Two-Dimensional Supramolecular Assemblies from pH-Responsive

Sep 28, 2017 - Amphiphilic block copolymers containing polypeptides can self-assemble into a variety of nonspherical structures arising from strong in...
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Two-Dimensional Supramolecular Assemblies from pH-Responsive Poly(ethyl glycol)‑b‑poly(L‑glutamic acid)‑b‑poly(N‑octylglycine) Triblock Copolymer Yunxia Ni,† Jing Sun,*,† Yuhan Wei,† Xiaohui Fu,† Chenhui Zhu,‡ and Zhibo Li*,† †

Key Laboratory of Biobased Polymer Materials, Shandong Provincial Education Department; School of Polymer Science and Engineering, Qingdao University of Science and Technology, Qingdao266042, China ‡ Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States S Supporting Information *

ABSTRACT: Amphiphilic block copolymers containing polypeptides can self-assemble into a variety of nonspherical structures arising from strong interactions between peptide units. Here, we report the synthesis of a pHresponsive poly(ethyl glycol)-block-poly(L-glutamic acid)-block-poly(N-octylglycine) (PEG-b-PGA-b-PNOG) triblock copolymers by sequential ringopening polymerization using amine-terminated poly(ethyl glycol) as the macroinitiator followed by selective deprotection of the benzyl protecting group. The obtained triblock copolymer can be directly dispersed in aqueous solution with hydrophilic PEG, pH-responsive PGA block, and hydrophobic PNOG. We present a systematic study of the influence of pH, molar fraction, and molecular weight on the self-assemblies. It was found that the PEG-bPGA-b-PNOG triblock tends to form two-dimensional nanodisks and nanosheet-like assemblies. The nanodisk-to-nanosheet transition is highly dependent on the pH and molar fraction despite the different molecular weights. We demonstrate that the dominant driving force of the nanodisks and nanosheets is the hydrophobicity of the PNOG blocks. The obtained bioinspired 2D nanostructures are potential candidates for applications in nanoscience and biomedicine.

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amide nitrogen instead of α-carbon. Such structure variation results in the lack of hydrogen-bonding sites and chirality in the main chains and offers a potentially flexible backbone. In contrast to the polypeptides with poor processability, polypeptoids with longer alkyl side chains are semicrystalline with tunable melting transitions.22−24 Further, polypeptoids exhibit great biocompatibility and potential bioactivities.20,21,25 Besides the step-by-step solution phase coupling strategy, the ring-opening polymerization (ROP) of N-substituted glycine N-carboxyanhydrides (NCA) offers an effective way to produce the polypeptoids with high molecular weights and large quantities.26,27 Diversity of the side-chain functionalities of polypeptoids is generally limited to alkyl moieties due to synthetic challenge.21 For example, Schlaad et al. reported the postmodification strategy by thiol-yne/ene click chemistry.28,29 The Zhang group reported pegylated polypeptoids that exhibit excellent protein-resistant property.30 Here, we intend to incorporate both polypeptoid and polypeptide blocks into one copolymer chain to form a pHresponsive triblock copolymer. The aim of such design is that the triblock copolymer can not only combine the structural

INTRODUCTION The self-assembly of amphiphilic block copolymers in aqueous solution has been of great interest over the past few decades.1−5 A variety of nanostructured morphologies have been identified, including spheres, vesicles, cylinders, and so forth. Recently, two-dimensional (2D) planar structures have received extensive attention due to their excellent properties.6 It has been reported that disk-like morphology facilitates cell surface binding with reduced cell uptake as compared to those of spheres.7 A couple nanodisk and nanosheet-like structures have been obtained from various amphiphilic block copolymers.8−12 A major focus is the design and preparation of a membrane that is difficult to bend into a vesicular structure as the free energy of bending into a vesicle is generally low.8 In nature, proteins can fold into highly ordered structures via intra- and intermolecular interactions, which enable high level control over the biological performance. Protein-mimetic polymers combine the structural advantages of proteins with improved stability and processability, which is beneficial for many applications.13−18 In particular, polypeptoids, or poly Nsubstituted glycines, are a promising class of peptidomimetic polymers that offer excellent unique properties for both fundamental research and applications in biotechnology.19−21 The chemical structure of a polypeptoid differs from a polypeptide only in that the side chains are attached to the © XXXX American Chemical Society

Received: July 18, 2017 Revised: September 1, 2017

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DOI: 10.1021/acs.biomac.7b01014 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules advantages of both blocks but finely tune the properties of the materials such as degradability. The polypeptoid-block-polypeptide copolymers have barely been reported, except for a few polysarcosine-based block copolymers.31−33 We designed and synthesized a series of PEG-b-PGA-b-PNOG triblock copolymers containing hydrophobic PNOG, hydrophilic PEG, and pH-responsive PGA block by the sequential ROP technique. The obtained triblock copolymers can be directly dispersed in water and self-assemble into nanodisk/nanosheet-like morphologies depending on the pH and molar fraction. We systematically investigated the influence of the pH, molar fraction, and chain length on the assemblies.

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hexane three times to obtain 5.8 g of solid white BLG-NCA. Yield: 52.3%. Synthesis of PEG-b-PBLG-b-PNOG Triblock Copolymers. mPEG-NH2 (Mn = 2000 g/mol) was heated at 50 °C and dried under a vacuum for 24 h and then dissolved in anhydrous DMF in a Schlenk flask. The solution of BLG-NCA (50 mg/mL in DMF) at the prescribed ratio was added to the flask. The polymerization was performed under a N2 atmosphere at 25 °C and monitored by FTIR spectrum until the characteristic peaks (1850 cm−1, 1790 cm−1) of NCA disappeared. Then, 1 mL of the polymer solution was taken out and precipitated in excess diethyl ether to obtain a white solid of the block copolymer, which was further used for 1H NMR and GPC testing. The remaining polymer solution was then heated to 50 °C followed by adding the solution of NNCA (25 mg/mL in THF). The solution of triblock polymer was eventually concentrated and precipitated in a 1:1 mixture (v/v) of hexane/diethyl ether after 24 h. After centrifugation, a yellow solid was obtained. Yield: 56%. The benzyl group on PEG-b-PBLG-b-PNOG was removed by reacting with 4 mol equiv of HBr (C = 33%, in HAc) with respect to BLG repeat units in CF3COOH (35 mg/mL) at 0 °C for 2.5 h. The final product poly(ethylene glycol)-b-poly(L-glutamic acid)-b-poly(N-octylglycine) (PEG-b-PGA-b-PNOG) was obtained after dialysis and lyophilization. Yield: 78%. Preparation of Polymer Solutions. The polymer was directly dissolved into aqueous solutions at a concentration of 2 mg/mL with the desired pH value, which was adjusted by HCl or NaOH solution. The pH value of the solutions was measured using a calibrated pH electrode. Characterizations. GPC analysis was conducted using an SSI pump connected to Wyatt Optilab DSP with 0.05 M LiBr in DMF as the eluent at a flow rate of 1.0 mL/min at 50 °C. All GPC samples were prepared at concentrations of ∼7 mg/mL. DSC studies were conducted using a TA DSC 2920 calorimeter. Powder samples enclosed in the aluminum pans were heated from −40 to 200 °C at 10 °C/min for three cycles. TEM experiments were conducted on FEI TECNAI 20. Ten microliters of the polymer solution (2 mg/mL) was pipetted onto the carbon-coated copper grid, which was pretreated in a plasma cleaner. The grid was blotted to remove any excess solution and then dyed using 2% uranyl acetate. The samples were dried and stored under ambient conditions before TEM testing. A FEI T20 cryoTEM was used to examine the vitrified specimens that were prepared using a Vitrobot (FEI, Inc.). A 5 μL droplet of the aqueous solution at a concentration of 2 mg/mL was deposited on the surface of glowdischarged grids with lacey carbon films. The droplet was blotted by filter paper for 2 s, followed by 2 s draining, and then plunged into liquid ethane to obtain a vitrified thin film. The thin films were examined at −185 °C and 200 kV acceleration voltage to obtain 2D projections. AFM studies were conducted using noncontact mode AFM under ambient conditions. Three microliters of the polymer solution (2 mg/mL) was placed on freshly cleaved mica, and then the samples were dried and stored under ambient conditions before AFM testing. For grazing incidence wide-angle X-ray scattering (GIWAXS measurements), the polymer solution (2 mg/mL) was deposited on Si wafers, dried, and stored under ambient conditions before testing. The measurement was performed at beamline 7.3.3, Advanced Light Source (ALS), Lawrence Berkeley National Lab (LBNL). X-ray energy was 10 keV and operated in top off mode. The scattering intensity was recorded on a 2D Pilatus 1 M detector (Dectris) with a pixel size of 172 μm. A silver behenate sample was used as a standard to calibrate the sample−detector distance and the beam position. Circular dichroism spectra (CD) analysis was recorded on MOS-450/AF-CD spectrometer. The polymer solutions (0.2 mg/mL) of various pH were placed into a quartz cell with a path length of 0.1 cm. Ellipticity ([θ] in deg cm2 dmol−1) = (millidegrees × mean residue weight)/(path length in millimeters × concentration of polymer in mg/mL). The α-helix contents of the block copolymers were calculated by the equation: % α-helix = (−θ222 + 3000)/39000.34

EXPERIMENTAL SECTION

Materials and Methods. Hexane, dichloromethane (CH2Cl2), and tetrahydrofuran (THF) were purified by passing through activated alumina columns prior to use. N,N-Dimethylformamide (DMF) was stored over calcium hydride (CaH2) and purified by reduced pressure distillation. α-Methoxy-ω-amino poly(ethylene glycol) (mPEG-NH2, Mn = 2000 g/mol) was purchased from JenKem Technology Co, Ltd. γ-Benzyl-L-glutamic acid was purchased from GL Biochem (Shanghai) Ltd. Glyoxylic acid and di-tert-butyl dicarbonate were purchased from Aladdin. All other chemicals were purchased from commercial suppliers and used without further purification. Synthesis of 2-(n-Octyl amino)acetic Acid Hydrochloride. Glyoxylic acid (15 g, 163 mmol) was stirred with 0.5 mol equiv noctylamine (13.5 mL, 81.5 mmol) in CH2Cl2 (400 mL) for 24 h at 25 °C until a clear solution was obtained. The solvent was evaporated to yield a yellow viscous liquid to which aqueous HCl (400 mL, 1 M) was added. The mixture was refluxed for no less than 24 h. The water was removed under vacuum to obtain a light yellow solid, which was further dissolved in methanol and precipitated in cold diethyl ether. Finally, 11.7 g of purified 2-(n-octyl amino)acetic acid hydrochloride, white in color, was obtained. Yield: 64%. Synthesis of 2-(N,N-tert-Butoxycarbonyl-N-Octylamino) Acetic Acid. A mixture of 2-(n-octyl amino) acetic acid hydrochloride (11.6 g, 51.9 mmol), di-tert-butyl dicarbonate (28.2 g, 129 mmol), and triethylamine (36 mL, 258 mmol) in deionized water was stirred at 25 °C until pellucid solution was achieved. The reaction mixture was extracted with hexane (3 × 200 mL) in a separating funnel and separated by partitioning. The water phase was then acidized with aqueous HCl (4M), and its pH value was monitor by pH-indicator papers. The acidized phase was then extracted with ethyl acetate (3 × 100 mL). The organic phase was separated, washed with saturated salt solution, and dried with anhydrous Na2SO4 overnight. After filtration and solvent removal, a pale yellow oil was obtained (9.0 g, 60% yield). Synthesis of N-Octyl-N-carboxyanhydride (Oct-NNCA). 2(N,N-tert-Butoxycarbonyl-N-octylamino) acetic acid (9.4 g, 32.8 mmol) was dissolved in anhydrous CH2Cl2 (200 mL) under a N2 atmosphere. The solution was cooled to 0 °C in an ice-bath. Soon thereafter, phosphorus trichloride (2.5 mL, 26.2 mmol) was added dropwise. The reaction mixture was stirred for 2 h followed by filtration. The solvent was then removed under vacuum to afford white solid. Three milliliters of anhydrous CH2Cl2 was added to the mixture under a N2 atmosphere, filtered again if necessary, followed by addition of hexane. After three cycles of recrystallization with anhydrous CH2Cl2/hexane, white crystals (2.8 g, 13.1 mmol) were obtained. Yield: 40%. Synthesis of γ-Benzyl-L-glutamate N-Carboxyanhydride (BLG-NCA). γ-Benzyl-L-glutamate (10 g, 42.2 mmol) (BLG) was suspended in anhydrous THF (100 mL) in a N2 atmosphere at 50 °C. Then, 1.05 mol equiv triphosgene (4.4 g, 14.7 mmol) was added to the reaction solution until a clear solution was achieved. Subsequently, the solvent was removed using the rotary evaporator to give a light yellow solid. Then, the solid was dissolved in ethyl acetate (100 mL) and further filtered. Ethyl acetate was removed under a vacuum to give a light yellow solid. In the glovebox, the solid was recrystallized in THF/ B

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Figure 1. Representative 1H NMR spectra of (a) PEG-b-PBLG64, (b) PEG-b-PBLG64-b-PNOG21, and (c) PEG-b-PGA64-b-PNOG21 in CDCl3/ CF3CO2D (v:v = 10:1) (* indicates CDCl3).

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RESULTS AND DISCUSSION The N-octyl-N-carboxyanhydride (Oct-NNCA) and γ-benzyl-Lglutamate N-carboxyanhydride (BLG-NCA) monomers were synthesized following reported methods.22,35 The chemical structure of the monomers was confirmed by 1H NMR spectroscopy (Figure S1). The triblock copolymers (PEG-bPBLG-b-PNOG) were then synthesized by sequential addition of BLG-NCA and Oct-NNCA using mPEG-NH2 (Mn = 2000 g/mol) as the macroinitiator (Scheme S1). The polymerization was monitored by FTIR. The disappearance of two characteristic νCO peaks of the monomer at 1790 and 1850 cm−1 was considered as complete conversion of NCA/NNCA monomers to polypeptides/polypeptoids (Figure S2).27 The 1H NMR spectra show that all peaks of the synthesized di- and triblock copolymers are well assigned, confirming their chemical structures (Figure 1a and b). A series of triblock copolymers were synthesized by varying the ratio of NCA/NNCA to PEGNH2 macroinitiator. The average DP of PNOG was held fixed at ∼10 and 20, and the average DP of PBLG was varied from

24 to 64. Table 1 summarized the molecular characteristics of all triblock copolymers PEG-b-PBLGm-b-PNOGn, where the subscripts m and n represent the average DP of PBLG and PNOG blocks, respectively. The molecular weight distribution of the corresponding di- and triblock copolymers were determined by GPC (Figure 2 and Figure S3), and 1H NMR spectroscopy was used to determine the molar ratio and molecular weight of the blocks. The GPC trace showed a narrow molecular weight distribution with dispersity (Đ) ≤ 1.24, indicating well-controlled polymerization. The benzyl protective groups of the triblock copolymers were removed to yield poly(ethylene glycol)-b-poly(L-glutamic acid)-b-poly(N-octylglycine) (PEG-b-PGA-b-PNOG).35 The chemical structure of deprotected triblock copolymer PEG-bPGA-b-PNOG was confirmed by 1H NMR, as shown in Figure 1c. The disappearance of peaks at 7.21 and 5.02 ppm suggests complete debenzylation. The thermal properties of PEG-b-PGA-b-PNOG were first investigated by DSC, as shown in Figure S4. The DSC C

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of PEG-b-PGA42-b-PNOG17 as a function of pH is shown in Figure 3a. The solution pH was adjusted by adding aqueous HCl or NaOH. The triblock copolymer adopts α-helix conformation at acidic conditions (pH 2.5 and 4.8), as indicated by two negative peaks at λ ≈ 210 and 222 nm.36 As the pH increases to 7.2, one strong negative signal at λ ≈ 198 nm indicates random coil conformation. We further plotted α-helix content as a function of pH (Figure 3b). In all cases, the α-helicity of the samples initially increased slightly and subsequently decreased largely with increasing pH. The initial increase may be attributed to the presence of large aggregates at pH 2.5, consistent with previous results. This will be discussed shortly. The α-helix content is highly dependent on the chain length of PGA at low pH (pH 4.8). The α-helix content was reduced from 66.2 to 33.1% as the chain length of PGA decreased from 64 to 24. As the pH increased to 7.2, the α-helix content reduced to 0%, suggesting complete helix-to-coil transition. All other samples showed similar results (Figure S5). To probe the nanostructure and morphology of the triblock copolymer, we performed transmission electron microscopy (TEM) and atomic force microscopy (AFM). Spherical-shape structure with an average diameter of 14.6 nm of PEG-bPGA42-b-PNOG17 self-assemblies at pH 7.2 is observed by negative-stained TEM (Figure 4a). Interestingly, most of the assemblies show irregular edges of the nanostructure. In addition, a few short fiber-like structures are observed (indicated by arrows), which is possibly because they lie orthogonal to the plane of the specimen.37 This may suggest a two-dimensional nanodisk-like morphology. The AFM image shows a spherical structure with a diameter of 37.5 ± 0.5 nm, more than 6-times the thickness (5.9 ± 0.4 nm), possibly denoting the formation of disks. To preclude the influence of sample preparation during the drying process, we further performed cryo-TEM (Figure S6a). In addition to sphere-like structures of 21.4 ± 0.6 nm in diameter, the appearance of short fibers are observed in the self-assemblies in the appearance of short fibers are observed, as they lie orthogonal to the plane of the image. This confirms the consistency of the morphology in both solution and dry state. The detailed structure of the triblock copolymer was further investigated by GIWAXS. Figure 5 shows the out-of-plane and in-plane line cuts. Scattering peak at q = q* = 3.0 nm−1 in plane is due to Bragg reflections of PNOG crystals, consistent with the previously reported results.22,23 The peak corresponds to the

Table 1. Molecular Parameters of Triblock Copolymers sample PEG-b-PBLG64b-PNOG21 PEG-b-PBLG42b-PNOG17 PEG-b-PBLG24b-PNOG17 PEG-b-PBLG24b-PNOG11

feed ratio (BLG/ NOG)a

m/nb

xc

Mnd (kDa)

Mne (kDa)

PDI

60/20

64/21

0.75

19.6

14.9

1.09

40/20

42/17

0.71

14.1

10.5

1.14

20/20

24/17

0.58

10.1

9.6

1.16

20/10

24/11

0.69

9.1

7.4

1.24

a

Feed molar ratio of BLG-NCA/Oct-NNCA. bCalculated from 1H NMR spectra. cMolar fraction of PGA calculated from 1H NMR spectra. dMn of triblock copolymer calculated from 1H NMR spectra; the DP of PBLG was calculated by f/(b1 + b2), and the DP of PNOG was calculated by l/(b1 + b2), as shown in Figure 1. eMn of triblock copolymer, as determined from GPC.

endotherms of PEG-b-PGA24-b-PNOG11 contain one strong peak at 25 °C, which is associated with the melting transition of PEG crystals. The PNOG block is completely devoid of melting peaks possibly due to the short chain length. As the DP of PNOG increases to 17, two small melting peaks are shown. One of the peaks is in the vicinity of 25 °C, which can be attributed to the melting transition of PEG. We associate the higher melting peak at around 124 °C to the melting of the PNOG crystals. Note that the crystallization of both blocks is largely suppressed.22 We then fixed the DP of PNOG at ∼20 and increased the DP of PGA to investigate the influence of the molar ratio of PGA to PNOG on the thermal properties of the system. As the DP of PGA is increased to 64, two melting peaks eventually disappeared, indicating a lack of crystalline pattern. Interestingly, all of the triblock copolymers can readily dissolve in deionized water at a concentration of 2 mg/mL. The solution appears bluish, which is typical of solutions containing aggregates. Note that water is a poor solvent for hydrophobic PNOG block, good solvent for PEG, and a selective solvent for the PGA block as a function of pH. PGA with a pKa of ∼4.3 is known to adopt neutral α-helix at low pH and negative-charged random coil at high pH.35 The triblock copolymers are thus expected to show pH-responsive behavior in aqueous solution considering the presence of PGA block. We first investigated the pH dependence of secondary conformation of the triblock copolymers by CD spectroscopy. A typical plot of CD spectra

Figure 2. Representative GPC chromatograms of (a) PEG-b-PBLG42 and PEG-b-PBLG42-b-PNOG17 and (b) all of the triblock copolymers. D

DOI: 10.1021/acs.biomac.7b01014 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 3. Representative CD spectrum of (a) PEG-b-PGA42-b-PNOG17 at a concentration of 0.2 mg/mL at different pH; (b) α-helicity of all the deprotected triblock copolymers as a function of pH.

Figure 4. (a) TEM and (b) AFM images of PEG-b-PGA42-b-PNOG17 at pH 7.2, (d) TEM and (e) AFM images of PEG-b-PGA42-b-PNOG17 at pH 4.8, and (c, f) the corresponding height profiles.

side chain packing, and the distance between adjacent backbones is given by d = 2π/q* = 2.1 nm. Higher-order peaks at 2q* and 3q* indicate the presence of a lamellar morphology. Note that the spacing is calculated to be twice the length of a fully extended octyl chain, indicating an end-to-end packing of the side chains (Scheme 1). An additional peak at q = q* = 13.7 nm−1 is very broad, indicative of the less ordered packing. This is consistent with the DSC results, confirming the suppressed crystallization of PNOG block. It is generally accepted that the PEG is well soluble in water, as confirmed by the absence of a typical crystalline peak. Note that merely two broad peaks are shown in the out-of-plane line cuts, indicating

the lack of ordered domains. The significant difference in both scattering patterns confirms that the triblock copolymer selfassembles into a 2D nanodisk-like structure with hydrophobic PNOG block as the core and the two hydrophilic segments PGA and PEG as the corona and shell, respectively (Scheme 1). As the solution pH is decreased to 4.8, apart from a few nanodisks, a majority of nanosheet-like structures of hundreds of nanometers in length are observed (Figure 4). Cryo-TEM confirms the nanosheet self-assemblies (Figure S6b). The average thickness of both nanodisks and nanosheets are very similar (∼10.2 ± 0.4 nm), as measured by AFM. With decreasing pH, the PGA block is protonated, and its secondary E

DOI: 10.1021/acs.biomac.7b01014 Biomacromolecules XXXX, XXX, XXX−XXX

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A series of triblock copolymers with fixed chain length of PNOG (DP ≈ 20) were synthesized for a deep understanding of the possible mechanism of the morphology. Table S1 summarizes the characteristics of the self-assemblies. As the DP of PGA increased to 64, the triblock copolymer solution remained a nanodisk-like structure at both pH 4.8 and 7.2 (Figure 6a and Figure S8). The thickness of the nanodisk was reduced by ∼4 nm with increasing pH due to the helix-to-coil transition (Table S1). Note that the triblock copolymer is completely amorphous by DSC results. Therefore, it is conceivable that the dominant driving force of the assemblies is the hydrophobicity of PNOG instead of crystallinity. In the case of polymer with shorter chain length of PGA (DP = 24), nanosheets are obtained with comparable thickness of 9−10 nm at both pHs (Figure 6b, Figure S8, Figure S9). This is possibly due to the low α-helix content of the short PGA at acidic pH indicated by the CD spectra. The triblock copolymer with higher molar ratio of PGA blocks excludes the formation of nanosheets at low pH. This may be attributed to the unfavorable dipole moments of the PGA helices.38 We further investigated the influence of molecular weight on self-assembly of the triblock copolymers by fixing the molar ratio of PGA to PNOG. A triblock copolymer PEG-b-PGA24-b-PNOG11 with similar molar ratio of PGA to PNOG but smaller molecular weight was synthesized. Similar to PEG-b-PGA42-b-PNOG17, PEG-b-PGA24-b-PNOG11 forms a disk-like structure at pH 7.2, whereas nanosheets are obtained as the solution pH is increased to 4.8 (Figure S10). Note that the thickness of the nanosheet is slightly lower than that of the nanodisk-like structure due to the low helix content. We can conclude that the transition of disk-to-sheet is largely dependent on the molar fraction instead of molecular weights.

Figure 5. GIWAXS out-of-plane and in-plane measurements for PEGb-PGA42-b-PNOG17 at pH 7.2.

conformation changes from charged random coil to rigid αhelix, indicated by an increase of ∼4 nm in thickness. The PGA blocks join PNOG as a part of the core, which largely reduces static repulsive interaction of PGA residues. We therefore assume that the extended dimension of the self-assemblies is due to the enhanced hydrophobicity and decreased repulsion of PGA blocks (Scheme 1). The hydrophilic PEG forms as the corona and the hydrophobic PNOG and PGA comprises two parts of the core, respectively (Scheme 1). Further decreasing the solution pH to 2.5 leads to large aggregates (Figure S7) consistent with the reduced CD signals. The nanodisk-tonanosheet transition is completely reversible. As the pH is increased back to 7.2 by adding NaOH solution, the nanodisklike morphology with similar size is shown again. Note that small amounts of sodium salts may be generated during this process, which barely influence the morphology.

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CONCLUSIONS In summary, we have successfully prepared two-dimensional nanodisk and nanosheet-like structures from a triblock copolymer containing both polypeptide and polypeptoid

Scheme 1. Nanodisk-to-Nanosheet Transition of the Triblock Copolymer at Different pH Levels

F

DOI: 10.1021/acs.biomac.7b01014 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 6. TEM images of (a)PEG-b-PGA64-b-PNOG21 with a concentration of 2 mg/mL at pH 4.8 and (b)PEG-b-PGA24-b-PNOG17 with a concentration of 2 mg/mL at pH 7.2. (2) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Micellar nanocontainers distribute to defined cytoplasmic organelles. Science 2003, 300, 615−618. (3) Pochan, D. J.; Chen, Z.; Cui, H.; Hales, K.; Qi, K.; Wooley, K. L. Toroidal Triblock Copolymer Assemblies. Science 2004, 306, 94. (4) Li, Z.; Kesselman, E.; Talmon, Y.; Hillmyer, M. A.; Lodge, T. P. Multicompartment Micelles from ABC Miktoarm Stars in Water. Science 2004, 306, 98−101. (5) Mai, Y.; Eisenberg, A. Self-assembly of block copolymers. Chem. Soc. Rev. 2012, 41, 5969−5985. (6) Jin, H.; Fang, J.; Daily, M. D.; Chen, Y.; Feng, Y.; Ding, Y. H.; Xin, Z.; Robertson, E. J.; Baer, M. D.; Chen, C. L. Highly stable and self-repairing membrane-mimetic 2D nanomaterials assembled from lipid-like peptoids. Nat. Commun. 2016, 7, 12252. (7) Zhang, Y.; Tekobo, S.; Tu, Y.; Zhou, Q.; Jin, X.; Dergunov, S. A.; Pinkhassik, E.; Yan, B. Permission to enter cell by shape: nanodisk vs nanosphere. ACS Appl. Mater. Interfaces 2012, 4, 4099−4105. (8) Shi, Y.; Zhu, W.; Yao, D.; Long, M.; Peng, B.; Zhang, K.; Chen, Y. Disk-Like Micelles with a Highly Ordered Pattern from Molecular Bottlebrushes. ACS Macro Lett. 2014, 3, 70−73. (9) Lin, Z.; Sun, J.; Zhou, Y.; Wang, Y.; Xu, H.; Yang, X.; Su, H.; Cui, H.; Aida, T.; Zhang, W. A Non-crystallization Approach Toward Uniform Thylakoids-like 2D ″Nano-coins″ and Their Grana-like 3D Supra-structures. J. Am. Chem. Soc. 2017, 139, 5883. (10) Shi, Y.; Zhu, W.; Yao, D.; Long, M.; Peng, B.; Zhang, K.; Chen, Y. Disk-Like Micelles with a Highly Ordered Pattern from Molecular Bottlebrushes. ACS Macro Lett. 2014, 3, 70−73. (11) Nam, K. T.; Shelby, S. A.; Choi, P. H.; Marciel, A. B.; Chen, R.; Tan, L.; Chu, T. K.; Mesch, R. A.; Lee, B. C.; Connolly, M. D.; Kisielowski, C.; Zuckermann, R. N. Free-floating ultrathin twodimensional crystals from sequence-specific peptoid polymers. Nat. Mater. 2010, 9, 454. (12) Zhou, Z.; Li, Z.; Ren, Y.; Hillmyer, M. A.; Lodge, T. P. Micellar shape change and internal segregation induced by chemical modification of a tryptych block copolymer surfactant. J. Am. Chem. Soc. 2003, 125, 10182. (13) Ilhan, F.; Galow, T. H.; Gray, M.; Gilles Clavier, A.; Rotello, V. M. Giant Vesicle Formation through Self-Assembly of Complementary Random Copolymers. J. Am. Chem. Soc. 2000, 122, 5895−5896. (14) Velonia, K. Protein-polymer amphiphilic chimeras: recent advances and future challenges. Polym. Chem. 2010, 1, 944−952. (15) Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.; Pochan, D.; Deming, T. J. Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles. Nature 2002, 417, 424. (16) Lu, H.; Wang, J.; Song, Z.; Yin, L.; Zhang, Y.; Tang, H.; Tu, C.; Lin, Y.; Cheng, J. Recent advances in amino acid N-carboxyanhydrides and synthetic polypeptides: chemistry, self-assembly and biological applications. Chem. Commun. 2014, 50, 139−155.

blocks. The triblock copolymer is synthesized by sequential ring-opening polymerization (ROP) using amine-terminated poly(ethyl glycol) (PEG-NH2) as the macroinitiator. The triblock copolymer can directly dissolve in water with hydrophilic PEG block, hydrophobic PNOG block, and pHresponsive PGA block. The influence of pH, molar fraction, and molecular weight on the self-assemblies has been systematically studied. We demonstrate that the nanodisk-to-nanosheet transition is mainly dependent on the pH and molar ratios despite the different molecular weights. The bioinspired 2D nanostructures show great potential for applications in nanoscience and biomedicine.

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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.biomac.7b01014. Detailed thickness of the assemblies at different pH, additional 1H NMR data, DSC results, CD spectra, GPC data, TEM images, and AFM images (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jing Sun: 0000-0003-1267-0215 Zhibo Li: 0000-0001-9512-1507 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51503115, 21434008 and 21674054), and the Taishan Scholars Program. The beamline 7.3.3 at the Advanced Light Source is supported by the Director of the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC0205CH11231.

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DOI: 10.1021/acs.biomac.7b01014 Biomacromolecules XXXX, XXX, XXX−XXX