JULY/AUGUST 2006 Volume 17, Number 4 © Copyright 2006 by the American Chemical Society
COMMUNICATIONS Annexin A5-Conjugated Quantum Dots with a Paramagnetic Lipidic Coating for the Multimodal Detection of Apoptotic Cells Geralda A. F. van Tilborg,*,† Willem J. M. Mulder,† Patrick T. K. Chin,‡ Gert Storm,§ Chris P. Reutelingsperger,| Klaas Nicolay,† and Gustav J. Strijkers† Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands, Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands, Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), 3508 TB, Utrecht, The Netherlands, and Department of Biochemistry, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Received February 24, 2006; Revised Manuscript Received April 25, 2006
Apoptosis, or programmed cell death, plays an important role in the etiology of a variety of diseases, including cancer. Visualization of apoptosis would allow both early detection of therapy efficiency and evaluation of disease progression. To that aim we developed a novel annexin A5-conjugated bimodal nanoparticle. The nanoparticle is composed of a quantum dot that is encapsulated in a paramagnetic micelle to enable its use both for optical imaging and MRI. Multiple recombinant human annexin A5 protein molecules were covalently coupled to the nanoparticle for targeting. In this study the specificity of the annexin A5-conjugated nanoparticles for apoptotic cells was demonstrated both with fluorescence microscopy and MRI, which confirms its potential for the detection of apoptosis with both imaging modalities in vivo.
Apoptosis, or programmed cell death, is an essential biological process that both occurs in normal tissue homeostasis and in diseased tissue (1). In pathophysiological situations the measurement of apoptotic activity may greatly enhance possibilities for staging the disease, and, equally important, apoptosis may serve as an early indicator for the effect of therapeutic * To whom correspondence should be addressed. Fax: +31 40 243 2598. E-mail:
[email protected]. † Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology. ‡ Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology. § Utrecht Institute for Pharmaceutical Sciences. | University of Maastricht.
interventions. In antitumor therapies the induction of apoptosis by, for example, cytotoxic drugs leads to an arrest of tumor proliferation, since the unrestrained division of tumor cells is related to insufficient apoptosis (2). Other disorders, like reperfusion injury after myocardial infarction or stroke, are rather associated with excessive apoptosis. Several biochemical markers of apoptosis have been identified and could in principle be exploited for the detection of apoptosis. The exposure of the normally asymmetrically distributed phosphatidylserine (PS) at the outer monolayer of the cell membrane of apoptotic cells is an especially attractive hallmark of apoptosis, since the protein annexin A5 binds with high affinity to this negatively charged phospholipid (3). For the visualization of apoptosis, annexin A5 may be used as a targeting ligand, which requires the protein
10.1021/bc0600463 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/16/2006
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Figure 1. Schematic representation of a TOPO/HDA quantum dot with a paramagnetic lipid coating, to which multiple annexin A5 proteins are conjugated (A5-pQD). The lipids drawn with the yellow and red headgroups represent PEG-DSPE and Gd-DTPA-BSA, respectively. Annexin A5 (indicated with A5) is conjugated to the maleimide group of Mal-PEG-DSPE.
to be conjugated to a contrast agent for the imaging modality of choice. Fluorescently labeled annexin A5 for optical imaging and annexin A5 conjugated with a radioactive label for nuclear imaging have been used successfully to visualize apoptosis in animal models (4) and in patients (5), respectively. Recently, annexin A5 conjugated to a magnetic nanoparticle was used for the detection of apoptosis in tumors and after myocardial infarction in mice with magnetic resonance imaging (MRI) (6). Although MRI excels in depicting opaque soft tissue with higher spatial resolution and better contrast as compared to, for example, nuclear methods, it is a relatively insensitive technique and it lacks the microscopic resolution of the optical methods. The bimodal approach of using MRI in combination with optical imaging would therefore make it possible to visualize apoptosis at the microscopic and macroscopic level. In this study we report on the development of a novel annexin A5-conjugated nanoparticle, which has excellent paramagnetic and fluorescent properties. The nanoparticle consists of a quantum dot (QD), which is encapsulated in a paramagnetic micelle. Quantum dots have received much attention lately as optical imaging agents because of their excellent photostability and sensitivity, and narrow and tunable emission wavelength (7). Because of the small size of this nanoparticle, less than 10 nm, compared to most other nanoparticulate MRI contrast agents, which are typically in the order of 50-250 nm (8, 9), quantum dots may distribute more easily into diseased tissue. This improves the bioavailability and can be expected to enhance the binding to apoptotic cells. CdSe/ZnS core/shell quantum dots were synthesized under a flow of argon by injection of the precursors into a hot coordinating solvent (10). The TOPO/HDA-capped quantum dots were coated with paramagnetic micelles according to an
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extended procedure described by Dubertret et al. (11). In short, TOPO/HDA-capped quantum dots were washed and suspended in chloroform. A mixture of 40 mol % 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))2000] (PEG-DSPE), 10 mol % 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[maleimide(poly(ethylene glycol))2000] (Mal-PEG-DSPE), and 50 mol % of the paramagnetic lipid Gd-DTPA-bis(stearylamide) (Gd-DTPA-BSA) dissolved in chloroform/methanol (20/1) was added to the quantum dots. The solvent was gently evaporated, and a solution of HEPES buffer (pH 6.7) that had been heated to 70 °C was added to the obtained mixed film. This mixture was rigorously heated and vortexed until a clear suspension was obtained. Empty micelles and micelles containing a quantum dot were separated with ultracentrifugation (1 h, 500000g). Both the gadolinium and the cadmium content of the annexin A5-pQD preparations were determined with inductively coupled plasma atomic emission spectrometry (ICP-AES). Furthermore it was measured with thermogravimetric analysis (TGA) that 56 mass % of the quantum dots consists of CdSe/ZnS. From these values an average value of 150-200 Gd-DTPA-BSA lipid molecules per nanoparticle could be derived. The ability of the annexin A5-conjugated nanoparticle (A5-pQD) to reduce the intrinsic T1 and T2 values of water are described by its molar relaxivities r1 and r2, which are expressed in mM-1 s-1. The ionic relaxivities of this nanoparticle at 37 °C and 60 MHz, were measured to be r1 ) 12.4 ( 0.7 mM-1s-1 and r2 ) 18.0 ( 0.1 mM-1s-1, respectively. The relaxivity per mM particles therefore is at least 150 times higher than the ionic relaxivity. Next, recombinant human annexin A5, containing a single free cysteine, was covalently linked to the maleimide group of Mal-PEG-DSPE via the thiol of the cysteine as described previously (12). A schematic representation of the annexin A5-conjugated nanoparticle (A5-pQD) is shown in Figure 1. The specificity of this contrast agent for apoptotic cells was obtained in vitro. For that purpose the T-lymphoma cell line Jurkat (ATCC) was grown in RPMI1640 medium (Gibco), which was supplemented with 10% fetal calf serum and a mixture of antibiotics (100 units ml-1 penicillin, 0.1 mg ml-1 streptomycin). Cells were grown at 37 °C in a humified atmosphere and 5% CO2. Apoptosis is naturally occurring without exposing the cells to apoptotic stimuli, typically involving 5 to 10% of the cells within the culture. To induce an increased number of apoptotic cells, the cells were resuspended to 106 cells ml-1 medium and treated with 200 ng ml-1 anti-Fas (CD95 human monoclonal antibody, Beckman Coulter B.V.) for 3 h, which initiates approximately 40 to 50% apoptosis. To allow fluorescence imaging and MRI of cell pellets, 5 million cells were treated with anti-Fas and subsequently incubated for 15 min with the annexin A5-conjugated nanoparticles at a final
Figure 2. Fluorescence microscopy of Jurkat cells incubated with annexin A5-conjugated pQDs. (A) Apoptotic cells incubated in the presence of EDTA, (B) vital cells with a naturally occurring percentage of apoptotic cells, and (C) apoptotic cells. +aF denotes stimulation with anti-Fas, while -aF denotes no stimulation. +Ca2+ indicates the presence of Ca2+, while -Ca2+ indicates incubation in the presence of EDTA to remove Ca2+.
Communications
Figure 3. Cell pellets of the different incubations illuminated with 254 nm UV light. From left to right: Control cells (sample 1), apoptotic cells incubated with A5-pQDs in the presence of EDTA to remove Ca2+ (sample 2), vital cells incubated with A5-pQDs (sample 3), and apoptotic cells incubated with A5-pQDs (sample 4).
concentration of 392.4 µM quantum dots in Ca2+-containing binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, pH 7.4). To further assess the binding specificity 5 × 106 cells that were not treated with antiFas were also incubated with A5-pQDs. Since annexin A5 binds to phosphatidylserine in a Ca2+-dependent manner, the removal of Ca2+ will prevent binding. Therefore, A5-pQDs that were incubated in HEPES-buffered saline that was supplemented with EDTA (25 mM HEPES, 140 mM NaCl and 5 mM EDTA, pH 7.4) were also used as control. Following the incubations, all cell samples were extensively washed in binding buffer or HEPES-buffered saline supplemented with EDTA, respectively. For fluorescence imaging and MRI of cell pellets, the 5 million cells of the different incubations were collected, fixed in a 4% paraformaldehyde solution, and put in small cups. Cells were allowed to settle as loosely packed cell pellets overnight at 4 °C Fluorescence microscopy of the apoptotic cells that were incubated with A5-pQDs in binding buffer showed bright fluorescence indicative for a massive association of the contrast agent with the cells (Figure 2C). On the contrary, the cells that were not stimulated with anti-Fas showed much less fluorescence (Figure 2B), because of the lower percentage of apoptotic cells. In the cell pellet that was incubated with A5-pQDs in the presence of EDTA almost no fluorescence was observed (Figure 2A). The observed low fluorescent intensity in sample 2 could not be induced by fluorescent quenching due to EDTA since the emission spectrum of paramagnetic quantum dots in HEPESbuffered saline was measured to remain unchanged after addition of EDTA to a final concentration of 5 mM. In Figure 3 the different cups with loosely packed cell pellets, illuminated with 254 nm UV light, are depicted. The green emitting cell pellet that originated from the incubation of annexin A5 nanoparticles with apoptotic cells (Figure 3, sample 4) can clearly be distinguished from the other incubations (Figure 3, samples 1-3).
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Following the above optical analysis, the cell pellets were measured with MRI. T1-weighted images (Figure 4A) revealed a strong signal enhancement of the apoptotic cells incubated with A5-pQDs in binding buffer (sample 4) as compared to the three control incubations (samples 1, 2, and 3). The cells that were not treated with anti-Fas (sample 3) also showed association of the contrast material. This accounts for the low level of apoptosis that is commonly seen in cell suspensions even in the absence of apoptotic stimuli. For absolute quantification of the T1 and T2 relaxation times an MRI inversion recovery experiment and a multi-echo experiment were performed, respectively. From these data the T1 and T2 relaxation times of the different cell pellets were calculated. Since the concentration of a paramagnetic agent is proportional with the relaxation rate R1 (1/T1) and R2 (1/T2), these values were calculated for the different cell pellets. In Figure 4B and 4C the R1 and R2 relaxation rates of the different cell pellets are depicted. Control cells that were not incubated with the contrast agent (sample 1) showed the lowest R1 and R2. Cell pellets of apoptotic cells incubated with A5-pQDs in binding buffer (sample 4) showed the highest R1 and R2 values. The cell pellet of anti-Fas treated cells that were incubated with A5-pQDs in the presence of EDTA (sample 2), as well as the untreated cell pellet incubated with A5-pQDs (sample 3), had much lower relaxation rates than the anti-Fas treated cells that were incubated with A5-pQDs in the presence of Ca2+ (sample 4). The novel annexin A5-conjugated nanoparticle presented in this study may be of great use for successive MRI and intravital microscopy of apoptosis in animal models of cardiac dysfunction or in tumor models. This allows the detection of apoptosis both at the macroscopic and the microscopic level in vivo. Dumont et al. have demonstrated the feasibility to detect apoptosis in the beating murine heart, following the administration of fluorescently labeled annexin A5 (4). A comparable setup could be used for intravital microscopy of subcutaneously grown tumors in mice. Recently, Stroh et al. have demonstrated simultaneous detection of multiple quantum dot species (13). The application of near-infrared quantum dots would additionally increase the penetration depth, which allows the real time investigation of tissue as deep as 1 cm (14). The technology developed in this study may also be applied to quantum dots with a different size and consequently with a different emission wavelength. This allows optimization of the nanoparticle both for the tissue of interest and the additional fluorescent dyes to be imaged. In conclusion, we have developed a novel nanoparticulate contrast agent for the detection of apoptosis, with excellent paramagnetic and fluorescent properties. The in vitro results demonstrated a high specificity of this contrast agent for apoptotic cells, suggesting its potential for the detection of apoptosis in different animal models with both intravital fluorescence microscopy and MRI in vivo. Because of the relatively small size of this QD-based contrast agent compared
Figure 4. (A) T1-weighted image of the different cell pellets. (B, C) The R1 and R2 relaxation rates of the different cell pellets with the highest values were measured for apoptotic cells incubated with A5-pQDs (black bar). The cells in sample 1 are untreated control cells. Sample 2, 3, and 4 are incubated with A5-pQDs.
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to most other MRI detectable nanoparticles the availability of the present contrast agent for apoptotic cells in the extravascular space may be greatly enhanced. This technology opens exciting new opportunities for multimodality molecular imaging of apoptotic processes in animal models of human disease processes.
ACKNOWLEDGMENT This study was funded in part by the EC - FP6-project DiMI, LSHB-CT-2005-512146-, and by the BSIK program entitled Molecular Imaging of Ischemic Heart Disease (project number BSIK03033). The authors thank Dr. Rolf Koole and Dr. Celso de Mello Donega´ for reading the manuscript and useful discussions.
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