Selenylsulfide Bond-Launched Reduction-Responsive

Letter www.acsami.org

Selenylsulfide Bond-Launched Reduction-Responsive Superparamagnetic Nanogel Combined of Acid-Responsiveness for Achievement of Efficient Therapy with Low Side Effect Yanan Xue,†,‡ Xiaoyang Xia,†,‡ Bo Yu,‡ Lijun Tao,‡ Qian Wang,§ Shi-Wen Huang,§ and Faquan Yu*,‡ ‡

Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory for Novel Reactor and Green Chemistry Technology, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China § College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China S Supporting Information *

ABSTRACT: With the objective to achieve in-between reduction-responsive drug release, selenylsulfide bond was first explored as a reduction cleavable linkage, compared with the most commonly employed disulfide and diselenide bonds. The reductive nanogel, with a combination of superparamagnetic and acid responsiveness, was fabricated. The expected release profiles were testified. Cytotoxicity assays illustrated the remarkable inhibition to the growth of HeLa cells, in contrast, high tolerance to L02 cells. In vivo investigation exhibited the obvious shrinkage in tumor but a healthy appearance. Hematoxylin-eosin staining and histological examination revealed the lower toxicity. The complex nanogels would have appeared highly promising in cancer therapy. KEYWORDS: nanogel, drug delivery, selenylsulfide, superparamagnetism, reduction-responsiveness, acid-responsiveness ffective delivery of antitumor therapeutic agents into tumor tissues and tumor cells for high therapeutic and low side effect is usually desired. Responsive nanogels are usually used as the delivery cargos. Disulfide bond (S−S) is the most popular reduction responsive unit1−3 in light of the significantly elevated concentration of glutathione (GSH), a reductive agent, in tumor compared to healthy tissues.4 This kind of linkage, however, expressed slow responsive release, taking 10 h to reach only approximate 305 or 25%6 accumulative release even at as high as 10 mM of GSH. The similar result was observed in our previous study.7 Diselenide linkage (Se−Se) was recently verified to function alike.8−12 In contrast, the Se−Se bond dependent nanogel took only 2−3 h to reach 90% of the equilibrium release even at as low as the concentration of 33 μM of GSH.8 Their direct comparison was also conducted.9 The quick release benefits efficient therapy but causes a systemic adverse effect, and vice versa for the S−S bond case.8 Selenylsulfide bond (Se−S) perhaps is an alternative that would achieve in-between release rate and integrate both advantages, though it has so far not been explored. These recognitions drove us to explore the Se−S bond as the reduction cleavable linkage. In another respect, pH-responsive cargos were frequently fabricated.13,14 Superparamagnetic nanogel exhibited versatile features in targeting delivery.15 Theoretically, the integration of reduction- and pH-responsiveness with superparamagnetism into one system would restrain the release intensively in healthy tissues and augment the targeting release.

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Herein, we launched the Se−S bond as the reductionresponsive linkage, preparing a doxorubicin (DOX)-loaded superparamagnetic nanogel for the achievement of reduction/ pH cotriggered release. The fabrication of magnetic DOXloaded Se−S alginate nanogel (MDSeSAN-gel) is depicted in Scheme 1. The synthesis process of 3,3′-diselanediyldipropanoic acid (DSeDPA) is displayed in Figure 1A. The exchange reaction of thiolated alginate (SA-SH) with DSeDPA formed selenylsulfide bond-modified alginate (SA-SSe-COOH), which is in agreement with the combined characteristic 1H NMR peaks of both SA-SH and DSeDPA (Figure 1B). The Raman band at 372 cm−1 was assigned to Se−S bond according to previous literature16,17 (Figure 1C). All these examinations confirmed the successful preparation of SA-SSe-COOH. The nanogel was obtained by virtue of the electrostatic interaction between thiolated SA and aminated magnetic nanoparticle (SPION-NH2).18,19 Upon the optimized fed ratio, an appropriate size was achieved at 105 nm with PDI of 0.165 and zeta potential of −44.2 ± 0.7 mV (Figure S1a, b). The magnetophoresis, superparamagnetic properties (Figure S1c, d) and long storage stability (Figure S2) were evaluated. DOX loading content and loading efficiency were determined at 18.2 and 95.6%, respectively, both of which are higher than before.7,20−22 Acidic medium would break the electrostatic Received: May 15, 2017 Accepted: July 10, 2017 Published: July 10, 2017 30253

DOI: 10.1021/acsami.7b06818 ACS Appl. Mater. Interfaces 2017, 9, 30253−30257

Letter

ACS Applied Materials & Interfaces Scheme 1. Illustration of the Fabrication Process and the Fate of MDSeSAN-gel after Entering Cell

Figure 1. (A) Synthesis route of SA-SSe-COOH. (B) 1H NMR spectra of SA, SA-SH, SA-SSe-COOH, and DSeDPA. (C) Raman analysis of DTDPA, SA-SSe-COOH, and DSeDPA.

linkages. Either reduction or acid cannot dissociate its fabrication or release its payload completely. The equilibrium release at pH 7.4 was 22.7%. In contrast, the value reached around 56.1% at pH 5.0, a pH simulating the cancerous intracellular acidic environments.7 As mentioned above, the acidic pH rendered partial disassembly or slack fabrication of nanogels, thereby led to the release of DOX. On the other hand, approximate 56.9% of DOX was liberated in 10 mM

interaction via the protonation process of SA as well as GSH would induce the cleavage of selenylsulfide bond. Both the mechanisms will bring about the disassembly of the nanogels (Figure S2). The feature of disassembly predicted the pH- and GSH-cotriggered release, observed in the following investigation. Figure 2 shows the in vitro DOX release profiles. The nanogel was fabricated by both reduction and acid responsive 30254

DOI: 10.1021/acsami.7b06818 ACS Appl. Mater. Interfaces 2017, 9, 30253−30257

Letter

ACS Applied Materials & Interfaces

attacked atom instead, the rate is still increased because selenol has a lower pKa relative to thiol. The leaving group atom must either be protonated by a general acid or have a pKa that is less than or equal to the solution pH.23 Furthermore, the high polarizability of selenium in contrast to sulfur also renders selenium to leave easily. In short, the Se−S bond is more easily attacked by GSH than the S−S bond. In the case of Se−Se bond as the reduced linkage, where Se acts as both the attacked atom and as the leaving group, the rate will, of course, be increased further compared with the Se−S in terms of the discussion above. In other words, Se−S would exhibit higher reaction activity than S−S or lower reactivity than Se−Se under the action of thiol groups. It was reported that the rate constant of Se−Se with RSH is 1.3 × 105 M−1 s−1; in contrast, the rate constant of Se−S with RSH is 11 M−1 s−1.24 Chemical bond energy is an optional indicator to judge the reactivity. The bond energy of diselenide bond (172 kJ mol−1) is significantly lower than that of disulfide bond (240 kJ mol−1). All these endorsed the result. The cytotoxicity assays showed retained metabolic ability even at a high dose of 50 μg mL−1 plain nanogels (Figure S3a). MDSeSAN-gel showed significantly dose-dependent (Figure S3b) and incubation-time-dependent cytotoxicity on HeLa cells (Figure S3c) but low cytotoxicity against L02 healthy cell lines. The external magnetic field intensified the cytotoxicity, revealing enhanced therapy effect (Figure S3d). Reverse microscopy displayed rapid endocytosis of MDSeSAN-gel by HeLa cells (Figure S4). CLSM analysis (Figure S5) indicated restricted release and subsequent transfer of released DOX inside the cytoplasm into the nuclei. Figure 3a showed the insignificant difference between the groups treated with MDSeSAN-gel and with PBS (the control group) in body weight. The lowest mean tumor volumes

Figure 2. Release of DOX from MDSeSAN-gels dependent on pH 7.4 and pH 5.0 with or without 10 mM or 10 μM GSH at 37 °C.

GSH, a characteristic cancerous level. The accumulative release reached far away from 100% just because the nanogel was still wrapped up by the acid responsive linkage. As anticipated, the release increased significantly to 97.0% at 10 mM GSH/pH 5.0, implying the concurrent dissociation of both the linkages. The feature will tremendously retain the release in healthy tissues and effectively lessen the systemic side effect. As for the rate, the release reached 75.8% after 10 h at 10 mM GSH/pH 5.0. The release is slower than the case of Se−Se bond;8−12 however, it is higher than the case of the S−S bond,5,6 even if here the acid-responsive linkage still limits the release. In the process that GSH or a thiol group functions as the electrophilic attacker of the Se−S bond, if the S part acts as the leaving group or the Se part as the attacked atom, the rate of the reaction is increased in comparison with attacking the S−S bond because selenium is more electrophilic than sulfur. If the Se part functions as the leaving group or the S part as the

Figure 3. In vivo antitumor efficacy of the nanogel in BABL/c mice harboring H22 liver tumor. (a) Variation in body weight over the treatment regimen, (b) tumor volume over the treatment regimen, (c) tumor weight and the inhibition rate of tumor growth. (d) Representative tumors under different treatment modes. 30255

DOI: 10.1021/acsami.7b06818 ACS Appl. Mater. Interfaces 2017, 9, 30253−30257

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ACS Applied Materials & Interfaces

Responsive Superparamagnetic Nanogel with Chemically Anchored DOX for Enhanced Anticancer Efficacy and Low Systemic Adverse Effects. J. Mater. Chem. B 2015, 3, 8949−8962. (4) Deng, B.; Ma, P.; Xie, Y. Reduction-Sensitive Polymeric Nanocarriers in Cancer Therapy: a Comprehensive Review. Nanoscale 2015, 7, 12773−12795. (5) Li, Y.; Xiao, K.; Luo, J.; Xiao, W.; Lee, J. S.; Gonik, A. M.; Kato, J.; Dong, T. A.; Lam, K. S. Well-Defined, Reversible Disulfide CrossLinked Micelles for On-Demand Paclitaxel Delivery. Biomaterials 2011, 32, 6633−6645. (6) Wen, H. Y.; Dong, H. Q.; Xie, W. J.; Li, Y. Y.; Wang, K.; Pauletti, G. M.; Shi, D. L. Rapidly Disassembling Nanomicelles with Disulfidelinked PEG Shells for Glutathione-Mediated Intracellular Drug Delivery. Chem. Commun. 2011, 47, 3550−3552. (7) Huang, J.; Xue, Y.; Cai, N.; Zhang, H.; Wen, K.; Luo, X.; Long, S.; Yu, F. Efficient Reduction and pH co-Triggered DOX-Loaded Magnetic Nanogel Carrier Using Disulfide Crosslinking. Mater. Sci. Eng., C 2015, 46, 41−51. (8) Ma, N.; Li, Y.; Xu, H.; Wang, Z.; Zhang, X. Dual Redox Responsive Assemblies Formed from Diselenide Block Copolymers. J. Am. Chem. Soc. 2010, 132, 442−443. (9) Wei, C.; Zhang, Y.; Song, Z.; Xia, Y.; Xu, H.; Lang, M. Enhanced Bioreduction-Responsive Biodegradable Diselenide-Containing Poly(ester urethane) Nanocarriers. Biomater. Sci. 2017, 5, 669−677. (10) Deepagan, V. G.; Kwon, S.; You, D. G.; Nguyen, V. Q.; Um, W.; Ko, H.; Lee, H.; Jo, D. G.; Kang, Y. M.; Park, J. H. In Situ DiselenideCrosslinked Polymeric Micelles for ROS-Mediated Anticancer Drug Delivery. Biomaterials 2016, 103, 56−66. (11) Xia, Y.; He, H.; Liu, X.; Hu, D.; Yin, L.; Lu, Y.; Xu, W. RedoxResponsive, Core-Crosslinked Degradable Micelles for Controlled Drug Release. Polym. Chem. 2016, 7, 6330−6339. (12) Zhai, S.; Hu, X.; Hu, Y.; Wu, B.; Xing, D. Visible Light-Induced Crosslinking and Physiological Stabilization of Diselenide-Rich Nanoparticles for Redox-Responsive Drug Release and Combination Chemotherapy. Biomaterials 2017, 121, 41−54. (13) Guan, X.; Li, Y.; Jiao, Z.; Chen, J.; Guo, Z.; Tian, H.; Chen, X. pH-Sensitive Charge-Conversion System for Doxorubicin Delivery. Acta Biomater. 2013, 9, 7672−7678. (14) Ding, J.; Xu, W.; Zhang, Y.; Sun, D.; Xiao, C.; Liu, D.; Zhu, X.; Chen, X. Self-Reinforced Endocytoses of Smart Polypeptide Nanogels for ″On-Demand″ Drug Delivery. J. Controlled Release 2013, 172, 444−455. (15) Wang, Y. X. J.; Hussain, S. M.; Krestin, G. P. Superparamagnetic Iron Oxide Contrast Agents: Physicochemical Characteristics and Applications in MR Imaging. Eur. Radiol. 2001, 11, 2319−2331. (16) Xu, J.; Yang, X.; Yang, Q.; Zhang, W.; Lee, C. S. Phase Conversion from Hexagonal CuSySe1‑y to Cubic Cu2‑xSySe1‑y: Composition Variation, Morphology Evolution, Optical Tuning, and Solar Cell Applications. ACS Appl. Mater. Interfaces 2014, 6, 16352− 16359. (17) Ishii, M.; Shibata, K.; Nozaki, H. J. Anion Distributions and Phase Transitions in CuS 1‑x Se x (x = 0−1) Studied by Raman Spectroscopy. J. Solid State Chem. 1993, 105, 504−511. (18) Zhang, H.; Xue, Y.; Huang, J.; Xia, X.; Song, M.; Wen, K.; Zhang, X.; Luo, X.; Cai, N.; Long, S.; Yu, F. Tailor-Made Magnetic Nanocarriers with pH-Induced Charge Reversal and pH-Responsiveness to Guide Subcellular Release of Doxorubicin. J. Mater. Sci. 2015, 50, 2429−2442. (19) Yu, F.; Huang, Y.; Cole, A. J.; Yang, V. C. The Artificial Peroxidase Activity of Magnetic Iron Oxide Nanoparticles and its Application to Glucose Detection. Biomaterials 2009, 30, 4716−4722. (20) Cheng, Y.; He, C.; Xiao, C.; Ding, J.; Ren, K.; Yu, S.; Zhuang, X.; Chen, X. Reduction-Responsive Cross-Linked Micelles Based on PEGylated Polypeptides Prepared via Click Chemistry. Polym. Chem. 2013, 4, 3851−3858. (21) Mu, C. F.; Balakrishnan, P.; Cui, F. D.; Yin, Y. M.; Lee, Y. B.; Choi, H. G.; Yong, C. S.; Chung, S. J.; Shim, C. K.; Kim, D. D. The Effects of Mixed MPEG-PLA/Pluronic Copolymer Micelles on the

(Figure 3b), the significant inhibition rates of tumor growth (Figure 3c), just as the representative photographs (Figure 3d) treated with MDSeSAN-gel intuitively indicated that the nanogel showed better antitumor activity and lower systemic adverse effect than DOX. Remarkable necrosis with the most extensive necrotic centers and the least viable tumor cells was observed in tumors in terms of H&E staining (Figure S6). As for vital organs (Figure S7), normal appearance was observed in MDSeSAN-gel groups just in the PBS. Obvious accumulation of MDSeSAN-gel (indicated in blue dots in Figure S8) was identified in the tumor tissue. In conclusion, selenylsulfide bond was successfully introduced in the fabrication of a nanogel for achieving mild reduction-responsive release. Se−S would exhibit reductive cleavability higher than S−S or lower than Se−Se. Upon the combination of reductive/acidic stimuli and superparamagnetism, MDSeSAN-gel exhibited multiple stimuli-responsiveness with targeted release. In vivo experiments, including body weight, tumor inhibition rate, and histological assessments, indicated the noticeable therapeutic efficacy with a low systemic adverse effect.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b06818. Experimental and characterization detail; structural properties, including the size distribution, TEM, magnetophoresis, and VSM measurement of MDSeSAN-gels; microscopic images of Prussian blue in cellular and tumor sections; histological assessments of tissue sections, CLSM image analysis and in vitro cytotoxicity (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (86-27) 87194980. Fax: (86-27) 87194465. ORCID

Shi-Wen Huang: 0000-0002-4017-7594 Faquan Yu: 0000-0002-5062-8731 Author Contributions †

Y.X. and X.X. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (21571147 and 81601605), as well as by Innovative Team Program of Natural Science Foundation of Hubei Province (2014CFA011), and by the Program of Hubei Provincial Department of Education, China (Q20151518).

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DOI: 10.1021/acsami.7b06818 ACS Appl. Mater. Interfaces 2017, 9, 30253−30257