ABSTRACT
PURPOSE: 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. METHODS: 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 α(V)ß(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 α(V)ß(3)-expressing cells. Molecular modeling was performed to support experimental results using MOE. RESULTS: Prodrugs recognized and bound to integrin α(V)ß(3)-expressing cells in integrin α(V)ß(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 α(V)ß(3)-expressing cells. CONCLUSIONS: Binding-induced conformation change of hairpin peptide can be used to design integrin-targeted drug delivery system.
Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Drug Delivery Systems/methods , Integrin alphaVbeta3/metabolism , Methotrexate/administration & dosage , Oligopeptides/chemistry , Prodrugs/administration & dosage , Amino Acid Sequence , Antimetabolites, Antineoplastic/chemistry , Antimetabolites, Antineoplastic/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Methotrexate/chemistry , Methotrexate/metabolism , Models, Molecular , Neoplasms/drug therapy , Oligopeptides/metabolism , Prodrugs/chemistry , Prodrugs/metabolism , Protein Binding , Protein ConformationABSTRACT
A model peptide-drug conjugate designed upon a beta-hairpin peptide with the alpha4beta1 integrin recognition sequence LDV appended to the N-terminus and a fluorescent model drug appended to the C-terminus. This model recognizes and binds to alpha4beta1 expressing cells and displays an enhanced rate of enzyme catalyzed hydrolytic model drug release in the presence of the cells compared to the rate in the absence of cells. The present work suggests that peptide-drug conjugate conformation change due to receptor binding may be a viable approach to targeted drug release.
Subject(s)
Coumarins/metabolism , Enzymes/metabolism , Peptides/metabolism , Amino Acid Sequence , Cell Line, Tumor , Fluorescent Dyes/chemistry , Humans , Hydrolysis , Indicators and Reagents/metabolism , Integrin alpha4beta1/metabolism , Models, Chemical , Peptides/chemistryABSTRACT
Novel amphiphilic conjugates consisting of an Arg-Gly-Asp (RGD) peptide binding motif and aliphatic fatty acids of varying chain length (C10-C18) were synthesized and evaluated for their ability to form micelles and bind specifically to alphaVbeta3 integrin over-expressing tumor cells. The aphilphiles were characterized by IR, proton NMR and mass spectrometry. The size and zeta potential of the resultant micelles were ranged from 178 to 450 nm and - 13.5 to 39.6 mV, respectively. The critical micellar concentration (CMC), drug loading efficiency and tumor cell binding of these amphiphiles were determined. The CMC values, determined by pyrene fluorescent probe method, ranged from 0.02 to 0.12 mM for C14-RGD, C16-RGD and C18-RGD. The C18-RGD micelles with lowest CMC were found to increase the solubility of taxol, a model anticancer drug, by 87%. C18-RGD amphiphiles also exhibited significantly higher (12.1 +/- 1.14%, P < 0.05) binding to alphaVbeta3 integrin over-expressing human breast cancer cells (HTB-129) when compared to normal human epidermal keratinocyte (NHEK) cells (6.68 +/- 0.34). The results from this study demonstrated the feasibility of designing RGD-fatty acid amphiphiles as micellar drug delivery carriers to target to cancer cells.
