Versatile Bioorthogonal Hydrogel Platform by Catalyst-Free Visible

Jun 13, 2017 - However, bioorthogonal photoclick reactions generally requires two components for cross-linking and, in many cases, the formation of a ...
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Versatile Bioorthogonal Hydrogel Platform by Catalyst-Free Visible Light Initiated Photodimerization of Anthracene Vinh X. Truong,*,† Fanyi Li,†,‡ and John S. Forsythe*,† †

Department of Materials Science and Engineering, Monash Institute of Medical Engineering, Monash University, Clayton, 3800 VIC, Australia ‡ CSIRO Manufacturing, Bayview Avenue, Clayton, VIC 3168, Australia S Supporting Information *

ABSTRACT: Recent developments in photochemistry have introduced new methods to prepare hydrogels initiated by nonharmful light which is essential for encapsulation of cells and bioactive components. However, bioorthogonal photoclick reactions generally requires two components for cross-linking and, in many cases, the formation of a reactive intermediate that may cross-react with nucleophiles in biological media. Here we report the utilization of a visible light triggered dimerization of electron-rich anthracene for polymer cross-linking to form bulk hydrogels and microgels. Incorporation of gelatin within the hydrogel enhanced cell attachment and viability after 7 days of culture and spatiotemporal conjugation of a bioactive component using photochemical dimerization of anthracene was demonstrated. This work therefore introduces a simple yet powerful tool for light modulated bioorthogonal polymer crosslinking, which can be utilized in various bioengineering applications.

P

hotochemical cross-linking has been widely employed in the fabrication of hydrogels, providing excellent spatial and

Scheme 1. Standard Photochemical Dimerization of Anthracene and Structure of 4-Arm PEG Conjugated with Anthracene and Electron-Rich Derivatives

temporal control over the gelation process.1−3 In recent years, advances in photochemistry have enabled material scientists to utilize low energy long wavelength UV light and visible light to initiate the gelation process. This in turn allows hydrogel materials to be formed under physiological conditions without significantly affecting living cells, a bioorthogonal process that can be utilized for advanced biological studies. For instance, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) absorbs light in the 330−420 nm region to produce free radicals and has been used to initiate acrylate4 or thiol−ene polymerization5,6 under visible light irradiation, enabling rapid gelation and encapsulation of cells. Lin and co-workers © XXXX American Chemical Society

Figure 1. (A) UV−vis spectra of the polymers investigated; (B) Under irradiation of visible light (400−500 nm, 20 mW cm−1), rapid decrease of the absorbance was observed for polymer P2 (each scan was collected after 2 min of irradiation) and (C) polymer P3 (each scan was collected after 10 s of irradiation); (D) Fluorescent spectra of the polymer solutions after being treated with visible light.

Received: April 26, 2017 Accepted: June 5, 2017

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DOI: 10.1021/acsmacrolett.7b00312 ACS Macro Lett. 2017, 6, 657−662

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Figure 2. (A) Scheme of polymer cross-linking under visible light; Rheological analysis of gelation under visible light irradiation (20 mW cm−2) and a polymer concentration of 6 wt % (3 mM) for (B) polymer P3, (C) polymer P2, and (D) polymer P1, the red arrow indicates when the solution was irradiated; (E) Effect of light irradiation on P2 solution (3 mM), the polymerization was halted when light was turned off; (F) Storage moduli values of hydrogels from different concentrations of polymer P2 at complete gelation (after 15 min of visible light irradiation).

photodimerization reactions require harmful UV light (250− 350 nm) and high irradiation intensity, rendering this approach unsuitable for cell encapsulation applications. In addition, none of the gelation methods by photochemical dimerization have been used for functionalization of bioactive components essential for cell interactions and subsequent proliferation in biological studies. To further advance research in bioengineering applications, we seek to develop a bioorthogonal hydrogel system with simplified preparation procedures and synthetic flexibility that allows for facile presentation of bioactive components within the gel structure. It came to our attention recently that a red shift in the absorption of anthracene can be induced by placing an electron rich group at the 9-position of the anthracene ring.24,25 The light-triggered dimerization of anthracene follows the [4 + 4] photodimerization mechanism in which the photoexcited diene forms a short-life excimer that undergoes a transition into the cyclooctane structure (Scheme 1).26 We surmised that the visible light absorption properties of electronrich anthracene can enhance the dimerization process by photons in the visible wavelength and can be employed for benign polymer cross-linking in the preparation of a universal hydrogel system. Herein we report the preparation of a versatile hydrogel platform from a poly(ethylene glycol) (PEG) precursor containing the anthracene moiety, which can be cross-linked, for the first time, by low intensity visible light without any catalyst. The bio-orthogonality of the cross-linking and utility of the system were demonstrated by the encapsulation and extended 3D culture of human mesenchymal stem cells, the fabrication of autofluorescent microgels, as well as the ligation of a bioactive molecule postgelation. To investigate the photochemical properties of electron rich anthracene and the subsequent utilization of the photochemistry in hydrogel cross-linking, we synthesized three

employed Eosin-Y as a photoinitiator for the free radical mediated thiol-norbornene addition, which can be triggered by green light (520−550 nm).7,8 In addition to the utilization of light at nonharmful wavelengths, several strategies have been developed to initiate photo-cross-linking in the absence of catalyst. In particular, the group of Zhu reported the synthesis of photoresponsive caged thiol compounds such as thiolspiropyran9 and coumarinconjugated thiol.10 Upon exposure to UV light at 365 nm, these precursors liberate reactive thiols that can participate in the nucleophilic addition to the maleimide group for polymer cross-linking. In a different approach, the photosensitive nitrobenzyl moiety was conjugated to polymer end-groups which, under UV light irradiation, can be converted to either a ketone or an aldehyde group for subsequent hydrazine click addition11 or imine bond formation with amine groups,12 respectively. The catalyst-free photoligation of tetrazole-alkene was also reported in the fabrication of hydrogel for protein delivery.13 A common feature in these photoclick reactions, with and without catalyst, is the generation of a reactive intermediate which may cross-react with nucleophiles such as thiols and amines present in the biological media.14 This is generally overcome by careful selection of a clickable counterpart having high selectivity and fast reaction rate with the intermediate. Photodimerization of chromophores such as anthracene,15−17 coumarin,18−20 thymine,21 and cinnamylidene22,23 has also been employed in polymer cross-linking for the preparation of hydrogels. This cross-linking method is highly attractive because it not only excludes the use of catalyst, but also requires only one polymer precursor for light-triggered cross-linking, thus, simplifying the synthesis procedure and increasing the reproducibility of the hydrogel materials in terms of gelation time and mechanical properties. Nevertheless, most 658

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Figure 3. Images (z-stack with 3-D construct) of live/dead staining of (A) cell-laden PEG hydrogel from P2 polymer after 1 and 7 days of culture and (B) cell-laden PEG-gelatin hydrogel after 7 days of culture (hydrogels were prepared by irradiation of visible light at the intensity of 20 mW cm−1 for 10 min, live cell = green, dead cell = red, scale bar = 100 μm); (C) quantified data for live cells after 7 days of culture in PEG only and PEGgelatin hydrogels.

different PEG precursors containing anthracene (P1), triazole anthracene (P2), and benzyl triazole anthracene (P3; Scheme 1). The anthracene moiety can be conjugated to the PEGamine end-group via ester bond formation, while the electronrich anthracene group was prepared by copper(I) catalyzed alkyne−azide cycloaddition (CuAAC) of 9-alkyne anthracene and PEG-azide. The CuAAC click condition resulted in a mixture of 1,4- and 1,5-triazole adducts with the 1,4-/1,5- ratio of about 1/3 from 1H NMR analysis. Compared to P1, UV−vis spectra of P2 and P3 show a red shift extending into the visible light region (Figure 1A). Irradiation of P2 and P3 in aqueous solutions (2 μM) with visible light (400−500 nm, 20 mW cm−2) resulted in rapid consumption of the peak in the region above 400 nm as well as a decrease in the absorption of the other peaks in the UV region (Figure 1B,C), while no change in UV−vis absorbance was observed for P1 under visible light irradiation was seen (Figure S14). The rate of decrease in absorbance for polymer P3 solution is significantly faster than polymer P2 solution (assuming first order reaction kinetics, the dimerization rate constants for P2 and P3 are 9.61 × 10−4 s−1 and 3.12 × 10−3 s−1, respectively), which suggests that the presence of a benzyl substitute on the triazole ring enhances the reactivity of the anthracene group under visible light illumination. Furthermore, irradiation of the dimer product

with UV light at 365 nm did not cause any change in the resultant UV−vis spectra of both P2 and P3. The dimerization adducts also display strong UV fluorescent properties (Figure 1D; excitation at λex = 350 nm, λem = 425 nm for P2 dimer and λem = 459 nm for P3 dimer). Having established that the electron-rich anthracene group indeed undergoes rapid dimerization in water under visible light, we next utilized the photodimerization of the 4-arm PEG anthracene in cross-linking for preparation of catalyst-free visible light curing hydrogels (Figure 2A). Rheological analysis of the aqueous solution containing polymer P2 and P3 (Figure 2B,C) displayed a crossover of storage modulus (G′) and loss modulus (G″), characteristic of the gelation process, under illumination of visible light (400−500 nm) at an intensity of 20 mW cm−2, followed by a sharp increase in the G′ value. A faster rate of gelation was observed for the P3 solution, which corresponds to faster photodimerization observed in the UV− vis study. Complete gelation times, defined as the time point when the G′ value reached a plateau, for P3 and P2 were 8 and 15 min, respectively. This fast gelation time is comparable to the rate of cross-linking observed in free radical step growth thiol−ene addition,4 thus, presenting a catalyst free rapid crosslinking procedure for hydrogel fabrication. Consistent with UV−vis analysis, irradiation of the hydrogels with UV light 659

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Figure 4. Images of microgels as prepared in (A) oil and (B) after washing PBS solution; (C) fluorescent image of microgels; (D) fluorescent image of microgel after conjugation with biotin and treatment with TRITC-labelled streptavidin, (E) image showing that only microgel with conjugated biotin binds to TRITC-labeled streptavidin; (F) image (z-stack, with 3D construct) of live/dead staining of hMSCs encapsulation in PEG-gelatin microgel after 7 days of culture, * mark indicates the region of cell protrusion (scale bar = 100 μm).

(365 nm and at an intensity of 100 mW cm−2) did not cause any change in storage modulus. We conclude that long wavelength UV light is not sufficient to revert the anthracene dimer in aqueous environment for hydrogel degradation. This observation is in contrast with a previous study that reported photoscission of the anthracene dimer in CH2Cl2 or MeCN using UV light at 365 nm wavelength.24 Gelation was not observed for the P1 solution under prolonged irradiation with either visible light or UV light (at 365 nm wavelength and intensity up to 100 mW cm−2). In a previous report for the preparation of PEG-anthracene hydrogel with the same end-group as polymer P1, a UV light power of 100 W was required to initiate cross-linking.16 Such high power irradiation is not practical for cell culture as we observe significant cell death for hMSCs when exposed to UV light (365 nm) at an intensity higher than 20 mW cm−1 (Figure S17). Although the P3 solution forms a gel faster under visible light irradiation, synthesis of P2 is easier to achieve, that is, direct CuAAC coupling of the azide end group with alkyne anthracene and is therefore more desirable in materials fabrication. Thus, we carried out further characterization of hydrogel materials prepared from P2. The effect of visible light irradiation on the cross-linking of P2 in aqueous solution was further illustrated by switching the light on and off at various time points. As seen from Figure 2E, the storage modulus of the system can be fine-tuned by controlling the time of irradiation as turning off the light effectively stalls the polymerization process. Visible light

initiated cross-linking is highly efficient as we were able to prepare hydrogel from P2 solution with a polymer concentration of 2 wt % (1 mM) producing a storage modulus of 550 Pa at complete gelation. Increasing the polymer precursor concentration results in a higher concentration of the crosslinking groups and subsequently a higher resultant G′ value. Thus, the storage modulus of the gels after curing, which varies from 0.55 to 3.21 kPa, can be tuned by varying the concentration of the polymer (Figure 2F). In addition, we recorded high reproducibility of the final modulus of the gels in rheological measurement with the error (standard deviation ×2) within 1%. Because the dimer product does not absorb light in the 400−500 nm wavelength, cross-linking is highly efficient and we were able to prepare gels with a thickness of 5 mm having a storage modulus similar to the storage modulus of the hydrogel in the rheological tests (thickness of 0.3 mm). To demonstrate the efficacy of our hydrogel system in 3D cell culture, we prepared cell-laden hydrogels from polymer P2 (6 wt %) by encapsulation of hMCSs within the gel structure during light initiated cross-linking (10 min of irradiation with 400−500 nm light at 20 mW cm−2). Cell culture media was used for hydrogel preparation to enhance cell survival during the encapsulation process and the storage modulus of the gel prepared under this condition was recorded at 1.12 kPa (Figure S16). The hMSCs show high viability (92 ± 4%, Figure 3A) after 1 day postencapsulation, which clearly shows the visible light regulated-dimerization of triazole anthracene is bioorthogonal. This observed viability is comparable with the 660

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viability (90 ± 4%) reported for hMSC encapsulation in peptide conjugated PEG hydrogel using catalyst-free UV lightinduced hydrazine-aldehyde ligation.11 After 7 days culture, the cell viability decreased to 64 ± 8%. This is due to PEG not having inherent binding motifs for cell attachment and spreading. To present a bioactive component for cell attachment, we synthesized gelatin-triazole anthracene from gelatin-azide27 and 9-ethynylanthracene via CuAAC click reaction (see Supporting Information) and incorporated into the hydrogel structure during cell encapsulation process. The hMSCs in the gelatin-PEG hydrogel indeed displayed a higher cell viability (83% ± 1%) compared to PEG only hydrogel after 7 days of encapsulation (Figure 3B,C). Beyond the fabrication of dynamic and bioorthogonal hydrogel materials, we are interested in the preparation of bioorthogonal microgels for culture and delivery of therapeutic stem cells. Using an efficient and low cost microfluidic approach,6 we were able to fabricate microgels (from polymer P2) with uniform diameter (Figure 4A,B). These microgels are autofluorescent due to the presence of the anthracene dimer, which may be used for imaging and tracking of microgels in delivery applications (Figure 4C). Furthermore, we were able to postfunctionalize the microgel using a biotin-PEG3-triazole anthracene. The biotin-conjugated PEG microgel displayed very high binding affinity to TRITC-labeled streptavidin which can be visualized by fluorescent confocal microscopy (Figure 4D,E). Finally, the microgels composed of PEG and gelatin could be used to support hMSCs in a 3D environment with very high cell viability (87 ± 1%) after 7 days of culture and some cell protrusions were observed (Figure 4F). This result indicates that microgels are a better cell culture environment for hMSCs compared to the bulk hydrogel. In summary, we demonstrated that fast dimerization of anthracene under irradiation of low intensity visible light (400− 500 nm) in aqueous environment can be induced by placing an electron-rich substituent group on the anthracene, further demonstrating the utility of the reaction following recent demonstration of visible light induced surface modification.24 This chemistry was subsequently applied in rapid polymer cross-linking to form bioorthogonal hydrogels suitable for cell encapsulation. The versatility of this hydrogel system was demonstrated by the incorporation of bioactive gelatin for enhancement of cell attachment and viability and the fabrication of microgels, as well as postmodification with a biomacromolecule. Microgels prepared by this method also display autofluorescent properties, which may be used for monitoring particles in vivo. The simplicity in material preparation combined with the synthetic flexibility of our hydrogel system is highly attractive for 3D cell culture studies. We envisage that our hydrogel materials will be widely employed in various cell biology studies and possible medical applications such as delivery of cells and therapeutics.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

John S. Forsythe: 0000-0003-2849-229X Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

The research was supported under the Australian Research Council’s Discovery Projects funding scheme (DP160101591). F.L. acknowledges support from the New Horizons MonashCSIRO Council Joint Ph.D. Scholarship Program. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Francesca Ercole is acknowledged for assistance with the fluorospectrometry measurements. The authors acknowledge the facilities and scientific and technical assistance of Monash Micro Imaging at Monash University.

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ABBREVIATIONS PEG, poly(ethylene glycol); CuAAC, copper-catalyzed alkyne azide cycloaddition; hMSC, human mesenchymal stem cell REFERENCES

(1) Grim, J. C.; Marozas, I. A.; Anseth, K. S. Thiol-ene and photocleavage chemistry for controlled presentation of biomolecules in hydrogels. J. Controlled Release 2015, 219, 95−106. (2) Buwalda, S. J.; Vermonden, T.; Hennink, W. E. Hydrogels for Therapeutic Delivery: Current Developments and Future Directions. Biomacromolecules 2017, 18, 316−330. (3) Blasco, E.; Wegener, M.; Barner-Kowollik, C. Photochemically Driven Polymeric Network Formation: Synthesis and Applications. Adv. Mater. 2017, 29, 1604005. (4) Fairbanks, B. D.; Schwartz, M. P.; Bowman, C. N.; Anseth, K. S. Photoinitiated polymerization of PEG-diacrylate with lithium phenyl2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials 2009, 30, 6702−6707. (5) Sawicki, L. A.; Kloxin, A. M. Design of thiol-ene photoclick hydrogels using facile techniques for cell culture applications. Biomater. Sci. 2014, 2, 1612−1626. (6) Li, F.; Truong, V. X.; Thissen, H.; Frith, J. E.; Forsythe, J. S. Microfluidic Encapsulation of Human Mesenchymal Stem Cells for Articular Cartilage Tissue Regeneration. ACS Appl. Mater. Interfaces 2017, 9, 8589−8601. (7) Shih, H.; Greene, T.; Korc, M.; Lin, C.-C. Modular and Adaptable Tumor Niche Prepared from Visible Light Initiated ThiolNorbornene Photopolymerization. Biomacromolecules 2016, 17, 3872− 3882. (8) Shih, H.; Lin, C.-C. Visible-Light-Mediated Thiol-Ene Hydrogelation Using Eosin-Y as the Only Photoinitiator. Macromol. Rapid Commun. 2013, 34, 269−273. (9) Liu, Z.; Liu, T.; Lin, Q.; Bao, C.; Zhu, L. Sequential Control over Thiol Click Chemistry by a Reversibly Photoactivated Thiol Mechanism of Spirothiopyran. Angew. Chem., Int. Ed. 2015, 54, 174−178. (10) Liu, Z.; Lin, Q.; Sun, Y.; Liu, T.; Bao, C.; Li, F.; Zhu, L. Spatiotemporally Controllable and Cytocompatible Approach Builds 3D Cell Culture Matrix by Photo-Uncaged-Thiol Michael Addition Reaction. Adv. Mater. 2014, 26, 3912−3917.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00312. Experimental procedures, NMR spectra of the synthesized compounds, additional rheology data, cell encapsulation procedure, microgel fabrication, and conjugation procedure (PDF). 661

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ACS Macro Letters (11) Azagarsamy, M. A.; Marozas, I. A.; Spaans, S.; Anseth, K. S. Photoregulated Hydrazone-Based Hydrogel Formation for Biochemically Patterning 3D Cellular Microenvironments. ACS Macro Lett. 2016, 5, 19−23. (12) Yang, Y.; Zhang, J.; Liu, Z.; Lin, Q.; Liu, X.; Bao, C.; Wang, Y.; Zhu, L. Tissue-Integratable and Biocompatible Photogelation by the Imine Crosslinking Reaction. Adv. Mater. 2016, 28, 2724−2730. (13) Fan, Y.; Deng, C.; Cheng, R.; Meng, F.; Zhong, Z. In Situ Forming Hydrogels via Catalyst-Free and Bioorthogonal “Tetrazole− Alkene” Photo-Click Chemistry. Biomacromolecules 2013, 14, 2814− 2821. (14) McKay, C. S.; Finn, M. G. Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation. Chem. Biol. 2014, 21, 1075− 1101. (15) Froimowicz, P.; Frey, H.; Landfester, K. Towards the Generation of Self-Healing Materials by Means of a Reversible Photo-induced Approach. Macromol. Rapid Commun. 2011, 32, 468− 473. (16) Zheng, Y.; Micic, M.; Mello, S. V.; Mabrouki, M.; Andreopoulos, F. M.; Konka, V.; Pham, S. M.; Leblanc, R. M. PEG-Based Hydrogel Synthesis via the Photodimerization of Anthracene Groups. Macromolecules 2002, 35, 5228−5234. (17) Wells, L. A.; Brook, M. A.; Sheardown, H. Generic, AnthraceneBased Hydrogel Crosslinkers for Photo-controllable Drug Delivery. Macromol. Biosci. 2011, 11, 988−998. (18) Nagata, M.; Yamamoto, Y. Photoreversible poly(ethylene glycol)s with pendent coumarin group and their hydrogels. React. Funct. Polym. 2008, 68, 915−921. (19) Kim, S. H.; Sun, Y.; Kaplan, J. A.; Grinstaff, M. W.; Parquette, J. R. Photo-crosslinking of a self-assembled coumarin-dipeptide hydrogel. New J. Chem. 2015, 39, 3225−3228. (20) Draper, E. R.; McDonald, T. O.; Adams, D. J. Photodimerisation of a coumarin-dipeptide gelator. Chem. Commun. 2015, 51, 12827− 12830. (21) Yang, K.; Zeng, M. Multiresponsive hydrogel based on polyacrylamide functionalized with thymine derivatives. New J. Chem. 2013, 37, 920−926. (22) Andreopoulos, F. M.; Deible, C. R.; Stauffer, M. T.; Weber, S. G.; Wagner, W. R.; Beckman, E. J.; Russell, A. J. Photoscissable Hydrogel Synthesis via Rapid Photopolymerization of Novel PEGBased Polymers in the Absence of Photoinitiators. J. Am. Chem. Soc. 1996, 118, 6235−6240. (23) Gattás-Asfura, K. M.; Weisman, E.; Andreopoulos, F. M.; Micic, M.; Muller, B.; Sirpal, S.; Pham, S. M.; Leblanc, R. M. NitrocinnamateFunctionalized Gelatin: Synthesis and “Smart” Hydrogel Formation via Photo-Cross-Linking. Biomacromolecules 2005, 6, 1503−1509. (24) Claus, T. K.; Telitel, S.; Welle, A.; Bastmeyer, M.; Vogt, A. P.; Delaittre, G.; Barner-Kowollik, C. Light-driven reversible surface functionalization with anthracenes: visible light writing and mild UV erasing. Chem. Commun. 2017, 53, 1599−1602. (25) Zdobinsky, T.; Sankar Maiti, P.; Klajn, R. Support Cuvature and Conformational Freedom Control Chemical Reactivity of Immobilized Species. J. Am. Chem. Soc. 2014, 136, 2711−2714. (26) Ihmels, H.; Luo, Jia The reversible [4 + 4] photocycloaddition of acridizinium derivatives. J. Photochem. Photobiol., A 2008, 200, 3−9. (27) Truong, V. X.; Tsang, K. M.; Simon, G. P.; Boyd, R. L.; Evans, R. A.; Thissen, H.; Forsythe, J. S. Photodegradable Gelatin-Based Hydrogels Prepared by Bioorthogonal Click Chemistry for Cell Encapsulation and Release. Biomacromolecules 2015, 16, 2246−2253.

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