ABSTRACT
PURPOSE: To understand the binding and internalization of novel RGD micelles in tumor cells that overexpress the αvß3 integrin receptor. METHODS: Peptide amphiphiles containing a C16 or C18 fatty-acid chain with one or two ADA units linked to an RGD motif were prepared, characterized, and assessed for their binding specificity to the αvß3 receptor. The internalization of the amphiphiles was evaluated by confocal microscopy and cytotoxicity studies in A2058 cells that overexpress the αvß3 integrin receptor. RESULTS: The CMC and size and of RGD micelles ranged from 9 to 30 µM and 130 to 300 nm, respectively. Micelles showed good in vitro stability by retaining their micellar integrity and good specificity by binding to the αvß3 integrin receptor in an RGD-dependent manner. Confocal studies showed higher intracellular fluorescence when FITC was delivered through the micelles compared with its free form and showed significantly higher FITC uptake at 37 °C versus 4 °C (p < 0.05). The lower IC50 values were obtained when paclitaxel was delivered to A2058 cells via the RGD-loaded carriers (3.6-4.87 nM) compared with unencapsulated drug (7.86 nM), further demonstrated micelle specificity to the αvß3 receptor. CONCLUSION: RGD micelles bound specifically to the αvß3 receptor and their uptake was mediated by an endocytic process.
Subject(s)
Drug Delivery Systems , Integrin alphaVbeta3/metabolism , Micelles , Oligopeptides/metabolism , Antineoplastic Agents, Phytogenic/administration & dosage , Fluorescence Polarization , Fluorescence Resonance Energy Transfer , Microscopy, Confocal , Paclitaxel/administration & dosage , Protein BindingABSTRACT
PURPOSE: To design and synthesize fatty acid-RGD peptide amphiphiles with ADA linker for their potential delivery of hydrophobic drugs like paclitaxel targeted to α(v)ß(3) integrin overexpressing tumors. METHODS: Four amphiphiles - C16 or C18 fatty acid-RGD peptide and ADA linker were designed and synthesized. CMC, size and zeta potential of the amphiphiles were determined. FITC loaded micelles uptake into A2058 melanoma cells was investigated at 4°C and 37°C using confocal microscopy. Paclitaxel was loaded into micelles, their encapsulation efficiency and cytotoxicity of micelles was evaluated. The stability of the micelles was determined using FRET method. RESULTS: Mass, (1)H NMR and HPLC analysis confirmed the formation of amphiphiles and their purity. Among the amphiphiles, C18-(ADA)(2)-RGD amphiphile exhibited lowest CMC (9.00 ± 1.73 µM) and its micelles had suitable size (194.63 ± 44.86 nm) and zeta potential (0.27 ± 1.96 mV) for targeting. The cellular uptake of the micelles was temperature dependent and the micelles were stable. The IC50 of paclitaxel loaded in micelles decreased 50% in α(v)ß(3) integrin overexpressing cells and showed a 4 fold increase in normal cells when compared to free paclitaxel. CONCLUSION: Amphiphiles of fatty acids-ADA-RGD were synthesized. These amphiphiles formed stable micelles and were effective as targeted delivery carriers to α(v)ß(3) integrin overexpressing tumors.
