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CT imaging of myocardial scars with collagen-targeting gold nanoparticles
POTENTIAL CLINICAL RELEVANCE Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 1067 – 1076
Research Article
nanomedjournal.com
CT imaging of myocardial scars with collagen-targeting gold nanoparticles Delia Danila, PhD⁎, Evan Johnson, MS, Patrick Kee, MD, PhD Department of Internal Medicine, Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX, USA Received 8 January 2013; accepted 24 March 2013
Abstract In the setting of myocardial ischemia, recovery of myocardial function by revascularization procedures depends on the extent of coronary disease and myocardial scar burden. Currently, computed tomographic (CT) imaging offers superior evaluation of coronary lesions but lacks the capability to measure the transmural extent of myocardial scar. Our work focuses on determining if collagen-targeting gold nanoparticles (AuNPs) can effectively target myocardial scar and provide adequate contrast for CT imaging. AuNPs were coated with a collagen-homing peptide, collagen adhesin (CNA35). Myocardial scar was created in mice by occlusion/reperfusion of the left anterior descending coronary artery. Thirty days later, un-gated CT imaging was performed. Over 6 h, CNA35-AuNPs provided uniform and prolonged opacification of the vascular structures (100-130 HU). In mice with larger scar burden, focal contrast enhancement was detected in the myocardium, which was not apparent within that of control mice. Histological staining confirmed myocardial scar formation and accumulation of AuNPs. From the Clinical Editor: This team of investigators presents a collagen-targeting gold nanoparticle-based approach that enables the imaging of myocardial scars via CT scans in a rodent model. This information would enable clinicians to judge the recovery potential of myocardium more accurately than the current CT-scan based approaches. Published by Elsevier Inc. Key words: Gold nanoparticles; Myocardial scars; CT imaging; Molecular imaging
Despite advances in the management of coronary artery disease, myocardial infarction is still prevalent. The development of myocardial scar leads to heart failure, cardiac arrhythmia and sudden cardiac death. 1 However, in a subset of patients with viable myocardial tissues in the peri-infarct zone, studies have clearly shown that revascularization procedures such as coronary artery bypass grafts and coronary angioplasty increase left ventricular ejection fractions and improve survival in those patients. 2,3 Clearly, identification of such patients will be important for disease management. Among the imaging techniques currently being investigated or utilized for evaluating myocardial scar burden in ischemic cardiomyopathy, MR imaging with delayed Gd enhancement shows enormous potential. Imaging modalities such as echocardiography, fluoroscopic left ventriculogram with contrast and CT cardiac imaging are at best semi-quantitative. However, MR All co-authors have seen and agreed with the contents of the manuscript and there is no financial interest to report. ⁎Corresponding author: Houston, TX, 77054, USA. E-mail addresses: [email protected] (D. Danila), [email protected] (E. Johnson), [email protected] (P. Kee).
imaging with Gd administration may not be appropriate for all subjects and obstacles to its use include patient preferences, the presence of metallic medical devices and the risk of nephrogenic systemic fibrosis associated with gadolinium-based MR contrast agent, especially in patients with renal impairment. CT coronary angiography has unrivaled advantages in terms of speed in image acquisition, relative availability, spatial resolution and tissue penetration. Hardware development in CT scanners has made a major leap over the years and the latest generation of CT scanners can provide excellent anatomic details of luminal abnormalities in association with obstructive coronary artery disease. However, the identification of viable myocardium and ischemic myocardial scar with CT remains challenging. Attempts have been made to replicate the technique of delayed Gd hyperenhancement in MR imaging with conventional iodinated contrast agent but the beamhardening artifact from contrast within the left ventricle and the short blood pool persistence of conventional CT contrast agent clearly hamper the advances in myocardial perfusion imaging with multidetector CT. 4,5 Conventional CT contrast agent has proven to be very safe but their formulations have remained relatively unchanged over the last 60 years. There is also a paucity of iodinated CT contrast agents that have been formulated for targeted imaging. 6-8 In the
1549-9634/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.nano.2013.03.009 Please cite this article as: Danila D, et al, CT imaging of myocardial scars with collagen-targeting gold nanoparticles. Nanomedicine: NBM 2013;9:1067-1076, http://dx.doi.org/10.1016/j.nano.2013.03.009
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face of this developmental plateau in CT contrast agents, there is a need to develop new metal-based agents for X-ray imaging. 9 Gold nanoparticles have been an attractive candidate not only for X-ray imaging due to their superior X-ray attenuation properties when compared with conventional contrast agents, they may also offer interesting applications for thermal therapy and chemical sensing via color changes and surface-enhanced Raman spectroscopy. Bare gold nanoparticles are unstable in vivo, however, their rapid aggregation and clearance can be reduced by anti-fouling coating with agents such as polyethylene glycol (PEG). 10-15 PEG-stabilized gold nanoparticles have been investigated as a contrast agent for blood pool imaging. 15 Depending on the size, small gold nanoparticles are readily excreted by the kidneys with little retention in the liver and spleen. 16 Antibody-functionalized gold nanoparticles, also known as “immunogold”, have been used by electron microscopy but they exhibit poor biocompatibility and aggregate via nonspecific adsorption of proteins in vivo. However, the proteinrepelling PEG matrix on the surface of gold nanoparticles can be readily modified by inclusion of heterobifunctional PEG for effective conjugation to peptides, folic acid or antibodies 17-25 Antibody-conjugated gold nanoparticles have been investigated as a targeted contrast agent for detecting tumors by CT imaging 26 and thermodynamic therapy. 27 Other high k-edge metals have been prepared for CT imaging. Bismuth is inherently toxic but has been stabilized with a polymer coating to improve its biocompatibility and retain its high X-ray attenuation properties and long circulation times in vivo. 28 Another candidate metal suitable for X-ray imaging is tantalum and has been stabilized with zwitterionic agents 29 and PEG 30 for in vivo blood pool imaging. Specific retention of contrast agents in myocardial scar can be enhanced by attaching collagen-homing ligands on their surface. Although antibodies are commonly used for molecular targeting, the use of homing peptides is thought to be more biocompatible and translatable. The collagen-homing ligand chosen for myocardial scar targeting is a truncated form of collagen adhesin, CNA35, which has a higher affinity for collagen than the wild type protein isolated from the cell surface of Staphylococcus aureus. The collagen-binding domains of CNA35 have excellent affinity for collagen I with a Kd ranging from 20 nM to 30 μM. 31,32 CNA35 is approximately five times smaller than antibodies, and this could be beneficial for tissue penetration and on-rate kinetics. CNA35 has been conjugated to fluorescent probes for histologic staining of myocardial fibrosis and fibrotic components in atherosclerotic arteries in apoE −/− mice. 33 Other collagen-homing ligands have also been reported. Glutathione S-transferase fusion protein of the I domain of human integrin subunit α1 was previously tested but its affinity was inferior to that of CNA35. 33 A proprietary collagentargeting peptide was discovered by phage display and conjugated to Gd-based MR contrast agent, EP-3533, which demonstrated excellent myocardial scar enhancement in vivo. However, this formulation is not commercially available. 34 We report here the development of a new CT imaging contrast agent for the non-invasive detection of myocardial scars. We developed a targeted radiocontrast agent based on gold nanoparticles (AuNPs) which has the advantages of offering
3-fold greater contrast per unit weight than iodine-based x-ray contrast agents, 35 and probably better safety profile than iodineor gadolinium-based agents in terms of renal and cutaneous toxicity. X-ray imaging has the advantages of providing superior tissue penetration and spatial resolution.
Materials and Methods Materials Sodium citrate, gold chloride and Picrosirius Red stain (Direct Red 80, Picric acid solution, and Hematoxylin solution A according to Weigert) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PEG-SH and OPSS-PEG-SVA were purchased from Laysan Bio (Arab, AL, USA). PES membranes (3,000 MWCO and 100,000 MWCO) were purchased from Fisher Scientific. PES membranes (10,000 MWCO) were purchased from Viva Products Inc. (Littleton, MA, USA). Silver enhancement staining kit was purchased from Structure Probe, Inc. (West Chester, PA, USA). Collagen I was purchased from BD Bioscience. CNA35 was kindly donated by Magnus Hook, and anti-His-HRP antibody was purchased from AbCam. Methods Gold Nanoparticles Synthesis In order to prepare gold nanoparticles, nanopure water (500 ml) was filtered through 0.22 μm filter and boiled in a 1 L conical flask. 5 ml of Gold Chloride (10%) was added to the boiling water followed by 4 ml of 1% sodium citrate solution. The solution was boiled for about an hour or until 200 ml of solution was left. The solution has a burgundy color. Next, the AuNPs were pegylated with polyethylene glycol derivatives in order to avoid aggregation in vivo. The AuNPs were incubated for 1 hour with a 100:1 molar ratio of PEG-SH to prevent aggregation and with 50:1 orthopyridyldisulfide-polyethyleneglycol-N-hydroxysuccinimide (OPSS-PEG-SVA) for the covalent coupling of the peptide. After pegylation, the AuNPs were further concentrated by centrifugation at 3000 rpm for 30 min. The pink supernatant was transferred to clean tubes and centrifuged again. This process was repeated 2 more times and all the concentrated fractions were combined and filtered through 0.22 μm filters. The AuNPs were further concentrated to ~ 4 mg/ml using PES membrane concentrators (MWCO 10,000). The AuNPs were characterized in terms of size and polydispersity by UV and Dynamic Light Scattering after appropriate dilution. Synthesis of CNA35-AuNPs CNA35 (36.5 μg) was incubated with 500 μl AuNPs (40 mg/ml) overnight at 4 °C. Next day, the excess CNA35 was removed by centrifugation using Vivaspin filters (MWCO 100,000). Flocculation assay Gold nanoparticles (2×10 13 AuNPs) were incubated with various molar ratios of PEG-SH. The following ratios of PEGSH:AuNPs were prepared: 0:1, 1:1, 2.5:1, 5:1, 10:1, 25:1, 50:1, 100:1, 250:1, 500:1, 1K:1, 10K:1. After one hour incubation at room temperature, NaCl (10%) was added and the solution incubated for 30 min at room temperature. The absorbance
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spectrum (400-600 nm) was then recorded for all of the above mentioned solutions. The disappearance or the shift in the signal indicates aggregation. Solid-phase binding assay 96-well plates were coated overnight with collagen I at 4 °C, followed by blocking the non-specific sites with BSA (50 mg/ml) for 1 h at 37 °C. Next, various forms of CNA35 ligands (CNA35, CNA35-AuNPs, and unconjugated AuNPs) were incubated for 2 h at 37 °C. After washing the unbound CNA35 ligands, anti-His-HRP antibody was added to specifically recognize CNA35, followed by color development with a substrate for horseradish peroxidase, for 30 min at room temperature. The absorbance readings at 405 nm indicate the binding of CNA35 to the various substrates. Animal experiments The animal studies were performed on female C57BL/6 mice (20 g) ordered from Jackson Laboratories (Bar Harbor, ME, USA). The animals were housed at the University of Texas Health Science Center at Houston (UTHSCH). The animal protocol was approved by the UTHSCH Center for Laboratory Animal Medicine and Care (protocol number: HSC-AWC-11150- “Pilot project: Myocardial Scar Imaging with Gold Nanoparticles”); experiments were performed in accordance with institutional guidelines, and all efforts were made to minimize suffering. Myocardial infarction was created in C57BL/6 mice by occlusion of the proximal left anterior descending (LAD) artery with a 8-0 Proline suture for 60 min followed by releasing the occlusive suture under general anesthesia to re-establish coronary perfusion. Thirty days after the LAD occlusion/ reperfusion surgery, a collagen-rich scar was well developed for CT imaging with CNA35-AuNPs. CT imaging protocol and analysis The animals were sedated with IP Avertin. 200 μl of CNA35AuNPs (40 mg/ml) was injected into the animals via the tail vein. Eight myocardial infarction mice and seven control mice were injected and CT imaging was performed with a GE Ultra flat panel CT scanner (General Electric, Milwaukee, WI) with the following acquisition settings: 80 kVp, 22 mA with 16 s rotation/exposure. Non-cardiac gated CT images were acquired at baseline, 1 min, 15 min, 30 min, 45 min, 60 min, 2 h, 4 h, 6 h and 24 h after AuNPs injection. At the end of the imaging, animals were perfused with buffered paraformaldehyde and the carcasses were imaged once more by the CT scanner. Imaging of the carcasses was performed due to the fact that the CT imaging was ungated and motion artifacts from cardiac contractions may reduce the sensitivity for contrast detection in the myocardium. Simple back projections were obtained for the 0.154 μm image reconstruction and exported as DICOM images. Image analysis was performed using the OsiriX software. Stacks of 3-4 consecutive sections were viewed with maximum intensity projection (MIP) and window levels were adjusted to identify any signal enhancement in the myocardium. The myocardium was divided into 3 sectors to identify the location of signal
Figure 1. Stabilization of AuNPs in saline environment (UV data). Three different flocculation assays demonstrating the reproducible relationship between the ratio of PEG-SH:AuNPs and size homogeneity of AuNPs. Discrete and uniform preparations of AuNPs with a diameter of 50 nm displays a peak absorbance at 530 nm. The magnitude of A530 drops precipitously when AuNPs aggregate as seen when PEG-SH:AuNPs decreases below 50.
enhancement and correlated with scar formation and AuNPs deposition by histological staining. Histological staining of myocardial scar formation and AuNPs deposition The excised heart was sectioned at 5 mm intervals and embedded in paraffin for histological analysis. Collagen formation and AuNPs retention in the myocardium were stained with Picrosirius Red and silver enhancement staining kit, respectively. Light microscopy was performed to determine the distribution of collagen deposition and AuNPs retention in the myocardium.
Results CNA35-AuNPs preparation and characterization The AuNPs prepared by citrate reduction had a hydrodynamic diameter of 40-70 nm with a polydispersity index of 0.213-0.480 (based on DLS data). Our UV-VIS data indicate that the AuNPs have a diameter of 50 nm, exhibiting an absorption maximum at 530 nm, characteristic for a uniform preparation of AuNPs of 50 nm. The amount of short PEG-SH needed to prevent aggregation of AuNPs in the presence of 1% NaCl solution was determined by flocculation assays. Figure 1 summarizes three separate runs of the flocculation assays. The 50 nm gold nanoparticles exhibit a maximum absorption peak at 530 nm and the magnitude of this peak decreases drastically as the particles aggregate in a saline environment. A ratio of 100:1 PEG-SH: AuNPs was sufficient to provide stability of the gold nanoparticles. By DLS analysis, the hydrodynamic diameter and polydispersity index of PEG-stabilized AuNPs were 0 nm for a 1:1 or 10:1 PEG-SH:AuNPs, 64.1 nm for a 100:1 PEG-SH: AuNPs and 68.6 nm for a 10K:1 PEG-SH:AuNPs, confirming
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Table 1 DLS analysis of PEG-stabilized AuNPs. PEG-SH:AuNPs
Diameter (nm)
Polydispersity Index (PDI)
1:1 10:1 100:1 500:1 1K:1 10K:1
0 0 64.1 62.8 64.5 68.6
1.0 1.0 0.473 0.522 0.519 0.478
Figure 2. Specificity of binding of CNA35-AuNPs to Collagen 1. Solid phase Collagen 1 binding assay was performed by incubating pure CNA35 or CAN35-AuNPs with Collagen 1. The amount of peptide bound to Collagen 1 was measured by a colorimetric method and the level of absorbance at 405 nm was proportional to the amount of bound peptide.
the lack of aggregation of PEG-stabilized AuNPs (Table 1). The PEG-stabilized AuNPs were functionalized with CNA35 via a long PEG. Collagen-binding capacity of CNA35 was relatively retained after conjugation to AuNPs as shown in the ELISA type assay (Figure 2). Pharmacokinetics and biodistribution of CNA35-AuNPs in vivo After CNA35-AuNPs were administered intravenously, peak blood pool enhancement of 130-150 HU was observed at up to 2 h, followed by a gradual time-dependent decline in contrast enhancement in the blood pool to 100-130 HU at 6 h (Figure 3). By 24 h, most of the CNA35-AuNPs disappeared from the blood pool. This illustrated the long circulating nature of CNA35AuNPs, which is one of the pre-requisites for effective targeted imaging. Substantial amount of contrast was detected in the liver and spleen and may represent the major sources of elimination of CNA35-AuNPs. Targeted imaging of collagen deposition in myocardial scar with CNA35-AuNPs and histological correlation During the first 6 h of CT imaging, MIP did not demonstrate any focal enhancement of contrast in the myocardium of control animals and animals subjected to LAD occlusion/ reperfusion surgery with smaller myocardial scar formation. However, in animals with larger myocardial scar formation, CT imaging revealed focal enhancement in the myocardium of the nonbeating heart (Figure 4).
Picrosirius Red staining confirmed the myocardial scar formation (the red staining in Figure 4, C) in animals subjected to LAD occlusion/ reperfusion surgery. Silver enhancement of AuNPs demonstrated the proximity between AuNPs retention and collagen deposition in the myocardium. Silver enhancement staining was seen mainly in the periphery of collagen formation but there was a good correlation between the amount of scar formation and silver enhancement in the myocardium. Interestingly, in the myocardial segments not injured by ischemia, scattered silver enhancement was also observed. In control mice (Figure 4, D), there was no evidence of collagen deposition or silver enhancement within the myocardium. We observed some variability in the transmural extent of the scar formation in the animals by measuring the scar burden using Picrosirius Red staining. Picrosirius Red staining revealed that the amount of collagen formation in the myocardial scar was variable even though all the animals were subjected to a similar surgical intervention (Figure 5). In animals with relatively small amount of collagen deposition in the myocardium (Figure 5, A and D), CT imaging was unable to detect AuNPs even in the non-beating heart but small focal enhancement was detectable in non-beating heart with relatively large amount of collagen deposition (Figure 4, A and C).
Discussion There are two ways to evaluate patients for viable or hibernating myocardium. One way is to detect the presence of metabolically active myocardium, which can be achieved by nuclear imaging techniques or low-dose dobutamine stress echocardiography. 36,37 However, those techniques interpret myocardial viability as an all-or-none phenomenon within a myocardial region and cannot assess the transmural extent of viability of the ventricular wall. The other way is to detect the thickness of myocardial scarring. Non-contrast enhanced MRI and CT imaging can detect thinning of the myocardial wall and subtle signal changes in the infarcted tissues. 38,39 Cardiac Magnetic Resonance (CMR) has emerged as a reliable noninvasive imaging method that allows a comprehensive assessment of myocardial anatomy and function. 40 However, a major breakthrough was reported in the seminal work by Kim et al, demonstrating that delayed hyper enhancement by Gd could accurately measure the extent of myocardial scarring and distinguish between reversible and irreversible myocardial injury. The amount of viable myocardium as detected by the lack of delayed Gd hyperenhancement strongly relates to the degree of improvement in the global mean wall-motion score and the ejection fraction after revascularization 41,42 and provide important prognostic information for rehospitalization for heart failure, sudden cardiac death or ventricular arrhythmias. 43 However, late Gd enhancement (LGE-CMR) has some limitations; Gd contrast agents are not specific markers for myocardial fibrosis. LGE identifies not only the extracellular matrix in myocardial fibrosis, but also inflammation or edema. Furthermore, the use of LGE for precise quantification of myocardial fibrosis quantification is variable in a clinical setting. 43 This may be related to the fact that LGE measures myocardial replacement
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Figure 3. Long circulating nature of CNA35-AuNPs in vivo. At baseline, pre-existing diet-related contrast was seen in the intestine and urinary bladder. At 30 min after AuNPs injection, blood pool contrast remained visible at around 200-250 HU. At 360 min after injection, blood pool contrast remained visible at 100-130 HU. At 25 h after injection, no blood pool contrast was observed.
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Figure 4. In vivo targeting of collagen I in myocardial scar with CNA35-AuNP. A hyperintense region was detected by CT scanning in the myocardial scar in a mouse 30 days after ischemia–reperfusion of the heart (A) which corresponded to the accumulation of AuNPs enhanced by silver staining (black) in the myocardial scar stained with Picrosirius Red (pink) (C). In contrast, there was a lack of corresponding hyperenhancement by CT imaging (B) or AuNPs retention (D) in a control mouse.
fibrosis and not the diffuse interstitial fibrosis. Thus, difference in signal intensity between fibrotic and normal myocardium can be inaccurate given that the process of ischemia is diffuse. An alternative strategy using diffusion spectrum imaging appears promising in generating a 3-dimensional tractogram of myofiber architecture at a microstructural level but this has so far been studied in ex vivo settings and clinical translation with most clinical scanners remains to be established. 44 Magnetic resonance imaging may not be suitable for all patients and obstacles to its use include patient preferences, the presence of metallic medical devices and the risk of nephrogenic systemic fibrosis associated with Gd-based MR contrast agent, especially in patients with renal impairment. As of today, the use of Gd for myocardial scar evaluation has not been approved by the FDA and is administered on an off-label basis. CT angiography is an excellent alternative to invasive coronary angiography in providing anatomic characterization of the coronary arteries. Attempts have been made to push the
boundary of that technology into the realm of myocardial scar quantitation by replicating the techniques used in MR imaging. Unfortunately, the diagnostic accuracy of delayed enhancement of myocardial scar by iodine-based agents has not been widely accepted. 4,5 Clearly, there is a need to completely revamp the CT contrast agents to improve their detection sensitivity and targeting capacity to overcome the shortcomings of conventional CT contrast agents. The innovative approaches in this study aimed at addressing a number of major deficiencies in the current imaging strategies. These include (1) specific targeting of myocardial scar with a homing peptide against collagen I which facilitates retention and concentration of contrast agents in the myocardial scar, (2) enhanced X-ray attenuation of AuNPs over conventional iodine-based contrast agents to compensate for the relative insensitivity of CT contrast agents, and (3) nanoparticulate nature of AuNPs which allows better tissue retention and concentration of AuNPs in the myocardium than iodine-based contrast agents.
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Figure 5. The extent of scar formation in different MI mice. Picrosirius Red (pink) staining revealed that the amount of collagen formation in the myocardial scar was variable even though all the animals were subjected to a similar surgical intervention.
There is extensive experience with the use of gold in the treatment of various inflammatory and infective conditions in humans such as rheumatoid arthritis. In human use, adverse reactions develop in about one-third of patients with rheumatoid arthritis treated with intramuscular gold. 45 The most common complications are dermatitis, stomatitis, transient hematuria and mild proteinuria. 45 The toxicity profile of AuNPs has been characterized in in vitro, animal and human studies. Incubation of citrate-stabilized gold colloids with plasma resulted in the adsorption of proteins to the surface of the colloidal particles but interestingly, was not associated with detectable platelet aggregation, change in plasma coagulation time or complement activation in vitro. 46 The size of AuNPs appears to affect their interactions with plasma proteins and cells as well as their biodistribution. Smaller AuNPs (30 nm) have larger surface area
and more protein adsorption than larger AuNPs (50 nm) when incubated with human plasma. 47 A number of in vitro cellular uptake studies with a variety of AuNPs and surface modifiers have not detected any evidence of cytotoxicity or activation. 48-50 However, a number of studies suggest that smaller AuNPs (b 2 nm) and cationic coating could induce membrane disruption and cellular necrosis. 51,52 It had been demonstrated that 50 nm gold nanoparticles are taken up by cells at a faster rate and higher concentration than other sizes. 53 The biodistribution of injected AuNPs in various organs may provide a source of end-organ toxicity. Clearance and retention of AuNPs by various organs are size dependent. Ten nanometer AuNPs are more likely to be retained in the kidneys and brain while 250 nm AuNPs are more likely to be retained in the liver and spleen. 54 The use of 5060 nm AuNPs may provide the right balance between
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minimizing their accumulation in the renal and reticuloendothelial system and achieving a size that is small enough for them to extravasate into the myocardial scar. Furthermore, targeting with collagen-homing peptide allows accumulation of the contrast agents in the myocardial scar and reduces the required dose. Mice intravenously injected with AuNPs at 2.7 g Au kg − 1 survived over a year without signs of illness. The LD50 for AuNPs is approximately 3.2 g Au kg − 1; a hefty dose that will not be required for diagnostic imaging. 55 Boote et al injected 85 mg/kg AuNPs as a CT contrast agent in juvenile swines and did not detect any deleterious effects in the animals. 56 We report the synthesis and characterization of CNA35AuNPs. AuNPs of ~ 50-60 nm in size (as determined by UV spectrophotometry and Dynamic Light Scattering) were prepared and the CNA35 peptide coupled onto the surface of these AuNPs. 57-60 The gold nanoparticles are very unstable in saline environment, thus we need to stabilize the gold nanoparticles and prevent their aggregation. 61 This was done by coating the surface of the gold nanoparticles with polyethylene glycol (PEG). The surface of gold nanoparticles was coated by the functionalized long PEG (with functional groups available for the covalent coupling to CNA35) and short PEG-SH. The longer bifunctional polyethylene glycol, orthopyridyldisulfide-polyethyleneglycolN-hydroxysuccinimide (OPSS-PEG-SVA) chain (MW = 5 kDa) was used to extend the PEGylated CNA35 away from the shorter 2K-PEG-SH stabilized gold nanoparticle surface thereby circumventing any potential steric hindrance to receptor binding that may arise due to the dense 2K-PEG-SH surface coating. 62 Terminal OPSS groups are used to conjugate the PEGylated CNA35 to the gold surface via the strong gold-thiolate bonds. By occupying any remnant sites on the nanoparticle surface left unoccupied by OPSS-PEG-CNA35, PEG-SH helps both to deter nonspecific protein adsorption onto the metal surface and to sterically stabilize the nanoparticles in a complex saline environment such as whole blood. Prior to conjugated PEGylated CNA35 to AuNPs, stability of PEGylated AuNPs was evaluated by the changes in particle size by dynamic light scattering in the flocculation assay in which aggregation in a 1% NaCl solution is measured by absence of a dramatic red shift in the characteristic plasmon absorption peak (500 to 600 nm) or any dramatic changes in size as measured by dynamic light scattering. The optimal formulation of PEGylated AuNPs containing the minimum amount of PEG-SH required for stabilization was determined and was incubated with PEGylated CNA35. We observed that non-gated CT imaging of the beating hearts in mice was not ideal for the detection of AuNPs in the myocardium. Even in the presence of sufficient local concentrations of contrast agent in the myocardium, the constant motion of the myocardium rendered it impossible for the CT scanner to detect sufficient X-ray attenuation. However, when imaging the non-beating hearts in mice, focal enhancement was detectable by CT imaging in mice with bigger scar formation. Picrosirius Red staining revealed that the amount of collagen formation in the myocardial scar was variable even though all the animals were subjected to a similar surgical intervention (Figure 5). This provided the opportunity to examine the detection limit of CT imaging for AuNPs. In animals with relatively small amount of
collagen deposition in the myocardium, CT imaging was unable to detect AuNPs even in the non-beating heart but small focal enhancement was detectable in non-beating heart with relatively large amount of collagen deposition. Furthermore, histology also provided more insight into the distribution of CNA35-AuNPs in the myocardium. Although there was a correlation between the amount of retained AuNPs and collagen I formation in the myocardium, AuNPs appeared to preferentially deposit at the periphery of the myocardial scar without penetrating into the interior of the myocardial scar. This may be related to the size of the AuNPs and the regional change in porosity of the collagen network in the myocardial scar. As a result, the amount of CNA35-AuNPs deposition may underestimate the true extent of the myocardial scar and overestimate the amount of viable myocardium. Future studies will determine if a reduction in the hydrodynamic parameter of CNA35-AuNPs could result in a different pattern of collagen detection by CT imaging.
Conclusion This is the first reported attempt to utilize AuNPs for myocardial scar detection with CT imaging. Our results demonstrated that targeted imaging of myocardial scar in a mouse model of myocardial infarction with CNA35-AuNPs was possible. However, a number of technical hurdles need to be overcome. Both the small size of the mouse heart and the even smaller size of the myocardial scar in the heart proved to be rather challenging even for an imaging modality with a high spatial resolution such as CT imaging. The rapid heart rate and ungated nature of CT image acquisition also reduced the sensitivity for CT detection of AuNPs. Thus, in the future, we plan to induce myocardial infarction in rats with a larger heart than mice and further evaluate of the utility of CNA35-AuNPs targeting of myocardial scar. Further manipulation of the size of CNA35-AuNPs may also facilitate collagen scar penetration for better quantitation of myocardial scar burden. This study demonstrated the feasibility of using CNA35AuNPs for vascular imaging and specific collagen targeting in the myocardial scar. This combined use of a nanomaterial with higher attenuation than traditional CT contrast agents and specific targeting mechanism for myocardial scar burden may expand the applications of cardiovascular CT imaging. Acknowledgments We would like to thank Dr. Magnus Hook for constructing and providing the CNA35 peptide. We would also like to thank Drs. David Cormode and Emil Martin for technical advice on the AuNPs synthesis and peptide synthesis.
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Research Article
nanomedjournal.com
CT imaging of myocardial scars with collagen-targeting gold nanoparticles Delia Danila, PhD⁎, Evan Johnson, MS, Patrick Kee, MD, PhD Department of Internal Medicine, Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX, USA Received 8 January 2013; accepted 24 March 2013
Abstract In the setting of myocardial ischemia, recovery of myocardial function by revascularization procedures depends on the extent of coronary disease and myocardial scar burden. Currently, computed tomographic (CT) imaging offers superior evaluation of coronary lesions but lacks the capability to measure the transmural extent of myocardial scar. Our work focuses on determining if collagen-targeting gold nanoparticles (AuNPs) can effectively target myocardial scar and provide adequate contrast for CT imaging. AuNPs were coated with a collagen-homing peptide, collagen adhesin (CNA35). Myocardial scar was created in mice by occlusion/reperfusion of the left anterior descending coronary artery. Thirty days later, un-gated CT imaging was performed. Over 6 h, CNA35-AuNPs provided uniform and prolonged opacification of the vascular structures (100-130 HU). In mice with larger scar burden, focal contrast enhancement was detected in the myocardium, which was not apparent within that of control mice. Histological staining confirmed myocardial scar formation and accumulation of AuNPs. From the Clinical Editor: This team of investigators presents a collagen-targeting gold nanoparticle-based approach that enables the imaging of myocardial scars via CT scans in a rodent model. This information would enable clinicians to judge the recovery potential of myocardium more accurately than the current CT-scan based approaches. Published by Elsevier Inc. Key words: Gold nanoparticles; Myocardial scars; CT imaging; Molecular imaging
Despite advances in the management of coronary artery disease, myocardial infarction is still prevalent. The development of myocardial scar leads to heart failure, cardiac arrhythmia and sudden cardiac death. 1 However, in a subset of patients with viable myocardial tissues in the peri-infarct zone, studies have clearly shown that revascularization procedures such as coronary artery bypass grafts and coronary angioplasty increase left ventricular ejection fractions and improve survival in those patients. 2,3 Clearly, identification of such patients will be important for disease management. Among the imaging techniques currently being investigated or utilized for evaluating myocardial scar burden in ischemic cardiomyopathy, MR imaging with delayed Gd enhancement shows enormous potential. Imaging modalities such as echocardiography, fluoroscopic left ventriculogram with contrast and CT cardiac imaging are at best semi-quantitative. However, MR All co-authors have seen and agreed with the contents of the manuscript and there is no financial interest to report. ⁎Corresponding author: Houston, TX, 77054, USA. E-mail addresses: [email protected] (D. Danila), [email protected] (E. Johnson), [email protected] (P. Kee).
imaging with Gd administration may not be appropriate for all subjects and obstacles to its use include patient preferences, the presence of metallic medical devices and the risk of nephrogenic systemic fibrosis associated with gadolinium-based MR contrast agent, especially in patients with renal impairment. CT coronary angiography has unrivaled advantages in terms of speed in image acquisition, relative availability, spatial resolution and tissue penetration. Hardware development in CT scanners has made a major leap over the years and the latest generation of CT scanners can provide excellent anatomic details of luminal abnormalities in association with obstructive coronary artery disease. However, the identification of viable myocardium and ischemic myocardial scar with CT remains challenging. Attempts have been made to replicate the technique of delayed Gd hyperenhancement in MR imaging with conventional iodinated contrast agent but the beamhardening artifact from contrast within the left ventricle and the short blood pool persistence of conventional CT contrast agent clearly hamper the advances in myocardial perfusion imaging with multidetector CT. 4,5 Conventional CT contrast agent has proven to be very safe but their formulations have remained relatively unchanged over the last 60 years. There is also a paucity of iodinated CT contrast agents that have been formulated for targeted imaging. 6-8 In the
1549-9634/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.nano.2013.03.009 Please cite this article as: Danila D, et al, CT imaging of myocardial scars with collagen-targeting gold nanoparticles. Nanomedicine: NBM 2013;9:1067-1076, http://dx.doi.org/10.1016/j.nano.2013.03.009
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face of this developmental plateau in CT contrast agents, there is a need to develop new metal-based agents for X-ray imaging. 9 Gold nanoparticles have been an attractive candidate not only for X-ray imaging due to their superior X-ray attenuation properties when compared with conventional contrast agents, they may also offer interesting applications for thermal therapy and chemical sensing via color changes and surface-enhanced Raman spectroscopy. Bare gold nanoparticles are unstable in vivo, however, their rapid aggregation and clearance can be reduced by anti-fouling coating with agents such as polyethylene glycol (PEG). 10-15 PEG-stabilized gold nanoparticles have been investigated as a contrast agent for blood pool imaging. 15 Depending on the size, small gold nanoparticles are readily excreted by the kidneys with little retention in the liver and spleen. 16 Antibody-functionalized gold nanoparticles, also known as “immunogold”, have been used by electron microscopy but they exhibit poor biocompatibility and aggregate via nonspecific adsorption of proteins in vivo. However, the proteinrepelling PEG matrix on the surface of gold nanoparticles can be readily modified by inclusion of heterobifunctional PEG for effective conjugation to peptides, folic acid or antibodies 17-25 Antibody-conjugated gold nanoparticles have been investigated as a targeted contrast agent for detecting tumors by CT imaging 26 and thermodynamic therapy. 27 Other high k-edge metals have been prepared for CT imaging. Bismuth is inherently toxic but has been stabilized with a polymer coating to improve its biocompatibility and retain its high X-ray attenuation properties and long circulation times in vivo. 28 Another candidate metal suitable for X-ray imaging is tantalum and has been stabilized with zwitterionic agents 29 and PEG 30 for in vivo blood pool imaging. Specific retention of contrast agents in myocardial scar can be enhanced by attaching collagen-homing ligands on their surface. Although antibodies are commonly used for molecular targeting, the use of homing peptides is thought to be more biocompatible and translatable. The collagen-homing ligand chosen for myocardial scar targeting is a truncated form of collagen adhesin, CNA35, which has a higher affinity for collagen than the wild type protein isolated from the cell surface of Staphylococcus aureus. The collagen-binding domains of CNA35 have excellent affinity for collagen I with a Kd ranging from 20 nM to 30 μM. 31,32 CNA35 is approximately five times smaller than antibodies, and this could be beneficial for tissue penetration and on-rate kinetics. CNA35 has been conjugated to fluorescent probes for histologic staining of myocardial fibrosis and fibrotic components in atherosclerotic arteries in apoE −/− mice. 33 Other collagen-homing ligands have also been reported. Glutathione S-transferase fusion protein of the I domain of human integrin subunit α1 was previously tested but its affinity was inferior to that of CNA35. 33 A proprietary collagentargeting peptide was discovered by phage display and conjugated to Gd-based MR contrast agent, EP-3533, which demonstrated excellent myocardial scar enhancement in vivo. However, this formulation is not commercially available. 34 We report here the development of a new CT imaging contrast agent for the non-invasive detection of myocardial scars. We developed a targeted radiocontrast agent based on gold nanoparticles (AuNPs) which has the advantages of offering
3-fold greater contrast per unit weight than iodine-based x-ray contrast agents, 35 and probably better safety profile than iodineor gadolinium-based agents in terms of renal and cutaneous toxicity. X-ray imaging has the advantages of providing superior tissue penetration and spatial resolution.
Materials and Methods Materials Sodium citrate, gold chloride and Picrosirius Red stain (Direct Red 80, Picric acid solution, and Hematoxylin solution A according to Weigert) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PEG-SH and OPSS-PEG-SVA were purchased from Laysan Bio (Arab, AL, USA). PES membranes (3,000 MWCO and 100,000 MWCO) were purchased from Fisher Scientific. PES membranes (10,000 MWCO) were purchased from Viva Products Inc. (Littleton, MA, USA). Silver enhancement staining kit was purchased from Structure Probe, Inc. (West Chester, PA, USA). Collagen I was purchased from BD Bioscience. CNA35 was kindly donated by Magnus Hook, and anti-His-HRP antibody was purchased from AbCam. Methods Gold Nanoparticles Synthesis In order to prepare gold nanoparticles, nanopure water (500 ml) was filtered through 0.22 μm filter and boiled in a 1 L conical flask. 5 ml of Gold Chloride (10%) was added to the boiling water followed by 4 ml of 1% sodium citrate solution. The solution was boiled for about an hour or until 200 ml of solution was left. The solution has a burgundy color. Next, the AuNPs were pegylated with polyethylene glycol derivatives in order to avoid aggregation in vivo. The AuNPs were incubated for 1 hour with a 100:1 molar ratio of PEG-SH to prevent aggregation and with 50:1 orthopyridyldisulfide-polyethyleneglycol-N-hydroxysuccinimide (OPSS-PEG-SVA) for the covalent coupling of the peptide. After pegylation, the AuNPs were further concentrated by centrifugation at 3000 rpm for 30 min. The pink supernatant was transferred to clean tubes and centrifuged again. This process was repeated 2 more times and all the concentrated fractions were combined and filtered through 0.22 μm filters. The AuNPs were further concentrated to ~ 4 mg/ml using PES membrane concentrators (MWCO 10,000). The AuNPs were characterized in terms of size and polydispersity by UV and Dynamic Light Scattering after appropriate dilution. Synthesis of CNA35-AuNPs CNA35 (36.5 μg) was incubated with 500 μl AuNPs (40 mg/ml) overnight at 4 °C. Next day, the excess CNA35 was removed by centrifugation using Vivaspin filters (MWCO 100,000). Flocculation assay Gold nanoparticles (2×10 13 AuNPs) were incubated with various molar ratios of PEG-SH. The following ratios of PEGSH:AuNPs were prepared: 0:1, 1:1, 2.5:1, 5:1, 10:1, 25:1, 50:1, 100:1, 250:1, 500:1, 1K:1, 10K:1. After one hour incubation at room temperature, NaCl (10%) was added and the solution incubated for 30 min at room temperature. The absorbance
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spectrum (400-600 nm) was then recorded for all of the above mentioned solutions. The disappearance or the shift in the signal indicates aggregation. Solid-phase binding assay 96-well plates were coated overnight with collagen I at 4 °C, followed by blocking the non-specific sites with BSA (50 mg/ml) for 1 h at 37 °C. Next, various forms of CNA35 ligands (CNA35, CNA35-AuNPs, and unconjugated AuNPs) were incubated for 2 h at 37 °C. After washing the unbound CNA35 ligands, anti-His-HRP antibody was added to specifically recognize CNA35, followed by color development with a substrate for horseradish peroxidase, for 30 min at room temperature. The absorbance readings at 405 nm indicate the binding of CNA35 to the various substrates. Animal experiments The animal studies were performed on female C57BL/6 mice (20 g) ordered from Jackson Laboratories (Bar Harbor, ME, USA). The animals were housed at the University of Texas Health Science Center at Houston (UTHSCH). The animal protocol was approved by the UTHSCH Center for Laboratory Animal Medicine and Care (protocol number: HSC-AWC-11150- “Pilot project: Myocardial Scar Imaging with Gold Nanoparticles”); experiments were performed in accordance with institutional guidelines, and all efforts were made to minimize suffering. Myocardial infarction was created in C57BL/6 mice by occlusion of the proximal left anterior descending (LAD) artery with a 8-0 Proline suture for 60 min followed by releasing the occlusive suture under general anesthesia to re-establish coronary perfusion. Thirty days after the LAD occlusion/ reperfusion surgery, a collagen-rich scar was well developed for CT imaging with CNA35-AuNPs. CT imaging protocol and analysis The animals were sedated with IP Avertin. 200 μl of CNA35AuNPs (40 mg/ml) was injected into the animals via the tail vein. Eight myocardial infarction mice and seven control mice were injected and CT imaging was performed with a GE Ultra flat panel CT scanner (General Electric, Milwaukee, WI) with the following acquisition settings: 80 kVp, 22 mA with 16 s rotation/exposure. Non-cardiac gated CT images were acquired at baseline, 1 min, 15 min, 30 min, 45 min, 60 min, 2 h, 4 h, 6 h and 24 h after AuNPs injection. At the end of the imaging, animals were perfused with buffered paraformaldehyde and the carcasses were imaged once more by the CT scanner. Imaging of the carcasses was performed due to the fact that the CT imaging was ungated and motion artifacts from cardiac contractions may reduce the sensitivity for contrast detection in the myocardium. Simple back projections were obtained for the 0.154 μm image reconstruction and exported as DICOM images. Image analysis was performed using the OsiriX software. Stacks of 3-4 consecutive sections were viewed with maximum intensity projection (MIP) and window levels were adjusted to identify any signal enhancement in the myocardium. The myocardium was divided into 3 sectors to identify the location of signal
Figure 1. Stabilization of AuNPs in saline environment (UV data). Three different flocculation assays demonstrating the reproducible relationship between the ratio of PEG-SH:AuNPs and size homogeneity of AuNPs. Discrete and uniform preparations of AuNPs with a diameter of 50 nm displays a peak absorbance at 530 nm. The magnitude of A530 drops precipitously when AuNPs aggregate as seen when PEG-SH:AuNPs decreases below 50.
enhancement and correlated with scar formation and AuNPs deposition by histological staining. Histological staining of myocardial scar formation and AuNPs deposition The excised heart was sectioned at 5 mm intervals and embedded in paraffin for histological analysis. Collagen formation and AuNPs retention in the myocardium were stained with Picrosirius Red and silver enhancement staining kit, respectively. Light microscopy was performed to determine the distribution of collagen deposition and AuNPs retention in the myocardium.
Results CNA35-AuNPs preparation and characterization The AuNPs prepared by citrate reduction had a hydrodynamic diameter of 40-70 nm with a polydispersity index of 0.213-0.480 (based on DLS data). Our UV-VIS data indicate that the AuNPs have a diameter of 50 nm, exhibiting an absorption maximum at 530 nm, characteristic for a uniform preparation of AuNPs of 50 nm. The amount of short PEG-SH needed to prevent aggregation of AuNPs in the presence of 1% NaCl solution was determined by flocculation assays. Figure 1 summarizes three separate runs of the flocculation assays. The 50 nm gold nanoparticles exhibit a maximum absorption peak at 530 nm and the magnitude of this peak decreases drastically as the particles aggregate in a saline environment. A ratio of 100:1 PEG-SH: AuNPs was sufficient to provide stability of the gold nanoparticles. By DLS analysis, the hydrodynamic diameter and polydispersity index of PEG-stabilized AuNPs were 0 nm for a 1:1 or 10:1 PEG-SH:AuNPs, 64.1 nm for a 100:1 PEG-SH: AuNPs and 68.6 nm for a 10K:1 PEG-SH:AuNPs, confirming
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Table 1 DLS analysis of PEG-stabilized AuNPs. PEG-SH:AuNPs
Diameter (nm)
Polydispersity Index (PDI)
1:1 10:1 100:1 500:1 1K:1 10K:1
0 0 64.1 62.8 64.5 68.6
1.0 1.0 0.473 0.522 0.519 0.478
Figure 2. Specificity of binding of CNA35-AuNPs to Collagen 1. Solid phase Collagen 1 binding assay was performed by incubating pure CNA35 or CAN35-AuNPs with Collagen 1. The amount of peptide bound to Collagen 1 was measured by a colorimetric method and the level of absorbance at 405 nm was proportional to the amount of bound peptide.
the lack of aggregation of PEG-stabilized AuNPs (Table 1). The PEG-stabilized AuNPs were functionalized with CNA35 via a long PEG. Collagen-binding capacity of CNA35 was relatively retained after conjugation to AuNPs as shown in the ELISA type assay (Figure 2). Pharmacokinetics and biodistribution of CNA35-AuNPs in vivo After CNA35-AuNPs were administered intravenously, peak blood pool enhancement of 130-150 HU was observed at up to 2 h, followed by a gradual time-dependent decline in contrast enhancement in the blood pool to 100-130 HU at 6 h (Figure 3). By 24 h, most of the CNA35-AuNPs disappeared from the blood pool. This illustrated the long circulating nature of CNA35AuNPs, which is one of the pre-requisites for effective targeted imaging. Substantial amount of contrast was detected in the liver and spleen and may represent the major sources of elimination of CNA35-AuNPs. Targeted imaging of collagen deposition in myocardial scar with CNA35-AuNPs and histological correlation During the first 6 h of CT imaging, MIP did not demonstrate any focal enhancement of contrast in the myocardium of control animals and animals subjected to LAD occlusion/ reperfusion surgery with smaller myocardial scar formation. However, in animals with larger myocardial scar formation, CT imaging revealed focal enhancement in the myocardium of the nonbeating heart (Figure 4).
Picrosirius Red staining confirmed the myocardial scar formation (the red staining in Figure 4, C) in animals subjected to LAD occlusion/ reperfusion surgery. Silver enhancement of AuNPs demonstrated the proximity between AuNPs retention and collagen deposition in the myocardium. Silver enhancement staining was seen mainly in the periphery of collagen formation but there was a good correlation between the amount of scar formation and silver enhancement in the myocardium. Interestingly, in the myocardial segments not injured by ischemia, scattered silver enhancement was also observed. In control mice (Figure 4, D), there was no evidence of collagen deposition or silver enhancement within the myocardium. We observed some variability in the transmural extent of the scar formation in the animals by measuring the scar burden using Picrosirius Red staining. Picrosirius Red staining revealed that the amount of collagen formation in the myocardial scar was variable even though all the animals were subjected to a similar surgical intervention (Figure 5). In animals with relatively small amount of collagen deposition in the myocardium (Figure 5, A and D), CT imaging was unable to detect AuNPs even in the non-beating heart but small focal enhancement was detectable in non-beating heart with relatively large amount of collagen deposition (Figure 4, A and C).
Discussion There are two ways to evaluate patients for viable or hibernating myocardium. One way is to detect the presence of metabolically active myocardium, which can be achieved by nuclear imaging techniques or low-dose dobutamine stress echocardiography. 36,37 However, those techniques interpret myocardial viability as an all-or-none phenomenon within a myocardial region and cannot assess the transmural extent of viability of the ventricular wall. The other way is to detect the thickness of myocardial scarring. Non-contrast enhanced MRI and CT imaging can detect thinning of the myocardial wall and subtle signal changes in the infarcted tissues. 38,39 Cardiac Magnetic Resonance (CMR) has emerged as a reliable noninvasive imaging method that allows a comprehensive assessment of myocardial anatomy and function. 40 However, a major breakthrough was reported in the seminal work by Kim et al, demonstrating that delayed hyper enhancement by Gd could accurately measure the extent of myocardial scarring and distinguish between reversible and irreversible myocardial injury. The amount of viable myocardium as detected by the lack of delayed Gd hyperenhancement strongly relates to the degree of improvement in the global mean wall-motion score and the ejection fraction after revascularization 41,42 and provide important prognostic information for rehospitalization for heart failure, sudden cardiac death or ventricular arrhythmias. 43 However, late Gd enhancement (LGE-CMR) has some limitations; Gd contrast agents are not specific markers for myocardial fibrosis. LGE identifies not only the extracellular matrix in myocardial fibrosis, but also inflammation or edema. Furthermore, the use of LGE for precise quantification of myocardial fibrosis quantification is variable in a clinical setting. 43 This may be related to the fact that LGE measures myocardial replacement
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Figure 3. Long circulating nature of CNA35-AuNPs in vivo. At baseline, pre-existing diet-related contrast was seen in the intestine and urinary bladder. At 30 min after AuNPs injection, blood pool contrast remained visible at around 200-250 HU. At 360 min after injection, blood pool contrast remained visible at 100-130 HU. At 25 h after injection, no blood pool contrast was observed.
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Figure 4. In vivo targeting of collagen I in myocardial scar with CNA35-AuNP. A hyperintense region was detected by CT scanning in the myocardial scar in a mouse 30 days after ischemia–reperfusion of the heart (A) which corresponded to the accumulation of AuNPs enhanced by silver staining (black) in the myocardial scar stained with Picrosirius Red (pink) (C). In contrast, there was a lack of corresponding hyperenhancement by CT imaging (B) or AuNPs retention (D) in a control mouse.
fibrosis and not the diffuse interstitial fibrosis. Thus, difference in signal intensity between fibrotic and normal myocardium can be inaccurate given that the process of ischemia is diffuse. An alternative strategy using diffusion spectrum imaging appears promising in generating a 3-dimensional tractogram of myofiber architecture at a microstructural level but this has so far been studied in ex vivo settings and clinical translation with most clinical scanners remains to be established. 44 Magnetic resonance imaging may not be suitable for all patients and obstacles to its use include patient preferences, the presence of metallic medical devices and the risk of nephrogenic systemic fibrosis associated with Gd-based MR contrast agent, especially in patients with renal impairment. As of today, the use of Gd for myocardial scar evaluation has not been approved by the FDA and is administered on an off-label basis. CT angiography is an excellent alternative to invasive coronary angiography in providing anatomic characterization of the coronary arteries. Attempts have been made to push the
boundary of that technology into the realm of myocardial scar quantitation by replicating the techniques used in MR imaging. Unfortunately, the diagnostic accuracy of delayed enhancement of myocardial scar by iodine-based agents has not been widely accepted. 4,5 Clearly, there is a need to completely revamp the CT contrast agents to improve their detection sensitivity and targeting capacity to overcome the shortcomings of conventional CT contrast agents. The innovative approaches in this study aimed at addressing a number of major deficiencies in the current imaging strategies. These include (1) specific targeting of myocardial scar with a homing peptide against collagen I which facilitates retention and concentration of contrast agents in the myocardial scar, (2) enhanced X-ray attenuation of AuNPs over conventional iodine-based contrast agents to compensate for the relative insensitivity of CT contrast agents, and (3) nanoparticulate nature of AuNPs which allows better tissue retention and concentration of AuNPs in the myocardium than iodine-based contrast agents.
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Figure 5. The extent of scar formation in different MI mice. Picrosirius Red (pink) staining revealed that the amount of collagen formation in the myocardial scar was variable even though all the animals were subjected to a similar surgical intervention.
There is extensive experience with the use of gold in the treatment of various inflammatory and infective conditions in humans such as rheumatoid arthritis. In human use, adverse reactions develop in about one-third of patients with rheumatoid arthritis treated with intramuscular gold. 45 The most common complications are dermatitis, stomatitis, transient hematuria and mild proteinuria. 45 The toxicity profile of AuNPs has been characterized in in vitro, animal and human studies. Incubation of citrate-stabilized gold colloids with plasma resulted in the adsorption of proteins to the surface of the colloidal particles but interestingly, was not associated with detectable platelet aggregation, change in plasma coagulation time or complement activation in vitro. 46 The size of AuNPs appears to affect their interactions with plasma proteins and cells as well as their biodistribution. Smaller AuNPs (30 nm) have larger surface area
and more protein adsorption than larger AuNPs (50 nm) when incubated with human plasma. 47 A number of in vitro cellular uptake studies with a variety of AuNPs and surface modifiers have not detected any evidence of cytotoxicity or activation. 48-50 However, a number of studies suggest that smaller AuNPs (b 2 nm) and cationic coating could induce membrane disruption and cellular necrosis. 51,52 It had been demonstrated that 50 nm gold nanoparticles are taken up by cells at a faster rate and higher concentration than other sizes. 53 The biodistribution of injected AuNPs in various organs may provide a source of end-organ toxicity. Clearance and retention of AuNPs by various organs are size dependent. Ten nanometer AuNPs are more likely to be retained in the kidneys and brain while 250 nm AuNPs are more likely to be retained in the liver and spleen. 54 The use of 5060 nm AuNPs may provide the right balance between
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minimizing their accumulation in the renal and reticuloendothelial system and achieving a size that is small enough for them to extravasate into the myocardial scar. Furthermore, targeting with collagen-homing peptide allows accumulation of the contrast agents in the myocardial scar and reduces the required dose. Mice intravenously injected with AuNPs at 2.7 g Au kg − 1 survived over a year without signs of illness. The LD50 for AuNPs is approximately 3.2 g Au kg − 1; a hefty dose that will not be required for diagnostic imaging. 55 Boote et al injected 85 mg/kg AuNPs as a CT contrast agent in juvenile swines and did not detect any deleterious effects in the animals. 56 We report the synthesis and characterization of CNA35AuNPs. AuNPs of ~ 50-60 nm in size (as determined by UV spectrophotometry and Dynamic Light Scattering) were prepared and the CNA35 peptide coupled onto the surface of these AuNPs. 57-60 The gold nanoparticles are very unstable in saline environment, thus we need to stabilize the gold nanoparticles and prevent their aggregation. 61 This was done by coating the surface of the gold nanoparticles with polyethylene glycol (PEG). The surface of gold nanoparticles was coated by the functionalized long PEG (with functional groups available for the covalent coupling to CNA35) and short PEG-SH. The longer bifunctional polyethylene glycol, orthopyridyldisulfide-polyethyleneglycolN-hydroxysuccinimide (OPSS-PEG-SVA) chain (MW = 5 kDa) was used to extend the PEGylated CNA35 away from the shorter 2K-PEG-SH stabilized gold nanoparticle surface thereby circumventing any potential steric hindrance to receptor binding that may arise due to the dense 2K-PEG-SH surface coating. 62 Terminal OPSS groups are used to conjugate the PEGylated CNA35 to the gold surface via the strong gold-thiolate bonds. By occupying any remnant sites on the nanoparticle surface left unoccupied by OPSS-PEG-CNA35, PEG-SH helps both to deter nonspecific protein adsorption onto the metal surface and to sterically stabilize the nanoparticles in a complex saline environment such as whole blood. Prior to conjugated PEGylated CNA35 to AuNPs, stability of PEGylated AuNPs was evaluated by the changes in particle size by dynamic light scattering in the flocculation assay in which aggregation in a 1% NaCl solution is measured by absence of a dramatic red shift in the characteristic plasmon absorption peak (500 to 600 nm) or any dramatic changes in size as measured by dynamic light scattering. The optimal formulation of PEGylated AuNPs containing the minimum amount of PEG-SH required for stabilization was determined and was incubated with PEGylated CNA35. We observed that non-gated CT imaging of the beating hearts in mice was not ideal for the detection of AuNPs in the myocardium. Even in the presence of sufficient local concentrations of contrast agent in the myocardium, the constant motion of the myocardium rendered it impossible for the CT scanner to detect sufficient X-ray attenuation. However, when imaging the non-beating hearts in mice, focal enhancement was detectable by CT imaging in mice with bigger scar formation. Picrosirius Red staining revealed that the amount of collagen formation in the myocardial scar was variable even though all the animals were subjected to a similar surgical intervention (Figure 5). This provided the opportunity to examine the detection limit of CT imaging for AuNPs. In animals with relatively small amount of
collagen deposition in the myocardium, CT imaging was unable to detect AuNPs even in the non-beating heart but small focal enhancement was detectable in non-beating heart with relatively large amount of collagen deposition. Furthermore, histology also provided more insight into the distribution of CNA35-AuNPs in the myocardium. Although there was a correlation between the amount of retained AuNPs and collagen I formation in the myocardium, AuNPs appeared to preferentially deposit at the periphery of the myocardial scar without penetrating into the interior of the myocardial scar. This may be related to the size of the AuNPs and the regional change in porosity of the collagen network in the myocardial scar. As a result, the amount of CNA35-AuNPs deposition may underestimate the true extent of the myocardial scar and overestimate the amount of viable myocardium. Future studies will determine if a reduction in the hydrodynamic parameter of CNA35-AuNPs could result in a different pattern of collagen detection by CT imaging.
Conclusion This is the first reported attempt to utilize AuNPs for myocardial scar detection with CT imaging. Our results demonstrated that targeted imaging of myocardial scar in a mouse model of myocardial infarction with CNA35-AuNPs was possible. However, a number of technical hurdles need to be overcome. Both the small size of the mouse heart and the even smaller size of the myocardial scar in the heart proved to be rather challenging even for an imaging modality with a high spatial resolution such as CT imaging. The rapid heart rate and ungated nature of CT image acquisition also reduced the sensitivity for CT detection of AuNPs. Thus, in the future, we plan to induce myocardial infarction in rats with a larger heart than mice and further evaluate of the utility of CNA35-AuNPs targeting of myocardial scar. Further manipulation of the size of CNA35-AuNPs may also facilitate collagen scar penetration for better quantitation of myocardial scar burden. This study demonstrated the feasibility of using CNA35AuNPs for vascular imaging and specific collagen targeting in the myocardial scar. This combined use of a nanomaterial with higher attenuation than traditional CT contrast agents and specific targeting mechanism for myocardial scar burden may expand the applications of cardiovascular CT imaging. Acknowledgments We would like to thank Dr. Magnus Hook for constructing and providing the CNA35 peptide. We would also like to thank Drs. David Cormode and Emil Martin for technical advice on the AuNPs synthesis and peptide synthesis.
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