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. Author manuscript; available in PMC: 2012 Oct 3.
Published in final edited form as: J Nucl Med. 2011 May 13;52(6):958–964. doi: 10.2967/jnumed.110.083220

Annexin A5-Conjugated Polymeric Micelles for Dual SPECT and Optical Detection of Apoptosis

Rui Zhang 1, Wei Lu 1, Xiaoxia Wen 1, Miao Huang 1, Min Zhou 1, Dong Liang 2, Chun Li 1
PMCID: PMC3463236  NIHMSID: NIHMS402767  PMID: 21571801

Abstract

Imaging of apoptosis can allow noninvasive assessment of disease states and response to therapeutic intervention for a variety of diseases. The purpose of this study was to develop and evaluate a multimodal nanoplatform for the detection of apoptosis.

Methods

To modulate the pharmacokinetics of annexin A5, a 36-kDa protein that binds specifically with phosphatidylserine, annexin A5 was conjugated to polyethylene glycol-coated, core-crosslinked polymeric micelles (CCPM) dually labeled with near-infrared fluorescence fluorophores and a radioisotope (indium 111). To evaluate the specificity of the binding of annexin A5-CCPM to apoptotic cells, both fluorescence microscopy and cell binding studies were performed in vitro. Pharmacokinetics, biodistribution, dual nuclear and optical imaging, and immunohistochemical studies were carried out in 2 xenografted tumor models to evaluate the potential applications of annexin A5-CCPM.

Results

In cell-based studies, annexin A5-CCPM exhibited strongly specific binding to apoptotic tumor cells. This binding could be efficiently blocked by annexin A5. In mice, annexin A5-CCPM displayed a mean elimination half-life of 12.5 h. The mean initial concentration in blood was predicted to be 22.4% of the injected dose/mL, and annexin A5-CCPM was mainly distributed in the central blood compartment. In mice bearing EL4 lymphoma treated with cyclophosphamide and etoposide and in mice bearing MDA-MB-468 breast tumors treated with poly(L-glutamic acid)-paclitaxel and cetuximab (IMC-C225) anti-EGFR antibody, the tumor apoptosis was clearly visualized by both single photon emission computed tomography and fluorescence molecular tomography. In contrast, there was little accumulation of this nanoradiotracer in the tumors of untreated mice. The biodistribution data were consistent with the imaging data, with tumor-to-muscle and tumor-to-blood ratios of 38.8 and 4.1, respectively, in treated mice, and 14.8 and 2.2, respectively, in untreated mice bearing EL4 lymphoma. Moreover, further studies demonstrated that the conventional Tc-99m-labeled HYNIC-annexin A5 and the plain CCPM control exhibited significantly lower uptake in the tumors of the treated mice than annexin A5-CCPM. Immunohistochemistry staining study showed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals co-localized with the region of tumor apoptosis.

Conclusions

Annexin A5-CCPM allowed visualization of tumor apoptosis by both nuclear and optical techniques. The complementary information acquired with multiple imaging techniques should be advantageous in assessing and validating early response to therapy.

Keywords: Apoptosis, Annexin A5, Polymeric Micelles, Nuclear Imaging, Fluorescence Optical Imaging

INTRODUCTION

Apoptosis, a form of programmed cell death, is critical to homoeostasis, normal development, and physiology. Dysregulation of apoptosis can lead to the accumulation of unwanted cells, such as occurs in cancer, and the removal of needed cells or disorders of normal tissues, such as heart, neurodegenerative, and autoimmune diseases (1–4). Because apoptosis is associated with many diseases, noninvasive detection of apoptosis has received much attention. To date, several biochemical features in cells have been identified and used for the visualization of apoptosis. Phosphatidylserine (PS), a component of cell membrane phospholipids in the inner plasma membrane, redistributes and externalizes to the outer surface of the membrane in the early stage of apoptosis (5,6). For the detection of PS on the surface of cells, annexin A5 can be used as a targeting ligand. Annexin A5 (molecular weight 36 KDa) is an endogenous human protein that consists of 319 amino acids. Annexin A5 binds strongly and specifically with PS residues (6–11). In past decades, a variety of annexin A5 derivatives have been developed for the detection of apoptosis with different imaging modalities, including fluorescence (12,13), positron emission tomography (14–17), single photon emission computed tomography (SPECT) (18–25), and magnetic resonance imaging (26,27). A few of these agents have shown promising results in imaging apoptosis in preclinical studies and clinical trials (18–21,23,25).

The timing of annexin A5 imaging after therapy has been shown to be critical (28). If peak apoptotic activity after administration of anticancer therapy varies from patient to patient and from treatment to treatment, then it will be necessary to determine for each patient the best time to scan after the start of chemotherapy. Because annexin A5 has a short blood half-life of only a few minutes, it has been suggested that an imaging protocol using multiple separate injections of technetium 99m (99mTc)-labeled annexin A5 and multiple radionuclide scans could be used to assess peak apoptotic activity (28). Such a protocol, however, might be difficult to implement in larger-scale trials. One approach to address this limitation is to modulate the pharmacokinetics of annexin A5 to ensure that the protein has sufficient time to reach its target over the course of action of apoptosis-inducing agents. In this work, we developed and evaluated micelle-modified annexin A5 with prolonged blood circulation time. We hypothesized that an imaging probe based on annexin A5 with a long blood half-life would make it possible to capture therapy-induced apoptotic cells over a prolonged course rather than a snapshot of the drug’s action. The micelles were also dually labeled with a gamma emitter, indium 111 (111In), and a near-infrared fluorescent indocyanine 7 (Cy7)-like dye to permit simultaneous visualization of apoptosis by both nuclear and optical imaging.

MATERIALS AND METHODS

Synthesis and Characterization of Annexin A5-CCPM

The synthesis, characterization, and radiolabeling procedures for 111In-labeled annexin A5-CCPM are described in the Supplementary Material.

Fluorescence Microscopy

DLD-1 human colorectal adenocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA). Cells were grown in Dulbecco’s modified Eagle’s medium and nutrient mixture F-12 Ham (DMEM/F12) (Gibco, Carlsbad, CA) supplemented with 10% fetal calf serum and a mixture of antibiotics (100 units mL-1 penicillin, 0.1 mg mL-1 streptomycin; Biochrom AG, Holliston, MA). Cells were grown at 37°C in a humidified atmosphere and 5% CO2. DLD-1 cells were treated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (Sigma-Aldrich) (200 μL, 150 ng/mL) for 2 h to induce apoptosis. The cells were then incubated with annexin A5-CCPM or CCPM in HEPES binding buffer (25 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) for 15 min at 37°C at a final concentration of 20 nM nanoparticles. Untreated DLD-1 cells were used as a control. Cells were washed 3 times with HEPES binding buffer and cell membrane stained with wheat germ agglutinin-Alexa Fluor 594 (Invitrogen, Carlsbad, CA). The cell samples were transferred onto Lab-Tek II chambered cover glass and visualized under a Zeiss Axio Observer.Z1 fluorescence microscope (Carl Zeiss MicroImaging GmbH, Thornwood, NY) equipped with Cy7 filters (wavelength 710/810 nm) and rhodamine filters (wavelength 570/620 nm).

Cell Binding Study

To study cell uptake of 111In-labeled annexin A5-CCPM, DLD-1 cells were grown in 6-cm petri dishes to subconfluent densities in DMEM/F12 containing 10% fetal bovine serum 1 day before experiments. DLD-1 cells were treated with TRAIL at doses of 1.5 ng/mL, 15 ng/mL, and 150 ng/mL for 2 h to induce apoptosis. After treatment with TRAIL, the medium was replaced with 2 mL of fresh medium containing 111In-labeled annexin A5-CCPM nanoparticles (~1.8 MBq/mL), and cells were incubated for 15 min. The cell monolayers were scraped and transferred into 5-mL tubes, and the tubes were briefly vortexed. Aliquots of DLD-1 cell suspension (100 μL) were transferred into a microcentrifuge tube containing 500 μL of a 75:25 mixture of silicon oil (density 1.05, Aldrich) and mineral oil (density 0.872, Acros). The mixture was centrifuged at 14,000 rpm for 5 min. After the tubes were frozen with liquid nitrogen, the bottom tips containing the cell pellet were cut off. The cell pellets and the supernatants were counted with a γ-counter (Perkin-Elmer). The protein content in 100 μL of cell suspension was quantified in a separate experiment using a Bio-Rad protein assay kit according to the manufacturer’s protocol. Activity ratios of the cell pellet to medium ([cpm/μg of protein in pellet]/[cpm/μg of medium]) were calculated and plotted against time. The experiments were performed in pentaplicate. In a separate study, aliquots of DLD-1 cell suspension (500 μL) after TRAIL treatment were transferred into microcentrifuge tubes, annexin A5-fluorescein isothiocyanate (FITC) was added to the tubes, and the percentage of apoptotic cells was analyzed using a Becton-Dickinson FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Pharmacokinetic Study

All animal studies were carried out in the Small Animal Imaging Facility at The University of Texas MD Anderson Cancer Center in accordance with institutional guidelines. For the pharmacokinetic study, healthy female Swiss mice (22–25 g; Charles River Laboratories, Wilmington, MA) (n = 8) were injected intravenously at a dose of 5 × 1013 nanoparticles per mouse (1.8 MBq/mouse). At predetermined intervals, blood samples (10 μL) were taken from the tail vein, and the radioactivity of each sample was measured with a Cobra Autogamma counter (Packard, Downers Grove, IL). The blood pharmacokinetic parameters for the radiotracer were analyzed using a noncompartmental model with WinNonlin 5.0.1 software (Pharsight Corp., Palo Alto, CA).

Tumor Models

Apoptosis of EL4 lymphoma was induced as described previously (30). Briefly, EL4 cells (1.0 × 106) were inoculated subcutaneously in the right flank of 6- to 8-week-old syngeneic C57BL/6 mice. Two weeks after inoculation, when tumors reached approximately 5–6 mm in diameter, mice were divided into 4 groups. Mice in group 1, 3, and 4 were given intraperitoneal injection of 25 mg/kg cyclophosphamide and 19 mg/kg etoposide. Mice in group 2 were not treated and were used as a control group. Mice in groups 1 and 2 received intravenous injection of 111In-labeled annexin A5-CCPM 1 day after drug treatment (n = 7 per group). Mice in group 3 received 99mTc-HYNIC annexin A5, mice in group 4 received 111In-labeled CCPM 1 day after drug treatment (n = 5 per group).

Induction of apoptosis in an MDA-MB-468 breast cancer model was achieved as described previously (31). Briefly, MDA-MB-468 cells (1 × 106) were inoculated subcutaneously in the right flank of 6- to 8-week-old female nude mice. Four weeks after inoculation, when tumors reached approximately 5–6 mm in average diameter, mice were divided into 2 groups (n = 2 per group). Mice in group 1 were given a single intravenous injection of poly(L-glutamic acid)-paclitaxel at a dose of 100 mg eq. paclitaxel/kg on day 1 and a single intraperitoneal injection of cetuximab (Imclone Systems, New York, NY), a monoclonal antibody directed against epidermal growth factor receptor, at a dose of 1 mg on day 4. Mice in group 2 were not treated and were used as a control group. 111In-labeled annexin A5-CCPM was injected intravenously via the tail vein 1 day after the injection of cetuximab.

Imaging and Biodistribution Studies

As described in the preceding section, 111In-labeled annexin A5-CCPM (1 × 1014 particles/mouse, 9.2 MBq/mouse) was injected intravenously via the tail vein into tumor-bearing mice after chemotherapy. At 48 h after administration of 111In-labeled annexin A5-CCPM, SPECT and computed tomography (CT) images were acquired using an xSPECT-CT scanner (Gamma Medica, Northridge, CA). SPECT scans (radius of rotation 3 cm, 32 projections, 20 s per projection) and CT scans (512 projections, 75 kv, 500 mA) were acquired and co-registered for image fusion and presentation of 3-dimensional anatomical localization of the tracer signal. Acquired SPECT and CT data sets were processed with AMIRA 5.1 (San Diego, CA). The whole-body optical imaging was performed using a fluorescent molecular tomography (Visen, Bedford, MA) equipped with a 760/790 nm excitation/emission wavelength filter. During each imaging session, mice were anesthetized with 2% isoflurane gas (Iso-Thesia, Rockville, NY) in oxygen.

Mice in group 3 injected with 99mTc-HYNIC annexin A5 were killed at 6 h after radiotracer injection. Mice in all the other three groups injected with 111In-labeled annexin A5-CCPM or 111In-labeled CCPM were killed at 48 h after administration of those particles. Various tissues were removed, weighed, and counted for radioactivity with a Cobra Autogamma counter (Packard). Uptake of the nanoparticles was calculated as the percentage of the injected dose per gram of tissue (%ID/g). In addition, fluorescence images of dissected tissues were acquired using an IVIS imaging system (Xenogen Corp., Alameda, CA) equipped with indocyanine green filter sets (excitation/emission, 710–760/810–875 nm). Fluorescence intensities were calculated using LIVINGIMAGE v.2.11 software (Xenogen). Uptakes of the nanoparticles in various tissues were calculated as flux (photons/s) per gram of tissue. Student’s t test was used to compare differences in tissue uptakes between different groups, and P values less than 0.01 were considered highly significant.

Autoradiography and Optical Imaging

Tumors harvested at the end of the imaging sessions were snap-frozen and sectioned into 5-μm sections. The sections were photographed and exposed on BAS-SR 2025 Fuji phosphorous films. The film was scanned with a FLA5100 Multifunctional Imaging System (Fujifilm Medical Systems USA, Stamford, CT).

After autoradiographic study, optical images of the sections were acquired by scanning at 800 nm using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).

Immunohistochemistry

Following autoradiographic study, 1 slice of each tumor was immunostained with anti-caspase-3 antibody by using a commercial kit (Sigma-Aldrich) according to the manufacturer’s protocol. The stained sections were counterstained with hematoxylin. Images were recorded using a Zeiss Axio Observer.Z1 microscope.

For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), a slide adjacent to those used for autoradiographic studies was stained with a TUNEL staining kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Cell nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). The cellular fluorescence was examined under a Zeiss Axio Observer.Z1 fluorescence microscope equipped with UV filter for DAPI, 494/517-nm filter for TUNEL staining, and 710/810-nm filter for Cy7.

Statistical Analysis

Statistical analysis was performed with GraphPad Prism v4.0 software (La Jolla, CA). Unless otherwise stated, group comparisons were made using standard ANOVA methods. Groups with P < 0.01 were considered highly significant.

Results

Characterization of Annexin A5-CCPM

The reaction scheme for the conjugation of annexin A5 to the surface of CCPM is shown in Suppl. Fig. 1A. Suppl. Fig. 1B is a transmission electron microscopic image of annexin A5-CCPM. The average diameter of the nanoparticles was 25 nm on the basis of transmission electron microscopy images. Each annexin A5-CCPM nanoparticle contained approximately 40 annexin A5 molecules on the surface calculated on the basis of the molar feed ratio. The excitation and emission light intensities for the annexin A5-CCPM nanoparticles peaked at 755 nm and 781 nm, respectively (Suppl. Fig. 1C). As indicated by instant thin-layer chromatography, the labeling efficiency of annexin A5-CCPM was greater than 98% without further purification. The specific activity was 185 MBq/nmol nanoparticles.

In Vitro Binding to Apoptotic Cells

Suppl. Fig. 2A shows fluorescence microscopic images of viable human DLD-1 colon cancer cells and apoptotic DLD-1 cells incubated with annexin A5-CCPM. The membrane of both TRAIL- and PBS-treated cells was stained red with wheat germ agglutinin-Fluor594. However, only the TRAIL-treated cells showed binding of annexin A5-CCPM to the cell membrane. Moreover, the binding of annexin A5-CCPM to the apoptotic cells was efficiently blocked by annexin A5 (Suppl. Fig. 2A).

TRAIL-induced apoptosis in DLD-1 cells was analyzed by flow cytometry at TRAIL concentrations of 1.5–150 ng/mL using FITC-annexin A5 (Suppl. Fig. 2B). There was a sharp increase in the apoptotic response when TRAIL concentration increased from 15 to 150 ng/mL, and the percentage of apoptotic cells increased from 3.76% to 24.5%. When 111In-labeled annexin A5-CCPM was incubated with TRAIL-treated DLD-1 cells, increasing amount of the radiotracer was bound to the cells in a TRAIL dose-dependent manner (Suppl. Fig. 2C). A significant increase in cell-associated radioactivity was detected at a TRAIL dose of 1.5 ng/mL. The binding of 111In-labeled annexin A5-CCPM to apoptotic cells was completely blocked by an excess of annexin A5 (Suppl. Fig. 2C). These results confirmed that 111In-labeled annexin A5-CCPM selectively bound to PS on the surface of apoptotic cells.

Pharmacokinetics

Suppl. Fig. 3 compares the activity-time profiles of 111In-labeled annexin A5-CCPM and plain CCPM. Data for CCPM were taken from reference (29). The plain CCPM showed a bi-exponential disposition, whereas 111In-labeled annexin A5-CCPM appeared to have a single exponential disposition following intravenous administration. Mean pharmacokinetic parameters are summarized in Table 1. Introduction of annexin A5 to CCPM resulted in a significantly shorter mean terminal elimination half-life, less systemic exposure (total area-under-the-bloodconcentration versus time curve), and smaller mean volume of distribution at steady-state as compared with unmodified CCPM. The mean systemic clearance was significantly slower with the unmodified CCPM (0.0902 mL/h) than with 111In-labeled annexin A5-CCPM (0.217 mL/h, P <0.001), suggesting that 111In-labeled annexin A5-CCPM was cleared more than twice as fast as the unmodified CCPM. This can be attributed to higher elimination by the liver and/or spleen of 111In-labeled annexin A5-CCPM than of CCPM. For both 111In-labeled CCPM and annexin A5-CCPM, mean volume of distribution at steady-state was similar to mean volume of distribution in the central compartment, indicating that both agents mainly distributed to the central compartment (systemic blood circulation).

Table 1.

Comparison of mean ± standard deviation pharmacokinetic parameters for plain CCPM and 111In-labeled annexin A5-CCPM in mice.

Parametera CCPM Annexin A5-CCPM P
Number of mice 8 7 -
T1/2 (h) 39.0 ± 8.4 12.5 ± 1.4 0.000
AUC (%ID/mL blood) 1148 ± 213 466 ± 53 0.000
Cmax (%ID/mL) 27.2 ± 3.1 22.4 ± 2.9 0.008
Vd (mL) 4.95 ± 0.9 3.91 ± 0.64 0.024
Vss (mL) 5.18 ± 0.65 4.44 ± 0.24 0.015
CL (mL/h) 0.0902 ± 0.019 0.217 ± 0.023 0.000
MRT (h) 58.7 ± 8.3 20.6 ± 1.9 0.000
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a

T1/2 = terminal biological half-life; AUC = total area under the blood concentration versus time curve; Cmax = predicted maximum drug concentration in blood; %ID = percentage of injected dose; Vd = apparent volume of distribution; Vss = steady-state volume of distribution; CL = total body clearance; MRT = mean residence time. P values were obtained using a 2-sample t test.

Biodistribution

Treatment of mice bearing EL4 lymphoma with cyclophosphamide and etoposide induces substantial apoptosis (30). This model was used to study the effect of apoptosis on the biodistribution of 111In-labeled annexin A5-CCPM (Fig. 1). At 48 h after injection, 111In-labeled annexin A5-CCPM showed significantly higher uptake in the tumors of the treated mice (8.01 %ID/g) than in the tumors of the untreated mice (3.2 %ID/g) (P <0.001) (Fig. 1A). The tumor-to-blood ratios were 2.2 in the untreated group versus 4.1 in the cyclophosphamide- and etoposide-treated group, and the tumor-to-muscle ratios were 14.8 in the untreated group versus 38.8 in the chemotherapy-treated group. The biodistribution was also determined by analysis of fluorescence signal intensities of the resected tissues (Suppl. Fig. 4). This data was consistent with the data obtained by the radioactivity count method. Spleens of the mice treated with chemotherapy showed significantly higher uptake of 111In-labeled annexin A5-CCPM than spleens in the control group (P <0.001), probably owing to cyclophosphamide-induced apoptosis of this tissue (20). 111In-labeled annexin A5-CCPM (8.01 %ID/g) also showed significantly higher uptake in the tumors of the treated mice than 99mTc-HYNIC annexin A5 (4.14 %ID/g) and 111In-labeled CCPM (2.81 %ID/g) (P <0.001) (Fig. 1B).

Fig. 1.

Fig. 1

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Biodistribution in mice bearing EL4 lymphoma. (A) Biodistribution 48 h after the administration of 111In-labeled annexin A5-CCPM. The mice in the treatment group were injected with 111In-labeled annexin A5-CCPM intravenously 24 h after administration of cyclophosphamide and etoposide. The mice in the control group were injected only with 111In-labeled annexin A5-CCPM. (B) Tumor uptake of 111In-labeled annexin A5-CCPM, 111In-labeled CCPM, and 99mTc-HYNIC annexin A5 in the EL4 tumor of mice treated with cyclophosphamide and etoposide. Data obtained using the radioactivity count method plotted as percentage of injected dose per gram of tissue (%ID/g). All the data are expressed as mean ± standard deviation. **P = 0.001.

Nuclear and Optical Imaging

Figure 2 compares μSPECT and fluorescent molecular tomography optical images of mice without treatment and mice treated with cyclophosphamide/etoposide regimen obtained 48 h after administration of 111In-labeled annexin A5-CCPM. The nuclear images were generally in concordance with the biodistribution data: there was relatively high accumulation in the liver and spleen. The apoptotic tumor was clearly visualized after chemotherapy in these mice with EL4 lymphoma (Fig. 2A). In contrast, there was little signal in the tumors of untreated mice (Fig. 2A). In optical images, the tumors in mice treated with chemotherapy were readily visualized in the mice after chemotherapy, whereas tumors in the untreated mice were not detected (Fig. 2B). Similar findings were observed in mice bearing subcutaneous MDA-MB-468 breast cancer (Suppl. Fig. 5).

Fig. 2.

Fig. 2

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Control group: Dual SPECT/CT and near-infrared fluorescence optical imaging of EL4 lymphoma apoptosis with 111In-labeled annexin A5-CCPM. Mice were injected intravenously only with 111In-labeled annexin A5-CCPM. (A) Representative SPECT/CT images. (B) Representative fluorescence molecular tomographic images. (C) Representative autoradiographs of excised tumors. (D) Fluorescence images of the same slides used in autoradiographic studies. (E and F) Immunohistochemical staining with caspase-3 (brown) of the same slides used in autoradiographic studies. All images were acquired 48 h after injection of 111In-labeled annexin A5-CCPM. Bar, 50 μm.

Autoradiography and Immunohistochemistry

Intratumoral distribution of 111In-labeled annexin A5-CCPM was shown in both autoradiographic images (Fig. 2C & Suppl. Fig. 5C) and fluorescence optical images of tumor sections (Fig. 2D & Suppl. Fig. 5D). Chemotherapy caused markedly increased radioactivity and fluorescent signal intensity in both tumor models. In the EL4 lymphoma model, localization of both radioactivity and fluorescence signal from 111In-labeled annexin A5-CCPM correlated with apoptotic cells stained with caspase-3 antibody (Figs. 2C–2F). In both the EL4 lymphoma model and the MDA-MB-468 models, the fluorescent signal from 111In-labeled annexin A5-CCPM co-localized with apoptotic cells detected with TUNEL assay (Fig. 3 & Suppl. Fig. 6).

Fig. 3.

Fig. 3

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Chemotherapy group: Dual SPECT/CT and near-infrared fluorescence optical imaging of EL4 lymphoma apoptosis with 111In-labeled annexin A5-CCPM. Mice in the chemotherapy group received an intravenous injection of 111In-labeled annexin A5-CCPM 24 h after treatment with cyclophosphamide 25 mg/kg by intraperitoneal (i.p.) injection and etoposide 19 mg/kg by i.p. injection. (A) Representative SPECT/CT images. (B) Representative fluorescence molecular tomographic images. (C) Representative autoradiographs of excised tumors. (D) Fluorescence images of the same slides used in autoradiographic studies. (E and F) Immunohistochemical staining with caspase-3 (brown) of the same slides used in autoradiographic studies. All images were acquired 48 h after injection of 111In-labeled annexin A5-CCPM. Bar, 50 μm.

Discussion

In this study, we found that injection of 111In-labeled annexin A5-CCPM allowed ready visualization of chemotherapy-induced apoptosis with both SPECT and near-infrared fluorescence imaging in 2 separate xenograft models in mice. Moreover, 111In-labeled annexin A5-CCPM permitted detection of apoptotic cells at the microscopic level by optical imaging.

Apoptosis is a dynamic process in which newly generated apoptotic cells are rapidly removed by phagocytic macrophages (32). Therefore, there is a short “time window” for detection of apoptotic cells, Owing to its short blood half-life (<7 min) (33), 99mTc-labeled annexin A5 exhibits limited exposure time and penetration into tumors, which compromise its sensitivity. We hypothesized that modulation of the pharmacokinetics of annexin A5 through the use of long-circulating nanoparticles would permit annexin A5-nanoparticles to penetrate deep into the tumor mass and to visualize apoptotic cells over a prolonged period, allowing improved detection of therapy-induced apoptosis. In this study, annexin A5 was conjugated to the surface of polyethylene glycol-coated CCPM. Although introduction of annexin A5 molecules to CCPM resulted in significant reduction in the blood half-life of the resulting annexin A5-CCPM as compared to the unmodified CCPM (Table 1), the mean half-life of 12.5 h was still much longer than that of annexin A5. We found that while conventional 99mTc-HYNIC annexin A5 showed an uptake value of 4.14 %ID/g in EL4 lymphoma of the treated mice, 111In-labeled annexin A5-CCPM showed an uptake value of 8.01 %ID/g, indicating that prolonging the blood half of annexin A5 could lead to increased uptake of the radiotracer in apoptotic tumors. The increased tumor uptake of 111In-labeled annexin A5-CCPM was not a result of increased permeability and retention effect, because 111In-labeled CCPM, which had longer blood half life than annexin A5-CCPM, displayed only 2.81 %ID/g in the treated tumors (Fig. 1B). Taken together, our data support the notion that increasing the half-life of annexin A5 improved the level of tumor uptake signal and was superior for noninvasive detection of apoptotic cells.

The overall strength of nanoparticle binding to target cells depends on both the affinity of the ligand-target interaction and the number of targeting ligands present on the nanoparticle surface. Multivalency effects have advantages over monovalency for binding interactions and can increase the avidity of interaction of ligands to their target receptor. This is a particularly useful feature for ligands that have a weak affinity to their target receptors in their monomer form. Nanoparticles containing multiple targeting ligands can provide multivalent binding to cell surface receptors (34). In our study, each annexin A5-CCPM nanoparticle contained approximately 40 annexin A5 molecules on the surface. Thus, the repertoire of annexin A5 was greatly expanded due to multiple, simultaneous interactions between the surface of the nanoparticle and the surface of the apoptotic cell. The in vitro studies of binding to apoptotic cells showed that 111In-labeled annexin A5-CCPM displayed higher detection sensitivity with radioactivity binding assay than monomeric FITC-annexin A5 with flow cytometry assay, possibly as a result of increased binding avidity (Suppl. Fig. 2).

Annexin A5 derivatives labeled with fluorescent dyes for optical imaging and with radioisotopes for nuclear imaging have been reported for detection of apoptosis in animal models and in patients, respectively (13,14,16–25,27,28,31,35). However, it is highly desirable that an imaging probe combining a radioisotope and a near-infrared fluorescent dye be available for dual nuclear and optical imaging (36). Nuclear imaging (SPECT/CT), an established clinical imaging modality, offers excellent sensitivity and covers the whole body. However, nuclear imaging techniques are limited by relatively poor spatial resolution. Optical imaging techniques have the potential to offer real-time, high-resolution images of tissues as long as they are accessible with near-infrared light. Importantly, the fluorescent signal from the imaging probes permits ex vivo analysis of excised tissues and validation of its binding to the molecular targets in vivo. The combination of 2 or more imaging techniques can therefore offer synergistic advantages over 1 modality alone in providing valuable diagnostic information. To date, several nanoparticle-based multimodal imaging probes (or contrast agents) have been developed and applied for multimodality functional imaging in living animals (27,35). These multimodal imaging systems have shown great promise in preclinical drug development and biomedical research. In this study, Cy-7 dye was entrapped in the core of CCPM and radioisotope chelator DTPA was conjugated on the surface of CCPM. Each micellar nanoparticle was loaded with multiple Cy7 dye molecules and 111In ions, providing a huge boost in signal intensity. As shown in both nuclear and optical imaging (Fig. 2 & Suppl. Fig. 5), 111In-labeled annexin A5-CCPM potentially can be used to locate apoptosis by whole-body nuclear and optical imaging. Histopathologically, 111In-labeled annexin A5-CCPM revealed apoptotic areas in tumor xenografts consistent with autoradiographic and fluorescent findings of tumor sections (Figs. 2, 3 & Suppl. Figs. 5, 6).

Conclusions

In this study, we evaluated the potential application of 111In-labeled annexin A5-functionalized core-crosslinked polymeric micelles for multimodality detection of drug-induced tumor apoptosis. In 2 tumor models, chemotherapy-induced apoptosis was readily visualized with 111In-labeled annexin A5-CCPM using both SPECT and near-infrared fluorescence imaging. Moreover, annexin A5-CCPM permitted detection of apoptotic cells at the microscopic level by optical imaging. Successful translation of 111In-labeled annexin A5-CCPM and other multimodal imaging probes into the clinic should improve the efficacy of detection of tumor apoptosis and the management of cancer patients after various therapeutic interventions.

Supplementary Material

supplementary

Fig. 4.

Fig. 4

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Fluorescence microscopy of EL4 lymphoma from mice treated with chemotherapy. The tumor sections were subjected to TUNEL staining (red). Signal from Cy7 loaded annexin A5-CCPM was pseudocolored green and cell nuclei were stained with DAPI (blue). Scale bar: 50 μm.

Acknowledgments

The authors thank Stephanie Deming for editing the manuscript and Qian Huang and Zhi Yang for helping with pharmacokinetic and biodistribution studies. This work was supported in part by National Institutes of Health grants (R01 CA119387 and RC2 GM092599), and the John S. Dunn Foundation. RZ is a recipient of the Harry S. & Isabel C. Cameron Foundation Fellowship. We also acknowledge the National Institutes of Health Cancer Center Support Grant CA016672 for support of MD Anderson’s Small Animal Imaging Facility and High-Resolution Electron Microscopy Core Facility.

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