Eyk Schellenberger

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.

Annexin V-CLIO: A Nanoparticle for Detecting Apoptosis by MRI

Annexin V, which recognizes the phosphatidylserine of apoptotic cells, was conjugated to crosslinked iron oxide (CLIO) nanoparticles, a functionalized superparamagnetic preparation developed for target-specific magnetic resonance imaging (MRI). The resulting nanoparticle had an average of 2.7 annexin V proteins linked per CLIO nanoparticle through disulfide bonds. Using camptothecin to induce apoptosis, a mixture of Jurkat T cells (69% healthy and 31% apoptotic) was incubated with annexin V-CLIO and was applied to magnetic columns. The result was an almost complete removal of the apoptotic cells (> 99%). In a phantom MRI experiment, untreated control cells (12% apoptotic cells, 88% healthy cells) and camptothecin-treated cells (65% apoptotic cells, 35% healthy cells) were incubated with either annexin V-CLIO (1.0, 0.5, and 0.1 microgram Fe/mL) or with unlabeled CLIO. A significant signal decrease of camptothecin-treated cells relative to untreated cells was observed even at the lowest concentration tested. Unmodified CLIO failed to cause a significant signal change of apoptotic cells. Hence, annexin V-CLIO allowed the identification of cell suspensions containing apoptotic cells by MRI even at very low concentrations of magnetic substrate. Conjugation of annexin V to CLIO affords a strategy for the development of a MRI imaging probe for detecting apoptosis.

Annexin V-CLIO: a nanoparticle for detecting apoptosis by MRI.

Annexin V, which recognizes the phosphatidylserine of apoptotic cells, was conjugated to crosslinked iron oxide (CLIO) nanoparticles, a functionalized superparamagnetic preparation developed for target-specific magnetic resonance imaging (MRI). The resulting nanoparticle had an average of 2.7 annexin V proteins linked per CLIO nanoparticle through disulfide bonds. Using camptothecin to induce apoptosis, a mixture of Jurkat T cells (69% healthy and 31% apoptotic) was incubated with annexin V-CLIO and was applied to magnetic columns. The result was an almost complete removal of the apoptotic cells (> 99%). In a phantom MRI experiment, untreated control cells (12% apoptotic cells, 88% healthy cells) and camptothecin-treated cells (65% apoptotic cells, 35% healthy cells) were incubated with either annexin V-CLIO (1.0, 0.5, and 0.1 microgram Fe/mL) or with unlabeled CLIO. A significant signal decrease of camptothecin-treated cells relative to untreated cells was observed even at the lowest concentration tested. Unmodified CLIO failed to cause a significant signal change of apoptotic cells. Hence, annexin V-CLIO allowed the identification of cell suspensions containing apoptotic cells by MRI even at very low concentrations of magnetic substrate. Conjugation of annexin V to CLIO affords a strategy for the development of a MRI imaging probe for detecting apoptosis.

Optical Imaging of Apoptosis as a Biomarker of Tumor Response to Chemotherapy

A rapid and accurate assessment of the antitumor efficacy of new therapeutic drugs could speed up drug discovery and improve clinical decision making. Based on the hypothesis that most effective antitumor agents induce apoptosis, we developed a near-infrared fluorescent (NIRF) annexin V to be used for optical sensing of tumor environments. To demonstrate probe specificity, we developed both an active (i.e., apoptosis-recognizing) and an inactive form of annexin V with very similar properties (to account for nonspecific tumor accumulation), and tested the agents in nude mice each bearing a cyclophosphamide (CPA) chemosensitive (LLC) and a chemoresistant LLC (CR-LLC). After injection with active annexin V, the tumor-annexin V ratio (TAR; tumor NIRF/background NIRF) for untreated mice was 1.22+/-0.34 for LLC and 1.43+/-0.53 for CR-LLC (n=4). The LLC of CPA-treated mice had significant elevations of TAR (2.56+/-0.29, P=.001, n=4), but only a moderate increase was obtained for the CR-LLC (TAR=1.89+/-0.19, P=.183). The in vivo measurements correlated well with terminal deoxyribosyl transferase-mediated dUTP nick end labeling indexes. When inactive Cy-annexin V was used, with or without CPA treatment and in both CCL and CR-CCL tumors, tumor NIRF values ranged from 0.91 to 1.17 (i.e., tumor were equal to background). We conclude that active Cy-annexin V and surface reflectance fluorescence imaging provide a nonradioactive, semiquantitative method of determining chemosensitivity in LLC xenografts. The method maybe used to image pharmacologic responses in other animal models and, potentially, may permit the clinical imaging of apoptosis with noninvasive or minimally invasive instrumentation.

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.

Protease-Specific Nanosensors for Magnetic Resonance Imaging

Imaging of enzyme activity is a central goal of molecular imaging. With the introduction of fluorescent smart probes, optical imaging has become the modality of choice for experimental in vivo detection of enzyme activity. Here, we present a novel high-relaxivity nanosensor that is suitable for in vivo imaging of protease activity by magnetic resonance imaging. Upon specific protease cleavage, the nanoparticles rapidly switch from a stable low-relaxivity stealth state to become adhesive, aggregating high-relaxivity particles. To demonstrate the principle, we chose a cleavage motif of matrix metalloproteinase 9 (MMP-9), an enzyme important in inflammation, atherosclerosis, tumor progression, and many other diseases with alterations of the extracellular matrix. On the basis of clinically tested very small iron oxide particles (VSOP), the MMP-9-activatable protease-specific iron oxide particles (PSOP) have a hydrodynamic diameter of only 25 nm. PSOP are rapidly activated, resulting in aggregation and increased T2*-relaxivity.

Linking Proteins with Anionic Nanoparticles via Protamine: Ultrasmall Protein‐Coupled Probes for Magnetic Resonance Imaging of Apoptosis

Magnetic resonance imaging (MRI) of a target in vivo depends on the surface, size, and particle relaxivity of the target-specific nanoparticles for MRI. Here a new method for decorating very small iron oxide particles (VSOPs) with target-specific ligands is described. The method is based on the electrostatic attraction of the strongly positively charged peptide protamine to the anionic citrate shell of the electrostatically stabilized VSOPs. The protamine coat allows linkage chemistry and chimera technology to functionalize VSOPs or other negative charged surfaces with biologics. Annexin A5 (anxA5)-VSOP utilizing thiol chemistry was generated to couple biologically active anxA5 to VSOPs for in vivo MRI of apoptosis. Annexin A5-VSOP comprises five anxA5 molecules per iron oxide nanoparticle with a high R2 particle relaxivity of 180 000 mM(-1) s(-1) yet small hydrodynamic diameter of only 14.7+/-2.9 nm beneficial for in vivo MRI of extravascular targets.

Comparison of the iron oxide-based blood-pool contrast medium VSOP-C184 with gadopentetate dimeglumine for first-pass magnetic resonance angiography of the aorta and renal arteries in pigs.

Rationale and Objectives:VSOP-C184 at a dose of 0.045 mmol Fe/kg has been shown to be an efficient blood pool contrast medium for equilibrium magnetic resonance angiography (MRA) that can be administered as a bolus. The present study was performed to determine whether VSOP-C184 is also suitable for first-pass MRA. Materials and Methods:Fifteen MRA examinations at 1.5 T were performed in minipigs using a fast 3D fast low-angle shot (FLASH) sequence (repetition time = 4.5 ms, echo time = 1.7 ms, excitation angle = 25°, matrix 256, body phased-array coil). The citrate-stabilized iron oxide preparation VSOP-C184 was investigated (total particle diameter: 7.0 ± 0.15 nm; core size: 4 nm) and compared with gadopentetate dimeglumine (Gd-DTPA). The following doses were tested: VSOP-C184: 0.015, 0.025, and 0.035 mmol Fe/kg; Gd-DTPA: 0.1 and 0.2 mmol Gd/kg; n = 3 examinations/dose. Data were analyzed quantitatively (signal enhancement (ENH) and vessel edge definition (VED)) and qualitatively. Results:First-pass MRA using the 3 doses of VSOP-C184 yielded the following ENH: aorta: 9.4 ± 2.6; 12.31 ± 1.2; 16.53 ± 1.7; renal arteries: 7.6 ± 2.2; 9.9 ± 1.0; 13.2 ± 0.5. The values for the 2 doses of Gd-DTPA were aorta: 12.9 ± 1.0; 16.8 ± 2.2; renal arteries: 11.2 ± 1.23; 11.3 ± 1.7. VED for the 3 doses of VSOP-C184 was aorta: 106.3 ± 31.0; 135.3 ± 58.8; 141.3 ± 71.0; renal arteries: 102.2 ± 24.3; 146.8 ± 63.0; 126.9 ± 37.6 and for the 2 doses of Gd-DTPA, aorta: 157.2 ± 47.8; 164.2 ± 36.8; renal arteries: 165.9 ± 30.4; 170.3 ± 38.2 respectively. The differences between VSOP-C184 and Gd-DTPA are clinically not relevant and statistically not significant (p ≥ .05). Qualitative evaluation of image quality, contrast, and delineation of vessels showed the results obtained with VSOP-C184 at doses of 0.025 and 0.035 mmol Fe/kg to be similar to those of Gd-DTPA at 0.1 and 0.2 mmol Gd/kg. Conclusion:VSOP-C184 is suitable for first-pass MRA at doses of 0.025 and 0.035 mmol Fe/kg and thus, in addition to its blood pool characteristics, allows for selective visualization of the arteries without interfering venous signal.

Coronary MR angiography using citrate-coated very small superparamagnetic iron oxide particles as blood-pool contrast agent: Initial experience in humans

To evaluate very small superparamagnetic iron oxide particles (VSOP‐C184) as blood‐pool contrast agent for coronary MR angiography (CMRA) in humans.

Optimal Modification of Annexin V with Fluorescent Dyes

The many uses of chemically modified annexin Vs necessitate an understanding of the optimal degree of modification and modification sites of the protein. When reacted with the N‐hydroxysuccinimide ester of Cy5.5, annexin V with one modification per mole of protein retained its affinity for phosphatidylserine of apoptotic cells, whereas modification with two dyes per mole of protein caused a complete loss of activity. A tryptic digest LC/MS method was used to identify the modification sites as either of two closely spaced lysine residues, in position 286 or 290. The crystal structure indicated the location of these lysines was distal to the phosphatidylserine binding sites on annexin V. These results can be used to develop active or inactive fluorescent control annexin V proteins and to suggest strategies for attaining higher levels of modification with retention of bioactivity.