Transmucosal Delivery of miR-30c-5p by Chitosan Nanoparticles for Oral Squamous Cell Carcinoma

Abstract

Background: Oral squamous cell carcinoma (OSCC) remains difficult to treat with current modalities. miR-30c-5p, a tumor-suppressive microRNA frequently downregulated in OSCC, inhibits cancer cell proliferation and migration. However, its clinical application is limited by poor stability and inefficient uptake. To address these issues, miR-30c-5p was encapsulated into chitosan nanoparticles (CS-NPs) using ionic gelation to enhance delivery and protect against degradation.

Methods: miR-30c-5p-loaded CS-NPs were characterized for particle size, zeta potential, morphology, and encapsulation efficiency. HSC-3 and OEC-M1 cells were treated with free miRNA, CS-NPs, or CS-miRNA-NPs at final concentrations of 5%, 10%, 25%, and 50% (v/v) in culture medium. Cellular uptake was assessed by confocal microscopy. Ex vivo porcine buccal membrane studies evaluated mucosal penetration. Cytotoxicity was determined using MTT assays, while gene regulation was analyzed via quantitative polymerase chain reaction and Western blotting.

Results: The prepared CS-NPs had particle sizes ranging from 434 to 452 nm and encapsulation efficiencies between 79% and 87%. Confocal imaging revealed significantly greater cytoplasmic uptake of CS-miRNA-FAM NPs versus free miRNA. Ex vivo studies showed that CS-miR-30c-5p-FAM NPs penetrated mucosa up to 80-160 μm with a 5.42-fold higher fluorescence intensity than free miR-30c-5p-FAM. Cytotoxicity testing showed high cell viability (>93%) for all treatments at concentrations ≤25% (v/v). At 50% (v/v) nanoparticle suspension, viability significantly decreased in OEC-M1 cells (84.41% for naked miRNA, 54.52% for CS-NPs, 61.10% for CS-miRNA-NPs; P < 0.001). After 48 h, greater reductions were observed at 50% (v/v), with cell viability in HSC-3 cells decreasing to 85.55% (free miRNA), 42.72% (CS-NPs), and 51.82% (CS-miRNA-NPs), and in OEC-M1 cells to 73.98%, 33.00%, and 39.89%, respectively. Functional assays showed vimentin mRNA reductions of 85% in HSC-3 and 30% in OEC-M1, with protein decreases confirmed by Western blot.

Conclusion: CS-NPs enhance miRNA delivery and gene-silencing efficacy in OSCC cells. These findings support CS-based systems for miRNA therapeutics in oral cancer.

Keywords: buccal delivery; chitosan nanoparticles; microRNA; oral squamous cell carcinoma; transmucosal.

Graphical abstract

Figure 1

Preparation and characterization of miR-30c-5p-loaded CS-NPs. (A) Schematic representation of the encapsulation process of miR-30c-5p within CS-NPs. (B) Representative TEM image showing the morphology of the miR-30c-5p-loaded CS-NPs. (C) Particle size distribution of CS-NPs encapsulating various concentrations of miRNA. (D) Gel electrophoresis retardation assay of miR-30c-5p-FAM encapsulated in CS-NPs. Agarose gel electrophoresis demonstrating the retardation effect of CS-NPs on miR-30c-5p-FAM at various concentrations. Lanes are as follows: M (size marker), free miR-30c-5p-FAM (unencapsulated control), and CS-NPs encapsulating 0 nM, 100 nM, 300 nM, and 500 nM of miR-30c-5p-FAM, showing the efficiency of miRNA encapsulation.

Figure 2

Cytotoxicity and cellular uptake of chitosan-based miRNA NPs in oral squamous carcinoma cells. (A) HSC-3 and OEC-M1 cells were treated with free miRNA, CS-NPs, or CS-miRNA-NPs at final concentrations of 0% (control), 5%, 10%, 25%, and 50% (v/v) for 24 or 48 hours. The y-axis represents the percentage of cell viability relative to the untreated control group. The error bars indicate the standard deviation of four replicate wells from three independent experiments. *P<0.05; **P<0.01; ***P<0.001. (B) CLSM images showing the intracellular uptake of free miRNA-FAM, CS-NPs, and CS-miRNA-FAM NPs in HSC-3 and OEC-M1 cells after 48 hours of treatment. The negative control group consisted of untreated cells. Cell nuclei were stained with DAPI (blue), and the cytoplasmic region was labeled using GAPDH immunofluorescence (red). Green fluorescence indicates FAM-labeled miRNA. Quantification was performed using ImageJ to count the number of green pixels colocalized with red fluorescence per cell. Bar graphs represent the mean ± SD from three representative CLSM images per treatment group. P < 0.05.

Figure 3

Characterization of TR146 cell multilayers as a model for buccal epithelium and the penetration of CS-miRNA NPs. (A) TEER values of TR146 cell multilayers over a period of 25 days. (B) The penetration ratio of fluorescently labeled CS-miRNA NPs into and through TR146 cell multilayers over time, with and without the presence of TR146 cells. *P<0.05; **P<0.01; n.s., no significance. (C) Visualization of the penetration of CS-miRNA-FAM NPs into TR146 cells and across the multilayer barrier. On day 25 of culture, the cell layers were exposed to CS-miRNA-FAM NPs for 48 h and then analyzed by CLSM. The top left image showed a three-dimensional reconstruction of the multilayered cell model with CS-miRNA-FAM NPs distributed across different layers (1 to 5). The adjacent images displayed corresponding two-dimensional sections at increasing depths, with the green fluorescence indicating the presence of CS-miRNA-FAM NPs, and blue fluorescence (DAPI) highlighting cell nuclei.

Figure 4

Histological and fluorescence analysis of miRNA-FAM nanoparticle penetration in porcine buccal membrane. (A) Schematic illustration of a histological cross-section of the porcine buccal mucosa stained to show the structural layers: oral epithelium, basement membrane, lamina propria, submucosa (containing blood vessels and nerves), and muscle layer. (B) Fluorescence microscopy images at 4x magnification showing the distribution of CS-miR-30c-5p-FAM NPs within the buccal membrane. The upper image in the left panel shows the CS-NPs treatment. The middle image indicates tissue treated with free miR-30c-5p-FAM only. The lower image demonstrates tissue treated with miR-30c-5p-FAM loaded nanoparticles. Highlighted regions (1, 2, 3) on the right panel provide close-up views, showing detailed localization of CS-miR-30c-5p-FAM NPs within the cells. Arrows in all images point to areas of CS-miR-30c-5p-FAM NPs delivery.

Figure 5

CLSM images of miR-30c-5p-FAM alone and CS-miR-30c-5p-FAM NPs in porcine buccal membrane after 24 h in situ buccal delivery. (A) Sequential xyz plane scans of porcine buccal membrane treated with aqueous miR-30c-5p-FAM solution and CS-miR-30c-5p-FAM NPs, with the full membrane thickness divided into 10 sections from the top surface (left to right). (B) Cumulative xyz images of all optical sections were merged across a 0–200 μm xyz scan. Fluorescence intensity profiles relative to buccal membrane depth were quantified from CLSM micrographs for both the free miR-30c-5p-FAM and CS-miR-30c-5p-FAM NPs groups. The enhancement ratio (#ER) was determined as the ratio of the integrated fluorescence intensity of CS-miR-30c-5p-FAM NPs to that of miR-30c-5p-FAM alone.

Figure 6

Impact of CS-miR30c-5p NPs-mediated transfection on vimentin expression. (A) The bar graph illustrates the relative mRNA expression of vimentin in HSC-3 and OEC-M1 cell lines following transfection with CS-NPs encapsulating either mimic NC or miR-30c-5p. (B) Western blot analysis showing vimentin protein levels with β-actin as the loading control. Both mRNA and protein levels are normalized to the mimic NC group, set at 1. Data are presented as mean±SD from three independent biological replicates. *P<0.05; ***P<0.001.