Bioactive Protein and Peptide Release from a Mucoadhesive Electrospun Membrane

Abstract

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.

Keywords: Bradykinin; Drug delivery; Electrospinning; Insulin; Mucoadhesion; Oral mucosa; Peptides.

Fig. 1

Characterisation of bradykinin-loaded mucoadhesive electrospun membranes. Bradykinin was incorporated into polymer fibres by mixing with ethanolic polymer solutions immediately before electrospinning and the resulting fibres imaged by SEM. SEM micrographs of a control electrospun fibres b electrospun fibres containing 0.1% w/w bradykinin (BK), scale bar = 20 µm. c Fibre diameter distribution presented as box and whisker plots displaying median, interquartile range and range (n = 30). Statistical analysis was performed by Student’s t-test

Fig. 2

Preparation of mucoadhesive electrospun fibres containing 0, 1, 3, 5% w/w insulin. Insulin was dissolved in 2% v/v acetic acid in PBS and mixed 1:4 v/v with an ethanolic polymer solution shortly before electrospinning. a SEM micrographs of electrospun fibres containing 0, 1 3, and 5% w/w insulin. b Fibre diameter distributions presented as median, interquartile range, and range. Data were analysed using one-way ANOVA with Tukey post hoc test. ***p < 0.001 (n = 30). c Surface pH of electrospun fibres presented as mean ± SD (n = 3) and analysed using one-way ANOVA with Tukey’s post hoc test

Fig. 3

Release of bradykinin and insulin from mucoadhesive membranes following immersion in PBS. Electrospun membrane samples were eluted in PBS at 37 °C. Bradykinin concentrations in the eluted PBS were measured by ELISA whilst insulin release was measured by BCA assay. a Percentage release of bradykinin over 240 minutes b cumulative insulin release per mg of membrane over 8 h. Data are presented as mean ± SD (n = 3). Statistical analysis for insulin was performed at the 8 h timepoint by one-way ANOVA with Tukey’s post hoc test

Fig. 4

Bradykinin functionality was preserved following release from electrospun mucoadhesive membranes. Intracellular calcium mobilisation in primary fibroblasts upon stimulation was determined over time using flow cytometry. Each dot represents a fluorescence measurement for an individual cell. Baseline fluorescence was acquired for 40 s before addition of samples (black arrow). a Placebo membrane eluent b recombinant bradykinin (BK) solution c BK electrospun membrane eluent d change in relative fluorescence units (RFU) were calculated by subtracting baseline median fluorescence from the maximal median fluorescence following sample addition. Data is presented as mean ± SD (n = 3) and analysed using one-way ANOVA with Tukey’s post hoc test. **p < 0.01, ***p < 0.001

Fig. 5

Membrane–eluted bradykinin increases intracellular calcium mobilisation in oral fibroblast monolayers. a Representative series of confocal micrographs showing the change in calcium mobilisation over time in primary fibroblast monolayers following treatment with PBS, placebo membrane eluent, bradykinin (BK) solution (2.5 ng/ml), or BK membrane eluent, scale bar = 500 μm. b Fold-change in fluorescence relative units (RFU) over time following addition of solutions. c Fold-change in RFU after 10 s for each solution. Data is presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. **p < 0.01, ***p < 0.001