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Abstract:
Introduction
Calcium responsive contrast agents could be considered of great interest in MRI as they could assess pathophysiological and biological processes in vivo at the cellular and molecular levels, with a remarkable spatial and temporal resolution.In this work liposomes carrying a Ca(II) sensitive Gd(III) complex have been prepared to improve both the overall sensitivity in the MRI detection of the probe and the Ca(II) responsiveness.The structure of the complex consisted of two coordination cages: one (macrocyclic, DO3A-like) selective for Gd(III), and the other (linear, EGTA-like) selective for Ca(II) linked to two C18 aliphatic chains (Fig.1). Similar ligands have been already demonstrated to act as Ca(II) sensors under the form of monomer, dimer, and dendrimer.1
Methods
Liposomes formulated with DPPC, DSPE-PEG2000, and the amphiphilic ligand I (85:5:10) were prepared by the film hydration method followed by sequential extrusion.Size and polydispersion index (PDI) of the nanovesicles were measured by Dynamic Light Scattering.Titration with Gd(III) was performed and NMRD profiles,reporting the longitudinal relaxivity as a function of the magnetic field strength in the range 0.01-70 MHz, were acquired.
Results
The hydrodynamic diameter of the liposomes was 140±8 nm with a PDI value around 0.1. Upon titrating liposomes with Gd(III), the relaxivity showed a non linear behavior.Three regions can be detected corresponding to the binding of the metal to: A) the DO3A-like cage, B) the EGTA-like cage, and C) the phosphatidyl heads exposed on the liposomes surface.This finding has been confirmed by performing the titration on liposomes lacking ligand I. To remove Gd(III) bound to sites B and C, a controlled excess of EDTA was added.The relaxivity of the Gd-I-liposomes, measured after exhaustive dialysis,was around 7.0 s-1mMGd-1, thus indicating that no water molecules are bound to the paramagnetic center. However, the NMRD profile of such a species shows a relaxivity hump consistent with relaxation contributions from outer- and second-sphere water protons (Fig.2). Upon addition of equimolar amount of Ca(II), a five-fold increase in relaxivity was observed.This unprecedented large enhancement reflected the change in the hydration state of Gd(III) (from 0 to 1) that occurs when one donor atom of the EGTA-like cage detaches from Gd(III) and moves to coordinate Ca(II). Moreover,the binding of Ca(II) rigidifies the overall structure,causing a further increase in relaxivity.
Conclusions
The herein presented results demonstrate that the incorporation of Ca(II) responsive Gd(III) complexes into liposomes can represent a valuable option to improve the overall performance of this class of smart MRI probes.
