Fred Reynolds

Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle

The ability to image cardiomyocyte apoptosis in vivo with high‐resolution MRI could facilitate the development of novel cardioprotective therapies. The sensitivity of the novel nanoparticle AnxCLIO‐Cy5.5 for cardiomyocyte apoptosis was thus compared in vitro to that of annexin V‐FITC and showed a high degree of colocalization. MRI was then performed, following transient coronary artery (LAD) occlusion, in five mice given AnxCLIO‐Cy5.5 and in four mice given an identical dose (2 mg Fe/kg) of CLIO‐Cy5.5. MR signal intensity and myocardial T2* were evaluated, in vivo, in hypokinetic regions of myocardium in the LAD distribution. Ex vivo fluorescence imaging was performed to confirm the in vivo findings. Myocardial T2* was significantly lower in the mice given AnxCLIO‐Cy5.5 (8.1 versus 13.2 ms, P < 0.01), and fluorescence target to background ratio was significantly higher (2.1 versus 1.1, P < 0.01). This study thus demonstrates the feasibility of obtaining high‐resolution MR images of cardiomyocyte apoptosis in vivo with the novel nanoparticle, AnxCLIO‐Cy5.5. Magn Reson Med, 2005.

Enzyme-Sensitive Magnetic Resonance Imaging Targeting Myeloperoxidase Identifies Active Inflammation in Experimental Rabbit Atherosclerotic Plaques

Background— Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase is a central inflammatory enzyme secreted by activated macrophages and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance agent can visualize myeloperoxidase activity in atherosclerotic plaques in a rabbit model. Methods and Results— We performed magnetic resonance imaging of the thoracic aorta of New Zealand White rabbits fed a cholesterol (n=14) or normal (n=4) diet up to 2 hours after injection of the myeloperoxidase sensor bis-5HT-DTPA(Gd) [MPO(Gd)], the conventional agent DTPA(Gd), or an MPO(Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hours after injection) showed focal areas of increased contrast (>2-fold) in diseased wall but not in normal wall (P=0.84) compared with both DTPA(Gd) (n=11; P<0.001) and bis-tyr-DTPA(Gd) (n=3; P<0.05). Biochemical assays confirmed that diseased wall possessed 3-fold elevated myeloperoxidase activity compared with normal wall (P<0.01). Areas detected by MPO(Gd) imaging colocalized and correlated with myeloperoxidase-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, P<0.0001). Although macrophages were the main source of myeloperoxidase, not all macrophages secreted myeloperoxidase, which suggests that distinct subpopulations contribute differently to atherogenesis and supports our functional approach. Conclusions— The present study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme to noninvasively provide both anatomic and functional information in vivo.

In Vivo Phage Display Selection Yields Atherosclerotic Plaque Targeted Peptides for Imaging

PurposeAtherosclerosis is a leading cause of morbidity and mortality in the Western world, yet specific imaging agents to detect and map inflammatory plaques are still lacking.ProceduresWe used in vivo phage display to interrogate early atherosclerotic lesions present in ApoE−/− mice with the goal of identifying plaque-associated endothelial cell internalized affinity ligands.ResultsWe identified 30 phage families with some of these families exhibiting homology to known atherosclerotic proteins, namely, leukemia inhibitory factor, transferrin, and VLA-4. VLA-4 homologous peptides [termed vascular cellular adhesion molecule-1 (VCAM-1) internalizing peptide-28 (VINP28)] bound to and were internalized by VCAM-1-expressing cells and were inhibited by soluble VCAM-1. In addition, a VINP28 modified multimodal nanoparticle showed high affinity for endothelial cells expressing VCAM-1 but low affinity for macrophages or smooth muscle cells.ConclusionThe identified peptides represent a set of probes to interrogate the cell surface repertoire and potentially allow early detection of atherosclerosis.

Transport Of Surface‐Modified Nanoparticles Through Cell Monolayers

We synthesized three peptides, a D‐polyarginyl peptide (r8(FITC)), a Tat peptide (Tat(FITC)), and a control peptide (Cp(FITC)) and attached each to amino‐CLIO, a nanoparticle 30 nm in diameter. We then examined the effective permeability, Peff, of all six materials through CaCo‐2 monolayers. The transport of peptide–nanoparticles was characterized by a lag phase (0–8 h) and a steady‐state phase (9–27 h). The steady‐state Peff values for peptides were in the order r8(FITC)>Tat(FITC)=Cp(FITC). When r8(FITC) and Tat(FITC) peptides were attached to the nanoparticle, they conferred their propensity to traverse cell monolayers onto the nanoparticle, whereas Cp(FITC) did not. Thus, when the r8(FITC) peptide was attached to the amino‐CLIO nanoparticle, the resulting peptide–nanoparticle had a Peff similar to that of this poly‐d‐arginyl peptide alone. The Peff of r8(FITC)–CLIO (MW∼1000 kDa) was similar to that of mannitol (MW=182 Da), a poorly transported reference substance, with a far lower molecular weight. These results are the first to indicate that the modification of nanoparticles by attachment of membrane‐translocating sequence‐based peptides can alter nanoparticle transport through monolayers. This suggests that the surface modification of nanoparticles might be a general strategy for enhancing the permeability of drugs and that high‐permeability nanoparticle‐based therapeutics can be useful in selected pharmaceutical applications.

Molecular MRI of Cardiomyocyte Apoptosis With Simultaneous Delayed-Enhancement MRI Distinguishes Apoptotic and Necrotic Myocytes In Vivo Potential for Midmyocardial Salvage in Acute Ischemia

Background—A novel dual-contrast molecular MRI technique to image both cardiomyocyte apoptosis and necrosis in vivo within 4 to 6 hours of ischemia is presented. The technique uses the annexin-based nanoparticle AnxCLIO-Cy5.5 (apoptosis) and simultaneous delayed-enhancement imaging with a novel gadolinium chelate, Gd-DTPA-NBD (necrosis). Methods and Results—Mice with transient coronary ligation were injected intravenously at the onset of reperfusion with AnxCLIO-Cy5.5 (n=7) or the control probe Inact_CLIO-Cy5.5 (n=6). T2*-weighted MR images (9.4 T) were acquired within 4 to 6 hours of reperfusion. The contrast-to-noise ratio between injured and uninjured myocardium was measured. The mice were then injected with Gd-DTPA-NBD, and delayed-enhancement imaging was performed within 10 to 30 minutes. Uptake of AnxCLIO-Cy5.5 was most prominent in the midmyocardium and was significantly greater than that of Inact_CLIO-Cy5.5 (contrast-to-noise ratio, 8.82±1.5 versus 3.78±1.1; P<0.05). Only 21±3% of the myocardium with accumulation of AnxCLIO-Cy5.5 showed delayed-enhancement of Gd-DTPA-NBD. Wall thickening was significantly reduced in segments with delayed enhancement and/or transmural accumulation of AnxCLIO-Cy5.5 (P<0.001). Fluorescence microscopy of AnxCLIO-Cy5.5 and immunohistochemistry of Gd-DTPA-NBD confirmed the presence of large numbers of apoptotic but potentially viable cardiomyocytes (AnxCLIO-Cy5.5 positive, Gd-DTPA-NBD negative) in the midmyocardium. Conclusions—A novel technique to image cardiomyocyte apoptosis and necrosis in vivo within 4 to 6 hours of injury is presented and reveals large areas of apoptotic but viable myocardium in the midmyocardium. Strategies to salvage the numerous apoptotic but potentially viable cardiomyocytes in the midmyocardium in acute ischemia should be investigated.

Surface-functionalized nanoparticle library yields probes for apoptotic cells

We have developed techniques for the efficient synthesis and screening of small libraries of surface‐functionalized nanoparticles for the recognition of specific types of cells. To illustrate this concept we describe the development of a nanoparticle that preferentially recognizes apoptotic Jurkat cells in a manner similar to the apoptosis‐recognizing protein annexin V. The nanoparticle, which is detectable by fluorescence or NMR relaxometry, was analyzed for the ability to recognize normal and apoptotic cells by fluorescence‐activated cell sorting (FACS) analysis and fluorescence microscopy. The capability to develop nanoparticles which interact with specific target cells could be applied to the design of materials for diverse applications including quantum dots, which serve as fluorescence tracers, colloidal gold, which serves as a tracer for electron micrographs, or the crystalline forms of drugs.

Human Breast Cancer Tumor Models: Molecular Imaging of Drug Susceptibility and Dosing during HER2/neu-targeted Therapy

PURPOSE To use near-infrared (NIR) optical imaging to assess the therapeutic susceptibility and drug dosing of orthotopic human breast cancers implanted in mice treated with molecularly targeted therapy. MATERIALS AND METHODS This study was approved by the institutional animal care and use committee. Imaging probes were synthesized by conjugating the human epidermal growth factor receptor type 2 (HER2)-specific antibody trastuzumab with fluorescent dyes. In vitro probe binding was assessed with flow cytometry. HER2-normal and HER2-overexpressing human breast cancer cells were orthotopically implanted in nude mice. Intravital laser scanning fluorescence microscopy was used to evaluate the in vivo association of the probe with the tumor cells. Mice bearing 3-5-mm-diameter tumors were intravenously injected with 0.4 nmol of HER2 probe before or after treatment. A total of 123 mice were used for all in vivo tumor growth and imaging experiments. Tumor fluorescence intensity was assessed, and standard fluorescence values were determined. Statistical significance was determined by performing standard analysis of variance across the imaging cohorts. RESULTS HER2 probe enabled differentiation between HER2-normal and HER2-overexpressing human breast cancer cells in vitro and in vivo, with binding levels correlating with tumor trastuzumab susceptibility. Serial imaging before and during trastuzumab therapy revealed a significant reduction (P < .05) in probe binding with treatment and thus provided early evidence of successful HER2 inhibition days before the overall reduction in tumor growth was apparent. CONCLUSION NIR imaging with HER2-specific imaging probes enables evaluation of the therapeutic susceptibility of human mammary tumors and of drug dosing during HER2-targeted therapy with trastuzumab. This approach, combined with tomographic imaging techniques, has potential in the clinical setting for determining patient eligibility for and adequate drug dosing in molecularly targeted cancer therapies.

Molecular MRI detects low levels of cardiomyocyte apoptosis in a transgenic model of chronic heart failure.

Background—The ability to image cardiomyocyte (CM) apoptosis in heart failure could facilitate more accurate diagnostics and optimize targeted therapeutics. We thus aimed to develop a platform to image CM apoptosis quantitatively and specifically in heart failure in vivo. The myocardium in heart failure, however, is characterized by very low levels of CM apoptosis and normal vascular permeability, factors thought to preclude the use of molecular MRI. Methods and Results—Female mice with overexpression of Gaq were studied. Two weeks postpartum, these mice develop a cardiomyopathy characterized by low levels of CM apoptosis and minimal myocardial necrosis or inflammation. The mice were injected with the annexin-labeled nanoparticle (AnxCLIO-Cy5.5) or a control probe (CLIO-Cy5.5) and imaged in vivo at 9.4 T. Uptake of AnxCLIO-Cy5.5 occurred in isolated clusters, frequently in the subendocardium. Myocardial T2* was significantly lower (7.6±1.5 versus 16.8±2.7 ms, P<0.05) in the mice injected with AnxCLIO-Cy5.5 versus CLIO-Cy5.5, consistent with the uptake of AnxCLIO-Cy5.5 by apoptotic CMs. A strong correlation (r2=0.86, P<0.05) was seen between in vivo T2* (AnxCLIO-Cy5.5 uptake) and myocardial caspase-3 activity. Conclusions—The ability of molecular MRI to image sparsely expressed targets in the myocardium is demonstrated in this study. Moreover, a novel platform for high-resolution and specific imaging of CM apoptosis in heart failure is established. In addition to providing novel insights into the pathogenesis of CM apoptosis, the developed platform could facilitate the development of novel antiapoptotic therapies in heart failure.

Simplified syntheses of complex multifunctional nanomaterials

Multifunctional probes are synthesized in a single step using peptide scaffold-based multifunctional single-attachment-point (MSAP) reagents.

A screening paradigm for the design of improved polymer-coated superparamagnetic iron oxide nanoparticles

Polymer-coated superparamagnetic iron oxide nanoparticles (NPs) have been used for a variety of biomedical applications, including as MRI contrast agents, for the treatment of iron anemias, and for ex vivo labeling of the cells used in cell-based therapies so that they can be tracked by MRI. Here we describe a three-stage screening paradigm to develop high potency (i.e., high relaxivity), stable, polymer-coated superparamagnetic iron oxide NPs. Each screen examined different facets of the interaction between iron oxides and polymers. First, an Ion Challenge Screen assessed whether a polymer interacted with the surface of the iron oxide. Second, a Synthesis Optimization Screen examined whether polymers that passed the Ion Challenge Screen were compatible with the synthetic method used to make NPs, and optimized the synthetic method for each polymer. Finally, a Heat Stress/Stability Screen assessed the stability of the optimized polymer-coated superparamagnetic iron oxides that passed the Synthesis Optimization Screen. A carboxymethyl dextran-coated superparamagnetic iron oxide nanoparticle (CMD-NP) with a transverse relaxivity (R2) of 271 mM−1 s−1 and a diameter of 47 nm by dynamic light scattering was obtained. A second NP, a carboxymethyl polyvinyl alcohol-coated nanoparticle (CMPVA-NP) had an R2 of 119 and diameter of 37 nm, but was less stable to heat stress than the CMD-NP. The CMD-NP is a polymer-coated superparamagnetic iron oxide NP with improved relaxivity and high stability achieved without the crosslinking procedure used in our cross-linked iron oxide (CLIO) NP.