FRET Phenomenon in Photoreversible Dual-Color Fluorescent Polymeric Nanoparticles Based on Azocarbazole/Spiropyran Derivatives

Article abstract of DOI:10.1021/acs.macromol.5b02401

Incorporation of chromophores into a polymer chain results in prominent photoreversibility and fatigue resistance, photostability over the long term, and restriction of the internal conversion (IC). Here, we report the copolymerization of two photoactive monomers with methyl methacrylate via emulsion polymerization in order to obtain photoswitchable dual-color fluorescent nanoparticles. For this purpose, azocarbazole ethyl acrylate (AzoCzEA) and spiropyran ethyl acrylate (SPEA) were synthesized and the fluorescence polymeric latex nanoparticles were prepared correspondingly. FT-IR and ¹HNMR spectra were used to confirm the structure of novel fluorescent AzoCzEA. UV-vis studies of the obtained nanoparticles displayed the spectral features of both AzoCzEA and SPEA under stimuli-irradiation and inclusion of these chromophores into the polymer particles. DSC analysis revealed an increase in T_g of the prepared copolymer, indicating covalently incorporation of the photoactive monomers into the polymer chains. The optimum ratio of two chromophores to achieve complete quenching and highest energy transfer was determined by UV-vis spectroscopy. DLS and SEM results demonstrated particle size distribution of 40-80 nm with spherical morphology. Fluorescence spectra revealed remarkable fluorescence resonance energy transfer (FRET) from AzoCzEA to SPEA after UV irradiation at 365 nm and dual-color characteristic of the prepared nanoparticles. Besides, an enhancement in the photoreversibility, photostability, prevention of IC, dye leakage, and aggregation were studied elaborately. The obtained results were attributed to the involvement of such chromophores into the polymeric matrix via covalent bonding. Labeling and tracking of living cells and rewriteable patterning are potential applications for such dual-color fluorescent nanoparticles.

Full text of DOI:10.1021/acs.macromol.5b02401

Article
pubs.acs.org/Macromolecules
FRET Phenomenon in Photoreversible Dual-Color Fluorescent
Polymeric Nanoparticles Based on Azocarbazole/Spiropyran
Derivatives
Jaber Keyvan Rad, Ali Reza Mahdavian,* Hamid Salehi-Mobarakeh, and Amin Abdollahi
Polymer Science Department, Iran Polymer & Petrochemical Institute, P.O. Box 14965/115, 14977-13115 Tehran, Iran
ABSTRACT: Incorporation of chromophores into a polymer chain results
in prominent photoreversibility and fatigue resistance, photostability over
the long term, and restriction of the internal conversion (IC). Here,
we report the copolymerization of two photoactive monomers with
methyl methacrylate via emulsion polymerization in order to obtain photo-
switchable dual-color fluorescent nanoparticles. For this purpose,
azocarbazole ethyl acrylate (AzoCzEA) and spiropyran ethyl acrylate
(SPEA) were synthesized and the fluorescence polymeric latex nano-
1
particles were prepared correspondingly. FT-IR and HNMR spectra were
used to confirm the structure of novel fluorescent AzoCzEA. UV−vis
studies of the obtained nanoparticles displayed the spectral features of both
AzoCzEA and SPEA under stimuli-irradiation and inclusion of these
chromophores into the polymer particles. DSC analysis revealed an
increase in Tg of the prepared copolymer, indicating covalently incorporation of the photoactive monomers into the polymer
chains. The optimum ratio of two chromophores to achieve complete quenching and highest energy transfer was determined by
UV−vis spectroscopy. DLS and SEM results demonstrated particle size distribution of 40−80 nm with spherical morphology.
Fluorescence spectra revealed remarkable fluorescence resonance energy transfer (FRET) from AzoCzEA to SPEA after UV
irradiation at 365 nm and dual-color characteristic of the prepared nanoparticles. Besides, an enhancement in the
photoreversibility, photostability, prevention of IC, dye leakage, and aggregation were studied elaborately. The obtained results
were attributed to the involvement of such chromophores into the polymeric matrix via covalent bonding. Labeling and tracking
of living cells and rewriteable patterning are potential applications for such dual-color fluorescent nanoparticles.
large band gap.20 Carbazole derivatives have a long π conjugation
that results in high fluorescent emission quantum yields. They
can easily be modified by different groups at 3 and/or 6 positions
1. INTRODUCTION
The fluorescent photoswitchable nanoparticles have received
increasing attention because of their exploitations in life science
of the carbazole ring to tune the optical and spectroscopic
such as selectively highlighting biological substances (e.g.,
properties. Copolymers containing carbazole derivatives could
proteins and living cells) and individually light addressable
devices.1,2 Organic photochromic spiropyran derivatives are
be used in drug delivery with fluorescence tracing capability
and have exhibited negligible cytotoxicity.12,21,22 Fluorescent
among the most convenient compounds which have isomeric
commoners can be introduced to polymeric nanoparticles by
forms of closed-ring (SP) and the colored conjugated open-ring
merocyanine (MC). This isomerization occurs under UV−vis
irradiation alternatingly and is widely used due to their fast
covalent linkage, entrapment, or self-assembly strategies that
make them very promising and versatile for their applications in
many systems.1,23
photochromic response and photoreversible fluorescence
modulation through energy transfer.3−5 Anchoring such photo-
The fluorescence resonance energy transfer (FRET) systems
consist of a donor and an acceptor dye in which the fluorescence
emission of a component can be switched on and off through a
chromic groups covalently into a polymer chain induces their
light-sensitive properties. However, light is an especially attrac-
nonradiative energy transfer between nearest chromophores
tive external stimulus and results in producing light responsive
polymers.6,7 Upon light irradiation, the chemical and physical
parameters of the photochromic polymeric nanoparticles would
be consequently altered.8−11
Polymers containing carbazole moieties are of considerable
interest due to their potential applications in highly fluorescent
probes and detectors,12−14 organic light emitting diode
materials,15 hollow particles,16 photorefractive materials,17,18
photovoltaic devices,19 and they are most frequently used as
polymeric hosts because of their hole transporting attribute and
(light-sensitive molecules) by UV−vis irradiation with the same
excitation wavelengths.1,24−29 FRET is a dynamic quenching
mechanism, because energy transfer occurs while the donor is in
the excited state.30,31 Energy transfer occurs on nanometeric
distance and depends on the precise spatial arrangement and
Received: November 4, 2015
Revised: December 7, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 1. Modification and isomerization of SPEA (a); and synthetic method for preparation of AzoCzEA (b).
approximately parallel transition dipoles in donor and acceptor
moieties.25,26,32 In recent years, materials with optically
modulated properties, like fluorescence resonance energy
transfer, have attracted much attention as a result of some
advantages over conventional fluorescence dyes in broad range of
scientific fields including biological diagnosis, cell labeling,
imaging, and sensing tools.23,33
nanoparticles by a facile one-pot RAFT-mediated miniemulsion
method.5 In their research, boron-dipyrromethene phenol
methacrylate and spiropyran-linked methacrylate dyes have
been covalently incorporated into the polymer backbone. The
results exhibited excellent structural stability, prominent photo-
stability, favorable photoreversibility and also lack of potential
dye leakage, and aggregation that restrict their biological
applications. The as-prepared photoswitchable fluorescent
polymeric nanoparticles demonstrated high-contrast fluores-
cence cell imaging and rewriteable fluorescence pattering
successfully. Wang et al.38 prepared carbazole-spironaphtoxazine
copolymers through radical polymerization. The reversibly
changes in fluorescence intensity by irradiation of UV−vis light
were attributed to the photoinduced electron transfer between
carbazole and open-ring form of spirooxazine moiety. The major
problem in this work was the absence of adequate overlap
between emission spectrum of the donor (carbazole derivative)
and absorption spectrum of the acceptor (spironaphtoxazine)
dyes for total quenching of the fluorescence intensity under UV
irradiation. On the other hand, photostability and fatigue
resistance fell due to the accessibility of the donor and acceptor
dyes to the surrounding environment. A donor−acceptor of
another couple based on spiropyran as a dopant in polyvinyl
carbazole has been reported in 2011 for development in erasable
holography.39
Photoswitchable dual-color fluorescents are appropriate
candidates especially due to their ability to distinguish fake
signals made by adventitious fluorescent biomolecules from
original ones.34−36 Zhu and co-workers37 integrated photo-
switchable spyropiran and fluorescent perylene diimide dyes via
covalently linkage into a polymeric nanoparticle to generate an
optically addressable dual-color fluorescent system by means of
emulsion polymerization. The prepared photoluminescent
nanoparticles exhibit superior resistance to photobleaching,
high contrast, and high intense fluorescence with potential
application in biological tracking and labeling. Incorporation of
noncovalently bonded chromophores into the nanoparticles may
have some disadvantages in long-term stability because of leakage
and aggregation of the dyes and decrease in photoresponsivity.
Chen et al.24 reported the reversible modulation of fluorescence
by spyropiran and a nitrobenzoxadizole (NBD) derivative dye via
miniemulsion polymerization. The spiropyran-based monomer
was covalently incorporated into the polymer nanoparticles and
doped (noncovalently) with NBD. The efficiency of FRET was
primarily good but might have some disadvantages in biological
system due to the unbounded NBD dye, leading to FRET
weakening and cells cytotoxicity. This group has already
synthesized amphiphilic photoswitchable fluorescent polymeric
To the best of our knowledge, there is no report on FRET
systems with coupling of azo-derivatives of carbazole as a donor
and spiropyran ethyl acrylate (SPEA) as an acceptor. In this
study, monomers of an acrylic-modified spiropyran derivative
(SPEA) and an azocarbazole derivative (AzoCzEA) were
B
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
air-dried and finally purified by the modified recrystallization method
from a 1:1 cyclohexane: toluene mixture to give white needle crystals
with 88.7% yield. 3-[(4-Nitrophenyl)] azo-9H-carbazole-9-ethanol
(NACzEtOH) was synthesized next. And finally, (4-nitrophenyl)-[3-
[N-[2- (acryloyloxy) ethyl] carbazolyl]] diazene (AzoCzEA) was
synthesized through the modification reaction of NACzEtOH with
acryloyl chloride in dry THF by using of triethylamine as the catalyst.
The resulting solid was purified by recrystallization from a 2:1 ethanol:
chloroform mixture (in 83.4% yields).
synthesized and then incorporated into the poly(methyl
methacrylate) (PMMA) chains inside nanoparticles via emulsion
polymerization. The obtained photoactive polymeric nano-
particles can protect dyes from environmental effects as internal
conversion (IC), beside sustained photostability in long-term
and photoreversibility with improving fatigue resistance. Finally,
the observed complete fluorescence quenching of the donor
moiety upon UV irradiation referred to the excellent overlap
between the two chromophores in the obtained nanoparticles.
2.4. Preparation of Solutions Containing SP−OH and
NACzEtOH. Several solutions with different amounts of NACzEtOH
and SP−OH were prepared in acetone (Table 1). Then, their spectral
features were investigated by UV−vis spectroscopy at 365 nm.
2.5. Emulsion Polymerization and Preparation of Dual-Color
Fluorescent Polymeric Nanoparticles. The proportion of each
component in preparation of different latexes has been given in Table 2.
An aqueous solution of SDS (0.06 g) and Triton X-100 (0.03 g) as
surfactants, NaHCO3 (0.03 g) as the buffer and KPS (0.03 g) as the
initiator was prepared, poured into a three-necked round-bottom
reactor and purged with nitrogen gas. Afterword, the solution tem-
perature reached to 70 °C. Then, solutions of SPEA in 5 mL of DI water
and AzoCzEA in MMA (3.2 mL) were added from two separate funnels
dropwise and simultaneously into the reactor within 20 min. Emulsion
polymerization reaction continued for 3h, to reach to conversions of
above 95%. The coagulation for each latex was less than 1 wt %.
2.6. Characterization. Identification of the synthesized products in
every step was carried out by FT-IR BRUKER-IFS48 spectropho-
tometer (Germany), using KBr pellets. To approve the precise synthesis
2. EXPERIMENTAL SECTION
2.1. Materials. 2,3,3-trimethylindolenine, and the surfactant sodium
dodecyl sulfate (SDS, 99%) were purchased from Sigma-Aldrich.
All of the solvents, 2-hydroxy-5-nitrobenzaldehyde, methyl metha-
crylate (MMA), potassium persulfate (KPS) as the initiator, sodium
bicarbonate as a buffer (NaHCO3), carbazole (Cz, 89%), sodium nitrite
(NaNO2), hydrochloric acid (HCl, 37%), 4-nitroaniline, triethylamine,
Triton X-100, 2-bromoethanol, and acryloyl chloride (AC) were
supplied by Merck Chemical Co. Tetrahydrofuran (THF) was dried
over sodium and distilled off. Other solvents and all reagents were used
without further purification. Deionized (DI) water was used in all
recipes.
2.2. Synthesis of 1′-(2-Acryloxyethyl)-3′, 3′-dimethyl-6-nitro-
spiro-(2-H-1-benzopyran-2,2′-indoline) (SPEA). 1′-(2-Acrylox-
yethyl)-3′, 3′-dimethyl-6-nitrospiro-(2-H-1-benzopyran-2,2′-indoline)
(SPEA) was synthesized in four sequential steps according to the
previously reported procedure by our group.40 The forward synthesis of
SPEA included: (1) Substitution nucleation reaction between 2,3,3-
trimethylindolenine and 2-bromoethanol. (2) Conducting isomerization
reaction of the product from step (1), resulting in (R/S)-9,9,9a-
trimethyl-2,3,9,9a-tetrahydrooxazolo[3,2-a]indole (R/S). (3) Conden-
sation reaction between the product of step (2) and 2-hydroxy-5-
nitrobenzaldehyde, giving (R/S)-2-(3′,3′-dimethyl-6-nitro-3′H-spiro-
[chromene-2,2′-indole]-1′-yl)ethanol (R/S) (SP−OH). (4) Modification
of SP−OH with acryloyl chloride for preparation of SPEA (Figure 1a).
2.3. Synthesis of (4-Nitrophenyl)-[3-[N-[2-(acryloyloxy) ethyl]
carbazolyl]] Diazene (AzoCzEA). (4-Nitrophenyl)-[3-[N-[2-
(acryloyloxy)ethyl]carbazolyl]] diazene was synthesized based on the
modified procedure reported before.41 Primarily, N-(2-hydroxyethyl)
carbazole (C-A) was synthesized by nucleation substitution reaction
between carbazole with 2-bromoethanol in dimethyl formamid (DMF)
in the presence of potassium hydroxide (KOH) at room temperature to
give C-A (Figure 1b). After 12h, the reaction mixture was poured into
water and the obtained white solid was filtered, washed with water,
1
of azocarbazole ethyl acrylate, HNMR spectrum was recorded on an
Avance 250 MHz instrument (BRUKER, Germany) in CDCl3 and using
tetramethyl silane (TMS) as the internal reference. Characterization of
the prepared copolymers was carried out by differential scanning
calorimetry (DSC) under N2 atmosphere at 10 °C/min heating rate by
NETZSCH Instruments Co. (DSC 200, F3Maia, Germany; 30−150 °C).
Also thermogravimetric analysis (TGA) was performed under N2
atmosphere at 10 °C/min heating rate by TGA-STA: PL-1500 (UK)
(30−600 °C). Dried latexes were used for these analyses directly.
Particle size and size distribution were measured by using SEMATECH
laser light scattering (LLS) (France). Size and morphology of the
polymeric particles were characterized by SEM, Tescan Vega II (Czeck
Republic). For this reason, a drop of the diluted latex was placed on a
sample holder and dried under vacuum at 25 °C. Then, they were put
under vacuum, evacuated, and a layer of gold was deposited under
flushing with argon by using EMITECH K450x sputter-coating
(England). Photochromic properties of the polymeric nanoparticles
were investigated by UV−vis analysis via Shimadzu-UV2550 UV−vis
Spectrophotometer (Japan) and the fluorescence behavior was studied
by JASCO FP-6500 Spectrofluorometer (Japan). For these analyses, the
initial latexes were diluted to 0.2 wt % for UV−vis spectroscopy and
1.0 wt % for fluorescence analysis. A UV lamp (365 nm), CAMAG
12VDC/VAC (50/60 Hz, 14VA, Switzerland), was used to stimulate
changes in structure and absorption bands of the photochromic moieties
and providing proper conditions for energy transfer between the two
existing chromophores in the polymeric nanoparticles. Also the source
for visible light was a common LED lamp with white light which was
used for recovery of fluorescence of the donor and monitoring of
photoreversibility in the nanoparticles. UV and vis irradiation times were
set at 4 and 7 min, respectively, in normal excitation and isomerization
for all the samples.
Table 1. Solutions with Different Amounts of NACzEtOH and
a
SP−OH
SP−
NACzEtOH
(mg)
CNACzEtOH
(mol/L)
OH
CSP−OH
(mol/L)
CSP−OH/
CNACzEtOH
sample
(mg)
DA1
DA2
DA3
DA4
DA5
0.2
0.4
0.8
1.6
3.3
1.85 × 10−5
3.71 × 10−5
7.42 × 10−5
1.48 × 10−4
3.06 × 10−4
5.3
5.3
5.3
5.3
5.3
5.02 × 10−4
5.02 × 10−4
5.02 × 10−4
5.02 × 10−4
5.02 × 10−4
27.1
13.5
6.8
3.4
1.6
a
The volume was reached to 30 mL with acetone for each sample.
Table 2. Emulsion Polymerization and Preparation of Dual-Color Fluorescent Latexes with Various Amounts of the
Chromophores
sample
SPEA (mg)
CSPEA (mol/L)
azoCzEA(mg)
CAzoCzEA (mol/L)
CSPEA/CAzoCzEA
NP
0.0
30
0.0
0.0
4.5
4.5
4.5
0.0
0.0
6.8
NP1
NP2
NP3
2.46 × 10−3
4.92 × 10−3
7.4 × 10−3
3.62 × 10−4
3.62 × 10−4
3.62 × 10−4
60
13.5
20.4
90
C
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 2. FT-IR spectra of the synthesized compounds in preparation of AzoCzEA (a) and 1H NMR spectrum of AzoCzEA in CDCl3 (the upper insets
reveal the corresponding expanded regions) (b).
AzoCzEA, were prepared and purified according to the
literature41 with some modification in the method. Then, the
new fluorescent dye (AzoCzEA) and SPEA were covalently
incorporated into the PMMA polymeric matrix in the latex
particles via emulsion polymerization. Such chemical attachment
can efficiently avoid undesired effects like dye leakage and dye
3. RESULTS AND DISCUSSION
One of the most promising employed strategies for preparation
of the reversible photoswitchable dual-color nanoparticles is
based on emulsion polymerization technique.37 In this study,
SP−OH and SPEA were synthesized according to our previous
report.40 C-A and NACzEtOH, as precursors in the synthesis of
D
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 3. Schematic representation for illustrating radiative or nonradiative energy transfer between the AzoCzEA and SPEA at ground and excited
states by UV irradiation.
aggregation, which occurs in physically doped polymeric systems
and particles.23,24
carbazole ring. The addition of acryloyloxy group was verified
by the appearance of a doublet of doublet at 5.80 and 6.28 ppm
(Ha, Hc) with a coupling constant of 123 Hz and a triplet at
6.02 ppm (Hb) relating to the vinylic protons. The protons of
nitrobenzene ring (Hl and Hk) could be found as two doublets at
8.07 and 8.20 ppm. The proton of position 4 of the carbazole ring
(Hi) has been appeared as a singlet at 8.77 ppm and also other
protons of the carbazole ring are at 7.2−7.6 ppm. The results of
1HNMR spectroscopy approve the modification of NACzEtOH
with acryloyl chloride and successful synthesis of AzoCzEA. For
better elucidation, some expanded regions have been given in the
upper part of Figure 2b.
3.2. UV−Vis Analysis. Fluorescence quenching refers to any
process which significantly reduces the fluorescence intensity of a
given material. A variety of environmental factors can result in
quenching, including energy transfer between fluorophores,
interaction between the fluorophore and surrounding solvent
molecules (dictated by solvent polarity), complex formation,
collisional energy transfer, temperature, pressure, pH and
localized concentration of the fluorescent species.33,42,43 Mostly,
the fluorescence intensity is influenced by the interactions with
local environment in the excited state lifetime. The excited
AzoCzEA fluorophore (AzoCzEA*) has two pathways to return
to its ground state after excitation at 410 nm: (i) direct fluo-
rescence emission, and (ii) nonradiation energy transfer to a
nearest appropriate acceptor (Figure 3). SPEA (SP form) can
3.1. Identification of AzoCzEA. At first, all synthesized com-
pounds were characterized by FT-IR spectroscopy (Figure 2a).
The synthesis of C-A was approved by the presence of char-
acteristic peaks at 3221 cm−1, 1079 cm−1 and 2863−2967 cm−1
relating to hydroxyl groups, C−O and C−H stretching vibrations
of the aliphatic moieties, respectively. In addition, the elimination
of N−H stretching vibration at 3416 cm−1 in carbazole ring
confirms the progress of reaction. The shift of hydroxyl group
stretching vibration in NACzEtOH to higher wavenumbers is
due to the weak hydrogen bonding because of spatial repulsions.
On the other hand, C−H bending vibration profile of carbazole
at 712 and 742 cm−1 changed due to the substitution nucleation
reaction on the carbazole ring with 4-nitrobenzenediazoniom
chloride. Also symmetric and asymmetric stretching vibrations of
NO2 group at 1331 and 1452 cm−1 are another indication for the
synthesis of NACzEtOH. AzoCzEA was obtained by ester-
ification reaction of NACzEtOH. FT-IR spectrum of AzoCzEA
demonstrates its characteristic peaks at 1722 and 1610 cm−1
that are related to the carbonyl and vinyl stretching vibrations
respectively, beside the elimination of hydroxyl groups at
3412 cm−1.
1
Figure 2b displays HNMR spectrum of AzoCzEA with two
doublets at 4.60 ppm (2H) and 4.67 ppm (2H), indicating
methylene groups (Hd, He) attached to the nitrogen atom in the
E
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 4. UV−vis analysis of DA1 (a), DA2 (b), DA3 (c), DA4 (d), and DA5 (e) solutions in acetone, before (- - -) and after () UV irradiation
at 365 nm.
absorb UV irradiation at the 365 nm and isomerizes to its MC
form. Then, MC isomer with absorption region of 500−650 nm
will be a proper acceptor to receive energy from AzoCzEA*
according to the significant band gap overlap and subsequently,
quenching process occurs before the fluorescence emission of
AzoCzEA at 530 nm. The orbital energy levels were calculated by
Spartan’14, version 1.1.0.
spectra were recorded before and after irradiation at 365 nm to
survey the possibility of spectral overlap and proper energy
transfer between the above two chromophores (Figure 4).
NACzEtOH has an absorption band in 400−550 nm. When
DA-series samples (Table 1) are irradiated at 365 nm, the
spiropyran moiety will change from the closed-ring isomer
(spiro, SP) to the open-ring one (merocyanine, MC) with a
strong absorption band at 400−700 nm (λmax of 560 nm).
SP−OH solution shows no absorption band before irradiation at
Primarily, solutions of SP−OH/NACzEtOH in acetone (DA-
series samples) were prepared and their UV−vis absorption
F
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
this region at all. The emission band of NACzEtOH lay in the
absorption region of MC isomer. Hence, there will be the
possibility of efficient overlap between MC absorption and
NACzEtOH emission. It provides the situation for quenching of
the fluorescent dye and correspondingly, the relevant absorption
at 410 nm will be disappeared. This could be observed for DA1,
DA2 and almost DA3 (Figure 4, parts a−c). But the quenching
phenomenon was not monitored for DA4 and DA5 (Figure 4,
parts d and e). It is noteworthy that the quenching process
depends on the concentration ratio of acceptor to donor in dual-
color polymeric systems.24 To have maximum quenching yield,
it is necessary that some acceptor chromophores surround each
donor and proceed this process sufficiently. The weak quenching
in DA4 and DA5 could be attributed to the high concentration of
donors (NACzEtOH) and lack of enough MC isomer as the
acceptor (quenching agent) in these solutions. Complete
quenching was observed for DA1 and DA2, but the absorption
intensity at 500−650 nm for DA2 is more than DA1 after
irradiation at 365 nm, owing to the higher energy transfer from
NACzEtOH to SP−OH in DA2. The comparison between DA2
and DA3 illustrates that despite similar absorption intensities
(500−650 nm), the complete quenching (400−550 nm) has
SPEA and AzoCzEA with identical molar ratios in DA2 were
copolymerized with MMA through emulsion polymerization to
give dual-color fluorescent nanoparticles. Typically, the
morphology and size distribution of photoswitchable fluorescent
NP2 nanoparticles were studied by scanning electron micros-
copy (SEM) and dynamic light scattering (DLS) (Figure 6).
Figure 6. Particle size and size distribution for NP2 dual-color
fluorescent nanoparticles by SEM (a) and DLS (b) analyses.
Both show a good agreement and particles with narrow size
distribution (40−80 nm) have been obtained. SEM micrograph
reveals the uniform spherical nanoparticles with a smooth
surface and narrow size distribution. Also DLS analysis reveals a
unimodal pattern and confirms the successive progress of
emulsion polymerization with one regulating nucleation
mechanism without existence of secondary particles.
UV−vis analysis of NP2 latex demonstrates a complete
quenching upon UV irradiation (Figure 7). In order to confirm
the previous results in the solution state for determining
optimum ratio of the two chromophores, NP1 with less and
NP3 with higher ratios relative to NP2 were prepared and ob-
served too. Disappearance of the absorption band at 400−550 nm
after UV irradiation in NP2 is quite evident here. The band
appeared at 350−400 nm after UV irradiation (365 nm) is
attributed to the π → π* electronic transition of the chromene
moiety. The highest absorption intensity at 500−650 nm for
NP2 approves the obtained optimized ratio for energy transfer
and synergistic effect between these two chromophores.
This was the starting point for our extensive studies on such dual-
color polymer particles and the results will be discussed in the
upcoming.
been accomplished for DA2. Therefore, DA2 with CSP−OH
/
CNACzEtOH of 13.5 was chosen as the optimum sample with the
best quenching and synergistic effect for observing FRET
phenomenon.
3.3. Dual-Color Fluorescent Polymeric Nanoparticles.
Here, the employed basic strategy for preparation of the novel
photoswitchable fluorescent polymeric nanoparticles was based
on emulsion polymerization. All of the nanoparticles were
made of SPEA−AzoCzEA−MMA copolymer chains. AzoCzEA
(the fluorescent dye) serves as the electron donor and SPEA (the
photochromic dye) acts as the electron acceptor moiety and
both are covalently bound to MMA in polymeric chains in the
nanoparticles (Figure 5).
3.4. Thermal Properties. Thermal properties of PMMA
(NP) and NP2 sample containing AzoCzEA and SPEA were
investigated by differential scanning calorimetry and thermog-
ravimetric analyses (Figure 8). A comparative study of glass
transition temperature (Tg) by DSC depicts an increment in
Tg from 102 °C for NP to 116 °C for NP2 as a result of
incorporation of AzoCzEA and SPEA into the polymeric chains
in the nanoparticles. The DSC thermogram of NP2 indicates a
single transition temperature at 116 °C that has a few raise with
respect to the amorphous structure of PMMA and could be
attributed to the incorporation of AzoCzEA and SPEA as
comonomers into the PMMA polymeric matrix. Introducing
these bulk chromophores as comonomers to the polymer
backbone leads to an increase in the restriction of mobility
and steric hindrance of PMMA chains. It is worth mentioning
that no phase separation was observed here according to the
appearance of single transition temperature in DSC thermogram
(Figure 8a).
Figure 5. Schematic representation of a SPEA−AzoCzEA−MMA
copolymer chain and FRET process accompanied by fluorescence color
change through UV−vis irradiation.
Covalently linked dyes and the polymer chains effectively
avoid dye leakage and remove the possibility of dye aggregation
in long-term, which are undesirable features in the noncovalently
introduced dyes into the polymeric matrix. To have an optimum
FRET phenomenon, in addition to photoluminescence proper-
ties and good overlap between donor and acceptor components
in the nanoparticles,24,37,44 particles size should reach to less than
100 nm.40 This could be attributed to the high scattering of light
beams by the particles, while they are equal or larger than half of
the incident light wavelength (λ/2). The strength point in
emulsion polymerization is the capability to tune particles size by
variation of some parameters such as surfactants concentration or
ionic strength of the polymerization environment.
Thermal resistance of the prepared latex nanoparticles was
studied by TGA. Figure 8b indicates that thermal stability of the
G
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 7. UV−vis analysis of the latexes containing dual-color fluorescent polymeric nanoparticles before (- - -) and after () UV irradiation
at 365 nm.
Figure 8. DSC (a) and TGA (b) thermograms of NP and NP2 samples (the inset in TGA thermogram shows corresponding DTGs).
copolymer containing two chromophores shows a slight increase
with respect to PMMA polymeric matrix because of the presence
of aromatic structures. It should be noted that both samples
degrade in similar single-step and profile. A multistep decom-
position and different degradation pattern would be expectable, if
SPEA and/or AzoCzEA generate their own aggregates during
polymerization. Totally, the observations from DSC and TGA
thermograms imply that SPEA and AzoCzEA have been success-
fully copolymerized with MMA and distributed in the copolymer
chains appropriately.
H
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 9. Fluorescence spectroscopy of the prepared dual-color latexes before () and after (- - -) UV irradiation at 365 nm for 4 min (the samples were
preirradiated at 410 nm).
Figure 10. (a) Fluorescence emission spectra of NP2 dual-color fluorescence polymeric nanoparticles in water (excited at 410 nm), before and after UV
irradiation at 365 nm. (b) Schematic representation of the absorption and fluorescence spectra of AzoCzEA and MC chromophores (the gray area
indicates the spectral overlap between fluorescence emission of AzoCzEA* as the donor and absorption band of MC as the acceptor).
3.5. Fluorescence and Photoreversibility of the
Polymeric Nanoparticles. As mentioned before, merocyanine
isomer of SPEA has a fluorescence emission at λmax of 630 nm
after excitation at 550 nm. Therefore, a reasonable overlap
I
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 11. Reversible fluorescence photoswitching of AzoCzEA-SPEA in NP2 latexes upon UV and visible irradiations for 10 cycles (a) and variation in
fluorescence intensity for a cycle of UV (4 min) and visible (7 min) irradiation (b).
between fluorescence emission of AzoCzEA and absorption of
MC isomer will result in a nonradiation energy transfer from the
excited AzoCzEA to MC and subsequent fluorescence emission
of MC at 630 nm just by excitation at 410 nm.
Figure 10b demonstrates the overlap between AzoCzEA
fluorescence emission and MC absorption in the prepared
polymeric nanoparticles in 450−700 nm schematically. Another
possibility would be providing the appropriate situation for good
stacking between carbazole and spiropyran rings in the polymeric
nanoparticles that triggers FRET process efficiently.
The analysis on DA2 sample did not show any fluorescence
emission for NACzEtOH and SP−OH in acetone solution,
although UV−vis spectroscopy (Figure 4) revealed the
possibility of overlap between these two chromophores. This
could be attributed to the nonradiative decay of the excited states
(SP−OH* and/or NACzEtOH*) by internal conversion (IC)
processes that were discussed in the preceding. These ICs
predominate over the radiative processes, causing insignificant
fluorescence emission. Inclusion of these moieties into a
polymeric matrix would be a good resolution to suppress these
IC processes and observing preferred radiative emissions. In
other words, the probable ICs in solution would become less
probable while entrapped in a polymeric phase. This is a great
privilege for immobilization of these chromophores into a
polymeric matrix and removing internal conversions possibility.
Among polymeric systems, nanoparticles are of high importance
owing to their dispersibility in diverse media and having these dyes
in the liquid state (specifically in water phase for hydrophobic
dyes) and observing their radiation-emission responses.
In this work and with respect to the above approach, the
prementioned polymeric latex nanoparticles were prepared to
restrict dye leaching and aggregation and also ICs. These
particles exhibited good structural stability, photostability and
excellent switching. Fluorescence spectroscopy of the prepared
dispersed nanoparticles in water was recorded and shown in
Figure 9. They showed a green emission at 530 nm after
excitation at 410 nm. Upon UV irradiation at 365 nm for 4 min,
fluorescence intensity of the dual-color nanoparticles reduced
remarkably. This is due to the occurrence of SPEA isomerization,
which results in nonradiative energy transfer between AzoCzEA*
to MC isomer.
Photoswitchable nature of the fluorescent NP2 nanoparticles
has been shown in Figure 11 upon exposure to alternating
illumination cycles of UV (365 nm, 4 min) and vis (520 nm,
7 min) lights. Diluted NP2 latex with 1 wt % solid content was
irradiated with repeated cycles of UV−vis lights and in each cycle.
The sample was excited at 410 nm and the fluorescence intensity
was recorded at 450−600 nm. This process is due to the optically
switching and conversion between SP and MC by using
alternating irradiation at 365 and 520 nm in which, the
fluorescence intensity of AzoCzEA is switched “off” and “on”
in these nanoparticles, respectively. Our investigations depict
that the incorporation of both SPEA and AzoCzEA chromo-
phores into the hydrophobic polymeric matrix by covalent
linkage substantially enhances the photoreversibility beside
photostability in these nanoparticles. These imply that light-
induced reversibility of fluorescence emission can be repeated
several times (Figure 11a) with remarkable fatigue resistance for
long time without any leaching with respect to other nonbonded
dyes in polymers. Typically, the variations in fluorescence inten-
sity with time upon UV irradiation at 365 nm and also visible
irradiation at 520 nm in a cycle have been given in Figure 11b.
4. CONCLUSION
In this study, preparation and characterization of new dual-color
fluorescent polymeric nanoparticles with average particle size of
40−80 nm were discussed by means of emulsion polymerization
in the aqueous phase. An acrylic azocarbazole derivative
(AzoCzEA) was synthesized and copolymerized with MMA
and an acrylic-functionalized spiropyran (SPEA) and corre-
sponding FRET process was investigated in the obtained stimuli
responsive photoreversible nanoparticles. AzoCzEA was used as
the fluorescent (donor) moiety and SPEA was exploited as the
photochromic (acceptor) group. At certain conditions and ratios,
the intermolecular interactions between light absorbing donor
and energy receiving acceptors were found to be the main
responsible phenomenon for energy transfer. Hence, the
Fluorimetry analysis approved that NP2 sample with an
optimized ratio of the two chromophores shows maximum
energy transfer with complete quenching after UV irradiation at
365 nm for 4 min. Hence, the fluorescence red emission of MC
at 630 nm was observed (Figure 10a). Totally, red and green
fluorescence emissions are visible reversibly with switching UV
light on and off at 365 nm, respectively after excitation at 410 nm.
J
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
(9) Zhu, M.; Zhu, L.; Han, J. J.; Wu, W.; Hurst, J. K.; Li, A. D. Q.
Spiropyran-Based Photochromic Polymer Nanoparticles with Optically
Switchable Luminescence. J. Am. Chem. Soc. 2006, 128, 4303−4309.
(10) Lv, Y.; Liu, H.; Zhao, B.; Tian, Z.; Li, A. D. Q. Tuning
Photoswitchable Dual-Color Fluorescence from Core-Shell Polymer
Nanoparticles. Isr. J. Chem. 2013, 53, 294−302.
(11) Tian, W.; Tian, J. An insight into the solvent effect on photo-,
solvato-chromism of spiropyran through the perspective of intermo-
lecular interactions. Dyes Pigm. 2014, 105, 66−74.
(12) Liu, Y.; Wu, J.; Meng, L.; Zhang, L.; Lu, X. Self-assembled,
fluorescent polymeric micelles of a graft copolymer containing carbazole
for thermo-controlled drug delivery in vitro. J. Biomed. Mater. Res., Part B
2008, 85B, 435−443.
(13) Chang, C.-C.; Kuo, I.-C.; Ling, I.-F.; Chen, C.-T.; Chen, H.-C.;
Lou, P.-J.; Lin, J.-J.; Chang, T.-C. Detection of quadruplex DNA
structures in human telomeres by a fluorescent carbazole derivative.
Anal. Chem. 2004, 76, 4490−4494.
(14) Palayangoda, S. S.; Cai, X.; Adhikari, R. M.; Neckers, D. C.
Carbazole-based donor-acceptor compounds: highly fluorescent
organic nanoparticles. Org. Lett. 2008, 10, 281−284.
(15) van Dijken, A.; Bastiaansen, J. J. A. M.; Kiggen, N. M. M.;
Langeveld, B. M. W.; Rothe, C.; Monkman, A.; Bach, I.; Stossel, P.;
Brunner, K. Carbazole Compounds as Host Materials for Triplet
Emitters in Organic Light-Emitting Diodes: Polymer Hosts for High-
Efficiency Light-Emitting Diodes. J. Am. Chem. Soc. 2004, 126, 7718−
7727.
(16) Yoon, S.-J.; Chun, H.; Lee, M.-S.; Kim, N. Preparation of poly (N-
vinylcarbazole) (PVK) nanoparticles by emulsion polymerization and
PVK hollow particles. Synth. Met. 2009, 159, 518−522.
(17) Morin, J.-F.; Leclerc, M. Syntheses of conjugated polymers
derived from N-alkyl-2, 7-carbazoles. Macromolecules 2001, 34, 4680−
4682.
fluorescence and UV−vis analysis showed that the fluorescence
emission of AzoCzEA (λmax = 530 nm) overlapped appropriately
with the absorption band of SPEA (λmax of 550 nm after UV
irradiation at 365 nm). The optimized NP2 latex revealed a green
emission at 530 nm after excitation at 410 nm. Upon UV
irradiation at 365 nm, the above green fluorescence emission was
quenched and a red fluorescence at 630 nm was observed.
Maximum efficiency of nonradiative energy transfer for
quenching of the fluorescence emission was obtained the in
these dual-color fluorescent nanoparticles. The covalent bonding
between AzoCzEA, SPEA, and MMA in the polymeric matrix
was identified by thermal analysis and this was responsible for the
structural stability and preventing dye leakage as the main
concern in such systems. However, internal conversions with
surrounding environment were controlled by inclusion of the
chromophores and their fixation in the polymer. Investigations
on reversibility and fluorescence photoswitching of the prepared
dual-color latexes depict their prominent photostability and
excellent fatigue resistance with no leaching and aggregation of
the chromophores. These dual-color photoswitchable fluores-
cent nanoparticles could be exploited in biological systems such
cell labeling and cell imaging with fluorescence tracing capability,
rewriteable patterning, and photosensitive displays.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: +9821 4478 7000. Fax: +9821 4478 7023. E-mail:
a.mahdavian@ippi.ac.ir.
Notes
(18) Tsutsumi, N.; Kinashi, K.; Ogo, K.; Fukami, T.; Yabuhara, Y.;
Kawabe, Y.; Tada, K.; Fukuzawa, K.; Kawamoto, M.; Sassa, T.; Fujihara,
T.; Sasaki, T.; Naka, Y. Updatable Holographic Diffraction of
Monolithic Carbazole-Azobenzene Compound in Poly (methyl
methacrylate) Matrix. J. Phys. Chem. C 2015, 119, 18567−18572.
(19) Grazulevicius, J. V.; Strohriegl, P.; Pielichowski, J.; Pielichowski,
K. Carbazole-containing polymers: synthesis, properties and applica-
tions. Prog. Polym. Sci. 2003, 28, 1297−1353.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We wish to express our gratitude to Iran Polymer and
Petrochemical Institute (IPPI) for financial support of this
work (Grant No. 24761172).
(20) Zhang, B.; Chen, Y.; Zhuang, X.; Liu, G.; Yu, B.; Kang, E.-T.; Zhu,
J.; Li, Y.L. Poly (N-vinylcarbazole) chemically modified graphene oxide.
J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 2642−2649.
(21) Adhikari, R. M.; Mondal, R.; Shah, B. K.; Neckers, D. C. Synthesis
and photophysical properties of carbazole-based blue light-emitting
dendrimers. J. Org. Chem. 2007, 72, 4727−4732.
(22) Liu, Y.; Nishiura, M.; Wang, Y.; Hou, Z. π-Conjugated aromatic
enynes as a single-emitting component for white electroluminescence. J.
Am. Chem. Soc. 2006, 128, 5592−5593.
(23) Chen, J.; Zeng, F.; Wu, S.; Chen, Q.; Tong, Z. A core-shell
nanoparticle approach to photoreversible fluorescence modulation of a
hydrophobic dye in aqueous media. Chem. - Eur. J. 2008, 14, 4851−
4860.
(24) Chen, J.; Zeng, F.; Wu, S.; Su, J.; Tong, Z. Photoreversible
Fluorescent Modulation of Nanoparticles via One-Step Miniemulsion
Polymerization. Small 2009, 5, 970−978.
(25) Li, C.; Zhang, Y.; Hu, J.; Cheng, J.; Liu, S. Reversible three-state
switching of multicolor fluorescence emission by multiple stimuli
modulated FRET processes within thermoresponsive polymeric
micelles. Angew. Chem., Int. Ed. 2010, 49, 5120−5124.
(26) Zadran, S.; Standley, S.; Wong, K.; Otiniano, E.; Amighi, A.;
Baudry, M. Fluorescence resonance energy transfer (FRET)-based
biosensors: visualizing cellular dynamics and bioenergetics. Appl.
Microbiol. Biotechnol. 2012, 96, 895−902.
REFERENCES
■
(1) Chen, J.; Zhang, P.; Fang, G.; Yi, P.; Yu, X.; Li, X.; Zeng, F.; Wu, Sh.
Synthesis and characterization of novel reversible photoswitchable
fluorescent polymeric nanoparticles via one-step miniemulsion
polymerization. J. Phys. Chem. B 2011, 115, 3354−3362.
(2) Wang, Y.; Hong, C. H.; Pan, C. P. Spiropyran-based hyperbranched
star copolymer: synthesis, phototropy, FRET, and bioapplication.
Biomacromolecules 2012, 13, 2585−2593.
(3) Sennett, K. A.; Lindner, B. K.; Kaur, N.; Fetner, S. M.; Stitzel, S. E.
Synthesis and photochromic properties of methacryloxy 6-nitro-
spirobenzopyrans. Dyes Pigm. 2013, 98, 437−441.
(4) Beyer, C.; Wagenknecht, H. A. Synthesis of spiropyrans as building
blocks for molecular switches and dyads. J. Org. Chem. 2010, 75, 2752−
2755.
(5) Chen, J.; Zhong, W.; Tang, Y.; Wu, Z.; Li, Y.; Yi, P.; Jiang, j.
Amphiphilic BODIPY-Based Photoswitchable Fluorescent Polymeric
Nanoparticles for Rewritable Patterning and Dual-Color Cell Imaging.
Macromolecules 2015, 48, 3500−3508.
(6) Achilleos, D. S.; Vamvakaki, M. Multiresponsive spiropyran-based
copolymers synthesized by atom transfer radical polymerization.
Macromolecules 2010, 43, 7073−7081.
(7) Son, S.; Shin, E.; Kim, B.-S. Light-responsive micelles of spiropyran
initiated hyperbranched polyglycerol for smart drug delivery. Bio-
macromolecules 2014, 15, 628−634.
(8) Ming, L.; Gu, L. Y.; Zhang, Q.; Xue, M. Z.; Liu, Y. G. Preparation
and study of photoswitchable fluorescence nanoparticles based on
spirobenzopyran. Chin. Chem. Lett. 2013, 24, 1014−1018.
(27) Cheng, P.-C. The contrast formation in optical microscopy in
Handbook of Biological Confocal Microscopy, 3rd ed.; Pawley, J. B., Ed.;
Springer: New York, 2006; pp 162−206.
K
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
(28) Rajdev, P.; Basak, D.; Ghosh, S. Insights into Noncovalently Core
Cross-Linked Block Copolymer Micelles by Fluorescence Resonance
Energy Transfer (FRET) Studies. Macromolecules 2015, 48, 3360−3367.
(29) Roy, R.; Hohng, S.; Ha, T. A practical guide to single-molecule
FRET. Nat. Methods 2008, 5, 507−516.
(30) Helms, V. Principles of Computational Cell Biology; Wiley-VCH
Verlag GmbH & Co. KGaA: Weinheim, Germany, 2008.
(31) Clegg, R. M. Fluorescence resonance energy transfer. Curr. Opin.
Biotechnol. 1995, 6, 103−110.
(32) Zheng, J. Spectroscopy-based quantitative fluorescence resonance
energy transfer analysis. Ion Channels 2006, 337, 65−77.
(33) Zhu, M.-Q.; Zhang, G.-F.; Hu, Z.; Aldred, M. P.; Li, C.; Gong, W.-
L.; Chen, T.; Huang, Z.-L.; Liu, s. Reversible Fluorescence Switching of
Spiropyran-Conjugated Biodegradable Nanoparticles for Super-Reso-
lution Fluorescence Imaging. Macromolecules 2014, 47, 1543−1552.
(34) Ando, R.; Mizuno, H.; Miyawaki, A. Regulated fast
nucleocytoplasmic shuttling observed by reversible protein highlighting.
Science 2004, 306, 1370−1373.
(35) Wiedenmann, J.; Ivanchenko, S.; Oswald, F.; Schmitt, F.; Rocker,
̈
C.; Salih, A.; Spindler, K.-D.; Nienhaus, G. U. EosFP, a fluorescent
marker protein with UV-inducible green-to-red fluorescence con-
version. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 15905−15910.
(36) Chudakov, D. M.; Verkhusha, V. V.; Staroverov, D. B.; Souslova,
E. A.; Lukyanov, S.; Lukyanov, K. A. Photoswitchable cyan fluorescent
protein for protein tracking. Nat. Biotechnol. 2004, 22, 1435−1439.
(37) Zhu, L.; Wu, W.; Zhu, M.-Q.; Han, J. J.; Hurst, J. K.; Li, A. D. Q.
Reversibly photoswitchable dual-color fluorescent nanoparticles as new
tools for live-cell imaging. J. Am. Chem. Soc. 2007, 129, 3524−3526.
(38) Wang, S.; Yu, C.; Choi, M.; Kim, S. Synthesis and switching
properties of photochromic carbazole − spironaphthoxazine copolymer.
J. Photochem. Photobiol., A 2007, 192, 17−22.
(39) Bertarelli, C.; Bianco, A.; Castagna, R.; Pariani, G. Photo-
chromism into optics: Opportunities to develop light-triggered optical
elements. J. Photochem. Photobiol., C 2011, 12, 106−125.
(40) Abdollahi, A.; Mahdavian, A. R.; Salehi-Mobarakeh, H.
Preparation of stimuli-responsive functionalized latex nanoparticles.
The effect of spiropyran concentration on size and photochromic
properties. Langmuir 2015, 31, 10672−10682.
(41) Ho, M. S.; Barrett, C.; Paterson, J.; Esteghamatian, M.;
Natansohn, A.; Rochon, P. Synthesis and optical properties of poly
{(4-nitrophenyl)-[3-[N-[2-(methacryloyloxy) ethyl]-carbazolyl]] dia-
zene}. Macromolecules 1996, 29, 4613−4618.
(42) Valeur, B., Berberan-Santos, M. N. Molecular fluorescence:
principles and applications, 2nd ed.; Wiley-VCH Verlag & Co, KGaA:
Weinheim, Germany, 2012.
(43) Zhu, M. Q.; Zhang, G. F.; Li, C.; Aldred, M. P.; Chang, E.; Drezek,
R. a.; et al. Reversible two-photon photoswitching and two-photon
imaging of immunofunctionalized nanoparticles targeted to cancer cells.
J. Am. Chem. Soc. 2011, 133, 365−372.
(44) Raymo, M. F.; Tomasulo, M. Electron and energy transfer
modulation with photochromic switches. Chem. Soc. Rev. 2005, 34,
327−336.
L
DOI: 10.1021/acs.macromol.5b02401
Macromolecules XXXX, XXX, XXX−XXX