Novel Integrin-Targeted Binding-Triggered Drug Delivery System for Methotrexate

To design a binding-induced conformation change drug delivery system for integrin-targeted delivery of methotrexate and prove the feasibility of using hairpin peptide structure for binding triggered drug delivery. Methotrexate prodrugs were synthesized using solid phase peptide synthesis techniques by conjugating methotrexate to Arg-Gly-Asp (RGD) or a hairpin peptide, RWQYV(D)PGKFTVQRGD (hairpin-RGD). Levels of integrin alpha(V)beta(3) in HUVEC were up-regulated using adenoviral system and knocked down using siRNA. Stability of prodrugs and methotrexate release from prodrugs were evaluated in plasma, in presence or absence of integrin alpha(V)beta(3)-expressing cells. Molecular modeling was performed to support experimental results using MOE. Prodrugs recognized and bound to integrin alpha(V)beta(3)-expressing cells in integrin alpha(V)beta(3) expression level-dependent manner. Prodrug with hairpin peptide could resist Streptomyces griseus-derived glutamic acid-specific endopeptidase (SGPE) and plasma enzyme hydrolysis. Drug release was triggered in presence of HUVEC cells and SGPE. Analysis of conformation energy supported that conformational change in MTX-hairpin-RGD led to exposure of labile link upon binding to integrin alpha(V)beta(3)-expressing cells. Binding-induced conformation change of hairpin peptide can be used to design integrin-targeted drug delivery system.