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RESEARCH ARTICLE | Updated:2024-08-06
    • Glucose-sensitive Membrane with PBA-based Contraction-type Linear Polymer as Chemical Valves Prepared by Surface Grafting

    • Zhang Chun-Peng

      a ,  

      Gao Si-Jia

      b ,  

      Luo Ying

      c ,  

      Zou Lei

      b ,  

      Zhang Yong-Jun

      bd ,  
    • Chinese Journal of Polymer Science   Vol. 42, Issue 8, Pages: 1067-1076(2024)
    • DOI:10.1007/s10118-024-3135-3    

      CLC:
    • Published:01 August 2024

      Published Online:17 May 2024

      Received:18 January 2024

      Revised:02 April 2024

      Accepted:07 April 2024

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  • Chun-Peng Zhang, Si-Jia Gao, Ying Luo, et al. Glucose-sensitive Membrane with PBA-based Contraction-type Linear Polymer as Chemical Valves Prepared by Surface Grafting. [J]. Chinese Journal of Polymer Science 42(8):1067-1076(2024) DOI: 10.1007/s10118-024-3135-3.

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    Abstract

    Glucose-sensitive membrane has potential application in self-regulating insulin release. Phenylboronic acid (PBA) is a well-known glucose reporter. Unfortunately, most PBA-based glucose-sensitive materials are expansion-type, which are not suitable as chemical valves in membrane pores for self-regulating insulin release. According to a new glucose-sensitive mechanism, we synthesized PBA-based contraction-type glucose-sensitive liner polymer and microgels. Herein, a glucose-sensitive membrane was prepared by grafting PBA-based contraction-type glucose-sensitive linear polymer on the membrane surface. Through adjusting the chain length and chain density, the glucose-sensitivity of the membrane was optimized. The membrane can reversibly regulate insulin release at physiologically relevant glucose concentrations in simulates body fluids and fetal bovine serum. The membrane also has good stability, anti-fouling and biocompatibility. It has potential application in self-regulating insulin release.

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    Keywords

    Phenylboronic acid; Glucose-sensitive; Membrane; Surface grafting; Insulin

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    INTRODUCTION

    Glucose-sensitive membrane has attracted attention because it has potential application in self-regulating insulin release system.[

    1−3] The main method for constructing glucose-sensitive membrane for insulin release is to modify the membrane surface or membrane channel with contraction-type glucose-sensitive microgels or linear polymer.[4−7] The increased glucose concentration causes the glucose-sensitive material to shrink, thus opening the channel of the membrane and allowing insulin to be released. The reduced glucose concentration causes the glucose-sensitive material to extend, thus closing the channel of the membrane and reducing insulin release.[8,9] The reported contraction-type glucose-sensitive materials were mainly based on glucose oxidase (GOD).[10,11] Phenylboronic acid (PBA), another commonly used glucose-sensitive component, exhibits much higher reliability than GOD.[12,13] However, PBA-based glucose-sensitive membrane for insulin release has rarely been reported.
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    The well-known glucose-sensitive mechanism of PBA is shown in Scheme 1(a). Combination of glucose and PBA will lead to increased degree of ionization and hydrophilicity of the polymer.[

    14,15] Therefore, PBA-based glucose-sensitive materials are mostly expansion-type, which are not suitable as chemical valves in membrane channels to regulate insulin release. Our group has proposed a new glucose-sensitive mechanism (Scheme 1b). Poly(N-isopropylacrylamide-co-2-(acrylamido) phenylboronic acid) (P(NIPAM-co-2-AAPBA) has both glucose-sensitivity and temperature-sensitivity.[16,17] Due to O and B can form coordination bond, the combination of glucose and PBA will reduce the distance between glucose and PNIPAM polymer chains, rather than increase hydrophilicity of the polymer.[18,19] The reduced distance between glucose and PNIPAM chain leads to the destruction of the structural water around NIPAM, thereby reducing the lower critical solution temperature (LCST) of the polymer chain, which causes the polymer to shrink.[20] Based on this glucose-sensitive mechanism, we synthesized PBA-based glucose-sensitive contraction-type microgels and linear polymer. And glucose-sensitive membranes with PBA-based contraction-type polymer as chemical valves were prepared by non-solvent induced phase separation (NIPS).[21,22] During the process of NIPS, it is necessary to introduce more microgels or linear polymers to improve the glucose-sensitivity of the membrane. However, increasing the content of microgels or linear polymers will lead to the enlargement of membrane pores, which will limit the improvement of glucose-sensitivity. In addition, the blended microgels or liner polymers will inevitably dissolve out during use.
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    Fig 1  (a) The traditional glucose-sensitive mechanism of PBA; (b) The new glucose-sensitive mechanism of PBA; (c) Preparation process of the glucose-sensitive membrane by surface-initiated RAFT polymerization; (d) Glucose-sensitive mechanism of the membrane.

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    In this work, we grafted contraction-type PBA-based glucose-sensitive polymers on the surface of PVDF membrane by surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization (Scheme 1c). Compared with blending, surface grafting has better stability. Furthermore, by adjusting the chain length and chain density, the membrane can perform controlled insulin release at physiologically relevant glucose concentrations (Scheme 1d).

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    EXPERIMENTAL

    Materials

    Poly(vinylidene fluoride) (PVDF) membrane (MWCO=100 kD) was purchased from AMFOR. Potassium hydroxide (KOH), potassium permanganate (KMnO4), methanol, dichloromethane, ethanol, acetone, n-hexane, potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4) and glucose were supplied by local suppliers. Bovine insulin and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) were provided by Yuanye. 2-Aminophenylboronic acid (2-APBA) and bovine serum albumin (BSA) were provided by Bi De Pharm. Fetal bovine serum (FBS) was obtained from Solarbio. Alizarin Red S (ARS) and fluorescein isothiocyanate (FITC) was purchased from Sigma-Aldrich. Azodiisobutyronitrile (AIBN) and cyano-4-(dodecyl thioalkyl thiocarbonyl) thioalkyl valerate (CDTPA) were purchased from Macklin. N-isopropylacrylamide (NIPAM) and N-isopropylmethylacrylamide (NIPMAM) were all obtained from TCI. NIPAM and NIPMAM were purified by recrystallization. Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12), penicillin/streptomycin (P/S) were purchased from Life Technologies. CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega. 2-Acrylamidophenylboronic acid (2-AAPBA) was synthesized according to the literature and characterized by 1H-NMR (Fig. S1 in the electronic supplementary information, ESI).

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    PVDF Membrane Modified with ―OH

    Before use, PVDF membrane was soaked in anhydrous ethanol for 10 min, cleaned with deionized water and vacuum dried at room temperature to remove surface impurities. Next, the cleaned PVDF membrane was immersed in 4 wt% KOH/alcohol solution at 60 °C for 6 min. Thoroughly clean the membrane with deionized water to terminate the reaction. This step allows the membrane surface to be modified with double bonds. Then, the membrane was immersed in 4 wt% KMnO4/30 wt% KOH aqueous at 25 °C for 10 min. Clean thoroughly with deionized water to terminate the reaction.[

    23,24] In this step, the double bonds were oxidized to hydroxyl group. After vacuum drying at room temperature, the membrane modified with hydroxyl group (M-OH) was obtained.
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    PVDF Membrane Modified with CDTPA

    CDTPA was modified on the membrane surface through esterification reaction. M-OH with a diameter of 5 cm was added into CDTPA/DCM solution. In order to adjust density of polymer chain, CDTPA concentrations were set as 0.07, 0.14 and 0.56 mmol·L−1. After 10 min, EDC/DCM solution (nEDC = 10nCDTPA) was added dropwise to the above solution. The reaction was carried out at 25 °C for 48 h. After the reaction, the membrane was washed with DCM, ethanol and water in turn.[

    25] After vacuum drying, the membrane modified with CDTPA (M-CDTPA) was obtained.
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    PVDF Membrane Modified with PBA-based Glucose-sensitive Linear Polymer

    Contraction-type PBA-based glucose-sensitive linear polymer was modified on the membrane surface through surface-initiated RAFT polymerization. In 250 mL Schlenk flask, 2-AAPBA, NIPAM and NIPMAM were dissolved in 20 mL of 95 vol% methanol/water. The ratio among NIPAM, NIPMAM, 2-AAPBA was set as 10:5:4. In order to control the chain length, the total monomer concentrations were adjusted to 1.2, 2.4 and 3.6 mmol·L−1. Then, M-CDTPA and the initiator AIBN (0.012, 0.024 and 0.036 mmol·L−1) were added. The reaction was carried out in N2 at 70 °C for 24 h. The membrane was washed with ethanol and water in turn. After vacuum drying, the membrane modified with glucose-sensitive polymer was obtained.[

    26] The prepared glucose-sensitive membrane was named as M-RAx-Py (Table 1).
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    Table 1  Dosage of monomer and CDTPA in glucose-sensitive membrane.
    CDTPA (mmol·L−1)NIPAM (mmol·L−1)NIPMAM (mmol·L−1)2-AAPBA (mmol·L−1)
    M-0 / / / /
    M-RA0.56-P1.2 a 0.56 12.45 6.20 4.95
    M-RA0.56-P2.4 0.56 24.90 12.40 9.90
    M-RA0.56-P3.6 0.56 37.35 18.60 14.85
    M-RA0.07-P2.4 0.07 24.90 12.40 9.90
    M-RA0.14-P2.4 0.14 24.90 12.40 9.90

    a The glucose-sensitive membrane is named as M-RAx-Py, where x represents the concentration of CDTPA during the reaction and y represents the total concentration of monomers during the polymerization.

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    Membrane Characterization

    The chemical structure of membrane surface was characterized by attenuated total refraction-fourier transform infrared spectrometer (ATR-FTIR, Nicolet iS50, ThermoFisher). The elemental composition of the membrane surface was characterized by X-ray photoelectron spectroscopy (XPS, K-alpha, ThermoFisher). Relative elemental content was analyzed by elemental analyzer (EA, Vario EL Cube, Elementar). The surface morphology was characterized by scanning electron microscopy (SEM, S-4800, Hitachi). The dynamic contact angle of the membrane was measured at room temperature using a contact angle measuring instrument (JYSP-180, Jinshengxin). The surface roughness of the membrane was detected by atomic force microscopy (AFM, Dimension Icon, Bruker) in tapping mode. The porosity of the membrane was characterized by wet-dry weight method. The mass of the membrane was measured by analytical balance (ME204E, Mettler Toledo).

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    Grafting Degree

    The grafting degree of monomers on the membrane is the increase of the mass of glucose-sensitive membrane in unit area compared to M-CDTPA.[

    27] Prior to the weight measurement, the samples were dried in a vacuum oven at 60 °C for 24 h. The total grafting degree (GD) could be calculated according to Eq. (1):
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    GD=W1W0Amath 1

    where w0 (μg) and w1 (μg) are the weight of the M-CDPTA and glucose-sensitive membrane, respectively, A (cm2) is the surface area of the membrane. Weight measurements were conducted for 3 times, and the average value was reported.

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    ARS as Fluorescent Probe to Detect PBA

    M-0 and M-RA0.56-P2.4 (1 cm × 1 cm) were immersed in 0.1 mg/mL ARS (50 mmol·L−1 pH 7.4 Tris-HCl) and incubated at 25 °C for 6 h. Then the membrane was washed for 3 times with 50 mmol·L−1 pH 7.4 Tris-HCl. After vacuum drying, the ARS stained membrane was observed by confocal laser scanning microscope (CLSM, FV3000, Olympus).

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    Glucose-sensitive Flux Test

    The cross-flow membrane filtration device was used to test the transmembrane flux at 37 °C and 0.1 MPa. The effective diameter of the membrane is 3 cm. Filtration at 0.1 MPa for 3 h to achieve a stable flux. Then the glucose-sensitivity of the membrane was tested by measuring the glucose solution flux at 37 °C. The glucose solution was prepared in 20 mmol·L−1 pH 7.4 PBS, and the glucose concentrations were 0, 10, 20, 30, 40 and 50 mmol·L−1, respectively. The flux (J) is calculated according to Eq. (2):

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    J=VA×tmath 2

    where V (L) is the volume of the permeate solution, A (m2) is the area of the membrane, and t (h) is the time. Through three parallel experiments, the average value is recorded as the flux of each membrane.

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    Permeability Test of Insulin through Membrane

    Insulin permeation was tested with a standard side-by-side diffusion cell at 37 °C (Fig. S2 in ESI). Insulin solution (3 mg·mL−1, 20 mmol·L−1 pH 7.4 PBS with different glucose concentrations) was added in donor cells. Glucose solution (20 mmol·L−1, pH 7.4 PBS) with different glucose concentrations (0, 0.001, 0.002, 0.004 g·mL−1) were added into receptor cell. The glucose concentration in the donor cell is equal to that in the receptor cell. The concentration of insulin in the receptor cell was determined by UV-Vis spectrophotometer (UH4150, Hitachi) at 276 nm.

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    The diffusion permeability coefficient (P) is calculated according to Eq. (3):

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    P=Mt×hS×Cd×t×100%math 3

    where Mt (mg) is the mass of drug permeation at time t (s), h (cm) is the thickness of the membrane, S (cm2) is the effective permeation area, Cd (mg/mL) is the insulin concentration in donor cells, and t (s) is the permeation time.

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    In order to study whether the inorganic salts and other metabolites in the blood have an effect on the glucose-sensitivity of the membrane, insulin permeation test was also carried out in simulated human body fluids (SBF) and fetal bovine serum (FBS). The formula for preparing SBF is as follows: NaCl (8.035 g), NaHCO3 (0.355 g), K2HPO4·3H2O (0.225 g), MgCl2·6H2O (0.311 g), 1.0 mol·L−1 HCl (39 mL), CaCl2 (0.292 g), Na2SO4 (0.072 g), Tris (6.118 g), 1.0 mol·L−1 HCl (1−5 mL) were sequentially added into 700 mL H2O at room temperature. The pH of solution was adjusted to 7.4, with 1.0 mol·L−1 HCl or 1.0 mol·L−1 NaOH. Then the insulin release test of the membrane in SBF was tested.

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    The insulin release of the membrane in FBS was tested according to this method. The PBS was replaced with FBS. Insulin was replaced by fluorescein isothiocyanate labeled insulin (FITC-insulin).[

    28] The concentration of FITC insulin in the receptor pool is tested by fluorescence spectrophotometer (UF-7000, Hitachi, Japan).
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    Anti-fouling Performance

    The membrane was pre-wetted with 20 mmol·L−1 pH 7.4 PBS. Then, it was immersed in 40 mL of 0.5 mg·mL−1 BSA solution (20 mmol·L−1 pH 7.4 PBS) for 12 h to achieve adsorption equilibrium. The membrane was washed with deionized water to remove BSA from the surface. Then, it was subjected to ultrasonic treatment in 40 mL of 20 mmol·L−1 pH 7.4 PBS for 1 h. The protein concentration of the eluent obtained after ultrasound was determined by UV-Vis. The same membrane was tested for 3 times, and the results were averaged. The adsorption capacity of BSA (Q, μg·cm−2) was determined by Eq. (4):

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    Q=ΔC×VS×100%math 4

    where ΔC is the difference between the initial concentration of BSA and the concentration after adsorption equilibrium, V (mL) is the volume of BSA solution, and S (cm2) is the area of the membrane.[

    29]
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    Biocompatibility of the Membrane

    Firstly, the membrane was cut into 1 cm × 1 cm samples, and then immersed in ethanol for 30 min for disinfection. At 37 °C, L929 mouse fibroblast-like cells were cultured in DMEM/F-12 medium containing 1% antibiotics and 10% FBS, and the external environment was humid atmiosphere containing 5% CO2. L929 cells were seeded in 24-well plates at a density of 5×104 cells per well. Cells cultured without the membrane were used as controls. Cell viability was evaluated after 24, 72 and 120 h of culture. MTS assay was used to evaluate cell viability. Absorbance at 490 nm was recorded with a VICTOR® NIVOTM mutlimode plate reader (PerkinElmer, USA).

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    Stability Test of Membrane

    In order to test the stability of glucose-sensitive membrane. The glucose-sensitive membranes were immersed in 20 mmol·L−1 pH 7.4 PBS at 37 °C and shaken for 7 h. Glucose-sensitive flux was measured before and after the treatment to evaluate the stability of the membrane. In addition, the glucose-sensitive membrane immersed in PBS for 7 days was subjected to a flux test in a glucose concentration environment of 0 and 50 mmol/L alternating cycles to further test the stability of the membrane.[

    30]
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    Rejection Rate Test

    The rejection rate of the membrane was tested by cross-flow membrane filtration device. The solutes were 500 mg·L−1 BSA (6.7×104 Da), ovalbumin (4.3×104 Da), and insulin (5.8×103 Da), respectively. Before the test, the protein solution concentration-absorbance standard curve was drawn. The membrane was first compacted with pure water for 30 minutes to eliminate any effect of compaction on flux decline. Then, the protein rejection rate of M-RA0.56-P2.4 in 0, 10, 20, 30, 40 and 50 mmol·L−1 glucose (in 100 mmol·L−1 pH 7.4 PBS) was tested at 37 °C. The rejection rate is calculated by Eq. (5):[

    31]
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    R=C0C1C0×100%math 5

    where C0 is the initial concentration of each protein and C1 is the concentration of each protein after passing through the membrane.

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    RESULTS AND DISCUSSION

    Preparation and Chemical Characterization of Glucose-sensitive Membranes

    Glucose-sensitive polymer was grafted on the membrane surface through surface-initiated RAFT polymerization. Briefly, PVDF membrane was treated with KOH and KMnO4 to introduce hydroxyl groups on the membrane surface. Then, the RAFT agent (CDTPA) was modified on the membrane surface through esterification reaction. Finally, glucose-sensitive polymer chain was grafted on the membrane surface by RAFT polymerization (Fig. S3 in ESI).

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    ATR-FTIR was used to characterize the chemical structure of the membrane surface. As shown in Fig. 1(a), ATR-FTIR spectrum of pristine PVDF membrane (M-0) shows three strong characteristic bands at 1400, 1167 and 880 cm−1, which can be attributed to ―CH2―, ―CF2― stretching and C―C bond skeleton vibration, respectively. After alkali treatment, a strong absorption band appears at 3400 cm−1. This is the characteristic band of ―OH, which proves that ―OH has been successfully introduced to the membrane surface. After esterification, the band strength of ―OH characteristic band decreases significantly, proving that CDTPA was successfully modified. The characteristic absorption band of amide bond (1550 cm−1) appears after surface-initiated RAFT polymerization, demonstrating that glucose-sensitive polymer was successfully modified on the membrane surface.[

    32]
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    Fig 1  (a) ATR-FTIR spectra of M-0, M-OH, M-CDTPA and M-RA0.56-P2.4; (b) XPS spectra of M-0, M-OH, M-CDTPA and M-RA0.56-P2.4.

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    The chemical structure of the membrane surface was further confirmed by XPS (Fig. 1b, Table S1 in ESI). The XPS of M-0 shows emission peaks corresponding to C 1s, N 1s, O 1s and F 1s at 285.27, 400.09, 532.07 and 688.23 eV, respectively. M-0 contains N and O, which may be due to the addition of additives in the process of membrane preparation. Compared with M-0, the content of F element of M-OH decreased from 37.38% to 8.13%. This is due to the removal of HF on the membrane surface after alkali treatment. Due to the introduction of hydroxyl group on the membrane surface, the content of oxygen element on the membrane surface increased from 3.29% to 15.75%. After CDTPA modification, S element (168.2 and 232 eV corresponding to S 2p and S 2s) appeared on the membrane surface, which proved that CDTPA was successfully modified on the membrane surface. After polymerization, the content of N element (400.04 eV) increased from 3.79% to 9.21%, while the content of S element decreased from 2.57% to 1.53%. This indicated that the polymer was successfully grafted onto the surface of the membrane. The XPS results were consistent with the ATR-FTIR results.

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    In order to further verify glucose-sensitive polymer was successfully grafted on the membrane surface, the membrane was stained with ARS and observed with CLSM. PBA can combine with diol on ARS to form PBA-ARS complex, which can improve the fluorescence intensity of ARS (Fig. S4 in ESI).[

    33] ARS-stained M-0 shows no fluorescence under CLSM. However, under the same observation conditions, ARS-stained glucose-sensitive membrane emits obvious fluorescence due to the formation of PBA-ARS complex (Fig. S5 in ESI). CLSM results proved that PBA monomer was successfully modified on the membrane surface.
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    Glucose-sensitive Membrane with Different Chain Length and Chain Density

    Polymer chain length and chain density have significant effect on glucose-sensitivity of the membrane.[

    34,35] By adjusting CDTPA and monomer concentration, we constructed a series of glucose-sensitive membrane with different chain lengths and chain densities. Thus, optimal glucose-sensitivity can be obtained.[36,37]
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    In order to adjust the polymer chain length, we fixed the concentration of CDTPA as 0.56 mmol·L−1, and changed the total concentration of monomer during polymerization. The total concentration of monomer was set as 1.2, 2.4 and 3.6 mmol·L−1. The resulting membrane was named as M-RA0.56-P1.2, M-RA0.56-P2.4 M-RA0.56-P3.6. Since the polymer was fabricated from CDTPA active sites, the density of the polymer chain on the membrane surface was equal to the density of CDTPA on the membrane surface before polymerization. Therefore, M-RA0.56-P1.2, M-RA0.56-P2.4, M-RA0.56-P3.6 have similar chain densities. Under the same density of CDTPA and the same polymerization conditions, with the increase of monomer concentration, the length of polymer chain increases. As a result, the grafting degree of the membrane increases (Fig. 2a). ATR-FTIR and XPS results confirmed that the amount of grafted polymer increased with the increase of monomer concentration (Fig. S6 in ESI). By adjusting the total concentration of monomer, we obtained membranes with similar density and different chain lengths.

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    Fig 2  (a) Grafting degree (GD) and (b) glucose-sensitive flux of M-0, M-RA0.56-P1.2, M-RA0.56-P2.4, M-RA0.56-P3.6.

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    The effect of polymer chain lengths on the glucose-sensitivity (glucose sensitivity coefficient R, the ratio of membrane flux to 0 mmol/L membrane flux in a 50 mmol·L−1 glucose environment) of the membrane was investigated (Fig. 2).[

    38] Due to the introduction of the polymer chain, the flux of the glucose-sensitive membrane is lower than that of M-0. As the polymer chain length increases, the flux of the membrane decreases. This is due to the fact that as the polymer chain length increases, part of the membrane pores become blocked. The flux of M-0 hardly changed with the change of glucose concentration (Fig. 2 and Fig. S8A in ESI). For glucose-sensitive membranes, with the increase of glucose concentration, the flux of membrane increases. This is due to the increase in glucose concentration, the polymer chain contracts, the membrane pore opens, and the flux increases (Fig. S8B in ESI).[39] Glucose-sensitive coefficient was used to quantitatively characterize the glucose-sensitivity of the membrane, which was defined as the ratio of membrane flux at glucose concentration of 50 mmol·L−1 to that at glucose concentration of 0 mmol·L−1. As shown in Fig. 2(b), for M-RA0.56-P1.2, M-RA0.56-P2.4, when the polymer chain length increases, the glucose-sensitive coefficient increases. However, when the polymer chain was further increased, for M-RA0.56-P3.6, the glucose-sensitive coefficient decreased. When the chain length is too long, even if the polymer chain is in a shrinking state, the membrane pores cannot be opened (Fig. S8C in ESI).[40] So it is necessary to select a suitable concentration of monomer to achieve the optimal glucose-sensitivity of the membrane. As a result, we chose the total monomer amount of 2.4 mmol·L−1 for subsequent experiments.
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    Next, the graft density of the polymer chain was adjusted by changing the concentration of CDTPA, which was set as 0.07, 0.14 and 0.56 mmol·L−1, respectively.[

    41] EA was used to characterized the amount of CDTPA modified on the membrane surface.[42] As shown in Table S2 (in ESI), M-0 itself does not contain the S element. After modified with CDTPA, M-CDTPA contains S element. The increase of S element mainly comes from the modification of CDTPA. When CDTPA concentration increased from 0.07 mmol·L−1 to 0.56 mmol·L−1, the content of S element in the M-CDTPA increased from 1.57% to 2.50%. This proved that the density of CDTPA modified on the membrane surface increased. The density of the polymer chain is equivalent to the density of CDTPA before polymerization. After polymerization, we obtained the membrane with different chain densities, which was named as M-RA0.07-P2.4, M-RA0.14-P2.4, M-RA0.56-P2.4, respectively. With the increase of chain density, the grafting degree of the membrane also increases (Fig. 3a). ATR-FTIR and XPS results also confirmed that the amount of polymer grafted on the membrane increased (Fig. S7 in ESI).
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    Fig 3  (a) Grafting degree (GD) and (b) glucose-sensitive flux of M-0, M-RA0.07-P2.4, M- RA0.14-P2.4, M-RA0.56-P2.4.

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    The effect of chain density on membrane surface on glucose-sensitivity was explored. As shown in Fig. 3(b), when the graft density of the polymer chain increases, the flux of the membrane decreases, while glucose-sensitivity of the membrane increases. As the density of the polymer chain increases, the number of polymer chains acting as chemical valves increases (Figs. S8B and S8D in ESI). By adjusting CDTPA and total monomer concentration, M-RA0.56-P2.4 has the optimal glucose-sensitivity. In order to more effectively prove the glucose sensitivity of the membrane, the rejection rate of the membrane was tested with BSA, ovalbumin and insulin. As shown in Fig. S9 (in ESI), at each glucose concentration, the rejection rate decreased with the decrease of molecular weight. For each protein, the rejection rate decreased with the increase of glucose concentration. These results were consisting with the flux results. The results confirmed that insulin can permeate through membrane under pressure.

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    Microstructure of the Membrane

    In order to observe the effect of surface grafting on membrane microstructure, the surface of the membrane was observed by SEM. Membrane pores can be clearly observed on the membrane surface of M-0 (Fig. 4a), M-OH (Fig. 4b), and M-CDTPA (Fig. 4c). Furthermore, the size of pores on the membrane surface does not change significantly. After polymerization, membrane surface became denser, and the size of pores decreases significantly (Figs. 4d−4i). With the increase of chain density, the size of pores on the membrane surface becomes smaller and the membrane surface becomes denser (Figs. 4d−4f). The same is true as the polymer chain length increases (Figs. 4g−4i). This is due to the fact that the grafted polymer covers the pores on the membrane surface. The roughness of the membrane was characterized by AFM. As the polymer chain density or chain length increases, the roughness of the membrane increases (Fig. S10 in ESI). The results were consistent with the SEM results.

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    Fig 4  SEM images the surface of (a) M-0, (b) M-OH, (c) M-CDTPA, (d) M-CDTPA0.07-P2.4, (e) M-CDTPA0.14-P2.4, (f) M-CDTPA0.56-P2.4, (g) M-CDTPA0.56-P1.2, (h) M-CDTPA0.56-P2.4, (i) M-CDTPA0.56-P3.6. Scale bar is 0.5 μm.

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    Hydrophilicity of the Membrane

    The hydrophilicity of the membrane was studied by dynamic contact angle (Fig. S11 in ESI). Compared with M-0, the hydrophilicity of M-OH was improved. This was due to the formation of ―OH on the membrane surface. After modification of CDTPA, the hydrophilicity of membrane was deteriorated, due to the hydrophobicity of dodecyl in CDTPA. After polymerization, the hydrophilicity of the membrane surface was improved due to the hydrophilicity of the glucose-sensitive polymer (Fig. S11A in ESI). With the increase of chain length or chain density, the initial contact angle of the membrane decreases gradually. The contact angle of the membrane gradually decreases over time, until the equilibrium is reached (Figs. S11B and S11C in ESI). With the increase of polymer chain length or chain density, the hydrophilicity of the membrane surface increases gradually.[

    44]
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    Release of Glucose-sensitive Insulin In vitro

    Standard side-by-side diffusion cell was used to study the permeability of insulin on the membrane. The glucose concentration was selected to simulate the blood glucose level in the human body, 0.001 g·mL−1 (normal pre-meal blood glucose in non-diabetic patients), 0.002 g·mL−1 (diabetic patients) and 0.004 g·mL−1 (maximum detectable value of a blood glucose test device and test strip).[

    45] M-RA0.56-P2.4 was used for insulin diffusion experiment. As shown in Figs. 5(a) and 5(d), due to its larger pore size (Figs. 4a and 4h), cumulative insulin release of M-0 was greater than M-RA0.56-P2.4 at all glucose concentrations. As the glucose concentration increased, the cumulative insulin release on M-0 was approximately the same. However, the cumulative release of insulin on M-RA0.56-P2.4 increased with the increase of glucose concentration. The diffusion permeability coefficient of insulin through the membrane was calculated according to Eq. (3) (Figs. 5b and 5e). The insulin diffusion permeability coefficients of M-RA0.56-P2.4 were 4.8×10−7, 5.7×10−7, 6.7×10−7 and 7.9×10−7 cm2·s−1 at 0, 0.001, 0.002, 0.004 g·mL−1 glucose concentrations, respectively, while the diffusion coefficient of M-0 was relatively constant. Glucose-sensitive insulin release was characterized by P200/P100 (the ratio of P at 0.002 g·mL−1 glucose to P at 0.001 g·mL−1 glucose) and P400/P100 (the ratio of P at 0.004 g·mL−1 glucose to P at 0.001 g·mL−1 glucose). Compared with M-0, M-RA0.56-P2.4 has better glucose-sensitive insulin release behavior (Figs. 5c and 5f). Due to the negatively charged of insulin, the effect of glucose concentration on the zeta potential of membrane surface should also be considered in addition to the conformational change of the polymer. According to new glucose-sensitive mechanism, glucose will not significantly alter the ionization degree of the PBA groups. Furthermore, addition of glucose will not significantly change the zeta potential of P(NIPAM-2-AAPBA) liner polymer (Fig. S12 in ESI).[17] Thus, glucose-induced changes in the zeta potential of the membrane surface will not significantly affect the permeability of insulin.
    transl

    fig

    Fig 5  Cummuiative insulin release through (a) M-0 and (d) M-RA0.56-P2.4 at different glucose concentrations; Diffusion permeability coefficient (P) of (b) M-0 and (e) M-RA0.56-P2.4 at different glucose concentrations; The ratio of insulin diffusion permeability coefficient of (c) M-0 and (f) M-RA0.56-P2.4.

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    Alternating release ability is one of the most important conditions for self-regulating insulin delivery. By alternating the glucose concentration between 0.004 and 0.001 g·mL−1, changes in the amount of insulin released can be observed. The amount of permeated insulin (Fig. 6a) and the permeability coefficient of insulin (Fig. 6b) increases with increasing glucose from 0.001 g·mL−1 to 0.004 g·mL−1 and decreases when glucose is reduced to 0.001 g·mL−1. Similar results can be observed in four consecutive cycles and P400/P100 hardly changed (Fig. 6c). These results demonstrated the reproducibility of the membrane.

    transl

    fig

    Fig 6  (a) Commutative insulin release and (b) P of M-RA0.56-P2.4 at different glucose concentrations in four alternating cycles; (c) The diffusion permeability coefficient ratio (P400/P100) in four alternating cycles.

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    In order to further test the release of insulin in the physiological environment, FBS and SBF were used to simulate the physiological environment. In FBS or SBF, the insulin release and P value of M-RA0.56-P2.4 still increased with the increase of glucose concentration, as shown in Figs. S13(a), S13(b), S13(d) and S13(e) (in ESI). The glucose sensitivity coefficients (P400/P100, P200/P100) in FBS are 1.43 and 1.25, respectively. The glucose sensitivity coefficients (P400/P100, P200/P100) in SBF are 1.39 and 1.21, respectively. The above results are similar to those in PBS. These results indicate that protein and some metabolite in human body fluids have no significant effect on the glucose-sensitivity of the membrane. The M-RA0.56-P2.4 prepared by this method has good self-regulated glucose-sensitive insulin release at physiologically relevant blood glucose concentrations.

    transl

    Antifouling and Cytotoxicity of Membrane

    BSA was used for static adsorption experiments to characterize the anti-fouling performance of the glucose-sensitive membrane (Fig. S14 in ESI).[

    43] BSA adsorption capacity of glucose-sensitive membrane was lower than that of PVDF membrane. With the increase of polymer chain length or chain density, the BSA adsorption amount of glucose-sensitive membrane decreased. This may be caused by the increased hydrophilicity of the membrane surface (Fig. S14 in ESI). Cytotoxicity of the membrane was evaluated by L929 cells. There was no significant decrease in cell activity after co-culture with membrane (Fig. S15 in ESI). The results showed that the membrane had low cytotoxicity and good biocompatibility to mammalian cells.
    transl

    Membrane Stability

    To observe the membrane stability, the glucose-sensitive flux of M-RA0.56-P2.4 was measured before and after rinsing. The glucose-sensitive flux of the membrane did not change significantly before and after rinsing (Fig. 7a). Furthermore, a reversible glucose-sensitive flux test was performed (Fig. 7b). These results further confirmed that the membrane still had good glucose-sensitivity after rinsing.

    transl

    fig

    Fig 7  (a) Glucose-sensitive flux of M-RA0.56-P2.4 before and after rinsing; (b) Reversible glucose-sensitive flux M-RA0.56-P2.4 after rinsing.

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    CONCLUSIONS

    In this work, glucose-sensitive membranes with contraction-type PBA-based glucose-sensitive linear polymer as chemical valves were prepared by surface-initiated RAFT polymerization. M-RA0.56-P2.4 with optimal glucose-sensitivity was obtained by adjusting chain length and chain density. M-RA0.56-P2.4 can reversibly regulate insulin release at physiologically relevant glucose concentration. At the same time, the small molecules and proteins in SFB or FBS had no significant effect on the release of insulin. Due to the improvement of hydrophilicity, M-RA0.56-P2.4 has good anti-fouling performance. Because the glucose-sensitive polymer was modified on the surface of the membrane by covalent bond, the glucose-sensitivity of the membrane was still well maintained even after washing for 7 days. The membrane also has good biocompatibility and has a promising application for self-regulating insulin release.

    transl

    References

    1

    Liu, Z; Wang, W; Xie, R; Ju, X. J; Chu, L. Y. Stimuli-responsive smart gating membranes.Chem. Soc. Rev.2015,45, 460−475.. [Baidu Scholar] 

    2

    Gholami, S; Zarkesh, I; Ghanian, M. H; Hajizadeh-Saffar, E; Hassan-Aghaei, F; Mohebi, M. M; Baharvand, H. Dynamically capped hierarchically porous microneedles enable post-fabrication loading and self-regulated transdermal delivery of insulin.Chem. Eng. J.2020,421, 127823.. [Baidu Scholar] 

    3

    Yu, J; Wang, J; Zhang, Y; Chen, G; Mao, W; Ye, Y; Kahkoska, A. R; Buse, J. B; Langer, R; Gu, Z. Glucose-responsive insulin patch for the regulation of blood glucose in mice and minipigs.Nat. Biomed. Eng.2020,4, 499−506.. [Baidu Scholar] 

    4

    Shen, D; Yu, H; Wang, L; Khan, A; Haq, F; Chen, X; Huang, Q; Teng, L. Recent progress in design and preparation of glucose-responsive insulin delivery systems.J. Control. Rel.2020,321, 236−258.. [Baidu Scholar] 

    5

    Yin, R; Tong, Z; Yang, D; Nie, J. Glucose and pH dual-responsive concanavalin a based microhydrogels for insulin delivery.Int. J. Biol. Macromol.2011,49, 1137−1142.. [Baidu Scholar] 

    6

    Wang, X; Li, Q; Guan, Y; Zhang, Y. Glucose oxidase-incorporated hydrogel thin film for fast optical glucose detecting under physiological conditions.Mater. Today. Chem. 2016 ,1−2, 7−14.. [Baidu Scholar] 

    7

    Guan, Y; Zhang, Y. Boronic acid-containing hydrogels: Synthesis and their applications.Chem. Soc. Rev.2013,42, 8106−8121.. [Baidu Scholar] 

    8

    Tang, Y. J; Cheng, K; Tan, L. F; Xie, X. T; Zhang, B; Su, W. M; Zhao, Y. D; Xu, Q. R. pH and non-enzymatic glucose response at ultra-low concentration using sub-microchannel heterogeneous membrane.Chem. Eng. J.2023,463, 142438.. [Baidu Scholar] 

    9

    Huang, Q; Wang, L; Yu, H; Ur-Rahman, K. Advances in phenylboronic acid-based closed-loop smart drug delivery system for diabetic therapy.J. Control. Rel.2019,305, 50−64.. [Baidu Scholar] 

    10

    Chu, L. Y; Li, Y; Zhu, J. H; Wang, H. D; Liang, Y. J. Control of pore size and permeability of a glucose-responsive gating membrane for insulin delivery.J. Control. Release2004,97, 43−53.. [Baidu Scholar] 

    11

    Sun, X; Ji, W; Zhang, B; Ma, L; Fu, W; Qian, W; Zhang, X; Li, J; Sheng, E; Tao, Y; Zhu, D. A theranostic microneedle array patch for integrated glycemia sensing and self-regulated release of insulin.Biomater. Sci.2022,10, 1209−1216.. [Baidu Scholar] 

    12

    Hu, D. N; Ju, X. J; Pu, X. Q; Xie, R; Wang, W; Liu, Z; Chu, L. Y. Injectable temperature/glucose dual-responsive hydrogels for controlled release of insulin.Ind. Eng. Chem. Res.2021,22, 8147−8158.. [Baidu Scholar] 

    13

    Matsumoto, A; Tanaka, M; Matsumoto, H; Ochi, K; Moro-oka, Y; Kuwata, H; Yamada, H; Shirakawa, I; Miyazawa, T; Ishii, H; Kataoka, K; Ogawa, Y; Miyahara, Y; Suganami, T. Synthetic “smart gel” provides glucose-responsive insulin delivery in diabetic mice.Sci. Adv.2017,3, 0723.. [Baidu Scholar] 

    14

    Xing, S; Guan, Y; Zhang, Y. Kinetics of glucose-induced swelling of P(NIPAM-AAPBA) microgels.Macromolecules2011,11, 4479−4486.. [Baidu Scholar] 

    15

    Chen, S; Matsumoto, H; Moro-oka, Y; Tanaka, M; Miyahara, Y; Suganami, T; Matsumoto, A. Microneedle-array patch fabricated with enzyme-free polymeric components capable of on-demand insulin delivery.Adv. Funct. Mater.2018,29, 1807369.. [Baidu Scholar] 

    16

    Tang, Z; Guan, Y; Zhang, Y. The synthesis of a contraction-type glucose-sensitive microgel working at physiological temperature guided by a new glucose-sensing mechanism.Polym. Chem.2018,9, 1012−1021.. [Baidu Scholar] 

    17

    Wang, Q; Fu, M; Guan, Y; James, T. D; Zhang, Y. Mechanistic insights into the novel glucose-sensitive behavior of P(NIPAM-co-2-AAPBA).Sci. China Chem.2020,63, 377−385.. [Baidu Scholar] 

    18

    Yang, X; Lee, M. C; Sartain, F; Pan, X; Lowe, C. R. Designed boronate ligands for glucose-selective holographic sensors.Chem. Eur. J.2006,33, 8491−8497.. [Baidu Scholar] 

    19

    Tang, Z; Guan, Y; Zhang, Y. Contraction-type glucose-sensitive microgel functionalized with a 2-substituted phenylboronic acid ligand.Polym. Chem.2013,5, 1782−1790.. [Baidu Scholar] 

    20

    Tang, Z; Weng, J; Guan, Y; Zhang, Y. Unexpected large depression of VPTT of a PNIPAM microgel by low concentration of PVA.Macromol. Chem. Phys.2017,218, 1700364.. [Baidu Scholar] 

    21

    Liu, J; Gao, S; Luo, Y; Zhang, C; Zhang, P; Wang, Z; Zou, L; Zhao, Z; Zhang, Y. Glucose-sensitive poly(ether sulfone) composite membranes blended with phenylboronic acid-based amphiphilic block copolymer for self-regulated insulin release.J. Mater. Chem. B2023,11, 5000−5009.. [Baidu Scholar] 

    22

    Huang, D; Gao, S; Luo, Y; Zhou, X; Lu, Z; Zou, L; Hu, K; Zhao, Z; Zhang, Y. Glucose-sensitive membrane with phenylboronic acid-based contraction-type microgels as chemical valves.J. Membr. Sci.2022,650, 120406.. [Baidu Scholar] 

    23

    Zheng, Z; Gu, Z; Huo, R; Luo, Z. Fabrication of self-cleaning poly(vinylidene fluoride) membrane with micro/nanoscaled two-tier roughness.J. Appl. Polym. Sci.2011,122, 1268−1274.. [Baidu Scholar] 

    24

    Dong, L; Liu, X; Xiong, Z; Sheng, D; Lin, C; Zhou, Y; Yang, Y. Fabrication of highly efficient ultraviolet absorbing PVDF membranesviasurface polydopamine deposition.J. Appl. Polym. Sci.2017,135, 45746.. [Baidu Scholar] 

    25

    Ye, J. L; Guo, M. Y; Han, C; Zhang, Y. F; Meng, J. Q. Multifunctionalization of RC membraneviacombining surface initiated RAFT polymerization with thiolactone chemistry for enhanced antibody recovery.Polymer2022,261, 125414.. [Baidu Scholar] 

    26

    Chen, Y; Sun, W; Deng, Q; Chen, L. Controlled grafting from poly(vinylidene fluoride) films by surface-initiated reversible addition-fragmentation chain transfer polymerization.J. Polym. Sci., Part A: Polym. Chem.2006,44, 3071−3082.. [Baidu Scholar] 

    27

    Shen, X; Yin, X; Zhao, Y; Chen, L. Improved protein fouling resistance of PVDF membrane grafted with the polyampholyte layers.Colloid. Polym. Sci.2015,293, 1205−1213.. [Baidu Scholar] 

    28

    Gaballa, H; Theato, P. Glucose-responsive polymeric micelles via boronic acid-diol complexation for insulin delivery at neutral pH.Biomacromolecules2019,2, 871−881.. [Baidu Scholar] 

    29

    Adelsberger, J; Kulkarni, A; Jain, A; Wang, W; Bivigou-Koumba, A. M; Busch, P; Pipich, V; Holderer, O; Hellweg, T; Laschewsky, A; Müller-Buschbaum, P; Papadakis, C. M. Thermoresponsive PS-b-PNIPAM-b-PS micelles: aggregation behavior, segmental dynamics, and thermal response.Macromolecules2010,5, 2490−2501.. [Baidu Scholar] 

    30

    Liu, J; Wang, N; Yu, L. J; Karton, A; Li, W; Zhang, W; Guo, F; Hou, L; Cheng, Q; Jiang, L; Weitz, D. A; Zhao, Y. Bioinspired graphene membrane with temperature tunable channels for water gating and molecular separation.Nat. Commun.2017,5, 02198.. [Baidu Scholar] 

    31

    Lin, Y. C; Tseng, H. H; Wang, D. K. Uncovering the effects of PEG porogen molecular weight and concentration on ultrafiltration membrane properties and protein purification performance.J. Membr. Sci.2021,618, 118729.. [Baidu Scholar] 

    32

    Sun, B; Lin, Y; Wu, P. Structure analysis of poly(N-isopropylacrylamide) using near-infrared spectroscopy and generalized two-dimensional correlation infrared spectroscopy.Appl. Spectrosc.2007,61, 765−771.. [Baidu Scholar] 

    33

    Chen, P. C; Wan, L. S; Ke, B. B; Xu, Z. K. Honeycomb-patterned film segregated with phenylboronic acid for glucose sensing.Langmuir2011,20, 12597−12605.. [Baidu Scholar] 

    34

    Lue, S. J; Hsu, J. J; Wei, T. C. Drug permeation modeling through the thermo-sensitive membranes of poly(N-isopropylacrylamide) brushes grafted onto micro-porous films.J. Membr. Sci.2008,321, 146−154.. [Baidu Scholar] 

    35

    Li, P. F; Xie, R; Jiang, J. C; Meng, T; Yang, M; Ju, X. J; Yang, L; Chu, L. Y. Thermo-responsive gating membranes with controllable length and density of poly(N-isopropylacrylamide) chains grafted by ATRP method.J. Membr. Sci.2009,337, 310−317.. [Baidu Scholar] 

    36

    Roghani-Mamaqani, H; Khezri, K. A grafting from approach to graft polystyrene chains at the surface of graphene nanolayers by RAFT polymerization: various graft densities from hydroxyl groups.Appl. Surf. Sci.2016,360, 373−382.. [Baidu Scholar] 

    37

    Takahashi, H; Nakayama, M; Yamato, M; Okano, T. Controlled chain length and graft density of thermoresponsive polymer brushes for optimizing cell sheet harvest.Biomacromolecules2010,8, 1991−1999.. [Baidu Scholar] 

    38

    Liu, H; Zhao, X; Jia, N; Sotto, A; Zhao, Y; Shen, J; Gao, C; van der Bruggen, B. Engineering of thermo-/pH-responsive membranes with enhanced gating coefficients, reversible behaviors and self-cleaning performance through acetic acid boosted microgel assembly.J. Mater. Chem. A2018,6, 11874−11883.. [Baidu Scholar] 

    39

    Liu, H; Yang, S; Liu, Y; Miao, M; Zhao, Y; Sotto, A; Gao, C; Shen, J. Fabricating a pH-responsive membrane through interfacial in-situ assembly of microgels for water gating and self-cleaning.J. Membr. Sci.2019,579, 230−239.. [Baidu Scholar] 

    40

    Shen, S; Hao, Y; Zhang, Y; Zhang, G; Zhou, X; Bai, R. B. Enhancing the antifouling properties of poly(vinylidene fluoride) (PVDF) membrane through a novel blending and surface-grafting modification approach.ACS Omega2018,12, 17403−17415.. [Baidu Scholar] 

    41

    Kavand, A; Blanck, C; Przybilla, F; Mély, Y; Anton, N; Vandamme, T; Serra, C. A; Chan-Seng, D. Investigating the growth of hyperbranched polymers by self-condensing vinyl RAFT copolymerization from the surface of upconversion nanoparticles.Polym. Chem.2020,11, 4313−4325.. [Baidu Scholar] 

    42

    Wang, W; Yu, Y; Wang, P; Wang, Q; Li, Y; Yuan, J; Fan, X. Controlled graft polymerization on the surface of filter paperviaenzyme-initiated RAFT polymerization.Carbohydr. Polym.2018,207, 239−245.. [Baidu Scholar] 

    43

    Chang, C. C; Beltsios, K. G; Lin, J. D; Cheng, L. P. Nano-titania/polyethersulfone composite ultrafiltration membranes with optimized antifouling capacity.J. Taiwan Inst. Chem. E2020,113, 325−331.. [Baidu Scholar] 

    44

    Liu, F; Zhu, B. K; Xu, Y. Y. Improving the hydrophilicity of poly(vinylidene fluoride) porous membranes by electron beam initiated surface grafting of AA/SSS binary monomers.Appl. Surf. Sci.2006,253, 2096−2101.. [Baidu Scholar] 

    45

    Gordijo, C. R; Shuhendler, A. J; Wu, X. Y. Glucose-responsive bioinorganic nanohybrid membrane for self-regulated insulin release.Adv. Funct. Mater.2010,20, 1404−1412.. [Baidu Scholar] 

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