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To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificia...
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Artificial Cell Membrane-Covered Nanoparticles Embedding Quantum Dots as Stable and Highly Sensitive Fluorescence Bioimaging Probes Yusuke Goto,† Ryosuke Matsuno,†,‡ Tomohiro Konno,†,‡ Madoka Takai,†,‡ and Kazuhiko Ishihara*,†,‡,§ Department of Materials Engineering and Department of Bioengineering, School of Engineering, and Center for NanoBio Integration, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8586, Japan Received July 22, 2008; Revised Manuscript Received September 22, 2008

To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificial cell membrane. These nanoparticles were designed by assembling phospholipid polar groups as a platform, and oligopeptide was immobilized as a bioaffinity moiety on the surface of the nanoparticles. The polymer nanoparticles showed resistance to cellular uptake from HeLa cells owing to the nature of the phosphorylcholine groups. When arginine octapeptide was immobilized at the surface of the nanoparticles, they were able to penetrate the membrane of HeLa cells effectively. Cytotoxicty of the nanoparticles was not observed even after immobilization of oligopeptide. Thus, we obtained stable fluorescent polymer nanoparticles covered with an artificial cell membrane, which are useful as an excellent bioimaging probe and as a novel evaluation tool for oligopeptide functions in the target cells.

Introduction Great attention has been paid to bioimaging technologies in the fields of medicine, pharmacology and biology. Fluorescence imaging with an adaptable fluorescence probe is widely used in life sciences because of its noninvasibility. Organic fluorescence molecules are easy to use, relatively inexpensive, and are capable of labeling cells in culture for a short time. However, organic dyes may have a photobleaching effect over time and lose fluorescence with concentration due to energy transfer among them. Therefore, they are inadequate for use in labeling cells for a substantial amount of time. In addition, nanometersized probes are very important for analyzing cell functions and the activity of biological molecules in the life sciences. Semiconductor nanocrystallites (quantum dots or QDs) have gained much interest as a promising alternative to organic dyes for biological imaging. QDs ranging in size between 2 and 6 nm have unique optical properties: material- and size-dependent emission spectra, a wide absorption spectrum, high quantum yields, simultaneous multicolor emissions, and especially excellent resistance to photobleaching.1,2 This photostability is a critical feature in most fluorescence applications, particularly for long-term monitoring of labeled substances, and is an area in which QDs have a singular advantage over organic dyes. As QDs themselves are hydrophobic, the key to developing QDs as a tool in biological systems is to achieve good dispersion ability in an aqueous medium, and compatibility with biological components including cells. In developing biofunctionalized QDs, researchers have employed various polymers and molecular assemblies for the encapsulation of QDs, such as amphiphilic block copolymers,3,4 a cross-linked polymer gel * To whom all correspondence should be addressed. Tel.: +81-3-58417124. Fax :+81-3-5841-8647. E-mail:[email protected]. † Department of Materials Engineering. ‡ Center for NanoBio Integration. § Department of Bioengineering.

matrix,5 polymer beads,6,7 or phospholipid liposomal vehicles.8 In spite of numerous efforts, the problems of cytotoxicity and the nonselective cellular uptake of QDs remain. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers have been synthesized by free radical copolymerization as biocompatible materials.9-14 The MPC polymers have found a number of biomedical applications 15-18 because they can provide an artificial cell membrane through coating, blending with other polymers, and grafting to the polymer substrates.11-13 By controlling the composition of the MPC units in the polymer and the molecular weight of the polymer, water-soluble MPC polymers, including poly(MPC-co-n-butyl methacrylate (BMA)co-ω-methacryloyloxy poly(ethylene oxide) oxycarbonyl 4-nitrophenol (MEONP) (PMBN), could be synthesized.19-22 The PMBN can suspend hydrophobic compounds solubilized well in an aqueous medium due to its amphiphilic nature and can immobilize with biomolecules covalently under very mild conditions due to active ester groups in the MEONP units. We have reported the preparation of artificial cell membrane-covered nanoparticles using amphiphilic PMBN and water-insoluble polymers such as poly(L-lactic acid) (PLA) and polystyrene as a core material and their applications in various biological fields.20,23-27 These nanoparticles are easy to produce and show the bioinert abilities to suppress nonspecific protein adsorption,23,27 to avoid phagocytosis from macrophage-like cells,15 and to sustain the activity of antibodies immobilized onto their surfaces.27 That is, the phosphorylcholine group coverings should reduce nonspecific uptake from each cell, and create a specific affinity by ligand molecules immobilized on the surface. Additionally, tremendous possibilities remain to gain various organic-inorganic hybrid nanocomposites by incorporating hydrophobic substances inside the PLA matrix. Thus, PMBN, PLA, and QDs are the best candidate-materials for preparing tools to overcome the above-mentioned problems. We report on the fabrication of polymer nanoparticles containing QDs (PMBN/PLA/QD, Figure 1) that have been

10.1021/bm800819r CCC: $40.75  2008 American Chemical Society Published on Web 10/09/2008

Fluorescence Bioimaging Probes

Figure 1. Schematic concept of PMBN/PLA/QD.

Figure 2. Chemical structure of poly(MPC-co-BMA-co-MEONP)(PMBN).

covered with an artificial cell membrane by a solvent evaporation technique that is simple and inexpensive. This method has been described previously, but we extend it by incorporating QDs inside the polymer nanoparticles. The ability of such PMBN/PLA/QD to protect against nonselective cellular uptake was tested in vitro on cultured HeLa cells. The PMBN/PLA/ QD immobilized with octaarginine (R8) was used as a model to investigate cell membrane permeation. The transfer of R8immobilized PMBN/PLA/QD (R8-PMBN/PLA/QD) into the HeLa cells was assessed by confocal microscopy. Cytotoxicity of PMBN/PLA/QD was also evaluated. To our knowledge, this is the first report regarding the preparation of polymerencapsulated QDs with excellent water-dispersion ability in an aqueous medium, as well as biocompatibility and cell-specificity.

Experimental Section Materials. The trioctylphoshine oxide (TOPO)-coated ZnS-shell CdSe-core QDs dispersed in toluene were purchased from Evident Technologies Co, Ltd. (Troy, U.S.A.). The specific fluorescence emission wavelength of these QDs was 543 nm, as a commercial value. Here, the core diameter of the QDs was 2.4 nm, as a commercial value. MPC was provided by NOF Co. Ltd. (Tokyo, Japan), and was synthesized by the previously reported method.9 BMA was purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and purified under reduced pressure; the fraction at 63 °C/24 mmHg was used. MEONP was synthesized by a previously reported method.21 2,2′-Azobisisobutyronitrile (AIBN) was purchased from Kanto Chemical Co., Ltd. (Tokyo, Japan). PLA (average molecular weight: 2.0 × 104) was purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). R8 was synthesized by solid phase peptide synthesis. Glycine was purchased from Sigma-Aldrich, Corp. (St. Louis, MO). Cell Counting Kit-8 (WST-8 assay) was purchased from Dojindo Molecular Technologies (Tokyo, Japan). Methods. Preparation of PMBN/PLA/QD. The PMBN/PLA/QD were prepared as follows. The PMBN (Figure 2) was synthesized by a conventional radical copolymerization of corresponding monomers using a previously reported method.21 An aqueous solution (20 mL) containing 200 mg of the PMBN (10 mg/mL) was placed in a glass bottle, and the solution was stirred at 400 rpm while being cooled in an ice bath. A total of 100 µL of TOPO-coated ZnS-shell CdSe-core QDs (the original QDs) suspended in toluene were dried under a vacuum. The remaining solidified QDs were suspended again in 1 mL of dichloromethane with 0.1 mg/mL PLA. The suspension was then added to the aqueous PMBN solution. This mixture was sonicated using

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a probe-type sonicator for 30 min under cool conditions and then it was maintained under reduced pressure for 20 min to evaporate the dichloromethane. Because the solution contained both the formed PMBN/PLA/QD and the remaining PMBN after evaporation, the nanoparticles in aqueous medium were ultracentifuged at 50000 rpm for 2 h. The supernatant was discarded and the PMBN/PLA/QDs as precipitate were resuspended in water. Characterization of PMBN/PLA/QD. Purification of PMBN/PLA/ QD was confirmed using gel permeation chromatography (GPC; Jasco, Tokyo, Japan) using a PU-2000 plus pump, a UV detector UV-2057 plus (Jasco), a refractive index detector RI-2031 plus (Jasco), and a fluorescence detector FP-2025 plus (Jasco) with a column (Shodex SB804 HQ, Showa Denko, Tokyo, Japan) under a flow rate of 0.1 mL/ min. As an eluent, 0.1 M LiBr aqueous solution was used. Purified PMBN/PLA/QD was stored at 4 °C. The morphology and the average diameter of the PMBN-PLA-QD were determined using atomic force microscopy (AFM; Nanoscope IIIa; Veeco, Tokyo, Japan) and dynamic light scattering (DLS; Zetasizer NanoZS; Malvern, Ltd., Worcestershire, U.K.). The existence of MPC units on the PMBN-PLA-QD surface was confirmed by X-ray photoelectron spectroscopy (XPS; AXIS-His; Shimadzu/Kratos, Kyoto, Japan). The takeoff angle of the photoelectrons was 90°. Incorporation of quantum dots into phospholipid polymer nanoparticles was confirmed using transmission electron microscopy (TEM; H-800; Hitachi, Tokyo, Japan). A small drop of PMBN-PLA-QD solution was placed on a copper grid (100 meshes) with carbon films and the excess solution was evaporated under vacuum. The optical properties of PMBN-PLAQD were measured by fluorescence spectrometer (FP-6500; Jasco, Tokyo, Japan) and UV/visible spectrometer (V-650; Jasco, Tokyo, Japan). The QD concentration was calculated using the Lambert-Beer law with mole absorption coefficient:  ) 7.2 × 104 M-1cm-1 at 525 nm as a commercial value. For evaluation of optical properties in aqueous solution after solubilization, 100 µL of TOPO-coated QD in toluene was diluted with toluene to make a standard solution. Bioconjugation of R8 Peptide to PMBN/PLA/QDs for Assessing Cell Penetration. The R8 was dissolved in PBS to prepare a 1 µM solution. After mixing 1 mL of the PMBN/PLA/QD solution (2 µM) and 1 mL of the R8 solution, the resulting solution was stored for 24 h at 4 °C. As a control, the active ester groups on PMBN/PLA/QD for bioconjugation were blocked with a glycine (3 mg/mL) aqueous solution to avoid reaction with the proteins in the cellular environment. Before dosing with these QDs, the QDs were put through a filter with a 0.45 µm pore diameter. Cell Culture, QD Treatment, Cellular Imaging, and Cytotoxicity Test. HeLa cells were grown at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (D-MEM; Gibco, Invitrogen, U.S.A.), supplemented with 10% fetal bovine serum (Gibco, Invitrogen, U.S.A.) and were passaged twice before the start of experiments. For confocal microscopy, HeLa cells were seeded into 3 cm glass bottom culture dishes at 6.0 × 104 cells/mL in 1 mL of culture medium and were grown to 40-60% confluency before QD treatment. The medium in the 3 cm glass bottom culture dishes was replaced with 1 mL of D-MEM containing 100 nM R8-conjugated-PMBN-PLA-QD or glycine-masked PMBN-PLA-QD and incubated for 24 h. After 24 h of dosing with QDs, the 3 cm culture dishes were rinsed three times with 1 mL of PBS and the medium was replaced with 1 mL of prewarmed PBS. QDs were imaged in HeLa cells treated by formalin using a laser scanning confocal microscope (LSM-510; Carl Zeiss, Oberkochen, Germany) with a 60× objective. QDs were excited with a 488 nm Ar laser line with filter-based emission channels of 520-550 nm. ConfocalDIC images of HeLa cells were obtained by excitation with a 633 nm HeNe laser line. Images were captured using Carl Zeiss software. The full cellular thickness (approximately 25 mm) was scanned in 0.3 µm steps. Uptake of QDs by HeLa cells was analyzed using a fluorescence microscope (IX71; Olympus, Tokyo, Japan) equipped with a 20× objective lens using Hg lamp as a light source. Fluorescence was collected by the same objective, passed through a filter ranging from 510 to 550 nm.

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Figure 3. Optical properties of PMBN/PLA/QD in PBS. PMBN/PLA/QD (solid) and original QD (broken) were measured by UV-vis spectrometer (left) and fluorescence spectrometer (right).

A WST-8 assay was used to assess cell viability after 24, 48, or 72 h of QD treatment. WST-8 assay is based on the cleavage of the tetrazolium salt to formazan by cellular mitochondrial dehydrogenase. Bioreduction of WST-8 produces a water-soluble formazan, which eliminates the need for an additional solubilization step. The amount of formazan produced is directly proportional to the number of living cells. The detection sensitivity of cell proliferation assays using WST-8 is higher than assays using the other tetrazolium salts such as MTT. A brief explanation of WST-8 assay is as follows. HeLa cells were seeded into 96-well plates at 1.0 × 104 cell/well in 100 µL of culture medium. After incubation of 24 h, the medium in the 96-well plates was replaced with 100 µL of QD-containing medium. The final QD concentration was 100 nM. QDs in culture medium were incubated with cells for 24, 48, or 72 h. After incubation, 10 µL of WST-8 reagent was added to all cell culture mediums. HeLa cells were returned to the incubator for 3 h. At the end of the incubation period, the microplates were read at 450 nm in a multilabel counter (Wallac ARVO SX 1420; PerkineElmer Inc., Waltham, U.S.A.). A total of 100 µL of D-MEM with cells and 100 µL of D-MEM without cells were used as the controls and background controls, respectively. Wells with 100 µL of D-MEM and QDs were as the background controls for the QD-treated samples. The average background absorbance from background control wells was subtracted from WST-8 treated samples and the data were plotted as a percentage of the appropriate control (100 µL of D-MEM with cells). The values of each sample were in the linear region of the standard curve of WST-8 assay. Eight replicates per data point were used for all treatments. Data are expressed as the means and SEs of three experiments.

Figure 4. Results of photobleaching tests of PMBN/PLA/QD (solid) and fluorescein-5-isothiocyanate (broken).

Results and Discussion Characterization of PMBN/PLA/QD. The PMBN/PLA/QD obtained has good dispersion ability in water and phosphate buffered saline (PBS, pH 7.4), and has maintained high fluorescence. The absorption and emission spectra were very similar to those of uncoated commercial QDs in toluene (Figure 3). We confirmed that the encapsulation of QDs provided extended stability, and we observed no change in solubility or fluorescence over one year (stored at 4 °C). These indicate that our PMBN/PLA/QD does not degrade at least one year. We also tested whether these encapsulations are stable at various pHs that may be found in the different cellular environments required in bioconjugation chemistries. It was found that the PMBN/PLA/QD is stable between pH 4.0 and pH 9.0. The results of a photobleaching test depicted in Figure 4 demonstrate that PMBN/PLA/QD was much more resistant to photobleaching compared with the typical organic dye, fluoresciene isothiocyanate. Overall, the encapsulation of QDs using PMBN and PLA did not have any influence on the optical properties of QDs. Next, the physical characterization of PMBN/PLA/QD was carried out. The results of the DLS measurements shown in Figure 5 shows that the hydrodynamic diameter of PMBN/PLA/

Figure 5. Hydrodynamic size revealed by the DLS measurement.

QD was around 20 nm, and the size distribution was small enough to use. As shown in Figure 6, atomic force microscopy (AFM) demonstrated that the morphology of PMBN/PLA/QD was spherical. The size by AFM observation was consistent with the hydrodynamic diameter determined by DLS measurement. Furthermore, a transmission electron microscope (TEM) image showed that several groups of QDs, appearing as dark spherically shaped objects due to the electron-dense QDs, were encapsulated within a single polymer nanoparticle (Figure 7). X-ray photoelectron spectroscopy (XPS) analysis indicated that the PMBN/PLA/QD had a specific signal at 403 eV, as shown in Figure 8. This signal was attributed to nitrogen from the ammonium group in the phosphorylcholine group of the MPC unit. This result revealed that the surface of PMBN/PLA/QD was covered with phosphorylcholine groups. Several lines of evidence indicated clearly that the QDs were completely included in the PLA core and that the nanoparticles were covered with the MPC polymer.

Fluorescence Bioimaging Probes

Figure 6. Morphology of PMBN/PLA/QD analyzed by AFM.

Figure 7. TEM image of PMBN/PLA/QD. Each broken line circle size is 20 nm.

Figure 8. XPS spectra of PMBN/PLA/QD (solid) and original QD (broken).

Hydrophobic interaction among materials plays a key role in the mechanism by which PMBN, PLA, and QDs can form into nanoparticles. PMBN could form polymer aggregates in an aqueous medium at concentrations greater than 0.1 mg/mL due to its amphiphilic nature.27 The core polymer PLA and QDs, which were dissolved in dichloromethane, are insoluble in water. Droplets of the PLA and QD mixture in dichloromethane were suspended in an aqueous solution with the PMBN aggregate. When evaporating the dichloromethane, hydrophobic PLA chains were precipitated with QDs at the interface of the aqueous medium. At the interface, the PMBN chains may form entanglements with the PLA chains. Thus, a stable PMBN layer was formed on the surface of the PLA core. That is, we were able to construct a phosphorylcholine platform with oligopeptide binding based on the MEONP units at the surface of the nanoparticles. Ability of PMBN/PLA/QD to Protect against Nonselective Cellular Uptake. One of the issues preventing a full unveiling of the functions of biomolecules is nonselective

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cellular uptake of the supporting materials for analysis, such as fluorescent nanoparticles or fluorescent proteins. Prediction of the exact locations of biomolecules is complicated by nonselective cellular uptake. Thus, there is a critical need to fabricate biocompatible materials, which have the abilities to protect against nonselective cellular uptake and to be traced constantly by noninvasive imaging technology. The ability of PMBN/PLA/QD to protect against nonselective cellular uptake was assessed using HeLa cells in vitro system. To avoid chemical reactions between active ester groups in the MEONP units and proteins in the culture medium, glycine, a simple amino acid, was immobilized to active ester groups on the surface of the PMBN/PLA/QD (glycine-PMBN/PLA/QD). A fluorescence image of the HeLa cells incubated with glycinePMBN/PLA/QD is shown in Figure 9, at top. However, we could not observe any fluorescence from the cells (Figure 9b, top). We also confirmed that the internalization of glycinePMBN/PLA/QDs was not observed even when they adapted to macrophage-like cells, J774.1 cells (data not shown). These results demonstrated that the PMBN/PLA/QD could protect against a nonspecific cellular uptake. This is in good agreement with the previously reported results using MPC polymer-coated polystyrene nanoparticles,15 and poly(MPC)-grafted QDs.28 Although nanoparticles with a diameter less than about 200 nm cannot avoid cellular uptake by the well-known endocyosis mechanism, the PMBN/PLA/QD was able to suppress cellular uptake regardless of its small hydrodynamic diameter. Cell Penetrating Ability of R8-immobilized PMBN/ PLA/QD. Subsequently, octarginine (R8) was immobilized to the PMBN/PLA/QD (R8-PMBN/PLA/QD) for evaluation of its cell penetrating ability. R8 is well-known as cell membrane penetration enhancer. It has the ability to translocate through cell membranes in a manner that does not involve the typical endocytic pathways of internalization.30,31 A fluorescence microscopy image of HeLa cells that had been incubated with R8-PMBN/PLA/QD (green-emitting) is depicted in Figure 9b, bottom. The R8-PMBN/PLA/QD clearly was associated with the HeLa cells and was found internalized at a perinuclear location. This assessment is consistent with the results of a confocal laser scan microscopy as shown in the movie S1 (see Supporting Information). Both microscopy scans (Figure 9b, top and bottom) were made with the same settings for laser power and photomultiplier sensitivity, allowing a direct comparison. For clarity, the corresponding phase contrast images are depicted in Figure 9c. The cell density for both incubations is comparable, and thus the difference in fluorescence intensity arose from the difference in the level of association of PMBN/ PLA/QD. Preliminary experiments were done using other amino acid and octapeptide sequences to confirm the selectivity of uptake to cells. However, we observed a small amount of cellular uptake only in the cases of octapeptide of glutamic acid (E8) and asparagine (N8). Other octapeptides of tyrosine (Y8) and histidine (H8) were not effective in causing uptake to the cells. The results indicated that R8 could function as a cellpenetrating peptide even in close proximity to phosphorylcholine groups, which reduce a translocation into cytosol. Thus, R8PMBN/PLA/QD is a most suitable material for conducting the kinetic analysis of cell membrane permeation. Cytotoxicity of PMBN/PLA/QD. Finally, we investigated the cytotoxicity of PMBN/PLA/QD. The use of QDs in biological cells always poses concerns about potential cytotoxicity. The unmodified TOPO-coated QDs are toxic in live cells,32,33 probably due to the release of Cd2+ or Se2- ions into the cell as the result of poor surface coverage. Proper modifica-

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Figure 9. Flureoscence microscopic images of HeLa cells incubated with glycine-PMBN/PLA/QD (upper) and R8-PMBN/PLA/QD (bottom) for 24 h: (a) phase contrast image, (b) fluorescence image, and (c) the merged images, respectively.

In conclusion, we have described the fabrication of polymer nanoparticles with embedded QDs in the core, covered with an artificial cell membrane. The optical properties of QDs were unchanged after incorporation of them into the polymer nanoparticles. Although the nanoparticles were fundamentally inert against nonselective cellular uptake from cells, when bioactive molecules were immobilized, the nanoparticles showed excellent affinity to the cells. From these findings, we conclude that the nanoparticles are candidates for the role of stable and highly sensitive fluorescence bioimaging probes in the field of biotechnology. Figure 10. Cytotoxicity of PMBN/PLA/QD with various peptide groups against HeLa cells after incubations of 24, 48, and 72 h.

tion, such as a poly(ethylene glycol), can reduce the cytotoxicity of QDs.34,35 However, immobilizing with specific biomolecules is difficult, and if a reactive group to biomolecules such as amino group in the one terminal of the polymer chain was introduced, the cytotoxicity reappeared.35 Figure 10 shows the cytotoxicity of three kinds of PMBN/PLA/QDs. The cell viability was almost 100%, and there was no significant difference from the control. We also confirmed that our QDs did not inhibit cell growth (Table S1, Supporting Information). In our experiments, the concentration of QDs (100 nM) was much higher than that of other studies (2∼20 nM). Nevertheless, none of the PMBN/ PLA/QD showed any cytotoxic effects, even after three days of incubation with live HeLa cells. This finding indicates that PMBN could survive having QDs completely inside the PLA matrix and suggests that MPC polymer coating is an effective way to make biocompatible QDs.

Conclusion Our results imply that the PMBN and PLA can integrate any kind of hydrophobic nanoparticles whose size matches the space. Nanocrystal-based organic-inorganic hybrid materials with exceptional properties have been explored in recent years because of their applications in nanobiotechnology.36,37 The incorporation of, for example, nanometer-sized magnetic38 or metallic particles39 will transform the abilities of nanoparticles and open up novel possibilities for manipulating them as individuals or in ensembles. These changes will have various applications for cellular targeting, targeted drug delivery, contrast agents, and so on.

Acknowledgment. We thank Dr. Fukumoto at the University of Tokyo for helpful discussions. Our synthesis of R8 was assisted by Dr. Yamaguchi and Prof. Nagamune at the University of Tokyo. Core Research for Evolutional Science and Technology (CREST), at the Japan Science and Technology Agency, supported part of this research. Supporting Information Available. The movie of confocal laser scan microscopy of HeLa cells incubated with R8-PMBN/ PLA/QD and the data of cell numbers in the WST-8 experiments. This information is available free of charge via the Internet at http://pubs.acs.org.

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