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Enhanced Photoacoustic Signal of Passion FruitLike Nano-Architectures in Biological Environment Cinzia Avigo, Domenico Cassano, Claudia Kusmic, Valerio Voliani, and Luca Menichetti J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b11799 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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The Journal of Physical Chemistry

Enhanced Photoacoustic Signal of Passion FruitLike Nano-Architectures in Biological Environment Cinzia Avigoa‡, Domenico Cassanob, c, Claudia Kusmica, Valerio Volianib‡*, Luca Menichettia, d* a Institute of Clinical Physiology, National Research Council, Via G. Moruzzi, 1 – 56124, Pisa, Italy. b Center for Nanotechnology Innovation@NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro, 12 - 56126, Pisa, Italy. c NEST-Scuola Normale Superiore, Piazza San Silvestro, 12 - 56126, Pisa, Italy. d Fondazione CNR/Regione Toscana G. Monasterio, via G. Moruzzi, 1- 56124, Pisa, Italy.

ABSTRACT

PhotoAcoustic Imaging (PAI) is a promising non-invasive technique for molecular and cellular characterization of cancer. Cancer detection by PAI is an area of active research, and the recent advent of contrast agents based on near infrared (NIR)-absorbing dyes or organic/inorganic nanoparticles tremendously improved the specificity and the signal contrast.

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Here, we report the PhotoAcoustic (PA) signal enhancement produced by biodegradable passion fruit-like nano-architectures in phantoms and ex vivo. By this approach, the synergistic interaction between commercial NIR-fluorophores and ultra-small metal nanoparticles is exploited to produce a PA signal enhancement in the biological window. Moreover, the degradation of the nano-architectures is investigated in physiological environment as reference matrix.

MAIN TEXT The development of non-invasive techniques for diagnostic purposes is a crucial step toward the reduction of patient’s pain, health risks and social costs. In this context, PhotoAcoustic Imaging (PAI) plays a pivotal role, allowing to obtain quick in vivo morphological, functional, and molecular information.1–5 Exogenous contrast agents are generally used in order to improve the spatial resolution and the imaging depth limit of PA images of tissues and organs.6,7 The most common agents are dyes or gold nanoparticles (GNPs). The first usually demonstrate a fast clearance by the renal pathway, but suffer from low specificity and poor PA signal.8–13 On the other hand, GNPs are very promising PA contrast agents because of their tunable Localized Surface Plasmon Resonance (LSPR) in the biological windows and strong extinction coefficient.14–18 However, their average size is usually above 20 nm,19 that leads to severe clearance issues, with accumulation of NPs in excretory organs and potential long-term toxicity.20–23 In order to overcome the issues of dyes and GNPs, many research groups recently developed innovative organic nanomaterials for PAI by various and elegant approaches, producing for example porphyrin nanoparticles,24–26 semiconducting polymers,3 melanin nanoparticles,27 or


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carbon nanotubes.28 However, in these organic nanomaterials are usually not included metal nanoparticles, missing their optical and synergistic features.29 In order to combine the intriguing behavior of metal nanoparticles with an increased organism excretion by the renal pathway,20,21 we have introduced a passion fruit-like biodegradable nanoarchitecture composed by 100 nm hollow silica nanospheres embedding arrays of 3 nm GNPs in polymeric matrices.30 In these nanocapsules the silica shell both protects the material in the inner cavity from the environment and provides an easily modifiable/functionalizable surface. GNPs confer the physical behavior needed for imaging applications, while the cationic polymer is covalently functionalizable with active molecules.31 Here, we report the PA signal produced by passion fruit-like nano-architectures in different media when a commercial NIR-dye is inserted in the inner cavity of the nanostructures. In particular, we observed that the PA signal is one-order of magnitude enhanced with respect to the one produced by commercial NIR-dyes. Thus, we have demonstrated a novel paradigm for production of exogenous PA probes. Indeed, the produced PA signal enhancement in the biological window is related to the synergistic interaction between NIR-dyes and ultra-small metal nanoparticles. Moreover, the degradation of the nano-architectures in human plasma is investigated by PAI, and results in a PA signal decrease in 32h.

Results and discussion


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Figure 1A shows the synthetic approach for the production of passion fruit-like nanoprobes (AuSiA). Briefly, poly(L-lysine) 15-30 kDa (PL) was covalently functionalized with AlexaFluor750-NHS ester on about 5% of amines and used for the synthesis of nanocapsules.30,31 Ultra-small gold nanoparticles showing a diameter of 2.8±0.4 nm (Figure S1) and coated by poly(sodium 4-styrene sulfonate) (PSS) were aggregated in spherical arrays by dye-modified poly(L-lysine) (PLa) by means of ionic interactions.

Figure 1. A) General scheme of the AuSiA synthesis: negatively charged gold nanoparticles are assembled in spherical arrays employing dye-modified poly(L-lysine). The dye (red clouds) is AlexaFluor-750. The arrays are then embedded in hollow silica shells employing a modified Stöber method. B) TEM images of AuSiA (left) and MFSiA (control without gold nanoparticles, right). Scalebar 40 nm. C) UV-vis spectra in PBS buffer of AuSiA (red) and MFSiA (blue).


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Silica shell was finally grown on this template by a modified Stöber method, resulting in nanoarchitectures of 97.6 ± 8.8 nm in diameter and 20.2 ± 1.6 nm of wall thickness (Figure 1B and S2). As control samples, nano-architectures (MFSiA) composed by hollow silica nanospheres not embedding gold nanoparticles were synthesized with the same protocol of AuSiA but replacing the gold nanoparticles arrays with polymeric arrays composed by PLa and PSS. The dye AlexaFluor750 (AF750) was inserted in the nano-architectures because of its relatively high molar extinction coefficient, low quantum yield, and narrow spectral bands.32 The intrinsic modularity of the nano-architectures results in easily bioconjugation to PL, in order to produce versatile nanomaterials with peculiar features for specific applications. In Figure 1C the optical behavior in phosphate buffer solution (PBS) of AuSiA and MFSiA are reported. The UV-vis absorbance spectrum of AuSiA shows a typical Rayleigh scattering background due to the silica shells including the absorption band of AF750 and the LSPR at around 540 nm of gold nanoparticles arrays. As expected, the LSPR is not present in the UV-vis spectrum of MFSiA. Photoacoustic measurements were performed by a commercial PAI system VevoLAZR from VisualSonics Inc. together with custom made cylindrical agar phantom with the inserted polyethylene tubes filled with different nano-architecture solutions (Figure 2A). The PA spectra were collected from PBS solutions of AF750, AuSiA, and MFSiA at the same final concentration of dye (Figure 2B and S3). The shape of the spectra is the same for all the samples, with a signal peak at about 760 nm. Only AF750 presents a hypsochromic shift of the PA band by decreasing its concentration (Figure S3). This behavior is related to the solvatochromism and concentration dependent absorbance of cyanines.33 Moreover, the PA signal of AuSiA, at the lowest measured


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dye concentration (2 µM), shows a contrast-to-background ratio well above one, contrary to the MFSiA sample (Table 1).

Figure 2. A) Experimental setup of the photoacoustic tests. The polyethylene tubes of the agar phantom were filled with solutions of samples at different concentrations and the PA signal was acquired with a specific probe equipped with an optical fiber bundle, and a 256 element RX linear array transducer, for the laser irradiation and the ultrasonic (US) response respectively. B) PA spectrum of AuSiA, MFSiA and AF750 in PBS solution. Final dye concentration 8µM for all the samples.

Table 1. Contrast-to-background ratio measured on the PA signal maximum of the dye in PBS solution.


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16 µM

8µM

4 µM

2 µM

AuSiA

/

21,57

9,37

3,79

AF750

2,82

1,85

1,46

/

MFSiA

2,76

1,18

0,63

0,42

The PA signal linearity with respect to dye concentration was verified for AuSiA, MFSiA, AF750 and Methylene Blue (MB) considering the PA signal amplitude at the wavelength of the signal peak (Figure S4). It is interesting to notice that the slope for AuSiA is one order of magnitude higher than that for MFSiA and AF750 and three order of magnitude higher than that of MB, that is considered as a reference dye for PA imaging. This suggests that the synergistic interaction of dye and gold nano-spheres results in an enhancement of the PA sensitivity. The AuSiA detection limit relative to this experimental setup, i.e. polyethylene tube of 1.05 mm2 section, calculated as three times the background noise, was estimated to be 0.5 µM dye concentration. These findings can be ascribed to the proximity of the dyes to the gold nanoparticle surface. Indeed, when dyes are next to a gold surface usually undergo to fluorescence quench effects, increasing non-radiative decays.34–36 In order to investigate this behavior, both AuSiA and MFSiA were treated with potassium cyanide (KCN). KCN is able to penetrate the silica shell and convert gold nanoparticles to a water-soluble aurocyanide coordination complex, [Au(CN)2]−, without affecting the structure of nano-architectures (Figures 3A and 3B for, respectively, AuSiA and MFSiA).37,38 As expected, the fluorescence emission band of the dye in AuSiA increased with time. This behavior is related to the dissolution of gold (Figure 3C), i.e. the quenching effect due to close proximity between AF750 and gold nanoparticles was lost. On the other hand, after the treatment with KCN, no


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effects were recorded on the fluorescence emission spectra of MFSiA (Figure 3C). Thus, the presence of gold nanoparticles in the nano-architectures is a key-requisite to induce peculiar optical features and enhancing PA signals.

Figure 3. TEM images of AuSiA and MFSiA treated with KCN (panel A and B, respectively) and their fluorescence emission (panel C) recorded by irradiation on the maximum of absorbance of the dye. Scalebar 50 nm. For a better comparison, the integral of the emission area of AuSiA (green triangle) and MFSiA (red triangle) are plotted on the right of panel C.

Moreover, a number of dyes or chromophores can be easily included in the nano-architectures, increasing the versatility of this system. It is worth to notice that the laser used for triggering these nano-architectures is on the absorbance maximum of the dye, 760 nm, far from the LSPR, 540 nm. Thus, this design allows to avoid nanoparticles reshaping and associates PA efficiency reduction due to the metal melting commonly related to the laser irradiation on the LSPR.39 Passion fruit-like nano-architectures undergo complete biodegradation in biological fluids and cellular environment to potentially renal clearable building blocks in less than 48h.30 Therefore, a decrease of the PA signal from AuSiA was expected during their degradation, due to the removal


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of the dye from the gold nanoparticles surface. PA spectra of AuSiA in human plasma were recorded at different times (Figure 4A) and compared, as reference, to MB (Figure 4B). During 32h the AuSiA/MB PA signal ratios decreased from 207% to 40%.

Figure 4. PA spectra of AuSiA (dye concentration 14 µM) in human plasma during 32h at 37 ˚C (A), the PA signal ratio between AuSiA and the reference dye (Methylene Blue, 4mM) (B), and representative TEM images of AuSiA during the experiment (C).

TEM images (Figure 4C) confirmed that the decrease in intensity of the PA response has to be ascribed to the degradation of the nano-architectures to their potentially renal clearable building blocks, i.e., silicic acid, PSS, PLa and ultra-small gold nanoparticles.20 Therefore, passion fruit-


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like nano-architectures behave as biodegradable PA probes, and PAI has demonstrated to be an effective modality to monitor their real-time biodegradation.

Figure 5. A) PA spectra of AuSiA in human blood (dye concentration 37 µM) and human blood at different time after the experimental setup preparation (at different oxygen saturation levels), without (red curves) and with (green curves) the TM-PDMS layer superimposed on the agar phantom. In table are reported the contrast-to-background ratio values measured at the PA peak of AuSiA or AuSiA/TMPDMS. B) Ex vivo PA spectrum of a mouse paw before (blue) and after (orange) the AuSiA bolus injection of AuSiA with a dye concentration of 11 µM.


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In Figure 5A are reported the PA acquisitions of AuSiA in whole human blood. The blood sample was acquired at different oxygen saturation levels (i.e. from venous at t=0 to arteriosus at t=40 minutes), representing typical patterns of the PA signal of biological health tissues. Initial PA spectrum of blood presents the specific pattern of the mixture of the de-oxygenated and the oxygenated hemoglobin, while after 40 minutes the shape of the spectrum is representative of the oxygenated hemoglobin. PA signal of AuSiA is always well-recognizable; also, if a tissuemimicking polydimethylsiloxane (TM-PDMS) layer is superimposed to the agar phantom in order to mimic a real tissue. The contrast-to-background ratio values are reported in Figure 5. These results demonstrate that the AuSiA can be detected by PAI in biological media, where the background signal is very high, due to the absorption of endogenous molecules such as hemoglobin, melanin, and lipids. It is interesting that PA signal from AuSiA is collectable also in a mixture of de-oxygenated and oxygenated hemoglobin, such as in highly hypoxic tissues, in which the de-oxygenated component of the blood is characterized by a PA peak at about 750 nm. It is worth to notice that the PA signal of the passion fruit-like nano-architectures is tunable over the biological windows reasonably by changing the dye covalently linked to PL. In order to investigate the PA signal in real biological environment, 50 µL of AuSiA with a dye concentration of 11 µM were injected in a paw of a healthy mouse ex vivo. The specific PA spectrum was acquired (Figure 5B) and the contrast-to-background ratio (measured on the PA signal peak) resulted to be well above the unit (≈ 2), supporting the use of the nano-architectures in vivo. Conclusion In summary, we demonstrated that biodegradable passion fruit-like nano-architectures have peculiar physical properties, among which the disassembling behavior, and can be detected by


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the emerging techniques of PAI through the enhancement effect realized by this composite architecture. This innovative paradigm employs the synergistic interaction between dyes/chromophores and ultra-small gold nanoparticles to obtain a collectable PA signal, and represents a relevant advantage for their use in biomedical applications. A novel family of PA probes is achievable by changing the organic molecules inside the cavity of the nanoarchitectures, demonstrating a high degree of versatility. Moreover, by including drugs in the cavity, these nanostructures might become highly attractive platforms for theranostics. In vivo experiments are hitherward ongoing.

Methods Nano-architectures (AuSiA and MFSiA) Synthesis of dye-modified poly(L-lysine) – PLa: 1.5 mg of poly(L-lysine) hydrobromide 15-30 kDa (PL) were dissolved in 780 μL of PBS and 200 μg of AlexaFluor750-NHS ester (10 mg/mL, DMSO solution) were added to the solution. The reaction mixture was kept under stirring overnight at RT, and it was used without further purification. By this approach, about 5% mol/mol of the amines of PL are functionalized by the dye. Synthesis of gold nanoparticles: ultra-small gold nanoparticles with a diameter of approximately 3 nm were prepared according to the following procedure. To 20 mL of milliQ water were added 10 μL of poly(sodium 4-styrene sulfonate) (30% aqueous solution, PSS) and 200 μL of HAuCl4 aqueous solution (10 mg/mL). During vigorously stirring, 200 μL of sodium borohydride (4 mg/mL in milliQ water) were added quickly, and the mixture was stirred vigorously for other 2-minute. After the addition of NaBH4, the solution underwent some color


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changes until becoming brilliant orange. Before its use the solution was aged for at least 30 minutes and employed without further purification. Synthesis of gold nanoparticles arrays: 1 mL of gold nanoparticles solution was added to a 2 mL plastic vial followed by 40 μL of the PBS solution of PLa, and the mixture was allowed to stir for 30-minute at room temperature. The as synthesized gold aggregates were collected by centrifugation (13400 rpm for 3-minutes), suspended in 100 μL of milliQ water and sonicated for maximum 4 minutes. Synthesis of nano architectures (AuSiA): In a 100 mL round bottomed flask was added 70 mL of absolute ethanol followed by 2.4 mL of ammonium hydroxide solution (30% in water), and 40 μL of tetraethyl orthosilicate (TEOS, 98%). The solution was allowed to stir for 20 minutes at RT. 2 mL of the gold nanoparticles arrays previously prepared were added to the reaction flask and the solution was allowed to stir for further 3h. The as-synthesized AuSiA nanoparticles were collected by 30-minute centrifugation at 4000 rpm, washed twice with ethanol to remove unreacted precursors and suspended in 1 mL of ethanol. A short spin centrifugation was employed in order to separate the structure over 150 nm from the supernatant, which was recovered as a pink- iridescent solution. The solution was centrifuged at 13400 rpm for 5 minutes, suspended in 500 μL milliQ water, sonicated for 5-minute and freeze-dried overnight. Usually, about 1.5 mg of a brilliant pink powder is obtained, which remains stable for at least 1 year if stored sealed in the dark at 10 ˚C. Synthesis of metal-free nano architectures (MFSiA): To 20 mL milliQ water were added, during ultrasonication, 10 µL of PSS and PLa (3 mg, 20 mg/mL PBS solution). After 1-minute of ultrasonication, the mixture was left under stirring for 15 minutes at RT and then centrifuged (13400 rpm for 3 minutes). The blue precipitate was recovered with 2 mL milliQ water and


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added to a 100 mL round bottomed flask containing a mixture of 70 mL of absolute ethanol, 2.4 mL of ammonium hydroxide (30% in water) and 40 µL of tetraethyl orthosilicate (TEOS, 98%). The mixture was allowed to stir for 3h at RT. The as-synthesized MFSiA were collected by 30minute centrifugation at 4000 rpm, washed twice with ethanol to remove unreacted precursors and suspended in 1 mL of ethanol. A short spin centrifugation was employed in order to separate the structure over 150 nm from the supernatant, which was recovered as a milky blue solution. The solution was centrifuged at 13400 rpm for 5 minutes, suspended in 500 µL milliQ water, sonicated for 5-minute and freeze-dried overnight. Usually, about 1.5 mg of a blue powder is obtained, which remains stable for at least 1 year if stored sealed in the dark at 10 °C. UV/Vis spectrophotometry Extinction spectra were collected by means of a double beam spectrophotometer Jasco V-550 UV/VIS equipped with quartz cuvettes of 1.5 mm path length and normalized to the maximum absorbance. PBS (1X) buffer was employed as solvent. Fluorescence spectrometry Fluorescence spectra were collected by means of a Cary Eclipse fluorimeter equipped with quartz cuvettes of 1.5 mm path length. Excitation and emission slits of 10 nm were employed and a photomultiplier voltage between 500 V and 700 V, depending on the concentration of the sample. All the samples were dispersed in PBS (1X) buffer before collecting the spectra. Gold dissolution experiments: AuSiA and MFSiA were suspended in 300 μL PBS buffer with a final AF750 concentration of 0.87 μM and 0.64 μM, respectively. At those concentrations, the intensities of the fluorescence emission spectra were similar for the two systems. Then, 5μl of KCN (10 mg/mL, PBS solution) were added and the fluorescence spectra of the two samples collected every 5 minutes for 45 minutes.


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Electron microscopy TEM observations of nanoparticles were carried out on a ZEISS Libra 120 TEM operating at an accelerating voltage of 120 kV, equipped with an in-column omega filter. The colloidal solutions were deposited on 300-mesh carbon-coated copper grids and observed after at least 5h. For biodegradation experiments in human plasma, TEM grids were washed with ethanol to remove the excess biological fluids before being imaged. Photoacoustic Imaging Experiments Photoacoustic measurements were performed with a commercial PAI system from VisualSonics Inc. (VevoLAZR, FUIJIFILM VisualSonics Inc., Toronto, ON, Canada) that produces PA images co-registered with B-mode images of the surrounding structures. A full description of the system design is available in reference.40 PA signals were excited by a pulsed (20 Hz, 6 - 8 ns pulse width) and tunable (680 to 970 nm) Nd:YAG laser with an optical parametric oscillator to illuminate the sample through two rectangular fiber-optic bundles placed on both sides of a linear array transducer (13 - 24 MHz, 23 mm × 30 mm field of view) at an angle of 30 deg with the imaging plane. The acquisition time for the absorption spectrum acquisitions was ~60 s for 146 wavelengths (2 nm step). For the photoacoustic tests a custom made 2% agar cylindrical phantom, with up to 5 polyethylene tubes inserted (PE 50, micro medical tubing, 0.58 mm I.D. X 0.99 mm O.D.) was used. In Figure 2A a scheme of the experimental setup is reported. A 3-mm layer of tissue mimicking material composed of polydimethylsiloxane (TM-PDMS) mixed with TiO2 (0.73 mg⁄mL) and India ink (0.25 mg⁄mL) was deposited on the phantom during some PA acquisitions. A full description of the tissue mimicking material design is available in reference.41


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Photoacoustic tests were performed with the agar phantom filled with: AlexaFluor-750 PBS solutions (16 µM, 8 µM and 4 µM), AuSiA PBS solutions (final dye concentrations: 8 µM, 4 µM and 2 µM) or MFSiA (nano-architectures without gold nanoparticles, final dye concentrations: 16 µM, 8 µM, 4 µM and 2 µM). PA spectra resulted from the average of 3 different measurements. The nanoparticle degradation in physiological environment was examined as follow. AuSiA in human plasma (final dye concentration 14 µM) was incubated at 37°C and stirred at 650 rpm for 32h. PA and transmission electron microscope (TEM) acquisitions of the sample were performed at 0h, 8h, 24h, 28h and 32h. PA spectra of Methylene Blue 4 mM were recorded as standard reference. PA spectra of AuSiA diluted in human blood with a final dye concentration of 37 µM were acquired up to 40 minutes after the preparation of the experimental setup, in order to collect signals with different levels of oxygen saturation. Moreover, the acquisition was performed both with and without the TM-PDMS layer superimposed on the agar phantom in order to simulate a blood vessel under a tissue layer. A bolus of 50 µL of AuSiA with a final dye concentration of 11 µM was injected into a mouse paw and the PA spectrum was acquired.

ASSOCIATED CONTENT Supporting Information. PA measurements and linearity, size-dispersion histograms. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Authors


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* [email protected] , [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interests. ‡These authors contributed equally. ACKNOWLEDGMENT We thank Dr. M. Gemmi, Dr. S. Luin and Prof. F. Beltram for their support. We would like to thank PhD – Ing. F. Faita and Ing. N. Di Lascio for their contribution to the image postprocessing. Project “INSIDE” POR CREO FESR 2014 – 2020 (CUP ST: 3389.30072014.067000001).

ABBREVIATIONS PAI, Photoacoustic Imaging; GNP, Gold Nano Particle; LSPR, Localized Surface Plasmon Resonance; AuSiA, passion fruit-like nano-architectures; PL, poly(L-lysine) 15-30 kDa; PSS, poly(sodium 4-styrene sulfonate); PLa, dye-modified poly(L-lysine); MFSiA, metal-free passion fruit-like nano-architectures; AF750, AlexaFluor750; PBS, Phosphate Buffer Solution; KCN, potassium cyanide; MB, Methylene Blue; TM-PDMS, tissue-mimicking polydimethylsiloxane; REFERENCES (1)

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