural proteins including keratins, small proline-rich and late cornified envelope proteins, 20 are related to metabolism and 16 encode proteases, peptidases, and their inhibitors including kallikrein-related peptidases (KLKs), and serine protease inhibitors (SERPINs). mRNAs were also identified to be differentially expressed only in K6 (81) or K16 (141) patient samples. Furthermore, 13 mRNAs were identified that may be involved in pain including nociception and neuropathy. Protein profiling, comparing three K6a plantar tape-stripping samples to non-PC controls, showed changes in the PC corneocytes similar, but not identical, to the mRNA analysis., Conclusion: Many differentially-expressed genes identified in PC-involved skin encode components critical for skin barrier homeostasis including keratinocyte proliferation, differentiation, cornification, and desquamation. The profiling data provide a foundation for unraveling the pathogenesis of PC and identifying targets for developing effective PC therapeutics., (Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

The development of siRNA-based gene silencing therapies has significant potential for effectively treating debilitating genetic, hyper-proliferative or malignant skin conditions caused by aberrant gene expression. To be efficacious and widely accepted by physicians and patients, therapeutic siRNAs must access the viable skin layers in a stable and functional form, preferably without painful administration. In this study we explore the use of minimally-invasive steel microneedle devices to effectively deliver siRNA into skin. A simple, yet precise microneedle coating method permitted reproducible loading of siRNA onto individual microneedles. Following recovery from the microneedle surface, lamin A/C siRNA retained full activity, as demonstrated by significant reduction in lamin A/C mRNA levels and reduced lamin A/C protein in HaCaT keratinocyte cells. However, lamin A/C siRNA pre-complexed with a commercial lipid-based transfection reagent (siRNA lipoplex) was less functional following microneedle coating. As Accell-modified "self-delivery" siRNA targeted against CD44 also retained functionality after microneedle coating, this form of siRNA was used in subsequent in vivo studies, where gene silencing was determined in a transgenic reporter mouse skin model. Self-delivery siRNA targeting the reporter (luciferase/GFP) gene was coated onto microneedles and delivered to mouse footpad. Quantification of reporter mRNA and intravital imaging of reporter expression in the outer skin layers confirmed functional in vivo gene silencing following microneedle delivery of siRNA. The use of coated metal microneedles represents a new, simple, minimally-invasive, patient-friendly and potentially self-administrable method for the delivery of therapeutic nucleic acids to the skin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Treatment of skin disorders with short interfering RNA (siRNA)-based therapeutics requires the development of effective delivery methodologies that reach target cells in affected tissues. Successful delivery of functional siRNA to the epidermis requires (1) crossing the stratum corneum, (2) transfer across the keratinocyte membrane, followed by (3) incorporation into the RNA-induced silencing complex. We have previously demonstrated that treatment with microneedle arrays loaded with self-delivery siRNA (sd-siRNA) can achieve inhibition of reporter gene expression in a transgenic mouse model. Furthermore, treatment of human cultured epidermal equivalents with sd-siRNA resulted in inhibition of target gene expression. Here, we demonstrate inhibition of CD44, a gene that is uniformly expressed throughout the epidermis, by sd-siRNA both in vitro (cultured human epidermal skin equivalents) and in vivo (full-thickness human skin equivalents xenografted on immunocompromised mice). Treatment of human skin equivalents with CD44 sd-siRNA markedly decreased CD44 mRNA levels, which led to a reduction of the target protein as confirmed by immunodetection in epidermal equivalent sections with a CD44-specific antibody. Taken together, these results demonstrate that sd-siRNA, delivered by microneedle arrays, can reduce expression of a targeted endogenous gene in a human skin xenograft model.

N,N-diethyl-3-methylbenzamide (DEET) is popular insect repellent which is considered safe and effective, yet is subject to considerable skin absorption. Skin absorption decreases effective repellency since less DEET is available for evaporation. We have investigated the extent to which DEET skin absorption can be reduced and evaporation sustained through encapsulation. DEET permeation through human skin in vitro was measured for an ethanolic solution standard and for two novel topical controlled-release formulations in which the DEET active material was temporarily sequestered within a permeable, charged-film microcapsule. Evaporation measurements were gathered using Tenax TA cartridges and a sampling pump drawing air over the skin. Three formulations were studied: a previously reported microcapsule formulation (Formulation A); a newly-developed microcapsule formulation (Formulation B); and a non-encapsulated ethanol control solution. Formulation B led to a 30% reduction in DEET permeation versus control. The two microcapsule DEET formulations exhibited 36-40% higher cumulative evaporation from the skin than did the control. The vapor trapping measurements in vitro show that Formulation B provided more than 48h of effective evaporation rate for repellency, while Formulation A provided less than 35h and the ethanol control less than 15h. This establishes a technical advantage for the controlled-release approach., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.

Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation.

The insect repellent N,N-diethyl-3-methylbenzamide (DEET) is widely used and is generally regarded as safe when used according to label instructions. Yet many studies have shown it to be absorbed through the skin. The objective of this study was to determine whether the skin absorption rate of DEET could be decreased while maintaining an evaporation rate consistent with effective repellency. To this end, an aqueous suspension containing 14C-DEET (15%w/w) entrapped in walled polysaccharide microcapsules was prepared and tested for skin absorption in vitro using modified Franz cells maintained in a fume hood. The control formulation was 15%w/w DEET in ethanol. Two doses (3 microL and 5 microL per 0.79 cm2 cell) of each formulation were applied to split-thickness human cadaver skin (n=8/dose), and permeation was monitored for 24h. The microencapsulated DEET formulation lead to a 25-35% reduction of radiolabel permeation compared to the ethanolic DEET formulation. Skin levels of radioactivity at 24h were comparable, indicating that DEET evaporation from the microencapsulated formulation was comparable to or greater than that from ethanol. Hence microencapsulation increased the ratio of DEET evaporation rate to skin penetration rate relative to unencapsulated control in this in vitro study.

The New York 1999 strain of West Nile virus (WNV) is nearly 100% fatal in the American crow (Corvus brachyrhynchos). We evaluated four WNV vaccine formulations in American crows, including intramuscular (i.m.) DNA vaccine, i.m. DNA vaccine with adjuvant, orally administered microencapsulated DNA vaccine, and i.m. killed vaccine. Neutralizing antibodies developed in approximately 80% of crows that received the DNA vaccine i.m. (with or without adjuvant), and in 44% that received the killed vaccine. However, no crows that received the oral microencapsulated DNA vaccine or the placebo developed WNV antibodies. All crows were challenged 10 wk after initial vaccination. No unvaccinated crows survived challenge, and survival rates were 44% (i.m. DNA vaccine), 60% (i.m. DNA vaccine with adjuvant), 0% (oral microencapsulated DNA vaccine), and 11% (killed vaccine). Peak viremia titers in the birds that survived were significantly lower as compared to titers in birds that died. Parenteral administration of a WNV DNA vaccine was associated with reduced mortality but did not provide sterile immunity.

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.

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 9-15, 2000)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 ionization constants in water for six 3-substituted azuloic acids were determined spectrophotometrically. Conversion of these physical constants to their pKa values allowed a set of Hammett-type sigma values for the substituents on these acids to be calculated. Determination of partition coefficients for nine 1-substituted azulenes allowed Hansch-type pi values to be determined, using azulene as the model compound.

This paper describes the acute toxicity of two azulylamides and six azulenecarboxamides and the nerve conduction-inhibiting properties of some of these compounds. The azulene derivatives are compared to their benzenoid prototypes, lidocaine and procainamide, as part of a continuing investigation of the biological properties of nonbenzenoid aromatic compounds.

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.

As part of a search for phencyclidine (PCP) antagonists, a series of eleven aryl-substituted PCP analogs were prepared and characterized. TLC and GC elution profiles of these derivatives are described in a number of systems allowing for the tentative identification of the PCP analogs. Further, MS, NMR, UV/vis and IR spectral tabulations are provided. Correlation of chromatographic character with physical organic structure parameters, such as those of Hammet, showed good predictability.

Azulenes reacted unexpectedly and readily with thionyl chloride to give sulfonic acid chlorides and bisthioethers. The sulfonic acids but not the thioethers have antibacterial activity.