Hybrid, metal oxide-peptide amphiphile micelles for molecular magnetic resonance imaging of atherosclerosis.
ABSTRACT: BACKGROUND:Atherosclerosis, a major source of cardiovascular disease, is asymptomatic for decades until the activation of thrombosis and the rupture of enlarged plaques, resulting in acute coronary syndromes and sudden cardiac arrest. Magnetic resonance imaging (MRI) is a noninvasive nuclear imaging technique to assess the degree of atherosclerotic plaque with high spatial resolution and excellent soft tissue contrast. However, MRI lacks sensitivity for preventive medicine, which limits the ability to observe the onset of vulnerable plaques. In this study, we engineered hybrid metal oxide-peptide amphiphile micelles (HMO-Ms) that combine an inorganic, magnetic iron oxide or manganese oxide inner core with organic, fibrin-targeting peptide amphiphiles, consisting of the sequence CREKA, for potential MRI imaging of thrombosis on atherosclerotic plaques. RESULTS:Hybrid metal oxide-peptide amphiphile micelles, consisting of an iron oxide (Fe-Ms) or manganese oxide (Mn-Ms) core with CREKA peptides, were self-assembled into 20-30 nm spherical nanoparticles, as confirmed by dynamic light scattering and transmission electron microscopy. These hybrid nanoparticles were found to be biocompatible with human aortic endothelial cells in vitro, and HMO-Ms bound to human clots three to five times more efficiently than its non-targeted counterparts. Relaxivity studies showed ultra-high r2 value of 457 mM-1 s-1 and r1 value of 0.48 mM-1 s-1 for Fe-Ms and Mn-Ms, respectively. In vitro, MR imaging studies demonstrated the targeting capability of CREKA-functionalized hybrid nanoparticles with twofold enhancement of MR signals. CONCLUSION:This novel hybrid class of MR agents has potential as a non-invasive imaging method that specifically detects thrombosis during the pathogenesis of atherosclerosis.
Project description:The leading causes of morbidity and mortality globally are cardiovascular diseases, and nanomedicine can provide many improvements including disease-specific targeting, early detection, and local delivery of diagnostic agents. To this end, we designed fibrin-binding, peptide amphiphile micelles (PAMs), achieved by incorporating the targeting peptide cysteine-arginine-glutamic acid-lysine-alanine (CREKA), with two types of amphiphilic molecules containing the gadoliniuim (Gd) chelator diethylenetriaminepentaacetic acid (DTPA), DTPA-bis(stearylamide)(Gd), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(poly(ethylene glycol) (PEG))-2000]-DTPA(Gd) (DSPE-PEG2000-DTPA(Gd)). The material characteristics of the resulting nanoparticle diagnostic probes, clot-binding properties in vitro, and contrast enhancement and safety for dual, optical imaging-magnetic resonance imaging (MRI) were evaluated in the atherosclerotic mouse model. Transmission electron micrographs showed a homogenous population of spherical micelles for formulations containing DSPE-PEG2000-DTPA(Gd), whereas both spherical and cylindrical micelles were formed upon mixing DTPA-BSA(Gd) and CREKA amphiphiles. Clot-binding assays confirmed DSPE-PEG2000-DTPA(Gd)-based CREKA micelles targeted clots over 8-fold higher than nontargeting (NT) counterpart micelles, whereas no difference was found between CREKA and NT, DTPA-BSA(Gd) micelles. However, in vivo MRI and optical imaging studies of the aortas and hearts showed fibrin specificity was conferred by the peptide ligand without much difference between the nanoparticle formulations or shapes. Biodistribution studies confirmed that all micelles were cleared through both the reticuloendothelial system and renal clearance, and histology showed no signs of necrosis. In summary, these studies demonstrate the successful synthesis, and the molecular imaging capabilities of two types of CREKA-Gd PAMs for atherosclerosis. Moreover, we demonstrate the differences in micelle formulations and shapes and their outcomes in vitro versus in vivo for site-specific, diagnostic strategies, and provide the groundwork for the detection of thrombosis via contrast-enhancing agents and concurrent therapeutic delivery for theranostic applications.
Project description:We created and evaluated a preclinical, multimodality imaging, and software platform to assess molecular imaging of small metastases. This included experimental methods (e.g., GFP-labeled tumor and high resolution multispectral cryo-imaging), nonrigid image registration, and interactive visualization of imaging agent targeting. We describe technological details earlier applied to GFP-labeled metastatic tumor targeting by molecular MR (CREKA-Gd) and red fluorescent (CREKA-Cy5) imaging agents. Optimized nonrigid cryo-MRI registration enabled nonambiguous association of MR signals to GFP tumors. Interactive visualization of out-of-RAM volumetric image data allowed one to zoom to a GFP-labeled micrometastasis, determine its anatomical location from color cryo-images, and establish the presence/absence of targeted CREKA-Gd and CREKA-Cy5. In a mouse with >160 GFP-labeled tumors, we determined that in the MR images every tumor in the lung >0.3?mm2 had visible signal and that some metastases as small as 0.1?mm2 were also visible. More tumors were visible in CREKA-Cy5 than in CREKA-Gd MRI. Tape transfer method and nonrigid registration allowed accurate (<11??m error) registration of whole mouse histology to corresponding cryo-images. Histology showed inflammation and necrotic regions not labeled by imaging agents. This mouse-to-cells multiscale and multimodality platform should uniquely enable more informative and accurate studies of metastatic cancer imaging and therapy.
Project description:Arterial and venous thrombosis are among the most common causes of death and hospitalization worldwide. Nanotechnology approaches hold great promise for molecular imaging and diagnosis as well as tissue-targeted delivery of therapeutics. In this study, we developed and investigated bioengineered nanoprobes for identifying thrombus formation; the design parameters of nanoparticle shape and surface chemistry, i.e. incorporation of fibrin-binding peptides CREKA and GPRPP, were investigated. Two nanoparticle platforms based on plant viruses were studied - icosahedral cowpea mosaic virus (CPMV) and elongated rod-shaped tobacco mosaic virus (TMV). These particles were loaded to carry contrast agents for dual-modality magnetic resonance (MR) and optical imaging, and both modalities demonstrated specificity of fibrin binding in vitro with the presence of targeting peptides. Preclinical studies in a carotid artery photochemical injury model of thrombosis confirmed thrombus homing of the nanoprobes, with the elongated TMV rods exhibiting significantly greater attachment to thrombi than icosahedral (sphere-like) CPMV. While in vitro studies confirmed fibrin-specificity conferred by the peptide ligands, in vivo studies indicated the nanoparticle shape had the greatest contribution toward thrombus targeting, with no significant contribution from either targeting ligand. These results demonstrate that nanoparticle shape plays a critical role in particle deposition at the site of vascular injury. Shaping nanotechnologies opens the door for the development of novel targeted diagnostic and therapeutic strategies (i.e., theranostics) for arterial and venous thrombosis.
Project description:To synthesize multi-component nanochains, we developed a simple 'one-pot' synthesis, which exhibited high yield and consistency. The nanochains particles consist of parent nanospheres chemically linked into a higher-order, chain-like assembly. The one-pot synthesis is based on the addition of two types of parent nanospheres in terms of their surface chemical functionality (e.g., decorated with PEG-NH2 or PEG-COOH). By reacting the two types of parent nanospheres at a specific ratio (?2?:?1) for a short period of time (?30 min) under rigorous stirring, nanochains were formed. For example, we show the synthesis of iron oxide nanochains with lengths of about 125 nm consisting of 3-5 constituting nanospheres. The chain-like shaped nanoparticle possessed a unique ability to target and rapidly deposit on the endothelium of glioma sites via vascular targeting. To target and image invasive brain tumors, we used iron oxide nanochains with the targeting ligand being the fibronectin-targeting peptide CREKA. Overexpression of fibronectin is strongly associated with the perivascular regions of glioblastoma multiforme and plays a critical role in migrating and invasive glioma cells. In mice with invasive glioma tumors, 3.7% of the injected CREKA-targeted nanochains was found in gliomas within 1 h. Notably, the intratumoral deposition of the nanochain was ?2.6-fold higher than its spherical variant. Using MR imaging, the precise targeting of nanochains to gliomas provided images with the exact topology of the disease including their margin of infiltrating edges and distant invasive sites.
Project description:The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1(+) cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1-coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[(18)F]fluorobenzoic acid ([(18)F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [(18)F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [(18)F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.
Project description:Molecular MRI is a promising in-vivo modality to detect and quantify morphological and molecular vessel-wall changes in atherosclerosis. The combination of different molecular biomarkers may improve the risk stratification of patients. This study aimed to investigate the feasibility of simultaneous visualization and quantification of plaque-burden and inflammatory activity by dual-probe molecular MRI in a mouse-model of progressive atherosclerosis and in response-to-therapy. Homozygous apolipoprotein E knockout mice (ApoE-/-) were fed a high-fat-diet (HFD) for up to four-months prior to MRI of the brachiocephalic-artery. To assess response-to-therapy, a statin was administered for the same duration. MR imaging was performed before and after administration of an elastin-specific gadolinium-based and a macrophage-specific iron-oxide-based probe. Following in-vivo MRI, samples were analyzed using histology, immunohistochemistry, inductively-coupled-mass-spectrometry and laser-inductively-coupled-mass-spectrometry. In atherosclerotic-plaques, intraplaque expression of elastic-fibers and inflammatory activity were not directly linked. While the elastin-specific probe demonstrated the highest accumulation in advanced atherosclerotic-plaques after four-months of HFD, the iron-oxide-based probe showed highest accumulation in early atherosclerotic-plaques after two-months of HFD. In-vivo measurements for the elastin and iron-oxide-probe were in good agreement with ex-vivo histopathology (Elastica-van-Giesson stain: y?=?298.2?+?5.8, R2?=?0.83, p?<?0.05; Perls' Prussian-blue-stain: y?=?834.1?+?0.67, R2?=?0.88, p?<?0.05). Contrast-to-noise-ratio (CNR) measurements of the elastin probe were in good agreement with ICP-MS (y?=?0.11x-11.3, R²?=?0.73, p?<?0.05). Late stage atherosclerotic-plaques displayed the strongest increase in both CNR and gadolinium concentration (p?<?0.05). The gadolinium probe did not affect the visualization of the iron-oxide-probe and vice versa. This study demonstrates the feasibility of simultaneous assessment of plaque-burden and inflammatory activity by dual-probe molecular MRI of progressive atherosclerosis. The in-vivo detection and quantification of different MR biomarkers in a single scan could be useful to improve characterization of atherosclerotic-lesions.
Project description:The tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) specifically homes to tumors by binding to fibrin and fibrin-associated clotted plasma proteins in tumor vessels. Previous results show that CREKA-coated superparamagnetic iron oxide particles can cause additional clotting in tumor vessels, which creates more binding sites for the peptide. We have used this self-amplifying homing system to develop theranostic nanoparticles that simultaneously serve as an imaging agent and inhibit tumor growth by obstructing tumor circulation through blood clotting. The CREKA nanoparticles were combined with nanoparticles coated with another tumor-homing peptide, CRKDKC, and nanoparticles with an elongated shape (nanoworms) were used for improved binding efficacy. The efficacy of the CREKA peptide was then increased by replacing some residues with nonproteinogenic counterparts, which increased the stability of the peptide in the circulation. Treatment of mice bearing orthotopic human prostate cancer tumors with the targeted nanoworms caused extensive clotting in tumor vessels, whereas no clotting was observed in the vessels of normal tissues. Optical and magnetic resonance imaging confirmed tumor-specific targeting of the nanoworms, and ultrasound imaging showed reduced blood flow in tumor vessels. Treatment of mice with prostate cancer with multiple doses of the nanoworms induced tumor necrosis and a highly significant reduction in tumor growth.
Project description:OBJECTIVES:To obtain compensatory ultra-short echo time (UTE) imaging and T2-weighted (T2W) imaging of Watanabe heritable hyperlipidemic (WHHL) rabbits following dextran-coated magnetic nanocluster (DMNC) injection for the effective in vivo detection of inflammatory vascular wall. METHODS:Magnetic nanoparticle was synthesized by thermal decomposition and encapsulated with dextran to prepare DMNC. The contrast enhancement efficiency of DMNC was investigated using UTE (repetition time [TR] = 5.58 and TE = 0.07 ms) and T2W (TR = 4000 and TE = 60 ms) imaging sequences. To confirm the internalization of DMNC into macrophages, DMNC-treated macrophages were visualized by cellular transmission electron microscope (TEM) and magnetic resonance (MR) imaging. WHHL rabbits expressing macrophage-rich plaques were subjected to UTE and T2W imaging before and after intravenous DMNC (120 ?mol Fe/kg) treatment. Ex vivo MR imaging of plaques and immunostaining studies were also performed. RESULTS:Positive and negative contrast enhancement of DMNC solutions with increasing Fe concentrations were observed in UTE and T2W imaging, respectively. The relative signal intensities of the DMNC solution containing 2.9 mM Fe were calculated as 3.53 and 0.99 in UTE and T2W imaging, respectively. DMNC uptake into the macrophage cytoplasm was visualized by electron microscopy. Cellular MR imaging of DMNC-treated macrophages revealed relative signals of 3.00 in UTE imaging and 0.98 in T2W imaging. In vivo MR images revealed significant brightening and darkening of plaque areas in the WHHL rabbit 24 h after DMNC injection in UTE and T2W imaging, respectively. Ex vivo MR imaging results agreed with these in vivo MR imaging results. Histological analysis showed that DMNCs were localized to areas of inflammatory vascular wall. CONCLUSIONS:Using compensatory UTE and T2W imaging in conjunction with DMNC is an effective approach for the noninvasive in vivo imaging of atherosclerotic plaque.
Project description:BACKGROUND:The role of local alterations in endothelial functional integrity in atherosclerosis remains incompletely understood. This study used nanoparticle-enhanced optical molecular imaging to probe in vivo mechanisms involving impaired endothelial barrier function in experimental atherothrombosis. METHODS AND RESULTS:Atherosclerosis was induced in rabbits (n=31) using aortic balloon injury and high-cholesterol diet. Rabbits received ultrasmall superparamagnetic iron oxide nanoparticles (CLIO) derivatized with a near-infrared fluorophore (CyAm7) 24 hours before near-infrared fluorescence imaging. Rabbits were then either euthanized (n=9) or underwent a pharmacological triggering protocol to induce thrombosis (n=22). CLIO-CyAm7 nanoparticles accumulated in areas of atheroma (P<0.05 versus reference areas). On near-infrared fluorescence microscopy, CLIO-CyAm7 primarily deposited in the superficial intima within plaque macrophages, endothelial cells, and smooth muscle cells. Nanoparticle-positive areas further exhibited impaired endothelial barrier function as illuminated by Evans blue leakage. Deeper nanoparticle deposition occurred in areas of plaque neovascularization. In rabbits subject to pharmacological triggering, plaques that thrombosed exhibited significantly higher CLIO-CyAm7 accumulation compared with nonthrombosed plaques (P<0.05). In thrombosed plaques, nanoparticles accumulated preferentially at the plaque-thrombus interface. Intravascular 2-dimensional near-infrared fluorescence imaging detected nanoparticles in human coronary artery-sized atheroma in vivo (P<0.05 versus reference segments). CONCLUSIONS:Plaques that exhibit impaired in vivo endothelial permeability in cell-rich areas are susceptible to subsequent thrombosis. Molecular imaging of nanoparticle deposition may help to identify biologically high-risk atheroma.
Project description:In our program to develop non-invasive magnetic resonance imaging (MRI) methods for the diagnosis of Alzheimer's disease (AD), we have synthesized antibody-conjugated, superparamagnetic iron oxide nanoparticles (SPIONs) for use as an in vivo agent for MRI detection of amyloid-? plaques in AD. Here we report studies in A?PP/PS1 transgenic mice, which demonstrate the ability of novel anti-A?PP conjugated SPIONs to penetrate the blood-brain barrier to act as a contrast agent for MR imaging of plaques. The conspicuity of the plaques increased from an average Z-score of 5.1 ± 0.5 to 8.3 ± 0.2 when the plaque contrast to noise ratio was compared in control AD mice with AD mice treated with SPIONs. The number of MRI-visible plaques per brain increased from 347 ± 45 in the control AD mice, to 668 ± 86 in the SPION treated mice. These results indicated that our SPION enhanced amyloid-? detection method delivers an efficacious, non-invasive MRI detection method in transgenic mice.