In Vitro and In Vivo Efficacy of Self-Assembling RGD Peptide Amphiphiles for Targeted Delivery of Paclitaxel.

PURPOSE: The objective of this work was to compare the efficacy of self-assembling cyclic and linear RGD peptide amphiphiles as carriers for delivering paclitaxel to αvß3 integrin overexpressing tumors. METHODS: Linear (C18-ADA5-RGD) and cyclic (C18-ADA5-cRGDfK) peptide amphiphiles were synthesized and characterized for CMC, aggregation number and micelle stability using fluorescence spectroscopy methods. Size and morphology of micelles was studied using TEM. Fluorescence polarization and confocal microscopy assays were established to compare binding and internalization of micelles. The targeting efficacy was studied in A2058 cells using cytotoxicity assay as well as in vivo in melanoma xenograft mouse model. RESULTS: The linear and cyclic RGD amphiphiles exhibited CMC of 25 and 8 µM, respectively, formed nano-sized spherical micelles and showed competitive binding to αvß3 integrin protein. FITC-loaded RGD micelles rapidly internalized into A2058 melanoma cells. Paclitaxel-loaded RGD micelles exhibited higher cytotoxicity compared with free drug in A2058 cells in vitro as well as in vivo. CONCLUSION: Cyclic RGD micelles exhibited better targeting efficacy but were less effective compared to linear RGD micelles as drug delivery vehicle due to lower drug solubilization capacity and lesser kinetic stability. Results from the study proved the effectiveness of self-assembling low molecular weight RGD amphiphiles as carriers for targeted delivery of paclitaxel.

Binding and uptake of novel RGD micelles to the αvß3 integrin receptor for targeted drug delivery.

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.

Fatty acid-RGD peptide amphiphile micelles as potential paclitaxel delivery carriers to α(v)ß3 integrin overexpressing tumors.

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.

Daily stepping in individuals with motor incomplete spinal cord injury.

BACKGROUND: In individuals with motor incomplete spinal cord injury (SCI), ambulatory function determined in the clinical setting is related to specific measures of body structure and function and activity limitations, although few studies have quantified the relationship of these variables with daily stepping (steps/day). OBJECTIVE: The aim of this study was to quantify daily stepping in ambulatory individuals with SCI and its relationship with clinical walking performance measures and specific demographics, impairments, and activity limitations. DESIGN: A cross-sectional study was performed to estimate relationships among clinical variables to daily stepping in self-identified community versus non-community (household) walkers. METHODS: Average daily stepping was determined in 50 people with chronic, motor incomplete SCI. Data for clinical and self-report measures of walking performance also were collected, and their associations with daily stepping were analyzed using correlation and receiver operating characteristic (ROC) analyses. Relationships between daily stepping and the measures of demographics, impairments, and activity limitations were identified using correlation and regression analyses. RESULTS: The ROC analyses revealed a significant discriminative ability between self-reported community and non-community walkers using clinical gait measures and daily stepping. Stepping activity generally was low throughout the sample tested, however, with an average of approximately 2,600 steps/day. Knee extension strength (force-generating capacity) and static balance were the primary variables related to daily stepping, with metabolic efficiency and capacity and balance confidence contributing to a lesser extent. LIMITATIONS: The small sample size and use of specific impairment-related measures were potential limitations of the study. CONCLUSIONS: Daily stepping is extremely limited in individuals with incomplete SCI, with a potentially substantial contribution of impairments in knee extension strength and balance.