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Mar 16, 2017 - Synthesis of Zwitterionic Diblock Copolymers with Cleavable Biotin Groups at the Junction Points and Fabrication of Bioconjugates by ...
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Synthesis of Zwitterionic Diblock Copolymers with Cleavable Biotin Groups at the Junction Points and Fabrication of Bioconjugates by Biotin−Streptavidin Coupling Jin-Tao Wang, Lin Wang, Xiaotian Ji, Li Liu,* and Hanying Zhao* Key Laboratory of Functional Polymer Materials, Ministry of Education, College of Chemistry, Nankai University, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China S Supporting Information *

ABSTRACT: Combinations of synthetic polymers and natural proteins provide a route to the synthesis of new biomaterials. The bioconjugates combining tunable properties of polymers with functionalities of proteins have found broad applications. One of the most challenging problems in this research field is the self-assembly behaviors of responsive polymer−protein bioconjugates. In this research, synthesis and self-assembly of bioconjugates composed of zwitterionic block copolymer and streptavidin were investigated. Block copolymers of poly(ethylene glycol) (PEG) and poly[3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate] (PDMAPS) with cleavable biotin groups at the junction points were synthesized. The zwitterionic block copolymers exhibit phase transitions with upper critical solution temperatures (UCSTs) in aqueous solutions. The concentration of sodium chloride exerts a significant influence on the UCST. The zwitterionic block copolymer chains selfassemble into vesicles in aqueous solution at a temperature below UCST. Bioconjugates comprising of streptavidin molecules and zwitterionic block copolymer chains were fabricated based on biotin−streptavidin coupling. Upon conjugation to the protein molecules, the UCST of the zwitterionic block copolymer decreases due to the screening effect of the protein molecules. The bioconjugates are able to make self-assembly into different structures, depending on the average number of block copolymer chains on a protein molecule. The bioconjugate molecules with average 1.3 block copolymer chains on a streptavidin selfassemble into rodlike structures, while those with average 2.9 chains on a streptavidin self-assemble into spherical micelles.

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INTRODUCTION

modified protein molecules. Before direct coupling to protein molecules, polymer chains are usually modified with functional groups which are capable of reacting with amino or thiol groups natively on amino acid residues in proteins.10−14 Alternatively, through bioengineering techniques, non-natural amino acids can be precisely introduced to the protein molecules, allowing subsequent site-specific conjugation.15,16 One benefit of the “grafting to” method is that the polymers can be fully characterized and functionalized before conjugation reactions. However, due to the high molecular weights of the polymers and proteins, the conjugation efficiency in the “grafting to” approach is lower than the “grafting from” method. In order to

Biohybrids synthesized by the conjugation of synthetic polymers to natural proteins combine tunable properties of polymers with biological activities of proteins. In 1977, Abuchowski and co-workers reported, for the first time, the synthesis of bioconjugates composed of poly(ethylene glycol) (PEG) and bovine serum albumin (BSA).1,2 Since then, a variety of bioconjugates have been synthesized, and the bioconjugates have found applications in bioseparations, drug/siRNA delivery, enzyme bioprocesses, and cell culture processes.3−8 In general, synthetic approaches toward polymer−protein conjugates can be catalogued into “grafting to” and “grafting from” methods.9 In the “grafting to” approach, polymer chains were directly conjugated to protein molecules through chemical reactions or specific interactions; in the “grafting from” method, polymer chains grow from the © XXXX American Chemical Society

Received: December 9, 2016 Revised: March 6, 2017

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DOI: 10.1021/acs.macromol.6b02665 Macromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Schemes for (a) the Synthesis of Pyridyl Disulfide-Functionalized Biotin (Biotin-SS-Py) and (b) the Synthesis of NPropargyloxycarbonyl-N′,N″,N‴-tri(tert-butyloxycarbonyl)cystamine

antifouling surfaces,27−33 have been widely investigated. In a previous research, Russell and co-workers made atom transfer radical polymerization (ATRP) of DMAPS on the surface of chymotrypsin and found that the enzyme−polymer conjugates retained temperature-dependent changes in conformation while still maintaining enzyme function.34 However, in their research, the self-assembly behavior was not studied. Most previous researches focused on the grafting of homopolymer chains to the surfaces of proteins. However, in the field of polymer science it is well documented that the architectures of multicomponent polymers exert significant effects on their self-assembled structures.35 We envision that the architecture of a polymer in a bioconjugate has a significant influence on the self-assembly of the bioconjugate. The targets of this research are to study the synthesis of bioconjugates comprising of protein molecules and zwitterionic block copolymer chains and demonstrate the effects of polymer architecture and the structure of bioconjugates on the selfassembly behaviors of the bioconjugates. In this research, PEGblock-PDMAPS with biotin group at the junction point (PEGb-PDMAPS-SS-Biotin) was used as a model polymer, streptavidin was used as a model protein, and bioconjugates were synthesized by coupling between biotin and streptavidin. The temperature-induced self-assemblies of the bioconjugates were investigated.

prepare well-defined polymer−protein conjugates, highly efficient coupling reactions or molecular recognitions with high efficiency of binding are required. A number of chemical reactions, such as reactions of N-hydroxysuccinimide (NHS) activated esters and amines, thiol−disulfide exchange reaction, Huisgen [3 + 2] 1,3-dipolar cycloaddition, Michael addition of a thiol to a maleimide, and thiol−ene reaction, have been applied in the synthesis of polymer−protein bioconjugates.17 In addition to the chemical reactions, molecular recognitions have been exploited to prepare polymer−protein conjugates. For example, biotin binds with high affinity to (strept)avidin, and the bioconjugates based on the strong and stable affinity between the two components have been synthesized.18−21 The molecular recognitions have many advantages in the fabrication of bioconjugates, including mild synthetic condition, high affinity, and high efficiency. Polymer−protein conjugates show comparable self-assembly properties to block copolymers. Depending on the hydrophobic/hydrophilic interactions, amphiphilic polymer−protein conjugates are able to self-assemble into nanostructures, such as micelles, capsules, and vesicles.22 However, due to the insolubility of the hydrophobic polymers in water, the synthesis of amphiphilic bioconjugates in aqueous solutions is difficult. One way to solve this problem is to attach stimuli-responsive polymers to proteins, which allows for tuning of the solubility and self-assembly of the resulting bioconjugates by applying an external stimulus.23,24 For example, bioconjugates comprising superfolder GFP (sfGFP) decorated with temperatureresponsive poly[(oligo ethylene glycol) methyl ether methacrylate] (POEGMA) chains self-assembled into core−corona structures at a temperature above lower critical solution temperature (LCST) of POEGMA.25 The driving force for the self-assembly process is the change of the polymer from hydrophilic to hydrophobic as temperature is above LCST. Poly[3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate] (PDMAPS) is a typical zwitterionic polymer with an ammonium cation and a sulfonic anion isolated with a propyl group on each repeating unit. PDMAPS exhibits an upper critical solution temperature (UCST) in water due to Coulombic attraction.26 The applications of PDMAPS in the preparation of smart materials, including thermoresponsive nanocomposite gels, nanospheres, layer-by-layer films, and

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METHODS

Materials. Epichlorohydrin (Tianjin Chemical Reagent Company, 98%) was distilled before use. N-Hydroxysuccinimide (NHS) (Heowns Biochem LLC, 97%) was purified by recrystallization from a mixture of ethyl acetate and ethanol and dried under reduced pressure. CuBr (99%), purchased from Guo Yao Chemical Company, was purified by washing with glacial acetic acid and dried under reduced pressure at 100 °C. Poly(ethylene glycol) monomethyl ether (CH3O-PEG-OH, Aldrich), sodium azide (Alfa Asear, 99%), ammonium chloride (Tianjin Chemical Reagent Company, 99%), cystamine dihydrochloride (Aldrich, 96%), di-tert-butyl dicarbonate (Heowns Biochem LLC, 97%), 2-bromoisobutyryl bromide (Alfa Asear, 97%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, Aldrich, 99%), propargyl alcohol (Aldrich, 99%), N,N′-carbonyldiimidazole (Heowns Biochem LLC, 99%), 4-(dimethylamino)pyridine (DMAP, Alfa Asear, 99%), biotin (Energy Chemical, 98%), 2,2′-dithiopyridine (Aldrich, 99%), cysteamine hydrochloride (Heowns Biochem LLC, 98%), 2-(4-hydroxyphenylazo)benzoic acid B

DOI: 10.1021/acs.macromol.6b02665 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules (HABA, Heowns Biochem LLC, 98%), DL-dithiothreitol (DTT, Aldrich, 99%), streptavidin (Aldrich), 3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate (DMAPS, Aldrich, 97%), and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, Heowns Biochem LLC, 98%) were used as received. Chemical structures and synthetic schemes for the synthesis of pyridyl disulfide-functionalized biotin (biotin-SS-Py) and N-propargyloxycarbonyl-N′,N″,N‴-tri(tert-butyloxycarbonyl)cystamine are shown in Scheme 1. Analysis and synthetic details for the two compounds are presented in the Supporting Information (Figures S1 and S2). Synthesis of Epoxide-Terminated PEG (Epoxy-PEG). A typical procedure for the synthesis of epoxy-PEG was briefly described as follows. In a dry round-bottom flask, PEG (10.0 g) was dissolved in 80 mL of dry toluene. After azeotropic distillation of 20 mL of toluene, sodium hydride (1.0 g, 25 mmol) was added into the solution under an argon atmosphere. After stirring at 35 °C for 24 h, epichlorohydrin (4.0 mL, 50 mmol) was added into the solution. The reaction was performed at 40 °C for 24 h. The inorganic salt produced in the reaction was removed by centrifugation, and the supernatant was concentrated on a rotary evaporator. The polymer solution was added into cold diethyl ether. After filtration and drying, 8.9 g of epoxy-PEG was obtained. Yield: 88%. Synthesis of Azide-Terminated PEG. In a dry round-bottom flask, epoxy-PEG (5.1 g) was dissolved in 30 mL of DMF, and sodium azide (0.33 g, 5.0 mmol) and ammonium chloride (0.27 g, 5.0 mmol) were added into the solution. The solution was stirred at 60 °C for 60 h. The solvent was removed on a rotary evaporator. The polymer was dissolved in dichloromethane, washed with water to remove any residual salt, and dried over anhydrous magnesium sulfate. The polymer solution was concentrated on a rotary evaporator and then added into cold diethyl ether. After filtration and drying under reduced pressure, azide-terminated PEG (4.8 g) was obtained. Yield: 93%. Synthesis of Disulfide-Terminated PEG. Disulfide-terminated PEG was synthesized by click reaction between azide-terminated PEG and N-propargyloxycarbonyl-N′,N″,N‴-tri(tert-butyloxycarbonyl)cystamine. Azide-terminated PEG (1.0 g, 0.20 mmol) and Npropargyloxycarbonyl-N′,N″,N‴-tri(tert-butyloxycarbonyl)cystamine (0.19 g, 0.56 mmol) were dissolved in 6 mL of dry DMF, and PMDETA (42 μL, 0.20 mmol) and CuBr (29 mg, 0.20 mmol) were added to the solution under an argon atmosphere. After three freeze− pump−thaw cycles, the solution was stirred at 35 °C for 48 h. DMF was removed by rotary evaporation, and the crude product was dissolved in dichloromethane. After removal of copper ions by passing through a neutral Al2O3 column, the polymer solution was added into cold diethyl ether. After filtration and drying under reduced pressure, disulfide-terminated PEG (0.92 g) was obtained. Yield: 86%. Synthesis of Bromide/Disulfide-Terminated PEG. A typical procedure for the synthesis of bromide/disulfide-terminated PEG was briefly described as follows. In a dry round-bottom flask, disulfideterminated PEG (0.40 g, 0.071 mmol) and triethylamine (0.22 mL, 1.6 mmol) were dissolved in 5 mL of dry dichloromethane, and 2bromoisobutyl bromide (0.14 mL, 1.1 mmol) was added dropwise into the solution at 0 °C. The solution was stirred at room temperature for 48 h. The polymer solution was washed with water, dried over anhydrous magnesium sulfate, and added into cold diethyl ether. After filtration and drying under reduced pressure, bromide/disulfideterminated PEG (0.3 g) was obtained. Yield: 73%. Synthesis of Bromide/Biotin-Terminated PEG. A typical procedure for the synthesis of bromide/biotin-terminated PEG was briefly described as follows. In a dry round-bottom flask, bromide/ disulfide-terminated PEG (0.40 g) was dissolved in 15 mL of THF, and 2.1 mL of tributylphosphine (PBu3) solution (10 wt % in hexane) was added into the polymer solution. After stirring at room temperature for 24 h, the polymer solution was concentrated on a rotary evaporator under reduced pressure and added into cold diethyl ether to remove PBu3. The precipitated polymer was dissolved in 15 mL of DMF, and biotin-SS-Py (42 mg, 0.35 mmol) was added into the solution under an argon atmosphere. The solution was stirred at room temperature for 24 h. After the thiol−disulfide exchange reaction, the polymer solution was transferred to a dialysis tubing (MWCO 3500

Da) and dialyzed against DMF for 2 days. After dialysis, DMF was removed by rotary evaporation. The crude product was dissolved in dichloromethane and added into cold diethyl ether. After filtration and drying under reduced pressure, bromide/biotin-terminated PEG (0.20 g) was obtained. Yield: 50%. Synthesis of Block Copolymers of PEG and PDMAPS with Cleavable Biotin Groups at the Junction Points (PEG-bPDMAPS-SS-Biotin). Bromide/biotin-terminated PEG (30 mg) and DMAPS monmer (87 mg, 0.31 mmol) were dissolved in 3 mL of PBS solution (100 mM at pH 6.5). The solution was degassed by three freeze−pump−thaw cycles. Water (1 mL), PMDETA (18 μL), and CuBr (12.5 mg) were added into a Schlenk flask, and after freeze− pump−thaw cycle the catalyst solution was stirred for 30 min at room temperature. Catalyst solution (67 μL) was added into the monomer/ macroinitiator solution. After three freeze−pump−thaw cycles, ATRP of DMAPS was conducted at 30 °C for 8 h and stopped by exposure of the solution to air. The block polymer solution was dialyzed in a dialysis tubing (MWCO 7000 Da) against deionized water for 2 days, and PEG-b-PDMAPS-SS-Biotin was obtained after freeze-drying. UCST Measurement. The UCSTs of the polymer at different salt concentrations were determined by measuring the change in transmittance at 600 nm on a UV−vis spectrometer at different temperatures. The temperature, where the transmittance of the solution decreased to the half of the original value, was determined as the UCST of the solution. Streptavidin/2-(4-Hydroxyphenylazo)benzoic Acid (HABA) Assay Studies. A standard curve in streptavidin/HABA assay was obtained by using the following method. HABA solution (7 mM) was prepared by dissolving HABA (8.5 mg) in 5 mL of NaOH solution (10 mM). Streptavidin (5 mg) was dissolved in 5 mL of PBS solution (50 mM, pH 6.0) containing 150 mM NaCl. To prepare streptavidin/ HABA solution, streptavidin solution (1.0 mL) was diluted with PBS solution to 2 mL, and HABA solution (71.2 μL) was added to the streptavidin solution under vortexing. Biotin (2.49 mg) was dissolved in 100 mL of PBS solution at pH 6.0, yielding a 0.1 mM biotin solution. The biotin solution (9 μL) was added to SAv/HABA solution (0.9 mL) under vortexing repeatedly. After each addition, the absorbance change of the solution at 500 nm was recorded on a UV− vis spectrometer. A standard curve is shown in Figure S3. Preparation of Protein−Polymer Conjugates. Streptavidin and block copolymer were dissolved in PBS solution (50 mM, pH 6.0) containing 150 mM NaCl and incubated at room temperature for 12 h. The average number of polymer chains conjugated to a streptavidin molecule was determined based on a standard curve of streptavidin/ HABA-biotin. The protein−polymer conjugates were purified by using Millpore ultrafiltration tube with a molecular weight cutoff (MWCO) of 100 kDa at 5000 rpm to remove any possible “free” polymer chains and salt. Characterization. 1H NMR and 13C NMR spectra were collected on a Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer at room temperature using DMSO-d6, D2O, or CDCl3 as the solvents. The apparent number-average molecular weights and dispersities of PEG and modified PEG were determined on a size exclusion chromatograph (SEC) equipped with a Hitachi L-2130 HPLC pump, a Hitachi L-2350 column oven operated at 50 °C, three Shodex columns with 5000−5K, 400−0.5K, and 5−0.15K molecular ranges, a Hitachi L-2490 refractive index detector, and a light scattering detector (Viscotek 270 dual detector, Malvern Instruments Ltd.). DMF was used as eluent at a flow rate of 1.0 mL/min. The apparent number-average molecular weights and dispersities of the polymers were calibrated on poly(methyl methacrylate) (PMMA) standards. The apparent molecular weights and dispersities of PEG-bPDMAPS-SS-Biotin were determined on a SEC equipped with a CoMetre 6000 LDI pump and a Schambeck SFD GmbH RI2000 refractive index detector. NaH2PO4 (300 mM) and acetic acid (1.0 M) were used as mobile phase at a flow rate of 0.5 mL/min. Polymer solutions were injected through Shodex SB-802.5, 803, and 804 HQ columns at 40 °C. PEG standards were used for calibration. In the measurements of the protein−polymer conjugates and streptavidin, ammonium acetate (10 mM) and sodium chloride (150 mM) at pH = C

DOI: 10.1021/acs.macromol.6b02665 Macromolecules XXXX, XXX, XXX−XXX

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Scheme 2. Synthetic Route for the Synthesis of Block Copolymers of Poly(ethylene glycol) (PEG) and Poly[3dimethyl(methacryloyloxyethyl)ammonium Propanesulfonate] (PDMAPS) with Cleavable Biotin Groups at the Junction Points (PEG-b-PDMAPS-SS-Biotin)

6.6 were used as mobile phase at a flow rate of 0.5 mL/min. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer Nano-S90 at a wavelength of 633 nm, and the results were analyzed in CONTIN mode. Transmission electron microscopy (TEM) images were collected on a Tecnai G2 F20 S-TWIN electron microscope operated at a voltage of 200 kV. Formvar and carbon sequentially coated copper EM grids were used in the observations. The TEM specimens were prepared by casting diluted aqueous solutions on the copper grids at room temperature and then moving the copper grids immediately onto the surface of an iron block which was previously immersed in liquid nitrogen. The TEM specimens were dried by lyophilization and stained under OsO4 atmosphere for 2 h. Atomic force microscopy (AFM) images were recorded on a Nanoscope IV atomic force microscope (Digital Instruments Inc.) operated in the tapping mode using Si cantilevers with a scan rate of 1.0 Hz and a resonance frequency of 320 kHz. The AFM specimens were prepared by depositing aqueous solutions of the polymer aggregates on the surfaces of mica, and water was evaporated in air at room temperature. Fourier transform infrared absorption spectra (FTIR) were obtained on a Bio-Rad FTS6000 system using diffuse reflectance sampling accessories. UV−vis spectra were collected on a Shimadzu UV-2450 spectrometer using a quartz cell of 1 cm path length. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass analysis was made on Bruker Autoflex III spectrometer equipped with a 337 nm nitrogen laser, and mass spectra were acquired in reflex mode at an acceleration voltage of +20 kV. αCyano-4-hydroxycinnamic acid was used as the matrix. HRMS measurements were conducted on an Agilent 6520 Q-TOF LC/MS equipped with an electrospray interface.

dipolar cycloaddition reaction (click reaction). (4) Bromide end groups were produced at the ends of the polymer chains after esterification reaction between the hydroxyl end groups and 2-bromoisobutyryl bromide. After the click reaction and the esterification, both bromide and disulfide groups were attached to the ends of PEG chains. (5) The disulfides were cleaved with PBu3, resulting in thiol groups at the ends of PEG chains. After thiol−disulfide exchange reaction between the thiols and biotin-SS-Py, biotin groups were attached to the ends of polymer chains. (6) PEG-b-PDMAPS-SS-biotin was synthesized by ATRP of DMAPS. Synthesis of Epoxy-PEG. PEG with average repeating unit number of 107 (PEG107), as determined by 1H NMR, was used in this research. The hydroxyl end group of PEG was converted into sodium alkoxide by reaction with sodium hydride. After reaction of the sodium alkoxide groups with excess epichlorohydrin, epoxy-PEG107 was synthesized.36 1H NMR result of epoxy-PEG107 is shown in Figure S4 (spectrum a). In the spectrum, the peaks at 3.16 (c), 2.79 (d), and 2.62 ppm (e) represent the protons on the epoxide rings, indicating that the epoxide groups have been successfully introduced to the ends of polymer chains. The peak at 3.38 ppm (a) represents the methyl protons of PEG. Based on the integral ratio of the peak at 3.16 ppm to the peak at 3.38 ppm, the end-capping efficiency was estimated to be around 96%. Synthesis of PEG with Terminated Hydroxyl and Azide Group (PEG107-N3/OH). PEG107-N3/OH was synthesized by opening of the end oxirane ring of epoxy-PEG with sodium azide.37,38 The 1H NMR spectrum of PEG-N3/OH in CDCl3 is shown in Figure S4 (spectrum b). The signals at 3.16, 2.79, and 2.62 ppm representing the protons on the epoxide rings disappear completely, demonstrating the ring-opening reaction by azide anions. After the reaction, new peaks at 3.95 ppm (c) representing the methine proton next to the hydroxyl group (−CH−OH) and at 3.35 ppm (d) representing methene protons connected to the azide group (−CH2N3) are observed. The opening of the oxirane rings was also demonstrated by FTIR results. As shown in Figure S5, an absorption peak at

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RESULTS AND DISCUSSION As shown in Scheme 2, the following steps are included in the synthesis of PEG-b-PDMAPS-SS-biotin block copolymer. (1) The hydroxyl end groups of PEG were reacted with sodium hydride and excess epichlorohydrin sequentially, and epoxyPEG was obtained. (2) Azide-terminated PEG was synthesized by opening of the epoxide rings of epoxy-PEG with sodium azide. Meanwhile, hydroxyl end groups were also produced in the ring-opening reaction. Herein, azide-terminated PEG was assigned as PEG-N3/OH. (3) Disulfide groups were introduced onto the polymer chains by Cu(I)-catalyzed Huisgen 1,3D

DOI: 10.1021/acs.macromol.6b02665 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules 2100 cm−1 representing the valence vibration of azide group can be clearly observed (spectrum a), which confirms the introduction of azide groups onto the polymer chains. Synthesis of PEG with Disulfide End Group. The disulfide bonds were introduced to the polymer ends by click reaction, and PEG chains with disulfide end groups and hydroxyl end groups (PEG107-SS/OH) were prepared (Scheme 2). Figure 1 shows the 1H NMR spectrum and peak

c). In the spectrum, featured peak signals (peak m, n, and o), corresponding to the protons on the biotin end group, are observed. By calculating the integral ratio of the peak corresponding to the methine proton on 1,2,3-triazole ring (peak f) to the peak corresponding to the protons on biotin (peak o), the exchange efficiency was estimated to be around 94%. The FTIR spectrum of biotin-modified PEG is shown in Figure S5 (spectrum c). Compared to the FTIR spectra of the precursors, the absorption peak at 3250 cm−1 representing the valence vibration of N−H bond is observed clearly. The elution chromatograms of PEG107-Br/biotin and the precursors are shown in Figure 2. All the SEC curves show

Figure 1. 1H NMR spectra of (a) PEG with a disulfide end group and a hydroxyl end group (PEG107-SS/OH), (b) PEG with a disulfide end group and an ATRP initiator (PEG107-SS/Br), and (c) bromide/ biotin-terminated PEG (PEG107-Br/biotin) in CDCl3.

assignments of PEG107-SS/OH (spectrum a). After the click reaction, new peaks at 7.85 ppm (f) representing the methine proton on 1,2,3-triazole ring and at 5.32 ppm (g) representing the two methylene protons next to the 1,2,3-triazole ring are observed. The peaks in the range between 4.36 and 4.56 ppm (e) corresponding to the methylene protons next to the 1,2,3triazole ring are also observed. By calculating the integral ratio of peak e to peak g, the “click” efficiency was estimated to be around 98%. In the spectrum, the appearance of the peak at 2.85 ppm (j) representing the methylene protons next to the disulfide bonds demonstrates the introduction of disulfides to the polymer chains. The FTIR spectrum of the polymer is shown in Figure S5 (spectrum b). Compared to the precursor (spectrum a), the absorption at 2100 cm−1 corresponding to the valence vibration of azide group disappears completely after the click reaction. Synthesis of Bromide/Biotin-Terminated PEG (PEG107Br/Biotin). ATRP initiators were introduced to the polymer chains by esterification of the hydroxyl end group of PEG107SS/OH with 2-bromoisobutyryl bromide. The 1H NMR spectrum and the peak assignments of PEG with a disulfide end group and an ATRP initiator (PEG107-SS/Br) are shown in Figure 1 (spectrum b). After the esterification, new peak at 1.87 ppm (k) representing the six methyl protons [−C(Br)− (CH3)2] is observed, indicating that 2-bromoisobutyryl group was attached to the ends of polymer chains. Based on 1H NMR result, the degree of esterification was calculated to be around 97%. After cleavage of the disulfide bonds on PEG107-SS/Br, thiols are produced at the ends of polymer chains. PEG107-Br/biotin was prepared by thiol−disulfide exchange reaction between the thiol groups and biotin-SS-Py (Scheme 1).39 The 1H NMR spectrum of PEG107-Br/biotin is shown in Figure 1 (spectrum

Figure 2. Size exclusion chromatography (SEC) results of (a) PEG107, (b) epoxy-PEG107, (c) PEG107-SS/OH, (d) PEG107-SS/Br, and (e) PEG107-Br/biotin. DMF was used as eluent in SEC measurements.

monomodal distributions, indicating that there are no intermolecular coupling reactions at each step reaction. It is worthy of note that the elution peak maximum of PEG is almost the same as epoxy-PEG due to the small size of the epoxy group; however, after click reaction and thiol−disulfide exchange reaction the SEC curve moves slightly to high molecular weight part due to the increase in the sizes of the end groups. Figure 3 shows chemical structure and MALDI-TOF mass spectrum of PEG107-Br/biotin. The repeating peaks in the spectrum have an interval of 44, representing the molecular weight of ethylene oxide unit. The expected molecular mass for PEG-Br/biotin plus Na+ was calculated to be 5573.40, which agreed well with the MALDI-TOF mass result (5573.25). This result demonstrated the successful synthesis of PEG107-Br/ biotin. Synthesis of PEG-b-PDMAPS-SS-Biotin Diblock Copolymer. PEG107-Br/biotin was used as macroinitiator to initiate ATRP of DMAPS, and CuBr/PMDETA was used as catalyst. The polymerization was conducted in phosphate buffer solution (PBS, 100 mM, pH = 6.5) at 30 °C. The 1H NMR spectrum and peak assignments of a block copolymer with biotin group at the junction point (PEG-b-PDMAPS-SS-biotin) are shown in Figure 4. After ATRP of DMAPS, following new peaks are observed: 0.86−1.26 ppm (e) representing the methyl protons [−C(CH3)−], 2.02 ppm (d) representing the methene protons (−C−CH2−C−) on the backbone, 2.30 ppm (j) representing the pendant methene protons (−CH2−CH2− CH2−), 3.01 ppm (k) representing the methene protons next to the sulfonate group (−CH 2−SO3−), 3.26 ppm (h) E

DOI: 10.1021/acs.macromol.6b02665 Macromolecules XXXX, XXX, XXX−XXX

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cm−1 (stretching vibration of the S−O), 1475 cm−1 [C−H stretching vibration of the −N+(CH3)2 group], and 1727 cm−1 (stretching vibration of CO group) are observed, which further demonstrate the synthesis of the block copolymer. As shown in Figure 2, the modifications of terminal groups of PEG only result in small changes in hydrodynamic volumes and slight shift of SEC trace. However, after ATRP of DMAPS, the size of the polymer increases significantly. SEC elution chromatograms of PEG107, PEG107-b-PDMAPS38-SS-biotin, and PEG107-b-PDMAPS53-SS-biotin are shown in Figure 5.

Figure 5. SEC curves of (a) PEG107, (b) PEG107-b-PDMAPS38-SSbiotin, and (c) PEG107-b-PDMAPS53-SS-biotin. Phosphate buffer solution was used as eluent in the measurements.

Figure 3. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum of PEG107-Br/Biotin. M = 15.02 + 44.03 × 107 + 824.18 = 5550.41. Matrix: α-cyano-4-hydroxycinnamic acid.

After ATRP, SEC trace moves to short retention time region (or high molecular weight part), demonstrating the synthesis of PDMAPS blocks. Meanwhile, the molecular weight dispersity increases from 1.03 for PEG107 to 1.32 for PEG107-bPDMAPS38-SS-biotin or to 1.35 for PEG107-b-PDMAPS53-SSbiotin. UCST of PEG-b-PDMAPS-SS-Biotin. PDMAPS exhibits a phase transition with an UCST in aqueous solution. At a temperature below the UCST, the polymer chains make a collapsed conformation as a result of intrachain/interchain associations of the zwitterionic groups. At a temperature above the UCST, the associations are disrupted, allowing the expansion and solubilization of the polymer chains.40,41 At a temperature around UCST, PDMAPS experiences a transition from the hydrophobic state to the hydrophilic state. With an increase in the molecular weight or polymer concentration, the UCST shifts to higher temperature. However, when salt is added to the solution, the UCST shifts to lower temperature due to the electrostatic screening effect. Herein, the effect of added salt on the UCST of PEG107-b-PDMAPS53-SS-biotin block copolymer was investigated. The transmittances of the polymer solutions at 600 nm were recorded on a UV−vis spectrophotometer at different temperatures.42 Figure 6a shows temperature dependence of the transmittance of the polymer solution at different concentrations of sodium chloride. The block copolymer exhibits an UCST at 55 °C in water. The transition temperature shifts to the lower temperature with increasing NaCl concentration and finally disappears when the salt concentration is above 80 mM. Figure 6b shows the relationship between UCST and the salt concentration. The UCST decreases from 55 to 35, to 22, to 12, and to 6 °C when the concentration of NaCl increases from 0 to 20, to 40, to 60, and to 80 mM. When the concentration of NaCl reaches 100 mM, the polymer solution keeps transparent even at 0 °C, indicating that the cloud point disappears at this salt concentration. PDMAPS is considered to be in a collapsed conformation in water below UCST due to the Coulombic

Figure 4. 1H NMR spectrum of PEG107-block-PDMAPS53 with the biotin group at the junction point (PEG107-b-PDMAPS53-SS-biotin) in D2O.

representing the methyl protons next to the nitrogen atom [−N(CH3)2−], 3.62 and 3.83 ppm (i, g) representing the methene protons next to the nitrogen atom [−CH2− N(CH3)2−CH2−], and 4.53 ppm (f) representing the methene protons next to the ester group (−COO−CH2−). Based on 1H NMR results, the degree of polymerization (DPn) of PDMAPS was calculated. In this research, two block copolymers with different PDMAPS block lengths were synthesized, and they were assigned as PEG107-b-PDMAPS53-SS-biotin and PEG107-bPDMAPS38-SS-biotin. The FTIR spectrum of PEG107-bPDMAPS53-SS-biotin is shown in Figure S5 (spectrum d). The characteristic absorption peaks of DMAPS units at 1043 cm−1 (symmetric stretching vibration of SO group), 1195 cm−1 (asymmetric stretching vibration of SO group), 930 F

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Driven by the intrachain/interchain associations of the zwitterionic groups, collapsed PDMAPS blocks are in the walls of the vesicles. The hydrophilic PEG blocks in the corona are directed preferentially to either the inside or the outside of the walls, and the biotin functional groups are at the interfaces. The self-assembled structures were also characterized by AFM. The AFM sample was prepared by casting the aqueous solution of the polymer onto mica surface and drying in air at 25 °C. Figure 7c shows tapping mode AFM image and a height profile of the structures. In the AFM image, the walls of the vesicles collapse forming a central cavity structure, demonstrating the formation of the hollow structures. In the height profile of two typical vesicles, the central part of the structure is lower than the periphery, demonstrating the collapse of the hollow structures in the dry state. Because of the collapse and deformation of the structures, the average height is only 5 nm, much smaller than the average lateral dimension (200 nm). Synthesis and Self-Assembly of Bioconjugates of Streptavidin and PEG107-b-PDMAPS53-SS-Biotin. It is well-known that biotin binds with high affinity to (strept)avidin (Ka = 1015 M−1), and the (strept)avidin−biotin interaction is the strongest known noncovalent biological recognition.43 In a previous research, we synthesized amphiphilic triblock copolymers with biotin groups at the junction points by a combination of click chemistry and ring-opening polymerization and studied the interaction between avidin molecules and the micelles formed by the triblock copolymers in aqueous solutions.44 Herein, zwitterionic block copolymer, PEG107-bPDMAPS53-SS-biotin, was conjugated to streptavidin by streptavidin−biotin interaction, and the self-assemblies of the bioconjugates in aqueous solutions were studied. The bioconjugates were prepared by simply dissolving two components in 150 mM NaCl and 50 mM PBS solution (pH 6.0) at two different molar ratios, and the solution was incubated at room temperature for 12 h. Streptavidin/2-(4hydroxyphenylazo)benzoic acid (HABA) competitive binding assays were used to quantify the average number of block copolymer chains conjugated to a streptavidin molecule.45 The complex formed by the binding of HABA to streptavidin exhibits an absorbance peak at 500 nm. When biotin or biotinylated polymers are added, biotin groups displace HABA from the complex, resulting in a decrease in the absorbance of the complex. The change in the absorbance can be used to calculate the concentration of biotin or biotinylated polymers in the solutions and the average number of biotin molecules (or biotinylated polymer chains) bound to a streptavidin molecule. Figure 8a shows UV−vis spectra of streptavidin/HABA complex before and after addition of PEG107-b-PDMAPS53SS-biotin. The streptavidin/HABA complex shows very strong absorbance at 500 nm, and the block copolymer does not show any absorbance. The absorbance of the complex at 500 nm decreases with the addition of the block copolymer, suggesting the displacement of HABA by biotin groups. A streptavidin molecule possesses four binding sites, but not all the binding sites, can be occupied by biotin groups due to the steric hindrance effect. The absorbance of the complex at 500 nm can still be observed, even if the molar ratio of streptavidin to biotin groups reaches 1:9 (curve c in Figure 8a). The average number of biotinylated polymer chains conjugated to a streptavidin was calculated based on a standard curve (Figure S3). Our calculation result indicated that bioconjugates with average 1.3 and 2.9 PEG107-b-PDMAPS53-SS-biotin chains on a streptavidin molecule were prepared in this research, and the

Figure 6. (a) Temperature dependence of the transmittance for aqueous solutions of PEG107-b-PDMAPS53-SS-biotin block copolymer at various NaCl concentrations. (b) Relationship between upper critical solution temperature (UCST) and concentration of NaCl in polymer solutions.

attraction between ammonium and sulfonate groups on the polymer chains. The added salt screens the Coulombic attraction of the PDMAPS blocks, and the block copolymer chains expand in the solution. Self-Assembly of PEG107-b-PDMAPS53-SS-Biotin in Aqueous Solution. Self-assembly of PEG107-b-PDMAPS53SS-biotin block copolymer in aqueous solution was investigated. Curves 1, 2, and 3 in Figure 7a represent the DLS curves of the block copolymer in 100 mM NaCl solution, in water at 65 °C, and in water at 25 °C, respectively. In water at 25 °C, the block copolymer self-assembled into aggregates with an average hydrodynamic diameter of 165 nm (curve 3), while upon heating to 65 °C or adding salt to 100 mM, the average size decreased to 13 nm (curve 2) or 11 nm (curve 1), indicating the dissociation of the aggregates. TEM was employed to study the morphology of the aggregates at a temperature below UCST. The TEM specimen was prepared by depositing diluted aqueous solution of the polymer on a copper grid at 25 °C, freezing on a metal immersed in liquid nitrogen, and followed by freeze-drying. Before TEM measurement, the specimen was stained under OsO4 atmosphere at room temperature. TEM result indicates that in water at 25 °C the block copolymer self-assembles into vesicles with an average size of around 100 nm (Figure 7b). G

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Figure 7. (a) Dynamic light scattering curves of PEG107-b-PDMAPS53-SS-biotin block copolymer in 100 mM NaCl solution (curve 1) and in water at 65 (curve 2) and 25 °C (curve 3). (b) TEM image of aggregates self-assembled by PEG107-b-PDMAPS53-SS-biotin in water at 25 °C. (c) Tapping mode AFM image and height profile of the aggregates self-assembled by PEG107-b-PDMAPS53-SS-biotin in water at 25 °C.

zwitterions by the electrostatic screening effect of the protein molecules. In order to demonstrate this, a control experiment was conducted. A block copolymer of PEG and PDMAPS without biotin group (PEG107-b-PDMAPS44) was synthesized by ATRP. The UCST of the block copolymer was determined to be around 31.5 °C, and it decreased to 29.5 °C upon mixing with streptavidin (Figure 8d), which demonstrated the dissociation of the zwitterionic pairs by protein molecules in aqueous solution. Similar to NaCl, the protein molecules in the bioconjugates exert a screening effect on the PDMAPS blocks, resulting in a decrease in UCST of the zwitterionic block copolymer. It is noted that streptavidin-bp2.9 has a broader transition range than streptavidin-bp1.3 (Figure 8c). This can be understood in terms of the structural diversity in streptavidinbp2.9. Although the average number of block copolymer chains bounded to a protein molecule is 2.9, bioconjugates with different numbers of block copolymer chains on a streptavidin were formed upon biotin−streptavidin coupling, which led to a broader transition in UCST measurement. The structural diversity in streptavidin-bp2.9 was also demonstrated by SEC. As

two bioconjugates were assigned as streptavidin-bp1.3 and streptavidin-bp2.9, respectively. In the synthesis of the bioconjugates, the “free” polymer chains, which were not coupled to the protein molecules, were removed by using Millipore ultrafiltration tubes with molecular weight cutoff (MWCO) of 100 kDa at 5000 rpm. SEC curves of streptavidin, streptavidin-bp1.3, and streptavidin-bp2.9 are shown in Figure 8b. Compared to native streptavidin, SEC curves of streptavidinbp1.3 and streptavidin-bp2.9 moved to higher molecular weight part, demonstrating the biotin−streptavidin coupling between streptavidin and PEG107-b-PDMAPS53-SS-biotin. Similar to the precursor block copolymer, streptavidin-bp1.3 and streptavidin-bp2.9 exhibited UCST-type thermosensitivity (Figure 8c); the transparencies of the solutions decreased with decreasing temperature. Upon conjugation to the protein molecules, the UCST measured in 10 mM NaCl aqueous solutions decreased from 41 °C for the precursor PEG107-bPDMAPS53-SS-biotin block copolymer to 35 °C for streptavidin-bp1.3 and to 28 °C for streptavidin-bp2.9. The decrease in UCST may be attributed to the dissociation of the pairing of H

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Figure 8. (a) UV−vis spectra of streptavidin/HABA complex (curve a), PEG107-b-PDMAPS53-SS-biotin diblock copolymer (d), and mixture of block polymer and streptavidin/HABA complex at molar ratios of 3:1 (curve b) and 9:1 (curve c). (b) SEC curves of native streptavidin (curve a), streptavidin-bp1.3 (curve b), and streptavidin-bp2.9 (curve c). (c) Temperature dependence of the transmittances for 10 mM NaCl aqueous solutions of PEG107-b-PDMAPS53-SS-biotin block copolymer, streptavidin-bp1.3 and streptavidin-bp2.9. (d) Temperature dependence of the transmittances for 10 mM NaCl aqueous solutions of PEG107-b-PDMAPS44 with and without streptavidin.

volume fraction of streptavidin-bp1.3 at a temperature below UCST became lower, which results in a structural change from vesicles to rodlike micelles. In order to demonstrate the effect of composition of polymer on the morphology of selfassembled structures of bioconjugate, a control experiment was performed. In the control experiment, PDMAPS-SS-biotin with an average repeating unit number of 46 (PDMAPS46-SSbiotin) was synthesized and conjugated to streptavidin. A scheme for the synthesis of PDMAPS46-SS-biotin is shown in Scheme S1, and characterizations of the polymer are presented in Figure S6. TEM result indicates that the conjugate formed by PDMAPS 46 -SS-biotin and streptavidin (streptavidinPDMAPS1.0) self-assembles into vesicles in aqueous solution at a temperature below UCST (Figure S7). At a temperature below UCST, the hydrophobic volume fraction of streptavidinPDMAPS1.0 is higher than streptavidin-bp1.3 due to absence of hydrophilic PEG blocks in streptavidin-PDMAPS1.0, so the morphological structure changes from rodlike micelles of streptavidin-bp1.3 to vesicles of streptavidin-PDMAPS1.0. Figure 9c shows TEM image of aggregates formed by streptavidin-bp 2.9 in water at room temperature. The bioconjugate self-assembles into spherical micelles with an average size of around 40 nm. The structure of streptavidinbp2.9 bioconjugate composed of a protein molecule with

shown in Figure 8b, compared with streptavidin-bp1.3, streptavidin-bp2.9 has a broader molecular weight distribution due to the formation of bioconjugates with different number of block copolymer chains on streptavidin molecules. Figures 9a,b show TEM images of self-assembled structures of streptavidin-bp1.3 in water at room temperature. SAv-bp1.3 self-assembled into rodlike micelles in water. The lengths of the structures are in the range 95−410 nm, and the average width is around 19 nm. At a temperature below UCST, the thermalsensitive PDMAPS blocks in the bioconjugate make a collapsed conformation as a result of associations of the zwitterions, driving a phase transition of the bioconjugate from unimers to rodlike micelles. The cores of the micelles are composed of collapsed PDMAPS blocks, and the hydrophilic PEG blocks and protein molecules are in the coronae. The self-assembled structure of streptavidin-bp1.3 is schemed in Scheme 3. In a given amphiphilic block copolymer system, the morphology of the self-assembled structures in aqueous solution is dependent on the hydrophobic volume fraction of the polymer. An increase in the hydrophobic volume fraction leads to a morphological evolution from spheres to rods to vesicles.46 Below UCST, PEG107-b-PDMAPS53-SS-biotin self-assembles into vesicles in water (Figure 7). After the block copolymer was conjugated to the hydrophilic streptavidin, the hydrophobic I

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Figure 9. TEM images of self-assembled structures of (a, b) streptavidin-bp1.3 and (c) streptavidin-bp2.9 in water at room temperature.

Scheme 3. Schematic Representation for the Self-Assembly of Bioconjugates Composed of Streptavidin and PEG107-bPDMAPS53-SS-Biotin Block Copolymer. Bioconjugates with Two Different Numbers of Polymer Chains on a Streptavidin Were Employed in the Self-Assembly Study

multiple block copolymer chains is similar to a miktoarm star polymer with two different star chains.47 In a selective solvent, the arrangement of the star polymer chains into spherical micelles, can reduce the entropic penalty in the self-assembly process. In a previous paper, Arms and co-workers reported that a star polymer with PDMAPS arms self-assembled into spherical micelles with PDMAPS cores.48 Compared to streptavidin-bp1.3, there are more PDMAPS blocks bound to a streptavidin molecule in streptavidin-bp2.9. In order to reduce the entropic penalty, the multicollapsed PDMAPS blocks selfassemble into the cores of the spherical micelles at a temperature below UCST, and the hydrophilic protein molecules and PEG blocks in the coronae stabilize the structures (Scheme 3). The biotin groups are grafted to the block copolymer chains via the disulfide bonds at the junction

points. The redox-responsive disulfides can be reduced to thiols by a reducing agent, leading to the cleavage of the disulfide bonds. Figure S8 shows a TEM image of the DTT-treated micelles. After treatment with DTT, aggregates of the cleaved protein molecules are observed. Protein molecules are attached to the assembled structures through biotin−streptavidin interaction. Upon cleavage of the disulfide bonds between the polymer chains and the biotin groups, the protein molecules were cleaved from the micellar structures and aggregated together.

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CONCLUSIONS Zwitterionic block copolymers with biotin groups at the junction points were synthesized by a combination of click chemistry, thiol−disulfide exchange reaction, and ATRP. The J

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(8) Huang, X.; Li, M.; Green, D. C.; Williams, D. S.; Patil, A. J.; Mann, S. Interfacial assembly of protein−polymer nano-conjugates into stimulus-responsive biomimetic protocells. Nat. Commun. 2013, 4, 3239. (9) Obermeyer, A. C.; Olsen, B. D. Synthesis and application of protein-containing block copolymers. ACS Macro Lett. 2015, 4, 101− 110. (10) Heredia, K. L.; Tolstyka, Z. P.; Maynard, H. D. Aminooxy endfunctionalized polymers synthesized by ATRP for chemoselective conjugation to proteins. Macromolecules 2007, 40, 4772−4779. (11) Li, H.; Bapat, A. P.; Li, M.; Sumerlin, B. S. Protein conjugation of thermoresponsive amine-reactive polymers prepared by RAFT. Polym. Chem. 2011, 2, 323−327. (12) Li, M.; De, P.; Li, H.; Sumerlin, B. S. Conjugation of RAFTgenerated polymers to proteins by two consecutive thiol−ene reactions. Polym. Chem. 2010, 1, 854−859. (13) Bontempo, D.; Heredia, K. L.; Fish, B. A.; Maynard, H. D. Cysteine-reactive polymers synthesized by atom transfer radical polymerization for conjugation to proteins. J. Am. Chem. Soc. 2004, 126, 15372−15373. (14) Heredia, K. L.; Grover, G. N.; Tao, L.; Maynard, H. D. Synthesis of heterotelechelic polymers for conjugation of two different proteins. Macromolecules 2009, 42, 2360−2367. (15) Kochendoerfer, G. G. Site-specific polymer modification of therapeutic proteins. Curr. Opin. Chem. Biol. 2005, 9, 555−560. (16) Deiters, A.; Cropp, T. A.; Summerer, D.; Mukherji, M.; Schultz, P. G. Site-specific PEGylation of proteins containing unnatural amino acids. Bioorg. Med. Chem. Lett. 2004, 14, 5743−5745. (17) Cobo, I.; Li, M.; Sumerlin, B. S.; Perrier, S. Smart hybrid materials by conjugation of responsive polymers to biomacromolecules. Nat. Mater. 2014, 14, 143−159. (18) Buller, J.; Laschewsky, A.; Lutz, J. F.; Wischerhoff, E. Tuning the lower critical solution temperature of thermoresponsive polymers by biospecific recognition. Polym. Chem. 2011, 2, 1486−1489. (19) Ding, Z.; Fong, R. B.; Long, C. J.; Stayton, P. S.; Hoffman, A. S. Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield. Nature 2001, 411, 59−62. (20) Kulkarni, S.; Schilli, C.; Grin, B.; Müller, A. H. E.; Hoffman, A. S.; Stayton, P. S. Controlling the aggregation of conjugates of streptavidin with smart block copolymers prepared via the RAFT copolymerization technique. Biomacromolecules 2006, 7, 2736−2741. (21) Bontempo, D.; Li, R. C.; Ly, T.; Brubaker, C. E.; Maynard, H. D. One-step synthesis of low polydispersity, biotinylated poly (Nisopropylacrylamide) by ATRP. Chem. Commun. 2005, 4702−4704. (22) Velonia, K.; Rowan, A. E.; Nolte, R. J. M. Lipase polystyrene giant amphiphiles. J. Am. Chem. Soc. 2002, 124, 4224−4225. (23) Boyer, C.; Huang, X.; Whittaker, M. R.; Bulmus, V.; Davis, T. P. An overview of protein−polymer particles. Soft Matter 2011, 7, 1599− 1614. (24) Palivan, C. G.; Fischer-Onaca, O.; Delcea, M.; Itel, F.; Meier, W. Protein-polymer nanoreactors for medical applications. Chem. Soc. Rev. 2012, 41, 2800−2823. (25) Moatsou, D.; Li, J.; Ranji, A.; Pitto-Barry, A.; Ntai, I.; Jewett, M. C.; O’Reilly, R. K. Self-assembly of temperature-responsive protein− polymer bioconjugates. Bioconjugate Chem. 2015, 26, 1890−1899. (26) Mary, P.; Bendejacq, D. D.; Labeau, M. P.; Dupuis, P. Reconciling low-and high-salt solution behavior of sulfobetaine polyzwitterions. J. Phys. Chem. B 2007, 111, 7767−7777. (27) Ning, J.; Li, G.; Haraguchi, K. Synthesis of highly stretchable, mechanically tough, zwitterionic sulfobetaine nanocomposite gels with controlled thermosensitivities. Macromolecules 2013, 46, 5317−5328. (28) Tian, M.; Wang, J.; Zhang, E.; Li, J.; Duan, C.; Yao, F. Synthesis of agarose-graft-poly [3-dimethyl (methacryloyloxyethyl) ammonium propanesulfonate] zwitterionic graft copolymers via ATRP and their thermally-induced aggregation behavior in aqueous media. Langmuir 2013, 29, 8076−8085. (29) Yusan, P.; Tuncel, I.; Bütün, V.; Demirel, A. L.; Erel-Goktepe, I. pH-Responsive layer-by-layer films of zwitterionic block copolymer micelles. Polym. Chem. 2014, 5, 3777−3787.

block copolymers exhibit UCST-type thermosensitivity, and the UCST is strongly dependent on the concentration of NaCl in the solution. Bioconjugates composed of streptavidin and zwitterionic block copolymer chains were fabricated based on biotin−streptavidin coupling. The UCSTs of the bioconjugates are lower than the zwitterionic block copolymer due to the electrostatic screening effect of the protein molecules. The bioconjugates make self-assembly in aqueous solutions at a temperature below UCST, and the morphology of the assembled structures is dependent on the average number of block copolymer chains grafted to a protein molecule. This research provides a new approach to the synthesis of polymer− protein bioconjugates with unique topological structures. More bioconjugates with different topological structures can be synthesized based on this method.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b02665. 1 H NMR and 13C NMR spectra and HRMS of precursor compounds and polymers, FTIR results of polymers, UV−vis results and TEM image (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(L.L.) E-mail [email protected]. *(H.Z.) E-mail [email protected]. ORCID

Hanying Zhao: 0000-0002-0706-9188 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (NSFC, 51473079, 51673098, and 21374047) and the National Basic Research Program of China (973 Program, 2012CB821500).

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REFERENCES

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