High-Density Lipoprotein–Based Contrast Agents for Multimodal Imaging of Atherosclerosis
Lipoproteins, natural nanoparticles, have a well-recognized biological role and are highly suitable as a platform for delivering imaging agents. The ease with which both the exterior and interior of the particles can be modified permits the creation of multifunctional nanoparticles for imaging as well as the delivery of therapeutics. Importantly, their endogenous nature may make them biocompatible and biodegradable and allows them to avoid the recognition of the reticuloendothelial system. In particular, high-density lipoproteins (HDL) are of interest, because of their small size they can easily cross the endothelium and penetrate the underlying tissue. We summarize here the progress in establishing HDL as a vector for delivering a variety of diagnostically active materials to vulnerable atherosclerotic plaques in mouse models of atherosclerosis. By loading various types of image-enhancing compounds into either the core or surface of HDL, they can be visualized by different imaging modalities (MRI, CT, optical). By rerouting of HDL away from plaque macrophages, imaging of biological processes in diseases besides atherosclerosis may also be achieved.
- cardiovascular imaging agents/techniques
- molecular imaging
- computerized tomography and magnetic resonance imaging
Series Editor: Stanley Hazen
ATVB In Focus
HDL Structure, Function, Therapeutics and Imaging
After Roentgen serendipitously discovered X-rays in the late 19th century, and the first simple images were produced, medical imaging has become an increasingly important part of medical practice and diagnostics. Although the underlying technology of projection X-ray imaging is very mature and therefore has advanced slowly the past decades, this technique is still the most commonly used imaging modality in clinical practice. Multiple new imaging modalities have been developed during the last century, and physicians as well as scientists now have a variety of imaging procedures to aid them, making it possible to diagnose, characterize, examine, and monitor disease processes noninvasively.
One of the most promising and fastest developing fields within medical diagnostics is molecular imaging.1–7 Molecular imaging is associated with several imaging techniques and is defined as the characterization and measurement of biological processes at the cellular and molecular level using in vivo imaging methods.3 Whereas conventional imaging technologies have traditionally been used for the visualization of anatomic structures, molecular imaging seeks to visualize fundamental cellular and even molecular processes, thereby providing knowledge of disease phenotype and increasing the understanding of the basic origin of a given process or disease. Techniques such as computed tomography (CT), MRI, and ultrasound (US) give excellent anatomic information, but because these modalities give relatively little information on cellular and molecular processes, their application as molecular imaging modality relies on the application of specially designed contrast agents.
Molecular imaging contrast agents are frequently nanoparticulate of nature and are usually made specific for a certain cell type, receptor, or other molecular component important in the investigated tissue, cell, process, or pathology. Their physical and functional properties, clearance profiles, biocompatibility, and long-term toxicity need to be carefully considered. Primarily because of advances in chemistry, nanoparticle contrast agents for molecular imaging are getting progressively more sophisticated, and currently include multimodal agents,8–10 “smart” or “activatable” agents,11–13 and also agents that transport, along with contrast generating materials, therapeutic payloads such as drugs,14,15 nucleotides,16,17 or proteins.18 For a more in-depth discussion on the state of the art of nanoparticle and probe design related to molecular imaging, we refer the reader to other recently published reviews.19–21
Biocompatibility is one of the most important challenges for therapeutic and diagnostic nanoparticles. The particle composition as well as the particle surface are of key importance. The surface of nanoparticles may be modified to enhance biocompatibility (eg, by using coatings such as polyethylene glycol,22 dextran,23 and phospholipids24). Another way to create biocompatible contrast agents is by using composites of diagnostically active materials and natural nanoparticles, such as viruses25 or lipoproteins. Because of their well-recognized roles in physiology as well as pathophysiology, lipoproteins have become very interesting carrier vehicles for contrast agents and therapeutics.
This review will first briefly describe the imaging modalities used in molecular imaging, followed by a discussion of the roles of lipoprotein nanoparticles in the body, and their applications for imaging. The primary focus will be on the smallest of the lipoprotein family, high-density lipoprotein (HDL), and its application in molecular imaging of atherosclerosis.
Molecular Imaging Modalities
Traditionally, nuclear imaging techniques, including scintigraphic imaging, positron emission tomography (PET), and single photon emission computed tomography were the primary choices for molecular imaging because of their inherent high sensitivity.26 Because these nuclear techniques lack anatomic definition and exhibit poor spatial resolution, a number of different other imaging techniques have been exploited for molecular imaging of atherosclerotic plaques. MRI is one of the most versatile techniques in both clinical and research settings, and has also been widely exploited for imaging of atherosclerosis. MRI gives excellent images of soft tissue with high spatial resolution. For molecular imaging purposes exogenous contrast agents need to be applied. There are 2 major classes of MRI contrast agents. One class includes the positive contrast agents that give rise to bright contrast in so-called T1-weighted images. These positive contrast agents are largely based on paramagnetic gadolinium and manganese ion complexes. The other class consists of agents based primarily on superparamagnetic iron oxide that cause darkening in T2/T2*-weighted images and therefore are termed negative contrast agents.
Computed tomography (CT) excels in the visualization of hard tissue, such as bones or plaque calcifications. However, application of iodinated contrast agents also allows for the visualization by CT of the vascular lumen (angiography), but CT agents can also be targeted to the arterial wall, as pioneered by Hyafil et al, where atherosclerotic lesions in the rabbit aorta were visualized using a clinical CT scanner.27 Contrast agents for CT imaging are typically based on iodine,27,28 gold,29 or bismuth.30
Optical imaging techniques exhibit superb spatial and temporal resolution, are very sensitive, and are capable of visualizing multiple species simultaneously. In addition to confocal microscopy, multi-photon microscopy, intravital microscopy, as well as near infrared imaging techniques are increasingly being used on both cells in vitro and small laboratory animals in vivo.31–33 In contrast to the abovementioned techniques most of the optical techniques are invasive, can only be applied to dissected tissues, or are limited for application to tissues on or near the surface of small animals.34
Lipoproteins are a family of plasma particles responsible for the transportation of lipids throughout the body. Chylomicrons, very low–density, and low-density (VLDL, LDL) lipoproteins possess a hydrophobic core, consisting of triglycerides and cholesteryl esters, enclosed in a monolayer of phospholipids. Embedded in the phospholipid monolayer are cholesterol and the apolipoproteins, which strongly contribute to structural integrity as well as the lipoprotein’s biological properties. High-density lipoproteins (HDL) can be either disc-like (no core) or spherical (with a core of triglycerides and cholesteryl esters). Chylomicrons transport intestinally-derived lipids. VLDL is the major lipoprotein produced by the liver, and it transports the triglycerides and cholesterol made there. After secretion, some VLDLs are converted to LDL, which carries most of the cholesterol in the plasma. HDL can be produced by both the liver and intestine. After their formation, they can cross the endothelium to pick up cholesterol from peripheral cells, and then deliver this cargo to the liver in a process named reverse cholesterol transport (RCT).35 Besides the different roles they play, lipoproteins vary greatly in size, from 1000 nm for chylomicrons to 10 nm for HDL.
It is well established that an elevated plasma level of LDL-cholesterol is associated with increased risk of coronary artery disease.36 On the other hand, the HDL-cholesterol plasma level is inversely related to the risk of cardiovascular disease.36,37 Though LDL and HDL play opposing roles in the pathophysiology of atherosclerosis,37 both enter the plaques and therefore have been used as carriers for contrast agents for molecular imaging of atherosclerosis.9,38–43 Importantly, their endogenous origin makes them biocompatible and biodegradable and allows them to avoid recognition by the reticuloendothelial system. Notably, the ease with which both the exterior and interior of these particles can be modified permits the creation of multifunctional nanoparticles for multimodality imaging as well as for the delivery of therapeutics.
When LDL is retained in the plaque, it undergoes a series of modifications that increase the interactions with macrophages.44 As a consequence, LDL cholesterol and cholesteryl esters enter the cells, which eventually results in the formation of foam cells.45,46 Moreover the aggregation and the modification of LDL contribute to inflammatory processes in the arterial wall.47,48 The accumulation of inflamed (activated) macrophages and foam cells coupled with a thin fibrous cap is believed to result in plaque vulnerability and rupture.49 Therefore, the inherent affinity of modified LDL for plaque macrophages37 and the central role macrophages play in the pathogenesis of atherosclerosis makes LDL an attractive candidate as a contrast agent for the visualization of plaques38,39 and historically modified LDL has been the most exploited lipoprotein as an imaging agent. In addition, LDL can be exploited as an imaging agent for tumor visualization. Certain neoplastic cells overexpress LDL receptors (LDLrs),50–52 and as a result LDL contrast agents can be used to visualize these neoplastic tumor cells.53,54
HDL Contrast Agents
The main protein component of HDL is apolipoprotein A-I (apoA-I), which is initially synthesized in the liver or the intestine. It is a particle that consists of a predominantly cholesteryl ester and triglyceride core that is covered with apolipoproteins and a monolayer of phospholipids. Despite the fact that HDL carries various apolipoproteins,55,56 the abundant protein components are apoA-I and apoA-II.56–58 The major component apoA-I, like most apolipoproteins, is composed of an amphiphatic α-helical structure,58,59 with its hydrophobic region embedded in the phospholipids and the hydrophilic region facing the surrounding aqueous milieu. The apolipoprotein is suggested to fold in different “belt” like conformations on the HDL particle and thus is thought to significantly reduce the surface pressure on the lipoprotein,60,61 thereby stabilizing the particle. HDL/apoA-I interacts with the enzyme lecithin cholesterol acyltransferase (LCAT),35,55 which catalyzes esterfication of free cholesterol into cholesteryl esters. Along with an increased volume of cholesteryl esters present in the hydrophobic core of the lipoprotein, the HDL particle changes shape from discoidal to spherical.62
As mentioned earlier, HDL can accept cholesterol from peripheral cells in the reverse cholesterol transport process. The efflux of cholesterol from plaque macrophages is thought to be an important protective effect on the development of atherosclerosis, but HDL is additionally believed to exhibit various other antiatherothrombotic properties, including inhibition of oxidation, inflammation, proliferation, and platelet function35,55,63,64 (Figure 1).
These characteristics of HDL make it a more desirable contrast agent to be administered compared to LDL. In addition, HDL may cross the endothelial barrier better than other lipoproteins because of its smaller size and perhaps because of specialized transport processes,65 allowing a higher degree of infiltration into atherosclerotic plaques and other tissues.
So far, 4 different kinds of molecular imaging HDL particles have been reported and can be classified according as: radio-labeled HDL,40 reconstituted HDL,41 synthetic HDL,43 and nanocrystal HDL.9 All these HDL imaging particles were applied as contrast agents for imaging the vulnerable plaque in atherosclerosis, although lipoproteins also have the potential to be rerouted to target other disease processes.66
The application of HDL as an imaging agent was suggested for the first time in 2001 by Shaish et al.40 They compared the use of 125I-labeled LDL, oxidized LDL (oxLDL), HDL, and bovine serum albumin (BSA) for imaging of atherosclerosis in mice. HDL and LDL used in the study were harvested from human plasma, modified by washing via ultracentrifugation, dialyzed and purified, and finally radio-iodinated. The radio-iodination allowed detection of the particle location in vivo with gamma cameras and hence the accumulation in atherosclerotic lesions as well as the biodistribution and pharmacokinetics (plasma clearance and tissue distribution) could be determined (Figure 2A and 2B). Shaish et al showed in an apoE knock out (KO) mouse model that both 125I-LDL, 125I-HDL, and 125I-oxLDL accumulated in the atherosclerotic plaques as seen in Figure 2C. The highest accumulation was found 24 hours postadministration for each radio-labeled lipoprotein, and was mainly associated with the aortic arch and abdominal aorta, which corresponds with regions of high prevalence of atherosclerosis in the mouse model used.67,68 A significant difference in the plasma clearance was found among the three types of lipoproteins. It is noteworthy to mention that about 30% of the injected LDL and HDL were still present in the blood plasma 24 hours after intravenous administration, whereas only 10% of oxLDL was present in the plasma 30 minutes after injection.
In 2004 Frias and colleagues published the synthesis of a reconstituted HDL (rHDL)-like particle for imaging of atherosclerosis by contrast enhanced MRI.41 ApoA-I, extracted from human plasma, was reconstituted with commercially available phospholipids, one of which incorporated a gadolinium (Gd)-chelate, making the particle visible for MRI (Figure 3A). These were spherical particles with cholesteryl esters and triglycerides in their core. Furthermore, a green-emitting and amphiphilic fluorophore (NBD-DPPE) was included in the lipid monolayer, to enable its detection with fluorescence techniques. MRI scans of the abdominal aorta of the mice after injection of rHDL into the tail vein of apoE KO mice revealed significant accumulation of rHDL in the aortic wall at 24, 48, 72, 96 hours (Figure 3B). It was found that the increase in signal intensity due to the injected agent was highest at 24 hours. The signal was substantially decreased after 48 hours, whereas injection of the rHDL into nonatherosclerotic mice did not give rise to any increase in signal intensity of the aortic wall.
Mice were euthanized 24 hours after intravenous administration, and histological examination of excised aorta sections was performed. Confocal microscopy showed that the agent was primarily associated with macrophages in the plaques. Moreover it was established after histological examination that MRI signal enhancement was related to plaque composition; the higher the macrophage burden, the more intense the MRI signal.
Frias et al extended the previously described study 2 years later, again using rHDL, however this time rHDL-discs were used.42 rHDL was prepared in different ways, where the first method relied on sonication for the formation of the rHDL discs (disc 1). For the second method, cholate dialysis with phospholipids and phospholipids conjugated with Gd-DTPA was performed. These rHDL discs were named disc 2. In addition, to permit fluorescence microscopy imaging, the fluorophore NBD-DPPE was included in both rHDL particle types. A schematic depiction of spherical and discoidal HDL-like particles are given in Figure 3A.
Discs 1 and 2 were intravenously administered to apoE KO mice as well as to control wild-type mice. It was found that both agents accumulated in the atherosclerotic vessel wall of apoE KO mice, visualized by increased MRI signal intensity in this tissue. By histological examination it was further observed that the increase in signal intensity was highest after 24 hours in macrophage-rich plaques, whereas late (72 hours) augmentation in signal intensity was found in advanced plaques containing fewer macrophages and an increased level of cholesteryl crystals. Overall, Frias et al have described the development of discoidal and spherical, gadolinium-labeled, rHDL and documented their use for plaque imaging with MRI in atherosclerotic apoE KO mice.
Further extending this approach, Cormode et al43 developed a fully synthetic HDL/apoA-I mimicking nanoparticle. “Synthetic HDL” refers to HDL-like nanoparticles that are composed of lipids and an apolipoprotein A-I–derived peptide. For the creation of HDL-like discs, an apoA-I mimicking peptide, 37pA, was used along with phospholipids. 37pA is an amphiphatic α-helical peptide based on part of the native apoA-I sequence, whose hydrophobic face nestles with the acyl chains of phospholipids, whereas the hydrophilic part faces the aqueous environment. Hence the properties of 37pA are very similar to those of apoA-I. As before, both Gd-chelates and a lipid-based fluorophore, rhodamine-PE, were incorporated into the phospholipid layer, making the particles suitable for MRI and fluorescence imaging. A particle of micellar structure, containing both Gd-chelates and rhodamine-PE, but no 37pA-peptide was synthesized as a control particle.
The particles were characterized in terms of size, zeta potential, relaxivity, and phosphate and protein content. In addition cholesterol efflux measurements were undertaken for the 37pA particle. As described earlier, inducing cholesterol efflux is a pivotal property of HDL. Portrayed in Figure 4B is the first investigation of cholesterol efflux properties of a HDL imaging agent, showing there was a similar level of cholesterol efflux from macrophage cells incubated with the 37pA particle as with native HDL.
In vivo studies on apoE KO mice and WT mice were carried out, with a comparison between the 37pA particle and the micellar particle, using the same methodology as described for the previous studies. The pre- and 24 hour postinjection images, after administration of the 37pA particle, are shown in Figure 4C. The results showed that the 37pA particle gave significantly more signal increase in the abdominal aortic wall than the micellar particle (Figure 4D), proving the effectiveness of the peptide-based HDL for imaging atherosclerosis. Furthermore, increases in signal intensity found on administration of the 37pA particle in the nonatherosclerotic mice were not significant, further indicating the specificity of this contrast agent for atherosclerotic tissue.
The same group reported a similar synthetic HDL nanoparticle where another apoA-I mimicking peptide, 18A, was used in place of 37pA (ref). This agent, termed 18A-Gd, was compared extensively to 37pA-Gd as to their efficacy with respect to macrophage imaging.43a No significant difference in contrast in the aorta of apoE KO mice produced by each agent at 24 hours was observed, and so it was concluded that the 2 agents are equally efficacious.
Another HDL particle for atherosclerotic plaque imaging was recently reported by Cormode et al.9 The refined synthesis enabled inorganic nanocrystals to replace the natural HDL core, while retaining the phospholipid coating. Therefore these HDL particles could carry at least 2 different contrast generating materials via modification of both the core and the corona, rendering them multimodal. A schematic representation of this multimodal platform is depicted in Figure 5A.
The nanocrystals in the core of the particle were either gold (Au) for CT, iron oxide (FeO) for MRI, or quantum dots (QD) for optical imaging. In the synthesis procedure, phospholipids were applied to the nanocrystals thereby creating spherical structures with a nanocrystal core and a monolayer coating of phospholipids. In the phospholipid surface layer, apoA-I, lipid-based Gd chelates, and fluorescent rhodamine-PE were included.
The particles were characterized with transmission electron microscopy (Figure 5B), relaxometry, phantom imaging, protein and phosphorus analyses, and other techniques which revealed these nanoparticles to be very uniform and mono-disperse and to have similar phosphate and protein content to natural HDL.
At first the HDL particles were applied to a murine macrophage cell line and the uptake was investigated using confocal laser scanning microscopy (Figure 5C), MRI, and TEM (Figure 5D). Results showed that all 3 compounds were extensively taken up by macrophage cells. In vivo MRI experiments were performed with apoE KO mice. MR images of the abdominal aorta of the mice are depicted in Figure 5E. As a consequence of the Gd-chelates accumulating in the atherosclerotic plaques, the Au- and QD-HDL agents caused a significant enhancement of the vessel wall, whereas a significant decrease in signal was found after the application of the FeO-HDL. Ex vivo imaging of both whole excised aortas as well as aortic sections disclosed the presence of these contrast agents in the aortic wall and the association with macrophages within the vessel wall, as displayed in Figure 5F.
Within the lipoprotein family, historically LDL has been most exploited as an imaging agent. The idea of HDL as an imaging agent is relatively new, and only a limited number of HDL-based nanoparticles for imaging have been reported. Even so, there are already examples of HDL imaging particles of very sophisticated design.
Our own work has focused primarily on the development of HDL-based contrast agent for the detection of macrophages in atherosclerotic plaques with MRI as well as with CT and optical techniques. To improve detectability and to allow a lower dose to be injected, several modifications have been introduced to enhance the contrast generating properties. For example, the nanocrystal HDL platform allows the inclusion of nanoparticulate diagnostically active materials. In addition, amphiphilic Gd chelates can be used that exhibit improved relaxivity (eg, by including a so-called q=2 MRI contrast agent as reported by Briley-Saebo et al69).
In addition to the potential imaging of the vulnerable atherosclerotic plaque, reconstituted HDL particles also hold the promise to be applied for the experimental investigation of HDL metabolism and the mechanism of HDL interactions with cells to mediate cholesterol efflux.
Interestingly, HDL as well as LDL exhibits the ability to be rerouted to other desired targets. The rerouting of lipoproteins from their natural receptors to folate receptors has been demonstrated in a number of studies by Zheng and coworkers. They conjugated folic acid to the apolipoproteins of LDL and HDL thereby targeting tumors that over expressed the folate receptor.53,54,66,70,71 Besides conjugating a target-specific molecule directly to the apolipoproteins, a different strategy was used by Chen et al. The authors chose to include a functional amphiphile (ie, an apoE-derived lipopeptide), P2A2,72 into their reconstituted HDL nanoparticle to enhance the uptake by atherosclerotic plaques of apoE KO mice as determined by in vivo MRI. The rerouting approach of HDL is still in its infancy, but similar to what has been demonstrated with liposomes and micelles,73 a variety of peptides, antibodies, or small molecules may be used to target these lipoprotein-based carriers to epitopes of interest.
Another interesting application of HDL to be further investigated is targeted drug delivery. The structure of lipoproteins means that hydrophobic and amphiphilic drugs can be included most simply. Drug delivery in combination with the possibility of rerouting these carriers to a variety of receptors and cells adds to the versatility of this platform. In an early study in 1996 this approach was demonstrated by Bijsterbosch et al,74 where reconstituted HDL was used as a carrier for a lipophilic prodrug to specifically target parenchymal liver cells. Recently several studies have been published using reconstituted HDL particles for the delivery of therapeutic compounds to cells that act as models for a variety of diseases in vitro.75,76
To conclude, lipoproteins are a highly versatile group of carriers, which, given their endogenous origin, are thought to be nonimmunogenic and biodegradable. Although HDL-based contrast agents are designed to mimic endogenous HDL, the introduction of exogenous materials may significantly alter the biocompatibility and biodegradability of the particle. Moreover, injected HDL may exhibit a different pharmacological profile from and may compete with endogenous HDL and thereby enact influence on the lipoprotein balance in the blood. The flexibility and ease with which their composition can be manipulated/modified makes them well suited for the creation of contrast agents in diagnostic imaging as well as delivery vehicles for therapeutics. Continued progress in their formulation and in understanding the mechanisms for their efficacy promises improvements in their application to the diagnosis and therapy of atherosclerosis and other diseases.
Partial support provided by NIH grants R01 HL71021, R01 HL78667 and R01 EB009638. We thank the AHA Founder’s Affiliate for Postdoctoral Fellowship 09POST2220194, the Danish Heart Association for studentship 07-10-A1655-22406 and Danish Cardiovascular Research Academy.
Received May 11, 2009; revision accepted September 28, 2009.
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