Molecular and therapeutic effects of bioactive compounds-incorporated mucoadhesive buccal patch targeting oral...

Oral potentially malignant disorders (OPMD) are associated with high risk of progression to oral squamous cell carcinoma (OSCC) and remain difficult to manage, due to limited effective localized therapies, underscoring the urgent need for novel, targeted drug delivery systems. A mucoadhesive buccal patch loaded bioactive components such as isotretinoin, bromelain, and limonene (IBL patch) was fabricated and evaluated for its anticancer potential against OPMD-associated OSCC. The cytotoxic study demonstrated that the IBL patch significantly reduced CAL-27 cells viability and found IC₅₀ value around 650 μg/mL, while exhibiting minimal cytotoxicity toward human gingival fibroblast (HGF) cells, indicating selective anticancer activity. In comparison to HGF cells, the IBL patch significantly reduced the migration and invasion of CAL-27 cells. Colony formation assays further confirmed that the IBL patch significantly suppressed the long-term proliferative and clonogenic potential of CAL-27 cells, without affecting HGF cells division. Genotoxicity analysis revealed increased micronucleus formation in CAL-27 cells, suggesting DNA damage-mediated apoptotic induction. Then IBL patch showed molecular effects by suppressing EMT and fibrosis associated TGF-β/SMAD signaling, inhibiting inflammatory (NF-κB), Wnt/β-Catenin pathways and Cytokeratin 17 (CK-17) and restoring epithelial identity through E-Cadherin and CK-18 upregulation. Furthermore, pathway analysis in both monolayer and co-culture inflammatory models demonstrated that the IBL patch effectively suppressed LPS-induced inflammatory markers, including TGF-β, MMP-2, IL-6, and TNF-α expression. Oral acute toxicity testing (OECD 423) confirmed safety of the IBL patch, with no mortality or adverse effects up to 2000 mg/kg (LD₅₀ > 2000 mg/kg; GHS Category 5). Collectively, these findings highlights that the IBL mucoadhesive buccal patch has therapeutic potential against OPMD and controlling pathological epithelial remodeling.