Molecular imaging requires the specific accumulation of contrast agents at the target. To exploit the superb resolution of MRI for applications in molecular imaging, gadolinium chelates, as the MRI contrast agents (CA), have to be conjugated to a specific vector able to recognize the epitope of interest. Several Gd(III)-chelates can be chemically linked to the same binding vector in order to deliver multiple copies of the CA (multimers) in a single targeting event thus increasing the sensitivity of the molecular probe. Herein three novel bifunctional agents, carrying one functional group for the bioconjugation to targeting vectors and four Gd(III)-AAZTA chelate functions for MRI contrast enhancement (AAZTA = 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), are reported. The relaxivity in the tetrameric derivatives is 16.4 ± 0.2 mM s at 21.5 MHz and 25 °C, being 2.4-fold higher than that of parent, monomeric Gd(III)-AAZTA. These compounds can be used as versatile building blocks to insert preformed, high relaxivity, and high density Gd-centers to biological targeting vectors. As an example, we describe the use of these bifunctional Gd(III)-chelates to label a fibrin-targeting peptide.

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Moving from nano‐ to micro‐systems may not just be a matter of scale, but it might imply changes in the properties of the systems that can open new routes for the development of efficient MRI contrast agents. This is the case reported in the present paper, where giant liposomes (giant unilamellar vesicles, GUVs) loaded with LnIII complexes have been studied as chemical exchange saturation transfer (CEST) MRI contrast agents. The comparison between nanosized liposomes (small unilamellar vesicles, SUVs) and GUVs sharing the same formulation led to differences that could not be accounted for only in terms of the increase in size (from 100–150 nm to 1–2 μm). Upon osmotic shrinkage, GUVs yielded a saturation‐transfer effect three order of magnitude higher than SUVs consistent with the increase in vesicles volume. Confocal microscopy showed that the shrinkage of GUVs resulted in multilamellar particles whereas SUVs are known to yield asymmetrical, discoidal shape.

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A new class of liposomes (LipHosomes) is designed to induce a change of pH upon releasing their content. pH‐readout reports on the number of LipHosomes in the specimen. LipHosomes were prepared by entrapping NaOH or bicarbonate buffer in the intravesicular compartment. The liposomes suspension was purified from unentrapped compounds and brought to pH 7.0. The pH gradient between intra‐ and extra‐liposomal compartments is maintained because the phospholipidic membrane works as a semipermeable membrane thus preventing diffusion of ions across the membrane. The release of the liposomal content triggers a quantifiable variation of the pH of the medium. This feature has been harnessed in analytical assays based on ligand/anti‐ligand molecular recognition by exploiting the biotin‐streptavidin binding scheme. A pH difference of 0.2 units was observed upon the release of the payload from biotinylated LipHosomes bound to streptavidinated plates. The test showed an excellent sensitivity being able to reveal a concentration of bound LipHosomes in the sub‐pM range.

Objectives: The targeting of tumor cells and their visualization with MRI is an important task in biomedicine. The low sensitivity of this technique is a significant drawback, and one that may hamper the detection of the imaging reporters used.To overcome this sensitivity issue, this work explores the synergy between two strategies: i) RGDfunctionalized Giant Unilamellar Vesicles (GUVs) loaded with Gd-complexes to accumulate large amounts of MRI contrast agent at the targeting site; and ii) the use of Magnetization Transfer Contrast (MTC), which is a sensitive MRI technique for the detection of Gd-complexes in the tumor region.Materials and Methods: GUVs were prepared using the gentle swelling method, and the cyclic RGD targeting moiety was introduced onto the external membrane. Paramagnetic Gd-containing complexes were both part of the vesicle membranes and were the payload within the inner aqueous cavity together with the fluorescent probe, rhodamine. GUVs that were loaded with the imaging reporters, but devoid of the RGD targeting moiety, were used as controls. U-87 MG human glioblastoma cells, which are known to overexpress the targets for RGD moieties, were used. In the in-vivo experiments, U-87 MG cells were subcutaneously injected into nu/nu mice and the generated tumors were imaged using MRI, 15 days after cell administration. MRI was carried out at 7 T, and T2W, T1W and MTC/Z-spectra were acquired. Confocal microscopy images and ICP-MS were used for result validation.Results: In-vitro results show that RGD GUVs specifically bind to U-87 MG cells. Microscopy demonstrates that: i) RGD GUVs were anchored onto the external surface of the tumor cells without any internalization; ii) a low number of GUVs per cell were clustered at specific regions; iii) there is no evidence for macrophage uptake or cell toxicity. The MRI of cell pellets after incubation with RGD GUVs and untargeted ctrl-GUVs was performed. No difference in T1 signal was detected, whereas a 15% difference in MT contrast is present between the RGD-GUV-treated cells and the ctrl-GUV-treated cells.MR images of tumor-bearing mice were acquired before and after (t=0, 4 h and 24 h) the administration of RGD GUVs and ctrl-GUVs. A roughly 16% MTC difference between the two groups was observed after 4 h. Immunofluorescence analyses and ICP-MS analyses (for Gddetection) of the explanted tumors confirmed the specific accumulation of RGD GUVs in the tumor region.Conclusions: RGD GUVs appear to be interesting carriers that can facilitate the specific accumulation of MRI contrast agents at the tumor region. However, the concentration achieved is still below the threshold needed for T1w-MRI visualization. Conversely, MTC proved to be sufficiently sensitive for the visualization of detectable contrast between pre-and post-targeting images.

Purpose Prostate cancer (PCa) is the most widespread tumor affecting males in Western countries. We propose a novel MRI molecular tetrameric probe based on the heptadentate gadolinium (Gd)‐AAZTA (6‐amino‐6‐methylperhydro‐1,4‐diazepinetetraacetic acid) that is able to in vivo detect PCa through the recognition of the fibrin–fibronectin (FB–FN) complex. Methods The peptide CREKA (Cys‐Arg‐Glu‐Lys‐Ala), targeting the FB–FN complex in the reactive stroma of the tumor, was synthesized by solid phase peptide synthesis (SPPS) and conjugated to the tetramer dL‐(Gd‐AAZTA)4. The resulting probe was characterized by 1H relaxometry, tested in vitro on FB clots and in vivo on an orthotopic mouse model of PCa. Results CREKA‐dL‐(Gd‐AAZTA)4 showed a remarkable relaxivity of 18.2 mMGd-1s−1 (0.47 T, 25°C) because of the presence of 2 water molecules (q = 2) in the inner coordination sphere of each Gd3+ ion, whose rotational motion (τR) is lengthened as the result of the relatively high molecular weight. The probe displayed a detectable affinity for plasma‐derived FB clots. On intravenous injection of the probe in an orthotopic mouse model of PCa, a significant increase in the prostate T1 contrast (~40%) was observed. The MRI signal appears statistically higher either with respect to the one observed for the control probes and to the one detected when CREKA‐dL‐(Gd‐AAZTA)4 was administered to healthy animals. Conclusions This study demonstrated the ability of the CREKA‐dL‐(Gd‐AAZTA)4 probe to specifically localize in prostate tumor after injection. The high relaxivity of the probe allows the reduction of the injected dose to 20 µmolGd/kg, yielding a good in vivo contrast enhancement in the region of prostate tumor.

Moving from nano‐ to micro‐systems may not just be a matter of scale, but it might imply changes in the properties of the systems that can open new routes for the development of efficient MRI contrast agents. This is the case reported in the present paper, where giant liposomes (giant unilamellar vesicles, GUVs) loaded with LnIII complexes have been studied as chemical exchange saturation transfer (CEST) MRI contrast agents. The comparison between nanosized liposomes (small unilamellar vesicles, SUVs) and GUVs sharing the same formulation led to differences that could not be accounted for only in terms of the increase in size (from 100–150 nm to 1–2 μm). Upon osmotic shrinkage, GUVs yielded a saturation‐transfer effect three order of magnitude higher than SUVs consistent with the increase in vesicles volume. Confocal microscopy showed that the shrinkage of GUVs resulted in multilamellar particles whereas SUVs are known to yield asymmetrical, discoidal shape.