Reports of Biochemistry
and Molecular Biology
Wed, Jun 4, 2025
[Archive]
Volume 13, Issue 3 (Vol.13 No.3 Oct 2024)
rbmb.net 2024, 13(3): 349-357 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Hassani S, Nedaei K, Jafari R, Bagherpour G. Tight Junction Modulatory Fusion Peptide (ADT-6) Enhances GFP Protein Permeability through the Paracellular Pathway in Caco-2 Cell Lines: An In-Vitro Study. rbmb.net 2024; 13 (3) :349-357
URL: http://rbmb.net/article-1-1479-en.html
URL: http://rbmb.net/article-1-1479-en.html
Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
Abstract: (578 Views)
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-6, to improve the penetration of these therapeutic agents.
Methods: We constructed a fusion protein of ADT-6 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-6 construct, which provides a solid foundation for our study's methodology.
Results: Our molecular simulations showed that the linker between GFP and ADT-6 maintains the fusion protein's integrity and provides flexibility in receptor interaction. Permeability experiments revealed that ADT-6 markedly reduced transepithelial electrical resistance (TEER) and significantly increased GFP transfection in Caco-2 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-6.
Conclusions: This study demonstrates the potential of ADT-6 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.
Methods: We constructed a fusion protein of ADT-6 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-6 construct, which provides a solid foundation for our study's methodology.
Results: Our molecular simulations showed that the linker between GFP and ADT-6 maintains the fusion protein's integrity and provides flexibility in receptor interaction. Permeability experiments revealed that ADT-6 markedly reduced transepithelial electrical resistance (TEER) and significantly increased GFP transfection in Caco-2 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-6.
Conclusions: This study demonstrates the potential of ADT-6 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.
Type of Article: Original Article |
Subject:
Molecular Biology
Received: 2024/09/17 | Accepted: 2024/11/10 | Published: 2025/04/12
Received: 2024/09/17 | Accepted: 2024/11/10 | Published: 2025/04/12
References
1. Otani T, Furuse M. Tight Junction Structure and Function Revisited. Trends Cell Biol. 2020;30(10):805-817. [DOI:10.1016/j.tcb.2020.08.004] [PMID]
2. Zihni C, Mills C, Matter K, Balda MS. Tight junctions: from simple barriers to multifunctional molecular gates. Nat Rev Mol Cell Biol. 2016;17(9):564-80. [DOI:10.1038/nrm.2016.80] [PMID]
3. Ma TY, Nighot P, Al-Sadi R. Tight junctions and the intestinal barrier. Physiology of the gastrointestinal tract: Elsevier; 2018: 587-639. [DOI:10.1016/B978-0-12-809954-4.00025-6] []
4. Rosenthal R, Günzel D, Piontek J, Krug SM, Ayala-Torres C, Hempel C, et al. Claudin-15 forms a water channel through the tight junction with distinct function compared to claudin-2. Acta Physiol (Oxf). 2020;228(1):e13334. [DOI:10.1111/apha.13334] [PMID]
5. Brunner J, Ragupathy S, Borchard G. Target specific tight junction modulators. Adv Drug Deliv Rev. 2021;171:266-288. [DOI:10.1016/j.addr.2021.02.008] [PMID]
6. Nasir Uddin S, Sultana A, Fatima A, Geethakumari AM, Biswas KH. Regulation of Tight Junction by Cadherin Adhesion and Its Implication in Inflammation and Cancer. Tight Junctions in Inflammation and Cancer: Springer; 2023:49-66. [DOI:10.1007/978-981-99-2415-8_2]
7. Barcelona-Estaje E, Dalby MJ, Cantini M, Salmeron-Sanchez M. You Talking to Me? Cadherin and Integrin Crosstalk in Biomaterial Design. Adv Healthc Mater. 2021;10(6):e2002048. [DOI:10.1002/adhm.202002048] [PMID]
8. Ulapane KR, Kopec BM, Siahaan TJ. Improving In Vivo Brain Delivery of Monoclonal Antibody Using Novel Cyclic Peptides. Pharmaceutics. 2019;11(11):568. [DOI:10.3390/pharmaceutics11110568] [PMID] []
9. Horowitz A. Membrane Trafficking of Integral Cell Junction Proteins and its Functional Consequences. 2021; ArXiv. https://arxiv.org/abs/2101.05221. [DOI:10.31219/osf.io/cmdqb]
10. Ganjare A, Bagul N, Kathariya R, Oberoi J. 'Cell junctions of oral mucosa'-in a nutshell. QScience Connect. 2015;2015(1):7. [DOI:10.5339/connect.2015.7]
11. Adil MS, Narayanan SP, Somanath PR. Cell-cell junctions: structure and regulation in physiology and pathology. Tissue Barriers. 2021;9(1):1848212. [DOI:10.1080/21688370.2020.1848212] [PMID] []
12. Kiptoo P, Sinaga E, Calcagno AM, Zhao H, Kobayashi N, Tambunan US, Siahaan TJ. Enhancement of drug absorption through the blood-brain barrier and inhibition of intercellular tight junction resealing by E-cadherin peptides. Mol Pharm. 2011;8(1):239-49. [DOI:10.1021/mp100293m] [PMID] []
13. Kim D, Jin L, Park EJ, Na DH. Peptide permeation enhancers for improving oral bioavailability of macromolecules. J Pharm Investig. 2023;53:59-72. [DOI:10.1007/s40005-022-00609-4]
14. Bocsik A, Walter FR, Gyebrovszki A, Fülöp L, Blasig I, Dabrowski S, et al. Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells with Tight Junction Modulator Peptides. J Pharm Sci. 2016;105(2):754-765. [DOI:10.1016/j.xphs.2015.11.018] [PMID]
15. Fiser A, Sali A. Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol. 2003;374:461-91. [DOI:10.1016/S0076-6879(03)74020-8] [PMID]
16. Kuntal BK, Aparoy P, Reddanna P. EasyModeller: A graphical interface to MODELLER. BMC Res Notes. 2010;3:226. [DOI:10.1186/1756-0500-3-226] [PMID] []
17. Huang J, MacKerell AD Jr. CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. J Comput Chem. 2013;34(25):2135-45. [DOI:10.1002/jcc.23354] [PMID] []
18. Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14(1):33-8, 27-8. [DOI:10.1016/0263-7855(96)00018-5] [PMID]
19. Soezi M, Memarnejadian A, Aminzadeh S, Zabihollahi R, Sadat SM, Amini S, et al. Toward the development of a single-round infection assay based on EGFP reporting for anti-HIV-1 drug discovery. Rep Biochem Mol Biol. 2015;4(1):1-9.
20. Rezaei S, Hadadian S, Khavari-Nejad RA, Norouzian D. Recombinant Tandem Repeated Expression of S3 and SΔ3 Antimicrobial Peptides. Rep Biochem Mol Biol. 2020;9(3):348-356. [DOI:10.29252/rbmb.9.3.348] [PMID] []
21. Kruger NJ. The Bradford method for protein quantitation. Methods Mol Biol. 1994;32:9-15. [DOI:10.1385/0-89603-268-X:9] [PMID]
22. Sinaga E, Jois SD, Avery M, Makagiansar IT, Tambunan US, Audus KL, Siahaan TJ. Increasing paracellular porosity by E-cadherin peptides: discovery of bulge and groove regions in the EC1-domain of E-cadherin. Pharm Res. 2002;19(8):1170-9. [DOI:10.1023/A:1019850226631] [PMID]
23. Bocsik A. Modulation of tight junctions by peptides to increase drug penetration across biological barriers: Szegedi Tudomanyegyetem (University of Szeged, Hungary, Doctoral thesis); 2016.
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
