Tight Junction Modulatory Fusion Peptide (ADT-۶) Enhances GFP Protein Permeability through the Paracellular Pathway in Caco-۲ Cell Lines: An In-Vitro Study

Background: The oral delivery of therapeutic peptides and proteins presents a significant challenge in pharmaceutical development due to barriers such as the intestinal epithelium and the blood-brain barrier (BBB). These barriers limit the passage of large, hydrophilic molecules through transcellular pathways and restrict paracellular transport due to intercellular tight junctions. This study investigates the potential of E- cadherin-modulating peptide, ADT-۶, to improve the penetration of these therapeutic agents. Methods: We constructed a fusion protein of ADT-۶ and green fluorescent protein (GFP) to evaluate its activity and transport through the epithelial cells' paracellular pathway. Using Escherichia coli strains for expression, we cloned the GFP-ADT-۶ construct, which provides a solid foundation for our study's methodology. Results: Our molecular simulations showed that the linker between GFP and ADT-۶ maintains the fusion protein's integrity and provides flexibility in receptor interaction. Permeability experiments revealed that ADT-۶ markedly reduced transepithelial electrical resistance (TEER) and significantly increased GFP transfection in Caco-۲ cell monolayers dose-dependently. Results of ELISA confirmed these findings, showing high GFP levels in the lower compartment of Transwell systems treated with GFP-ADT-۶. Conclusions: This study demonstrates the potential of ADT-۶ to deliver proteins from the paracellular route, enhance the bioavailability of pharmaceutical drugs by altering cell-cell interactions, and provide new opportunities for oral drug delivery strategies.

کلیدواژه ها:

Caco-۲ cells ، Tight Junctions ، Permeability ، Recombinant fusion protein.