Bioactive Protein and Peptide Release from a Mucoadhesive Electrospun Membrane.

Protein-based biologics constitute a rapidly expanding category of therapeutic agents with high target specificity. Their clinical use has dramatically increased in recent years, but administration is largely via injection. Drug delivery across the oral mucosa is a promising alternative to injections, in order to avoid the gastrointestinal tract and first-pass metabolism. Current drug delivery formulations include liquid sprays, mucoadhesive tablets and films, which lack dose control in the presence of salivary flow. To address this, electrospun membranes that adhere tightly to the oral mucosa and release drugs locally have been developed. Here, we investigated the suitability of these mucoadhesive membranes for peptide or protein release. Bradykinin (0.1%) or insulin (1, 3, and 5%) were incorporated by electrospinning from ethanol/water mixtures. Immersion of membranes in buffer resulted in the rapid release of bradykinin, with a maximal release of 70 ± 12% reached after 1 h. In contrast, insulin was liberated more slowly, with 88 ± 11, 69.0 ± 5.4, and 63.9 ± 9.0% cumulative release of the total encapsulated dose after 8 h for membranes containing 1, 3, and 5% w/w insulin, respectively. Membrane-eluted bradykinin retained pharmacological activity by inducing rapid intracellular calcium release upon binding to its cell surface receptor on oral fibroblasts, when examined by flow cytometry. To quantify further, time-lapse confocal microscopy revealed that membrane-eluted bradykinin caused a 1.58 ± 0.16 fold-change in intracellular calcium fluorescence after 10 s compared to bradykinin solution (2.13 ± 0.21), relative to placebo. In conclusion, these data show that electrospun membranes may be highly effective vehicles for site-specific administration of biotherapeutic proteins or peptides directly to the oral mucosa for either local or systemic drug delivery applications.

Electrospinning polymersomes into bead-on-string polyethylene oxide fibres for the delivery of biopharmaceuticals to mucosal epithelia.

Fibrous mucoadhesive polymer membranes prepared using electrospinning demonstrate many advantages for mucosal drug delivery compared to other formulations. Previous electrospun membrane formulations have been developed mainly for the delivery of small molecule drugs. There remains great potential to further develop the technology for the delivery of vesicular vectors that allow administration of advanced therapeutic agents. However, there are no previous reports demonstrating the release of intact drug delivery vesicles from electrospun materials. Here, we describe incorporation and release of protein-loaded polymersomes from polyethylene oxide (PEO)-based electrospun membranes. Polymersomes comprising a copolymer of glycerol monomethacrylate (GMA) and hydroxypropyl methacrylate (HPMA) were prepared using polymerization-induced self-assembly and incorporated within PEO membranes using bead-on-string electrospinning at approximately 40 % w/w by polymer mass. Super-resolution fluorescence imaging showed that the vesicles remained intact and retained their encapsulated protein load within the fibre beads. Transmission electron microscopy and dynamic light scattering demonstrated that polymersomes retained their morphology following release from the polymer fibres. F(ab) antibody fragments were encapsulated within polymersomes and then electrospun into membranes. 78 ± 13 % of the F(ab) remained encapsulated within polymersomes during electrospinning and retained functionality when released from electrospun membranes, demonstrating that the formulation is suitable for the delivery of biologics. Membranes were non-irritant to the oral epithelium and fluorescence microscopy detected accumulation of polymersomes within the epithelia following application. This innovative drug delivery approach represents a novel and potentially highly useful method for the administration of large molecular mass therapeutic molecules to diseased mucosal sites.

Electrospun patch delivery of anti-TNFα F(ab) for the treatment of inflammatory oral mucosal disease.

Chronic ulcerative oral mucosal inflammatory diseases, including oral lichen planus and recurrent aphthous stomatitis, are painful and highly prevalent, yet lack effective clinical management. In recent years, systemic biologic therapies, including monoclonal antibodies that block the activity of cytokines, have been increasingly used to treat a range of immune-mediated inflammatory conditions such as rheumatoid arthritis and psoriasis. The ability to deliver similar therapeutic agents locally to the oral epithelium could radically alter treatment options for oral mucosal inflammatory diseases, where pro-inflammatory cytokines, in particular tumour-necrosis factor-α (TNFα), are major drivers of pathogenesis. To address this, an electrospun dual-layer mucoadhesive patch comprising medical-grade polymers was investigated for the delivery of F(ab) biologics to the oral mucosa. A fluorescent-labelled F(ab) was incorporated into mucoadhesive membranes using electrospinning with 97% v/v ethanol as a solvent. The F(ab) was detected within the fibres in aggregates when visualised by confocal microscopy. Biotinylated F(ab) was rapidly eluted from the patch (97 ± 5% released within 3 h) without loss of antigen-binding activity. Patches applied to oral epithelium models successfully delivered the F(ab), with fluorescent F(ab) observed within the tissue and 5.1 ± 1.5% cumulative transepithelial permeation reached after 9 h. Neutralising anti-TNFα F(ab) fragments were generated from whole IgG by papain cleavage, as confirmed by SDS-PAGE, then incorporated into patches. F(ab)-containing patches had TNFα neutralising activity, as shown by the suppression of TNFα-mediated CXCL8 release from oral keratinocytes cultured as monolayers. Patches were applied to lipopolysaccharide-stimulated immune-competent oral mucosal ulcer equivalents that contained primary macrophages. Anti-TNFα patch treatment led to reduced levels of active TNFα along with a reduction in the levels of disease-implicated T-cell chemokines (CCL3, CCL5, and CXCL10) to baseline concentrations. This is the first report of an effective device for the delivery of antibody-based biologics to the oral mucosa, enabling the future development of new therapeutic strategies to treat painful conditions.

Mucoadhesive Electrospun Fibre-Based Technologies for Oral Medicine.

Oral disease greatly affects quality of life, as the mouth is required for a wide range of activities including speech, food and liquid consumption. Treatment of oral disease is greatly limited by the dose forms that are currently available, which suffer from short contact times, poor site specificity, and sensitivity to mechanical stimulation. Mucoadhesive devices prepared using electrospinning offer the potential to address these challenges by allowing unidirectional site-specific drug delivery through intimate contact with the mucosa and with high surface areas to facilitate drug release. This review will discuss the range of electrospun mucoadhesive devices that have recently been reported to address oral inflammatory diseases, pain relief, and infections, as well as new treatments that are likely to be enabled by this technology in the future.

Incorporation of lysozyme into a mucoadhesive electrospun patch for rapid protein delivery to the oral mucosa.

The delivery of biopharmaceuticals to the oral mucosa offers a range of potential applications including antimicrobial peptides to treat resistant infections, growth factors for tissue regeneration, or as an alternative to injections for systemic delivery. Existing formulations targeting this site are typically non-specific and provide little control over dose. To address this, an electrospun dual-layer mucoadhesive patch was investigated for protein delivery to the oral mucosa. Lysozyme was used as a model antimicrobial protein and incorporated into poly(vinylpyrrolidone)/Eudragit RS100 polymer nanofibers using electrospinning from an ethanol/water mixture. The resulting fibrous membranes released the protein at a clinically desirable rate, reaching 90 ± 13% cumulative release after 2 h. Dual fluorescent fibre labelling and confocal microscopy demonstrated the homogeneity of lysozyme and polymer distribution. High encapsulation efficiency and preservation of enzyme activity were achieved (93.4 ± 7.0% and 96.1 ± 3.3% respectively). The released lysozyme inhibited the growth of the oral bacterium Streptococcus ratti, providing further evidence of retention of biological activity and illustrating a potential application for treating and preventing oral infections. An additional protective poly(caprolactone) backing layer was introduced to promote unidirectional delivery, without loss of enzyme activity, and the resulting dual-layer patches displayed long residence times using an in vitro test, showing that the adhesive properties were maintained. This study demonstrates that the drug delivery system has great potential for the delivery of therapeutic proteins to the oral mucosa.