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

Abstract

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.

Figure 1

Short name: Schematic representation of the paramagnetic Gd-chelate containing gold glyconanoparticle (Gd-GNPs). Detailed legend: Schematic representation of the paramagnetic Gd-chelate containing gold glyconanoparticle (Gd-GNPs) tested in this work. The gold core (Au) is coated with Gd:DO3A derivatives and self-assembled monolayer of saccharide conjugates. Thiol-ended alkane linkers are used as spacers to attach the conjugates to the gold core. Figure not drawn to scale. Glc = glucose. See also Table 1, text and reference [7] for further details.

Figure 2

Short name: Ex vivo MRI and example of Gd-GNPs evaluated; Boxplot of relative contrast enhancement. Detailed legend: A) Representative ex vivo T₁-weighted image and example of ROIs used (manually drawn as circles, green (ipsilateral) and yellow (contralateral) lines) for signal enhancement calculation for GNP (E_2). B) Enlarged central row (white discontinuous rectangle) C) Boxplot of relative contrast enhancement for saline solution, gadoterate meglumine (G.M.) and GNPs obtained from ROIs of all CAs studied. RCE was calculated using Equation 1. The limits of the box represent the quartiles 1 (Q1) and 3 (Q3) of the distribution, the central line corresponds to the median (quartile 2) and the whiskers represent the maximum and minimum value in each distribution. The data represented were included in the range [Q1-1.5 IQR - Q3 + 1.5 IQR], being IQR the interquartile range.

Figure 3

Short name: Coronal T₂-weighted images, DCE-MRI images and RCE maps of two mice bearing a GL261 glioma. Detailed legend: From left to right, representative coronal T₂-weighted images, DCE-MRI images (T₁-weighted reference prior to contrast (T₁-ref) and at the maximum contrast enhancement point (T₁-max)), and RCE maps of two mice bearing a GL261 glioma. One animal was studied with gadoterate meglumine (top), and the other with GNP (E_2) (bottom). T₁-ref images were acquired before injecting the contrast agent bolus while T₁-max images correspond to the point of maximum contrast enhancement after gadoterate meglumine or GNP administration (see Figure 4). Colour coded bars on top of RCE provide intensity range shown in figures. Black pixels correspond to instances with values above or below the user-established %RCE postprocessing limits as described in methods.

Figure 4

Short name: RCE time-course curves obtained from the quantification of DCE-MRI images. Detailed legend: RCE time-course curves obtained from the quantification of DCE-MRI images. Each curve displays the average contrast enhancement obtained for each group of mice. Values correspond to the results obtained from 3 animals/group and 3 slices/animal, i.e. n = 9 measurements/group. Gadoterate meglumine, open symbols; GNP (E_2), filled symbols. Tumor is coded by circles and contralateral non-tumoral areas by triangles. Bars show +/−SD.

Figure 5

Short name: Boxplot of %RCE obtained both ex vivo and in vivoat the T₁-max time. Detailed legend: Boxplot for comparison of %RCE obtained both ex vivo (red boxes) and in vivo (green boxes, at the T₁-max time for each CA being studied) for gadoterate meglumine and GNP (E_2).

Figure 6

Short name: Gold (Au) biodistribution as measured by ICP-MS 24 h post-administration of GNP (E_2). Detailed legend: Gold (Au) biodistribution as measured by ICP-MS 24 h post-administration of GNP (E_2). Inset shows tumor Au accumulation at a different scale, compared to element concentrations in plasma and contralateral brain. The difference of Au accumulation between contralateral brain and tumor is nearly significant for GNP (E-2), p =0.056. Values are shown as mean parts per million (ppm) and SD (bar). Statistical comparisons correspond to unpaired t-tests.

Figure 7

Short name: Coronal in vivoT₁-weighted MRI of mice bearing GL261 gliomas, with superimposed K_ep colour-coded maps. Detailed legend: A) Representative coronal in vivo T₁-weighted images, with superimposed K_ep colour-coded maps of two mice bearing a GL261 glioma, administered with Gadoterate Meglumine (left), and GNP (E_2) (right). The black pixels correspond to instances with values above or below the user-established K_ep limits (see Methods). The color scale on top shows the range of values found for K_ep in each case, as well as their color coding. B) Boxplot for comparison of K_ep (min⁻¹) obtained with all in vivo studied animals, using the same contrast agents shown in A). Significant differences (p < 0.05) are marked with a triangle. The limits of the box represent the quartiles 1 (Q1) and 3 (Q3) of the distribution, the central line corresponds to the median (quartile 2) and the whiskers represent the maximum and minimum value in each distribution. The data represented were included in the range [Q1-1.5 IQR - Q3 + 1.5 IQR], being IQR the interquartile range. Outliers are represented outside the whiskers in two ways: as a circle if its value is in the range [Q1-1.5 IQR > value > Q1- 3.0 IQR or Q3 + 1.5 IQR < value < Q1- 3.0 IQR], or as an asterisk if values are above or below this threshold.