Preparation and Characterization of an Oral Vaccine Formulation Using Electrosprayed Chitosan Microparticles.

Chitosan particles loaded with the antigen ovalbumin (OVA) and the adjuvant Quil-A were produced by electrospray, using mixtures of water/ethanol/acetic acid as a solvent. Three different chitosans designed as HMC+70, HMC+85, and HMC+90 (called as 705010, 855010, and 905010) were tested and its efficacy to be used in oral vaccine delivery applications was investigated. The morphology, size, and zeta potential of the produced particles were investigated, together with the encapsulation efficiency and release of OVA from the three chitosan formulations. Moreover, the mucoadhesion and cytotoxicity of the chitosan microparticles was examined. All the three formulations with OVA and Quil-A were in the micrometer size range and had a positive zeta potential between 46 and 75 mV. Furthermore, all the three formulations displayed encapsulation efficiencies above 80% and the release of OVA over a period of 80 h was observed to be between 38 and 47%. None of the developed formulations exhibited high mucoadhesive properties, either cytotoxicity. The formulation prepared with HMC+70, OVA, and Quil-A had the highest stability within 2 h in buffer solution, as measured by dynamic light scattering. The electrosprayed formulation consisting of HMC+70 with OVA and Quil-A showed to be the most promising as an oral vaccine system.

Development of electrosprayed mucoadhesive chitosan microparticles.

The efficacy of chitosan (CS) to be used as drug delivery carrier has previously been reported. However, limited work has been pursued to produce stable and mucoadhesive CS electrosprayed particles for oral drug delivery, which is the aim of this study. Various CS types with different molecular weight (MW), degree of deacetylation (DD), and degree of polymerization (DP) were assessed. In addition, the effect of the solvent composition was also investigated. Results showed that stable CS electrosprayed particles can be produced by dissolving 3% w/v of low MW CS in mixtures of aqueous acetic acid and ethanol (50/50% v/v). The stable CS particles displayed diameters of approximately 1 µm as determined by dynamic light scattering. The zeta potential of these particles was found to be approximately 40 mV confirming the mucoadhesion properties of these CS electrosprayed particles and its potential to be used as drug delivery carrier.

Innovative Methods and Applications in Mucoadhesion Research.

The present review is aimed at elucidating relatively new aspects of mucoadhesion/mucus interaction and related phenomena that emerged from a Mucoadhesion workshop held in Munster on 2-3 September 2015 as a satellite event of the ICCC 13th-EUCHIS 12th. After a brief outline of the new issues, the focus is on mucus description, purification, and mucus/mucin characterization, all steps that are pivotal to the understanding of mucus related phenomena and the choice of the correct mucosal model for in vitro and ex vivo experiments, alternative bio/mucomimetic materials are also presented. Then a selection of preparative techniques and testing methods are described (at molecular as well as micro and macroscale) that may support the pharmaceutical development of mucus interactive systems and assist formulators in the scale-up and industrialization steps. Recent applications of mucoadhesive systems (including medical devices) intended for different routes of administration (oral, gastrointestinal, vaginal, nasal, ocular, and intravesical) and for the treatment of difficult to treat pathologies or the alleviation of symptoms are described.

Interactions between Surfactants in Solution and Electrospun Protein Fibers: Effects on Release Behavior and Fiber Properties.

Intermolecular interaction phenomena occurring between endogenous compounds, such as proteins and bile salts, and electrospun compounds are so far unreported, despite the exposure of fibers to such biorelevant compounds when applied for biomedical purposes, e.g., tissue engineering, wound healing, and drug delivery. In the present study, we present a systematic investigation of how surfactants and proteins, as physiologically relevant components, interact with insulin-loaded fish sarcoplasmic protein (FSP) electrospun fibers (FSP-Ins fibers) in solution and thereby affect fiber properties such as accessible surface hydrophilicity, physical stability, and release characteristics of an encapsulated drug. Interactions between insulin-loaded protein fibers and five anionic surfactants (sodium taurocholate, sodium taurodeoxycholate, sodium glycocholate, sodium glycodeoxycholate, and sodium dodecyl sulfate), a cationic surfactant (benzalkonium chloride), and a neutral surfactant (Triton X-100) were studied. The anionic surfactants increased the insulin release in a concentration-dependent manner, whereas the neutral surfactant had no significant effect on the release. Interestingly, only minute amounts of insulin were released from the fibers when benzalkonium chloride was present. The FSP-Ins fibers appeared dense after incubation with this cationic surfactant, whereas high fiber porosity was observed after incubation with anionic or neutral surfactants. Contact angle measurements and staining with the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid indicated that the FSP-Ins fibers were hydrophobic, and showed that the fiber surface properties were affected differently by the surfactants. Bovine serum albumin also affected insulin release in vitro, indicating that also proteins may affect the fiber performance in an in vivo setting.

Bioactive protein-based nanofibers interact with intestinal biological components resulting in transepithelial permeation of a therapeutic protein.

Proteins originating from natural sources may constitute a novel type of material for use in drug delivery. However, thorough understanding of the behavior and effects of such a material when processed into a matrix together with a drug is crucial prior to further development into a drug product. In the present study the potential of using bioactive electrospun fish sarcoplasmic proteins (FSP) as a carrier matrix for small therapeutic proteins was demonstrated in relation to the interactions with biological components of the intestinal tract. The inherent structural and chemical properties of FSP as a biomaterial facilitated interactions with cells and enzymes found in the gastrointestinal tract and displayed excellent biocompatibility. More specifically, insulin was efficiently encapsulated into FSP fibers maintaining its conformation, and subsequent controlled release was obtained in simulated intestinal fluid. The encapsulation of insulin into FSP fibers provided protection against chymotrypsin degradation, and resulted in an increase in insulin transport to around 12% without compromising the cellular viability. This increased transport was driven by interactions upon contact between the nanofibers and the Caco-2 cell monolayer leading to the opening of the tight junction proteins. Overall, electrospun FSP may constitute a novel material for oral delivery of biopharmaceuticals.

Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes.

Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 ± 3 nm, and the zeta potential was -42 ± 0.3 mV. The NCXs were stable in water, gastric buffer, intestinal buffer and HEPES buffered glycose, and at all pH values from 2 to 9 except pH 3, where they aggregated. When proteolytic enzymes were present in the buffer, the NCXs were degraded. Only at high concentrations the NCXs caused a decreased viability in HeLa and U2OS cell lines. The simple processing procedure and the high stability of the NCXs, makes them excellent candidates for use in the food and pharmaceutical industry.

Bioactive electrospun fish sarcoplasmic proteins as a drug delivery system.

Nano-microfibers were made from cod (Gadus morhua) sarcoplasmic proteins (FSP) (Mw<200kDa) using the electrospinning technique. The FSP fibers were studied by scanning electron microscopy, and the fiber morphology was found to be strongly dependent on FSP concentration. Interestingly, the FSP fibers were insoluble in water. However, when exposed to proteolytic enzymes, the fibers were degraded. The degradation products of the FSP fibers proved to be inhibitors of the diabetes-related enzyme DPP-IV. The FSP fibers may have biomedical applications, among others as a delivery system. To demonstrate this, a dipeptide (Ala-Trp) was encapsulated into the FSP fibers, and the release properties were investigated in gastric buffer and in intestinal buffer. The release profile showed an initial burst release, where 30% of the compound was released within the first minute, after which an additional 40% was released (still exponential) within the next 30min (gastric buffer) or 15min (intestinal buffer). The remaining 30% was not released in the timespan of the experiment.

Identification of catechols as histone-lysine demethylase inhibitors.

Identification of inhibitors of histone-lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA-based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity in the low µM range (1, a catechol), a subset of structurally related compounds was selected and tested against a panel of HDMs. In this subset, two inhibitors (2 and 10) had comparable affinities towards KDM4C and KDM6A but no effect on PHF8. One inhibitor restored H3K9me3 levels in KDM4C transfected U2-OS cells.