Reversible photoswitching aggregation and dissolution of spiropyran-functionalized copolymer and light-responsive FRET process

Article abstract of DOI:10.1016/j.cclet.2013.12.014

Well-defined, reversibly light-responsive amphiphilic diblock copolymer grafted with spiropyran, was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. The copolymer self-assembles into polymeric micelles in water and exhibits reversible dissolution and re-aggregation characteristics upon ultraviolet (UV) and visible (Vis)-light irradiation. The fluorescence response of spiropyran immobilized onto the copolymer was light switchable. When nitrobenzoxadiazolyl derivative (NBD) dyes are encapsulated into the core of the micelles, a reversible, light-responsive, dual-color fluorescence resonance energy transfer (FRET) system is constructed and processed, which is well regulated by alternatively UV/vis irradiation. We anticipate these photoswitchable and FRET lighting up nanoparticles will be useful in drug delivery and cell imaging or tracking synchronously.

Full text of DOI:10.1016/j.cclet.2013.12.014

Chinese Chemical Letters 25 (2014) 389–396
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Reversible photoswitching aggregation and dissolution of spiropyran-
functionalized copolymer and light-responsive FRET process
*
*
Lei-Xiao Yu, Yang Liu, Si-Chong Chen , Yue Guan, Yu-Zhong Wang
Center for Degradable and Flame-Retardant Polymeric Materials, State Key Laboratory of Polymer Materials Engineering, College of Chemistry,
National Engineering Laboratory of Eco-Friendly Polymeric Materials (Sichuan), Sichuan University, Chengdu 610064, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Well-defined, reversibly light-responsive amphiphilic diblock copolymer grafted with spiropyran, was
prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization. The copolymer
self-assembles into polymeric micelles in water and exhibits reversible dissolution and re-aggregation
characteristics upon ultraviolet (UV) and visible (Vis)-light irradiation. The fluorescence response of
spiropyran immobilized onto the copolymer was light switchable. When nitrobenzoxadiazolyl
derivative (NBD) dyes are encapsulated into the core of the micelles, a reversible, light-responsive,
dual-color fluorescence resonance energy transfer (FRET) system is constructed and processed, which is
well regulated by alternatively UV/vis irradiation. We anticipate these photoswitchable and FRET
lighting up nanoparticles will be useful in drug delivery and cell imaging or tracking synchronously.
ß 2013 Si-Chong Chen and Yu-Zhong Wang. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
Received 11 November 2013
Received in revised form 5 December 2013
Accepted 6 December 2013
Available online 24 December 2013
Keywords:
RAFT polymerization
Spiropyran
Light-responsive
Self-assemble
Fluorescence resonance energy transfer
(FRET)
1. Introduction
light-responsive polymer. Upon light irradiation, the chemical
(cross-linking, isomerization, etc.) or physical parameters (polarity,
Inspired by fantastic natural phenomena, stimuli–responsive
systems were established and extensively developed in the past
two decades. Among them, ever-increasing attention has been paid
to the field of responsive polymers due to their adjustable
molecular structure and polymorphism of morphologies [1–3],
which can exhibit reversible or irreversible changes in physical
properties and/or chemical structures in response to external
stimuli, such as pH [4,5], temperature [6,7], ionic [8], light [1–
3,9,10], electric and magnetic fields [11,12], redox potential
[13,14], CO2 [15,16] or a combination of them, to name a few
[16,17]. Of these stimuli, light is an especially attractive external
stimulus and has been used in various areas. The use of light to
trigger a desired reaction of a polymer enables more temporal and
positional control and have been frequently employed in specific
applications, such as optically rewritable data storage [18], optical
switching [19], chemical sensing [8], drug or enzyme delivery
systems [20], smart surfaces [21], cell imaging [22] and bio-
separation [23].
hydrophilicity, etc.) of the photochromic segment would be
responsively altered, inducing the change of polymer conforma-
tions. Driven by the potential application in controlled drug
delivery, the principles of amphiphilic block copolymer (BCP)
design for photo-dissociable core–shell micelles or vesicles
(polymersomes) have received increasing attention. This kind of
light-responsive BCP assembles can be regrouped into four types
based on their photo-induced structural changes [24]. The first
approach based on optically shifting the hydrophilic–hydrophobic
balance of BCPs is the most studied one. The other three
approaches are light induced breaking block junction, main chain
degradation and reversible cross-linking. Azobenzenes [1,25],
spiropyrans (SPs) [9,10,13,26], diazonaphthoquinone (DNQ) [27],
o-nitrobenzyl [28] and coumarin [29] compounds are extensively
studied photochromers. Compared to other photochromic mole-
cules, spiropyrans are demandable photochromic compounds,
which can easily and reversibly be inter-converted between ring-
closed, colorless spiropyran (SP) and colored, ring-opened
merocyanine (MC) forms with UV (365 nm) and visible light
(550 nm), respectively. The SP and MC states have remarkably
different chemical structures and physical properties. Consequent-
ly, with an incorporated spiropyran compound into polymer chain
as a hydrophobic segment, the hydrophilic-hydrophobic balance of
the copolymer would responsively be shifted with the isomeriza-
tion of SP to MC. The polymer assembles were also process
Anchoring photochromic groups into a polymer chain to enable
its light-sensitive property is a common strategy to construct a
*
Corresponding authors.
E-mail addresses: chensichong@scu.edu.cn (S.-C. Chen), yzwang@scu.edu.cn
(Y.-Z. Wang).
1001-8417/$ – see front matter ß 2013 Si-Chong Chen and Yu-Zhong Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.12.014

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L.-X. Yu et al. / Chinese Chemical Letters 25 (2014) 389–396
disrupted and regeneration. This property enables this functional
copolymer to be an excellent light triggered delivery system for
drugs. Because spiropyran has a light switchable fluorescence
property, it can be used as a fluorescence probe at the same time.
In this work, amphiphilic copolymer, PEG-b-PSPMA, was
synthesized via one step RAFT polymerization, as one of powerful
methods to give well-defined polymers. A PEG based macro-chain
transfer agent was prepared and used as the modulator to
manipulated the polymerization of 10-(2-methacryloxyethyl)-
30,30-dimethyl-6-nitro-spiro(2H-1-benzo-pyran-2,20-indoline)
(SPMA). This SP-based copolymer was self-assembled into
micelles, which can be disrupted by UV irradiation and reversibly
regenerated by irradiating with visible light. The light switchable
responsiveness was very fast and not very dependent on the
photochromic concentration. Moreover, the copolymer containing
device with THF as eluent at a flow rate of 0.6 mL minꢀ1 at 40 8C.
UV/vis absorption was acquired on a VARIAN Cary 50 Probe
spectrophotometer. The measurements of fluorescence spectra
were carried out on
a VARIAN Cary Eclipse luminescence
spectrometer at room temperature. TEM observations were
conducted on a Tecnai G2 F20 electron microscope (FEI Co.) at
an acceleration voltage of 80 kV. The average size and size
distribution of micelles in aqueous solution were determined by
dynamic light scattering (DLS) after filtrated through a 0.45
Millipore filter. The measurements were carried out on
m
m
a
Brookhaven model BI-200SM spectrometer and 9000AT correlator
using an Innova304 He–Ne laser (1 W, = 532 nm) at a fixed
scattering angle ( ) of 908 for during of 2 min. The temperature was
l
u
set at 25 8C. For irradiation experiments, the samples were
irradiated with UV light at 365 nm, or visible light at 550 nm.
The UV and visible light source is a 300 W high-pressure Xe lamp
(CEL-HXUV300, AULTT, Co., China).
grafted spiropyran displayed
a photo-switchable, dual-color,
fluorescence property in aqueous solution, which is suitable to
image the cancer cell tissue during anti-cancer drug release.
However, the fluorescence intensity of MC is so weak that it cannot
be distinguished from background signals. To address this
problem, a fluorescence resonance energy transfer (FRET) process
was introduced to increase the fluorescence signal. Therefore,
a hydrophobic fluorescent dye, nitrobenzoxadiazolyl derivative
(NBD), a good FRET donor to MC, was encapsulated within the
hydrophobic cavities of the polymeric micelles. The PEG-b-PSPMA
copolymer can also reduce the cytotoxicity and incompatibility
within aqueous media of NBD dye. Exposed to alternative UV/vis
irradiation, the photo-switching between orange and red fluores-
cence of this system can be facilely achieved. These light trigger
responsivities and photo-switchable FRET processes enable this
copolymer to have a potential application in drug delivery and cell
imaging or tracking synchronously.
2.1. RAFT polymerization of SPMA using PEG-CTA as macro RAFT
agent
A typical procedure of RAFT polymerization is as follows: Into a
10 mL polymerization tube, PEG-CTA (0.047 mmol, 0.25 g), SPMA
(4.7 mmol, 2 g), AIBN (0.048 mmol, 8 mg) and THF (5 mL) were
added. After bubbled N2 for 30 min, the tube was sealed and
immersed into an oil bath thermostated at 75 8C. The polymeriza-
tions were conducted 24 h and 48 h, respectively, and after which
the tubes were cooled to room temperature rapidly, and the
polymer was obtained after re-precipitation three times from
diethyl ether. The resulting product was dried overnight in a
vacuum oven at 40 8C temperature. (Mn,GPC = 6435, Mw/Mn = 1.22,
SP moiety was 9 for 24 h sample and Mn,GPC = 9617, Mw/Mn = 1.52,
SP moiety was 18 for 48 h sample).
2. Experimental
2.2. Micellization of the amphiphilic light-responsive copolymers
Poly(ethylene glycol, Mn = 5000) (PEG), 4-chloro-7-nitrobenzo-
furazan (NBD-Cl, 99%), 2,3,3-trimethylindolenine (98%),
2-iodoethanol (99%) and 5-nitrosalicylaldehyde (98%) were
purchased from Alfa and used as received. Methacryloyl chloride
was purchased from TCI and storage at ꢀ10 8C before use. S-
PEG-b-PSPMA (Mn,GPC = 6435, 10 mg) was dissolved in THF
(3 mL). Under vigorously stirring, deionized water was added
slowly. Then the dispersion was slowly stirred for another 5 h and
THF was removed by dialysis [molecular weight (Mw) cut off:
3000 Da] against deionized water for 48 h and then diluted to a
1-Dodecyl-S-(
a
,
a0-dimethyl-a00-acetic
acid)
trithiocarbonate
(DBATC) was synthesized according to Ref. [30] in 40.6% yield.
Monomer 10-(2-methacryloxyethyl)-30,30-dimethyl-6-nitro-spiro
(2H-1-benzopyran-2,20-indoline) (SPMA) was synthesized by
esterification of SPOH with methacryloyl chloride according to
Ref. [31] with slight modification in 40% yield. 1H NMR (SPMA)
preset concentration. The solution was filtered through a 0.45 mm
filter before use. The critical micellization concentrations (CMC) of
the copolymers were determined on a luminescence spectrometer
with pyrene as fluorescent probe at a fixed exciting wavelength (l)
of 334 nm. Complex PEG-b-PSPMA core–shell micelles encapsu-
lated with NDB dyes were prepared by a similar procedure.
(400 MHz, CDCl3):
d 1.16 (–CH3), 1.28 (–CH3), 1.91 (–CH3), 3.37–
3.62 (–N–CH2–), 4.3 (–O–CH2–), 5.56 and 6.07 (55CH2), 5.87
(–CH55), 6.67–6.80 (Ar–H and –CH55), 6.86–6.95 (2Ar55H), 7.06–
7.13 (Ar–H), 7.17–7.24 (Ar–H), 7.97–8.05 (2Ar–H). Poly(ethylene
glycol)-based chain transfer agent (PEG-CTA) was synthesized
according to the literature procedure [32] in a yield of 80%. 1H NMR
3. Results and discussion
3.1. Preparation of amphiphilic light-responsive copolymer PEG-
b-PSPMA
(400 MHz, DMSO-d6): d 0.9 (–CH3), 1.23 (–CH2–), 1.65 (–CH3), 3.42
(–CH2–S–), 3.28 (–O–CH3), 3.45–4.25 (–O–CH2–CH2–O–), 3.68
(–CO–O–CH2–CH2–), 4.5 (–CO–O–CH2–). 2,20-Azobisisobutyroni-
trile (AIBN) was purified by recrystallization from ethanol, and
then dried under vacuum. The water used in this work was double
distilled water, which was further purified with a Milli-Q system.
Tetrahydrofuran (THF, A.R.), dichloromethane (DCM, A.R.) and
triethylamine (A.R.) were distilled over CaH2 and then distilled just
before use. All other reagents were purchased from Sinopharm
Chemical Reagent Co., Ltd. and used as received.
The 1H NMR spectra were recorded in CDCl3 and DMSO-d6
solution, on a Bruker spectrometer operating at a frequency of
400 MHz for protons. The molecular weight and molecular
distribution measurements were performed using a Waters GPC
Similar to the common thermo RAFT polymerization proce-
dure, the synthetic strategy in this research is schematically
illustrated in Scheme 1. The macro RAFT agent, PEG-CTA, was
synthesized by esterification of mPEG with DBATC, a well known
trithiocarbonate chain transfer agent used in RAFT polymeriza-
tion, and catalyzed by DCC and DMAP. The successful esterifica-
tion was unambiguously demonstrated by the disappearance of
the characteristic resonance peak of the proton of carboxyl, and, at
the same time, the observed appearance of the peaks at
d 4.5 and
d
3.45–4.25, attributed to the protons of –CO–O–CH2– and PEG
segment, respectively, as illustrated in Fig. 1. The light-sensitive
monomer, SPMA was polymerized under RAFT conditions
manipulated by the rationally designed RAFT agent at 75 8C in

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L.-X. Yu et al. / Chinese Chemical Letters 25 (2014) 389–396
391
(a)
S
S
DCC
OH
O
n
O
n
O
OH2
C12H25
S
S
C
O
+
C12H25
S
S
C
O
O
DMAP
O
N
m
O
NO2
O
N
O
NO2
Polymerization
S
O
n
O
C12H25
S
S
C
O
O
+
O
O
S
O
C
O
C
O
O
C
O
C12H25 S
S
n
PEG-co-PSPMA
HO
(b)
N
NO2
O
NH
O
NO2
O
O
O
S
O
C
S
O
C
UV (λ = 350 nm)
vis (λ = 590 nm)
O
O
n
O
n
O
C
O
C12H25
S
S
m
C
O
C12H25
S
S
m
PEG-co-PSPMA
PEG-co-PMEMA
Scheme 1. Synthesis of diblock copolymer PEG-b-PSPMA (a) and photo-induced isomerization between SP and MC (b).
THF for a preset time. The number-average molecular weight of
the amphiphilic copolymer determined by conventional GPC was
6435 Da and the polydispersity index (PDI) was 1.22. The GPC
traces of PEG-b-PSPMA and macro-CTA precursor are shown in
Fig. 2. It can be seen that the molecular weight of PEG-PSPMA
[(Fig._1)TD$FIG]
shifts toward the high molecular weight region without any trace
of the macro RAFT agent. This shows that PEG-CTA is an efficient,
macro, chain transfer agent for the polymerization of SPMA with
low polydispersity. The content of SP units was calculated by
following equation:
Fig. 1. 1H NMR spectra of (a) SPMA in CDCl3, (b) PEG-CTA in DMSO-d6 and (c) PEG-b-PSPMA in DMSO-d6 at room temperature.

[(Fig._2)TD$FIG]
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L.-X. Yu et al. / Chinese Chemical Letters 25 (2014) 389–396
concentration (CMC) of the PEG112-b-PSPMA9 determined by
Mn = 4127
fluorescence spectrometer using pyrene as fluorescent probe was
0.01 mg mLꢀ1. TEM and DLS measurements were performed to
determine the morphology and size of the self-assemblies. Fig. 3A
shows the dimension of assembles measured by DLS. The micelles
have a number-average hydrodynamic diameter (Dh) of 157.3 nm
and a polydispersity of 0.188. The PDI of the micellar size was a
little larger compared to other self-assembled micelles. However,
Mw/Mn = 1.05
PEG-PSPMA,48h
PEG-PSPMA,24h
PEG-CTA
Mn = 6435
Mw/Mn = 1.22
Mn = 9617
Mw/Mn = 1.52
this is
a common phenomenon for spiropyran containing
copolymers because of the dynamic equilibrium between SP and
MC. As shown in Fig. 3B, the PEG112-b-PSPMA9 self-assembled into
spherical micelles in water with a diameter of about 100 nm.
Due to the light-responsive SP groups in the copolymer, the
micellar morphology was enabled to be light-switchable because
of the shift of hydrophobic–hydrophilic balance. It is well known
that the SP moieties display two stable states, which could be
reversibly inter-converted upon irradiation with UV and visible
light. Generally, the absorption spectrum of the SP form in aqueous
solution does not show any absorption band larger than 400 nm,
whereas the MC form displays a characteristic absorption band at
about 560 nm [39]. Therefore, the reversible inter-conversion of
the SPMA units incorporated in the polymer chains can be studied
via their UV/vis absorption spectra. First, 365 nm UV light was
employed to irradiate the micelles solution (0.1 mg mLꢀ1) at
different times. As shown in Fig. 4A, the micellar solution
transformed from colorless to purple. At the same time, the peak
at 565 nm was gradually enhanced with increasing UV irradiation
time, indicating that the closed, colorless SP form isomerized into
the colored, opened MC form. Moreover, it can be found in Fig. 4A
that the two curves almost coincide with each other after
irradiating for 6 min and 7 min, demonstrating that the solution
had reached a light-stationary state after 6 min of UV irradiation.
Then, the ‘‘excited’’ micellar solution was exposed to 550 nm
visible light for 70 min. The UV/vis absorption spectra were
recorded at pre-arranged time intervals. In this process, the
solution converted back to colorless with a decreasing of the
absorption peak at 565 nm as shown in Fig. 4B. These aforemen-
tioned results indicated the reversible conversion between SP
and MC. The t1/2 of the MC back to SP process is 13.5 min and
7.5
8.0
8.5
9.0
9.5
Elution time (min)
Fig. 2. GPC curves of PEG-b-PSPMA and the corresponding precursor PEG-CTA.
112 ꢁ 4 ꢁ I5:9
D p ¼
I3:45ꢀ4:25
where I3.45–4.25 is the intensity of the methylene protons of PEG
(–(CH2–CH2)112–). And the intensity I5.9 is the methine protons of
benzopyran phenyl group next to the spiro tetrahydral linkage
(–CH55).
The Dps of SP units in copolymers polymerized 24 h and 48 h
were 9 and 18, respectively. The results obtained by 1H NMR are in
good agreement with the value estimated by GPC, and suggested
that the polymerization of SPMA proceeds in a well-controlled
manner. However, The PDIs of the photo-responsive copolymers
ranged between 1.22 and 1.52, which are somewhat higher than
those reported for the controlled polymerization of styrene and
methacrylate monomers [33,34], but lower compared to those
spiropyran-based copolymers synthesized by conventional free
radical polymerization [35,36]. The slightly higher molecular
weight distributions of the copolymers are attributed to the low
molecular weights of the polymers and the polymerization of
highly functional monomers (SPMA) [37] and are similar to those
reported in the literature for P4VP-co-PSPMA copolymer analogs
synthesized by RAFT [10] and PMMA-co-PSPMA by ATRP [38].
18.3 min for polymer concentration 0.1 mg mLꢀ1 and 0.5 mg mLꢀ1
,
respectively. Increasing the SP moiety immobilized in polymer
chain induced just a little increase in isomerization kinetic
(t1/2 = 15.2 min for PEG112–b-PSPMA18 with
a concentration
3.2. Reversible light-responsiveness of the copolymer micelles
0.1 mg mLꢀ1). To test the cyclic reversible response ability, the
sample was alternatively irradiated with 365 nm UV light and
550 nm visible light. The change of MC absorbance, monitored by
UV spectroscopy is shown in Fig. 5. The reversible isomerization
can take place very well in the beginning several cycles. However,
Owing to the light-switchable hydrophobic PSPMA segment
and hydrophilic PEG segment, the PEG112-b-PSPMA9 copolymer
can self-assemble into micelles in water. The critical micellization
[(Fig._3)TD$FIG]
Fig. 3. Characterizations of PEG112-b-PSPMA9 micelles: (A) DLS size distribution and (B) TEM image.

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L.-X. Yu et al. / Chinese Chemical Letters 25 (2014) 389–396
393
Fig. 4. Photo-isomerization process of PEG112-b-PSPMA9 micelles (0.1 mg mLꢀ1) after irradiating with different lights tracked by UV–vis spectroscopy: (A) 365 nm UV light for
7 min (B) subsequent 550 nm visible light for 70 min.
there was a decline of the reversibility with the irradiation cycles,
which was caused by an inevitable degradation of the dyes [40].
DLS and TEM measurements were also conducted to monitor
the hydrophobic–hydrophilic balance shifting process. Fig. 6
shows the diameter change of the micelles after irradiated by
UV/vis light at different times. The results show that the average
diameter of assembles decreased significantly after irradiating
with UV light. Adjusting the light to 550 nm visible light for 50 min
and the diameter gradually increased. TEM was used to further
confirm the evolution of the assemble dimension. The TEM images
in Fig. 7 displayed, after irradiation for 7 min under UV light, that
most of the sphere micelles dissociated, but a few smaller micelles
with diameters of about 40 nm still existed. Since the isomeriza-
tion is a dynamic equilibrium, the SP moiety cannot totally switch
to MC moiety, therefore, the spherical micelles were not totally
dissociated after UV irradiating. When subsequently irradiated the
micelles with visible light for 50 min, the diameter increased to
about 100 nm, which is comparable to that of before irradiation.
However, it is noteworthy that these re-assembled micelle sizes
are not uniform and some aggregates existed. The H-stack between
[(Fig._5)TD$FIG]
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Upon UV
planar MC molecules, whose
entire molecular framework, is
p
-bonding network extends over the
plausible reason for this
a
phenomenon [41]. These results indicate that the diameter of
the micelles can be reversibly regulated by light.
3.3. The phototropy of the spiropyran-containing micelles and photo
reversible modulation (switching) fluorescence of the NBD dye
Upon vis
0
1
2
3
4
5
6
7
8
Cycles
Fluorescence resonance energy transfer (FRET) is a non-
irradiation energy transfer process between light-absorbing
donors and light-receiving acceptors occurring in nano-scale.
It is required that the emission band of the donors should overlap
significantly with the absorption bands of the acceptor and the
Fig. 5. Reversible photo-switching process of PEG112-b-PSPMA9 micelles
(0.1 mg mLꢀ1
alternatively irradiating with UV light (6 min) and vis light
(70 min) for several cycles.
)
[(Fig._6)TD$FIG]
A
B
160
150
140
130
120
110
100
90
365 nm
UV 0 min
UV 5 min
UV 7 min
Vis 10 min
Vis 30 min
Vis 50 min
550 nm
80
1
10
100
1000
0
5
10 15 20 25 30 35 40 45 50 55 60
Irradiation time (min)
Hydrodynamic diameter (nm)
Fig. 6. Size distributions of PEG112-b-PSPMA9 micelles after irradiation with UV/visible light for different times (A) and size evolvement after irradiation with UV/visible light
for different times (B).

3
F
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L.-X. Yu et al. / Chinese Chemical Letters 25 (2014) 389–396
Fig. 7. TEM images of PEG112-b-PSPMA9 micelles after irradiation with: (A) 365 nm UV light irradiation for 7 min and (B) subsequent 550 nm visible light irradiation for
50 min.
distance between them is within the Fo¨rster radius (generally
1–10 nm) [42]. As we mentioned above, the spiropyran molecules
have two stable states: SP form and MC form. The SP form exhibits
no fluorescence emission, but the MC form has a strong emission
between 600 nm to 700 nm. The nitrobenzoxadiazolyl derivative
dyes are a good ‘‘donor’’ to MC [43,44]. When the NBD dye was
loaded in the PEG112–b-PSPMA9 copolymer micelles, the micelles
containing both the donor (NBD dye) and acceptor (spiropyran)
may serve as the scaffold for the FRET process. Fig. 8A shows the
absorption and fluorescence spectra of the aqueous micellar
[(Fig._8)TD$FIG]
Fig. 8. (A) Absorption and fluorescence spectra (lex = 570 nm) of PEG112-b-PSPMA9 micelles dispersion without NBD. (B) Absorption and fluorescence spectra (lex = 492 nm)
of NBD-loaded micelle dispersion upon UV- and vis-light irradiation. (C) The fluorescence color changes of the dispersions with (C2) and without (C1) NBD dye upon UV and
vis irradiation.

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L.-X. Yu et al. / Chinese Chemical Letters 25 (2014) 389–396
395
350
300
250
200
150
100
50
A
350
300
250
200
150
100
50
B
365nm
Upon vis
550nm
20
Upon UV
0
0
0
1
2
3
4
5
6
7
8
9
10
11
0
10
30
40
50
60
70
Cycles
Time (min)
Fig. 9. Fluorescence response (excited at 492 nm and emitted at 530 nm) of the NBD-loaded micelles of PEG112-b-PSPMA9 to the irradiation with UV (365 nm) and visible
(550 nm) light (A), and the fluorescence intensity change for the NBD-loaded micelles of copolymer under alternative irradiation by UV and visible light (B). (The
concentration of NBD in the dispersion is 1.25 ꢁ 10ꢀ5 mol Lꢀ1).
solution upon visible (550 nm) and UV (365 nm) light irradiation.
Upon visible light, SP moieties in the micelles exhibited no
absorption from 450 nm to 700 nm, whereas under UV irradiation,
a new absorption band at ca. 565 nm appeared corresponding to
However, with repeated irradiation cycles, the fluorescence
intensity of the ‘‘off state’’ (upon UV irradiation) increased, while
the fluorescence intensity of the ‘‘on state’’ (upon vis irradiation)
decreased, because of the irreversible photodamage of some NBD
molecules and spiropyran reversibly fluorescence ‘‘turn on’’ and
‘‘turn off’’ was monitored by fluorescence spectrometer based on
the emission of NBD at 530 nm. The reversible behavior of the
complex micelles displayed well even after several irradiation
cycles (Fig. 9). However, with repeat irradiation cycles, the
fluorescence intensity of the ‘‘off state’’ (upon UV irradiation)
increased, while the fluorescence intensity of moieties under
repeated UV irradiation [43–45].
the MC form (Fig. 8A1). Under irradiation at
lex = 570 nm, the
spiropyran molecules in MC form emitted strong fluorescence as
shown in Fig. 8A2. The absorption spectrum and fluorescence
intensity of the NBD in the micelle systems upon visible and UV
light irradiation are illustrated in Fig. 8B. Upon visible light,
spiropyran moieties in the SP form exhibited no absorption from
500 nm to 700 nm, with only an absorbance band of NBD at
460 nm observed. Otherwise, there was only one emission peak of
NBD at 550 nm in the fluorescence spectra. Upon UV (365 nm)
irradiation, the absorbance of MC at about 560 nm was observed.
And a new emission band at 675 nm, corresponding to MC form,
appeared in the fluorescence spectra after 3 min of UV irradiation.
The fluorescence color of the NBD-loaded micelle solution changed
to red from orange (Fig. 8C). However, the characteristic
fluorescence emission intensity of NBD decreased significantly
compared to that before UV irradiation. This phenomenon is
ascribed to the quenching of NBD fluorescence by the MC during
the FRET process. Otherwise, there was a 20 nm blue-shift of the
NBD characteristic fluorescence emission during this process.
Moreover, the fluorescence of the complex micellar solution can be
reversibly quenched and recovered by alternating irradiation
between the visible and UV light. The color of the micellar solution
was reversibly changed correspondingly (Fig. 8C). From the
perspective of an energy-level match, the energy of the first-
excited singlet state of NBD was estimated to be 2.50 eV based on
its absorption and emission maxima. Similar calculations gave a
first-excited singlet-state energy of 3.65 eV for the SP form and
2.05 eV for the MC form of the spiropyran moiety [44]. Therefore,
before UV irradiation, no fluorescence quenching of NBD was
observed. However, upon UV irradiation, the SP converted into MC
and then the energy transfer from NBD to the spiropyran became
efficient. These effects validate the energy transfer from NBD to SP
is impossible, but is possible to the MC.
4. Conclusion
Amphiphilic copolymers have been successfully prepared by
one-step RAFT polymerization of SPMA, using PEG-CTA as the
macro-RAFT agent. The self-assembling of the copolymer in water
formed spherical micelles with PSPMA segment in the core and
PEG segment as the shell. The diameter of the micelles has a
reversible, light-switchable behavior due to the shift of hydropho-
bic–hydrophilic balance induced by the isomerization between the
SP form and MC form. As a well matched ‘‘donor’’ to MC in the FRET
process, NBD dyes encapsulated into the core of the micelles
formed the light-responsive fluorescence emission system. Upon
UV irradiation, the latent fluorescence moiety converts to the MC
form, and then the FRET process was turn-on. But when the light
changed to visible, concomitant with the MC isomerizing back to
SP, the FRET process was reversibly turned off. The photo-
switching fluorescence between orange and red can be facilely
achieved and repeated over many cycles by alternative UV/vis
irradiation. These photochromic properties and the photo-switch-
able FRET process of this amphiphilic copolymer enable the
potential application in drug delivery and cell imaging and tracking
synchronously.
Acknowledgments
Lastly, the reversibility of fluorescence modulation of NBD
loaded micelles via alternatively cycled irradiation with UV and
visible light was studied. The light triggered cycle of reversible
fluorescence ‘‘turn on’’ and ‘‘turn off’’ was monitored by
fluorescence spectrometer based on the emission of NBD at
530 nm. The reversible behavior of the complex micelles
performed well even after several irradiation cycles (Fig. 9).
This work was financially supported by the National Natural
Science Foundation of China (Nos. 51073106 and 51121001) and
Program for Changjiang Scholars and Innovative Research Team in
University of Ministry of Education of China (No. IRT1026). The
Analytical and Testing Center of Sichuan University provided TEM
analysis.

396
L.-X. Yu et al. / Chinese Chemical Letters 25 (2014) 389–396
[24] Y. Zhao, Light-responsive block copolymer micelles, Macromolecules 45 (2012)
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