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The magnetic resonance imaging (MRI) performance of two liposome formulations incorporating amphiphilic 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA)‐like GdIII complexes has been investigated both in vitro and in vivo. The complexes differ in one donor group of the coordination cage (carboxylate versus carboxoamide), and in the length (C12 versus C18) and the point of attachment of the aliphatic chains to the chelators. The in vitro 1H relaxometric characterisation of the systems, performed with a newly developed relaxation model that takes into account the contributions of the GdIII chelates pointing in‐ and outwards of the liposome, indicates that their efficacy is optimal in the range 0.5–1.5 T. The tetracarboxylic C12‐containing liposomes (LIPO‐GdDOTA(GAC12)2; GA=glutaric acid) are four‐fold more efficient than the monoamide C18‐based analogue (LIPO‐GdDOTAMA(C18)2). Such a difference is also found in vivo at 1 T in a melanoma tumour model on mice. A few hours after intravenous injection, the T1 contrast enhancement in the organs where the nanovesicles typically distribute (liver, spleen, kidneys and tumour) is much higher for LIPO‐GdDOTA(GAC12)2. Interestingly, after about 7 h post‐injection the contrast enhancement observed for the more efficient liposomes decreases rapidly and becomes lower than for LIPO‐GdDOTAMA(C18)2. The relaxometric data and the quantification of the GdIII complexes in the organs, determined ex vivo by inductively coupled plasma mass spectrometry, indicate that: 1) the differences in the contrast enhancement can be attributed to the different rate of water exchange and rotational dynamics of the Gd complexes, and 2) the rapid contrast decrease is caused by a faster clearance of GdDOTA(GAC12)2 from the organs. This is also confirmed by using a newly synthesised amphiphilic cyanine‐based fluorescent probe (Cy5‐(C16)2). As one of the main limitations for the clinical translation of liposomes incorporating amphiphilic imaging agents is related to their very long persistence in the body, the results reported herein suggest that the clearance of the probes can be accelerated, without compromising their role, by a proper selection of the lipophilic portion of the incorporated compound as well as of the ligand site at which the aliphatic tails are linked. Then, GdDOTA(GAC12)2 complex may represent a good candidate for the development of improved MRI protocols based on paramagnetically labelled lipidic nanoparticles.

Murine melanoma B16 cells display on the extracellular side of the plasma membrane a large number of reactive protein thiols (exofacial protein thiols, EPTs). These EPTs can be chemically labeled with Gd-DO3A-PDP, a Gd(III)-based MRI contrast agent bearing a 2-pyridinedithio chemical function for the recognition of EPTs. Uptake of gadolinium up to 10(9) Gd atoms per cell can be achieved. The treatment of B16 cells ex vivo with a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) results in an increase by 850% of available EPTs and an increase by 45% of Gd uptake. Blocking EPTs with N-ethylmaleimide (NEM) caused a decrease by 84% of available EPTs and a decrease by 55% of Gd uptake. The amount of Gd taken up by B16 cells is therefore dependent upon the availability of EPTs, whose actual level in turn changes according to the extracellular redox microenvironment. Then Gd-DO3A-PDP has been assessed for the labeling of tumor cells in vivo on B16.F10 melanoma tumor-bearing mice. Gd-DO3A-PDP (or Gd-DO3A as the control) has been injected directly into the tumor region at a dose level of 0.1 μmol and the signal enhancement in MR images followed over time. The washout kinetics of Gd-DO3A-PDP from tumor is very slow if compared to that of control Gd-DO3A, and 48 h post injection, the gadolinium-enhancement is still clearly visible. Therefore, B16 cells can be labeled ex vivo as well as in vivo according to a common EPTs-dependent route, provided that high levels of the thiol reactive probe can be delivered to the tumor.

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd-DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd-K11) or through a hydrophilic spacer (compd Gd-K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd-K11 and Gd-K11N have a good affinity for β-cyclodextrins (K(D) 310 and 670 µ m respectively) and for serum albumin (K(D) 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd-K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs.