Multimodal clinical imaging to longitudinally assess a nanomedical anti-inflammatory treatment in experimental atherosclerosis

Abstract

Atherosclerosis is an inflammatory disease causing great morbidity and mortality in the Western world. To increase the anti-inflammatory action and decrease adverse effects of glucocorticoids (PLP), a nanomedicinal liposomal formulation of this drug (L-PLP) was developed and intravenously applied at a dose of 15 mg/kg PLP to a rabbit model of atherosclerosis. Since atherosclerosis is a systemic disease, emerging imaging modalities for assessing atherosclerotic plaque are being developed. (18)F-Fluoro-deoxy-glucose positron emission tomography and dynamic contrast enhanced magnetic resonance imaging, methods commonly used in oncology, were applied to longitudinally assess therapeutic efficacy. Significant anti-inflammatory effects were observed as early as 2 days that lasted up to at least 7 days after administration of a single dose of L-PLP. No significant changes were found for the free PLP treated animals. These findings were corroborated by immunohistochemical analysis of macrophage density in the vessel wall. In conclusion, this study evaluates a powerful two-pronged strategy for efficient treatment of atherosclerosis that includes nanomedical therapy of atherosclerotic plaques and the application of noninvasive and clinically approved imaging techniques to monitor delivery and therapeutic responses. Importantly, we demonstrate unprecedented rapid anti-inflammatory effects in atherosclerotic lesions after the nanomedical therapy.

Figures

Fig. 1. Pharmacokinetics, imaging and treatment scheme

The organ distribution and pharmacokinetic profile of liposomal PLP and free PLP were evaluated in 6 atherosclerotic rabbits. Two healthy rabbits and 6 atherosclerotic rabbits (top, left) were used to quantify the mean uptake values of 18F-FDG using PET/CT imaging. The treatment groups consisted of a group of 14 atherosclerotic rabbits that received a single dose of liposomal PLP and a group of 8 atherosclerotic rabbits that received a single dose of free PLP (bottom). All animals underwent baseline scanning, multimodality imaging was performed at different time points post treatment to visualize liposome accumulation in the vessel wall and to quantify inflammation of the abdominal aorta. Immunofluorescence and histological analyses were performed on randomly selected animals at the indicated time points.

Fig. 2. Images of delivery and localization of liposomal PLP by MRI and confocal laser scanning microscopy

(A) In vivo MRI of the abdominal aorta before (left) and two days after (right) the administration of liposomes. A marked signal intensity increase was observed throughout the atherosclerotic lesion. (B) NIRF images of an atherosclerotic aorta excised from a rabbit injected with liposomes (left) and untreated aorta (right). The color bar represents photon count. (C) CLSM of liposomes (red), cell nuclei (blue), and macrophages. (D) A high degree of co-localization of liposomes with macrophages was observed. (E) Although liposomes were found throughout the entire lesion areas, a vessel wall reconstruction of multiple CLSM images revealed heterogeneous accumulation of liposomes. F) The corresponding MRI slice of the histological section depicted in (E) revealed a similar heterogeneous distribution.

Fig. 3. Non-invasive imaging of therapeutic effects as determined by 18F-FDG-PET and DCE-MRI

(A) A representative coronal CT, 18F-FDG-PET, and fused imaging slice throughout the abdominal aorta of an atherosclerotic rabbit before and 1 week post administration of liposomal PLP. (B) The mean SUV for the different time points pre and post-injection of liposomal PLP and free PLP are given. (C) Relative changes in SUV for animals treated with liposomal and free PLP. (D) Overlays on anatomical images of AUC maps obtained with DCE-MRI before (left) and two days post (right) treatment with liposomal PLP.

Fig. 4. Correlation between histology and non-invasive 18F-FDG-PET imaging

Representative histological slices of aortic sections stained with Masson’s trichrome and stained for macrophages with RAM-11. (A) Section from a free PLP treated animal, 7 days after intravenous administration. (B) Macrophage density is significantly reduced 7 days post-treatment with liposomal PLP and was (C) back to baseline after 21 days. (D) The mean SUVs (left) for the rabbits that underwent histological quantification of macrophage density (right). (E) Correlation between macrophage density determined histologically and SUV determined by non-invasive imaging (FDG-PET).