Detection of U-87 Tumor Cells by RGD-Functionalized/Gd-Containing Giant Unilamellar Vesicles in Magnetization Transfer Contrast Magnetic Resonance Images
Authors: Ferrauto Giuseppe , Tripepi Martina , Di Gregorio Enza , Bitonto Valeria , Aime Silvio , Delli Castelli Daniela 
Published: December 2020

Objectives
The targeting of tumor cells and their visualization with magnetic resonance imaging (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 2 strategies: (1) arginine, glycine, aspartic acid peptide (RGD)-functionalized giant unilamellar vesicles (GUVs) loaded with Gd complexes to accumulate large amounts of MRI contrast agent at the targeting site; and (2) 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
Giant unilamellar vesicles were prepared using the gentle swelling method, and the cyclic RGD targeting moiety was introduced onto the external membrane. Paramagnetic Gd-containing complexes and the fluorescent probe rhodamine were both part of the vesicle membranes and Gd-complexes were also the payload within the inner aqueous cavity. Giant unilamellar vesicles 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. Magnetic resonance imaging was carried out at 7 T, and T
2W, T
1W, and MTC/Z-spectra were acquired. Confocal microscopy images and Inductively Coupled Plasma Mass Spectrometry (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 (1) RGD GUVs were anchored onto the external surface of the tumor cells without any internalization; (2) a low number of GUVs per cell were clustered at specific regions; and (3) 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 T
1 signal was detected, whereas a 15% difference in MT contrast is present between the RGD GUV–treated cells and the ctrl-GUV–treated cells.
Magnetic resonance imaging scans of tumor-bearing mice were acquired before and after (t = 0, 4 hours and 24 hours) the administration of RGD GUVs and ctrl-GUVs. A roughly 16% MTC difference between the 2 groups was observed after 4 hours. Immunofluorescence analyses and ICP-MS analyses (for Gd-detection) of the explanted tumors confirmed the specific accumulation of RGD GUVs in the tumor region.

Conclusions
RGD GUVs seem 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 T
1w-MRI visualization. Conversely, MTC proved to be sufficiently sensitive for the visualization of detectable contrast between pretargeting and posttargeting images.