Tailoring the Temperature-Induced Phase Transition and Coacervate

Aug 12, 2016 - Polyrotaxanes (PRXs) composed of threading methylated cyclodextrins exhibit a temperature-dependent reversible phase transition across ...
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Tailoring the Temperature-Induced Phase Transition and Coacervate Formation of Methylated β‑Cyclodextrins-Threaded Polyrotaxanes in Aqueous Solution Kei Nishida, Atsushi Tamura, and Nobuhiko Yui* Department of Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan ABSTRACT: Polyrotaxanes (PRXs) composed of threading methylated cyclodextrins exhibit a temperature-dependent reversible phase transition across the lower critical solution temperature (LCST) in water. In this study, a variety of methylated β-cyclodextrin (β-CD)-threaded PRXs (Me-PRXs) with different number of methyl groups modified on the β-CD and with different compositions of axle polymers (i.e., Pluronic, PEG-b-PPG-b-PEG) were synthesized, and the relationship between the LCST value and structural parameters was investigated. The LCST values of the Me-PRXs decreased with an increasing the methylation degree of the threading β-CD. Differential scanning calorimetric measurements demonstrated that the temperature-induced phase transition of the Me-PRXs occurred due to the dehydration of the threading methylated βCD moieties and the subsequent hydrophobic interactions. Interestingly, the Me-PRXs formed coacervate droplets above their LCST. Altogether, we concluded that the LCST values of the Me-PRXs varied according to their structural parameters and could be applied as temperature-responsive biomaterials or therapeutics.

1. INTRODUCTION Polyrotaxanes (PRXs) consisting of an inclusion complex of cyclodextrins (CDs) and a linear polymer capped with bulky stopper molecules exhibit interesting properties in the design of functional materials including biomedical applications.1−3 The threading CDs are allowed to move freely along the polymer chain because they are localized onto a linear polymer through noncovalent interactions in PRXs. Using this mobile characteristic of PRXs, a variety of applications including flexible and tough hydrogels for wearable devices, ligand-modified PRXs for enhanced molecular recognition, and as cell culture surfaces for the regulation of cellular differentiation have been developed.4−6 Additionally, when the cleavable linkages are introduced in the axle polymer of the PRXs, the supramolecular interlocked structure of the PRXs can be dissociated in response to environmental stimuli such as pH, light irradiation, and reaction with specific molecules.7−9 On the basis of this unique property, we have demonstrated that cleavable PRXs have great potential for biomaterials applications, such as biodegradable scaffolds, delivery carriers for bioactive molecules (e.g., nucleic acids and proteins), and dental resins.3,8−10 Recently, we developed β-cyclodextrin (β-CD)/Pluronicbased stimuli-labile PRXs as potential therapeutics for Niemann−Pick type C (NPC) disease,11−13 which is a congenital metabolic disorder characterized by the lysosomal accumulation of cholesterols.14 The stimuli-labile PRXs showed superior ability to reduce lysosomal cholesterol content compared to 2-hydroxypropyl-β-CDs (HP-β-CDs), making the stimuli-labile PRXs a promising candidate for the treatment © 2016 American Chemical Society

of NPC disease due to the lysosomal local release of threading β-CDs from the PRXs.11−13 Additionally, we recently found that the inclusion complexation ability of released β-CDs from the stimuli-labile PRXs with cholesterol is a predominant factor that determines the cholesterol reducing effect in NPC disease,13 suggesting that the modulation of inclusion complexation ability of released β-CDs is an essential strategy to reinforce the therapeutic effect on NPC disease. In general, the inclusion complexation ability of β-CD can be modulated via chemical modification; a variety of chemically modified β-CD derivatives have been developed for pharmaceutical applications.15,16 Among various β-CD derivatives, methylated β-CDs have the greatest ability to form an inclusion complex with cholesterols and adamantanes.16−18 Accordingly, methylated β-CDs-threaded PRXs (Me-PRXs) that can release threading methylated β-CDs in response to lysosomal pH are promising for further progressing the therapeutic potential of PRXs. However, previously reported methylated CDs-threaded PRXs composed of methylated α-CDs and poly(ethylene glycol) (PEG) exhibited a temperature-dependent phase transition across the lower critical solution temperature (LCST) in water.19,20 The LCST value for methylated CDsthreaded PRXs with a high methylation degree of threaded αCDs is below the physiological temperature (37 °C). Additionally, the pseudopolyrotaxanes formed from heptakisReceived: July 13, 2016 Revised: August 4, 2016 Published: August 12, 2016 6021

DOI: 10.1021/acs.macromol.6b01493 Macromolecules 2016, 49, 6021−6030

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Scheme 1. Synthesis of Methylated β-CDs-Threaded PRXs (Me-PRXs)

(2,6-di-O-methyl)-β-cyclodextrins (DM-β-CDs) and poly(propylene glycol) (PPG) precipitated in water at the temperature of 35 °C.21 Consequently, it seems to be difficult to consider the pharmacological application of Me-PRXs due to aggregation at a physiological temperature. In this study, to gain knowledge regarding the temperaturedependent phase transition behaviors of the methylated β-CDsthreaded PRXs, a variety of β-CD/Pluronic-based Me-PRXs with different numbers of methyl groups on β-CD and with different compositions of axle polymers were synthesized, and their temperature-dependent phase transition behavior was investigated. Additionally, the relationship between the phase transition temperature and the chemical compositions of the Me-PRXs was investigated to better understand the phase transition behavior of Me-PRXs and to determine the predominant factor for regulating the LCST. This comprehensive study could provide important insight into the application of the Me-PRXs as therapeutics or temperatureresponsive biomaterials.

2. MATERIALS AND METHODS 2.1. Materials. β-CD was obtained from Nihon Shokuhin Kako (Tokyo, Japan). Pluronic P105, an ABA-type triblock copolymer of poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG) (PEG-b-PPG-b-PEG), (Mn: 6400, PEG segment Mn,PEG: 1610 × 2, PPG segment Mn,PPG: 3180) was kindly provided by Dai-ichi Kogyo Seiyaku (Kyoto, Japan). Pluronic P84 (Mn: 4330, PEG segment Mn,PEG: 850 × 2, PPG segment Mn,PPG: 2630) and Pluronic P103 (Mn: 4870, PEG segment Mn,PEG: 810 × 2, PPG segment Mn,PPG: 3250) were kindly provided by Adeka (Tokyo, Japan). Pluronic P123 (Mn: 6350, PEG segment Mn,PEG: 1100 × 2, PPG segment Mn,PPG: 4150), Pluronic L121 (Mn: 4400, PEG segment Mn,PEG: 325 × 2, PPG segment Mn,PPG: 3750), Pluronic F127 (Mn: 12 600, PEG segment Mn,PEG: 4550 × 2, PPG segment Mn,PPG: 3500), N-(triphenylmethyl)glycine (Trt-Gly-OH), randomly methylated β-cyclodextrin (RM-βCD), heptakis(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD), and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin were obtained from Sigma-Aldrich (Milwaukee, WI). Sodium hydroxide (NaOH) and methyl iodide (MeI) were obtained from Wako Pure Chemical Industries (Osaka, Japan). N-Hydroxysuccinimide (NHS) was obtained from Acros Organics (Geel, Belgium). Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Other solvents were obtained from Kanto Chemicals (Tokyo, Japan). 2.2. Characterization of Polyrotaxanes. Size exclusion chromatography (SEC) was carried out using an HLC-8120 system (Tosoh, Tokyo, Japan) equipped with a combination of TSKgel α4000 and α-2500 columns (Tosoh). The system was eluted with dimethyl sulfoxide (DMSO) containing 10 mM LiBr at a flow rate of 0.3 mL/min at 60 °C. 1H nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance III 400 MHz spectrometer (Bruker BioSpin, Rheinstetten, Germany). Polydispersity index (Mw/ Mn) was calculated from a calibration curve of standard PEGs (Agilent Technologies, Wilmington, DE). The wide-angle X-ray diffraction (XRD) measurements were performed using a D8 Advance (Bruker AXS, Oestliche Rheinbrueckenstr, Germany) with Cu Kα radiation (λ = 1.542 Å). 2.3. Synthesis of Methyl Group-Modified β-CD/Pluronic Polyrotaxanes. The β-CD/Pluronic-based polyrotaxanes bearing N-triphenylmethyl (N-Trt) end groups (PRX) were synthesized according to our previous report (Scheme 1).13 The typical procedure for the synthesis of methyl group-modified PRXs (14.1Me-P123 in Table 1) is as follows: PRX (200 mg, 9.44 μmol of PRX, 0.11 mmol of threading β-CDs) was dissolved in dehydrated DMSO (15 mL). Powdered NaOH (204.9 mg, 5.12 mmol, 2.2 mol equiv to hydroxyl groups of threading β-CDs) and MeI (106.5 mg, 1.71 mmol, 0.73 mol equiv to hydroxyl groups of threading β-CDs) were successively added

to the solution, and the reaction mixture was vigorously stirred for 1 h at room temperature. After the reaction, the reaction mixture was poured into diethyl ether, and the precipitate was collected. Then, the precipitate was dissolved in water and dialyzed against water (Spectra/ Por 3, molecular weight cutoff of 3500 Da) (Spectrum Laboratories, Los Angeles, CA) for 2 days at 4 °C. The recovered solution was freeze-dried to obtain the Me-PRX (14.1Me-P123) (128.9 mg, 55.7% yield). Other Me-PRXs with different axle polymers were synthesized in the same manner (Table 1). Because the peaks of methyl groups on threaded β-CDs in the Me-PRXs were overlapped with the peaks of the Pluronic main chain (−CH2CH2O− and −CH(CH3)−CH2−) and the threading β-CDs (H2, H3, H4, H5, and H6 protons) in the 1H NMR spectra of Me-PRXs, the number of methyl groups was calculated by subtracting the integral ratio at 3.0−4.0 ppm in the precursor PRXs from the integral ratio at 3.0−4.0 ppm in the MePRXs. The Mn,NMR of Me-PRX was calculated based on the number of threaded β-CDs and methyl groups. 1H NMR (400 MHz, D2O): δ = 1.10 ppm (−CH2−CH(CH3)−O− of Pluronic), 3.0−4.0 (m, −CH2CH2O− and −CH(CH3)−CH2− of Pluronic main chain, H2, H3, H4, H5, and H6 protons of β-CD, and −OCH3 of methyl group), 4.9−5.3 (m, H1 proton of β-CD), 7.21 (t, Trt group), 7.30 (t, Trt group), 7.42 (d, Trt group). 2.4. Transmittance Measurements. The Me-PRXs were dissolved in phosphate-buffered saline (PBS, 1.47 mM KH2PO4, 8.1 mM Na2PO4, 2.68 mM KCl, 137 mM NaCl, pH 7.4) at a concentration of 0.1−10.0 mg/mL. Temperature-dependent optical transmittance at 600 nm of the Me-PRX solutions was recorded using a V-550 UV/vis spectrophotometer (Jasco, Tokyo, Japan) equipped with an ETC-505T peltier-type thermostatic cell holder (Jasco) at a heating or cooling rate of 1.0 °C/min. The LCST values (Tcl) of the solutions were defined as the temperature at which the initial transmittance was reduced to 50%. 2.5. Differential Scanning Calorimetric Measurements. MePRXs, Pluronic P123, and β-CD derivatives dissolved in PBS (10 mg/ mL for Me-PRXs and Pluronic P123, 20 mg/mL for β-CD derivatives) were degassed for 10 min just before measurements. DSC thermograms were recorded using a MicroCal VP-DSC (Malvern Instruments, Westborough, MA) at a heating rate of 1.0 °C/min. The phase transition temperature (Tendo) was determined from the middle point of the peak in the DSC thermogram. The degree of transition enthalpy change (ΔH) was determined from the area of the phase transition peak. The degree of transition entropy change (ΔS) was calculated using the equation ΔH − TΔS = 0 due to the Gibbs energy change ΔG = 0 at Tendo. 2.6. Formation of Coacervate Droplets. The Me-PRXs were dissolved in PBS (1−10 mg/mL), and the solutions were incubated at 6022

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Macromolecules Table 1. Characterization of the Me-PRXs Used in This Study codea

axle polymer

6.3Me-P123 14.1Me-P123 21.0Me-P123 15.2Me-L121 15.3Me-P84 12.2Me-P103 13.8Me-P105 13.1Me-F127 10.8Me-PEG

Pluronic P123 Pluronic P123 Pluronic P123 Pluronic L121 Pluronic P84 Pluronic P103 Pluronic P105 Pluronic F127 poly(ethylene glycol)

no. of threaded β-CDsb 11.8 11.8 11.8 11.9 10.4 12.8 12.7 9.44 40.0

(33.7%) (33.7%) (33.7%) (39.6%) (52.0%) (42.6%) (45.4%) (27.0%) (37.0%)

no. of methyl groups on PRXc 74.3 166.4 247.8 180.9 159.1 156.2 175.3 123.7 432.0

(6.30) (14.1) (21.0) (15.2) (15.3) (12.2) (13.8) (13.1) (10.8)

Mnd

Mw/Mne

21 900 23 300 24 500 18 900 18 900 22 200 24 000 25 200 55 900

1.05 1.05 1.07 1.11 1.10 1.03 1.19 1.03 1.09

Abbreviated as XMe-Y, where X and Y depict the number of methyl groups on threading β-CD and the axle polymer in the Me-PRXs, respectively. The values in parentheses depict the threading β-CDs ratio against the theoretical maximum number of threaded β-CDs onto the PPG segment of Pluronic. cThe values in parentheses depict the number of methyl groups on threading β-CD in the Me-PRXs. dCalculated from the molecular composition determined using 1H NMR. eDetermined by SEC in DMSO containing 10 mM LiBr at 60 °C. a b

the prescribed temperature for 2 h to equilibrate the temperature. Then, optical microscopic images were acquired using an IX-71 (Olympus, Tokyo, Japan) equipped with a DP-80 dual CCD camera (Olympus) and a Lucplfn 40× objective lens (Olympus). The diameters of the coacervate droplets were determined with an ImageJ software ver. 1.49 (National Institutes of Health, Bethesda, MD) and expressed as a Feret diameter, which is defined as the distance between two tangents on opposite sides of a particle. For fluorescence microscopic observation, fluorescein isothiocyanate (FITC)-labeled Me-PRXs (FITC-14.1Me-PRX) were synthesized in accordance with a previous report.11 The solution of FITC-14.1Me-P123 (5.0 mg/mL) was observed via fluorescence microscopy as described above.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Methyl Groups-Modified β-CD/ Pluronic Polyrotaxanes (Me-PRXs). The synthetic methods for the methylated CDs-threaded PRXs are classified into two major methodologies. One is the inclusion complexation between methylated CDs and an axle polymer, followed by the end-capping with bulky molecules.21−23 Although this method is convenient to prepare methylated β-CDs-threaded PRXs, the degree of methylation in the methylated β-CDsthreaded PRXs is limited because only DM-β-CDs forms pseudopolyrotaxanes with PPG and Pluronic. The other is the chemical modification of the PRXs by using methylation reagents.5,19 In this study, to prepare the methylated β-CDsthreaded PRXs with different degrees of methylation, the methylated β-CDs-threaded PRXs were synthesized via the methylation of the β-CD/Pluronic P123-based PRXs capped with N-triphenylmethyl end groups (Scheme 1). The methylation of the β-CDs-threaded PRXs was performed using MeI and powdered NaOH in DMSO for 1 h to avoid the degradation of the PRXs.24 By varying the feed molar ratios of NaOH and MeI to the PRXs, the Me-PRXs with different numbers of methyl groups were synthesized. SEC charts of the Me-PRXs revealed that the peaks of MePRXs with different numbers of methyl groups were unimodal and that the peak tops were slightly shifted to a high molecular weight region compared unmodified PRX (Figure 1a). This result suggests that the Me-PRXs were successfully synthesized without the degradation of their interlocked structure. In the 1 H NMR spectra of the Me-PRXs, the peaks assignable to methyl groups were observed in the Me-PRXs, indicating successful methylation of the threading β-CDs in the PRXs (Figure 1b). In the 1H NMR spectra of 14.1Me-P123, the peaks of methyl groups appeared at 3.2 and 3.6 ppm, which were similar to the peak of DM-β-CDs. This result suggests that

Figure 1. (a) SEC charts of β-CD, Pluronic P123, unmodified PRX (the number of threaded β-CDs of 11.8), and the Me-PRXs with different numbers of methyl groups on β-CD. (b) 1H NMR spectra of DM-β-CD, TM-β-CD, unmodified PRX, and the Me-PRXs with different numbers of methyl groups on β-CD in D2O.

methyl groups were initially introduced to the C2-OH and C6OH positions of the threading β-CDs on the PRXs. By further increasing the feed methylation reagents, the C3-OH position of the threading β-CDs on the PRXs was converted into a 6023

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upon increasing temperature to a physiological condition (37 °C), clearly showing that the Me-PRXs exhibit LCST-type phase transition. The detailed temperature-dependent phase transition behaviors of the Me-PRXs with different numbers of methyl groups were investigated via transmittance measurements at various concentrations. In this study, PBS was employed as a solvent to understand the LCST values under physiological salt concentration. The temperature-dependent phase transition of the Me-PRXs (14.1Me-P123) was reversible over heating-and-cooling cycles (Figure 3b). The transmittance curves of the Me-PRX solutions were shifted to lower temperature with increasing the concentration of the MePRXs (Figure 3c−e). The temperature-dependent transmittance reduction of the Me-PRX solutions occurred within narrow temperature range when the concentration of the MePRXs was high (e.g., 10 mg/mL). With a decreasing concentration, the temperature range necessary to complete transmittance reduction became wider, especially for 6.3MeP123 and 14.1Me-P123. However, in the case of 21.0Me-P123, temperature-dependent transmittance reduction occurred within a narrow temperature range regardless of the concentration. This result suggests that the number of methyl groups in the Me-PRXs strongly affects the temperature-induced association behavior of the Me-PRXs. Indeed, the LCST values of all the Me-PRX solutions decreased with an increasing concentration and an increasing number of methyl groups on the Me-PRXs (Figure 3f). Although these results were consistent with those of the previously reported methylated α-CD/PEG-based PRXs, the LCST values of the present methylated β-CD/Pluronic P123based PRXs were altered within a narrow temperature range (30.5−37.5 °C, Table 2) compared to that of the previously reported methylated α-CD/PEG-based PRXs (22−53 °C).19 This fact suggests that not only the number of methyl groups in the Me-PRXs but also the constituent molecules of the MePRXs (i.e., axle polymer or cyclic molecules) affect the temperature-induced phase transition behavior of the MePRXs. 3.4. Effect of Additives on the LCST of the Me-PRXs Aqueous Solutions. The representative temperature-responsive polymers, such as poly(N-isopropylacrylamide) (PNIPAM), exhibit temperature-dependent phase transition behavior due to the temperature-induced dehydration and subsequent association by hydrophobic interaction or intramolecular hydrogen bond formation.28,29 The dominant force that induces the phase transition can be confirmed by the addition of salt because salt promotes the dehydration and reduce intermolecular hydrogen bonding, leading to decreases of the LCST, in line with the Hofmeister series.30,31 Additionally, the addition of chaotropic salts, such as urea, into the system disturbs the hydrogen bond formation and reduces intermolecular hydrophobic interaction, leading to increase of the LCST.32 To verify what type of molecular interactions are involved in the temperature-dependent phase transition of the Me-PRXs, the effects of NaCl and urea on the LCST value of 14.1Me-P123 were investigated (Figure 4). In the presence of NaCl (0−1.0 M), the phase transition temperatures decreased with an increasing concentration of NaCl (Figure 4a,b). This result indicates that dehydration and intermolecular hydrogen bonding are essential to induce the phase transition of MePRXs. In the presence of urea (0−10 M), the phase transition temperatures slightly increased as the concentration of urea increased (Figure 4c,d). According to these results, both

methyl group. Consequently, the Me-PRXs with different numbers of methyl groups were successfully synthesized as summarized in Table 1. 3.2. Wide-Angle X-ray Diffraction of the Me-PRXs. The PRXs composed of α-CD/PEG and β-CD/PPG exhibit an Xray diffraction (XRD) pattern of a hexagonal channel-type crystalline structure due the intermolecular hydrogen bonding of the PRXs.25−27 To investigate the crystalline structure of the Me-PRXs, wide-angle XRD of the Me-PRXs was performed (Figure 2). The unmodified β-CDs, DM-β-CDs, and TM-β-

Figure 2. Wide-angle X-ray diffraction patterns of (a) β-CD, (b) RMβ-CD, (c) DM-β-CD, (d) TM-β-CD, (e) unmodified PRX, (f) 6.3MeP123, (g) 14.1Me-P123, and (h) 21.0Me-P123.

CDs showed many sharp diffraction, whereas RM-β-CDs showed no diffraction peaks. Because the chemical structures of unmodified β-CDs, DM-β-CDs, and TM-β-CDs are single and uniform, it is clear that they form crystalline structures. However, it is considered that they exist as an amorphous rather than a crystalline structure because the RM-β-CDs are a mixture of methylated β-CDs with different degree of methylation.20 In the case of polyrotaxane, the unmodified PRXs, 6.3Me-P123, and 14.1Me-P123 showed weak and broad peaks at 2θ = 19−20°, of which the peak at 2θ = 19.7° is associated with a channel-type crystalline structure due to packing of the PRXs.25−27 Therefore, it is suggested that some fractions of β-CD/Pluronic PRXs and Me-PRXs (6.3Me-P123 and 14.1Me-P123) formed the channel-type crystalline structure through the intermolecular hydrogen bonding of polyrotaxanes. By contrast, 21.0Me-P123 exhibited no diffraction peaks. Because the hydroxyl groups of threading βCDs are completely methylated in 21.0Me-P123, intermolecular hydrogen bonding between polyrotaxanes might be diminished. Therefore, entirely methylated PRXs lack the ability to form crystalline structure, resulting in the formation of an amorphous structure. 3.3. Effect of Methylation Degree of Me-PRXs on LCST. The PRXs composed of methylated α-CDs and PEG exhibit temperature-dependent reversible phase transition across their lower critical solution temperature (LCST) in the temperature range of 22−53 °C, which is dependent on the degree of methylation.19,20 The Me-PRXs (14.1Me-P123) were soluble in water and PBS at 25 °C, yielding a transparent solution (Figure 3a). However, the solution became turbid 6024

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Figure 3. (a) Photograph of the temperature-induced reversible phase transition of the Me-PRXs (14.1Me-P123) in PBS at 10 mg/mL. (b) Temperature-dependent transmittance change for the 14.1Me-P123 in PBS (10 mg/mL) over heating-and-cooling cycles. (c−e) Temperaturedependent transmittance range for the Me-PRXs with different numbers of methyl groups in PBS at various concentrations. (f) Concentration dependency of the LCST values of the Me-PRXs with different numbers of methyl groups.

Table 2. Tcl, Tendo, and Thermodynamic Parameters of β-CD Derivatives and the Me-PRXs in PBS code

concn (mg/mL)

DM-β-CD TM-β-CD 6.3MeP123 14.1-P123 21.0Me-P123

20 20 10 10 10

Tcla (°C)

Tendob (°C)

ΔHb (kJ/mol)

ΔSb (J/(mol K))

37.5 33.2 30.5

84.6 92.8 47.8 46.7 41.8

7.78 12.4 35.9 215.0 599.7

21.7 33.9 111.9 672.2 1904.1

a

Determined by transmittance measurements in PBS. bDetermined by DSC measurements in PBS. The units for ΔH and ΔS are expressed based on the mole of threaded β-CDs in the Me-PRXs.

intermolecular hydrogen bonding and the intermolecular hydrophobic interaction were involved in the process of temperature-induced association of the Me-PRXs. 3.5. Thermodynamics of the Me-PRXs via Differential Scanning Calorimetry (DSC). It is known that the constituent molecules for the Me-PRXs also show a temperature-responsive phase transition. For example, methylated βCDs, especially DM-β-CDs and TM-β-CDs, exhibit a temperature-dependent solubility change due to temperature-induced dehydration and subsequent crystallization in water (i.e., the Tendo of DM-β-CDs (20 mg/mL) and TM-β-CDs (20 mg/mL) were 84.6 and 92.8 °C, respectively, as shown in Figure 5).33−35 Additionally, Pluronic polymers show a cloud point that is dependent on their chemical composition (e.g., cloud point of Pluronic P123 is 90.1 °C).36 To further understand the temperature-dependent phase transition of the Me-PRXs, a

Figure 4. (a, c) Temperature-dependent transmittance change of the 14.1Me-P123 (10 mg/mL) in the presence of (a) NaCl and (c) urea at various concentrations. (b, d) Relationship between LCST values of the 14.1Me-P123 and the concentration of (b) NaCl and (d) urea.

thermodynamic analysis of the Me-PRXs and their constituent molecules was carried out using DSC (Figure 5). In general, temperature-responsive polymers such as PNIPAM show a 6025

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The ΔH and ΔS values increased with the methylation degree of the Me-PRXs, suggesting that temperature-induced dehydration and hydrophobic interactions were elevated by increasing the methylation degree in the Me-PRXs. Therefore, it is considered that Me-PRXs with a high methylation degree (i.e., 21.0Me-P123) show sharp transmittance change as observed in Figure 3e. Additionally, the Me-PRXs showed higher ΔH and ΔS values than the crystallization of DM-β-CDs and TM-β-CDs. Because the threading methylated β-CDs in the Me-PRXs were closely ordered along the polymer chain, the hydrogen bonding among threading β-CDs was likely to strengthen in the Me-PRXs, leading to higher ΔH and ΔS values than for the DM-β-CDs and TM-β-CDs. This structural difference between the Me-PRXs and methylated β-CDs could also be related to the reduction in the phase transition temperature of the Me-PRXs. Apart from the multimodal endothermic peak at approximately 40 °C, the 6.3Me-P123 and 14.1Me-P123 exhibited unimodal endothermic peaks at approximately 20 °C, whereas no peak appeared near this temperature for the 21.0Me-P123. It is known that Pluronic P123 forms polymeric micelles in water through dehydration and followed by hydrophobic interactions of intermediate PPG segments which is accompanied by an endothermic peak at 20.3 °C.40 Therefore, the peak at approximately 20 °C observed for the 6.3Me-P123 and 14.1Me-P123 is potentially derived from the molecular association of PPG segments in the axle polymer. The MePRXs with low methylation degree form polymeric micelle-like structure below LCST. By contrast, completely methylated βCDs threaded in the 21.0Me-P123 may randomly localize along the axle polymer to weaken the hydrophobic interactions of the PPG segment. Indeed, we have reported that the association behavior of the unmodified β-CDs/Pluronic-based PRXs is changed by the localization of the threading β-CDs in the PRXs.41 Therefore, the 21.0Me-P123 did not exhibit the

Figure 5. Differential scanning calorimetric diagrams of (a) DM-β-CD and (b) TM-β-CD in PBS (20 mg/mL) and (c) Pluronic P123, (d) 6.3Me-P123, (e) 14.1Me-P123, and (f) 21.0Me-P123 in PBS (10 mg/ mL).

unimodal endothermic peak.37 However, the Me-PRXs showed a broad and multimodal endothermic peak that appeared in the range of 30−50 °C, suggesting that several molecular interactions were involved in the temperature-dependent phase transition of the Me-PRXs. Therefore, the phase transition mechanism for the Me-PRXs was essentially different from the crystallization of methylated β-CDs because their Tendo values were significantly higher than that for the MePRXs. The enthalpy (ΔH) and the entropy (ΔS) for the phase transition of the Me-PRXs and the methylated β-CDs are summarized in Table 2. The enthalpy change represents the average potential energy of molecular interaction, such as the dissociation of intra- or intermolecular hydrogen bonding; the entropy change represents the order or intermolecular correlation, such as intermolecular hydrophobic interaction.38,39

Figure 6. Temperature-dependent transmittance change of the Me-PRXs with different axle polymers in PBS at various concentrations: (a) 15.2MeL121, (b) 15.3Me-P84, (c) 12.2Me-P103, (d) 13.8Me-P105, (e) 13.1Me-F127, and (f) 10.8Me-PEG. (g) Relationship between the LCST values and the concentration of the Me-PRXs with different axle polymers. (h) Relationship between the weight ratio of hydrophobic PPG segments in an axle polymer and the LCST values of the Me-PRXs with different axle polymers in PBS (10 mg/mL). 6026

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Figure 7. Optical microscopy images for the coacervate droplets of the 15.2Me-L121, 14.1Me-P123, and 13.1Me-F127 in PBS (5.0 mg/mL) at various incubation temperatures.

molecular association of PPG segments in the axle polymer or the formation of a polymeric micelle-like structure. 3.6. Effect of Molecular Composition of the Axle Polymer of Me-PRXs on LCSTs. Because the hydrophobic interaction of the Me-PRXs is one of the dominant forces that induces phase transition, it is predicted that the chemical composition of an axle polymer should affect the LCST value. To validate this hypothesis, a variety of Me-PRXs with different molecular compositions of axle polymers (e.g., Pluronic L121, P103, P123, P84, P105, F127, and PEG/α-CD) were synthesized (Table 1). Temperature-dependent transmittance changes of these Me-PRXs are shown in Figure 6a−f, and the LCSTs were plotted against concentration of the Me-PRXs (Figure 6g). All the Me-PRXs exhibited temperature-dependent transmittance reduction in a concentration-dependent manner. Notably, the 15.2Me-L121 showed a LCST at 18.7 °C (10 mg/ mL), which was a higher value than the cloud point of the axle polymer (14 °C, 10 mg/mL), confirming that the threading of methylated β-CDs altered the intra- and intermolecular interactions of axle polymers. Furthermore, the LCST of the Me-PRXs seemed to be a low value when the hydrophobic segment ratio (PPG ratio) in the axle polymer was high. The relationship between the LCSTs of these PRXs solutions (10 mg/mL) and the PPG ratio in the axle polymer is shown in Figure 6h. Although the degree of methylation and the threading ratio of β-CDs are not completely identical among

the tested Me-PRXs, it is clear that the LCST of the Me-PRXs decreased almost proportionally with increasing PPG ratios in the axle polymer. It is suggested that the axle polymers (Pluronic) with longer PPG segments or higher PPG molar ratio strengthen the hydrophobicity of the Me-PRXs. Indeed, in the case of the representative temperature-responsive polymers such as PNIPAM, the copolymerization hydrophobic monomer results in the reduction of the LCST values.43 Therefore, the phase transition temperature of the Me-PRXs is strongly affected by the hydrophobicity of the axle polymer through the hydrophobic interactions between dehydrated methylated βCDs threaded onto the Me-PRXs and the hydrophobic segment of axle polymer of the Me-PRXs. 3.7. Coacervate Droplet Formation of the Me-PRXs. Finally, we observed the Me-PRX solutions (15.2Me-L121, 14.1Me-P123, and 13.1Me-F127) via microscopy at various temperatures (Figure 7). The 15.2Me-L121 was aggregated in this temperature range because the LCST was approximately 25 °C. Interestingly, coacervate-like droplets with sizes of approximately 10−20 μm were observed for the 14.1MeP123 at 35 °C, which was above the LCST value of the 14.1MeP123. Moreover, the coacervate formation was also reversible (data not shown), similar to the reversibility of the temperature-dependent transmittance change (Figure 3). In the case of the 13.1Me-F127, negligible coacervate droplets formation or aggregation were observed because the observation temper6027

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Figure 8. (a) Optical microscopy images for the coacervate droplets of the 14.1Me-P123 in PBS at various concentrations at 37 °C. (b) Diameter distribution for the coacervate droplets of the 14.1Me-P123 at various concentrations (0.1, 5.0, and 10 mg/mL). (c) Optical and fluorescence images for FITC-labeled Me-PRXs (5.0 mg/mL) in PBS at 37 °C.

Figure 9. Proposed mechanism for the temperature-induced phase transition process of the Me-PRXs.

ature was below the LCST. According to these results, some type of the Me-PRXs forms coacervate droplets above the LCST. In general, coacervate is defined as spherical aqueous droplets comprising dense macromolecular solutions.42 The formation of coacervate is classified into two cases: complex coacervation and simple coacervation. Complex coacervation is formed by electrostatic interactions between oppositely charged polyelectrolytes, whereas simple coacervation is formed from the polymer−solvent interaction change induced by the temperature or additives. Because temperature-induced changes in the interaction with the solvent triggered the formation of coacervate droplets, coacervation of the Me-PRXs was classified as simple coacervation. The Me-PRXs would associate with holding water molecules in their hydrophilic segments above the LCST to form coacervate droplets because the hydrophilic

segment (PEG) in the axle polymer is considered to hydrate even at temperatures above the LCST. Indeed, when the molar ratio of hydrophilic segments in the axle polymer was low (e.g., 15.1Me-L121), the PRXs did not form coacervate droplets, resulting in simple aggregation. Further observation of the coacervation of the 14.1Me-P123 was carried out by changing the concentration of the Me-PRXs (Figure 8a). The size of the coacervate droplets was relatively small below the concentration of 1.0 mg/mL, and they seemed to interact with each other at the boundary phase of the droplets. However, the size of coacervate droplets increased as the concentration increased above 1.0 mg/mL. Indeed, the feret diameter for the coacervate droplets clearly increased with the concentration (0.1 mg/mL: 2.1 ± 1.3 μm; 5.0 mg/mL: 29.8 ± 14.4 μm; 10 mg/mL: 55.3 ± 31.7 μm) (Figure 8b). This result 6028

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indicates that the coacervate droplets of the 14.1Me-P123 became large presumably via the fusion of coacervate droplets. To confirm whether the coacervate droplets were formed from the Me-PRXs, a FITC-labeled 14.1Me-P123 (FITC14.1Me-P123) solution was observed above LCST via fluorescence microscopy (Figure 8c). Green signals derived from the FITC-14.1Me-P123 were distributed throughout the coacervate droplets and the optical images were completely merged with the fluorescence image, clearly demonstrating that the coacervate droplets were formed from the assembly of MePRXs. Altogether with the results of this study, the mechanism of the temperature-responsive phase transition of the Me-PRXs is proposed as shown in Figure 9. Below the LCST, the Me-PRXs are soluble in water due to the hydration of threaded methylated β-CD moieties and the hydrophilic PPG segment of the Pluronic axle polymer. With increasing temperature, they are dehydrated and elicit hydrophobic character. Then, the MePRXs are assembled via intermolecular hydrophobic interaction. The assembled Me-PRXs eventually form an aggregation or coacervate droplets, depending on the hydrophilic segment ratio of the axle polymer.

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4. CONCLUSIONS To obtain a comprehensive knowledge of the temperaturedependent phase transition of the Me-PRXs, the effects of their molecular compositions on phase transition behavior were investigated. It is indicated that the temperature-dependent phase transition of Me-PRXs was caused by the dehydration of threading methylated β-CD moieties and the hydrophobic segments of the axle polymer and the subsequent intermolecular interactions among them. The LCST values of MePRXs could be varied by regulating the number of methyl groups modified on β-CD and molecular composition of the Pluronic axle polymer. Above the LCST, the Me-PRXs were found to form coacervate droplets. According to this study, the temperature-responsive Me-PRXs with tailored LCSTs could be applied as a variety of biomaterials, such as temperatureresponsive biomaterials, therapeutics for inherent metabolic disorders, and regulators of cellular uptake via temperature change.44 Further studies on the coacervation and biomaterials application of the Me-PRXs are currently underway in our laboratory and will be reported elsewhere.

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AUTHOR INFORMATION

Corresponding Author

*(N.Y.) Tel +81-3-5280-8020, Fax +81-3-5280-8027, e-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Grant-in-Aid for Scientific Research on Innovative Areas “Nanomedicine Molecular Science” from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (No. 23107004 to N.Y.): Grant-in-Aid for Young Scientists (A) from Japan Society for the Promotion of Science (JSPS) (No. 16H05910 to A.T.); Grant-in-Aid for Young Scientists (B) from JSPS (No. 26750155 to A.T.); and Grant-in-Aid for JSPS fellows from JSPS (No. 16J09364 to K.N.). 6029

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