111In-Labeled annexin A5-polyethylene glycol–coated core-cross-linked polymeric micelle-Cy7


Leung K.

Publication Details



In vitro Rodents



Apoptosis (programmed cell death) plays an important role in the pathophysiology of many diseases, such as cancers, neurodegenerative disorders, vascular disorders, atherosclerosis, and chronic hepatitis, as well as in the biology of normal cells like epithelial cells and immune cells (1). During apoptosis, there is rapid redistribution of phosphatidylserine (PS) from the inner membrane leaflet to the outer membrane leaflet, exposing the anionic head group of PS. PS is also accessible for annexin V (or annexin A5) binding in necrosis because of disruption of the plasma membrane. Annexin V, a 36-kDa protein, binds to PS with high affinity (Kd = 7 nM). Annexin V derivatives have been developed for imaging apoptosis as well as necrosis with different imaging modalities such as single-photon emission computed tomography (SPECT) (2-4), positron emission tomography (5), near-infrared (NIR) fluorescence (6), and magnetic resonance imaging (7).

Cy5.5-Annexin V has been evaluated as an optical probe in small animals. Annexin V-CLIO-Cy5.5 (AnxCLIO-Cy5.5) is a multimodal agent that consists of CLIO nanoparticles labeled with annexin V and Cy5.5 (6). van Tilborg et al. (7) incorporated annexin A5 into di-stearoyl-polyethylene glycol-phosphatidylethanolamine 2000 (DSPE-PEG2000) micelles labeled with gadolinium-labeled diethylenetriamine pentaacetic acid (Gd-DTPA) and Cy5.5 (annexin A5-Gd-micelles-Cy5.5) for multimodality imaging of atherosclerotic plaques. Zhang et al. (8) conjugated A5 to PEG-coated core-cross-linked polymeric micelles (CCPMs) labeled with Cy7 and 111In (111In-A5-CCPM) for multimodality detection of apoptosis in tumor-bearing mice.



CCPM nanoparticles were obtained from Carestream Health (Rochester, NY). There were ~21 Cy7 dye molecules, 180 amino groups, and 19 DTPA moieties per CCPM nanoparticle. Annexin A5 was conjugated to the maleimide-CCPM by sulfhydryl-maleimide bonding. A5-CCPMs were purified with column chromatography (8). The average hydrodynamic diameter of A5-CCPMs was determined to be ~25 nm. There were 40 annexin A5 molecules per nanoparticle. The maximum excitation and emission intensities were 755 nm and 781 nm, respectively. 111In-A5-CCPM was prepared by incubation of A5-CCPM with 111InCl3 in sodium acetate buffer (pH 5.2) for 30 min at room temperature with a labeling efficiency of >98%. The specific activity was 185 MBq/nmol (5 mCi/nmol). Control 111In-CCPMs without annexin A5 were prepared similarly with similar quality control metrics.

In Vitro Studies: Testing in Cells and Tissues


Zhang et al. (8) performed cell-binding assays with 111In-A5-CCPM and 111In-CCPM in apoptotic and normal viable human DLD-1 colon cancer cells with the use of fluorescence microscopy and radioactivity measurements at 2 h after incubation. The apoptotic cells (25% apoptotic) incubated with 111In-A5-CCPM (1.8 MBq/ml) showed clear cell-associated fluorescence and radioactivity signals, whereas negligible signals were obtained with control 111In-CCPM. Normal cells (3% apoptotic) showed little signal with 111In-A5-CCPM. Both signals in the apoptotic cells treated with 111In-A5-CCPM were blocked to background levels by 100-fold excess A5.

Animal Studies



Zhang et al. (8) performed ex vivo biodistribution studies in mice bearing EL-4 lymphoma tumors with or without cyclophosphamide and etoposide treatment to induce apoptosis. 111In-A5-CCPM (1 x 1014 nanoparticles, 9.2 MBq/mouse), 111In-CCPM, or 99mTc-HYNIC-A5 was injected intravenously in the mice at 24 h after drug treatment (n = 5-7/group). At 48 h after injection, 111In-A5-CCPM exhibited significantly higher accumulation in the tumors (8.0% injected dose/gram (ID/g)) and spleens (20% ID/g) of the treated mice than in the tumors (3.2% ID/g) and spleens (13% ID/g) of untreated mice (P < 0.001). The tumor/muscle ratios were 14.8 and 38.8 for the untreated mice and treated mice, respectively. The livers of both groups accumulated ~9% ID/g. The accumulation levels in other normal tissues (<2% ID/g) were similar in both treated and untreated mice. 111In-A5-CCPM showed significantly (P < 0.001) higher tumor accumulation in the tumors of the treated mice than 111In-CCPM (2.8% ID/g, 48 h after injection) and 99mTc-HYNIC-A5 (4.1% ID/g, 6 h after injection). Autoradiography and immunohistochemistry showed that the fluorescence and nuclear signals of 111In-A5-CCPM co-localized with caspase-3 in the tumor sections from mice treated with cyclophosphamide and etoposide. No blocking studies were performed.

SPECT and optical imaging scans were performed in mice (n = 2-4/group) bearing EL-4 lymphoma tumors with or without cyclophosphamide and etoposide treatment at 48 h after injection of 9.2 MBq (0.25 mCi) 111In-A5-CCPM. The apoptotic tumors were clearly visualized in the treated mice with SPECT and NIR fluorescence imaging, but little signal was detected in the tumors of untreated mice with these imaging modalities. No blocking studies were performed.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

R01 CA119387, RC2 GM092599, CA016672


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