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To investigate the imaging performance of an elastin-specific molecular magnetic resonance imaging (MRI) probe with respect to the extracellular matrix (ECM) in an experimental hepatic cancer model. Twelve rabbits with hepatic VX2 tumors were examined using 3 T MRI 14, 21, and 28 days after tumor implantation for two subsequent days (gadobutrol, day 1; elastin-specific probe, day 2). The relative enhancement (RE) of segmented tumor regions (central and margin) and the peritumoral matrix was calculated using pre-contrast and delayed-phase T1w sequences. MRI measurements were correlated to histopathology and element-specific and spatially resolved mass spectrometry (MS). Mixed-model analysis was performed to assess the performance of the elastin-specific probe. In comparison to gadobutrol, the elastin probe showed significantly stronger RE, which was pronounced in the tumor margin (day 14–28: P ≤ 0.007). In addition, the elastin probe was superior in discriminating between tumor regions (χ2(4) = 65.87; P < 0.001). MRI-based measurements of the elastin probe significantly correlated with the ex vivo elastinstain (R = .84; P <0 .001) and absolute gadolinium concentrations (ICP-MS: R = .73, P <0 .01). LA-ICP-MS imaging confirmed the colocalization of the elastin-specific probe with elastic fibers. Elastin-specific molecular MRI is superior to non-specific gadolinium-based contrast agents in imaging the ECM of hepatic tumors and the peritumoral tissue.
Atherosclerosis is a progressive inflammatory vascular disease characterized by endothelial dysfunction and plaque burden. Extracellular matrix (ECM)-associated plasma proteins play an important role in disease development. Our magnetic resonance imaging (MRI) study investigates the feasibility of using two different molecular MRI probes for the simultaneous assessment of ECM-associated intraplaque albumin deposits caused by endothelial damage and progressive inflammation in atherosclerosis. Male apolipoprotein E-deficient (ApoE-/-)-mice were fed a high-fat diet (HFD) for 2 or 4 months. Another ApoE-/--group was treated with pravastatin and received a HFD for 4 months. T1- and T2*-weighted MRI was performed before and after albumin-specific MRI probe (gadofosveset) administration and a macrophage-specific contrast agent (ferumoxytol). Thereafter, laser ablation inductively coupled plasma mass spectrometry and histology were performed. With advancing atherosclerosis, albumin-based MRI signal enhancement and ferumoxytol-induced signal loss areas in T2*-weighted MRI increased. Significant correlations between contrast-to-noise-ratio (CNR) post-gadofosveset and albumin stain (R2 = 0.78, p < 0.05), and signal loss areas in T2*-weighted MRI with Perls’ Prussian blue stain (R2 = 0.83, p < 0.05) were observed. No interference of ferumoxytol with gadofosveset enhancement was detectable. Pravastatin led to decreased inflammation and intraplaque albumin. Multi-target MRI combining ferumoxytol and gadofosveset is a promising method to improve diagnosis and treatment monitoring in atherosclerosis.
Molecular MRI is a promising in-vivo modality to detect and quantify morphological and molecular vessel-wall changes in atherosclerosis. The combination of different molecular biomarkers may improve the risk stratification of patients. This study aimed to investigate the feasibility of simultaneous visualization and quantification of plaque-burden and inflammatory activity by dual-probe molecular
MRI in a mouse-model of progressive atherosclerosis and in response-to-therapy. Homozygous apolipoprotein E knockout mice (ApoE−/−) were fed a high-fat-diet (HFD) for up to four-months prior to MRI of the brachiocephalic-artery. To assess response-to-therapy, a statin was administered for the same duration. MR imaging was performed before and after administration of an elastin-specific gadolinium-based and a macrophage-specific iron-oxide-based probe. Following in-vivo MRI, samples were analyzed using histology, immunohistochemistry, inductively-coupled-mass-spectrometry and laser-inductively-coupled-mass-spectrometry. In atherosclerotic-plaques, intraplaque expression
of elastic-fibers and inflammatory activity were not directly linked. While the elastin-specific probe demonstrated the highest accumulation in advanced atherosclerotic-plaques after four-months of HFD, the iron-oxide-based probe showed highest accumulation in early atherosclerotic-plaques after two months of HFD. In-vivo measurements for the elastin and iron-oxide-probe were in good agreement
with ex-vivo histopathology (Elastica-van-Giesson stain: y = 298.2 + 5.8, R2 = 0.83, p < 0.05; Perls‘ Prussian-blue-stain: y = 834.1 + 0.67, R2 = 0.88, p < 0.05). Contrast-to-noise-ratio (CNR) measurements of the elastin probe were in good agreement with ICP-MS (y = 0.11x-11.3, R² = 0.73, p < 0.05). Late stage atherosclerotic-plaques displayed the strongest increase in both CNR and gadolinium concentration (p < 0.05). The gadolinium probe did not affect the visualization of the iron-oxide-probe and vice versa. This study demonstrates the feasibility of simultaneous assessment of plaque-burden.
Molecular MRI is a promising in-vivo modality to detect and quantify morphological and molecular vessel-wall changes in atherosclerosis. The combination of different molecular biomarkers may improve the risk stratification of patients. This study aimed to investigate the feasibility of simultaneous visualization and quantification of plaque-burden and inflammatory activity by dual-probe molecular MRI in a mouse-model of progressive atherosclerosis and in response-to-therapy. Homozygous apolipoprotein E knockout mice (ApoE−/−) were fed a high-fat-diet (HFD) for up to four-months prior to MRI of the brachiocephalic-artery. To assess response-to-therapy, a statin was administered for the same duration. MR imaging was performed before and after administration of an elastin-specific gadolinium-based and a macrophage-specific iron-oxide-based probe. Following in-vivo MRI, samples were analyzed using histology, immunohistochemistry, inductively-coupled-mass-spectrometry and laser-inductively-coupled-mass-spectrometry. In atherosclerotic-plaques, intraplaque expression of elastic-fibers and inflammatory activity were not directly linked. While the elastin-specific probe demonstrated the highest accumulation in advanced atherosclerotic-plaques after four-months of HFD, the iron-oxide-based probe showed highest accumulation in early atherosclerotic-plaques after two-months of HFD. In-vivo measurements for the elastin and iron-oxide-probe were in good agreement with ex-vivo histopathology (Elastica-van-Giesson stain: y = 298.2 + 5.8, R2 = 0.83, p < 0.05; Perls‘ Prussian-blue-stain: y = 834.1 + 0.67, R2 = 0.88, p < 0.05). Contrast-to-noise-ratio (CNR) measurements of the elastin probe were in good agreement with ICP-MS (y = 0.11x-11.3, R² = 0.73, p < 0.05). Late stage atherosclerotic-plaques displayed the strongest increase in both CNR and gadolinium concentration (p < 0.05). The gadolinium probe did not affect the visualization of the iron-oxide-probe and vice versa. This study demonstrates the feasibility of simultaneous assessment of plaque-burden and inflammatory activity by dual-probe molecular MRI of progressive atherosclerosis. The in-vivo detection and quantification of different MR biomarkers in a single scan could be useful to improve characterization of atherosclerotic-lesions.
The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.