Fabrication of Dual-Redox Responsive Supramolecular Copolymers

Jul 6, 2016 - Self-assembly of amphiphilic block copolymers into well-defined nanostructures as drug delivery systems for the treatment of cancer has ...
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Fabrication of Dual-Redox Responsive Supramolecular Copolymers Using a Reducible β‑Cyclodextran-Ferrocene Double-Head Unit Cai Zuo, Xianyin Dai, Sijie Zhao, Xiaoning Liu, Shenglong Ding, Liwei Ma, Mingzhu Liu, and Hua Wei* State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China S Supporting Information *

ABSTRACT: Self-assembly of amphiphilic block copolymers into well-defined nanostructures as drug delivery systems for the treatment of cancer has been a hot subject of research. However, sequential polymerizations synthesized amphiphilic block copolymers with covalent links suffered mainly from multistep synthesis and purification procedures as well as repeated optimization of polymer composition to form aggregates with well-defined structures. To overcome these drawbacks, supramolecular amphiphilic block copolymers with noncovalent links were developed to provide simplicity as required. Herein, we designed and prepared a reducible β-cyclodextran (β-CD)-ferrocene (Fc) double-head unit from which a dual-redox responsive supramolecular amphiphilic copolymer was fabricated together with a traditional polymer block through supramolecular induced polymerization. Typically, well-defined supramolecular micelles and vesicles were fabricated, respectively. Due to the integration of oxidation-sensitive noncovalent β-CD/Fc connections and reduction-sensitive covalent disulfide bridges in the polymer backbone, the resulting supramolecular micelles and vesicles showed structural deformation and accelerated drug release in response to both intracellular reducing and oxidizing environments, thus, presenting a new platform for both reactive oxygen species (ROS) and glutathione (GSH)-triggered anticancer drug delivery.

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tions. To this end, supramolecular amphiphilic block copolymers with noncovalent links were designed and developed to provide simplicity as required. It is facile to control composition as well as self-assembly behavior of supramolecular amphiphilic block copolymers by altering the mixed ratios of different supramolecular moieties.15−19 Notably, Huang et al. reported the pioneering work regarding the construction of supramolecular aggregates with controlled morphologies through coself-assembly of a traditional polymer block and a supramolecular polymer block mediated by host− guest interactions.15 Moreover, supramolecular polymers, compared to traditional polymers, showed some distinctive and intriguing functionalities contributed by the noncovalent interactions.20 The supramolecular connections located either along the backbone or on the side chain may respond to various biorelevant stimuli (e.g., pH,21,22 light,23,24 certain molecule25) and allow the dissociation and recombination of each joint. Cyclodextrins (CDs), a family of cyclic oligosaccharides composed of α-(1 → 4)-linked D-glucopyranoside units, are good candidates to construct such a joint given their excellent biocompatibility and host capacity.20,26,27 β-CD is universally

olymeric nanoparticles are a promising class of controlled delivery systems for the treatment of various diseases due to their capacity to encapsulate therapeutic agents, thus, improving significantly the solubility and bioavailability of loaded cargoes, as well as prolonging their circulation time.1−3 The self-assembly of amphiphilic block copolymers into polymeric nanoparticles with diverse morphologies has been therefore a hot subject of research for both theoretical and practical investigations.4−6 Classical studies have revealed that the self-assembled morphology of amphiphilic block copolymers depends primarily on the weight fraction of the hydrophilic block.4−7 Of these self-assemblies, micelle and vesicle represent two of the most important structures due to their biomimicking characteristics. Higher hydrophilic weight fractions are apt to generate a micelle consisting of a hydrophobic core and a hydrophilic shielding corona,8−10 whereas lower values tend to produce a vesicle with a hydrophilic hollow cavity surrounded by a hydrophobic membrane wall.11−13 Amphiphilic block copolymers with covalent links are generally prepared by sequential polymerizations, suffering mainly from multistep synthesis and purification procedures, as well as repeated optimization of polymer composition to form aggregates with well-defined structures.14 There is thus considerable scope for the development of amphiphilic block copolymers with a facilely controlled chain length as well as hydrophilic weight fraction, tailor-made for practical applica© XXXX American Chemical Society

Received: June 16, 2016 Accepted: July 4, 2016

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DOI: 10.1021/acsmacrolett.6b00450 ACS Macro Lett. 2016, 5, 873−878

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ACS Macro Letters Scheme 1. Synthesis of Fc-SS-β-CD and Fc-P(OEGMA)

Considering the characteristic levels of ROS and GSH in cancer cell lines, many studies have focused on the construction of ROS or GSH-responsive drug/gene delivery systems for cancer therapy, 36,37 but polymeric carriers capable of responding to both ROS and GSH stimuli, to the best of our knowledge, are rare. Herein, we reported on the design and preparation of a reducible β-cyclodextran (β-CD)-ferrocene (Fc) double-head unit, from which a dual-redox responsive supramolecular amphiphilic copolymer was fabricated, together with a traditional polymer block through supramolecularinduced polymerization for anticancer drug delivery. Due to the integration of a reduction-sensitive covalent disulfide link and oxidation-sensitive noncovalent β-CD/Fc joint in the polymer backbone, the resulting supramolecular self-assemblies with well-defined structures can respond to both intracellular reducing and oxidizing environments for structural disassembly and accelerated drug release, thus, providing a new platform for both GSH and ROS-triggered anticancer drug delivery. To fabricate dual-redox responsive supramolecular amphiphilic block copolymers, β-CD/Fc pair and disulfide bridge were introduced into the polymer structure simultaneously for ROS and GSH-triggered responsiveness, respectively. The resultant supramolecular amphiphilic block copolymers contain a supramolecular hydrophobic segment with both β-CD and Fc termini connected by a central disulfide link (Fc-SS-β-CD) and

utilized to build efficient drug delivery systems due to the facile self-assembly process and appropriate association strength in binding conjugates through host−guest interaction. One wellrecognized host−guest pair is β-CD and ferrocene (Fc).28,29 The hydrophobic Fc group is strongly bound to the cavity of βCD, while the oxidized state of Fc binds very weakly to β-CD because of its cationic nature.30 Therefore, reactive oxygen species (ROS) that have been found to play an important role in cancer development can be used as a biorelevant stimulus to trigger the dissociation and subsequent drug release from β-CD and Fc-based delivery systems.31,32 Since several tumor cell lines could consecutively produce certain amounts of ROS during their growth,28 the direct consumption of ROS by this type of delivery system can result in a decreased cellular ROS level, compromising the proliferation of cancer cells simultaneously. In addition to the characteristic ROS level, tumor cells also exhibit several times higher concentrations of glutathione (GSH) than normal cells.33 The disulfide bonds are relatively stable in the extracellular fluids (10 μM of GSH), but can quickly degrade to free thiols by an elevated concentration of GSH (approximately 1−11 mM in the cytoplasm) within cells.34,35 Therefore, GSH can be used as another biorelevant stimulus to facilitate deformation and rapid drug release from delivery systems containing disulfide links. 874

DOI: 10.1021/acsmacrolett.6b00450 ACS Macro Lett. 2016, 5, 873−878

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ACS Macro Letters a conventional hydrophilic homopolymer P(OEGMA) with Fc terminus (Fc-P(OEGMA); Scheme 1). The three main steps in the synthesis of supramolecular block, Fc-SS-β-CD are (i) synthesis of mono-6-azido-β-CD (N3-β-CD), (ii) functionalization of 3,3′-dithiodipropionic acid with both Fc and alkyne termini by two-consecutive DCC/ DMAP coupling to prepare a hetero double-head agent with a central disulfide bridge, Fc-SS-CCH, and (iii) generation of target supramolecular unit with both Fc and β-CD termini by Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAc) between N3-β-CD and Fc-SS-CCH. The successful synthesis of all the intermediate and target molecules was confirmed by a NMR study (Figures S1−S7). Next, the conventional hydrophilic homopolymer with Fc terminus, Fc-P(OEGMA) was synthesized by RAFT polymerization of OEGMA using a Fc-modified CTA, Fc-(4cyanopentanoic acid dithiobenzoate) (Fc-CPADB; Figures S8 and S9). Our previous studies have shown that the hydrophilic block with a molecular weight (MW) of ∼5.0 kDa can provide sufficient extracellular colloidal stability for carriers,38 therefore RAFT polymerization was performed at various polymerization time summarized in Table S1 to screen a 5.0 kDa P(OEGMA) block. Notably, the MWs determined by GPC match well with the data calculated from 1H NMR analysis (Table S1). The increase of MW with polymerization time together with the narrow polydisersity index (PDI) recorded for all the polymers demonstrate well-controlled RAFT process with living characteristics. P(OEGMA) synthesized by 80 min polymerization shows unimodal and narrowly distributed GPC elution trace (Figure S10) with a Mn of 5.7 kDa, was thus chosen as the hydrophilic block to construct the supramolecular amphiphilic copolymers. Due to the well-documented host−guest interaction between β-CD and Fc, supermolecular amphiphilic block copolymers with noncovalent β-CD/Fc junctions were prepared by mixing hydrophobic Fc-SS-β-CD and hydrophilic Fc-POEGMA in solution. The MW, composition, and hydrophilic weight fraction of the resultant supermolecular amphiphilic block copolymers could be adjusted easily by changing the molar mixed ratio of these two moieties. An increase of the Fc-SS-βCD/Fc-POEGMA molar mixed ratio from 1:1 to 3:1 leads to an increase of MW with decreased hydrophilic weight fraction, resulting in the formation of different preferred self-assemblies. We have examined the solubility of a pure supramolecular polymer block composed of Fc-SS-β-CD in water and found that this block is insoluble in water even at a very low concentration of 1 μg/mL. Moreover, we tried the formation of supramolecular copolymers at a higher Fc-SS-β-CD/FcPOEGMA molar mixed ratio of 4:1, and observed obvious formation of precipitates in solution during dialysis, indicating that supramolecular copolymers prepared at this molar mixed ratio and higher values are unable to form stable self-assemblies due to the unbalanced hydrophobic/hydrophilic ratio. In other words, the hydrophobicity of supramolecular block overwhelms the hydrophilicity of traditional polymer block, leading to the precipitation of overall supramolecular copolymers at higher molar feed ratios of hydrophobic supramolecular unit Fc-SS-βCD. The successful formation of β-CD/Fc inclusion complex was first confirmed by the 2D NOESY spectrum of Fc-SS-β-CD and Fc-P(OEGMA) (Figure 1). The cross-peaks highlighted using red squares are attributed to the dipolar interactions between the signals in the range of 3.34−3.28 ppm assigned to the

Figure 1. 2D NOESY NMR spectrum of Fc-SS-β-CD and FcP(OEGMA) in DMSO-d6.

protons of β-CD and the peaks at 4.20 and 4.78 ppm ascribed to Fc, indicating the Fc molecules are embedded in the cavities of β-CD and the successful formation of β-CD/Fc inclusion complex. Here TEM observation was applied to identify the selfassembly of supermolecular amphiphilic block copolymers with different Fc-SS-β-CD/Fc-POEGMA molar mixed ratios into well-defined nanostructures. As shown in Figure 2, 1:1 molar mixed ratio gives birth to uniform core−shell micelles with regularly spherical shape. The dark black core observed in the central is feasibly attributed to the presence of Fc moiety with high atomic number. However, vesicle formation, visualized at a higher molar mixed ratio of 3:1, clearly shows different architectures from micelles. Such evolution of self-assembled morphologies is primarily due to the decrease hydrophilic weight fractions, demonstrating supermolecular amphiphilic block copolymers follow the same classical rules of self-assembly applied to traditional amphiphilic copolymers.4−6 The average diameter of micelles and vesicles are determined to be 147.1 and 381.2 nm, respectively, by dynamic light scattering (DLS), consistent with TEM observation. The schematic illustration for supramolecular micelles and vesicles formation was presented in Scheme 2. To investigate whether the self-assembled nanostructures formed using supermolecular amphiphilic block copolymers would show dual-redox sensitivity as expected, the particle size of supramolecular micelles and vesicles in the presence and absence of water-soluble reducing agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl; 10 mM) or oxidizing agent NaClO39 (1 mM) were monitored over time. Micelles and vesicles both were found to aggregate dramatically from nano- to microscale in size due to the dissociation and subsequent aggregation of supermolecular hydrophobic block, Fc-SS-β-CD, either resulting from the reduction-triggered cleavage of central disulfide bridges by TCEP or the oxidation-promoted disconnection of terminal β-CD/Fc host−guest joints by NaClO, whereas both structures were highly stable in size without any additives (Figure 3). Hence, the supermolecular micelles and vesicles developed herein may have excellent colloidal stability in the circulation and still be readily destabilized in the tumor cell cytosol, facilitating drug release. 875

DOI: 10.1021/acsmacrolett.6b00450 ACS Macro Lett. 2016, 5, 873−878

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Figure 2. TEM images and size distributions of Fc-SS-β-CD/Fc-P(OEGMA) supramolecular self-assemblies formed at molar mixed ratios of 1:1 (a, b) and 3:1 (c, d).

vesicles. The drug-loaded nanoparticles were incubated in PBS (pH 7.4) with or without 10 mM TCEP or 1 mM NaClO at 37 °C and DOX release was quantified by dialysis method (Figure 4). Incubation with 10 mM TCEP resulted in over 60% and 52% DOX release from micelles and vesicles, respectively, after 72 h, consistently higher than the control groups without any additive (40% for micelles and 20% for vesicles) during the same period. Similar trends were recorded in the presence of oxidant. Treating with 1 mM NaClO resulted in over 54% and 43% DOX release from micelles and vesicles, respectively, after 72 h. The overall results confirm the dual-redox sensitivity, i.e., both reduction and oxidation-triggered structural deformation of supramolecular micelles and vesicles, thus promoting DOX release. Moreover, lower concentration of NaClO at 0.1 mM decelerated drug release with ∼46% and 33% DOX release from micelles and vesicles over 72 h (Figure S12), probably demonstrating slower and less completed dissociation of β-CD/ Fc host−guest connections with a lower concentration of

Scheme 2. Formation of Supramolecular Amphiphilic Block Copolymer and Its Self-Assembled Micelle and Vesicle

To further investigate the dual-redox dependent kinetics of drug release for potential cancer therapy, DOX and DOX·HCl were encapsulated into the supramolecular micelles and

Figure 3. Size change of supramolecular micelles (a) and vesicles (b) over time under various conditions. 876

DOI: 10.1021/acsmacrolett.6b00450 ACS Macro Lett. 2016, 5, 873−878

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Figure 4. In vitro DOX release from supramolecular micelles and vesicles under various conditions.

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oxidant treated. Taken together, it is reasonable to postulate that the drug encapsulated can be released in responsive to two respective triggers, ROS or GSH, on-demand. In summary, considering the characteristic high concentrations of ROS and GSH in cancer cells, we have designed and developed herein a novel dual-redox responsive supramolecular amphiphilic block copolymer with β-CD/Fc host−guest joints based on a supramolecular hydrophobic unit, Fc-SS-β-CD and a traditional hydrophilic block, Fc-P(OEGMA). By simply changing the molar mixed ratios of Fc-SS-β-CD and FcP(OEGMA), supramolecular amphiphilic block copolymers with tunable MWs and hydrophilic weight fractions were readily prepared. Typically, well-defined supramolecular micelles and vesicles were fabricated under the molar mixed ratios of 1:1 and 3:1, respectively. Due to the presence of noncovalent β-CD/Fc connections and covalent disulfide bridges, the resulting supramolecular micelles and vesicles showed accelerated drug release in response to both reductant TCEP or oxidant NaClO. This work thus provides a new platform for achieving both ROS and GSH-triggered anticancer drug delivery.

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(1) Wang, X.; Hall, J. E.; Warren, S.; Krom, J.; Magistrelli, J. M.; Rackaitis, M.; Bohm, G. G. A. Macromolecules 2007, 40, 499−508. (2) Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O. C. Mol. Pharmaceutics 2008, 5, 505−515. (3) Wong, E. H. H.; Lam, S. J.; Nam, E.; Qiao, G. G. ACS Macro Lett. 2014, 3, 524−528. (4) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967−973. (5) Wei, H.; Yu, C. Y.; Chang, C.; Quan, C. Y.; Mo, S. B.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Chem. Commun. 2008, 4598−4600. (6) Mai, Y.; Eisenberg, A. Chem. Soc. Rev. 2012, 41, 5969−5985. (7) Ray, J. G.; Johnson, A. J.; Savin, D. A. J. Polym. Sci., Part B: Polym. Phys. 2013, 51, 508−523. (8) Zhao, P.; Liu, L.; Feng, X.; Wang, C.; Shuai, X.; Chen, Y. Macromol. Rapid Commun. 2012, 33, 1351−1355. (9) Müllner, M.; Lunkenbein, T.; Schieder, M.; Gröschel, A. H.; Miyajima, N.; Förtsch, M.; Breu, J.; Caruso, F.; Müller, A. H. E. Macromolecules 2012, 45, 6981−6988. (10) Ji, X.; Li, J.; Chen, J.; Chi, X.; Zhu, K.; Yan, X.; Zhang, M.; Huang, F. Macromolecules 2012, 45, 6457−6463. (11) Osada, K.; Cabral, H.; Mochida, Y.; Lee, S.; Nagata, K.; Matsuura, T.; Yamamoto, M.; Anraku, Y.; Kishimura, A.; Nishiyama, N.; Kataoka, K. J. Am. Chem. Soc. 2012, 134, 13172−13175. (12) Chi, X.; Yu, G.; Ji, X.; Li, Y.; Tang, G.; Huang, F. ACS Macro Lett. 2015, 4, 996−999. (13) Duan, Q.; Cao, Y.; Li, Y.; Hu, X.; Xiao, T.; Lin, C.; Pan, Y.; Wang, L. J. Am. Chem. Soc. 2013, 135, 10542−10549. (14) Wang, Y.-C.; Li, Y.; Sun, T.-M.; Xiong, M.-H.; Wu, J.; Yang, Y.Y.; Wang, J. Macromol. Rapid Commun. 2010, 31, 1201−1206. (15) Ji, X.; Dong, S.; Wei, P.; Xia, D.; Huang, F. Adv. Mater. 2013, 25, 5725−5729. (16) Jie, K.; Zhou, Y.; Ji, X. Polym. Chem. 2015, 6, 218−222. (17) Ji, X.; Li, Y.; Wang, H.; Zhao, R.; Tang, G.; Huang, F. Polym. Chem. 2015, 6, 5021−5025. (18) Ji, X.; Wang, H.; Li, Y.; Xia, D.; Li, H.; Tang, G.; Sessler, J.; Huang, F. Chemical Science 2016, DOI: 10.1039/C6SC01851C. (19) Dong, S.; Zheng, B.; Zhang, M.; Yan, X.; Ding, X.; Yu, Y.; Huang, F. Macromolecules 2012, 45, 9070−9075. (20) Wei, H.; Yu, C. Y. Biomater. Sci. 2015, 3, 1050−1060. (21) Wang, C.; Kang, Y.; Liu, K.; Li, Z.; Wang, Z.; Zhang, X. Polym. Chem. 2012, 3, 3056−3059. (22) Zhang, Z.; Ding, J.; Chen, X.; Xiao, C.; He, C.; Zhuang, X.; Chen, L.; Chen, X. Polym. Chem. 2013, 4, 3265−3271. (23) Wang, C.; Chen, Q.; Xu, H.; Wang, Z.; Zhang, X. Adv. Mater. 2010, 22, 2553−2555. (24) Liu, Y.; Yu, C.; Jin, H.; Jiang, B.; Zhu, X.; Zhou, Y.; Lu, Z.; Yan, D. J. Am. Chem. Soc. 2013, 135, 4765−4770. (25) Liu, J.; Chen, G.; Guo, M.; Jiang, M. Macromolecules 2010, 43, 8086−8093.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00450. Experimental materials and methods, data on characterization of Fc-SS-β-CD and Fc-P(OEGMA), and additional results from an in vitro drug release study are available in Table S1 and Figures S1−S12 (PDF).

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Science Foundation of China (51473072 and 21504035) and the Fundamental Research Funds for the Central Universities (lzujbky-2015-k05 and lzujbky-2016-ct05). 877

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ACS Macro Letters (26) Wang, L.; Li, L. l.; Fan, Y. S.; Wang, H. Adv. Mater. 2013, 25, 3888−3898. (27) Zhang, J.; Ma, P. X. Adv. Drug Delivery Rev. 2013, 65, 1215− 1233. (28) Peng, L.; Feng, A.; Zhang, H.; Wang, H.; Jian, C.; Liu, B.; Gao, W.; Yuan, J. Polym. Chem. 2014, 5, 1751−1759. (29) Yan, Q.; Cai, Z.; Xin, Y.; Kang, Y.; Yuan, J. J. Am. Chem. Soc. 2010, 132, 9268−9270. (30) Du, P.; Liu, J.; Chen, G.; Jiang, M. Langmuir 2011, 27, 9602− 9608. (31) Kang, Y.; Ma, Y.; Zhang, S.; Ding, L. S.; Li, B. J. ACS Macro Lett. 2015, 4, 543−547. (32) Kumar, M.; Rhim, W. K.; Lim, D. K.; Nam, J. W. ACS Nano 2013, 7, 2221−2230. (33) Wei, H.; Zhuo, R.-X.; Zhang, X.-Z. Prog. Polym. Sci. 2013, 38, 503−535. (34) Sun, H.; Guo, B.; Cheng, R.; Meng, F.; Liu, H.; Zhong, Z. Biomaterials 2009, 30, 6358−6366. (35) Liu, J.; Pang, Y.; Huang, W.; Zhu, Z.; Zhu, X.; Zhou, Y.; Yan, D. Biomacromolecules 2011, 12, 2407−2415. (36) Wei, H.; Volpatti, L. R.; Sellers, D. L.; Maris, D. O.; Andrews, I. W.; Hemphill, A. S.; Chan, L. W.; Chu, D. S. H.; Horner, P. J.; Pun, S. H. Angew. Chem., Int. Ed. 2013, 52, 5377−5381. (37) Gupta, M. K.; Martin, J. R.; Werfel, T. A.; Shen, T.; Page, J. M.; Duvall, C. L. J. Am. Chem. Soc. 2014, 136, 14896−14902. (38) Wei, H.; Pahang, J. A.; Pun, S. H. Biomacromolecules 2013, 14, 275−284. (39) Nakahata, M.; Takashima, Y.; Yamaguchi, H.; Harada, A. Nat. Commun. 2011, 2, 511.

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DOI: 10.1021/acsmacrolett.6b00450 ACS Macro Lett. 2016, 5, 873−878