A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study

Background Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. Results The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. Conclusion We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations.

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study 1st revision, 26/02/2014 At established time-points after i.v. injection GNP or vehicle (Phosphate Buffered Saline, PBS) administration animals (n=3 per group, as mentioned in methods: "MRI studies -in vivo studies") were sacrificed by cervical dislocation. Required tissues (liver, kidney, spleen, brain and tumor among others) as well as urine and blood samples were collected and stored at -80ºC. Samples were analyzed by

Superparamagnetic iron oxide nanoparticle (SPION) synthesis:
The negative contrast agents evaluated in this work consisted in different TEGor DMSA-coated SPIONs. Water-dispersible TEG-coated SPIONs were produced through a synthesis pathway described by Cai and Wan [2], with slight modifications [3]. In these experiments, a mixture of iron acetyl acetonate [Fe(acac)3] and triethylene glycol was heated at 180ºC, leading to the partial decomposition of the reactants and the formation of an intermediate alkoxy-acetylacetonate-Fe 3+ . After that, the heating of this mixture at 280ºC produced the reduction and subsequent decomposition of these complexes leading to the nucleation and final growth of the iron oxide nanoparticles. The resulting particles were washed with a mixture of ethyl acetate and ethanol, collected with the help of a magnet and transferred to a phosphate buffered saline (PBS) solution. This so-obtained colloidal dispersion presented particle size homogeneity (∼ 5 nm) and good particle size distribution, with a mean hydrodynamic aggregate of 16 nm measured using dynamic light scattering (DLS) in a 90 Plus apparatus (Brookhaven). DMSA-coated SPIONs were obtained by replacing TEG coating for DMSA molecules, using a ligand-exchange reaction process described previously [4]. In this process, the A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study

T Studies
A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study  NA, 1; NR, 1; TAT, 3 min 12 sec. In both cases, the animals were anesthetized and handled as described for "in vivo studies". After that, animals were sacrificed and contrast administered as described in the "ex vivo post-mortem studies" section in the main text. After this, the T2 weighted image acquisition was repeated as above.

Processing and post-processing of MR data
The processing and post-processing or MR data were done essentially as described in the main text. The only remarkable difference is that for negative contrast agents, all slices with noticeable RCE effect were taken into account for RCE calculation.

Iv vitro results
The r2 relaxivity values for the negative CAs were measured at 1. Ferumoxtran-10 (20 nm) [6]. As expected, the increase of the SPIONs aggregate size markedly increases r2 at 1.4 T from 48.0 to 119.6 mM -1 s -1 , which is coincident with the results previously reported by Muller and collaborators [7] related to the variation of the relaxivity of superparamagnetic iron oxide MRI contrast agents in function of their aggregate size.

Ex vivo postmortem results
A representative T2-weighted image for SPIONs, and typical ROIs selected for analysis, are shown in Figure 3,

Discussion
The negative contrast agents showed differences between the r2 in vitro