NANO LETTERS
Properties of a Versatile Nanoparticle Platform Contrast Agent To Image and Characterize Atherosclerotic Plaques by Magnetic Resonance Imaging
2006 Vol. 6, No. 10 2220-2224
Juan C. Frias,†,‡ Yanqing Ma,§ Kevin Jon Williams,⊥ Zahi A. Fayad,*,† and Edward A. Fisher*,§ Zena and Michael A. Wiener CardioVascular Institute and the Marie-Josee and Henry R. KraVis CardioVascular Health Center, The Mount Sinai School of Medicine, New York, New York 10029, Leon H. Charney DiVision of Cardiology, Department of Medicine, New York UniVersity School of Medicine, New York, New York 10016, and Thomas Jefferson UniVersity, Philadelphia, PennsylVania 19107 Received June 29, 2006
ABSTRACT The need for more specific and selective contrast agents for magnetic resonance imaging motivated us to prepare a new nanoparticle agent based on high-density lipoproteins (HDL). This second generation contrast agent can be prepared in three different ways. The HDL nanoparticles (rHDL) were fully characterized by FPLC and gel electrophoresis. The flexibility of the platform also allows us to incorporate optical probes into rHDL for localization ex vivo by confocal fluorescence microscopy. The contrast-agent-containing nanoparticles were injected into mice that develop atherosclerotic lesions. Magnetic resonance imaging of the animals showed clear enhancement of the atherosclerotic plaques.
Atherosclerosis remains a major cause of morbidity and mortality in industrialized nations and throughout the world. The accumulation of harmful lipoproteins in the vessel wall and the resulting local responses are responsible for the development of atherosclerotic plaques. Clinical sequellae, such as angina, myocardial infarction, and stroke, are the consequences of both large, stable plaques that impede blood flow and smaller unstable plaques that give rise to acute events upon rupture.1-4 Given these severe health threats of atherosclerosis, noninvasive detection and characterization of plaques may be useful to detect patients at risk or to monitor the efficacies of therapies.5-8 Magnetic resonance imaging (MRI) is a powerful tool for visualizing tissues owing to superb spatial resolution that is achieved without ionizing radiation or invasive procedures. Image quality can be further enhanced by the administration of contrast agents. The presence of the contrast agent causes a large increase in the water proton relaxation rate, thereby * Corresponding authors. E-mail: E.A.F.,
[email protected]; Z.A.F.,
[email protected]. † The Mount Sinai School of Medicine. ‡ Current address: Instituto de Ciencia Molecular, ICMOL University of Valencia. § New York University School of Medicine. ⊥ Thomas Jefferson University. 10.1021/nl061498r CCC: $33.50 Published on Web 07/26/2006
© 2006 American Chemical Society
adding further detail to the anatomical resolution. Currently, more than 35% of all clinical MRI scans make use of clinically approved contrast agents (Magnevist, Prohance, Dotarem), based mainly on gadolinium (Gd3+) complexes.9,10 Contrast-enhanced imaging of atherosclerotic plaques using Gd-chelates either in steady state11 or during dynamic uptake12 has shown the potential for some plaque composition characterization, compared to nonenhanced imaging, but has limited specificity for components of atheroma.13 Therefore, major efforts are underway to design more specific and targeted MRI contrast agents.14-17 Given the wide clinical impact of coronary artery disease (CAD), there is a need for contrast agents that can image the presence and biologic activities of specific plaque components in vivo. The assessment of molecular information in vivo requires high-affinity, target-specific contrast agents with marked signal amplification. Most of the available contrast agents are not capable of delivering enough Gd into the arterial wall to induce a large MR signal. To minimize this disadvantage, several strategies have been devised to increase the efficacy (as defined by its relaxivity, r1, the increment of the total paramagnetic relaxation rate enhancement of water protons, R1p, per millimolar concentration of contrast agent) by incorporating the contrast agents into linear poly-
Figure 1. Different types of HDL-like nanoparticle contrast agents and the gadolinium complexes used in their preparation.
mers,18 dendrimers,19 micellar structures,20 and protein bound chelates21 that increase the effective local concentration. To achieve both high efficacy and the capability for specific targeting of atherosclerotic plaques in vivo, we chose to develop a family of high-density lipoprotein (HDL)-like nanoparticle contrast agents. HDL is a heterogeneous class of plasma lipoproteins that have a density between 1.063 and 1.21 g/mL and a Stoke’s diameter of 5-17 nm.22 HDL particles have a hydrophobic core that is surrounded by a monolayer of phospholipids, unesterified cholesterol, and apolipoproteins (Figure 1). The major apolipoprotein of HDL is apoA-I, although other apolipoproteins may also be present.23 In epidemiological studies, the concentration of cholesterol carried by HDL in plasma correlates inversely with the risk of CAD, with a number of mechanisms invoked for this relationship, particularly the ability of HDL to promote cholesterol efflux from plaque foam cells.24-28 From the standpoint of engineering a new MRI contrast nanoplatform, attractive attributes of HDL include (1) the ease of reconstitution of selected components into HDL-like nanoparticles (Figure 1); (2) their size, which is small enough to allow HDL and HDL-like nanoparticles to cross the endothelium and thereby enter and exit plaques, yet large enough for us to attach adequate amounts of MRI contrast materials; (3) their protein components, which are endogenous and biodegradable and do not trigger immunoreactions; and (4) their lack of atherogenic effects, in distinction to LDL.29,30 We prepared HDL-like nanoparticle contrast agents Nano Lett., Vol. 6, No. 10, 2006
Figure 2. FPLC gel permeation profiles of the different nanoparticle preparations (native incubated [incubated HDL], sonicated discoidal [rHDL-disc 1], and dialyzed discoidal [rHDL-disc 2]).
following well-established protocols for HDL re-constitution31,32 but modified for our purposes. Three strategies were used to prepare nanoparticle contrast agents; in each case, the contrast agent was incorporated into the phospholipid layer of the nanoparticle (more details are provided in the Supporting Information; see also Figure 1): (1) human plasma HDL was delipidated by standard methods to obtain total HDL apolipoproteins (apo-HDL), which consist mainly of apoA-I (Figure 3A). The apo-HDL was then reconstituted into a discoidal form (sonicated discoidal, or disk 1) by sonication with a molar excess of a single species of phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), along with the contrast agent gadolinium diethylenetriamine pentaacetic acid bis-stearylamide (Gd2221
Figure 3. (A) SDS-PAGE (4-12%) analysis of plasma HDL (“HDL”) and the sonicated discoidal contrast agent (“disc 1”). (B) Nondenaturing gradient gel electrophoresis in acrylamide (4-20%) of indicated FPLC fractions of the dialyzed discoidal nanoparticle contrast agent (A280 profile shown is taken from Figure 2). The analyses shown in each panel are representative of at least three preparations.
DTPA-bSA); (2) apo-HDL was reconstituted into a discoidal form (dialyzed discoidal, or disk 2) using the cholate dialysis method31 with lower molar amount of phospholipids (soy PC) that was mixed with gadolinium chelated to a DTPA moiety covalently attached to a phospholipid (gadolinium 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine diethylenetriamine pentaacetic acid, Gd-DTPA-DMPE); and (3) purified, intact native HDL, complete with core triglycerides and cholesteryl esters, was incubated in the presence of GdDTPA-bSA. The flexibility of the HDL-like nanoplatform has allowed us to also include a fluorescent phospholipid analogue, 1,2-dipalmitoyl-sn-glycero-3-phosphoethenolamineN-(7-nitro-2-1,3-benzoxadiazol-4-yl), NBD-DPPE (λex ) 460 nm, λem ) 534 nm) for confocal fluorescence microscopic localization of the nanoparticles in postmortem tissue samples.33,34 Characterization of our nanoparticles by fast protein liquid chromatography (FPLC; exact conditions in Supporting Information) afforded clear evidence that the material had the expected physical features of HDL particles (Figure 2). On the basis of the profile of absorbance at 280 nm of the column eluates, the majority of the signal for the incubated native HDL and disk 2 particles was found in fractions 5270, which corresponds to the size range of normal human28,35 and mouse36 plasma HDL. Some material was also detected in a peak that spanned fractions ∼30-40 (Figures 2 and 3), which is where particles of the size of very low density lipoprotein (VLDL, ∼70 nm) or larger would elute. For the discoidal particles prepared by sonication (disk 1), relatively more material was found in fractions 30-40, as expected from the lower protein:phospholipid ratio that was used in the reconstitution (Supporting Information). Further analysis of fractions 30-40 for the two discoidal nanoparticle forms confirmed the presence of apoA-I and either Gd-DTPA-bSA or Gd-DTPA-DMPE, depending on which contrast agent was used. Thus, fractions 30-40 appear to contain either vesicles or aggregates. On the basis of the A280 profiles, the amount 2222
of apo-HDL that was in fractions 30-40 was up to ∼55% for disk 1, but no more than ∼13% for disk 2, and essentially none for the native HDL that had been incubated with GdDTPA-bSA. Only the authentic HDL peak material (i.e., pooled fractions 52-70) was used for imaging purposes. The pooled material was concentrated using Amicon Ultra-15 Centrifugal Filter Devices (membrane cutoff range: 10 000 kDa; Millipore, Billerica, MA). In addition to the FPLC analyses, further studies to estimate the size of the contrast nanoparticles were performed by nondenaturing gradient gel electrophoresis37 using samples from the FPLC fractions of interest. As shown in Figure 3B, the gel migration of disk 2 particles from the various FPLC fractions corresponded with the size range for native HDL, as well as for HDL disks reconstituted in similar procedures by others.28 The gel and FPLC sizing results were independently corroborated by light scattering measurements (Supporting Information). From these different physical methods, the size range (expressed as hydrated diameter in nm) for the discoidal particles in fractions 52-70 was 8-13 nm, and for the incubated HDL particles, 14-17 nm. Thus, the contrast nanoparticles retain key physical features of HDL, and in particular, the incorporation of Gd moieties does not appear to substantially alter the size or other properties that are discernible by gel permeation, electrophoresis, or light scattering. Relaxivity measurements of the nanoparticles were done on a 60 MHz Bruker Minispeq (Bruker Medical BmbH, Ettingen) operating at 40 °C. The values were 9.45, 10.40, and 10.00 mM-1 s-1 for the disk 1, disk 2, and incubated HDL particles, respectively. In contrast, the relaxivity of the Gd-DTPA alone (i.e., without bSA) was ∼3 mM-1 s-1. We could not similarly determine the relaxivity of Gd-DTPADMPE because it is insoluble in water. All the preparations were stored in a saline with 1% sucrose at -20 °C, were stable for months, and did not show further aggregation. Nano Lett., Vol. 6, No. 10, 2006
Figure 4. In vivo MR images of apoE-/- mice injected with the two types of discoidal HDL-like nanoparticle contrast agents. (A) Preand postcontrast MR images at the indicated time points after injection of 2.77 µmol (of apo-HDL)/kg of disc 1 nanoparticles into an apoE-/- mouse. (B) Pre- and postcontrast MR images at the indicated time points after injection of 7.33 µmol/kg of disc 2 nanoparticles into an apoE-/- mouse. In both cases, the maximum enhancement was found at 72 h postinjection. The corresponding histopathology stained with hematoxylin and eosin of the aortic segment imaged in panel A showed a complex plaque with cholesterol crystals and a relatively low content of intact macrophage foam cells. (C) Images obtained in vivo after injection of disc 1 nanoparticles into an apoE-/mouse. Note the maximal enhancement is at 24 h postinjection. The corresponding histopathology now revealed a high content of macrophage foam cells in an uncomplicated plaque. The images are representative of 6 apoE-/- mice.
We have previously reported29 the successful use of spherical HDL nanoparticles that contain core lipids and Gd to enhance images of atherosclerotic plaques of apoE knockout mice (apoE-/-; a standard model of human atherosclerosis38). To now test the imaging capabilities of the two types of discoidal nanoparticles in vivo, we injected FPLC-purified material into the tail veins of wild-type mice, as well as into 13-month-old apoE-/- mice that had been fed on a high cholesterol diet to accelerate plaque progression (see Supporting Information for more experimental details). The animals underwent magnetic resonance microscopy (MRM) of the abdominal aorta in vivo using a 9.4 T MR system (Bruker Instruments, Billerica, MA). Prior to each imaging time point, mice were anesthetized with continuously inhaled isofluorane (1.5-2%) and placed headup in a vertical 30 mm birdcage coil. The injection dose was 200 µL of a 2 mM gadolinium solution of a discoidal contrast agent. Sequential MRI (Figure 4) showed that both types of discoidal contrast nanoparticles localized predominantly in atherosclerotic plaques of the apoE-/- mice. Moreover, the enhancement observed was related to plaque Nano Lett., Vol. 6, No. 10, 2006
composition, as determined by subsequent histologic analysis: early enhancement in vivo (24 h) was observed (panel C) in uncomplicated, macrophage-rich plaques (fatty streaks), whereas advanced plaques full of cholesterol crystals, but relatively fewer macrophages, showed late enhancement in vivo (72 h; panels A and B). In both uncomplicated and advanced plaques, the nanoparticle signals persisted for 24 h after the peak, with essentially complete wash-out by 48 h and 96 h, respectively, after injection. Wild-type animals (n ) 3), which have no arterial lesions, showed no MRI signal enhancement at any time (data not shown). As we previously reported,29 confocal fluorescence microscopy of histological samples revealed that the nanoparticles were internalized by macrophage foam cells located mainly in the intimal layer (data not shown), presumably because of the endocytosis pathway for HDL in macrophages (e.g., see ref 39). This localization of the HDL optical label to macrophages provides a ready basis for the observed correlation of the rate of enhancement of the MRI signal with macrophage content. 2223
In conclusion, we have characterized by FPLC chromatography, gel electrophoresis, and light scattering a new group of contrast nanoparticles based on HDL. They target atherosclerotic plaques and enhance the MRI signal in a manner dependent on plaque macrophage content. The phospholipids on the surface of the nanoparticles provide broad flexibility to the platform, which can be exploited not only for MRI but also for CT and NIR imaging. Also, modification of the protein components can preferentially direct the nanoparticles toward different receptors or other determinants within plaques or other targets.40 Acknowledgment. We thank Prof. E. Garcı´a-Espan˜a and Dr M. T. Albelda (Department of Inorganic Chemistry, University of Valencia, Valencia, Spain) for their assistance with the collection of the excitation and emission spectra. We thank Dr. J. G. S. Aguinaldo and Dr. V. Amirbekian for their help with the injections and MR imaging. We thank Dr. Karen C. Briley-Saebo for discussions and review of the studies. This work was supported by grants from the NIH/ NHLBI (R01 HL071021, R01 HL078667, R01 HL084312). The Mount Sinai Microscopy Shared Facility is supported with funding from NIH/NCI (R24 CA098523) and NSF (DBI-924504). Note Added after ASAP Publication. There was a clarification in the nomenclature used throughout the text, “spherical hollow HDL” changed to “disk 1 nanoparticles” and the term for the other discoidal preparation changed to “disk 2 nanoparticles”, and a corresponding change in the drawings of the structures in Figure 1 in the version published ASAP July 26, 2006; the corrected version was published ASAP August 11, 2006. Supporting Information Available: A description of the preparation of the different types of HDL is reported. Also the conditions for FPLC, gel electrophoresis and MRI scanning are provided. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Gaziano, T. A. Circulation 2005, 112, 3547-3553. (2) Lusis, A. J. Nature 2000, 407, 233-241. (3) Williams, K. J.; Tabas, I. Arterioscler. Thromb. Vasc. Biol. 1995, 15, 551-561. (4) Williams, K. J.; Tabas, I. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 1536-1540. (5) Choudhury, R. P.; Fuster, V.; Fayad, Z. A. Nat. ReV. Drug DiscoV. 2004, 3, 913-925. (6) Leiner, T.; Gerretsen, S.; Botnar, R.; Lutgens, E.; Cappendijk, V.; Kooi, E.; van Engelshoven, J. Eur. Radiol. 2005, 15, 1087-1099. (7) Yuan, C.; Kerwin, W. S. J. Magn. Res. Imaging 2004, 19, 710719.
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NL061498R
Nano Lett., Vol. 6, No. 10, 2006
