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A pseudopolyrotaxane for glucose-responsive insulin release: The effect of binding ability and spatial arrangement of phenylboronic acid group Tomohiro Seki, Keigo Abe, Yuya Egawa, Ryotaro Miki, Kazuhiko Juni, and Toshinobu Seki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00599 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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Molecular Pharmaceutics

A pseudopolyrotaxane for glucose-responsive insulin release: The effect of binding ability and spatial arrangement of phenylboronic acid group Tomohiro Seki∗, Keigo Abe, Yuya Egawa, Ryotaro Miki, Kazuhiko Juni, Toshinobu Seki ∗

Faculty of Pharmaceutical Sciences, Josai University, Keyakidai, Sakado, Saitama 350-0295,

Japan Email: [email protected], Tel: +81-49-271-7686, Fax: +81-49-271-7714

ABSTRACT. A pseudopolyrotaxane (PPRX) comprising 3-carboxy-5-nitrophrnylboronic acidmodified γ-cyclodextrin (NPBA-γ-CyD) and naphthalene-modified polyethylene glycol (NaphPEG) as a sugar-responsive supramolecular structure is prepared. The binding of sugar by the NPBA group induced disintegration of the Naph-PEG/NPBA-γ-CyD PPRX, allowing the components to be dissolved. The Naph-PEG/NPBA-γ-CyD PPRX exhibited better sensitivity

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compared to that of a PPRX based on 4-carboxyphenylboronic acid-modified γ-cyclodextrin (PBA-γ-CyD). We have previously reported the unique structure of Naph-PEG/PBA-γ-CyD PPRX, which formed an inclusion complex with a single-stranded PEG chain being threaded through the γ-CyD rings, with the remaining internal space being occupied by the sugar-sensing PBA moiety from a neighboring ring, thus shielding it from sugar molecules and reducing the sugar sensitivity of the PPRX. In contrast, structural analyses in this study revealed that the sugar-sensing NPBA moiety in the Naph-PEG/NPBA-γ-CyD PPRX is not included in the neighboring NPBA-γ-CyD. This spatial arrangement and the high affinity of NPBA for sugar contributed to the improved sugar responsivity. The enhanced NPBA-γ-CyD was then applied to a PPRX containing Naph-PEG-appended insulin (Naph-PEG-Ins) that showed an improved response for glucose-induced insulin release.

KEYWORDS. Cyclodextrin; Pseudopolyrotaxane; Polypseudorotaxane; Boronic acid; Stimuliresponsive; Insulin

INTRODUCTION. Stimuli-responsive molecular machines show great promise as a new generation of drug delivery systems. These systems release drugs in response to fluctuations in the concentration of biomolecules related to pathological conditions in the body.1 Recently, several stimuli-responsive controlled release systems that exhibit the ability to control the timing of drug release have been reported.1–3 The mechanisms of drug release in these systems are based on structural features that react to changes in the surrounding environment. One such system that has attracted increasing attention is cyclodextrin (CyD)-based molecular machines.4–8 CyDs are common cyclic oligosaccharides comprising 1,4-linked glucopyranoside units. A typical CyD molecule possesses a hydrophilic exterior and a hydrophobic cavity where hydrophobic


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molecules may be enclosed to form an inclusion complex. CyDs are capable of forming inclusion complexes not only with small molecules but also with polymers. An inclusion complex wherein many CyDs are threaded onto a linear polymer is called a pseudopolyrotaxane (PPRX). If both end groups of the polymer included in the PPRX have been modified with bulky stoppers, the structures are then called polyrotaxanes (PRXs). Harada et al. first reported that αCyD forms a crystalline complex with polyethylene glycol (PEG), where the PEG molecule is threaded through the cavities of many α-CyD molecules.9,10

There have been many attempts to build drug delivery systems with PPRXs since their discovery.11–18 Moreover, the use of PEG for the development of drug delivery systems is reasonable because it is biocompatible and has been approved as a pharmaceutical additive. Furthermore, PEGylation technologies have been widely used to improve the therapeutic efficacies of many protein drugs, imbuing them with improved circulation life, increased stability against proteolysis, and fewer immunogenic side effects.19–21 A drug-controlled release system using α- or γ-CyD-based PPRXs and PEGylated insulin (PEG-Ins) has been demonstrated by Higashi et al., where they combined CyDs and the PEG chain of PEG-Ins to form PPRXs. The PEG-Ins/CyD PPRXs showed a sustained release of PEGIns from the PPRXs in vitro and in vivo. These results indicate that the PEG-Ins/CyD PPRX is capable of forming a nanostructure for a sustained drug release system. However, the PEGIns/CyD PPRXs were not designed to be a sugar-responsive controlled release system. Sugarresponsive insulin release systems are required to increase the convenience of self-administered injections for diabetic patients because insulin self-injection treatments involve difficulties


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concerning the control of blood sugar levels and the risk of hypoglycemia. Sugar-responsive insulin release systems with supra molecular assemblies have been reported to meet the issue.22,23 We have previously reported a sugar-responsive PPRX comprising phenylboronic acidmodified γ-CyD (PBA-γ-CyD) and naphthalene-modified PEG (Naph-PEG).24 The PBA reacts with the diol of a sugar to form a boronate ester, initiating the collapse of the complex.25–29 In addition, we have previously designed a PPRX using Naph-PEG modified insulin (Naph-PEGIns) and PBA-γ-CyD. The PBA-γ-CyD PPRX is insoluble in water; however, the PPRX collapses when sugar is added to the solution, and the disintegration results in the release of Naph-PEG or Naph-PEG-Ins, depending on the sugar concentration. This system was intended to benefit diabetic patients who use insulin self-injection treatments. However, the sugar response of the Naph-PEG-Ins release from the PPRX was not sufficient to work appropriately in the dynamic range of blood glucose (Glc) levels in the body. We considered the possibility that the response of the PPRX of PBA-γ-CyD is limited by the low accessibility of the sugar-sensing group because the PBA moiety of the PPRX may be included in a neighboring CyD cavity, as shown in Figure 1b. Consequently, we envisioned a PPRX system containing a PBA derivative that is too bulky to be included in the cavity of the neighboring CyD ring (Figure 1a) in order to improve the sugar-responsivity of the Naph-PEG release. Consequently, we have adopted 3-carboxy-5-nitrophenylboronic acid (NPBA) as an improved PBA derivative with increased steric hindrance (Figure 1a). In addition, NPBA has a better affinity for Glc than PBA owing to its electron withdrawing group. As shown in Figure 1c, Naph-PEG was used as the building block for the axle molecule in the PPRX because our previous work showed that modification of the PEG terminus with Naph is


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advantageous for the formation of PPRXs with modified CyDs.24 We prepared a Naph-PEG /NPBA-γ-CyD PPRX and investigated it from the viewpoint of the spatial arrangement of the NPBA moiety. We then applied the NPBA-γ-CyD PPRX to Naph-PEG-Ins to fabricate a sugarresponsive Ins-release PPRX having an outward-facing sugar-sensing moiety (Figure 1d).

Figure 1. Schematic illustrations of (a) NPBA-γ-CyD PPRX with an outward-facing sugar sensor and (b) PBA-γ-CyD PPRX with its sugar sensor included in the CyD cavity. (c) Chemical structures of the building blocks of sugar-responsive PPRXs. (d) Schematic illustration of Naph-PEG-Ins/NPBA-γ-CyD PPRX and its sugar-responsive insulin release.

EXPERIMENTAL SECTION Materials γ-CyD was obtained from Junsei Chemical Co., Ltd. (Tokyo, Japan). Polyethylene glycol (MW 2,000), thionyl chloride, and insulin (human, recombinant) were purchased from Wako Pure Chemical Industries (Osaka, Japan). 4-Carboxyphenylboronic acid and streptozocin (STZ) were purchased from Sigma Aldrich Japan (Tokyo, Japan). 2-Naphthoyl chloride, 3-carboxy-5nitrophenylboronic acid, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride


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(EDC) were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2-[4-(2Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was purchased from Dojindo Laboratories (Kumamoto, Japan). Sunbright® PA-020HC (H2N-(CH2)3-O-PEG-(CH2)5-COOH, MW 2,000) was obtained from NOF Corporation (Tokyo, Japan). All other chemicals were of reagent grade and were used as received. Apparatus 1

H NMR spectra were measured on a Varian 400-MR (Agilent Technologies, California, USA).

Fluorescence spectroscopy was performed using a RF-5300PC spectrofluorophotometer (Shimadzu Corporation, Kyoto, Japan). Turbidity was monitored with a V-530 UV-Vis spectrometer (JASCO Corporation, Tokyo, Japan) using absorbance at 700 nm. The release rate of Naph-PEG was monitored using a Spectra Max M5e multiplate reader (Molecular Devices Japan, Tokyo, Japan). Powder X-ray diffraction (XRD) patterns were measured using a Mini FlexII (Rigaku Corporation, Tokyo, Japan) using CuKα radiation. Diffraction was performed at 30 kV and 15 mA at a scanning speed of 4°/min over the measurement range of 2θ = 3–40°. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS spectra were recorded using an AXIMA-CFR plus spectrometer (Shimadzu Corporation, Kyoto, Japan). Circular dichroic spectra were measured with a J-720WI spectropolarimeter (JASCO Corporation, Tokyo, Japan). Synthesis 3-Carboxy-5-nitrophenylboronic acid-modified γ-cyclodextrin (NPBA-γ-CyD)


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To protect the boronic acid moiety, 2,2-dimethyl-1,3-propanediol (2.82 g, 27.1 mmol) and 3carboxy-5-nitrophenylboronic acid (5.10 g, 24.2 mmol) were dissolved in tetrahydrofuran (100 mL), and the solution was heated to reflux at 70 °C. After 24 h, the solvent was evaporated, and the residue was dried in vacuo (7.90 g). The resulting residue was dissolved in thionyl chloride (50 mL) and a few drops of anhydrous dimethylformamide were added to the solution as a catalyst. After the solution was heated to reflux for 6 h, the solvent was evaporated under reduced pressure. The activated NPBA derivative (3.95 g, 12.1 mmol) was dissolved in 50 mL of anhydrous pyridine, and then added to a solution of γ-CyD (12.97 g, 10.0 mmol) in anhydrous pyridine (1.0 L). The solution was stirred at 50 °C, and the reaction was monitored using silica gel thin-layer chromatography (TLC) with n-butanol:EtOH:water (5:4:3 v/v/v) as the eluent and anisaldehyde as the detecting agent. After di-modified NPBA-γ-CyD began to appear, the reaction was stopped by the addition of a small amount of water. The reaction solution was evaporated to a concentrate, and 50 mL of the concentrated solution was added to 1.0 L of acetone, with the resulting precipitate being filtered using a glass filter. The white powder was dissolved in 1.0 L of water, and the solution was filtered to remove insoluble matter. The filtrate was then loaded onto a borate-specific chelating resin (Amberlite®IRA743, Aldrich Chemicals Co., Tokyo, Japan), and water was used to wash out the unmodified γ-CyD. After all the unmodified γ-CyD was removed, NPBA-γ-CyD and the di-modified NPBA-γ-CyD were eluted using 3.0 L of 5% aqueous AcOH solution. The eluate was then loaded onto a highly porous polystyrene gel (Diaion®HP-20, Mitsubishi Chemical, Japan) for the second separation, and a 3:7 mixed MeOH:water solution was used to elute monomodified NPBA-γ-CyD. The eluates containing NPBA-γ-CyD were gathered and evaporated to a concentrate. The concentrated solution (20 mL) was added to 300 mL of acetone, and the


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resulting precipitate was filtered using a glass filter. The white powder was washed three times with a small amount of acetone and dried in vacuo (1.87 g, 12.6%). 1

H NMR (400 MHz, DMSO-d6, Figure S1) δ 9.71-9.52 (s, 1H, BOH), 8.99-8.80 (m, 1H, ArHx),

8.77-8.72 (m, 1H, ArHy), 8.70-8.63 (m, 1H, ArHz), 6.21-5.66 (m, 16H, CyD OH-2, OH-3), 5.05-4.73 (m, 8H, CyD H-1), 4.67-4.36 (m, 7H, CyD OH-6), 3.86-3.46 (m, 32H, CyD H-3, H-5, H-6a, b), 3.45-3.02 (m, CyD H-2, H-4 overlaps with HOD). MS (FAB, negative mode, matrix: glycerol) m/z: 1544.5 ([M + glycerol - 2H2O - H]- requires 1544.5). The NPBA-γ-CyD was detected as an ester composed of NPBA-γ-CyD and glycerol. Anal. Calcd for C55H84BNO45+8H2O: C, 40.42; H, 6.17; B, 0.66; N, 0.86 O, 51.89. Found: C, 40.33; H, 6.04; N, 0.98. Phenylboronic acid-modified γ-cyclodextrin (PBA-γ-CyD) PBA-γ-CyD was synthesized as previously reported.24 4-Carboxyphenylboronic acid (4.02 g, 24.2 mmol) and 2,2-dimethyl-1,3-propanediol (2.82 g, 27.1 mmol) were dissolved in tetrahydrofuran (100 mL) to protect the boronic acid moiety. The stirred solution was heated to reflux at 70 °C. After 24 h, the solvent was removed, and the residue was dried in vacuo. The residue was then dissolved in thionyl chloride (50 mL) and a few drops of anhydrous dimethylformamide were added the solution. The solution was heated to reflux for 6 h. The reaction solution was then cooled to room temperature and evaporated under reduced pressure. The activated PBA derivative was dissolved in 50 mL of anhydrous pyridine, and then added to a solution of γ-CyD (12.97 g, 10.0 mmol) in anhydrous pyridine (1.0 L). The solution was stirred at 90 °C, and the reaction was monitored using silica gel TLC with n-butanol:EtOH:water (5:4:3


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v/v/v) as the eluent and anisaldehyde as the detecting agent. After the first appearance of dimodified PBA-γ-CyD, the reaction was stopped by adding a small amount of water. The reaction solution was evaporated to a concentrate, and 50 mL of the concentrated solution was added to 1.0 L of acetone. The resulting precipitate was filtered using a glass filter. The white powder was dissolved in 1.0 L of water, and the solution was filtered to remove insoluble matter. This filtrate was then applied to a column containing a borate-specific chelating resin, and water was used to wash out the unmodified γ-CyD. After all the unmodified γ-CyD was removed, PBA-γ-CyDs and the di-modified PBA-γ-CyD were eluted using 3.0 L of a 5% aqueous AcOH solution. The eluate was applied to a column containing a highly porous polystyrene gel (Diaion®HP-20, Mitsubishi Chemical, Japan) for the second separation. A mixed 1:1 MeOH:water solution was used to elute the mono-modified PBA-γ-CyD. The eluates containing PBA-γ-CyD were combined and evaporated to a concentrate. The concentrated solution (40 mL) was added to 400 mL of acetone, and the resulting precipitate was filtered using a glass filter. The white powder was washed three times with a small amount of acetone and dried in vacuo (3.79 g, 26.1%). Naphthalene-modified polyethylene glycol (Naph-PEG) Naph-PEG was prepared as previously reported.24 PEG (MW 2,000, 10.0 g) was dissolved in a mixed solution of anhydrous dichloromethane (300 mL) and anhydrous pyridine (22.5 mL). The solution was stirred at 0 °C, and 2-naphthoyl chloride (6.4 g, 34 mmol) was added to the solution. The solution was then stirred at 0 °C under a nitrogen atmosphere for 3 days. A small amount of water was then added in order to stop the reaction, and the solution was filtered with a glass filter to remove insoluble matter. The filtrate was evaporated, and the residue was recrystallized from EtOH.


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One-terminal naphthalene-modified polyethylene glycol (One-Naph-PEG) One-Naph-PEG was prepared as previously reported.24 H2N-(CH2)3-O-PEG-(CH2)5-COOH (MW 2000, 300 mg) was dissolved in a mixed solution of anhydrous dichloromethane (10 mL) and anhydrous pyridine (0.70 mL). The solution was stirred at 0 °C, and 2-naphthoyl chloride (286 mg, 1.50 mmol) was added to the solution. The solution was then stirred at 0 °C under a nitrogen atmosphere for 4 days. The solvent was then evaporated, and water (40 mL) was added to the residue. The solution was filtered with a glass filter to remove insoluble matter, and the filtrate was dialyzed against water using a dialysis tube (MWCO 1000). The resulting solution was lyophilized, and One-Naph-PEG was obtained (229 mg, 69.3 %) Naph-PEG-appended insulin (Naph-PEG-Ins) Naph-PEG-Ins was prepared as previously reported.24 Insulin (208 mg, 48.2 µmol) and OneNaph-PEG (0.208 g, 96.3 µmol) were dissolved in DMSO (28 mL), and EDC (18.0 mg, 96.3 µmol) was added. The solution was stirred at room temperature for 48 h, and water (80 mL) was added. The solution was then dialyzed against water using a dialysis tube (MWCO 3500). The resulting solution was lyophilized, and Naph-PEG-Ins was obtained (348 mg, 66.3%). The stoichiometry of PEG/Ins in the Naph-PEG-Ins was calculated to be 2.2. The bioactivity of Naph-PEG-Ins was evaluated in the STZ-induced diabetic rats. By comparing the decrease in the blood Glc level of Naph-PEG-Ins with that of native insulin, Naph-PEG-Ins was determined to retain 71.5% of the bioactivity of native insulin. Binding constants of NPBA-γγ-CyD and PBA-γγ-CyD to Glc


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The abilities of NPBA-γ-CyD and PBA-γ-CyD to bind to Glc were investigated with fluorescence measurements using alizarin red S (ARS).30 ARS (0.10 mM) solution was prepared with a HEPES buffer solution (20 mM, pH 7.4). NPBA-γ-CyD or PBA-γ-CyD solutions of varying concentrations (0.0–5.0 mM) were prepared with the ARS solution and analyzed with a fluorescent spectrometer. The ARS binding constants were calculated with reference to the fluorescent intensities at λem (556 nm) using KaleidaGraph (Figure S2). Solutions containing constant amounts of CyD (1.0 mM: NPBA-γ-CyD or PBA-γ-CyD) and ARS (0.10 mM) and a range of amounts of Glc (0.0–1000.0 mM) were prepared for fluorescence measurements (Figure S3). The Glc binding constants were calculated using a previously reported method.30 The calculated binding constants were 50.1 M-1 for NPBA-γ-CyD and 8.0 M-1 for PBA-γ-CyD. Preparation of PPRXs Naph-PEG/NPBA-γ-CyD PPRX Naph-PEG (7.4 mg, 3.0 µmol) and NPBA-γ-CyD (100 mg, 67.1 µmol) were dissolved in water (500 µL), and the solution was kept at room temperature. After 28 days, the resulting PPRX precipitate was filtered and dried under reduced pressure (67.6 mg). The Naph-PEG/NPBA-γ-CyD PPRX was dissolved in DMSO-d6 and analyzed with 1H NMR (Figure S4). When the integration of H-1 of CyD was set to 8.0, the integration of the PEG peaks was 11.8, as shown in Figure S2. However, this value is slightly inflated by background signals due to NPBA-γ-CyD. Thus, the exact value was obtained by comparing Figure S1 and S2, and the exact value for the PEG signals was calculated to be 8.1. From this result, the stoichiometry between the ethylene glycol monomer (EG) unit and NPBA-γ-CyD was calculated to be 2.0:1.0.


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Naph-PEG/PBA-γ-CyD PPRX Naph-PEG/PBA-γ-CyD was prepared as previously reported.24 Naph-PEG (7.4 mg, 3.0 µmol) and PBA-γ-CyD (100 mg, 69.2 µmol) were dissolved in water (500 µL), and the solution was kept at room temperature. After 28 days, the resulting PPRX precipitate was filtered and dried under reduced pressure (84.7 mg). Naph-PEG-Ins/NPBA-γ-CyD PPRX Naph-PEG-Ins (25.0 mg, 2.31 µmol) and NPBA-γ-CyD (100 mg, 67.1 µmol) were dissolved in water (1000 µL), and the solution was kept at room temperature. After 28 days, the resulting PPRX precipitate was filtered and dried under reduced pressure (73.0 mg). The Naph-PEGIns/NPBA-γ-CyD PPRX formed as a crystalline precipitate. The Naph-PEG-Ins/NPBA-γ-CyD PPRX was dissolved in DMSO-d6 and analyzed with 1H NMR (Figure S5). When the integration of H-1 of CyD was set to 8.0, the integration of the PEG peaks was 14.8. By the same method as that described in the preparation of Naph-PEG/NPBA-γ-CyD PPRX, the difference in the values between the spectrum of Naph-PEG-Ins/NPBA-γ-CyD and the spectrum of NPBA-γ-CyD was calculated to be 11.1. From this result, the stoichiometry between the EG unit and PBA-γ-CyD was calculated to be 2.8:1.0. Naph-PEG-Ins/PBA-γ-CyD PPRX Naph-PEG-Ins/PBA-γ-CyD was prepared as previously reported.24 Naph-PEG-Ins (50 mg, 4.62 µmol) and PBA-γ-CyD (200 mg, 138.4 µmol) were dissolved in water (1000 µL), and the solution was kept at room temperature. After 28 days, the resulting PPRX precipitate was filtered


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and dried under reduced pressure (117.2 mg). The Naph-PEG-Ins/PBA-γ-CyD PPRX formed as a crystalline precipitate. Monitoring of PPRX formation by turbidity measurements The CyD derivative (100 mg, PBA-γ-CyD, NPBA-γ-CyD or γ-CyD) was dissolved in water (1.3 mL) and the solution was put into a cell holder of an absorption spectrometer. The solution was kept at 25 °C and stirred, and the turbidity was monitored by absorbance at 700 nm. After 5 min, Naph-PEG (7.4 mg) dissolved in 0.2 mL of water was added to the CyD solution. Evaluation of the sugar responsiveness of the PPRXs Turbidity measurements A buffer solution (20 mM HEPES, pH 7.4, 2.0 mL) was placed into a cell of an absorption spectrometer, and the buffer solution was stirred at 37 °C. The PPRX (Naph-PEG/PBA-γ-CyD PPRX or Naph-PEG/NPBA-γ-CyD PPRX, 6.0 mg) was suspended in the stirred buffer solution, and the turbidity was monitored by absorbance at 700 nm. After the turbidity reached a constant level, small amounts of a stock sugar solution (1.00 M) were added at 10 min intervals in order to increase the sugar concentration of the suspended solution. Assessment of the release of PEG chains using the fluorescence of the naphthalene moiety A buffer solution with and without sugar (20 mM HEPES, pH 7.4, 1.0 mL) was placed in a centrifuge microtube and kept at 37 °C. The PPRX (Naph-PEG/PBA-γ-CyD PPRX or NaphPEG/NPBA-γ-CyD PPRX, 3.0 mg) was added to the solution. After a predetermined time, the microtube was centrifuged and a small amount of the supernatant was collected. The


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fluorescence derived from the naphthoyl moiety was monitored with a microplate reader (λex = 280 nm, λem = 420 nm). In a similar manner, the release of Naph-PEG-Ins from the PPRX (Naph-PEG-Ins/PBA-γ-CyD PPRX or Naph-PEG-Ins/NPBA-γ-CyD PPRX, 3.0 mg) was evaluated by monitoring the fluorescence of the naphthoyl moiety on the PEG chains. RESULTS AND DISCUSSION Binding abilities of NPBA-γγ-CyD and PBA-γγ-CyD We successfully synthesized NPBA-γ-CyD, and it shows a higher binding constant (50.1 M-1) than PBA-γ-CyD (8.0 M-1). It is known that PBA derivatives with electron withdrawing groups tend to exhibit improved binding to Glc.31 Accordingly, the nitro group on the PBA ring of NPBA-γ-CyD contributes to the higher binding constant of NPBA-γ-CyD.

Preparation of the Naph-PEG/PBA-γγ-CyD PPRX

NPBA-γ-CyD and PBA-γ-CyD were used in the preparation of the different PPRXs. Previously, we found that modification of the PEG terminals with naphthalene groups facilitated the precipitation of a PPRX consisting of PBA-γ-CyD and PEG, whereas the use of unmodified PEG leads to the slow formation of a PPRX with a relatively low yield because the PBA moiety can occupy a CyD cavity.24 After confirming that the synthesized NPBA-γ-CyD and Naph-PEG are sufficiently water soluble, Naph-PEG (7.4 mg, 3.1 µmol) and NPBA-γ-CyD (100 mg, 67.1 µmol) were dissolved in water (500 µL) to form the Naph-PEG/NPBA-γ-CyD PPRX, and the solutions were kept at room temperature. Naph-PEG (7.4 mg, 3.1 µmol) and PBA-γ-CyD (100 mg, 69.2 µmol) were also used to prepare a Naph-PEG/PBA-γ-CyD PPRX in a similar manner.


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In the preparation of PPRX using PBA-γ-CyD, it takes several hours before a white precipitate is observed. In contrast, an observable white precipitate appears within 5 min when using NPBAγ-CyD. The faster formation of the PPRX in this case indicates that the NPBA moiety in NPBAγ-CyD does not inhibit the threading of the PEG chain through the NPBA-γ-CyD cavities. After 28 days, the resulting precipitate, which later analysis showed to be the PPRX of NPBA-γ-CyD and Naph-PEG, was filtered and dried under reduced pressure (67.6 mg). Likewise, the precipitate of PPRX of PBA-γ-CyD and Naph-PEG was obtained (84.7 mg). Although both precipitations were fast, following the preparation method of the previous type of PPRXs comprising PBA-γ-CyD and PEG chains not modified with Naph, we collected the sample with the preparation time for 28 days. The observed precipitation of NPBA-γ-CyD PPRX was faster than that of PBA-γ-CyD PPRX; however, the yield of NPBA-γ-CyD PPRX was slightly lower than that of PBA-γ-CyD PPRX. This reason is considered as the equilibrium of NPBA-γ-CyD PPRX derived from the rate constants of the fast forward reaction and the fast reverse reaction. The forward reaction rate of NPBA-γ-CyD with Naph-PEG is faster than that of PBA-γ-CyD with Naph-PEG, resulting in the fast attainment of equilibrium state and fast precipitation of NPBA-γ-CyD PPRX. However, reverse reaction rate from the NPBA-γ-CyD PPRX is relatively higher than that of PBA-γ-CyD PPRX, resulting in slightly lower yield of NPBA-γ-CyD PPRX than of PBA-γ-CyD PPRX. In order to obtain further information on the formation process, we used turbidity measurements to evaluate the difference of the PPRX-formation ability of the modified CyDs. As shown in Figure 3, the NPBA-γ-CyD solution mixed with Naph-PEG shows a more rapid increase in turbidity


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than the PBA-γ-CyD solution. This indicates that there must be sufficient space in the NPBA-γCyD cavity for the PEG chain to easily thread through it. In order to discuss the difference in the formation of the PPRXs with regard to differences in the available cavity space for the PEG chain, molecular orientation analysis of the NPBA and PBA moieties of the CyDs was performed. We measured the induced circular dichroism (ICD) spectra of NPBA-γ-CyD (0.15 mM) and PBA-γ-CyD (0.15 mM) in acidic water (pH 3.5–4.5), considering that both NPBA-γ-CyD and PBA-γ-CyD exist in the monomolecular state in these dilute solutions, and that the NPBA and PBA moieties are not ionized in acidic solutions. The transition moments of the NPBA moiety and the PBA moiety were calculated using density functional theory (DFT) calculations at the B3LYP/6-31+G (2d) level of theory, as shown in Figure S6. As shown in Figure S7a, two negative ICDs, one at 221 nm, and the other at 275 nm, are observed. The former is attributed to dipole moment (I), and the latter is attributed to dipole moment (III). A positive ICD at 245 nm assigned to dipole moment (II) is also observed. Harata and Uedaira reported that a positive ICD is observed when the transition moment of an included guest molecule or a modified moiety is in parallel to the molecular axis of the CyD cavity, and a negative ICD is observed when the transition moment is perpendicular to the molecular axis of the CyD cavity32. According to this rule, the angles to the CyD cavity axis of dipole moments (I) and (III) are close to 90°, and that of dipole moment (II) is close to 0° (Figure S8a). From the results for the molecular orientation of the NPBA moiety as shown in Figure S8b, we infer that it does not cover the CyD cavity and thus does not inhibit the threading of a PEG chain, leading to the faster formation of the NPBA-γ-CyD PPRX. Conversely, a negative ICD at 241 nm assigned to the transition moment (I’) of the PBA moiety is observed, as shown in Figure S7b. This confirms that the PBA moiety is tilted with respect to the axis of the CyD cavity (Figure S8c),


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indicating that the PBA moiety covers the CyD cavity (Figure S8d) and inhibits the threading of a PEG chain, causing the slower formation of the PBA-γ-CyD PPRX.

Figure 2. PPRX-formation ability of the CyDs revealed by monitoring turbidity changes after Naph-PEG addition to CyD solutions.

Structural analysis of the Naph-PEG/NPBA-γ-CyD PPRX The XRD spectrum of the Naph-PEG/NPBA-γ-CyD PPRX exhibits the same pattern as that of the PPRX formed with a native γ-CyD, where the γ-CyDs are stacked in a head-to-head and tailto-tail sequence (Figure 3b and 3c).33–35 In particular, the peaks at 2θ = 5.9°, 11.9°, 17.9°, and 23.9° observed in the XRD pattern of Naph-PEG/NPBA-γ-CyD PPRX are indexed on the hkl (001, 002, 003, 004) of the c axis (= 14.8 Å) of a tetragonal unit cell corresponding to the length of two γ-CyD units (Table 1). In contrast, the crystalline structure of the Naph-PEG/PBA-γ-CyD PPRX is different from that of the Naph-PEG/NPBA-γ-CyD PPRX, which is known to be distinct from PPRXs formed with native γ-CyD (Figure 3a). This result indicates that the NaphPEG/PBA-γ-CyD PPRX is not assembled in a head-to-head but head-to-tail arrangement,


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indicating that the PBA moiety is contained in the wider side of the adjacent CyD cavity, as confirmed by previous research.24

Figure 3. XRD patterns of (a) Naph-PEG/PBA-γ-CyD PPRX, (b) Naph-PEG/NPBA-γCyD PPRX, and (c) Naph-PEG/γ-CyD PPRX. The crystalline packing structure is illustrated on the right.

Table 1. Crystallographic characteristics of Naph-PEG/NPBA-γ-CyD PPRX,a calculated assuming a tetragonal unit cell with a = b = 23.76 Å, c = 14.77 Å

2 θ obs

(hkl)

(deg) 5.96

(001)

dobs

dcal

(Å)

(Å)

14.83

14.77


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7.44

(200)

11.88

11.88

8.26

(210)

10.7

10.63

10.36

(220)

8.54

8.4

11.98

(002)

7.39

7.39

14.02

(202)

6.32

6.27

14.88

(400)

5.95

5.94

15.84

(330)

5.59

5.6

16.66

(420)

5.32

5.31

16.76

(132)

5.29

5.27

17.92

(003)

4.95

4.92

21.08

(521)

4.21

4.23

21.9

(530)

4.06

4.07

22.44

(600)

3.96

3.96

23.74

(004)

3.75

3.69

Harada et al. reported that the PEG/α-CyD PPRX is single stranded, and the stoichiometry between its EG unit and α-CyD is 2:1.9,36 Conversely, the PEG/γ-CyD PPRX is double stranded, and the stoichiometry between its EG unit and γ-CyD is 4:1.10 The 1H NMR spectrum of NaphPEG/NPBA-γ-CyD (Figure S4) indicates that the stoichiometry between the EG unit and NPBAγ-CyD is 2.0:1.0. The value for Naph-PEG/NPBA-γ-CyD indicates that NPBA-γ-CyD forms a PPRX including only one Naph-PEG chain. Previously, we reported that Naph-PEG/PBA-γ-CyD


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was a new type of γ-CyD PPRX, where only one PEG chain is included.24 In terms of the number of PEG chains included in the PPRXs, the Naph-PEG/NPBA-γ-CyD PPRX is similar to the Naph-PEG/PBA-γ-CyD PPRX. In order to investigate the molecular interactions between the PBA moieties and CyD cavities, we performed two-dimensional nuclear Overhauser effect spectroscopy (NOESY) where a nuclear Overhauser effect (NOE) cross-peak is detected when two protons are closely located in space (< 5.0 Å). We measured the NOESY spectra of NPBA-γ-CyD and PBA-γ-CyD (5.0 mM). The assignment of protons in NPBA-γ-CyD was carried out using COSY, total correlation spectroscopy (TOCSY), and heteronuclear multiple-quantum correlation (HMQC) (Figure S9– S11). In the NOESY spectrum of NPBA-γ-CyD shown in Figure S12a, there is no NOE correlation between the protons of NPBA and the interior protons of the CyD cavity (H-3 and H5). This indicates that the NPBA moiety is not included in the CyD cavity. Instead, several weak NOE correlations between NPBA and the H-6 protons at the edge of the CyD cavity are observed. This indicates that the NPBA moiety is located on the edge of the primary hydroxyl groups of the CyD ring (Figure S12a). The NOESY spectrum indicates that the NPBA moiety is perpendicular to the axis of the CyD cavity because each proton in NPBA shows NOE correlation with H-6 of the CyD ring. In addition, the XRD pattern indicates that NaphPEG/NPBA-γ-CyD PPRX forms in a head-to-head channel structure where the narrow sides of the NPBA-γ-CyD rings face each other and the wide sides of the NPBA-γ-CyD rings face each other. Taking this head-to-head sequence into consideration, we propose that the NPBA moiety in the formed PPRX is perpendicular to the axis of the CyD cavity (Figure S12a). It is quite possible that the molecular orientation of the NPBA moiety changes as more molecular


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interaction occurs upon increasing the concentration of NPBA-γ-CyD, although the ICD results indicate that the NPBA moiety is parallel to the axis of the CyD cavity in dilute solutions. Conversely, NOE correlations between the PBA protons and the H-3 and H-5 protons of the CyD interior are observed in the NOESY spectrum of PBA-γ-CyD (Figure S12b), indicating that the PBA protons are close to H-5 of the intra CyD cavity and H-3 of the inter CyD cavity, i.e., the PBA moiety is included in the adjacent CyD cavity, as previously reported by our group. These results indicate that the NPBA moiety in the Naph-PEG/NPBA-γ-CyD PPRX is not included in the neighboring NPBA-γ-CyD unit, and that the NPBA moiety is on the edge of the primary hydroxyl groups of the CyD ring, which contrasts with the fact that the PBA moiety is included in the adjacent PBA-γ-CyD in the Naph-PEG/PBA-γ-CyD PPRX. This further indicates that the effective space of the narrow CyD rings for a PEG chain to thread into the CyD cavities is limited by the NPBA moieties on the narrow rings of the CyDs, and this limitation results in the single-stranded NPBA-γ-CyD PPRX in which only one PEG chain is allowed to thread into the CyD cavity. Sugar response of the Naph-PEG/NPBA-γ-CyD PPRX and Naph-PEG/PBA-γ-CyD PPRX The sugar responses of the Naph-PEG/NPBA-γ-CyD PPRX and the Naph-PEG/PBA-γ-CyD PPRX were evaluated with respect to solubility changes using turbidity measurements (Figure 4) and the release of Naph-PEG by monitoring the fluorescence of the terminal naphthoyl groups in Naph-PEG (Figure 5). A solid form of Naph-PEG/NPBA-γ-CyD PPRX (6.0 mg) was suspended in a buffer solution (2.0 mL, 20 mM HEPES buffer pH 7.4, 37 °C). After the turbidity stabilized, a stock Glc solution was added to the suspension. The turbidity decreases as the sugar concentration increases, which suggests that the solid-state PPRX disintegrates and dissolves


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under the effect of Glc (Figure 4). Moreover, the turbidity of Naph-PEG/NPBA-γ-CyD decreases when the Glc concentration in the cell reaches 20 mM, whereas the turbidity of Naph-PEG/PBAγ-CyD starts decreasing at 50 mM. These results indicate that NPBA-γ-CyD PPRX has a higher sugar-responsive Naph-PEG release rate compared with that of the PBA-γ-CyD PPRX. As shown in Figure 5, the release rates of Naph-PEG from both the PPRXs are accelerated at a Glc level of 100 mM. The release profiles also show that the NPBA-γ-CyD PPRX has a higher response to sugar than the PBA-γ-CyD PPRX. This improvement is due to the higher Glcbinding ability of NPBA-γ-CyD compared to that of PBA-γ-CyD. Moreover, the release profile of Naph-PEG from Naph-PEG/NPBA-γ-CyD is totally different to that of Naph-PEG/PBA-γCyD, and the initial release of Naph-PEG from Naph-PEG/NPBA-γ-CyD is much faster than that from Naph-PEG/PBA-γ-CyD. This faster initial release in response to Glc is due to the spatial arrangement of the NPBA moiety, i.e., it is not included in the adjacent CyD ring and is capable of binding with Glc directly. For the rapid Glc-response of NPBA-γ-CyD in the PPRX, the faster initial release of Naph-PEG due to the spatial arrangement of NPBA moiety is more important than the improved Glc-binding ability of NPBA moiety. The response mechanism of NaphPEG/NPBA-γ-CyD can be explained as follows: Naph-PEG/NPBA-γ-CyD PPRX is a stable crystalline structure in water, with NPBA moieties being located on the narrow ring of the CyD cavity. In the presence of Glc, the NPBA moiety changes to Glc-binding form that is sterically bulky, and this disrupts the crystalline structure. The Naph-PEG/NPBA-γ-CyD PPRX may then dissolve in the solution and Naph-PEG is released. When taking into consideration the release profile of Naph-PEG from the NPBA-γ-CyD PPRX in the absence of Glc, in which a small amount causes a rapid increase, it might be possible that another reason for the higher Glc response is the relative instability of the NPBA-γ-CyD PPRX. This instability could be due to the


22

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NPBA-γ-CyD being able to slide on the Naph-PEG chain, and more easily dissociate from it when the NPBA moiety binds with Glc.

Figure 4. Turbidity changes in (a) the Naph-PEG/NPBA-γ-CyD PPRX and (b) the Naph-PEG/PBA-γ-CyD PPRX depending on the Glc concentration (pH 7.4, 37 °C).

Figure 5. Release profiles of Naph-PEG from (a) the Naph-PEG/NPBA-γ-CyD PPRX and (b) the Naph-PEG/PBA-γ-CyD PPRX in the absence and presence of Glc (pH 7.4, 37 °C).


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Preparation of the Naph-PEG-Ins/NPBA-γ-CyD PPRX and the Naph-PEG-Ins/PBA-γ-CyD PPRX Naph-PEG-Ins was prepared using heterobifunctional PEG (H2N-(CH2)3-O-PEG-(CH2)5-COOH). The amine end was modified with a naphthoyl group, and the carboxylic group was linked to the amino group of insulin (Figure 1c). The PEG/Ins ratio was calculated to be 2.2, as reported in our previous research.24 The bioactivity of Naph-PEG-Ins was evaluated using STZ-induced diabetic rats and was determined to retain 71.5% of the bioactivity of native insulin. Naph-PEG-Ins was mixed with NPBA-γ-CyD in water, and Naph-PEG-Ins/NPBA-γ-CyD PPRX was obtained as a precipitate. Although the precipitate occurred within 1 h, we collected the sample after 28 days, following the preparation method that we previously used. A Naph-PEG-Ins/PBA-γ-CyD PPRX was also prepared by the same method. The formation of Naph-PEG-Ins/NPBA-γ-CyD PPRX was faster than that of Naph-PEG-Ins/PBA-γ-CyD, which is consistent with the rapid precipitation of the Naph-PEG/NPBA-γ-CyD PPRX. The results of 1H NMR (Figure S5) and XRD (Figure S13) analysis are similar to those of the Naph-PEG/NPBA-γ-CyD PPRX, indicating that the Naph-PEG-Ins/NPBA-γ-CyD PPRX was successfully prepared. Sugar response of the Naph-PEG-Ins/NPBA-γ-CyD PPRX and Naph-PEG-Ins/PBA-γ-CyD PPRX We evaluated the sugar response of the release of Naph-PEG-Ins from Naph-PEG-Ins/NPBA-γCyD and Naph-PEG-Ins/PBA-γ-CyD. Figure 6 shows that both the release rates are accelerated in the presence of Glc, and that the Naph-PEG-Ins/NPBA-γ-CyD has a higher responsiveness to Glc. Naph-PEG-Ins undergoes slow release from both the Naph-PEG-Ins/NPBA-γ-CyD PPRX and the Naph-PEG-Ins/PBA-γ-CyD PPRX, indicating that the PPRXs are disintegrated in water


24

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by dilution. Although these systems do not show an absolute on/off response for sugar, the slow release of Naph-PEG-Ins in the absence of sugar may potentially be applied to imitate basal insulin secretion. Recent guidelines in diabetes treatment recommend a post-meal plasma Glc level of < 10 mM and a fasting plasma Glc level of 3.9–7.2 mM.37 PPRX systems using NPBA-γ-CyD that exhibit improved sugar responsiveness still require a higher concentration of Glc than the recommended blood Glc level. Further, for in vivo application, by controlling the particle size and stability of dispersion in further study, this system can be prescribed as a suspension, which is injectable at a local area with a steady dose. In this study, we have demonstrated that a PPRX where a modified PBA derivative is not included in the adjacent CyD cavity exhibits improve sugar responsiveness. Our next target is to further increase the Glc responsiveness of PPRX systems. It is encouraging that we have successfully designed a PPRX that has a direct sugar-binding sensor and a PBA derivative that shows a higher affinity for Glc.


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Figure 6. Release profiles of Naph-PEG-Ins from (a) Naph-PEG-Ins/NPBA-γ-CyD PPRX and (b) Naph-PEG-Ins/PBA-γ-CyD PPRX in the absence and presence of Glc (pH 7.4, 37 °C).

CONCLUSION In this study, we modified γ-CyD with NPBA and prepared PPRXs with it. It was demonstrated that NPBA-γ-CyD PPRX released Naph-PEG-Ins with a much higher Glc responsiveness than that of PBA-γ-CyD PPRX reported in our previous work. CD spectra and 2D NMR measurements showed that the bulky NPBA moiety is situated on the edge of the CyD ring. From these observations, we proposed that the spatial arrangement of NPBA is effective in improving the sugar responsiveness of PPRXs. This study represents the first successful attempt to improve the stimuli-response of a PPRX by using a sensor group that may not be included in the CyD cavity, and confirms the importance of the spatial arrangement of the PBA moiety.


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ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. 1

H NMR spectrum of NPBA-γ-CyD in DMSO-d6 (Figure S1), binding abilities of NPBA-γ-CyD

and PBA-γ-CyD determined by the ARS method (Figure S2, S3), Naph-PEG/NPBA-γ-CyD (Figure S4), Naph-PEG-Ins/NPBA-γ-CyD (Figure S5), transition moments of NPBA-γ-CyD and PBA-γ-CyD calculated using DFT calculations (Figure S6), ICD spectra of NPBA-γ-CyD and PBA-γ-CyD (Figure S7), proposed orientations of the NPBA moiety and the PBA moiety (Figure S8), assignment of protons of NPBA-γ-CyD by COSY, TOCSY, and HMQC (Figure S9–S11), NOESY spectra of NPBA-γ-CyD and PBA-γ-CyD in D2O (Figure S12), and XRD patterns of Naph-PEG/NPBA-γ-CyD, Naph-PEG/PBA-γ-CyD and Naph-PEG/γ-CyD (Figure S13). AUTHOR INFORMATION Corresponding Author *(T.S.) Telephone: +81-49-271-7686, FAX: +81-49-271-7714, E-mail: [email protected] The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by JSPS KAKENHI Grant Number 25860027. REFERENCES (1)

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“For Table of Contents Use Only” A pseudopolyrotaxane for glucose-responsive insulin release: The effect of binding ability and spatial arrangement of phenylboronic acid group Tomohiro Seki∗, Keigo Abe, Yuya Egawa, Ryotaro Miki, Kazuhiko Juni, Toshinobu Seki


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Figure 1. Schematic illustrations of (a) NPBA-γ-CyD PPRX with an outward-facing sugar sensor and (b) PBAγ-CyD PPRX with its sugar sensor included in the CyD cavity. (c) Chemical structures of the building blocks of sugar-responsive PPRXs. (d) Schematic illustration of Naph-PEG-Ins/NPBA-γ-CyD PPRX and its sugarresponsive insulin release. Figure 1 174x58mm (300 x 300 DPI)


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Figure 2. PPRX-formation ability of the CyDs revealed by monitoring turbidity changes after Naph-PEG addition to CyD solutions. Figure 2 77x51mm (300 x 300 DPI)



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Figure 3. XRD patterns of (a) Naph-PEG/PBA-γ-CyD PPRX, (b) Naph-PEG/NPBA-γ-CyD PPRX, and (c) NaphPEG/γ-CyD PPRX. The crystalline packing structure is illustrated on the right. Figure 3 76x80mm (300 x 300 DPI)


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Figure 3. XRD patterns of (a) Naph-PEG/PBA-γ-CyD PPRX, (b) Naph-PEG/NPBA-γ-CyD PPRX, and (c) NaphPEG/γ-CyD PPRX. The crystalline packing structure is illustrated on the right. Figure 3 50x57mm (300 x 300 DPI)



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Figure 4. Turbidity changes in (a) the Naph-PEG/NPBA-γ-CyD PPRX and (b) the Naph-PEG/PBA-γ-CyD PPRX depending on the Glc concentration (pH 7.4, 37 °C). Figure 4 77x66mm (300 x 300 DPI)


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Figure 5. Release profiles of Naph-PEG from (a) the Naph-PEG/NPBA-γ-CyD PPRX and (b) the NaphPEG/PBA-γ-CyD PPRX in the absence and presence of Glc (pH 7.4, 37 °C). Figure 5 79x66mm (300 x 300 DPI)



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Figure 5. Release profiles of Naph-PEG from (a) the Naph-PEG/NPBA-γ-CyD PPRX and (b) the NaphPEG/PBA-γ-CyD PPRX in the absence and presence of Glc (pH 7.4, 37 °C). Figure 5 79x66mm (300 x 300 DPI)


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Figure 6. Release profiles of Naph-PEG-Ins from (a) Naph-PEG-Ins/NPBA-γ-CyD PPRX and (b) Naph-PEGIns/PBA-γ-CyD PPRX in the absence and presence of Glc (pH 7.4, 37 °C). Figure 6 79x70mm (300 x 300 DPI)



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Figure 6. Release profiles of Naph-PEG-Ins from (a) Naph-PEG-Ins/NPBA-γ-CyD PPRX and (b) Naph-PEGIns/PBA-γ-CyD PPRX in the absence and presence of Glc (pH 7.4, 37 °C). Figure 6 78x70mm (300 x 300 DPI)


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Graphic TOC 56x35mm (300 x 300 DPI)


A Pseudopolyrotaxane for Glucose-Responsive ... - ACS Publications

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