Emerging Nanoassembly of Polyrotaxanes Comprising Acetylated α

Letter Cite This: ACS Macro Lett. 2019, 8, 826−834

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Emerging Nanoassembly of Polyrotaxanes Comprising Acetylated α‑Cyclodextrins and High-Molecular-Weight Axle Polymer Asato Tonegawa, Atsushi Tamura,* and Nobuhiko Yui Department of Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan

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S Supporting Information *

ABSTRACT: Acetylated α-cyclodextrin (α-CD)/poly(ethylene glycol) (PEG)-based polyrotaxanes (Ac-PRXs) with varying degrees of acetylation (DA) and molecular weight of axle PEG were synthesized and their solubility in aqueous solutions was investigated. Ac-PRXs with low DA (less than 35%) were dissolved in aqueous solutions without considering the molecular weight of axle PEG, whereas Ac-PRXs with high DA (more than 40%) and low molecular weight of axle PEG (less than 35000) were precipitated into the solutions. Interestingly, Ac-PRXs with high DA and high molecular weight of axle PEG (100000) exhibited a colloidal dispersion in aqueous solutions. It is considered that the threaded acetylated α-CDs formed hydrophobic microenvironments via hydrophobic interactions and the noncovered segments of axle PEGs provided colloidal stability. Furthermore, the potential application of Ac-PRX100k as a drug carrier was examined and it was established that Ac-PRX100k can encapsulate a hydrophobic drug. Accordingly, acetylation of PRXs is a viable approach to promote solubility in aqueous solutions and prepare self-assembled nanoparticles.

M

To improve their solubility in aqueous solutions, the chemical modification of the hydroxy groups of threaded CDs in PRXs with ionic (e.g., amino group,6−9 carboxy group,1,19,20 sulfonate group10) and nonionic functional groups (e.g., hydroxypropyl group,1,11 oligoethylene glycol21) is necessary. In the chemistry of cellulose, various functional groups, such as methyl, carboxymethyl, and hydroxypropyl groups, are modified to modulate the solubility in aqueous solutions.22 These chemically modified celluloses are utilized in many industrial fields.23,24 Among the various chemical modifications, the methylation of cellulose is a unique method that mitigates hydrogen bonding, resulting in enhanced solubility in aqueous solutions. Accordingly, the methylation of PRXs was performed and methylated PRXs (Me-PRXs) were dissolved in aqueous solutions.25−28 Similarly, the acetylation of cellulose is also known to modulate solubility in a variety of solvents, including water.29,30 Acetylation is a widely utilized method for

aterials applications of polyrotaxanes (PRXs), which are a representative supramolecular polymer composed of cyclic molecules threaded along a linear polymer chain capped with bulky stopper molecules, have recently received considerable attention due to the unique properties of PRXs. PRXs that contain cyclodextrins (CDs) as a cyclic molecule are most widely used for material application. The interlocked CDs in PRXs can slide and rotate along a polymer axle. This mobility of CDs offers new functions in the design of materials such as tough and stretchable hydrogels,1 elastic binders,2 cell culture surfaces,3,4 materials for tissue regeneration,5 drug delivery carriers,6−15 and diagnostic agents.16 However, because the solubility of unmodified PRXs is limited to several solvents (e.g., dimethyl sulfoxide, alkaline solution), this occasionally hampers the fabrication and application of PRXs.17,18 The insolubility of PRXs is attributed to the formation of strong intramolecular hydrogen bonding between hydroxy groups of threaded CDs. In general, the chemical modification of hydroxy groups of CDs threaded in PRXs modulates their solubility in a variety of organic solvents.18 In particular, the solubilization of PRXs in aqueous solutions is important to their biomedical and pharmaceutical applications. © 2019 American Chemical Society

Received: April 15, 2019 Accepted: May 22, 2019 Published: June 19, 2019 826

DOI: 10.1021/acsmacrolett.9b00280 ACS Macro Lett. 2019, 8, 826−834

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ACS Macro Letters

Figure 1. Solubility of acetylated α-CD/PEG polyrotaxanes (Ac-PRXs) in aqueous solutions. (A) Schematic illustration of the synthesis of AcPRXs. (B) Images of the aqueous solutions of Ac-PRXs with various molecular weights of PEG axle and degree of acetylation (DA) at a concentration of 2 mg/mL (in distilled water).

Table 1. Reaction Conditions and Characterization of Ac-PRX100ks sample codea

feed molar ratio of [Ac2O]/[OH in PRX]

No. of acetyl groups on PRXb

DAc (%)

Mnd

Mw/Mne

PRX100k 6Ac-PRX100k 19Ac-PRX100k 26Ac-PRX100k 44Ac-PRX100k 47Ac-PRX100k 57Ac-PRX100k 75Ac-PRX100k 80Ac-PRX100k

0.1 0.5 1 2 3 5 10 15

336 (1.04) 1120 (3.39) 1520 (4.73) 2530 (7.85) 2720 (8.46) 3270 (10.2) 4360 (13.5) 4660 (14.5)

5.8 19.4 26.3 43.6 47.0 56.5 75.2 80.4

414000 428000 461000 478000 517000 528000 551000 597000 607000

2.18 2.46 2.42 2.21 2.30 2.27 2.38 2.31 2.30

a

Abbreviated as XAc-PRXY, where X and Y denote the degree of acetylation and Mn of PEG axle, respectively. bDetermined by 1H NMR in DMSOd6. The values in parentheses denote the average number of acetyl groups modified per threaded α-CD in Ac-PRXs. cDA: degree of acetylation. d Calculated based on the chemical composition of the Ac-PRXs determined by 1H NMR. eDetermined from SEC in DMSO using the calibration curve of standard PEG.

solutions and that Ac-PRXs may exhibit unique properties for material applications. In this study, a variety of Ac-PRXs with different degrees of acetylation (DA) and molecular weights of axle PEG were prepared to evaluate the solubility and solution properties of Ac-PRXs in aqueous media. Four series of α-CD/PEG-based PRXs with various molecular weights of axle PEG (Mn = 10000, 20000, 35000, and 100000) were synthesized (Supporting Information, Table S1),38 in which the percentage of threaded α-CDs was adjusted to almost comparable values (31.7 to 39.8%). Ac-PRXs were prepared from α-CD/PEG-

modulating the chemical properties of various carbohydrates, such as starch, dextran, and pullulan.31−34 Therefore, it is considered that the acetylation is potentially an effective method for improving the solubility of PRXs in aqueous solutions. There are few reports on the acetylation of PRXs and the improved solubility of acetylated α-CD/PEG-based PRXs (Ac-PRXs) in a variety of organic solvents.35−37 However, the solubility and properties of Ac-PRXs in aqueous solutions have not been studied in detail. Considering previous investigations on cellulose acetate,26,29,30 we hypothesized that the acetylation of PRXs might promote solubility in aqueous 827

DOI: 10.1021/acsmacrolett.9b00280 ACS Macro Lett. 2019, 8, 826−834

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Figure 2. Self-assembly of Ac-PRX100ks in aqueous solutions. (A) Relationship between the degree of acetylation of Ac-PRX100ks and the scattering intensity in distilled water (1 mg/mL). The data are expressed as mean ± SD (n = 3). (B) Distribution of the diameter of Ac-PRX100ks in distilled water (1 mg/mL). (C) Relationship between the degree of acetylation of Ac-PRX100ks and the number-average diameter (circles) of polydispersity index (squares) in distilled water (1 mg/mL). The data were expressed as mean ± SD (n = 3). (D) TEM image of 44Ac-PRX100k. (scale bar = 200 nm). (E) Histogram of the diameter distribution for PTX/44Ac-PRX100k determined from TEM image (n = 359). (F) I338/I333 ratios of pyrene in the presence of Ac-PRXs (6Ac-PRX100k: triangles, 44Ac-PRX100k: diamonds, 80Ac-PRX100k: circles).

based PRXs using acetic anhydride (Ac2O; Figure 1A),36 and they were identified using 1H NMR, Fourier transform infrared (FT-IR) spectroscopy, and size exclusion chromatography (SEC; Supporting Information, Figures S1−S3). The characterization results of Ac-PRXs are summarized in Table 1 (AcPRX100ks) and Supporting Information, Table S2 (Ac-PRX10ks, Ac-PRX20k, and Ac-PRX35k). By varying the feed molar ratio of Ac2O to hydroxy groups of threaded α-CDs in PRXs, Ac-PRXs with various DAs were prepared. Because the number-averaged molecular weight (Mn) of Ac-PRX100ks were difficult to be determined from SEC (Supporting Information, Table S3), the Mn values of Ac-PRX100ks were calculated based on the chemical composition of Ac-PRX100ks determined by 1H NMR. The solubility of Ac-PRXs in aqueous solutions was assessed by dissolving in distilled water at a concentration of 2 mg/mL (Figure 1B). Although unmodified PRX (DA 0%) was precipitated in water, it was determined that Ac-PRXs with low DAs (less than 36% for Ac-PRX10ks and 26% for AcPRX100ks) dissolved in water and yielded transparent solutions. However, in the case of Ac-PRX10ks, Ac-PRXs with high DA (DA more than 48%) precipitated into the solutions. Although the solubility of Me-PRXs in water did not change with the degree of methylation,25,27 the solubility of Ac-PRXs in aqueous solutions was different from that of the previously reported water-soluble PRXs. It is considered that the large number of acetyl groups increased the hydrophobicity and precipitated into the solutions, similar to the DA dependency of cellulose acetate.29,30 Consequently, the solubility of AcPRXs in water is largely dependent on the DA, and Ac-PRXs

with low DAs can be dissolved in water. On the contrary, it is interesting to note that Ac-PRXs with a high molecular weight axle PEG (Ac-PRX100ks) and high DAs (more than 43%) dissolved in water and resulted in colloidal dispersion. These colloidal dispersions were not observed for Ac-PRX20k and AcPRX35k with a DA of approximately 40%. Therefore, these results suggest that the molecular weight of axle PEG also strongly affects the solubility of Ac-PRXs in aqueous solutions. It is considered that Ac-PRX100ks with high DA were assembled into colloidal nanoparticles and dissolved in aqueous solutions. The scattered light intensity of the AcPRX100k solutions increased when the DA exceeded the threshold value (more than 43%; Figure 2A). This result indicates that Ac-PRX100ks with high DA form nanoparticles in aqueous solutions because the scattered light intensity is dependent on the sixth power of the diameter in Rayleigh scattering.39 The diameter of Ac-PRX100ks was determined using dynamic light scattering (DLS), and it was determined that the values with DA of less than 26% are 15−17 nm (Figure 2B,C), which is roughly comparable to the hydrodynamic diameter of PEG100k estimated based on the previous study (approximately 20 nm).40,41 Given that PEG has an expanded random coil conformation in water,40 Ac-PRX100ks with low DAs are considered to behave as a flexible random coil polymer in water. On the contrary, a larger diameter was confirmed for Ac-PRX100ks with high DA (Figure 2B). Additionally, the diameter of Ac-PRX100ks increased when the DA exceeded the threshold value (more than 43%), accompanied by a decrease in the polydispersity index (PDI; 828

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Figure 3. Contribution of hydrogen bonding and hydrophobic interaction in the self-assembly of Ac-PRX100ks. (A) FT-IR spectra of Ac-PRX100ks ranging from 2500 to 4000 cm−1. (B) Relationship between the degree of acetylation of Ac-PRX100ks and the maximum wavenumber of residual hydroxy groups. The dotted line denotes the linear fit of each plot. (C) Fluorescence spectra of TNS in the aqueous solution of Ac-PRX100k s (1 mg/mL). (D) Relationship between the degree of acetylation of Ac-PRX100ks and the fluorescence intensity of TNS. The data were expressed as mean ± SD (n = 3).

In the FT-IR spectra of Ac-PRX100ks, the peaks corresponding to the residual hydroxy groups are proportionally shifted to higher wavenumbers with increasing DA (Figure 3A,B). This result indicates that the formation of intramolecular hydrogen bonding among Ac-PRX100ks is reduced with increasing DA.46 This was also confirmed by X-ray diffraction analysis of AcPRX100ks (Supporting Information, Figure S5). Although unmodified PRX100k exhibited a strong diffraction peak at 2θ = 19.9° due to the formation of a hexagonal columnar structure via intramolecular hydrogen bonding,47,48 this typical peak disappeared after the modification of the acetyl groups. This result also suggests that the hydrogen bonding in Ac-PRX100ks was mitigated. The reduction of hydrogen bonding in AcPRX100ks is the main reason for acquiring the solubility in aqueous solutions of Ac-PRXs with low DA, which is a similar mechanism to that of the methylated PRXs.26 However, this does not adequately explain the self-assembly of Ac-PRX100ks with high DA. Next, the contribution of hydrophobic interactions of acetyl groups in Ac-PRX100ks was examined using 6-(p-toluidino)-2naphthalenesulfonic acid (TNS; Figure 3C,D), which emits strong fluorescence in a nonpolar environment.49 In the case of Ac-PRX100ks with DA of less than 26%, negligible fluorescence of TNS was observed. This indicates that Ac-PRX100ks with low DA dissolved in water without forming hydrophobic microenvironments. In contrast, the fluorescence intensity of TNS increased when the DA of Ac-PRX100ks was over 44%, and the intensity gradually increased with DA. It is expected that acetylated α-CDs threaded in Ac-PRX100ks are associated with

Figure 2B,C). For further confirmation, transmission electron microscopy (TEM) was performed for 44Ac-PRX100k, and the spherical nanoparticles were clearly observed (Figure 2D). From the TEM images, the average diameter of the 44AcPRX100k nanoparticles was determined to be 71.7 ± 19.1 nm (coefficient of variation: 0.26, n = 359; Figure 2E), which was consistent with the diameter determined by DLS (67.1 ± 29.4 nm). Additionally, Ac-PRX100ks with high DA showed critical micelle concentration (292 μg/mL for 44Ac-PRX100k, 258 μg/ mL for 80Ac-PRX100k; Figure 2F), which was determined using pyrene as a probe. On the other hand, Ac-PRX100ks with low DA (6Ac-PRX100k) showed no critical micelle concentration. These results clearly indicate that nanoparticles are formed by the self-assembly of Ac-PRX100ks. Furthermore, the diameter and PDI of Ac-PRX100ks (26Ac-, 44Ac-, and 80Ac-PRX100ks) varied with the feed concentration, but remained unchanged at a feed concentration over 2 mg/mL (Supporting Information, Figure S4). According to these results, Ac-PRX100ks with high DA form nanoparticles in aqueous solutions, which is likely attributable to the self-assembly of Ac-PRX100ks. Thus far, some reports have described the formation of nanoparticles via the self-assembly of PRXs.12,13,42−45 For example, hydrophilic polymer-conjugated PRXs form selfassembled nanoparticles via the intramolecular hydrogen bonding between the hydroxy groups of threaded CDs.12,13,42,43 According to these previous studies, it is considered that the formation of hydrogen bonding or hydrophobic interactions between acetyl groups are the main force to induce the self-assembly of Ac-PRX100ks with high DA. 829

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Figure 4. Predicted structure and colloidal stability of Ac-PRX100ks in aqueous solutions. (A) 1H NMR spectra of Ac-PRX100k s in D2O. (B) Schematic illustration of the potential structure of the self-assembled nanoparticles of Ac-PRX100ks in aqueous solutions. (C) Zeta-potential of AcPRX100ks in distilled water (1 mg/mL). The data were expressed as mean ± SD (n = 3). (D) Effect of the concentration of NaCl on the diameter (closed symbols) and polydispersity index (PDI, open symbols) of Ac-PRX100ks (44Ac-PRX100k: diamonds, 80Ac-PRX100k: circles). The data were expressed as mean ± SD (n = 3).

almost zero, regardless of the DA (Figure 4C), and the diameter and PDI of the Ac-PRX100k nanoparticles remained unchanged even at a NaCl concentration of 500 mM (Figure 4D). Collectively, threaded acetylated α-CDs in Ac-PRX100ks are associated via hydrophobic interactions and outer noncovered segments of axle PEGs, and the PEG layer is hydrated to provide excellent colloidal stability. However, it is still unclear why Ac-PRXs with the high-molecular-weight axle PEG assemble into nanoparticles in aqueous solutions. Because it is reported that the length of axle PEG and the threading ratio of α-CDs in PRX affects the colloidal stability of PTXbased self-assembled nanoparticles,44 the length of the noncovered segments of axle PEGs or the flexibility of axle PEG are related to the excellent colloidal stability of AcPRX100k nanoparticles. Finally, the encapsulation of hydrophobic molecules in the core of Ac-PRX100ks was investigated to evaluate the potential applications of Ac-PRX100k nanoparticles as a drug carrier. In this experiment, paclitaxel (PTX), which is regarded as a poorly soluble drug with a 1-octanol/water partition coefficient of Log Pow = 4.4,54 was selected as a model drug. The encapsulation of PTX in three series of Ac-PRX100ks (6Ac-, 44Ac-, and 80Ac-PRX100ks) was performed to verify the effects of DA on the encapsulation of PTX. PTX was successfully encapsulated in 44Ac- and 80Ac-PRX100ks, and the loading content of PTX increased with the feed content of PTX (Figure 5A). The maximum loading content of PTX was 19.5 ± 5.3% and 17.0 ± 3.1% for 44Ac- and 80Ac-PRX100ks, respectively, which was nearly equal to the level of the other

forming hydrophobic microenvironments, especially for DAs of Ac-PRX100ks in excess of 44%. Consequently, the hydrophobic interactions between the acetyl groups modified on AcPRX100ks are critical for the formation of self-assembled nanoparticles. Figure 4A shows the 1H NMR spectra of Ac-PRX100ks in D2O, in which the signals of threading α-CDs (approximately 3.8 to 4.1 ppm) and terminal adamantyl groups (1.55, 1.75, and 2.16 ppm) are clearly observed for 6Ac-, 19Ac-, and 26AcPRX100ks. On the contrary, these signals completely disappeared for Ac-PRX100ks with DAs in excess of 44%, although these peaks can be observed clearly in DMSO-d6 (Supporting Information, Figure S1). In the 1H NMR spectra of AcPRX100ks with DAs in excess of 44%, only the signals of the PEG axle and acetyl groups are observed. These results provide important insights into the structure of the Ac-PRX100k nanoparticles; threaded acetylated α-CDs and hydrophobic adamantyl groups are associated with form a hydrophobic core that is surrounded by the noncovered segments of axle PEGs (Figure 4B). If this hypothesis is correct, the colloidal properties of Ac-PRX100k nanoparticles should be the same as that of PEGylated nanoparticles, such as liposomes, gold nanoparticles, and polymeric micelles.50−53 In this regard, the zeta-potential of the nanoparticles and the effect of salt concentration were examined, because most of the PEGylated nanoparticles exhibited almost neutral zeta-potential and good dispersion stability, even under high salt concentration due to the steric stabilization of the outer PEG layer. Actually, the zeta-potential values of the Ac-PRX100k nanoparticles were 830

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Figure 5. Loading of PTX in Ac-PRX100ks and their cytotoxic effects. (A, B) Effect of feed loading content of paclitaxel (PTX) on the (A) actual loading content of PTX and (B) loading efficiency in Ac-PRX100ks (6Ac-PRX100k: triangles,44Ac-PRX100k: diamonds, 80Ac-PRX100k: circles) in distilled water. The data were expressed as mean ± SD (n = 3). (C) Number-average diameter of PTX-loaded Ac-PRX100ks in distilled water. The data were expressed as mean ± SD (n = 3). (D) Viability of HeLa cells treated with PTX (squares) and PTX-loaded Ac-PRX100ks (44Ac-PRX100k: diamonds, 80Ac-PRX100k: circles) at various PTX concentrations for 24 h. The data were expressed as mean ± SD (n = 6). (E) Cell cycle of HeLa cells treated with PTX or PTX-loaded Ac-PRX100ks (44Ac-PRX100k, 80Ac-PRX100k) at PTX concentrations of 5, 10, and 50 ng/mL for 24 h. The data were expressed as mean ± SD (n = 3, *P < 0.05 and **P < 0.001 vs PBS-treated cells).

drug carriers such as polymeric nanoparticles and micelles.55 However, the loading efficiency was only 5.8 to 16.8% (Figure 5B), which was remarkably low compared to that of other polymeric nanoparticles.55 Because the critical micelle concentrations of Ac-PRX100k nanoparticles were relatively high (Figure 2F), the hydrophobic interaction of acetyl groups is considered to be weak. Therefore, the low loading efficiency of PTX in Ac-PRX100k nanoparticles may result from the weak hydrophobic interaction of acetyl groups. The diameter of PTX-loaded Ac-PRX100k nanoparticles ranged from 132 to 194 nm with a relatively narrow distribution, as determined by DLS, which was almost unchanged with the loading content and DA value (Figure 5C, and Supporting Information, Figures S7 and S8). The formation of nanoparticles was also confirmed by TEM observation (Supporting Information, Figure S9). Interestingly, 6Ac-PRX100k, which does not form nanoparticles in aqueous solutions, failed to encapsulate PTX. This was probably because the hydrophobicity was insufficient to encapsulate hydrophobic PTX. Additionally, the diameter of PTX/44Acand PTX/80Ac-PRX100k nanoparticles in physiological conditions (in PBS at 37 °C) was investigated by DLS (Supporting Information, Figure S10). The diameter of PTX/80Ac-PRX100k nanoparticles was unchanged upon incubation in physiological conditions for 96 h, whereas the diameter of PTX/44Ac-PRX100k nanoparticles slightly in-

creased in physiological conditions, suggesting that the degree of acetylation affects the stability of nanoparticles. PTX-loaded Ac-PRX100ks exhibited toxic effects in human cervical cancer cell lines (HeLa cells), but to a lower extent compared to free PTX (IC50 of free PTX, PTX/44Ac-PRX100k, and PTX/80Ac-PRX100k were 9.9 ± 0.8, 23.8 ± 2.5, and 35.7 ± 3.6 ng/mL, respectively; Figure 5D). It is predicted that the differences in IC50 values are related to the cellular uptake and intracellular release rate of PTX. Additionally, the arrest of cell cycle at G2/M phased and increase of the sub-G1 populations were observed at concentrations of 50 and 5 ng/mL, respectively, for both free PTX and PTX-loaded Ac-PRX100ks (Figure 5E and Supporting Information, Figure S11). This represents typical behaviors of PTX-treated cells.56,57 Accordingly, PTX loaded in Ac-PRX100k nanoparticles can exhibit toxic effects with a mechanism of cell death similar to that of free PTX. In summary, the aqueous solubility of Ac-PRXs with various DA and molecular weight of axle PEG was investigated and it was determined that Ac-PRXs with low DA can be dissolved in aqueous solutions regardless of the molecular weight of axle PEG. Additionally, Ac-PRXs with high DA and high molecular weight of axle PEG (Mn = 100 000) were dissolved in water via the formation of self-assembled nanoparticles, whereas AcPRXs with high DA and low molecular weight of axle PEG (less than 35 000) were precipitated in the solutions. It is considered that threaded acetylated α-CDs in Ac-PRX100ks 831

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viable material via integration of biology and engineering” from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

form hydrophobic microenvironments via hydrophobic interactions that are surrounded by noncovered segments of axle PEGs. Moreover, the outer hydrated PEG layer provides excellent colloidal stability. Furthermore, we examined the potential applications of Ac-PRX100k as a drug carrier and confirmed that Ac-PRX100k can encapsulate hydrophobic PTX and exhibits cytotoxic effects. Although the advantage of AcPRX100k nanoparticles as a drug carrier was not fully evaluated in this study, we considered that the PRX architecture is advantageous in facilitating supramolecular degradation. When we introduce cleavable linkers in the axle PEG, the resulting PRX can degrade into their constituent molecules in response to physical and chemical stimuli (e.g., pH, light, reduction).58,59 Therefore, when the cleavable linkers are introduced in Ac-PRX100k, Ac-PRX100k nanoparticles can be exploited as a new class of biodegradable drug carriers that can be degraded using a specific stimulus. Further studies on the properties of Ac-PRX100k nanoparticles as a drug carrier and the design of biodegradable Ac-PRX100k nanoparticles are currently underway.

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(1) Imran, A. B.; Esaki, K.; Gotoh, H.; Seki, T.; Ito, K.; Sakai, Y.; Takeoka, Y. Extremely Stretchable Thermosensitive Hydrogels by Introducing Slide-Ring Polyrotaxane Cross-Linkers and Ionic Groups into the Polymer Network. Nat. Commun. 2014, 5, 5124. (2) Choi, S.; Kwon, T. W.; Coskun, A.; Choi, J. W. Highly Elastic Binders Integrating Polyrotaxanes for Silicon Microparticle Anodes in Lithium Ion Batteries. Science 2017, 357, 279−283. (3) Singh, A.; Zhan, J.; Ye, Z.; Elisseeff, J. H. Modular Multifunctional Poly(ethylene glycol) Hydrogels for Stem Cell Differentiation. Adv. Funct. Mater. 2013, 23, 575−582. (4) Arisaka, Y.; Yui, N. Polyrotaxane-Based Biointerfaces with Dynamic Biomaterial Functions. J. Mater. Chem. B 2019, 7, 2123− 2129. (5) Zhao, X.; Song, W.; Li, W.; Liu, S.; Wang, L.; Ren, L. Collagen Membranes Crosslinked by β-Cyclodextrin Polyrotaxane Monoaldehyde with Good Biocompatibilities and Repair Capabilities for Cornea Repair. RSC Adv. 2017, 7, 28865−28875. (6) Ooya, T.; Choi, H. S.; Yamashita, A.; Yui, N.; Sugaya, Y.; Kano, A.; Maruyama, A.; Akita, H.; Ito, R.; Kogure, K.; Harashima, H. Biocleavable Polyrotaxane−Plasmid DNA Polyplex for Enhanced Gene Delivery. J. Am. Chem. Soc. 2006, 128, 3852−3853. (7) Li, J.; Yang, C.; Li, H.; Wang, X.; Goh, S. H.; Ding, J. L.; Wang, D. Y.; Leong, K. W. Cationic Supramolecules Composed of Multiple Oligoethylenimine-Grafted β-Cyclodextrins Threaded on a Polymer Chain for Efficient Gene Delivery. Adv. Mater. 2006, 18, 2969−2974. (8) Yu, S.; Zhang, Y.; Wang, X.; Zhen, X.; Zhang, Z.; Wu, W.; Jiang, X. Synthesis of Paclitaxel-Conjugated β-Cyclodextrin Polyrotaxane and Its Antitumor Activity. Angew. Chem., Int. Ed. 2013, 52, 7272− 7277. (9) Yu, G.; Yang, Z.; Fu, X.; Yung, B. C.; Yang, J.; Mao, Z.; Shao, L.; Hua, B.; Liu, Y.; Zhang, F.; Fan, Q.; Wang, S.; Jacobson, O.; Jin, A.; Gao, C.; Tang, X.; Huang, F.; Chen, X. Polyrotaxane-Based Supramolecular Theranostics. Nat. Commun. 2018, 9, 766. (10) Terauchi, M.; Inada, T.; Kanemaru, T.; Ikeda, G.; Tonegawa, A.; Nishida, K.; Arisaka, Y.; Tamura, A.; Yamaguchi, S.; Yui, N. Potentiating Bioactivity of BMP-2 by Polyelectrolyte Complexation with Sulfonated Polyrotaxanes to Induce Rapid Bone Regeneration in a Mouse Calvarial Defect. J. Biomed. Mater. Res., Part A 2017, 105, 1355−1363. (11) Liu, Z.; Lin, T. M.; Purro, M.; Xiong, M. P. Enzymatically Biodegradable Polyrotaxane−Deferoxamine Conjugates for Iron Chelation. ACS Appl. Mater. Interfaces 2016, 8, 25788−25797. (12) Zhang, X.; Ke, F.; Han, J.; Ye, L.; Liang, D.; Zhang, A. Y.; Feng, Z. G. The Self-Aggregation Behaviour of Amphotericin B-Loaded Polyrotaxane-Based Triblock Copolymers and Their Hemolytic Evaluation. Soft Matter 2009, 5, 4797−4803. (13) Jiang, L.; Gao, Z. M.; Ye, L.; Zhang, A. Y.; Feng, Z. G. A pHSensitive Nano Drug Delivery System of Doxorubicin-Conjugated Amphiphilic Polyrotaxane-Based Block Copolymers. Biomater. Sci. 2013, 1, 1282−1291. (14) Tamura, A.; Yui, N. Polyrotaxane-Based Systemic Delivery of βCyclodextrins for Potentiating Therapeutic Efficacy in a Mouse Model of Niemann-Pick Type C Disease. J. Controlled Release 2018, 269, 148−158. (15) Tamura, A.; Ohashi, M.; Nishida, K.; Yui, N. Acid-Induced Intracellular Dissociation of β-Cyclodextrin-Threaded Polyrotaxanes Directed Towards Attenuating Phototoxicity of Bisretinoids Through Promoting Excretion. Mol. Pharmaceutics 2017, 14, 4714−4724. (16) Zhou, Z.; Mondjinou, Y.; Hyun, S. H.; Kulkarni, A.; Lu, Z. R.; Thompson, D. H. Gd3+-1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic-2-hydroxypropyl-β-cyclodextrin/Pluronic Polyrotaxane as a Long Circulating High Relaxivity MRI Contrast Agent. ACS Appl. Mater. Interfaces 2015, 7, 22272−22276.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.9b00280.

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REFERENCES

Additional text with details on the synthesis of Ac-PRXs, experimental methods, characterization of PRXs (Table S1), characterization of Ac-PRXs with the Mn of axle PEG of 10000, 20000, and 35000 (Table S2), 1H NMR spectra of Ac-PRX100ks (Figure S1), FT-IR spectra of AcPRX100ks (Figure S2), size exclusion chromatograms of Ac-PRX100ks (Figure S3 and Table 3), effects of the feed concentration of Ac-PRX100ks (Figure S4), X-ray diffractograms of Ac-PRX100ks (Figure S5), calibration curve for PTX (Figure S6), size distribution of PTXloaded Ac-PRX100ks (Figure S7), polydispersity indices of PTX-loaded Ac-PRX100ks (Figure S8), TEM image of PTX-loaded Ac-PRX100k (Figure S9), diameters of PTXloaded Ac-PRX 100k under physiological condition (Figure S10), and cell cycle analysis of HeLa cells treated with PTX-loaded Ac-PRX100ks (Figure S11) (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Atsushi Tamura: 0000-0003-0235-7364 Nobuhiko Yui: 0000-0001-5212-1371 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the following grants: Grant-in-Aid for Young Scientists (A) from Japan Society for the Promotion of Science (JSPS; JSPS KAKENHI Grant No. JP16H05910 to A.T.); Challenging Research (Exploratory) from JSPS (JSPS KAKENHI Grant No. JP18K19904 to A.T.); and Cooperative project among medicine, dentistry, and engineering for medical innovation “Construction of creative scientific research of the 832

DOI: 10.1021/acsmacrolett.9b00280 ACS Macro Lett. 2019, 8, 826−834

Letter

ACS Macro Letters

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DOI: 10.1021/acsmacrolett.9b00280 ACS Macro Lett. 2019, 8, 826−834

Letter

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DOI: 10.1021/acsmacrolett.9b00280 ACS Macro Lett. 2019, 8, 826−834