Nature-inspired nanoformulations for contrast-enhanced in vivo MR imaging of macrophages

Abstract

Magnetic resonance imaging (MRI) of macrophages in atherosclerosis requires the use of contrast-enhancing agents. Reconstituted lipoprotein particles that mimic native high-density lipoproteins (HDL) are a versatile delivery platform for Gd-based contrast agents (GBCA) but require targeting moieties to direct the particles to macrophages. In this study, a naturally occurring methionine oxidation in the major HDL protein, apolipoprotein (apo) A-I, was exploited as a novel way to target HDL to macrophages. We also tested if fully functional GBCA-HDL can be generated using synthetic apo A-I peptides. The fluorescence and MRI studies reveal that specific oxidation of apo A-I or its peptides increases the in vitro macrophage uptake of GBCA-HDL by 2-3 times. The in vivo imaging studies using an apo E-deficient mouse model of atherosclerosis and a 3.0 T MRI system demonstrate that this modification significantly improves atherosclerotic plaque detection using GBCA-HDL. At 24 h post-injection of 0.05 mmol Gd kg(-1) GBCA-HDL containing oxidized apo A-I or its peptides, the atherosclerotic wall/muscle normalized enhancement ratios were 90 and 120%, respectively, while those of GBCA-HDL containing their unmodified counterparts were 35 and 45%, respectively. Confocal fluorescence microscopy confirms the accumulation of GBCA-HDL containing oxidized apo A-I or its peptides in intraplaque macrophages. Together, the results of this study confirm the hypothesis that specific oxidation of apo A-I targets GBCA-HDL to macrophages in vitro and in vivo. Furthermore, our observation that synthetic peptides can functionally replace the native apo A-I protein in HDL further encourages the development of these contrast agents for macrophage imaging.

Keywords: apolipoprotein A-I; atherosclerosis; biomimetic peptide; gadolinium; lipoprotein nanoparticle; macrophage; magnetic resonance imaging; oxidation; vulnerable plaque.

Figure 1

Schematic representation of the proposed concept of targeting high density lipoproteins (HDL) to macrophages by using specific naturally occurring modifications of the HDL major protein, apolipoprotein (apo) A-I. In the human body, native unmodified HDL (depicted by blue) function to deliver excess cholesterol from peripheral tissues to the liver and are not normally uptaken by macrophages. In contrast, synthetic HDL containing oxidized (depicted by red) apo A-I (or apo A-I peptides) are uptaken by macrophages, thus delivering the incorporated agent(s) directly to the cells of interest. Macrophage uptake is illustrated using the example of intraplaque macrophages.

Figure 2

Oxidation of apo A-I or synthetic apo A-I peptides does not affect HDL size and size distribution. EM images of paramagnetic and fluorescent HDL either with unmodified (A) and oxidized (B) apo A-I or unmodified (C) and oxidized (D) apo A-I peptides. Similar disc-shaped particles often in characteristic rouleaux (depicted by rectangles and zoomed in the right panels) are found in HDL particles containing apo A-I or synthetic apo A-I peptides in either unmodified or oxidized form. Unmodified apo A-I protein/peptides: black letters on a white background; oxidized apo A-I protein/peptides: white letters on a black background. Abbreviations: apo, apolipoprotein; high density lipoproteins, HDL; Gd, Gd-based contrast agent; Rho-B, fluorescently labeled lipid.

Figure 3

Oxidation of apo A-I or synthetic apo A-I peptides enhances macrophage uptake of paramagnetic and fluorescent HDL in vitro. (A) Mean fluorescence intensities of cell lysates normalized to cell protein content (mean ± SD, n = 3): J774 macrophages were incubated with medium only (white bars) or with 0.05 mM Gd paramagnetic and rhodamine B-labeled HDL(A-I) or HDL(H4+H6) for 4 h at 37°C. (B) Mean T₁ values of cell pellets (mean ± SD, n = 3): J774 macrophages were incubated with medium only (white bars) or with medium containing 0.05 mM Gd paramagnetic and Rho B-labeled HDL(A-I) or HDL(H4+H6) for 4 h at 37°C. The inset: exemplary T1-weighted images of cell pellets after incubation with medium only (M), HDL(A-I) with either unmodified (1) or oxidized (1*) apo A-I, and HDL(H4+H6) with either unmodified (2) or oxidized (2*) peptides. (C) Normalized enhancement ratio (NER) of cell pellets: values (mean ± SD, n = 3) are calculated from the corresponding T₁-weighted images and are relative to cells incubated with medium only. (D) Contrast-to-noise ratio (CNR) values of cell pellets: values (mean ± SD, n = 3) are calculated from the corresponding T₁-weighted images and are relative to cells incubated with medium only. Abbreviations: apo, apolipoprotein; Gd, Gd-based contrast agent; high density lipoproteins, HDL; HDL(A-I), HDL with apo A-I; HDL(H4+H6), HDL with a 1:1 mixture of synthetic peptides H4 and H6 that correspond to apo A-I helixes 4 and 6, respectively. Unmodified and oxidized protein/peptide species are depicted by light and dark gray bars, respectively.

Figure 4

Oxidation of synthetic apo A-I peptides enables targeted in vivo imaging of macrophages in aortic atherosclerotic lesions. Representative axial T₁-weighted images of wild type (WT) and apo E knockout (KO) mice, as collected before (left panels) and 24 h after (right panels) contrast agent injection: WT (A) and apo E KO (B, C) mice were administered with the equivalent of 0.05 mmol Gd/kg of paramagnetic and fluorescent HDL containing either oxidized (A, C) or unmodified (B) peptides H4 and H6. The insets: original images were cropped to select the aorta. Abbreviations: apo, apolipoprotein; high density lipoproteins, HDL; HDL(H4+H6), HDL with a 1:1 mixture of synthetic peptides H4 and H6 that correspond to apo A-I helixes 4 and 6, respectively; Gd, Gd-based contrast agent; Rho-B, fluorescently labeled lipid. Unmodified peptides: black letters on a white background; oxidized peptides: white letters on a black background.

Figure 5

Oxidation of apo A-I or synthetic apo A-I peptides enhances intraplaque macrophage uptake of paramagnetic and fluorescent HDL in vivo. (A) Normalized enhancement ratio (NER) of the apo E KO mouse aortic wall 24 h following injection of the equivalent of 0.05 mmol/kg Gd of paramagnetic and fluorescent HDL(A-I) or HDL(H4+H6): values (mean ± SD, n=4) are calculated from the corresponding T₁-weighted images and are relative to muscle. (B) Contrast-to-noise ratio (CNR) of mouse aorta wall 24 h following injection of the equivalent of 0.05 mmol/kg Gd of paramagnetic and fluorescent HDL(A-I) or HDL(H4+H6): values (mean ± SD, n=4) are calculated from the corresponding T₁-weighted images and are relative to muscle. Abbreviations: apo, apolipoprotein; Gd, Gd-based contrast agent; high density lipoproteins, HDL; HDL(A-I), HDL with apo A-I; HDL(H4+H6), HDL with a 1:1 mixture of synthetic peptides H4 and H6 that correspond to apo A-I helixes 4 and 6, respectively; KO, knockout. Unmodified and oxidized protein/peptide species are depicted by light and dark gray bars, respectively.

Figure 6

Paramagnetic and fluorescent HDL with oxidized peptides H4 and H6 colocalize with macrophages in an atherosclerotic plaque. (A) Differential interference contrast (DIC) microscopy image of an aorta section from an apo E KO mouse 24 h following injection of the equivalent of 0.05 mmol/kg Gd of paramagnetic and fluorescent HDL containing a 1:1 mixture of oxidized peptides H4 and H6. Confocal laser scanning microscopy of a zoomed-in area (depicted by rectangle) of the section shown in (A) and stained with DAPI (blue) (B) and an antibody against a macrophage surface marker, CD68, (green) (C) or analyzed in the rhodamine channel (red) for GBCA-HDL uptake (D). The merged image (E) demonstrates uptake of the rhodamine B-labeled HDL formulations by intraplaque macrophages. The autofluorescent elastic lamina is observed as multiple wave-like structures and is disrupted in an atherosclerotic lesion. (F) Hematoxylin and eosin (H&E) staining of the matched histopathological aorta section. Abbreviations: apo, apolipoprotein; Gd, Gd-based contrast agent; high density lipoproteins, HDL; KO, knockout.