Bioconjugate Chem. 2010, 21, 1939–1942
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Caspase Sensitive Gold Nanoparticle for Apoptosis Imaging in Live Cells In-Cheol Sun,† Seulki Lee,‡ Heebeom Koo,† Ick Chan Kwon,† Kuiwon Choi,† Cheol-Hee Ahn,*,§ and Kwangmeyung Kim*,† Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul, 136-791, Korea, Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), 31 Center Drive, Suite 1C14, Bethesda, Maryland 20892-2281, United States, and Research Institute of Advanced Materials (RIAM), Department of Materials Science and Engineering, Seoul National University, San 56-1, Sillim, Gwanak, Seoul, 151-744, Korea. Received July 12, 2010; Revised Manuscript Received October 1, 2010
We developed a new apoptosis imaging probe with gold nanoparticles (AuNPs). A near-infrared fluorescence dye was attached to AuNP surface through the bridge of peptide substrate (DEVD). The fluorescence was quenched in physiological conditions due to the quenching effect of AuNP, and the quenched fluorescence was recovered after the DEVD had been cleaved by caspase-3, the enzyme involved in apoptotic process. The adhesion of DEVD substrates on AuNP surface was accomplished by conjugation of the 3,4-dihydroxy phenylalanine (DOPA) groups which are adhesive to inorganic surface and rich in mussels. This surface modification with DEVD substrates by DOPA groups resulted in increased stability of AuNP in cytosol condition for hours. Moreover, the cleavage of substrate and the dequenching process are very fast, and the cells did not need to be fixed for imaging. Therefore, the real-time monitoring of caspase activity could be achieved in live cells, which enabled early detection of apoptosis compared to a conventional apoptosis kit such as Annexin V-FITC. Therefore, our apoptosis imaging has great potential as a simple, inexpensive, and efficient apoptosis imaging probe for biomedical applications.
Apoptosis is an important physiological mechanism to maintain homeostasis of multicellular organisms by elimination of infected or damaged cells and regulation of cell number (1, 2). Various imaging techniques were developed to image this process, because it could provide a monitoring tool for therapeutic effect of cancer treatment, efficacy of new drugs, or quality of protein therapeutics produced in mammalian cell culture. In particular, the TUNEL (TdT-mediated dUTP nick end labeling) assay is a typical method to label DNA fragmentation which occurs during apoptosis. However, many reaction steps are involved in this method and internucleosomal DNA breaks were not always associated with apoptosis (3, 4). Annexin V/FITC (fluorescein isothiocyanate) conjugate is another widely used method for apoptosis imaging due to its binding affinity to negatively charged phosphatidylserine (PS) exposed on the apoptotic cell membrane. However, this binding is not specific to apoptosis, because PS exposure can occur in other biological situations like myotube formation and necrosis (5, 6). To overcome the limitation of conventional imaging methods and to enable the early diagnosis of diseases or fast evaluation of therapeutic effect, it is necessary to develop a caspase enzymesensitive imaging probe, because activation of caspases is the initial stage of apoptotic process and responsible for the cell changes during apoptosis such as DNA fragment, nuclear chromatin condensation, and plasma membrane blebbing (7). For this reason, we synthesized a caspase-sensitive fluorescence imaging probe using gold nanoparticles (AuNPs). Caspase-3 cleavable motifs (8) combined with mussel-inspired adherent peptides, 3,4-dihydroxy phenylalanines (DOPA), and lysines (Gly-Asp-Glu-Val-Asp-Ala-Pro-DOPA-Lys-DOPA-Lys* To whom correspondence should be addressed. Phone: (+82) 2-958-5916, (+82) 2-880-5791, Fax: (+82) 2-958-5909, (+82) 2-8838197. E-mail: (K.K.)
[email protected]; (C.-H.A.)
[email protected]. † Korea Institute of Science and Technology. ‡ NIBIB, NIH. § Seoul National University.
DOPA, the cleavage site is between Asp and Ala) were purchased, and a near-infrared fluorescence (NIRF) dye, Cy5.5, was conjugated to the N-terminus of peptide sequence. DOPAs and lysines (DOPAK) at the C-terminus of caspase-3 cleavable motifs played an important role in adhesion of peptides on the surface of AuNP. The Cy5.5-conjugated peptide (Cy5.5-DEVDDOPAK) was used to modified the surface of citrated-stabilized AuNPs by a simple mixing method (Scheme 1). At the N-terminus of DEVD, three 3,4-dihydroxy phenylalanines and lysines (DOPAKs) were chemically conjugated to increase the adherence between the surface of AuNP and peptide. DOPA has been known to be rich in mussel foot protein and has excellent adhesive properties to the surface of various materials (9). AuNP carried out a role as a quencher of Cy5.5 for its strong surface energy transfer (10, 11). This nanoprobe facilitated detection and monitoring of enzymatic activity of caspase, because its NIRF emission was selectively turned on after degradation of DEVD sequence by caspase-3. Our probe has unique features such as sensitive and fast imaging, direct imaging of caspase-3 enzyme activity, and real-time imaging of apoptotic cells. For the biological application, the stability of AuNPs in the physiological condition should be guaranteed because aggregation of them impedes their long circulation and full function in the body. The stability of Cy5.5-DEVD-DOPAK coated AuNPs (DEVD-AuNPs) was confirmed through UV-vis absorption spectroscopy in caspase buffer condition (Figure 1A; see the Supporting Information for details of caspase buffer). The spectrum showed a unique surface plasmon resonance peak of DEVD-AuNPs at 523 nm, and it was no different from that of the uncoated AuNPs even in the caspase-containing buffer. However, in the case of the uncoated AuNPs, they aggregated and the UV-vis absorbance was reduced after incubation in caspase buffer. The stability of DEVD-AuNPs was also verified by observation of the morphology in the TEM images (Figure 1B). As seen in Figure 1B, DEVD-AuNPs were uniformly dispersed and no aggregation was found. The size of DEVD-
10.1021/bc1003026 2010 American Chemical Society Published on Web 10/11/2010
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Scheme 1. Schematic Diagram of Cy5.5 and DEVD Peptide Conjugated Gold Nanoparticles (DEVD-AuNPs)a
a
Blue spheres indicated quenched state of Cy5.5 and red ones represented recovered fluorescence.
Figure 1. (A) UV-vis absorption spectrum of bare AuNP in water (blue), caspase buffer (red), and DEVD-AuNP in buffer (black). (B) TEM images of DEVD-AuNP. (C) Particle size distribution measured with DLS (D) Optical image of AuNP and DEVD-AuNP.
AuNP in the buffer was measured with dynamic light scattering, and it showed the size distribution of 37.8 ( 14.1 nm (Figure 1C). The increment of diameter was due to the hydrophilicity of peptide and not caused by aggregation of AuNPs. The enhanced stability of DEVD-AuNP was also examined by the naked eye, because it retained the unique red color during the coating process (Figure 1D). From these results of DEVDAuNPs, it was inferred that DEVD peptide on the surface did not affect the stability of AuNPs but also stabilized AuNPs even in the caspase buffer environment. In addition, the amount of DEVD substrate bound to the AuNP surface was calculated through the measurement of UV-vis absorption of unreacted
Cy5.5-DEVD during the coating process. The UV-vis absorbance at 675 nm was calibrated with a standard curve of Cy5.5, and we estimated that about 1.2 × 102 molecules of DEVD were attached to each AuNP (0.095 49 molecules/nm2). The feasibility of DEVD-AuNP for optical imaging probe of apoptosis was investigated. The difference of NIRF intensity of Cy5.5 was measured before and after incubation of DEVDAuNP with caspase-3. NIRF was quenched before the addition of caspase-3 due to the nonradiative energy transfer properties of AuNP (12). The quenched fluorescence was recovered after the DEVD substrates on the AuNPs were degraded by caspase3. The cleavage of DEVD increased the distance between fluorophore and AuNP, and the NIRF signal was recovered because the quenching effect of AuNP was reduced. These results were imaged with NIRF imaging system (Kodak Image Station 4000MM, New Haven, CT, USA), a function of concentration of DEVD-AuNP (Figure 2A). Due to the strong quenching effect of AuNPs, the detection of recovered fluorescence was corrupted after incubation with caspase-3 when the concentration of DEVD-AuNP had been greatly increased. For comparison, the NIRF images were obtained from the decanted solution after incubation with caspase-3 followed by centrifugation. The quantitative measurement of NIRF using a spectrofluorometer at a fixed excitation wavelength of 675 nm showed that the signal-to-background ratio could be increased by concentration of DEVD-AuNP up to 1.0 mg/mL and NIRF signal saturation appeared due to the self-quenching of Cy5.5 dyes detached from DEVD-AuNP (Figure 2B). The recovered NIRF intensity was also dependent on concentration of caspase-3 as shown in Figure 2C, and DEVD-AuNP could detect and generate fluorescence in response to only about 1 nM of caspase3. Figure 2D shows that the fluorescence recovery of DEVD-
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Figure 2. (A) NIRF images of DEVD-AuNP in 96-well microplate containing various concentrations of DEVD-AuNP with or without caspase-3. (B) Fluorescence spectra of DEVD-AuNP as a function of probe concentration incubated with caspase-3. (C) NIRF images of DEVD-AuNP with various concentrations of caspase-3. (D) Fluorescence spectra of DEVD-AuNP as a function of incubation time with caspase-3.
AuNP was made very quickly in response to caspase-3 and had already occurred at 10 min. For real-time apoptosis imaging with DEVD-AuNPs in live cells, apoptosis was induced with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HeLa cells. TRAIL has been known to induce apoptosis effectively in various cell lines include HeLa cells (13). After DEVD-AuNPs were taken up by HeLa cells by coincubation for 1 h, TRAIL was treated and the cells were observed with microscopy to obtain real-time fluorescent images. Even before the formation of apoptotic body, the NIRF signal of Cy5.5 appeared after 30 min of TRAIL treatment (Figure 3A). This signal was condensed after the apoptotic body was formed in 120 min. In contrast, NIRF signal was not observed for 120 min when the cells were not treated with TRAIL. In addition, it was seen that cell death was delayed and the NIRF intensity was reduced when the cells were exposed to the inhibitor of caspase-3 (Z-DEVD-FMK, R&D Systems, Minneapolis, MN, USA) for 30 min prior to TRAIL treatment. This result was further investigated with DAPI (4′,6-diamidino2-phenylindole) staining, which binds strongly to DNA and forms a fluorescent complex in nuclei. It is well-known that caspase cascade is activated in the cytosol area (1, 8), so the recovered fluorescence of Cy5.5 in the cytosol should not overlap with that of DAPI in the nuclei as shown in Figure 3B. In addition, the existence of DEVD-AuNP was monitored through a dark-field microscope. The signal of DEVD-AuNP in the dark-field image was detected only in the cytosol area of HeLa cells, which was coincident with previous results (Figure 3C). These results clearly showed that the DEVD-AuNP probe has enhanced optical functionality and specificity against caspase enzyme and it was activated only through the caspase cascade occurring in the cytoplasm.
Figure 3. Microscopic images of HeLa cells after uptake of DEVDAuNP and induction of apoptosis with TRAIL. (A) DIC and NIRF (Cy5.5) microscopic images of nonapoptotic (no TRAIL) and apoptotic (+TRAIL, +TRAIL+inh) cells treated with DEVD-AuNP. (B) DIC and fluorescence images of DEVD-AuNP treated apoptotic cells after DAPI staining (C) Dark-field images of apoptotic cell. (D) AnnexinV/FITC treated apoptotic HeLa cells before (no trail) and after (+TRAIL) treatment of TRAIL.
To show the feasibility of DEVD-AuNP for the rapid visualization and real-time detection of early apoptosis, the same experimental condition was applied to Annexin V assay (Figure 3D). The commercially available Annexin V-FITC assay was treated after induction of apoptosis in HeLa cells, but the realtime imaging was not possible because the cells should be fixed before treatment of annexin V. Even before the TRAIL was treated, a faint background fluorescenct signal of FITC was observed. After 30 min of TRAIL treatment, the morphology of the cells was slightly changed but a strong FITC signal was not detected. Strong fluorescence of FITC was seen after 60 min of TRAIL when some cells already formed apoptotic bodies. A strong FITC signal was emitted exclusively from them, which was clear 120 min after TRAIL treatment when all of the HeLa cells died from apoptotic cell death. The morphology of FITC fluorescence from the apoptotic bodies was different from that of DEVD-AuNP in Figure 3A of apoptotic HeLa cells. The NIRF of DEVD-AuNP emitted from the inside of the cell due to the uptake by HeLa cells, whereas fluorescence of annexin V was concentrated around the cell membrane because annexin V is combined with phosphatidylserine, which was exposed to the external cellular environment in apoptosis. In conclusion, the apoptosis was successfully monitored in live cells with a molecular optical imaging technique using AuNP-based NIRF probe. It allowed rapid visualization and
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detection of early apoptosis, which was not possible through the conventional apoptosis kits such as Annexin V-FITC. Moreover, the real-time monitoring of caspase activity was enabled because the fluorescence of DEVD-AuNP was activated only by caspase enzyme. In light of its simple, inexpensive, and efficient properties for an apoptosis imaging probe, DEVDAuNP has great potential in biomedical applications.
ACKNOWLEDGMENT This research was financially supported by the Real-Time Molecular Imaging Project, GRL Program, M.D.-Ph.D. Program (2010-0019863, 2010-0019864) of MEST, and by a grant (A062254) of the Korea Health 21 R&D Project. Supporting Information Available: Experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.
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