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Self-Assembly of a Double Hydrophilic Block Glycopolymer and the Investigation of Its Mechanism Takahiro Oh, Masanori Nagao, Yu Hoshino, and Yoshiko Miura* Department of Chemical Engineering, Kyushu University, 744 Motooka, Nishiku, Fukuoka 819-0395, Japan

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S Supporting Information *

ABSTRACT: We report the self-assembly of a double hydrophilic block glycopolymer (DHBG) via hydrogen bonding and coordinate bonding. This DHBG, composed of poly(ethylene)glycol (PEG) and glycopolymer, self-assembled into a welldefined structure. The DHBG was prepared through the controlled radical polymerization of trimethylsilyl-protected propargyl methacrylate using a PEG-based reversible addition−fragmentation chain transfer reagent, followed by sugar conjugation using click chemistry. The DHBG self-assembly capability was investigated by transmission electron microscopy and dynamic light scattering. Interestingly, the DHBG self-assembled into a spherical structure in aqueous solution. Hydrogen bonding and coordinate bonding with Ca2+ were identified as the driving forces for self-assembly.

1. INTRODUCTION Living things have self-assembled structures as the basic unit. Cells are among the most fundamental self-assemblies and can form internal functional microspace. Hydrophobic interactions are the main driving force for cellular membrane formation in animal and plant cells. The imitation of cellular membranes and development of nanosized structures have received much attention in various applications, such as drug delivery systems and nanoreactors.1 Many studies have focused on hydrophobic interactions and the formation of various structures using amphiphilic block copolymers to control the ratio of hydrophilicity and hydrophobicity.2−6 Hydrophobic interactions are thought to be an essential factor for self-assembly, though various other interactions, such as van der Waals forces, electrostatic interaction, and hydrogen bonding can be utilized for self-assembly.7 Although both plant and animal cells contain cellular membrane self-assemblies, they are very different. The major difference is that plant cells contain a cell wall that surrounds the cellular membrane. These cell walls are usually composed of cellulose, a hydrophilic polymer (polysaccharide), which forms a strong structure through hydrogen bonding and makes the plant cells robust.8 In nature, hydrophilic polymers, such as polysaccharides, can self-assemble through various interactions, such as hydrogen bonding. Therefore, hydrophilic synthetic polymers have the potential to self-assemble into a well-defined structure. Despite numerous reports on the self-assembly of amphiphilic block copolymers through hydrophobic inter© XXXX American Chemical Society

actions, the self-assembly of a hydrophilic polymer through hydrogen bonding has rarely been reported. Recently, self-assemblies of double hydrophilic block copolymers through interactions other than hydrophobic interactions have been reported. For example, Kataoka et al. have developed polyion complex micelles through selfassembly of a double hydrophilic block copolymer, synthesized from poly(ethylene glycol) (PEG) and a charged block, in water via electrostatic interactions.9,10 Furthermore, some studies have focused on the immiscibility of some polysaccharides (such as dextran and pullulan) with PEG despite both being hydrophilic, which is known as phase separation.11,12 These two hydrophilic polymer phases separated when linked together. Therefore, the double hydrophilic block glycopolymer (DHBG) composed of a glycopolymer and another hydrophilic polymer self-assembled into a polymer vesicle.13−16 Hydrogen bonding is formed between some sugars, such as in cellulose. Recently, Abeyratne-Perera et al. reported that mannose surfaces exhibited self-latching via strong hydrogen bonding.17 The self-assembly of DHBGs using hydrogen bonding between sugars has yet to be investigated. We propose that DHBGs composed of PEG and a mannose block could Received: May 9, 2018 Revised: June 29, 2018 Published: June 29, 2018 A

DOI: 10.1021/acs.langmuir.8b01527 Langmuir XXXX, XXX, XXX−XXX

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Langmuir Table 1. Polymerization of the Polymer Backbone (P45Tn) P45T33 P45T60 P45T107

TMS-PrMA (mmol)

PEG-CTA (mmol)

AIBN (mmol)

toluene (mL)

Conv.a (%)

DPb

Mn theo.

0.47 1.00 6.28

0.02 0.02 0.08

0.04 0.04 0.016

0.5 0.5 2

72 80 71

33 60 107

8800 14 000 23 000

MnNMRb MnGPCc Mw/Mnc (PDI) 8700 14 000 23 000

7000 15 000 22 000

1.19 1.21 1.13

a

Determined by 1H NMR before deprotection. bDetermined by 1H NMR after purification. cDetermined by GPC analysis calibrated for poly(methyl methacrylate). polymerization (DP) were determined by 1H NMR (Figures S5−S7). The polydispersity of the polymer was determined by GPC (Table 1). 1 H NMR (400 MHz, CDCl3): δ (ppm) 4.60 (s, −CH2−CC), 3.65 (s, −(CH2−CH2−O)45−), 3.38 (s, −CH2−CH2−O−CH3), 1.76−2.16 (br dd, −C(CH3)−CH2−), 0.88−1.16 (−C(CH3)− CH2−), 0.19 (s, −Si−(CH3)3). 2.4. Deprotection of the Polymer Backbone. P45Tn (1 equiv of TMS groups in polymer backbone) was suspended in dry tetrahydrofuran (molar concentration of TMS groups, 0.05 M) and TBAF (1.5 equiv) and acetic acid (1.5 equiv) were added with a plastic conical tube. The mixture was stirred for 5 h at room temperature. The solution was concentrated under reduced pressure and the resultant residue was purified by dialysis against DMSO for 24 h, Milli-Q (pH = 4) for 24 h, and finally Milli-Q (pH = 7) for 24 h. The final solution was then freeze-dried to afford P45Prn and deprotection was confirmed by 1H NMR (Figures S8−S10). 1 H NMR (400 MHz, CDCl3): δ (ppm) 4.62 (s, −CH2−CCH), 3.66 (s, −(CH2−CH2−O)45−), 3.38 (s, −CH2−CH2−O−CH3), 2.51 (s, −CCH), 1.81−2.12 (br dd, −C(CH3)−CH2−), 0.82−1.15 (−C(CH3)−CH2−). 2.5. Synthesis of the DHBG via Cu-Catalyzed Alkyne−Azide Cycloaddition Reaction (Huisgen Reaction). The syntheses of mannose azide and trimethylsilyl-protected mannose azide are described in the Supporting Information. As the Cu-catalyzed alkyne−azide cycloaddition (CuAAC) reaction was influenced by the block length of TMS-PrMA, the reaction conditions were different for the three different block lengths, as described below. P45Pr33 (1 equiv of the alkyne group in the polymer backbone), TMS-protected mannose azide (5 equiv), CuBr (0.4 equiv), tris[(1benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (0.4 equiv), and TEA (0.24 equiv) were dissolved in dry dimethylformamide (DMF) (alkyne concentration, 0.07 M). The reaction mixture was stirred for 72 h at room temperature under a nitrogen atmosphere. The mixture was then dialyzed against DMSO for 24 h, Milli-Q (pH = 4) for 24 h, and Milli-Q (pH = 7) for 24 h. The final solution was freeze-dried to obtain the DHBG. The sugar incorporation ratio was confirmed by 1 H NMR (Figure S11). P45Pr60 (1 equiv of the alkyne group in the polymer backbone), Dmannose azide (5 equiv), CuSO4 (0.4 equiv), sodium-L-ascorbate (2 equiv), and TBTA (0.4 equiv) were dissolved in a mixture of water and CH3CN (1:1, v/v; alkyne concentration, 0.07 M). The reaction mixture was stirred for 72 h at room temperature, after which the mixture was dialyzed against DMSO for 24 h, Milli-Q (pH = 4) for 24 h, and Milli-Q (pH = 7) for 24 h. The final solution was freeze-dried to obtain the DHBG. The sugar incorporation ratio was confirmed by 1 H NMR (Figure S12). P45Pr107 (1 equiv of the alkyne group in the polymer backbone), Dmannose azide (5 equiv), CuBr (0.4 equiv), TBTA (0.4 equiv), and TEA (0.24 equiv) were dissolved in dry DMF (alkyne concentration, 0.07 M). The reaction mixture was stirred for 168 h at room temperature under a nitrogen atmosphere. The mixture was then dialyzed against DMSO for 24 h, Milli-Q (pH = 4) for 24 h, and MilliQ (pH = 7) for 24 h. The final solution was freeze-dried to obtain the DHBG. The sugar incorporation ratio was confirmed by 1H NMR (Figure S13). 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 8.32 (triazole), 5.9−6.1 (anomer of mannose) 3.66 (s, −(CH2−CH2−O)45−), 1.40−2.20 (br dd, −C(CH3)−CH2−), 0.50−1.05 (−C(CH3)−CH2−). 2.6. DLS Measurement. DLS was performed on ZETASIZER NANO-ZS (Malvern, UK). The particle size was measured at 25 °C

self-assemble into nanostructures through hydrogen bonding between mannose blocks. Herein, we have synthesized a well-controlled DHBG composed of a sugar block and a PEG block using reversible addition−fragmentation chain transfer (RAFT) polymerization. RAFT polymerization allowed each DHBG block length to be easily controlled. The self-assembly capability of this DHBG was investigated by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The driving force of the DHBG self-assembly has been investigated with regard to the contributions of hydrophobic interactions, coordinate bonding with Ca2+, and hydrogen bonding. The effect of the “block structure” and PEG moiety on self-assembly has also been investigated using DLS.

2. EXPERIMENTAL SECTION 2.1. Materials. Triethyl amine (TEA) (99%), 2,2′-azobis(isobutyronitrile) (AIBN) (98%), acetic acid, calcium chloride (95%), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (99%), toluene (99.5%), and deuterium oxide (D2O, 99.8%) were purchased from Wako. Sodium-L-ascorbate (98%), sodium chloride (99%), magnesium chloride hexahydrate (99%), potassium chloride (99.5%), copper(II)sulfate (99.5%), dry dichloromethane (99.5%), dry N,N-dimethylformamide (99.5%), hexane (95%), acetone (99%), and 2,2′,2″,2‴-(1,2-ethanediyldinitrilo)tetraacetic acid (EDTA) were purchased from Kanto Chemical. Poly(ethylene glycol)4-cyano-4-(phenylcarbonothioylthio)pentanoate (PEG-CTA), tetrabutylammonium fluoride trihydrate (TBAF) (97%), copper(I) bromide, and 4-cyano-4(phenylcarbonothioylthio)pentanoic acid (CPADB) (97%) were purchased from Aldrich. Acetonitrile was purchased from SigmaAldrich. Dimethyl sulfoxide (DMSO) (98%) was purchased from Kishida. 2.2. Characterization. 1H NMR spectra were recorded on a JNM-ECZ400 spectrometer (JEOL, Tokyo, Japan) using CDCl3, DMSO-d6, or D2O as a solvent. Gel permeation chromatography (GPC) was performed on a HLC-8320 GPC Eco-SEC system equipped with a TSKgel Super AW guard column and TSKgel Super AW (4000, 3000 and 2500) columns (Tosoh, Tokyo Japan). GPC analyses were performed by injecting 20 μL of a polymer solution (1 g L−1) in DMAc buffer containing 10 mM LiBr. The buffer solution was also used as the eluent at a flow rate of 0.5 mL/min. The GPC system was calibrated using a poly(methyl methacrylate) standard (Shodex). 2.3. General Procedure for RAFT Polymerization of TMSPrMA (Synthesis of P45Tn). The synthesis of trimethylsilyl-protected propargyl methacrylate (TMS-PrMA) is described in the Supporting Information. TMS-PrMA was introduced into a glass tube and mixed with a toluene solution of PEG-CTA as the RAFT agent and AIBN as the radical initiator. The [TMS-PrMA]/[RAFT agent]/[initiator] ratios are shown in Table 1. The tube was degassed with freeze− pump−thaw cycles, sealed under vacuum, and transferred to an oil bath at 60 °C. After heating for 15 h, polymerization was stopped by cooling the solution with liquid nitrogen. Then, the polymer was precipitated in n-heptane to remove the nonactive PEG-CTA (Figures S20 and S20) and the supernatant was collected by centrifugation to remove inactive PEG-CTA. The supernatant liquid was concentrated in vacuo and dialyzed against acetone. The resultant polymer solution was then concentrated in vacuo and the conversion and degrees of B

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Langmuir Scheme 1. Synthesis of the DHBG

Figure 1. Schematic illustration of the DHBG self-assembly (top) and TEM images and volume distributions of the DHBG self-assemblies ((1) P45M33; (2) P45M60; and (3) P45M107) in HEPES buffer (bottom). The TEM images indicate that all DHBGs self-assembled into a spherical structure. in HEPES buffer at concentration from 0.01 to 10 g L−1. DLS measurements were carried out at least three times to confirm the reproducibility. To confirm the important interaction for selfassembly, DLS measurement was carried out in several aqueous HEPES buffer (HEPES, 10 mM; NaCl, 136.9 mM; KCl, 2.68 mM; CaCl2, 1.80 mM; MgCl2·6H2O, 0.49 mM), without Ca2+ (HEPES, 10 mM; NaCl, 137.4 mM; KCl, 2.68 mM; CaCl2, 0 mM; MgCl2·6H2O,

1.80 mM), without Mg2+ (HEPES, 10 mM; NaCl, 137.4 mM; KCl, 2.68 mM; CaCl2, 1.80 mM; MgCl2·6H2O, 0 mM), without metal ion (HEPES: 10 mM, NaCl: 139.2 mM, KCl: 2.68 mM, CaCl2: 0 mM, MgCl2·6H2O: 0 mM), adding guanidine hydrochloride (HEPES: 10 mM, NaCl: 136.9 mM, KCl: 2.68 mM, CaCl2: 1.80 mM, MgCl2· 6H2O: 0.49 mM, guanidine hydrochloride: 6 M). C

DOI: 10.1021/acs.langmuir.8b01527 Langmuir XXXX, XXX, XXX−XXX

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Langmuir

Figure 2. DLS measurement of DHBGs under several conditions: (a) DHBGs (P45M33 and P45M60: 1 g/L, P45M107: 3 g/L) with different molecular weights (P45M33, P45M60, and P45M107); (b) P45M107 (3 g/L) with different temperatures (4 and 25 °C); (c) P45M107 (3 g/L) with and without Ca2+ and Mg2+; and (d) P45M107 with and without guanidine hydrochloride. 2.7. TEM Observation. TEM observation of each DHBG was performed using an FEI TECNAI-20 system (Thermo). The TEM grid was hydrophilized by plasma treatment for 30 s and then 2 μL of each polymer solution was dropped onto the grid. The grid was incubated for 1 min, excess solution was removed with a paper, and then uranyl acetate solution was dropped onto the TEM grid and incubated for 30 s. Excess solution was again removed with a paper and then TEM observation was conducted at 120 kV. All DHBGs were dissolved in HEPES buffer (HEPES, 10 mM; NaCl, 136.9 mM; KCl, 2.68 mM; CaCl2, 1.80 mM; MgCl2·6H2O, 0.49 mM).

P45Pr33, the proton peak of the TMS group should appear at 0.12 ppm in the 1H NMR but had almost disappeared instead (Figure S11). Therefore, the TMS group had been hydrolyzed during dialysis. The deprotection rate was over 99%, as calculated from the 1H NMR spectrum of P45M33. Using the Huisgen cycloaddition reaction, mannose azide was introduced into the polymer backbone. 3.2. Self-Assembly of DHBGs. The self-assembly capabilities of the prepared DHBGs (P45M33, P45M60, and P45M107) were investigated using TEM and DLS. The DHBGs were dissolved in HEPES buffer containing Ca2+ and Mg2+ and stained with uranyl acetate. Spherical structures were observed by TEM in all cases, and the average particle sizes for P45M33, P45M60, and P45M107 were 188, 94, and 134 nm, respectively. The volume distribution was also confirmed by TEM (Figure 1). P45M107 showed the narrowest polydispersity. P45M60 comprised many small particles surrounding larger particles (Figure S22). DLS measurements were also conducted to confirm selfassembly in HEPES buffer containing Ca2+ and Mg2+. The scattering of large particles was larger than that of small particles, which greatly influenced the intensity distribution. Therefore, the volume distribution was evaluated. The volume distribution corresponded with that of TEM. The average particle sizes of P45M33, P45M60, and P45M107 were 141 ± 4 (PDI: 0.23), 18 ± 7 (PDI: 0.30), and 149 ± 1 nm (PDI: 0.26), respectively (Figure 2a). The diameter of P45M60 was smaller than those of other DHBGs. P45M60 comprised many small particles (Figure S22), which greatly influenced the DLS measurements. Moreover, all DHBGs could not self-assemble in the aqueous solution at low concentration (Figure S24). Therefore, it is considered that the overlap concentration is in the range of 0.01−3 g L−1 and the overlap concentration affects the self-assembly. Because the DHBGs are block copolymers and have a large side chain on the glycopolymer block, it is difficult to estimate the overlap concentration. We regarded all DHBGs as composed of pullulan and estimated the overlap

3. RESULT AND DISCUSSION 3.1. Synthesis of the DHBG. 3.1.1. Synthesis of the Polymer Backbone. The DHBGs (PEG45-poly(mannose)n diblock copolymer), denoted as P45Mn (n = 33, 60, and 107), were synthesized as shown in Scheme 1. Polymer backbones with different DP were obtained by RAFT polymerization with TMS-PrMA and PEG-CTA. The feed ratios of [TMS-PrMA]/[PEG-CTA] were 23.5, 50, and 78.5, for n = 33, 60, and 107, respectively, taking into account the purity of PEG-CTA. The monomer conversion and DP of TMS-PrMA were calculated by 1H NMR. The conversion was over 70% and the DP were 33, 60, and 107 (Table 1). The polydispersity index (PDI) of each polymer backbone (P45Tn), as determined by GPC analysis, was narrow (99% by 1H NMR for all DHBGs (Figures S11−S13). Furthermore, after the reaction with D

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Langmuir concentrations based on previous reports.18,19 The details are shown in the Supporting Information. The TEM images confirmed the self-assembly structures of the DHBGs, all of which formed spherical molecular assemblies. The TEM image of P45M60 showed a large number of small self-assemblies, which was different to those of P45M33 and P45M107. DLS measurements also supported the selfassembly of DHBGs. The diameter of P45M60 was much smaller than those of the other DHBGs, and the volume distribution obtained by DLS measurements corresponded well with that obtained by TEM (Figures 1, 2 and S23). Some hydrophilic block copolymers have been reported to form loosely aggregated self-assemblies, though the majority of the polymer was unimolecularly dissolved.20 In contrast, the majority of DHBGs can be considered to self-assemble by the volume and number distributions (Figure S23). These results strongly supported the self-assembly capability of the DHBGs in aqueous solution. Though the polymers were composed of hydrophilic blocks, all DHBGs showed spherical structures, suggesting that the DHBG assemblies were facile and strong in aqueous solution. The detailed structure (micelle or vesicle) of the DHBGs will be further investigated using techniques such as small-angle X-ray scattering (SAXS) and cryo-TEM in the near future. 3.3. Driving Force of Self-Assembly. We investigated the contribution of three interactions to self-assembly, namely, hydrophobic interactions, coordinate bonding, and hydrogen bonding. P45M107 was used for these investigations because it was the most stable among the three DHBGs in DLS measurements. 3.3.1. Hydrophobic Interaction. To investigate the contribution of hydrophobic interactions, DLS measurements were conducted at different temperatures (4, 25, and 60 °C). The peak profiles at 4 and 25 °C were overlapped. At 60 °C, the P45M107 self-assembly was dissociated (Figure 2b). The self-assembly was stable between 4 and 35 °C but was dissociated in the solution at above 35 °C (Figure S25). If the driving force of self-assembly was hydrophobic interactions, the self-assembly would be stable even at high temperatures. As hydrophobic interactions are caused by the entropy change of water, they are strongly affected by temperature, such as being stronger at high temperatures and weaker at low temperatures.21 As the self-assembly of P45M107 was dissociated at high temperature, this indicated that the contribution of hydrophobic interactions to the self-assembly was small. This showed that, unlike conventional self-assemblies, which mimic animal cells using hydrophobic interactions, DHBG selfassembly formation was driven by other interactions. The driving force of DHBG self-assembly was certainly different to that of amphiphilic block copolymer self-assemblies. Considering the chemical structure of the glycopolymer, the polymer backbone was hydrophobic and the sugar moiety was hydrophilic, which suggested the amphiphilicity in the glycopolymer. Therefore, the glycopolymer was thought to behave like a polysoap. The amphiphilicity of glycopolymers has also been reported previously.22 Despite the possibility that glycopolymers aggregate, it is thought that macroscopic micelles are not formed as shown by DLS measurements (Figure 3a). 3.3.2. Coordinate Bonding. Coordinate bonding between the glycopolymer and two divalent metal cations (Ca2+ and Mg2+) was investigated using DLS measurements. The average particle size of P45M107 was 148 nm with both Mg2+ and Ca2+

Figure 3. DLS measurements of the (a) homopolymer (P45 and M105) and block polymer (P45M107); (b) linear-type DHBG (P45M107) and side-chain-type DHBG (Teg41M103); and (c) chemical structure of Teg41M103.

but became much smaller, at around 7 nm, without Ca2+ (PDI: 0.49; Figure 2c) or without both divalent cations (PDI: 0.50). Interestingly, in the absence of Mg2+, the average particle size remained almost the same, at 157 ± 1 nm (PDI: 0.36). Furthermore, in the presence of EDTA (2.5 mM), the average particle size was around 10 nm (PDI: 0.32) (Figure S26). When Ca2+ was removed by adding EDTA, the self-assembly (average particle size, 148 nm) was dissociated. The Ca2+ concentration dependence was also investigated. Interestingly, as the calcium concentration was increased, the particle size increased, and DHBG was precipitated when the Ca2+ concentration was 10 mM (Figures S27 and S28). Therefore, the self-assembly of P45M107 required Ca2+ and showed divalent cation selectivity and Ca2+ concentration dependence. This result strongly supported that Ca2+ had an influence on the self-assembly capability of DHBGs. To our knowledge, this is the first report of a DHBG self-assembly requiring divalent metal cations. Ca2+ has been reported to coordinate with carbohydrates,23,24 which can contribute to the self-assembly of DHBGs. Furthermore, Ca2+ chelation has been reported with several sugars, with the binding energy between Ca2+ and sugars at about 15−17 kJ mol−1.25,26 Densely packed hydroxyl groups in DHBGs are thought to enable Ca2+ coordination. 3.3.3. Hydrogen Bonding. The contribution of hydrogen bonding between the glycopolymers was investigated by adding guanidine hydrochloride, a reagent that cleaves hydrogen bonding. In the presence of guanidine hydrochloride, the average particle size became much smaller (14 nm; PDI: 0.53) than without guanidine hydrochloride, despite the salt strength being the same (Figure 2d). In addition to cleaving hydrogen bonding, guanidine hydrochloride might also displace Ca2+ and prevent coordination. However, in this study, the self-assembly capability showed divalent cation selectivity, indicating that the DHBG recognized the Ca2+ ionic radius.24 As the ionic radius of guanidine hydrochloride was E

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Langmuir larger than that of Ca2+, guanidine hydrochloride would not be able to displace Ca2+. As we mentioned, the self-assembly has an upper critical solution temperature behavior (Figure S25). Because the mobility of molecules is decreased at low temperature, the intramolecular hydrogen bonding is enhanced. On the other hand, the hydrogen bonding became weak at high temperature. Therefore, the hydrogen bonding weakens as temperature increases. Finally, all hydrogen bondings were cleaved at around 35 °C. Figure S25 supported that the self-assembly of DHBG is driven by hydrogen bonding. This result showed that hydrogen bonding is required for self-assembly of the DHBG, with hydrogen bonding thought to be formed between the glycopolymer blocks. The sugars have many hydroxyl groups per unit volume, with hydroxyl groups present at a high packing density in the glycopolymer. Therefore, it is possible to create strong hydrogen bonding interactions between glycopolymers. Similarly, AbeyratnePerera et al. reported that mannose surfaces exhibited selflatching via strong hydrogen bonding.17 The binding energy of OH···O hydrogen bonding was 14−20 kJ mol−1, which is also strong enough to cause self-assembly.8 The self-assembly of the DHBG into a nanostructure was due to an exquisite balance of hydrogen bonding and coordinate bonding in aqueous solution. Therefore, polymer materials such as sugars, polysaccharides, and glycopolymers bearing a large number of hydroxyl groups tend to form hydrogen bonding, making it easy to obtain an enthalpic gain through self-assembly. This study showed that DHBGs self-assembled into a nanostructure in a fashion similar to that of the plant cell wall. 3.4. Influence of the DHBG Structure. The self-assembly capability is strongly influenced by the block copolymer structure.27 When the block structure and steric hindrance of the block change, the volume of the block also change. When DHBG self-assembles into a structure, the degree of packing of the core is considered to change depending on the volume. We investigated the self-assembly capability of DHBG with respect to the block structure and steric hindrance. 3.4.1. Comparison of “Homo” and “Diblock” Polymers. We initially focused on the “diblock” structure of the DHBGs in comparison with the homopolymer. The influence of the “diblock” structure on self-assembly was investigated by DLS using each polymer block constituting the DHBG, namely, homo mannose polymer (M105) and PEG monomethyl ether (P45). The average particle sizes of M105 and P45 were 9 nm and less than 1 nm, respectively (Figure 3a). Therefore, these hydrophilic homopolymers (M105 and P45) did not have selfassembly capabilities in aqueous solution, despite P45M107 forming a spherical structure. This indicated that when two hydrophilic polymers were bonded, phase separation promoted self-assembly. In the prediction of miscibility using the Flory−Huggins interaction parameter and Hildebrand solubility parameter, PEG (Mw, 6000) and some sugars (such as glucose) have been reported to be immiscible.28 Furthermore, there is the possibility of complexation between PEG and glycopolymers, which self-assemble in aqueous solution.29 However, the mixture of two hydrophilic homopolymers showed no scattering at around 100 nm by DLS measurement (Figure S29), which showed that there was no complexation between the two hydrophilic homopolymers. Phase separation between the two hydrophilic blocks was also an important factor.13−16,30 When diblock polymers self-assemble into a

structure, a thermodynamically stable structure is obtained for polymers of the same type that are adjacent.31 When the DHBGs self-assemble into nanostructures via hydrogen bonding and coordinate bonding, the glycopolymer block and the PEG form a core and shell, respectively, to avoid thermodynamically unfavorable mixing of the two phases. It has been reported that the interface is presented between two immiscible polymers, including block copolymers.31,32 As a result of phase separation, it is considered that an interface between PEG and glycopolymer is produced (Figure S30).9,10,33−35 When the double hydrophilic block copolymer self-assembles, the association number increases to lower the free energy of the interface between the less solvated and the more solvated blocks. Simultaneously, the densities of blocks increase and their conformational entropy of the corona blocks becomes smaller, resulting in a tendency to reduce the association number of the self-assembly. Therefore, we believe that the possibility of the thermodynamically stable selfassembly of the DHBG is dependent on the balance between the interfacial free energy and the conformational entropy, as Kataoka et al. mentioned (Figure S30).9,10,33−35 3.4.2. Comparison of Linear-Type (P45M107) and SideChain-Type (Teg41M103) DHBGs. The self-assembly capabilities of linear-type (P45M107) and side-chain-type (poly(triethylene glycol monoethyl ether)41-poly(mannose)103 diblock copolymer; Teg41M103) DHBGs were compared using DLS measurements. The synthesis of Teg41M103 is shown in the Supporting Information (1H NMR spectrum shown in Figure S17). The average particle size of Teg41M103 was 40 ± 9 nm (PDI: 0.42), which was much smaller than that of the P45M107 self-assembly (Figure 3b). Teg41M103 self-assembled into a small structure and the morphology of the self-assembly was affected by the polymer structure. This showed that when the volume of the TEG block was large, steric hindrance on the outside of the structure increased and influenced self-assembly. Although the polymer based on TEG-polymerizable derivatives (TEG-MA) had a similar chemical property based on nonionic hydrophilicity with the ethylene glycol unit, the conformation and mobility of TEG and PEG were considered to be greatly different. The morphology of the polymer self-assembly varied depending on the volume of the PEG moiety.34 As mentioned above, the balance between interfacial free energy and conformational entropy determined the thermodynamically stable size of the micelles. For Teg41M103, the conformational entropy of the Teg block was higher than that of the PEG block. As a result, Teg41M103 self-assembled into a smaller structure with a low association number because of the balance between interfacial free energy and conformational entropy. From these results, it is thought that various self-assemblies can be formed by controlling the volume of the other hydrophilic block.

4. CONCLUSIONS DHBGs were successfully synthesized using RAFT polymerization and the Huisgen cycloaddition reaction. Interestingly, DHBGs self-assembled into spherical structures in aqueous solution through hydrogen bonding and coordinate bonding with Ca2+. To self-assemble, the DHBGs required a “block” structure to become phase separated. The morphology of the DHBG self-assembly was influenced by the structure of the other hydrophilic block (such as PEG). Driven by hydrogen bonding and coordinate bonding, these DHBGs formed a selfassembly different from conventional amphiphilic block F

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Langmuir

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copolymer self-assemblies. Therefore, DHBGs have the potential to fabricate new supramolecular systems and materials that could reflect the functions and properties of plant cell systems.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b01527.

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Experimental method and materials; synthesis of the monomers and Teg41M103; 1H NMR spectra of monomer and polymers; GPC data of polymer backbone; TEM image of P45M60; DLS result of each DHBG; and overlap concentration estimation (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yu Hoshino: 0000-0001-9628-6979 Yoshiko Miura: 0000-0001-8590-6079 Funding

This work was supported by a Grant-in-Aid for Scientific Research (B) (JP15H03818) and a Grant-in-Aid for Scientific Research on Innovative areas (JP18H04420). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. A. Kishimura (Kyushu University) for helpful discussion regarding TEM measurements. ABBREVIATIONS AIBN, 2,2′-azobis(2-methylpropionitril); Conv., conversion; CPADB, cyano-4-(phenylcarbonothioylthio)pentanoic acid; DP, degree of polymerization; D2O, deuterium oxide; DMSO, dimethyl sulfoxide; DLS, dynamic light scattering; EDTA, 2,2′,2″,2‴-(1,2-ethanediyldinitrilo)tetraacetic acid; GPC, gel permeation chromatography; HEPES, 2-[4-(2hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; Mn theo., theoretical molecular weight; SAXS, small-angle X-ray scattering; TBAF, tetrabutylammonium fluoride trihydrate; TBTA, synthesis of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; TEA, triethyl amine; TEG, tri(ethylene)glycol; TEG-MA, triethylene glycol monoethyl ether monomethacrylate; TEM, transmission electron microscopy; TMS-PrMA, trimethylsilyl propargyl methacrylate; PEG, poly(ethylene)glycol; PEGCTA, poly(ethylene glycol) 4-cyano-4(phenylcarbonothioltyio)pentanoate; PDI, polydispersity index; RAFT, reversible addition−fragmentation chain transfer

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REFERENCES

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DOI: 10.1021/acs.langmuir.8b01527 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.8b01527 Langmuir XXXX, XXX, XXX−XXX