Boosting buccal drug absorption: Mechanistic insights into bilosome-mediated delivery

Abstract

Effective buccal drug delivery is limited by the barrier properties of the mucosa, necessitating innovative systems to enhance permeability without compromising tissue integrity. In this study, bilosomes composed of sodium glycodeoxycholate and phosphatidylcholine were evaluated as a nanoparticulate platform for buccal drug delivery. Their in vitro uptake was investigated using the TR146 buccal cell line. The bilosomes demonstrated stable physicochemical properties and no aggregation. Functional assays indicated that they transiently opened cell-cell junctions, promoting transport across the mucosal barrier while minimizing toxicity. Quartz crystal microbalance with dissipation monitoring confirmed specific interactions with barrier components, supported by observed modulation of desmosomal junctions and cellular uptake. Ex vivo studies using porcine buccal mucosa further showed concentration-dependent distribution. Collectively, these results suggest that bilosomes are a safe and effective platform for enhancing buccal drug absorption.

Keywords: Bile salts; Ex vivo mucosal model; Nanoparticles; Permeation enhancement; QCM-D; TR146 cells.

Graphical abstract

Fig. 1

Workflow of the studies designed to investigate the mechanistic interactions of bilosomes, combining physicochemical characterization, in vitro assays and ex vivo permeability studies.

Fig. 2

Bilosome characterization using DLS and cryo-TEM; A) Particle size (nm) (mean ± SD) and PDI (mean ± SD) and B) zeta potential (mV) for bilosomes fabricated using different cholesterol amounts. C) Particle size (nm) of bilosomes (mean ± SD) incubated for 1 h in MQ water at varying pH and serum-free media, D) Cryo-TEM images of bilosomes in MQ water. The final concentration of bilosomes was 2 mg/mL, scale bar represents 100 nm. E) Size distribution of bilosomes incubated at different pH for 1 h. F) Size distribution of bilosomes incubated at different pH for 24 h.* indicates statistically significant differences (*p < 0.05, **p < 0.01, and ***p < 0.001. Statistical significance was determined by one-way ANOVA, followed by Tukey's multiple comparisons test for graphs A and B, and Dunnett's multiple comparisons test for graph C (using mQ water as the control).

Fig. 3

Bilosome interaction with mucin and phosphatidylcholine layers using Quartz Crystal Microbalance with Dissipation (QCM-D). Interaction of bilosomes with a mucin layer on a gold sensor, showing changes in frequency Δf (blue) and dissipation ΔD (red) for the seventh overtone as a function of time at 37 °C upon incubating bilosomes with A) a mucin layer on a gold sensor and B) a phosphatidylcholine layer on a silicon dioxide sensor. Phase I: formation of a physically adsorbed mucin or phosphatidylcholine layer. Phase II: rinsing with ultrapure water. Phase III: addition of bilosome formulation. Phase IV: rinsing with ultrapure water. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Cell viability (%), LDH release (%) of TR146 treated with bilosomes for 1 h. The percentages shown (% mean ± SD) refer to the corresponding controls.

Fig. 5

TR146 cells (control: D) after incubation with bilosomes at concentrations of A) 10 μg/mL, B) 50 μg/mL and C) 100 μg/mL after 1 h. For fluorescence microscopy images, the cytoskeleton was stained with Alexa Fluor 488 Phalloidin Cytoskeleton Dye (green), cell nuclei were stained with Hoechst 33342 (blue) and bilosomes with rhodamine B (red). Arrows indicate stress fibers. Scale bars represent 50 μm. Z-scans of A') 10 μg/mL, B') 50 μg/mL, C') 100 μg/mL, D′) control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Imaging of rhodamine labelled bilosomes in TR146 cells after incubation at 4 °C with bilosomes of A) 10 μg/mL, B) 50 μg/mL, pretreatment with C) chlorpromazine, D) dynasore, E) EIPA and F) genistein before incubation with 10 μg/mL bilosomes. The cytoskeleton was stained with Alexa Fluor 488 Phalloidin (green), and nuclei were counterstained with Hoechst 33342 (blue). NP were labelled with rhodamine-B (red). Scale bars represent 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Loss of cell-cell contact investigation. Incubation with bilosomes of 10 μg/mL for A) 1 h and B) 3 h and 50 μg/mL for C) 1 h and D) 3 h, bile salts of 10 μg/mL for E) 1 h and F) EDTA as positive control for 1 h. The nuclei were counterstained with Hoechst 33342 (blue), and the cell-junction opening is indicated via the red fluorescence (localization of desmoglein 3). Scale bars represent 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Coherent Anti-Stokes Raman Spectroscopy (CARS) imaging of rhodamine B labelled bilosomes A) 50 μg/mL, B) 100 μg/mL, C) 500 μg/mL in contact with ex vivo porcine buccal mucosa. Images of lipid-rich buccal mucosa (in green) by CARS at a Raman shift of 2850 cm⁻¹, and rhodamine-B bilosomes (in red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)