Enhanced Cellular Uptake Of Phenamil Through Inclusion Complex With Histidine Functionalized β-Cyclodextrin As Penetrative Osteoinductive Agent

Abstract

Vahid Jahed,1 Ebrahim Vasheghani-Farahani,1 Fatemeh Bagheri,2 Ali Zarrabi,3 Trine Fink,4 Kim Lambertsen Larsen5 1Biomedical Engineering Division, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran; 2Department of Biotechnology, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran; 3Sabanci University Nanotechnology Research and Application Center (SUNUM), Tuzla 34956, Istanbul, Turkey; 4Department of Health Science and Technology, Faculty of Health, Aalborg University, Aalborg, Denmark; 5Department of Chemistry and Bioscience, Faculty of Chemistry, Aalborg University, Aalborg, DenmarkCorrespondence: Ebrahim Vasheghani-FarahaniBiomedical Engineering Division, Faculty of Chemical Engineering, Tarbiat Modares University, Jalal ale Ahmad Highway, P.O. Box 14115-114, Tehran, IranTel [phone]Email [email protected] Lambertsen LarsenDepartment of Chemistry and Bioscience, Faculty of Chemistry, Aalborg University, 7H Fredrik Bajers Vej — 1340, Aalborg 9220, DenmarkTel [phone]Email [email protected]: Phenamil (PH) is a small molecule that induces bone formation through upregulation of the TRB3 gene in the bone-regeneration process. β-Cyclodextrins (βCDs) with hydrophilic surfaces and a relatively hydrophobic cavity can form inclusion complexes with primarily hydrophobic small molecules such as PH, and increase their apparent solubility and dissolution rate. The hydrophilic surface of βCDs prevents their interaction with the hydrophobic lipids of the cell membrane for penetration. Therefore, binding of penetrative groups, such as lysine, arginine, and histidine (His), to βCDs for cell penetration is required.Aim: The aim of this study was to investigate the effect of His-conjugated βCD on cellular uptake of PH for bone differentiation.Methods: In this study, His-βCDs were synthesized and used to prepare an inclusion complex of His-βCD-PH. A hydroxypropyl–βCD-PH (HP-βCD-PH) inclusion complex for increasing PH solubility without a penetrative group was prepared for comparison. 3-D geometry of βCD derivatives and PH-inclusion complexes was investigated by Fourier-transform infrared spectroscopy and molecular docking. Alizarin red staining and real-time PCR were performed to compare bone differentiation of His-βCD-PH and HP-βCD-PH.Results: The results suggested that the benzene ring of PH was inserted into the wide side of both His-βCD and HP-βCD. Alizarin red staining at 14 days postculture in the presence of His-βCD-PH at total concentration of 50 μM for PH showed that bone-matrix mineralization increased significantly compared with free PH and HP-βCD-PH. Real-time PCR confirmed this result, and showed gene expression increased significantly (OPN 1.84-fold, OCN 1.69-fold) when stem cells were cultured with His-βCD-PH.Conclusion: The overall results indicated that His-βCD-PH is a promising carrier for osteoinductive PH with possible penetration ability and sustained release that reduces BMP2 consumption for differentiation of mesenchymal stem cells to bone tissue.Keywords: phenamil, β-cyclodextrin, inclusion complex, histidine, cell penetration, bone regeneration

Keywords

• Phenamil
• β-cyclodextrin
• Inclusion Complex
• Histidine
• Cell penetration
• Bone Regeneration.