Two-Dimensional Supramolecular Assemblies from pH-Responsive Poly(ethyl glycol)-b-poly(l -glutamic acid)-b-poly(N-octylglycine) Triblock Copolymer

Article abstract of DOI:10.1021/acs.biomac.7b01014

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 pH-responsive poly(ethyl glycol)-block-poly(l-glutamic acid)-block-poly(N-octylglycine) (PEG-b-PGA-b-PNOG) triblock copolymers by sequential ring-opening 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-b-PGA-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.

Full text of DOI:10.1021/acs.biomac.7b01014

Article
pubs.acs.org/Biomac
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 pH-
responsive poly(ethyl glycol)-block-poly(L-glutamic acid)-block-poly(N-octyl-
glycine) (PEG-b-PGA-b-PNOG) triblock copolymers by sequential ring-
opening 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-b-
PGA-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.
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
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 N-
substituted 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
Here, we intend to incorporate both polypeptoid and
polypeptide blocks into one copolymer chain to form a pH-
responsive triblock copolymer. The aim of such design is that
the triblock copolymer can not only combine the structural
Received: July 18, 2017
Revised: September 1, 2017
© XXXX American Chemical Society
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hexane three times to obtain 5.8 g of solid white BLG-NCA. Yield:
52.3%.
advantages of both blocks but finely tune the properties of the
materials such as degradability. The polypeptoid-block-poly-
peptide 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 copoly-
mers 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 morphol-
ogies depending on the pH and molar fraction. We system-
atically investigated the influence of the pH, molar fraction, and
chain length on the assemblies.
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%.
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.
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.
Synthesis of 2-(n-Octyl amino)acetic Acid Hydrochloride.
Glyoxylic acid (15 g, 163 mmol) was stirred with 0.5 mol equiv n-
octylamine (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) Ace-
tic 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%.
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 cryo-
TEM 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 glow-
discharged 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
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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1
Figure 1. Representative H 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).
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
RESULTS AND DISCUSSION
■
The N-octyl-N-carboxyanhydride (Oct-NNCA) and γ-benzyl-L-
glutamate 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-b-
PBLG-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 character-
istic νCO peaks of the monomer at 1790 and 1850 cm−1 was
considered as complete conversion of NCA/NNCA monomers
1
determined by GPC (Figure 2 and Figure S3), and H 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-b-
PGA-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.
to polypeptides/polypeptoids (Figure S2).27 The H NMR
1
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 PEG-
NH2 macroinitiator. The average DP of PNOG was held fixed
at ∼10 and 20, and the average DP of PBLG was varied from
The thermal properties of PEG-b-PGA-b-PNOG were first
investigated by DSC, as shown in Figure S4. The DSC
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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-b-
PGA42-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,
Table 1. Molecular Parameters of Triblock Copolymers
feed ratio
d
e
(BLG/
Mn
Mn
a
b
c
sample
NOG)
m/n
x
(kDa) (kDa) PDI
PEG-b-PBLG64-
b-PNOG21
60/20
64/21
42/17
24/17
24/11
0.75
0.71
0.58
0.69
19.6
14.1
10.1
9.1
14.9
10.5
9.6
1.09
1.14
1.16
PEG-b-PBLG42-
b-PNOG17
40/20
20/20
20/10
PEG-b-PBLG24-
b-PNOG17
PEG-b-PBLG24-
b-PNOG11
7.4
1.24
a
b
1
Feed molar ratio of BLG-NCA/Oct-NNCA. Calculated from H
c
NMR spectra. Molar fraction of PGA calculated from 1H NMR
spectra. Mn of triblock copolymer calculated from H NMR spectra;
d
1
the DP of PBLG was calculated by f/(b1 + b2), and the DP of PNOG
e
was calculated by l/(b1 + b2), as shown in Figure 1. Mn 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
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
Figure 2. Representative GPC chromatograms of (a) PEG-b-PBLG42 and PEG-b-PBLG42-b-PNOG17 and (b) all of the triblock copolymers.
D
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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 self-
assembles 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
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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 PEG-
b-PGA42-b-PNOG17 at pH 7.2.
conformation changes from charged random coil to rigid α-
helix, 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-to-
nanosheet transition is completely reversible. As the pH is
increased back to 7.2 by adding NaOH solution, the nanodisk-
like 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.
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
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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.
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 pH-
responsive 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.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.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)
(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 two-
dimensional 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.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: jingsun@qust.edu.cn.
*E-mail: zbli@qust.edu.cn.
ORCID
Jing Sun: 0000-0003-1267-0215
Zhibo Li: 0000-0001-9512-1507
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(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.
■
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-AC02-
05CH11231.
(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.
REFERENCES
■
(1) Walker, S. A.; Kennedy, M. T.; Zasadzinski, J. A. Encapsulation of
bilayer vesicles by self-assembly. Nature 1997, 387, 61−64.
G
DOI: 10.1021/acs.biomac.7b01014
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
(17) Sheng, L.; Chen, H.; Fu, W. X.; Li, Z. B. Synthesis and Solution
Self-assembly of p H-responsive ABC Triblock Copolymers. Acta
Polym. Sin. 2015, 982−988.
(18) Guang, N. E.; Liu, S. X.; Li, X.; Tian, L.; Mao, H. G.
Micellization and gelation of the double thermoresponsive ABC-type
triblock copolymer synthesized by RAFT. Chin. J. Polym. Sci. 2016, 34,
965−980.
(37) Lodge, T. P.; Hillmyer, M. A.; Zhou, Z.; Talmon, Y. Access to
the Superstrong Segregation Regime with Nonionic ABC Copolymers.
Macromolecules 2004, 37, 6680−6682.
(38) Kim, K. T.; Park, C.; Vandermeulen, G. W. M.; Rider, D. A.;
Kim, C.; Winnik, M. A.; Manners, I. Gelation of helical polypeptide-
random coil diblock copolymers by a nanoribbon mechanism. Angew.
Chem., Int. Ed. 2005, 44, 7964.
(19) Sun, J.; Zuckermann, R. N. Peptoid polymers: a highly
designable bioinspired material. ACS Nano 2013, 7, 4715−4732.
(20) Zhang, D.; Lahasky, S. H.; Guo, L.; Lee, C. U.; Lavan, M.
Polypeptoid Materials: Current Status and Future Perspectives.
Macromolecules 2012, 45, 5833−5841.
(21) Gangloff, N.; Ulbricht, J.; Lorson, T.; Schlaad, H.; Luxenhofer,
R. Peptoids and Polypeptoids at the Frontier of Supra- and
Macromolecular Engineering. Chem. Rev. 2016, 116, 1753.
(22) Lee, C. U.; Li, A.; Ghale, K.; Zhang, D. Crystallization and
Melting Behaviors of Cyclic and Linear Polypeptoids with Alkyl Side
Chains. Macromolecules 2013, 46, 8213−8223.
(23) Rosales, A. M.; Murnen, H. K.; Zuckermann, R. N.; Segalman,
R. A. Control of Crystallization and Melting Behavior in Sequence
Specific Polypeptoids. Macromolecules 2010, 43, 5627−5636.
(24) Fetsch, C.; Luxenhofer, R. Thermal Properties of Aliphatic
Polypeptoids. Polymers 2013, 5, 112−127.
(25) Knight, A. S.; Zhou, E. Y.; Francis, M. B.; Zuckermann, R. N.
Sequence Programmable Peptoid Polymers for Diverse Materials
Applications. Adv. Mater. 2015, 27, 5665.
(26) Fetsch, C.; Grossmann, A.; Holz, L.; Nawroth, J. F.; Luxenhofer,
R. Polypeptoids from N-Substituted Glycine N-Carboxyanhydrides:
Hydrophilic, Hydrophobic, and Amphiphilic Polymers with Poisson
Distribution. Macromolecules 2011, 44, 6746−6758.
(27) Guo, L.; Zhang, D. Cyclic poly(alpha-peptoid)s and their block
copolymers from N-heterocyclic carbene-mediated ring-opening
polymerizations of N-substituted N-carboxylanhydrides. J. Am. Chem.
Soc. 2009, 131, 18072−18074.
(28) Robinson, J. W.; Schlaad, H. A versatile polypeptoid platform
based on N-allyl glycine. Chem. Commun. 2012, 48, 7835.
(29) Secker, C.; Robinson, J. W.; Schlaad, H. Alkyne-X modification
of polypeptoids. Eur. Polym. J. 2015, 62, 394−399.
(30) Xuan, S.; Gupta, S.; Xin, L.; Bleuel, M.; Schneider, G. J.; Zhang,
D. Synthesis and Characterization of Well-Defined PEGylated
Polypeptoids as Protein-Resistant Polymers. Biomacromolecules 2017,
18, 951−964.
(31) Birke, A.; Huesmann, D.; Kelsch, A.; Weilbacher, M.; Xie, J.;
̈
Bros, M.; Bopp, T.; Becker, C.; Landfester, K.; Barz, M. Polypeptoid-
block-polypeptide Copolymers: Synthesis, Characterization, and
Application of Amphiphilic Block Copolypept(o)ides in Drug
Formulations and Miniemulsion Techniques. Biomacromolecules
2014, 15, 548.
(32) Tao, X.; Deng, C.; Ling, J. PEG-amine-initiated polymerization
of sarcosine N-thiocarboxyanhydrides toward novel double-hydro-
philic PEG-b-polysarcosine diblock copolymers. Macromol. Rapid
Commun. 2014, 35, 875.
(33) Gkikas, M.; Iatrou, H.; Thomaidis, N. S.; Alexandridis, P.;
Hadjichristidis, N. Well-defined homopolypeptides, copolypeptides,
and hybrids of poly(l-proline). Biomacromolecules 2011, 12, 2396−
2406.
(34) Morrow, J. A.; Mc, L. K. S. P.; Knapp, M.; Kh, R. B. W.; Segall,
M. L. Differences in stability among the human apolipoprotein E
isoforms determined by the amino-terminal domain. Biochemistry
2000, 39, 11657−11666.
̌
(35) Sun, J.; Cernoch, P.; Volkel, A.; Wei, Y.; Ruokolainen, J.;
̈
Schlaad, H. Aqueous Self-Assembly of a Protein-Mimetic Ampholytic
Block Copolypeptide. Macromolecules 2016, 49, 5494−5501.
(36) Kukula, H.; Schlaad, H.; Antonietti, M.; Forster, S. The
̈
formation of polymer vesicles or ″peptosomes″ by polybutadiene-
block-poly(L-glutamate)s in dilute aqueous solution. J. Am. Chem. Soc.
2002, 124, 1658.
H
DOI: 10.1021/acs.biomac.7b01014
Biomacromolecules XXXX, XXX, XXX−XXX