Introduction: Magnetic resonance imaging (MRI) is a common diagnostic imaging modality of disease evaluation (i.e. cancer, atherosclerosis) and nanocarriers with complexes of gadolinium(Ⅲ) (Gd) can be used to passively target diseased tissue. However, low relaxivity and specificity limit this approach. Previously, we developed fluorescently-labeled, peptide amphiphile micelles (PAM) that can enhance diseased tissue by binding to surface markers. In order to integrate the selectivity of PAMs and the diagnostic performance of MRI, we mixed fibrin-binding peptides conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(poly(ethylene glycol)-2000 (DSPE-PEG2000) with 1 ,2-distearoyl-sn-glycerc-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt) (18:0 PE-DTPA (Gd)) or DTPA-bis(stearylamide) (Gd) (DTPA-BSA (Gd)). The physicochemical properties, biocompatibility and targeting abilities were assessed. Materials and Methods: The fibrin-binding peptide, CREKA, was synthesized using Fmoc-mediated solid phase peptide synthesis methods. Peptides were purified by reverse-phase HPLC and characterized by MALDI-TOF/TOF. Peptides were conjugated to DSPE-PEG(2000)-maleimide. Gd-containing PAMs were assembled by dissolving DSPE-PEG(2000)-CREKA or DSPE-PEG(2000)-maleimide, and 18:0 PE-DTPA (Gd) or DTPA-BSA (Gd). 100 pM PAM solutions were used for TEM and zeta potential measurements. Reiaxivity of PAMs were measured at 1.5T and 3T. The T1 -mapping sequence consisted of FSE-IR acquisition parameters: TR = 12 s; TE = 15 ms; TI = 50,100,250,500,750, 1000,2000,3000,4000,5000 ms; acquisition voxel size of 1 mm × 1 mm and 3 mm slice thickness. The T1 and T2 relaxation times were measured at 1.5T. A FSE-IR sequence (same as above) was acquired to measure T1. The T2's were measured with a SE sequence with varying TEs. The SE were acquired with: TR = 100 ms; TE's = 8, 10,15,20,30,40,50 ms; acquisition voxel size of 1 mm × 1 mm and 3 mm slice thickness. NIH/3T3 fibre-blasts were cultured with 100 μM PAMs and cell viability determined by Live/Dead and Presto Blue assays. Clots were incubated with 1 mM PAMs for 3 hours, washed, digested with nitric oxide, and analyzed using ICP-MS. Results and Discussion: DSPE-PEG(2000)-maleimide or DSPE-PEG(2000)-CREKA was combined with 18:0 PE-DTPA (Gd) or DTPA-BSA (Gd) in a 75:25 molar ratio and the presence of both spherical and cylindrical micelles were confirmed via TEM (Figure 1). Zeta potentials of control and CREKA-PAMs using 18:0 PE-DTPA were -10.5 ± 0.1 and -24.1 ± 0.1 mV. T1 and T2 measurements of control and CREKA-Gd PAMs were 330.0 ± 3.3 and 72.0 ± 3.6 ms, and 399.0 ±4.5 and 92.0 ± 4.5 ms. With varying 18:0 PE-DTPA (Gd) in micelles, an increase in Gd led to a decrease in T1 measurements via both 1.5T and 3T. In vitro, fibroblasts cultured with all PAMs were viable for up to 3 days. When clots were incubated with CREKA PAMs consisting of 18:0 PE-DTPA (Gd) or DTPA-BSA (Gd), micelles with DTPA-BSA (Gd) had enhanced targeting abilities compared to its nontargeting counterpart, conferring nonspecific binding of 18:0 PE-DTPA (Gd) (Figure 2). Conclusions: The fibrin-binding PA was combined with 18:0 PE-DTPA (Gd) or DTPA-BSA (Gd) to construct MRI contrast agents. Ongoing studies include micelles with DSPE-PEG(2000)-DTPA (Gd). Future in vivo studies will determined our micelles as safe and potent nanocarriers for MRI.
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