Well-Defined Biomimicking Brush-Polymer Self-Assemblies Revealing Cholesterol- and Phosphorylcholine-Enriched Surface

Article abstract of DOI:10.1021/acs.macromol.7b00805

We have newly synthesized a series of well-defined brush polyethers bearing cholesterol (Chol) and phosphorylcholine (PC) moieties in various compositions which can mimic cell membrane. They were thermally stable up to at least 230 °C and soluble in common solvents, showing good solution processability. Excitingly, they all favorably self-assembled, forming multibilayer structures with 2₁ chain conformation; in comparison, the brush polyether bearing only PC-bristles formed orthorhombically packed cylinder (OPC) structure with 12₅ helical chain conformation. Such multibilayer structure formations could be driven by a strong self-assembling ability of the Chol-bristle in extended conformation; the multibilayer structure formation was further promoted by the presence of PC-bristles. The OPC structure formation could be driven by a lateral packing ability of the brush polymer chain in the helical confirmation resulted from the minimization of repulsive interactions in the neighbored zwitterionic PC-bristles. Because of such the self-assembling natures, all brush polymers always revealed Chol- and PC-enriched surface. Overall, all brush polyethers of this study successfully mimicked cell membrane features (Chol- and PC-surface based on self-assembling). They are very suitable for uses in the fields required cell membrane surface characteristics.

Full text of DOI:10.1021/acs.macromol.7b00805

Article
pubs.acs.org/Macromolecules
Well-Defined Biomimicking Brush-Polymer Self-Assemblies
Revealing Cholesterol- and Phosphorylcholine-Enriched Surface
Changsub Kim,† Kyungho Kwon,† Jongchan Lee,† Heesoo Kim,*,‡ Keun-Hwa Chae,§
and Moonhor Ree*,†
†Division of Advanced Materials Science, Department of Chemistry, Pohang Accelerator Laboratory, and Polymer Research Institute,
Pohang University of Science and Technology, Pohang 37673, Republic of Korea
‡Department of Microbiology and Dongguk Medical Institute, Dongguk University College of Medicine, Gyeongju 38066, Republic
of Korea
§Advanced Analysis Centre, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea
S
* Supporting Information
ABSTRACT: We have newly synthesized a series of well-
defined brush polyethers bearing cholesterol (Chol) and
phosphorylcholine (PC) moieties in various compositions
which can mimic cell membrane. They were thermally stable
up to at least 230 °C and soluble in common solvents, showing
good solution processability. Excitingly, they all favorably self-
assembled, forming multibilayer structures with 21 chain
conformation; in comparison, the brush polyether bearing
only PC-bristles formed orthorhombically packed cylinder
(OPC) structure with 125 helical chain conformation. Such
multibilayer structure formations could be driven by a strong
self-assembling ability of the Chol-bristle in extended
conformation; the multibilayer structure formation was further
promoted by the presence of PC-bristles. The OPC structure formation could be driven by a lateral packing ability of the brush
polymer chain in the helical confirmation resulted from the minimization of repulsive interactions in the neighbored zwitterionic
PC-bristles. Because of such the self-assembling natures, all brush polymers always revealed Chol- and PC-enriched surface.
Overall, all brush polyethers of this study successfully mimicked cell membrane features (Chol- and PC-surface based on self-
assembling). They are very suitable for uses in the fields required cell membrane surface characteristics.
biosynthesis of other biomolecules. Furthermore, they can
increase activities of enzymes.1,2 Cholesterol is also necessary to
build and maintain the integrity and mechanical stability of cell
membranes.1−4 It is present in a wide range over a few to 50%,
depending on the cells. Cholesterol is known to regulate
membrane permeability and fluidity over a wide range of
temperatures and anchor molecules, like proteins, in the
membrane.1−4
In this study, we have aimed to mimic cell membrane in the
molecular design and synthesis of new functional high
performance polymers. In particular, we have tried to mimic
three unique features, namely (i) phospholipid, (ii) cholesterol,
and (iii) self-assembling ability, of cell membrane into the
development of a novel nanostructured polymer system which
can always provide cholesterol (Chol) and phosphorylcholine
(PC) enriched surface. A series of poly(oxy(4-(13-cholenoate-
nonyl)-1,2,3-trizoyl-1-methyl)ethylene-ran-oxy(4-(13-
INTRODUCTION
■
The cell membrane, which is known as plasma membrane or
cytoplasmic membrane, is a layer around the cells of all living
things.1,2 Its first important role is to separate the inside of cells
from the outside environment, protecting the cells. Its second
role is to selectively permit ions and organic molecules and
control the movement of substances in and out of cells. The
cell membrane is further involved in a variety of cellular
processes such as cell adhesion, ion conductivity, and cell
signaling and serves as the attachment surface for several
extracellular structures.1,2
In general, the cell membrane is known to consist of lipids
(phospholipid and glycolipid), cholesterols, proteins, and
carbohydrates (predominantly glycoproteins).1,2 In particular,
lipids are the major component to build cell membrane as a
bilayer. The lipid bilayer primarily serves as a containment unit
of living cells. In most cell membranes, lipids are present in
approximately 50% of the total mass. The lipid bilayer also
forms a matrix in which membrane proteins reside. In addition
to such the structural roles, lipids function as regulatory agents
in cell growth and adhesion and further participate in the
Received: April 23, 2017
Revised: August 15, 2017
Published: August 28, 2017
© 2017 American Chemical Society
6489
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
Scheme 1. Synthetic Route of PGA-CholmPCn Polymers (m = 0−100 mol % Chol-Bristle; n = 100−0 mol % PC-Bristle)
phosphorylcholinenonyl)-1,2,3-trizoyl-1-methyl)ethylene)s
(PGA-CholmPCn: m = 0−100 mol % Chol-containing bristle; n
= 100−0 mol % PC-containing bristle) in various compositions
were designed and successfully synthesized as a self-assembling
brush polymer system bearing Chol- and PC-moieties (Scheme
1). The obtained polymers were stable up to 230 °C or higher
temperatures depending on the compositions. They were
soluble in various organic solvents including chloroform
(CHCl3) and methanol (MeOH). They were investigated in
detail by synchrotron grazing incidence X-ray scattering
(GIXS) analysis in the aspect of thin film morphology. They
all nicely demonstrated self-assembling behaviors, forming
multibilayer structure with extended chain conformation or
orthorhombic cylinder structure with helical chain conforma-
tion depending on the compositions. Based on such self-
assembled structures, they always exhibited Chol- and PC-
enriched surface. Overall, these polymers successfully mimicked
cell membrane features. Therefore, they are potential candidate
materials for applications in the fields required cell membrane
surface characteristics.
EXPERIMENTAL SECTION
■
Synthesis. All materials were purchased from Sigma-Aldrich (St.
Louis, MO) and Tokyo Chemical Industry (TCI, Tokyo, Japan) and
used as received unless otherwise noted. 1-Cholenylundec-10-yne (R1,
Scheme 1) as ethyne-functionalized Chol-bristle was synthesized in a
two-step reaction by using the synthetic information in the
literature.5−7 In the first step, 10-undecyn-1-ol (1 g, 5.94 mmol) and
carbon tetrabromide (CBr4, 2.17 g, 6.54 mmol) were dissolved in
anhydrous dichloromethane (CH2Cl2, 5 mL) and then followed by
stirring with ice bath for 30 min. To the mixture under stirring,
triphenylphosphine (PPh3, 1.72 g, 6.54 mmol) in CH2Cl2 (2 mL) was
slowly added for 1 h. Then, the mixture was continuously stirred at
room temperature for an additional 1 h. The reaction mixture was
concentrated and then poured into cyclohexane. After filtration, the
solution was concentrated and further purified by flash column
chromatography with a mixture of cyclohexane and ethyl acetate (50:1
in volume). The obtained product in colorless liquid was identified in
deuterated chloroform (CDCl3) using Bruker nuclear magnetic
resonance (NMR) spectrometers (models AV300 and AVANCE-III
600, Rheinstetten, BW, Germany) with proton (1H) and carbon (13C)
probes. The target product, 1-bromo-10-undecyne, was obtained in
70.3% yield (0.965 g). 1H NMR (300 MHz, CDCl3, δ (ppm)): 3.42 (t,
2H, −CH2Br), 2.20 (m, 2H, −CH2CCH), 1.96 (t, 1H, −CH2C
CH), 1.87 (m, 2H, −CH2CH2Br), 1.60−1.25 (br, 12H, −(CH2)6−)
6490
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
(Figure S1a in Supporting Information). 13C NMR (75 MHz, CDCl3,
δ (ppm)): 84.8, 68.2, 34.1, 32.9, 29.4, 29.1, 28.8, 28.7, 28.6, 28.2, 18.5.
In the second step, 3β-hydroxy-Δ5-cholenic acid (1 g, 2.67 mmol)
was dissolved in anhydrous dimethylformamide (DMF, 4 mL).
Cesium carbonate (Cs2CO3, 1.30 g, 4.00 mmol) was added to the
solution, and then the mixture was stirred at room temperature for 1 h.
To the mixture, 11-bromoundec-1-yne (1.40 g, 5.34 mmol) was added
and followed by stirring at room temperature. After 20 h, the reaction
mixture was quenched by adding CHCl3 and 0.1 M HCl solution and
washed with 0.1 M HCl solution and deionized, distilled water. The
combined organic layers were dried over anhydrous magnesium sulfate
(MgSO4) and concentrated in a vacuum. The concentrated residue
was purified by silica gel column chromatography with a mixture of
ethyl acetate and hexane (7:3 in volume), giving the target product 1-
cholenyl-10-undecyne (R1) in white solid. Yield: 75%. R1 was
determined to reveal two melting transitions at 13.3 and 50.7 °C in
differential scanning calorimetry (DSC) analysis with 10 °C/min
ramping rate. Taking into consideration the chemical structure, the
first melting point at 13.3 °C could originate from the transition of
crystalline state to liquid crystalline state (Tc−lc), where the second
melting point at 50.7 °C could be assigned for the transition of liquid
crystalline state to fully disordered state (Tlc−do). 1H NMR (300 MHz,
CDCl3, δ (ppm)): 5.36 (m, 1H, −CCH−), 4.04 (t, 2H,
−COOCH2−), 3.53 (m, 1H, −CHOH), 2.44−2.10 (m, 6H,
−CH2CCH, cholesteric acid protons), 2.05−1.91 (m, 7H,
−CH2CCH, cholesteric acid protons, bristle linker protons),
1.68−0.79 (m, 35H, cholesteric acid protons, bristle linker protons),
slowly added to epichlorohydrin. The mixture was stirred at room
temperature for 2 days. The crude polymer in the reaction solution
was purified by precipitating into MeOH several times, followed by
drying in a vacuum at 40 °C for 12 h. The obtained PECH was
colorless viscous liquid. Yield: 65.4% (30.921 g). 1H NMR (300 MHz,
CDCl3, δ (ppm)): 3.89−3.49 (m, 5H, −CH2CHO−, −CH2CHO−,
−CH2Cl) (Figure S3a). 13C NMR (75 MHz, DMSO-d6, δ (ppm)):
78.8, 69.6, 44.1 (Figure S3b).
In the second step, PECH was converted to poly(glycidyl azide)
(PGA) as follows. A mixture of PECH (1 g, 10.8 mmol) and sodium
azide (2.10 g, 32.4 mmol) in DMF (40 mL) was stirred at 90 °C for 24
h. After filtration, the used DMF solvent was removed by evaporation.
The residue was dissolved in CHCl3 and washed three times with
saturated NaCl aqueous solution. The organic layer was dried over
anhydrous magnesium sulfate (MgSO4) and concentrated in a vacuum,
1
giving the product in liquid. Yield: 90.1% (0.964 g). H NMR (300
MHz, CDCl3, δ (ppm)): 3.78−3.63 (m, 3H, −CH2CHO−,
−CH2CHO−), 3.50−3.32 (m, 2H, −CH2N3) (Figure S4a). 13C
NMR (75 MHz, CDCl3, δ (ppm)): 78.8, 69.6, 51.8 (Figure S4b).
PGA-CholmPCn polymers were prepared from the azide−alkyne
click reactions of PGA with R1 and R2 in various mole fractions. For
typical synthetic examples, the preparations of PGA-Chol50PC50
polymer, PGA-Chol100, and PGA-PC100 were described as follows.
PGA (100 mg, 1.00 mmol) and R1 (394 mg, 0.75 mmol) in CHCl3
were added to R2 (250 mg, 0.75 mmol) in a mixture of MeOH and
deionized distilled water; here the mixture of CHCl3, MeOH, and
water was adjusted to 4:2:1 in volume. To the solution, copper(II)
sulfate pentahydrate (13 mg, 5 mol %) and sodium ascorbate (30 mg,
15 mol %) were added and then stirred at room temperature for 48 h.
The resulting product was purified by dialysis in a mixture of CHCl3
and MeOH for 3 day and then precipitated several times into cold
diethyl ether, followed by drying in vacuum at room temperature for 1
day. The target product PGA-Chol50PC50 was obtained in white solid.
Yield: 68.2% (361 mg). 1H NMR (300 MHz, CD3OD/CDCl3, δ
(ppm)): 8.00−7.40 (br, 2H, −CCH− in triazole), 5.36 (m, 1H,
−CCH−), 4.50−3.10 (br, 10H, −CH2CHO−, −CH2CHO−,
−CH2−triazole in backbone), 4.26 (m, 2H, −POCH2CH2N−), 4.04
(m, 2H, −COOCH2−), 3.85 (m, 2H, −CH2OP−), 3.67 (m, 2H,
−POCH2CH2N−), 3.46 (m, 1H, −CHOH), 3.25 (s, 9H, −N(CH3)3),
2.65 (m, 4H, −CH2−triazole in bristle linker), 2.42−2.13 (m, 4H,
cholesteric acid protons, bristle linker protons), 2.10−0.85 (m, 55H,
cholesteric acid protons, bristle linker protons), 0.69 (s, 3H, −CH3)
(Figure S5a). 13C NMR (150 MHz, CD3OD/CDCl3, δ (ppm)):
174.81, 148.31, 140.90, 122.82, 121.32, 77.95, 71.13, 68.64, 66.35,
65.79, 64.46, 63.36, 58.86, 56.69, 55.73, 54.07, 50.77, 50.08, 42.31,
41.82, 39.70, 37.23, 36.44, 35.30, 31.84, 31.80, 31.24, 31.11, 30.99,
30.77, 29.50, 29.36, 29.25, 28.58, 28.03, 25.89, 25.77, 25.56, 24.17,
20.99, 19.23, 18.18, 11.74 (Figure S5b).
1
0.67 (s, 3H, −CH3) (Figure S1b). H NMR (300 MHz, dimethyl
sulfoxide-d6 (DMSO-d6), δ (ppm)): 5.20 (m, 1H, −CCH−), 4.55
(d, 1H, −CHOH), 3.92 (t, 2H, −COOCH2−), 3.45−2.96 (m, 1H,
−CHOH), 2.66 (t, 1H, −CH2CCH), 2.34−0.77 (m, 47H,
cholesteric acid protons, bristle linker protons), 0.67 (s, 3H, −CH3)
(Figure S1b). 13C NMR (75 MHz, CDCl3, δ (ppm)): 174.44, 140.76,
121.65, 84.74, 71.76, 68.09, 64.41, 56.72, 55.78, 50.07, 42.36, 42.28,
39.73, 37.24, 36.48, 35.34, 31.87, 31.64, 31.32, 31.04, 29.35, 29.18,
29.01, 28.71, 28.64, 28.45, 28.10, 25.92, 24.25, 21.06, 19.39, 18.39,
18.30, 11.86 (Figure S1c).
10-Undecynylphosphorylcholine (R2, Scheme 1) as ethyne-
functionalized PC-bristle was synthesized in a two-step reaction
manner according to the methods in the literature.8−12 10-Undecyn-1-
ol (1 g, 5.94 mmol) and triethylamine (0.99 mL, 6.54 mmol) were
dissolved in anhydrous tetrahydrofuran (THF, 10 mL) under a dry
nitrogen atmosphere and stirred at 0 °C for 30 min. To the solution, 2-
chloro-2-oxo-1,3,2-dioxaphospholane (0.60 mL, 6.54 mmol) in THF
(2 mL) was added slowly for 1 h, followed by stirring at room
temperature for 4 h. The reaction mixture was filtered to remove the
precipitated triethylamine hydrochloride and then followed by removal
of the used THF solvent at 35−40 °C using an evaporator under
reduced pressure. The filtrate was put into a Smith Process vial and
dissolved together with trimethylamine (1.76 g, 30.0 mmol) in
anhydrous acetonitrile (20 mL). The solution was heated to 60 °C and
stirred for 20 h. Then, the reaction mixture was cooled at 2 °C
overnight, precipitating the target product. The precipitated product
was filtered and then washed three times with acetone. The white solid
product was dried and collected. Yield: 65.3% (1.293 g). R2 was
determined to exhibit a melting point at 112.0 °C (= Tm) in DSC
PGA-Chol100 was prepared from PGA (100 mg, 1.00 mmol) and R1
(787 mg, 1.5 mmol) in a mixture of DMSO and CHCl3 (3:1 in
volume). To the PGA and R1 mixture, copper(I) bromide (7.2 mg, 5
mol %) was added and then degassed by three freeze−pump−thaw
cycles. The solution was stirred at 55 °C for 24 h, followed by
distillation under vacuum. The residue was dissolved in CHCl3 and
then passed through aluminum oxide (activated) to remove copper
catalyst. The resulting product was purified by precipitating several
times into cold diethyl ether. The product was white solid. Yield:
1
analysis with 10 °C/min ramping rate. H NMR (300 MHz, CD3OD,
δ (ppm)): 4.25 (br, 2H, −POCH2CH2N−), 3.88 (m, 2H,
−CH2OP−), 3.62 (m, 2H, −POCH2CH2N−), 3.21 (s, 9H, −N-
(CH3)3), 2.18 (m, 3H, −CH2CCH, −CH2CCH), 1.72−1.58 (br,
2H, −CH2CH2OP−), 1.58−1.20 (br, 12H, −(CH2)6−) (Figure S2a).
13C NMR (75 MHz, CD3OD, δ (ppm)): 85.10, 69.54, 67.05, 66.97,
60.40, 54.67, 31.86, 30.57, 30.36, 30.12, 29.73, 29.67, 26.85, 18.99
(Figure S2b).
1
70.5% (440 mg). H NMR (300 MHz, CDCl3, δ (ppm)): 8.00−7.40
(br, 1H, -CCH- in triazole), 5.36 (m, 1H, -CCH-), 4.50−3.10
(br, 5H, −CH2CHO-, −CH2CHO-, −CH2-triazole in backbone), 4.04
(m, 2H, −COOCH2-), 3.53 (m, 1H, −CHOH), 2.65 (m, 2H, −CH2-
triazole in bristle), 2.42−2.13 (m, 4H, cholesteric acid protons, bristle
linker protons), 2.10−0.86 (m, 41H, cholesteric acid protons, bristle
linker protons), 0.71 (s, 3H, −CH3) (Figure S6a). 13C NMR (150
MHz, CDCl3, δ (ppm)): 173.96, 148.22, 141.01, 122.14, 121,34, 77.95,
71.61, 68.78, 64.21, 56.85, 56.02, 50.77, 50.32, 42.46, 42.43, 39.88,
37.39, 36.56, 35.28, 32.03, 31.89, 31.76, 31.37, 31.12, 29.47, 29.38,
29.31, 29.24, 27.99, 25.94, 25.71, 24.23, 21.13, 19.31, 18.35, 11.86
(Figure S6b).
Poly(glycidyl azide) (PGA) was synthesized in a two-step reaction
according to the method in the literature.13−15 In the first step,
poly(epichlorohydrin) (PECH) was prepared from epichlorohydrin as
follows. Epichlorohydrin (40 mL, 512 mmol) was cooled to −5 °C
under a nitrogen atmosphere. Triphenylcarbenium hexafluorophos-
phate (TCHP) (0.1 g, 0.256 mmol) was dissolved in CH2Cl2 and
6491
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
PGA-PC100 was prepared from PGA (100 mg, 1.00 mmol) in
CHCl3 and R2 (500 mg, 1.50 mmol) in a mixture of MeOH and
deionized water; the mixture of CHCl3, MeOH, and water was
adjusted to 2:2:1 in volume. To the PGA and R2 mixture, copper(II)
sulfate pentahydrate (12 mg, 5 mol %) and sodium ascorbate (30 mg,
15 mol %) were added and then stirred at room temperature for 48 h.
The resulting product was purified by dialysis in a mixture of CHCl3
and methanol for 3 days and then concentrated under vacuum. The
product was obtained in green solid. Yield: 68.4% (296 mg). 1H NMR
(300 MHz, CD3OD/CDCl3, δ (ppm)): 8.00−7.40 (br, 1H, −C
CH− in triazole), 4.50−3.10 (br, 5H, −CH2CHO−, −CH2CHO−,
−CH2−triazole in backbone), 4.26 (m, 2H, −POCH2CH2N−), 3.85
(m, 2H, −CH2OP−), 3.67 (m, 2H, −POCH2CH2N−), 3.25 (s, 9H,
−N(CH3)3), 2.65 (m, 2H, −CH2−triazole in bristle linker), 1.90−0.85
(m, 14H, −(CH2)7−) (Figure S7a). 13C NMR (150 MHz, CD3OD/
CDCl3, δ (ppm)): 148.09, 123.53, 77.79, 76.64, 70.21, 68.70, 66.06,
65.49, 58.97, 53.34, 50.91, 30.62, 29.43, 29.23, 25.65, 25.29 (Figure
S7b).
Thin Film Preparation. The individual polymers were dissolved in
either CHCl3, MeOH or CHCl3/MeOH mixtures and filtered using
disposable syringes equipped with polytetrafluoroethylene filter
membranes of 0.22 μm pore size, producing polymer solutions with
a concentration of 0.1−1.0 wt %. Each polymer solution was deposited
on precleaned silicon (Si) substrates via spin-coating process and
annealed with using various solvents, such as MeOH, THF, and
CHCl3, at room temperature for 0.5−4 h, then followed by drying in a
vacuum at room temperature for 24 h. The obtained polymer films
were determined to have a thickness of 60−80 nm by using a
spectroscopic ellipsometer (Model M-2000, Woollam, Lincoln, NE).
An average surface roughness (i.e., root-mean square roughness) was
additionally measured by ellipsometry.
Measurements. Molecular weight and distribution were deter-
mined by using a gel permeation chromatography (GPC) system
(model PL-GPC 210, Polymer Laboratories, Amherst, MA) equipped
with two PL-Gel Mixed-C columns calibrated with polystyrene
standards; in the measurements, CHCl3 was used as an eluent.
Thermal stability and phase transitions were measured with a rate of
10.0 °C/min under a nitrogen atmosphere by thermogravimetry
(TGA; model TG/DGA-6300, Seiko Instrument, Tokyo, Japan) and
differential scanning calorimetry (DSC) (model DSC-220CU, Seiko
Instrument, Tokyo, Japan).
Synchrotron GIXS measurements were performed at 3C and 9A
beamlines of the Pohang Accelerator Laboratory (PAL), Pohang
University of Science & Technology (POSTECH)), Pohang,
Korea.16−18 Scattering data were normally collected for 10−30 s
using X-ray radiation sources with a wavelength λ of 0.1117 and
0.1290 nm and a two-dimensional (2D) charge-coupled detector
(CCD) (model Rayonix 2D MAR, Evanston, IL). The sample-to-
detector distance (SDD) was 234.9 mm for grazing incidence wide-
angle X-ray scattering (GIWAXS) measurements and 2929.6 mm for
grazing incidence small-angle X-ray scattering (GISAXS) measure-
ments. The incidence angle αi of X-ray beam with respect to the
sample specimen surface was set in the range 0.130°−0.160°, which is
between the critical angle of the polymer film and the silicon substrate
(αc,f and αc,s). Scattering angles were corrected according to the
positions of the X-ray beams reflected from the silicon substrate as well
as by using precalibrated sucrose standard (TCI, Tokyo, Japan).
Aluminum foils were used as a semitransparent beamstop because the
intensity of the specular reflection from the substrate is much stronger
than the intensity of GIXS near the critical angle.
Water contact angles were measured at room temperature using a
contact angle meter (KSV Instruments, Tokyo, Japan). At least five
sessile drops of water were measured for each polymer film to get
reproducibility.
RESULTS AND DISCUSSION
■
In this study, we have aimed to develop a new, novel polymer
system mimicking three key features (i.e., phospholipid,
cholesterol, and self-assembling ability) of cell membranes.
To meet this mission, we have considered poly(ethylene glycol)
(PEG) as a base polymer backbone because it possesses
excellent biocompatibility and thus is widely used in the
biomedical fields. In general, linear PEG possesses only two
hydroxyl groups at the chain ends. Because of such lack of
functionality, PEG itself could not be useful for our study.
Thus, we have chosen PECH, which is a functionalized PEG
analogue, as a base polymer for our mission. To mimic the
biofunctionality and self-assembling nature of phospholipid, we
have considered the chemical combination of phosphorylcho-
line and functionalized alkane. And we have tried to add a self-
assembling ability to the biofunctionality of cholesterol via the
chemical combination of cholesterol and functionalized alkane.
Finally, we have made much effort to find a best way to
incorporate such phospholipid and cholesterol mimics into
PECH. To achieve such chemical incorporations, there are two
major possible options: (i) as a bottom-up approach, the
modifications of epichlorohydrin to include phospholipid and
cholesterol mimics and their copolymerization; (ii) as a top-
down approach, the preparation of PECH and its post-
modifications to incorporate phospholipid and cholesterol
mimics. The bottom-up approach was faced to serious
problems in the monomer modifications and their copoly-
merization due to the reactive hydroxyl group of cholesterol
unit as well as the zwitterionic nature of phosphorylcholine
unit. Indeed, we have decided to take the top-down approach in
this study. For the top-down approach there are basically two
options possible. The first option is to incorporate
phospholipid and cholesterol mimics into PECH via the
coupling reactions using the chloro groups of the polymer. The
second option is to modify the chloro groups of PECH to other
proper functional groups and use them to incorporate
phospholipid and cholesterol mimics into the polymer. For
the first option, we have realized some serious problems due to
the presences of hydroxyl group in the cholesterol unit and
relatively less stable phosphate linkage in the phosphorylcholine
unit as well as the quite different solubility nature between
cholesterol and phosphorylcholine units. In comparison, the
second option has more flexibility in finding proper chemical
reactions and their conditions. As a result of these efforts, we
have developed an ethyne-functionalized alkane containing
cholesterol, an ethylene-functionalized alkane bearing phos-
phorylcholine, and a fully azide-functionalized PECH, namely
PGA. From these materials, we have finally succeeded to
synthesize a well-defined brush random copolymer mimicking
the three key features of cell membranes.
X-ray photoelectron spectroscopy (XPS) measurements were
carried out on an X-ray photoelectron spectrometer (model PHI
5000 Versa Probe, Ulvac-PHI, Kanagawa, Japan) with monochrom-
atized Al Kα radiation (1486.6 eV). The pressure inside the analyzer
was maintained at 2.0 × 10−7 Pa. A spot size of 100 μm × 100 μm was
examined; the detection limit was 0.5 at.%. A wide scan pass energy of
117.4 eV and a narrow scan pass energy of 46.95 eV were used. The
electron takeoff angle was 45° with respect to the film surface. All
binding energies were referenced to the C1s peak (284.6 eV) of
adventitious carbon component.
1-Cholenylundec-10-yne (R1, ethyne-functionalized Chol-
bristle) as a cholesterol mimic was synthesized from 10-
undecyn-1-ol in a two-step reaction manner, as shown in
Scheme 1. 10-Undecyn-1-ol was converted to 11-bromoundec-
1-yne in a reasonably high yield via the reaction with CBr4 with
the aid of PPh3 catalyst. 11-Bromoundec-1-yne was further
reacted with 3β-hydroxy-Δ5-cholenic acid in the aid of Cs2CO3,
producing R1 in a good yield (Figure S1).
6492
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
Table 1. Characteristics of the Synthesized Brush Polymers
nanoscale thin film
c
h
composition
thermal properties
composition at film surface
surface
roughness
(nm)
d
e
f
g
chol-bristle
PC-bristle
(mol %)
Td
Tg
Tm
Chol-bristle
(mol %)
PC-bristle
water contact
a
b
i
polymer
PECH
Mw
38500
PDI
1.68
(mol %)
(°C)
(°C)
(°C)
(mol %)
angle (deg)
300
195
280
270
−33
−38
82
PGA
k
PGA-Chol100
≥99
76
0
121
171
0.43
0.25
100.0
78.8
0.0
96.8 (0.5)
PGA-
Chol75PC25
24
95
21.2
77.8 (0.7)
61.2 (0.8)
59.7 (0.7)
PGA-
Chol50PC50
48
24
0
52
76
240
230
230
85
165
1.01
0.97
0.41
59.0
31.2
0.0
41.0
68.8
j
PGA-
Chol25PC75
−16 to 92
j
PGA-PC100
a
≥99
−7 to 125
100.0
c
soluble
b
1
Weight-average molecular weight determined by GPC analysis. Polydispersity index determined by GPC analysis. Determined by H NMR
spectroscopy analysis. Onset degradation temperature measured by TGA analysis. Glass transition temperature (middle point) measured by DSC
analysis. Melting transition temperature (at the maximum of endothermic peak) measured by DSC analysis. Root-mean-square roughness
determined by spectroscopic ellipsometry. Calculated from the atomic concentration ratio of P and C based on the P2p and C1s peaks measured for
d
e
f
g
h
i
j
k
the film surface by XPS. Measured at room temperature. Transition range. Standard deviation.
10-Undecynylphosphorylcholine (R2, ethyne-functionalized
PC-bristle) as a phospholipid mimic was synthesized, as shown
in Scheme 1. 10-Undecyn-1-ol was reacted with 2-chloro-2-oxo-
1,3,2-dioxaphospholane in the first step and then treated with
trimethylamine in the second step, producing R2 in a
reasonably good yield. After the reaction was completed, the
unreacted 10-undecyn-1-ol could not be removed easily from
the target product R2 through conventional distillation under
reduced pressure because of its high boiling point. Acetonitrile
was found to be a good solvent to dissolve either both the
unreacted 10-undecyn-1-ol and the target product or only one
of them depending on the temperature. In this solvent, R2 was
soluble at 60 °C but insoluble at 2 °C, whereas 10-undecyn-1-ol
was completely soluble over the temperature range 0−60 °C.
Using the temperature-dependent solubility differences, R2 was
successfully purified (Figure S2).
With R1 and R2 a series of the PGA-CholmPCn polymers in
various compositions were synthesized in a three-step manner,
as shown in Scheme 1, as a novel brush random copolymer
system mimicking three key features (phospholipid, cholesterol,
and self-assembling ability) of cell membranes. In the first step,
epichlorohydrin was undergone cationic ring-opening polymer-
ization with the aid of TCHP initiator, producing PECH, a base
polymer. PECH was identified by 1H and 13C NMR
spectroscopy analysis (Figure S3). This base polymer was
characterized to have a weight-average molecular weight ( Mw)
of 38 500 and a polydispersity index (PDI) of 1.68 by GPC
analysis (Figure S8).
reaction we have been faced to a serious problem. The
problem was caused from the significantly different chemical
natures in solvent of R1 having an amphiphilicity, R2
possessing both an amphiphilicity and a zwitterionic character-
istic, and PGA with a weak amphiphilicity. As a result of testing
a number of solvent systems, a mixture of CHCl3, MeOH, and
deionized distilled water (4:2:1 in volume) was found as a best
solvent system for all components in the click reaction. But
there were required special cares for making such cosolvent
system as follows. At the first, R1 and PGA were dissolved in
CHCl3 while R2 was dissolved in a small amount of MeOH.
Then the R2 solution was added into the solution of R1 and
PGA under stirring. When the addition was completed, the
mixture appeared turbid. Thus, more MeOH was added to the
mixture until the turbidity disappeared, followed by adding a
proper amount of water. Because of the cosolvent containing
water, we additionally chose copper(II) sulfate pentahydrate
combined with sodium ascorbate as a catalyst system, rather
than copper(I) bromide being widely used in organic solvents.
After the click reaction was completed, the target brush
polymer products were purified through the two steps as
follows. R2 in excess and the used catalyst components were
removed out from the reaction mixture by dialysis in water for
3 days. The residues were again dissolved in a mixture of
chloroform and methanol and poured into diethyl ether in an
ice water bath, precipitating the brush polymer product. The
precipitate was filtered and again dissolved in a mixture of
chloroform and methanol. The solution was poured into cold
diethyl ether, precipitating the polymer product. The
precipitate was filtered and dried, giving the target brush
polymer. In similar manner, PGA-PC100 was synthesized from
PGA and R2. In comparison, PGA-Chol100 was prepared in a
mixture of DMSO and CHCl3 (3:1 in volume) from PGA and
R1 with the aid of copper(I) bromide rather than copper(II)
sulfate pentahydrate/sodium ascorbate.
In the second step, PECH was converted to PGA via the
1
reaction of the chloro side groups with sodium azide. The H
and 13C NMR and FTIR spectroscopy analyses confirmed that
all chloro groups of PECH were completely replaced by azido
groups (Figures S3, S4, and S9). In particular, the IR
spectroscopy analysis confirmed that the vibrational character-
istic band at 750 cm−1 of the −CH2−Cl groups of PECH
completely disappeared while the incorporated azido groups
newly appeared at 2100 cm−1 as a very strong vibrational band
(Figure S9a,b).
All brush polymer products were identified by NMR and IR
spectroscopy analyses (Figures S5, S6, S7, and S9). In
particular, the vibrational peak of the azido groups at 2100
cm−1 disappeared completely, but the chemical shift of the
azide−alkyne click reaction product triazolyl linkages (−C
CH−) newly appeared at 8.00−7.40 ppm. These results
In the last step, PGA was undergone azide−alkyne click
reaction with R1 and R2 in various compositions, producing
the target PGA-CholmPCn polymers. For conducting this
6493
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
confirmed that all azido groups of PGA were completely
undergone click reactions with R1 and/or R2. For each brush
polymer product, the chemical composition, namely the molar
fraction of Chol- and PC-bristles, was determined by analyzing
the integrals of the chemical shifts at 8.00−7.40 ppm (−C
CH− in triazole), 5.36 ppm (−CCH− in Chol-bristle), 3.25
ppm (−POCH2CH2N− in PC-bristle), and 2.65 ppm (−CH2−
1
triazole in bristle) in the H NMR spectrum. The obtained
chemical compositions are summarized in Table 1.
PGA-Chol100 was thermally stable up to 280 °C (= Td, onset
degradation temperature). PGA-PC100 revealed Td = 230 °C.
The relatively lower thermal stability of PGA-PC100 may be
attributed to the presence of phosphate linkage in the bristle.
The PGA-CholmPCn polymers exhibited Td over the range
230−270 °C; the copolymer bearing higher Chol-bristle
content showed higher Td (Figure 1a and Table 1). All brush
Figure 2. 2D GIWAXS patterns of PGA-CholmPCn polymers in thin
films which were measured with SDD = 234.9 mm using an X-ray
beam with λ = 0.1117 nm: (a) PGA-Chol100 (αi = 0.141°); (b) PGA-
Chol75PC25 (αi = 0.150°); (c) PGA-Chol50PC50 (αi = 0.150°); (d)
PGA-Chol25PC75 (αi = 0.150°); (e) PGA-PC100 (αi = 0.156°). 2D
scattering patterns reconstructed with the determined structural
parameters (Table 2) using the GIXS formulas derived for lamellar
and orthorhombic cylinder structure models (Supporting Informa-
tion): (f) PGA-Chol100; (g) PGA-Chol75PC25; (h) PGA-Chol50PC50;
(i) PGA-Chol25PC75; (j) PGA-PC100
.
shows representatives of the measured 2D GIWAXS patterns of
the polymer films, which were annealed with MeOH at room
temperature for 4 h, then followed by drying in a vacuum at
room temperature for 24 h. Their one-dimensional (1D)
profiles are shown in Figure 3, which were extracted along the
αf direction at 2θf = 0° and along the 2θf direction at αf =
0.160°; here αf and 2θf are the out-of-plane and in-plane exit
angle of the X-ray scattering beam, respectively.
Figure 1. (a) TGA and (b) DSC thermograms of PGA-CholmPCn
polymers, which were measured at a rate of 10.0 °C/min under a
nitrogen atmosphere.
random copolymers revealed excellent solubility in common
organic solvents (CHCl3, MeOH, DMF, DMSO, etc.), giving
good quality films (0.25−1.01 nm surface roughness) in
conventional solution casting processes (Table 1). In particular,
PGA-PC100 was soluble in water.
Nanoscale thin films (60−80 nm thick) of the PGA-
CholmPCn polymers were investigated by synchrotron GIXS
analysis. The as-cast films were confirmed to reveal poorly
developed structures. Thus, the as-cast films were undergone to
anneal with the aids of various solvents (MeOH, THF, and
CHCl3) to achieve more ordered morphological structures. As
shown in Figure S10, the brush polymers revealed more
featured GIXS images in the films annealed with MeOH rather
than the other solvents. These results inform that the brush
polymers tend to form higher ordered and oriented
morphology structures in the MeOH-annealed films rather
than in the other solvent-annealed films. With these results, the
MeOH-annealing process was further optimized. Figure 2
Figure 3. (a) Out-of-plane and (b) in-plane scattering profiles
extracted along the αf direction at 2θf = 0° and 2θf direction at αf =
0.160°, respectively, from the 2D scattering patterns in Figure 2a−e.
The open circles are the measured data points, and the red solid lines
were obtained by fitting the data with the GIXS formulas derived for
lamellar and orthorhombic cylinder structure models (Supporting
Information).
6494
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
Table 2. Structural Parameters of PGA-CholmPCn Polymers in Thin Films Determined by Quantitative GIXS Analysis
PGA-
Chol75PC25
PGA-
Chol50PC50
PGA-Chol100
PGA-Chol25PC75
PGA−PC100
structural
parameter
lamellar
structure
lamellar
structure
lamellar
structure
pseudo-lamellar
structure
lamellar
structure
pseudo- lamellar
structure
orthorhombic cylindrical
structure
a
dL (nm)
3.18
3.25
3.25
2.44
3.37
2.62
b
z
d1 (nm)
1.82 (0.08)
1.83 (0.21)
1.42
0.98 (0.10)
2.27
0.64 (0.05)
1.80
1.00 (0.13)
2.37
0.80 (0.10)
1.82
c
d2 (nm)
1.36
0.51
0.62
0.81
0.06
0.01
0 (4.20)
0.992
62
d
dr1 (nm)
0.50
0.50
0.50
0.49
0.49
e
dr2 (nm)
0.58
0.58
0.58
0.56
0.56
f
dr3 (nm)
0.81
0.81
0.81
0.81
0.81
g
g33
0.06
0.08
0.08
0.08
0.08
h
grr
0.01
0.01
0.01
0.01
0.01
i
φ (deg)
0 (5.99)
0.984
100
0 (9.17)
0.963
51
0 (10.94)
0.947
53
̅
j
OS
k
ϕL,h (%)
49
47
l
ϕL,v (%)
19
m
ϕL,r (%)
19
n
dv (nm)
3.43
o
dh (nm)
5.16
p
Rv (nm)
2.15
q
Rc,v (nm)
0.48 (0.05)
1.67 (0.30)
2.58
r
Rs,v (nm)
s
Rh (nm)
t
Rc,h (nm)
1.00 (0.03)
1.58 (0.21)
0.07
u
Rs,h (nm)
x
g
y
dr1 (nm)
0.48
i
φ (deg)
0 (3.31)
̅
j
OS
0.995
a
b
c
d
e
Long period. Thickness of denser sublayer. Thickness of less dense sublayer. Mean interdistance between the nearest bristles. Mean
f
g
interdistance between the nearest backbones. Mean interdistance between the nearest bristles along the polymer backbone. Paracrystal distortion
factor along a direction normal to the in-plane of multibilayer structure. Paracrystal distortion factor of the bristles along the lateral packing
direction. Mean polar angle (that is orientation angle) between the orientation n vector of nanostructure (horizontal multibilayer structure or
horizontal OPC structure) and the out-of-plane direction of the film. Second-order orientation factor. Relative volume fraction of horizontal
h
i
j
k
l
m
multibilayer structure. Relative volume fraction of vertical multibilayer structure. Relative volume fraction of randomly oriented multibilayer
n
o
structure. Mean interdistance between the stacks of the horizontal cylinder arrays. Mean center-to-center distance in the horizontal cylinder array.
p
q
r
Mean height of core−shell ellipsoidal cylinder. Mean height of the core part of core−shell ellipsoidal cylinder. Mean height of the shell part of
s
t
u
core−shell ellipsoidal cylinder. Mean width of core−shell ellipsoidal cylinder. Mean width of the core part of core−shell ellipsoidal cylinder. Mean
x
y
width of the shell part of core−shell ellipsoidal cylinder. Positional distortion factor of core−shell ellipsoidal cylinders. Mean interdistance between
z
the nearest bristles along the polymer backbone. Standard deviation.
The PGA-Chol100 film revealed four weak scattering rings
(i.e., semicircular rings) with a regular angle interval: 2.01°,
4.03°, 6.03°, and 8.05° (Figure 2a). In addition, a set of
scattering spots appeared with a regular angle interval along the
αf direction at 2θf = 0°; the positions in scattering angle of the
individual spots were well matched with those of the four weak
scattering rings (Figures 2a and 3A-a). The first three of such
four scattering spots were also discernible weakly along the 2θf
direction at αf = 0° or 0.160° (Figures 2a and 3B-a). The
scattering peaks were found to have relative scattering vector
lengths from the specular reflection position of 1, 2, 3, and 4,
indicating that they are in a relationship of the first-, second-,
third-, and fourth-order scattering peaks originating from a
same reflection plane. These results collectively inform that
lamellar structures in three different orientations are present in
the film; namely, horizontally oriented lamellae were formed as
a major structural component while vertically and randomly
oriented lamellae were formed as minor structural components.
The relative fractions of the lamellae in such orientations were
estimated by the quantitative analysis of the azimuthal
scattering profile extracted at 6.03° (Figure S11): 62%
horizontally oriented lamellar structure (= ϕL,h), 19% vertically
oriented lamellar structure (= ϕL,v), and 19% randomly
oriented lamellar structure (= ϕL,r). The polymer film showed
an additional scattering around 12.58°, which was broad and
weak. In fact, this scattering ring is anisotropic rather than
isotropic. Its intensity is relatively stronger along the meridian
direction and much stronger along the equatorial direction. Its
d-spacing was estimated to be 0.51 nm (= dr1). Taking into
consideration the chemical structure and lamellar structure
formation characteristics of the brush polymer, the dr1 value can
be assigned to the mean interdistance between the nearest
bristles in extended conformation. With these results, the GIXS
pattern was further analyzed in a quantitative manner by using
the GIXS formula derived for a lamellar structure model
composed of two sublayers; details of the GIXS formula are
given in the Supporting Information. The out-of-plane and in-
plane scattering profiles were successfully well fitted with the
GIXS formula, as shown in Figures 3A-a and 3B-a. In particular,
the azimuthal scattering profile extracted at 4.03° was analyzed
in order to get orientation details of the horizontal lamellar
6495
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
Figure 4. Schematic representations of molecular chain conformations and packing orders in films of PGA-CholmPCn polymers: (a) PGA-Chol100
;
(b) PGA-Chol75PC25; (c) PGA-Chol50PC50 and PGA-Chol25PC75; (d) PGA-PC100
.
structure (Figure S12a). The GIXS analysis results are
summarized in Table 2.
digitation and lateral packing of the Chol-bristles could be
feasible when the polymer backbone chains behave extended
conformation, namely 21 conformation. Because of the
complete interdigitated bristles, the lamellar structure can be
classified as a multibilayer structure. Considering this
interdigitation characteristic and the chemical structure, the
denser sublayer is made of the interdigitated cyclic parts, the
triazolyl units with inner parts of the alkylenyl linkers, and the
polymer backbones; the less dense sublayer is made of the rest
parts of the alkylenyl linkers. In fact, the formation of this
multibilayer structure is remarkable in regard to some obstacles
such as the triazolyl unit causing a kink and the bulky cyclic
cholesterol part in the Chol-bristle. It turns out that the self-
assembling ability of the Chol-bristle overrides possible
negative contributions of the triazolyl kink and bulky cyclic
cholesterol part, leading to the formation of multibilayer
structure. The multibilayer structure is formed in a mixture of
The GIWAXS analysis results collectively inform that the
PGA-Chol100 molecules favorably self-assemble, forming
lamellar structure with characteristic features in the following.
The long period dL is 3.18 nm, which consists of a denser
sublayer of 1.82 nm thick (= d1) and a less dense sublayer of
1.36 nm thick (= d2). The base polymer PECH of PGA-Chol100
is known fully amorphous. The dL value is close to the length of
the bristles in fully extended conformation. The d1 value is close
to twice the length of the cyclic parts in the cholesterol unit of
the Chol-bristle. Taking these facts into account, the lamellar
structure in the film might result from the favorable self-
assembling, namely lateral packing of the Chol-bristles in
extended conformation. In the lamellar structure, the bristles
might be completely interdigitated between the lamellae in
contact along the stacking direction. Such complete inter-
6496
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
horizontal, vertical, and random orientations. In particular, the
horizontal multibilayer structure is formed as the major
structural component. Its positional distortion factors (g33
and grr) are very small, 0.01−0.06, indicative of high
dimensional stability of the horizontal multibilayer structure.
Furthermore, the structure reveals high second order
The orthorhombically packed cylinder (OPC) structure
formed in the PGA-PC100 film is characterized with key features
as follows. The individual cylinders are preferentially oriented
in the film plane, elliptic, and consist of two phases, namely a
core part and a shell part; Rv = 2.15 nm (radius in the out-of-
plane direction, namely short radius): Rc,v = 0.48 nm (core
radius) and Rs,v = 1.67 nm (shell thickness); Rh = 2.58 nm
orientation factor Os (0.992) where the mean polar angle φ
̅
is 0° and the deviation of polar angle σφis 4.20°. Taking into
(radius in the in-plane direction, namely long radius): Rc,h =
1.00 nm (core radius) and Rs,h = 1.58 nm (shell thickness). The
cross section of the cylinders is comparable to that of a single
PGA-PC100 chain in helical conformation, suggesting that each
cylinder is made of a single helical PGA-PC100 chain. Taking
into consideration the helical conformation and chemical
structure of PGA-PC100, the core part with a relatively higher
electron density of the elliptic cylinder could be composed of
the triazolyl units, polymer backbone, and inner parts of the
alkylenyl linkers in the bristles, whereas the shell part could
consist of the PC end groups and outer parts of the alkylenyl
linkers in the bristles. The mean center-to-center distance of
the cylinders lain in a direction parallel to the film plane is 5.16
nm (= dh). The mean interdistance between the stacks of the
horizontal cylinder arrays is 3.43 nm (= dv). The dv/dh ratio is
calculated to be 0.665, which is somewhat far from that (= √3/
2) for a regular HPC structure. This dv/dh ratio value again
confirms that the horizontal helical PGA-PC100 cylinders were
closely packed together, forming a highly distorted HCP
structure, namely OPC structure. The positional distortion
factor g is very small, 0.07, whereas the orientation factor Os is
account the multibilayer structure with the complete
interdigitation of extended bristles, we could further estimated
the mean interdistance between the nearest backbones (= dr2)
and the mean interdistance between the nearest bristles along
the polymer backbone (= dr3) from the mean interdistance of
the nearest bristles (dr1 = 0.51 nm) determined from the
scattering ring at 12.58°. From the structural details determined
above, the molecular chain conformation and self-assemblies of
PGA-Chol100 in the film could be drawn in a schematic manner,
as shown in Figure 4a.
Surprisingly, a quite different scattering pattern was observed
for PGA-PC100 in films. As shown in Figure 2e, the PGA-PC100
film reveals several scattering spots in the angle region of <6°
and a relatively broad, isotropic scattering ring at 13.35°. Two
spots appear at 3.75° (1.71 nm d-spacing) and 5.64° (d = 1.14
nm) along the meridian line. These spots were estimated to
have relative scattering vector lengths from the specular
reflection position of 2 and 3, indicating that they are the
second- and third-order scattering peaks originating from a
same reflection plane. The first-order peak of these spots is
expected to appear at 1.88° (d = 3.43 nm), which is heavily
overlapped with the parasitic scattering from the reflected main
X-ray beam. The observation of these spots suggests that in the
film arrays with a mean interdistance of 3.43 nm are present
along a direction normal to the film plane. Three scattering
spots additionally appear in the out of meridian line. One weak
scattering spot is discernible at αf = 1.42° and 2θf = 1.05° (d =
3.61 nm) while another scattering spot is shown at αf = 2.88°
and 2θf = 2.19° (d = 1.79 nm). Their relative scattering vector
lengths from the specular reflection position are 1 and 2,
indicating that they are the first- and second-order scattering
peaks originating from a same reflection plane. A relatively
strong spot appears at αf = 3.06° and 2θf = 1.42° (d = 1.90 nm).
All scattering spots could be assigned with taking into
consideration horizontally oriented cylinders and their closed
packing lattice (i.e., hexagonal lattice), as shown in Figure S13;
the (01), (02), (03), (10), (20), and (11) reflections appear.
However, both the (10) and (20) reflections (αf = 1.42° and
2θf = 1.05° and at αf = 2.88° and 2θf = 2.19°) are found to
position at an azimuthal angle μ of 37° rather than 30°,
suggesting that in the film the horizontal cylinders formed a
hexagonally packed cylinder (HPC) structure with a certain
degree of distortion, rather than a regular HPC structure. The
out-of-plane and in-plane scattering profiles could be
successfully fitted by using the GIXS formula derived with an
orthorhombic lattice model (which is a distorted HPC lattice)
consisting of elliptical cylinders with two phases (Figures 3A−e
and 3B−e); the details of GIXS analysis are given in the
Supporting Information. In addition, the azimuthal scattering
profile extracted at 3.95° was analyzed in detail (Figure S12e).
The broad, isotropic scattering ring at 13.35° is estimated to
have a small d-spacing value, 0.48 nm. Considering such small
d-spacing value, the scattering ring might originate from the
mean interdistance of the nearest-neighbored bristles (= dr1).
The analysis results are listed in Table 2.
0.995 with φ = 0° and its standard deviation σφis 3.31°. These
̅
factors are the evidence that the OPC structure formed in the
film is well oriented in the film plane and very stable
dimensionally. Taking into account the dimensional parameters
of the molecular cylinder in the OPC structure and the dr1
value, the molecular PGA-PC100 cylinder has been confirmed to
behave 125 helical conformation by molecular simulations using
the Cerius2 software package (Accelrys, San Diego, CA). From
the structural analysis details above, schematic representations
of the molecular chain conformation and self-assemblies of
PGA-PC100 in the film could be depicted, as shown in Figure
4d.
Quantitative GIWAXS analysis was extended for the
scattering patterns of PGA-Chol75PC25, PGA-Chol50PC50, and
PGA-Chol25PC75 films. The analysis results are presented in
Table 2, Figure 3, and Figure S12. These brush random
copolymers in films were confirmed to form only horizontal
multibilayer structures rather than orientationally mixed
multibilayer structures. However, their structural dimension
parameters were found to vary somewhat with the chemical
compositions. As the content of Chol-bristle is decreased, dL as
well as d2 is discernibly increased, whereas d1 is decreased. In
cases of PGA-Chol50PC50 and PGA-Chol25PC75 films, horizon-
tally oriented pseudo-lamellar structure (namely, slightly less
ordered multibilayer structure) was found to form in addition
to the more ordered multibilayer structure (Figure S14). The
pseudo-multibilayer structures reveal relatively smaller dimen-
sional parameters than those of the more ordered multibilayer
structures. These dimensional changes as well as the additional
pseudo-multibilayer structure formations might be attributed to
compositional inhomogeneities of the bristles that make critical
roles in the self-assembly structure formation. In fact, the length
of the PC-bristle is relatively shorter than that of the Chol-
bristle. Taking this fact into account, the more ordered
multibilayer structures behaving a relatively larger dL value
6497
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
might be made of the polymer chain parts enriched with Chol-
bristles, whereas the pseudo-multibilayer structures revealing a
relatively smaller dL value might be formed with the polymer
chain parts enriched by PC-bristles and/or their mixture with
Chol-bristles. Taking into account the structural details in
Table 2, schematic representations of the molecular chain
conformation and self-assemblies of PGA-CholmPCn copoly-
mers in the film could be given, as presented in Figures 4b,c.
The brush polymer films were additionally examined by
GISAXS analysis. However, they showed featureless GISAXS
patterns (see some representative images in Figure S15),
suggesting that no discernible microstructures were formed in
the films.
For the brush polymer films, XPS analysis was conducted in
order to get information on the chemical composition at the
film surface. Considering atomic components of the polymers,
P and C elements were measured; P2p and C1s spectra were
obtained, as shown in Figure S16. For each film data,
quantitative XPS analysis was performed by the determination
of P2p and C1s peak areas corrected with the relative sensitivity
factors (0.525 for P2p and 0.314 for C1s). As a result, the
concentration ratio of P and C atomic components at each film
surface was obtained from the P2p and C1s peak areas. From the
P and C atomic concentration ratios, the composition ratios of
Chol- and PC-bristles were calculated. The content of PC-
bristle at the film surface was obtained to be 0 mol % for the
PGA-Chol100 film, 21.2 mol % for the PGA-Chol75PC25 film,
41.0 mol % for the PGA-Chol50PC50 film, 68.8 mol % for the
PGA-Chol25PC75 film, and 100 mol % for the PGA-PC100 film;
from the results, the Chol-bristle contents at the film surfaces
were further estimated (Table 1). Overall, the XPS analysis
confirmed the presence of PC bristles at the copolymer film
surfaces. The PC-bristle content at the film surface was
increased with its chemical loading into the copolymer.
However, the PC-bristle content of each copolymer at the
film surface is slightly lower than that determined by NMR
spectroscopy analysis (Table 1). Such lower contents of PC-
bristle at the film surfaces might be mainly attributed to the
multibilayer structures consisted of vertically oriented bristles in
which the PC-bristle is ca. 0.74 nm shorter in length than the
Chol-bristle. Also, such discrepancies might be caused in part
from the differences in the sensitivities and resolutions of XPS
and NMR spectroscopy analyses.
The brush polymer films were further subjected to water
contact angle measurements. The results are listed in Table 1.
The PGA-Chol film exhibits a high contact angle, 96.8°. For the
PGA-CholmPCn films, the water contact angle is decreased to
59.7° from 96.8° as the PC-bristle content is increased. The
PGA-PC film is expected to reveal a water contact angle lower
than that (59.7°) of the PGA-Chol25PC75 film. However, its
water contact angle could not be measured because it was
soluble in water.
These water contact angle results can be understood with
considering the thin film morphologies discussed above. The
water contact angle of PGA-Chol films is about 14° lower than
that (110.7°) of poly(oxy(decyltriazolylmethyl)ethylene)
(PGA-C10) films which also reveal horizontal multibilayer
structure (Figure S17) in which the surface is enriched with
nonpolar methyl end groups. In general, the methyl group is
more nonpolar than that of hydroxyl group. Taking this fact
into account, the relatively lower water contact angle of the
PGA-Chol film could be attributed to the film surface enriched
with the hydroxyl end groups in the Chol-bristles in support
with the multibilayer structures formed in the film. Higher PC-
bristle content in PGA-CholmPCn films exhibited lower water
contact angle, which could result from higher water affinity of
the zwitterionic PC-bristles in structural support with the
multibilayer structures formed in the films.
Self-assembling PGA-CholmPCn polymers and their homo-
polymers were further investigated in bulk state by DSC
analysis. The measured DSC thermograms are shown in Figure
1b and Table 1. PGA-Chol100 reveals two phase transitions: A
glass transition weakly appears at 82 °C (= Tg, glass transition
temperature at the middle point of the transition), and a
melting transition is observed at 121 °C (= Tm, melting
temperature at the transition peak maximum). PECH and PGA
(which are the base polymers for PGA-Chol100) were found to
be completely amorphous, revealing Tg = −33 and −38 °C
(Table 1 and Figure S18). Taking these facts into account, the
relatively high glass transition is originated from the Chol-
bristles bearing rigid triazolyl linker and rigid, bulky
cholesterolyl moiety. Regarding the chemical structure and
components, the observed Tm value seems too high. This high
Tm value might be attributed to the multibilayer structure with
partially interdigitated Chol-bristles discussed above.
Glass and melting transitions are also observed for PGA-
Chol75PC25 and PGA-Chol50PC50. Surprisingly, both of the
copolymers, however, exhibit higher Tg as well as much higher
Tm, respectively, compared to those of PGA-Chol100; in
particular, PGA-Chol75PC25 shows the highest Tg as well as
the highest Tm. In contrast, PGA-Chol25PC75 shows only a
broad, weak transition over 55−95 °C. These results
collectively inform that the PC-bristles make critical roles for
multibilayer structure formation in the PGA-CholmPCn
copolymer. In fact, the cholesterolyl moiety is bulky, and thus
the lateral packing efficiency of the Chol-bristles may not be
high in the multibilayer structure formation. Such limited lateral
packing could be solved in a constructive way by aids of the less
bulky, zwitterionic PC-bristles in the copolymer that are able to
relieve steric hindrances present between the Chol-bristles. The
results suggest that such positive, constructive contribution to
the Chol-bristles’ packing and resulting multibilayer structure
formation could be achievable over the PC-bristle content of
≤50 mol %. However, the PC-bristle content of >50 mol %
causes negative contributions on the Chol-bristles’ packing and
resulting multibilayer structure formation. These contributions
of the PC-bristle are discernible in the GIWAXS patterns, as
shown in Figure 2; PGA-Chol75PC25 and PGA-Chol50PC50 in
thin films revealed a more distinguishable and intense X-ray
scattering pattern, respectively, and however, PGA-Chol25PC75
showed a less distinguishable and weak scattering pattern
compared to that of PGA-Chol100
.
Interestingly, PGA-PC100 exhibits only a broad, weak
transition. A broad, weak transition is also observed for PGA-
Chol25PC75, as mentioned above. These phase transitions are
exceptionally too broad. PGA-PC100 was confirmed to have a
helical conformation and self-assemble, forming OPC structure
in films, as discussed above. On the other hand, PGA-
Chol25PC75 was found to have an extended conformation and
self-assemble, forming multibilayer structure in films; but the
perfectness and overall crystallinity of the structure are
relatively low. These structural features may have resulted
from the zwitterionic nature of the PC-bristles that causes
repulsive interactions rather than attractive interactions when
they are in close contact. Such repulsive interaction character-
istics of the PC-bristles could cause a significant reduction of
6498
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
cohesive energy gain in the OPC structure formation with
helical PGA-PC100 chains as well as in the multibilayer structure
formation with PGA-Chol25PC75 chains in extended con-
formation. Therefore, the single broad, weak transitions
observed in PGA-PC100 and PGA-Chol25PC75 might originate
from the order−disorder transitions of such self-assembled
structures with low cohesive energies in addition to the glass
transitions.
NMR, IR, GPC, GIXS, and XPS data and GIXS data
analysis (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail hskim@dongguk.ac.kr, Tel +82-54-770-2417, Fax +82-
54-770-2447 (H.K.).
*E-mail ree@postech.edu, Tel +82-54-279-2120, Fax +82-54-
279-3399 (M.R.).
■
CONCLUSIONS
■
ORCID
In this study a series of well-defined PGA-CholmPCn polymers
was successfully synthesized as a novel cell membrane
mimicking system. This study demonstrated that the azide−
alkyne click chemistry is a very powerful tool to effectively
incorporate even bulky, heavy biomolecules as well as
zwitterionic biomolecules into a polymer as side groups or
bristles in a controlled, well-defined manner. The cell
membrane mimicking polymers were found to be soluble in
common solvents, showing good solution processability. They
were thermally stable up to 230 °C or higher temperatures,
depending on the bristle compositions.
Moonhor Ree: 0000-0001-5562-2913
Author Contributions
C.K. and K.K. equally contributed to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by the National Research Foundation
of Korea (Haeksim Program 2015R1A2A2A2A01005642). The
synchrotron X-ray scattering measurements at the Pohang
Accelerator Laboratory were supported by MSIT, POSTECH
Foundation, and POSCO Company.
Morphology details of the cell membrane mimicking
polymers in nanoscale thin films were investigated by
synchrotron GIXS analysis. Very interestingly, all PGA-
CholmPCn polymers as well as PGA-Chol100 favorably formed
multibilayer structures with 21 chain conformation, whereas
PGA-PC100 formed OPC structure with 125 helical chain
conformation. Such multibilayer structure formations could be
driven by a strong self-assembling ability of the Chol-bristle in
extended conformation which overrides possible obstacles such
as the kink unit caused by trizolyl unit and the bulky cholesterol
unit in the bristle. In comparison, the OPC structure formation
could be driven by a lateral packing ability of the brush polymer
chain in the helical confirmation resulted from the
minimization of repulsive interactions in the neighbored
zwitterionic PC-bristles. The GIXS and DSC analyses
confirmed that the enthalpy gains in the Chol-bristle driven
multibilayer structure are much larger than that in the PC-
bristle driven OPC structure. Another interesting finding was
that in PGA-Chol75PC25 and PGA-Chol50PC50 the PC-bristles
could promote to properly mobilize the overall brush polymer
chains in the self-assembling process, thereby leading to the
multibilayer structure formation with larger enthalpy gain.
Because of such the self-assembled nanostructure formations,
PGA-CholmPCn polymers and their homopolymers always
provided the film surface enriched with Chol and/or PC
moieties which are mimicking biological cell membrane surface.
The presences of PC- and Chol-bristles at the film surfaces
were confirmed by XPS analysis. Higher Chol-enriched film
surface showed lower water affinity, whereas higher PC-
enriched film surface exhibited higher water affinity.
REFERENCES
■
(1) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P.
Molecular Biology of the Cell, 6th ed.; Garland Science: New York,
2014.
(2) Gennis, R. B. Biomembranes − Molecular Structure and Function;
Springer-Verlag: New York, 1989.
(3) Krause, M. R.; Regen, S. L. The Structural Role of Cholesterol in
Cell Membranes: From Condensed Bilayers to Lipid Rafts. Acc. Chem.
Res. 2014, 47, 3512−3521.
(4) Goluszko, P.; Nowicki, B. Membrane Cholesterol: a Crucial
Molecule Affecting Interactions of Microbial Pathogens with
Mammalian Cells. Infect. Immun. 2005, 73, 7791−7796.
(5) Neef, A. B.; Schultz, C. Selective Fluorescence Labeling of Lipids
in Living Cells. Angew. Chem., Int. Ed. 2009, 48, 1498−1500.
̌
̌ ́ ́
(6) Ikonen, S.; Macíckova-Cahova, H.; Pohl, R.; Sanda, M.; Hocek,
M. Synthesis of Nucleoside and Nucleotide Conjugates of Bile Acids,
and Polymerase Construction of Bile Acid-Functionalized DNA. Org.
Biomol. Chem. 2010, 8, 1194−1201.
(7) Li, W.; Li, X.; Zhu, W.; Li, C.; Xu, D.; Ju, Y.; Li, G. Topochemical
Approach to Efficiently Produce Main-Chain Poly(bile acid)s with
High Molecular Weights. Chem. Commun. 2011, 47, 7728−7730.
(8) Nakaya, T.; Li, Y. J. Phospholipid Polymers. Prog. Polym. Sci.
1999, 24, 143−181.
(9) Yaseen, M.; Wang, Y.; Su, T. J.; Lu, J. R. Surface Adsorption of
Zwitterionic Surfactants: n-Alkyl Phosphocholines Characterised by
Surface Tensiometry and Neutron Reflection. J. Colloid Interface Sci.
2005, 288, 361−370.
(10) Pepys, M. B.; Hirschfield, G. M.; Tennent, G. A.; Gallimore, J.
R.; Kahan, M. C.; Bellotti, V.; Hawkins, P. N.; Myers, R. M.; Smith, M.
D.; Polara, A.; Cobb, A. J. A.; Ley, S. V.; Aquilina, J. A.; Robinson, C.
V.; Sharif, I.; Gray, G. A.; Sabin, C. A.; Jenvey, M. C.; Kolstoe, S. E.;
Thompson, D.; Wood, S. P. Targeting C-Reactive Protein for the
Treatment of Cardiovascular Disease. Nature 2006, 440, 1217−1221.
(11) Milne, S. B.; Tallman, K. A.; Serwa, R.; Rouzer, C. A.;
Armstrong, M. D.; Marnett, L. J.; Lukehart, C. M.; Porter, N. A.;
Brown, H. A. Capture and Release of Alkyne-Derivatized Glycer-
ophospholipids Using Cobalt Chemistry. Nat. Chem. Biol. 2010, 6,
205−207.
Overall, we have developed novel PGA-CholmPCn polymers,
as a new cell membrane mimicking material system, which can
favorably self-assemble multibilayer structure and provide
biomolecular Chol- and PC-enriched surface. The PGA-
CholmPCn polymers are very suitable for uses in the fields
required cell membrane surface characteristics.
ASSOCIATED CONTENT
* Supporting Information
■
S
(12) Yu, X.; Liu, Z.; Janzen, J.; Chafeeva, I.; Horte, S.; Chen, W.;
Kainthan, R. K.; Kizhakkedathu, J. N.; Brooks, D. E. Polyvalent
Choline Phosphate as a Universal Biomembrane Adhesive. Nat. Mater.
2012, 11, 468−476.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.macro-
mol.7b00805.
6499
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500

Macromolecules
Article
(13) Jung, J. H.; Lim, Y. G.; Lee, K. H.; Koo, B. T. Synthesis of
Glycidyl Triazolyl Polymers Using Click Chemistry. Tetrahedron Lett.
2007, 48, 6442−6448.
(14) Liu, D.; Zheng, Y.; Steffen, W.; Wagner, M.; Butt, H. J.; Ikeda,
T. Glycidyl 4-Functionalized-1,2,3-Triazole Polymers. Macromol.
Chem. Phys. 2013, 214, 56−61.
(15) Song, S.; Ko, Y.-G.; Lee, H.; Wi, D.; Ree, B. J.; Li, Y.;
Michinobu, T.; Ree, M. High Performance Triazole-Containing Brush
Polymers via Azide-Alkyne Click Chemistry: A New Functional
Polymer Platform for Electrical Memory Devices. NPG Asia Mater.
2015, 7, e228−e240.
(16) Lee, B.; Park, Y.-H.; Hwang, Y.-T.; Oh, W.; Yoon, J.; Ree, M.
Ultralow-k Nanoporous Organosilicate Dielectric Films Imprinted
with Dendritic Spheres. Nat. Mater. 2005, 4, 147−150.
(17) Ree, M. Probing the Self-Assembled Nanostructures of
Functional Polymers with Synchrotron Grazing Incidence X-Ray
Scattering. Macromol. Rapid Commun. 2014, 35, 930−959.
(18) Kim, Y. Y.; Ree, B. J.; Kido, M.; Ko, Y.-G.; Ishige, R.; Hirai, T.;
Wi, D.; Kim, J.; Kim, W. J.; Takahara, A.; Ree, M. High Performance n-
Type Electrical Memory and Morphology-Induced Memory-Mode
Tuning of A Well-Defined Brush Polymer Bearing Perylene Diimide
Moieties. Adv. Electronic Mater. 2015, 1, 1500197.
6500
DOI: 10.1021/acs.macromol.7b00805
Macromolecules 2017, 50, 6489−6500