MRI-assessed therapeutic effects of locally administered PLGA nanoparticles loaded with anti-inflammatory siRNA in a murine arthritis model.

Rheumatoid arthritis is characterized by systemic inflammation of synovial joints leading to erosion and cartilage destruction. Although efficacious anti-tumor necrosis factor α (TNF-α) biologic therapies exist, there is an unmet medical need for safe and more efficient treatment regimens for disease remission. We evaluated the anti-inflammatory effects of poly(dl-lactide-co-glycolide acid) (PLGA) nanoparticles loaded with small interfering RNA (siRNA) directed against TNF-α in vitro and in vivo. The siRNA-loaded PLGA nanoparticles mediated a dose-dependent TNF-α silencing in lipopolysaccharide-activated RAW 264.7 cells in vitro. The severity of collagen antibody-induced arthritis in DBA/1J mice was assessed by paw scoring and compared to the degree of magnetic resonance imaging (MRI)-quantified joint effusion and bone marrow edema. Two intra-articular treatments per joint with nanoparticles loaded with TNF-α siRNA (1 µg) resulted in a reduction in disease activity, evident by a significant decrease of the paw scores and joint effusions, as compared to treatment with PLGA nanoparticles loaded with non-specific control siRNA, whereas the degree of bone marrow edema in the tibial and femoral head remained unchanged. When the siRNA dose was 5 or 10 µg, there was no difference between the specific and the non-specific siRNA treatment groups. These findings suggest that MRI is a promising method for evaluation of early disease progression and treatment in murine arthritis models. In addition, proper siRNA dosing seems to be important for a positive therapeutic outcome in vivo. However, further studies are needed to fully clarify the mechanism(s) underlying the observed anti-inflammatory effects of the siRNA-loaded nanoparticles.

Evaluation of infarcted murine heart function: comparison of prospectively triggered with self-gated MRI.

Measurement of cardiac function is often performed in mice after, for example, a myocardial infarction. Cardiac MRI is often used because it is noninvasive and provides high temporal and spatial resolution for the left and right ventricle. In animal cardiac MRI, the quality of the required electrocardiogram signal is variable and sometimes deteriorates over time, especially with infarcted hearts or cardiac hypertrophy. Therefore, we compared the self-gated IntraGateFLASH method with a prospectively triggered FLASH (fast low-angle shot) method in mice with myocardial infarcts (n = 16) and in control mice (n = 21). Mice with a myocardial infarct and control mice were imaged in a vertical 9.4-T MR system. Images of contiguous 1-mm slices were acquired from apex to base with prospective and self-gated methods. Data were processed to calculate cardiac function parameters for the left and right ventricle. The signal-to-noise and contrast-to-noise ratios were calculated in mid-ventricular slices. The signal-to-noise and contrast-to-noise ratios of the self-gated data were higher than those of the prospectively gated data. Differences between the two gating methods in the cardiac function parameters for both left and right ventricle (e.g. end-diastolic volumes) did not exceed the inter-observer variability in control or myocardial infarcted mice. Both methods gave comparable results with regard to the cardiac function parameters in both healthy control mice and mice with myocardial infarcts. Moreover, the self-gated method provided better signal-to-noise and contrast-to-noise ratios when the acquisition time was equal. In conclusion, the self-gated method is suitable for routine use in cardiac MRI in mice with myocardial infarcts as well as in control mice, and obviates the need for electrocardiogram triggering and respiratory gating. In both gating methods, more than 10 frames per cardiac cycle are recommended.

Characterization and in vitro and in vivo testing of CB2-receptor- and NGAL-targeted paramagnetic micelles for molecular MRI of vulnerable atherosclerotic plaque.

PURPOSE: Atherosclerotic plaque macrophages express the peripheral cannabinoid receptor (CB2-R) and promote fibrous cap degradation by secretion of neutrophil gelatinase-associated lipocalin 2 (NGAL). In this study, we report the preparation, characterization, and in vitro and in vivo testing of double-labeled (MR and fluorescent) CB2-R- and NGAL-targeted micelles. PROCEDURES/RESULTS: Specific CB2-R agonists or antibodies directed to 24p3 (mouse homolog of NGAL) were incorporated into di-oleoyl-polyethylene glycol-phosphatidylethanolamine 1000 (DOPE-PEG1000) micelles or di-stearoyl-polyethylene glycol-phosphatidylethanolamine 2000 (DSPE-PEG2000) micelles. The hydrodynamic diameter, determined by dynamic light scattering, was 16.5 and 19.0 nm for CB2-R-targeted DOPE-PEG1000 and DSPE-PEG2000 micelles, respectively, and 23.0 nm for Ab-conjugated DSPE-PEG2000 micelles. In vitro and in vivo MRI and fluorescence microscopy showed specific binding of CB2-R-targeted and 24p3-targeted micelles to in vitro systems and to aortic plaque in apoE(-/-)/eNOS(-/-) mice, respectively. CONCLUSIONS: CB2-R- and NGAL-targeted micelles show promise as tools for in vivo characterization of vulnerable plaque.

Recent developments and new perspectives on imaging of atherosclerotic plaque: role of anatomical, cellular and molecular MRI Part I and II.

Atherosclerotic plaque disruption accounts for the major part of cardiovascular mortality and the risk of disruption appears to depend on plaque composition. Carotid plaques in patients, scheduled for endarterectomy, have been successfully characterised with MRI. MRI has the advantage of combining information about morphology and function. Unfortunately, the tortuosity and size of the coronary arteries, and the respiratory and cardiac motion hinder the in vivo characterisation of human coronary plaque. In addition to plaque composition several molecular markers of the different processes involved in atherosclerosis, such as integrins, matrix metalloproteinases and fibrin seem to correlate with risk of plaque rupture and clinical outcome. These molecular markers can be targeted with antibodies coupled to carriers, which are loaded with gadolinium for detection (molecular MRI). Several cellular/molecular MRI studies in animal models and some in human patients have been conducted with varying levels of success. The advent of clinical high field magnets, the development of contrast agent carriers with high relaxivity and the development of relatively new MR contrast techniques are promising in the field of plaque imaging. Future MRI studies will have to focus on the molecular target of the atherosclerotic process, which has the highest prognostic value with regard to acute coronary syndromes and on the most suitable contrast agent to visualize that target.

Recent developments and new perspectives on imaging of atherosclerotic plaque: role of anatomical, cellular and molecular MRI part III.

Atherosclerotic plaque disruption accounts for the major part of cardiovascular mortality and the risk of disruption appears to depend on plaque composition. Carotid plaques in patients, scheduled for endarterectomy, have been successfully characterised with MRI. MRI has the advantage of combining information about morphology and function. Unfortunately, the tortuosity and size of the coronary arteries, and the respiratory and cardiac motion hinder the in vivo characterisation of human coronary plaque. In addition to plaque composition several molecular markers of the different processes involved in atherosclerosis, such as integrins, matrix metalloproteinases and fibrin seem to correlate with risk of plaque rupture and clinical outcome. These molecular markers can be targeted with antibodies coupled to carriers, which are loaded with gadolinium for detection (molecular MRI). Several cellular/molecular MRI studies in animal models and some in human patients have been conducted with varying levels of success. The advent of clinical high field magnets, the development of contrast agent carriers with high relaxivity and the development of relatively new MR contrast techniques appear to be promising in the field of plaque imaging. Future MRI studies will have to focus on the molecular target of the atherosclerotic process, which has the highest prognostic value with regard to acute coronary syndromes and on the most suitable contrast agent to visualize that target.

Negative MR contrast caused by USPIO uptake in lymph nodes may lead to false positive observations with in vivo visualization of murine atherosclerotic plaque.

OBJECTIVE: USPIOs are used clinically as contrast agent for magnetic resonance imaging (MRI) of lymph nodes, and in research settings for MRI of macrophages in atherosclerotic lesions. However, T2* weighted (T2*w) imaging can lead to "blooming" with overestimation of the area occupied by USPIOs. In this study, plaque uptake of USPIOs in atherosclerotic mice was investigated in the presence and absence of circulating monocytes. The influence of peri-aortic lymph node uptake on the interpretation of T2*w images of the aortic wall was studied. METHODS: Atherosclerotic mice were fed an atherogenic diet and were randomized to total body irradiation or non-irradiation. After 2 days, T2*w MRI of the abdominal aorta was performed, followed by intravenous administration of 100mumol/kg USPIOs (t=0). At t=3 and 5 days MRI of the abdominal aorta was repeated. Animals were sacrificed and histological evidence for iron uptake by aortic wall and lymph nodes was compared with the degree of focal signal loss on in vivo MR images. RESULTS: Aortic walls in irradiated and non-irradiated mice, but also in healthy wild-type mice, showed signal loss on T2*w MRI. Signal loss however did not correspond with histological evidence of USPIO uptake by aortic wall but by peri-aortic lymph nodes. CONCLUSIONS: The versatility of USPIOs as a negative MR contrast agent for both lymph node staging and atherosclerosis may limit the use for detection of atherosclerotic lesions in vessels where lymph nodes are highly prevalent.