A core-shell nanoparticle approach to photoreversible fluorescence modulation of a hydrophobic dye in aqueous media

Article abstract of DOI:10.1002/chem.200701994

Amphiphilic core-shell nanoparticles containing spiropyran moieties have been prepared in aqueous media. The nanoparticles consist of hydrophilic and biocompatible poly(ethyleneimine) (PEI) chain segments, which serve as the shell, and a hydrophobic copolymer of methyl methacrylate (MMA), a spiropyran-linked methacrylate, and a cross-linker, which forms the core of the nanoparticles. A hydrophobic fluorescent dye based on the nitrobenzoxadiazolyl (NBD) group was introduced into the nanoparticles to form NBD-nanoparticle complexes in water. The nanoparticles not only greatly enhance the fluorescence emission of the hydrophobic dye NBD in aqueous media, probably by accommodating the dye molecules in the interface between the hydrophilic shells and the hydrophobic cores, but also modulate the fluorescence of the dye through intraparticle energy transfer. This biocompatible and photoresponsive nanoparticle complex may find applications in biological areas such as biological diagnosis, imaging, and detection. In addition, this nanoparticle approach will open up possibilities for the fluorescence modulation of other hydrophobic fluorophores in aqueous media.

Full text of DOI:10.1002/chem.200701994

FULL PAPER
DOI: 10.1002/chem.200701994
A Core–Shell Nanoparticle Approach to Photoreversible Fluorescence
Modulation of a Hydrophobic Dye in AqueousMedia
[
a]
Jian Chen, Fang Zeng, Shuizhu Wu,* Qiming Chen, and Zhen Tong
Abstract: Amphiphilic core–shell nano-
particles containing spiropyran moiet-
ies have been prepared in aqueous
media. The nanoparticles consist of hy-
drophilic and biocompatible poly(eth-
was introduced into the nanoparticles
to form NBD–nanoparticle complexes
in water. The nanoparticles not only
greatly enhance the fluorescence emis-
sion of the hydrophobic dye NBD in
aqueous media, probably by accommo-
dating the dye molecules in the inter-
face between the hydrophilic shells and
the hydrophobic cores, but also modu-
late the fluorescence of the dye
through intraparticle energy transfer.
This biocompatible and photorespon-
sive nanoparticle complex may find ap-
plications in biological areas such as
biological diagnosis, imaging, and de-
tection. In addition, this nanoparticle
approach will open up possibilities for
the fluorescence modulation of other
hydrophobic fluorophores in aqueous
media.
A
C
H
T
R
E
U
N
G
yleneimine) (PEI) chain segments,
A
C
H
T
R
E
U
N
G
which serve as the shell, and a hydro-
phobic copolymer of methyl methacry-
late (MMA), a spiropyran-linked meth-
acrylate, and
a cross-linker, which
Keywords: chromophores · fluores-
cence · nanoparticles · polymers ·
spiro compounds
forms the core of the nanoparticles. A
hydrophobic fluorescent dye based on
the nitrobenzoxadiazolyl (NBD) group
Introduction
rescence modulation of fluorophores in aqueous media, for
example, by chemical modification of fluorophores to pro-
[6,7]
Materials with properties that can be optically modulated
are of high interest in a broad range of scientific fields, in-
cluding biotechnology and advanced optics.
duce dyad or triad molecules.
However, the fluorophore
moieties in the dyad or triad molecules still tend to aggre-
gate in aqueous media, which usually leads to fluorescence
quenching. In addition, they usually lack the necessary bio-
compatibility for applications in biotechnology.
Recently, fluorescent nanoparticles have attracted increas-
ing attention as a result of their advantages over conven-
tional dyes for labeling and sensing. They hold the most
promise as three-dimensional scaffolds for the development
of new tunable and versatile sensing devices because the se-
lectivity and operating range can be monitored, without any
synthetic effort, by changing the receptor and/or the dye.
[1–3]
Fluorescence
techniques are rapidly becoming a leading methodology in
many biological diagnosis, imaging, and detection applica-
tions, primarily because of their versatility and ease of use.
The modulation of fluorescence emission in aqueous media
represents a new trend in the application of fluorescence in
biological studies because it can be used to selectively high-
[4]
light cells, organelles, or proteins. Photochromic com-
pounds such as spiropyran, which can be interconverted re-
versibly by light between two states with different spectro-
scopic properties, can be used to achieve photoreversible
Hence, they are widely used as novel luminescent probes in
[5]
[8,9]
fluorescence modulation through energy transfer.
In
biological and environmental fields.
The fluorescent
recent years, various attempts have been made to enhance
the fluorescence emission and achieve photoreversible fluo-
nanoparticles are not only very bright and exhibit improved
photostability, but also feature versatility in their design and
synthesis. They can be prepared by incorporating fluorescent
dyes into various quantum dots and nanoparticles by cova-
lent linkage, entrapment, or self-assembly strategies. Thus,
the inclusion of fluorescent labels or probes within nanopar-
ticles is an attractive strategy for the application of fluores-
cence techniques in aqueous systems. In many of the appli-
cations that involve the use of fluorescent nanoparticles,
fluorescence resonance energy transfer (FRET), which is an
[10]
[
a] J. Chen, Prof. F. Zeng, Prof. S. Wu, Q. Chen, Prof. Z. Tong
College of Materials Science and Engineering
South China University of Technology, Guangzhou 510640 (China)
Fax : (+86)20-8711-4649
E-mail: shzhwu@scut.edu.cn
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2008, 14, 4851 – 4860
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4851

attractive mechanism because it does not directly produce
redox-active ions that could lead to photodamage or other
undesirable processes, has been widely employed to realize
the various functions that operate in, for example, sensors,
nanoparticle system. This fluorescent nanoparticle complex
possesses several features.
First, the nanoparticles with PEI chain segments as the
shell are hydrophilic and free from other surfactant mole-
cules; the particle matrix can protect the biological environ-
ment (for example, the intracellular environment) from any
toxic dyes. Branched PEI was used in this study because
PEI is a well-studied and commercially available polyamine
with defined molecular weights. Also, a series of nanoparti-
cles could be obtained by choosing PEIs with different mo-
lecular weights. As a weak, polybasic aliphatic amine with a
high concentration of amino groups, PEI has been widely
used in the fields of biochemistry and medicine as a result
of its unique features, such as its ability to bind to biomacro-
molecules. On the one hand, PEI can form complexes with
biological substances and exert positive effects on the latter,
for example, PEI as a polycation can interact with proteins
through long-range coulombic forces and increase the stabil-
ity of the proteins as well as enhance the rate of enzyme-cat-
[11]
[12]
[13]
probes,
optical switches,
and biological imaging.
In
addition, FRET can be applied experimentally to character-
[14]
ize the nanoparticles.
Among their various potential applications, fluorescent
nanoparticles can be utilized for fluorescence modulation.
Park and co-workers synthesized a new class of fluorescent
and photochromic compounds and used one of these to
form fluorescent nanoparticles that exhibited high-contrast
on/off fluorescence switching properties upon UV/Vis irradi-
[9a]
ation. Very recently, Zhu et al. incorporated a spiropyran
and a fluorescent dye into polymer nanoparticles to gener-
ate optically addressable two-color fluorescent systems, and
[15]
they used light to change the color of the nanoparticles.
Herein, we demonstrate a new strategy using a class of
spiropyran-containing amphiphilic core–shell nanoparticles
to realize photoreversible fluorescence modulation
[17]
alyzed reactions. Moreover, PEI can form polyplexes with
DNA through covalent conjugation or coulombic interaction
(
Scheme 1). The nanoparticles can incorporate hydrophobic
[18]
fluorescent dyes and reversibly switch their fluorescence
emission upon UV or visible-light irradiation. In this way,
various hydrophobic dyes can be easily incorporated into
the nanoparticles without additional synthetic effort. In the
and thus enhance the transfection efficiency. On the other
hand, the interaction of PEI with proteins often leads to
[19]
negative effects such as unspecific bindings and, accord-
ingly, cytosolic protein chemistry can interfere with the
[20]
nanoparticles, the hydrophilic and biocompatible poly(eth
A
H
R
U
G
above-mentioned polyplexes. Various measures have been
taken to reduce or avoid these unspecific bindings, for ex-
ACHTREUNG
eneimine) (PEI) chain segments act as the shell and a hy-
[21]
drophobic copolymer of methyl methacrylate (MMA), a spi-
ropyran-linked methacrylate (SPMA), and a cross-linker
forms the core of the nanoparticles. In this study, a hydro-
phobic fluorescent probe based on a hydrophobic nitroben-
ample, branched PEI and certain PEI derivatives can be
[21a]
used to avoid unspecific binding
and a natural anionic
mucopolysaccharide can be deposited onto the cationic sur-
face of DNA–PEI complexes to recharge the surface poten-
tial and reduce the number of unspecific interactions with
[16]
zoxadiazolyl dye (NBD) was loaded onto the nanoparti-
cles in aqueous media to form a photoswitchable fluorescent
[21b]
proteins.
In this approach to nanoparticle preparation,
many amine-containing water-soluble polymers can be used
to initiate the formation of
nanoparticles. Thus, the un-
modified branched PEI used in
this study, which may cause un-
specific binding in biological
applications, could eventually
be replaced by other amine-
containing polymers such as
modified PEIs or other biomac-
romolecules so that the unspe-
cific binding can be reduced or
avoided.
Second, many dyes are hy-
drophobic and in aqueous
media their fluorescence emis-
sion is very weak as a result of
their poor solubility. Moreover,
for those dyes that undergo
photoinduced
intramolecular
charge transfer (ICT) upon ex-
citation and emit fluorescence
as a result of the charge recom-
[22]
Scheme 1. Photoreversible fluorescence modulation of the NBD–nanoparticle complex.
bination process, high polari-
4852
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 4851 – 4860

Photoreversible Fluorescence Modulation of a Dye
FULL PAPER
ty solvents like water often shift the fluorescence spectra of
ditional spiropyran-linked methacrylate (SPMA) monomer
was used in the reaction and covalently incorporated into
the nanoparticles. In this preparation, a water-soluble initia-
tor, tert-butyl hydroperoxide (TBHP), was used to generate
macromolecular free radicals by reacting with the amine
groups in the PEI chains to initiate the polymerization of a
mixture of methyl methacrylate (MMA)/acrylate-linked spi-
ropyran (SPMA)/ethylene dimethacrylate (EDMA) in
water, thereby forming a copolymer of PEI-graft-(MMA-co-
SPMA-co-EDMA). As the copolymer of MMA, SPMA,
and EDMA is hydrophobic, whereas the PEI chains remain
hydrophilic, the amphiphilic copolymers consequently form
the core–shell particles in water and the PEI chain segments
the shell. The average diameters of the prepared core–shell
particles are in the range of around 80–130 nm, depending
on the PEIs used and the ratio of PEI to MMA. Specifically,
[22b,c]
those dyes strongly to the red.
In addition, for dyes that
can form hydrogen bonds with water molecules, hydrogen-
bonding often leads to nonradiative dissipation of the excita-
tion energy into hydrogen-bonding vibrations, and hence sig-
[23]
nificantly quenches the fluorescence emission of the dyes.
Thus, for the strategy described herein, by solubilizing the
hydrophobic fluorescent dyes and providing them with a hy-
drophobic environment, the amphiphilic core–shell nanopar-
ticles not only retain the high fluorescence emission intensi-
ty of the dye, but also prevent the unfavorable effects of
dye–water interactions. Moreover, this approach using am-
phiphilic nanoparticles can be applied to other hydrophobic
fluorophores as the NBD dye herein can be readily replaced
by other hydrophobic dyes without covalently connecting
the dyes to the nanoparticles.
Finally, the nanometer size of the particles not only mini-
mizes physical perturbation in biological substances, but also
confines a considerable number of the fluorescent dye mole-
cules (donor) and spiropyran moieties (acceptor) within the
Fçrster radius. Thus the fluorescence of the former can be
photoreversibly modulated by the latter through energy
transfer.
for sample NP-A1, with a 1:4 weight ratio of PEI (M =
w
1800) to MMA, we can obtain a stable dispersion of nano-
particles with an average diameter of 82 nm, as determined
by atomic force microscopy (AFM) and dynamic light scat-
tering (DLS) (Figure 1). The average particle sizes of the
samples determined by DLS are listed in Table 1.
The fluorescent dye/nanoparticle prepared in this study
may find applications in biological diagnosis, imaging, and
detection. For example, like the polymer nanoparticles pre-
[15]
pared by Zhu et al., the amphiphilic core–shell nanoparti-
cles can also be transported into cells by using liposomes as
delivery vehicles and be used to highlight the cells; this
highlighting of the cells can be controlled by light irradia-
tion. In addition, for the detection (or diagnosis) of cancer
cells, the nanoparticle complexes, with amine groups on
their surfaces, can be readily conjugated (through covalent
bonds or coulombic interactions) with a specific antibody to
form bioconjugated nanoparticles (antibody-labeled nano-
particles) and can accordingly be used for the detection of
cancer cells.
Results and Discussion
Amphiphilic nanoparticles with spiropyran moieties can ac-
commodate a hydrophobic fluorescent dye in aqueous
media, thereby enhancing its fluorescence emission. In addi-
tion, the nanoparticles can photoreversibly modulate the
fluorescence emission of the incorporated hydrophobic dye,
as illustrated in Scheme 1.
Preparation of amphiphilic spiropyan-containing nanoparti-
cles: To synthesize nanoparticles with covalently linked spi-
ropyran groups, we first synthesized SPCOOH, a carboxy-
Figure 1. A) AFM image of spiropyran-containing nanoparticles (sample
NP-A1). B) Size distribution for the same nanoparticle sample deter-
mined by light scattering.
[24]
containing spiropyran, according to a literature procedure.
We obtained a spiropyran-containing methacrylate (SPMA)
monomer by esterification of SPCOOH with 2-hydroxyethyl
methacrylate (HEMA). The core–shell nanoparticles were
prepared by a procedure similar to that previously reported
The absorption spectra of spiropyran-linked methacrylate
(SPMA) in toluene as well as the spiropyran-containing (spi-
ropyran-doped) nanoparticles in water upon UV or visible-
[25]
[26]
by us following a literature strategy, except that an ad-
Chem. Eur. J. 2008, 14, 4851 – 4860
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4853

S. Wu et al.
Table 1. Spiropyran-containing nanoparticle samples prepared in this
work.
mer, after visible-light irradiation, the spiropyran moieties
assumed the SP form and exhibited no absorption band
from 400 to 700 nm. However, after UV irradiation, a new
absorption band at 580 nm appeared owing to the formation
of the McH form, as shown in Figure 2A. The absorption
behavior of the spiropyran-containing nanoparticles is simi-
lar to that of the methacrylate-linked spiropyran monomer
(Figure 2B) except that, because of a light-scattering effect
of the nanoparticles, the intensity of the absorption decreas-
es from low wavelength to high wavelength. This absorption
behavior of the amphiphilic nanoparticles indicates that the
spiropyran-containing nanoparticles can also exhibit photo-
chromic behavior in aqueous media.
[
a]
[b]
Sample
M
w
of PEI
SP feed
g]
Diameter
(by DLS) [nm]
[
ACHTREUNG
NP-A1
NP-A2
NP-A3
NP-A4
NP-B1
NP-B2
NP-B3
NP-C0
NP-C1
NP-C2
NP-C3
1800
1800
1800
0.10
82
92
86
97
106
104
111
123
119
116
124
0.050
0.025
0.0125
0.10
0.050
0.025
0.00
0.10
0.050
0.025
1800
10000
10000
10000
70000
70000
70000
70000
[
[
a] The MMA and EDMA feeds are 3.0 and 0.03 g, respectively.
b] Weight ratio of PEI/MMA is 1:4.
Introduction of a hydrophobic fluorescent dye into spiropyr-
an-containing nanoparticles: A fluorescent dye with a nitro-
benzoxadiazolyl (NBD) group and a C alkyl tail (structure
8
light irradiation are shown in Figure 2. It is well known that
spiropyran molecules can assume one of two stable states:
The open-ring state, known as the protonated merocyanine
is shown in Scheme 1) was synthesized and used in this
study as the model hydrophobic dye. This NBD dye is hy-
drophobic as the nitrogen atom undergoes a strong electron-
withdrawing effect from the nitrobenzoxadiazolyl group.
Figures 3 and 4 show the absorption spectra and fluores-
cence emission of NBD in pure water and in nanoparticle
(
(
McH) form, and the closed-ring state, known as the spiro
SP) form. Upon irradiation with UV light, the spiropyran
molecules adopt the McH form, whereas with visible light,
[3,27]
they adopt the SP form.
Similarly, for the SPMA mono-
Figure 3. Absorption spectra of a saturated water solution of NBD, NBD
in a nanoparticle dispersion (sample NP-C0), and the neat nanoparticle
dispersion (sample NP-C0). To avoid interference of the absorption band
by spiropyran moieties, no spiropyran monomer was incorporated into
the nanoparticles.
dispersions, respectively. The NBD dye, owing to its hydro-
phobic nature, exhibits very low solubility in pure water. Its
solution remains almost colorless with a very weak fluores-
cence emission at 541 nm (inset of Figure 4), and no reliable
UV/Vis absorption spectrum could be obtained from its sa-
turated water solution.
Although the NBD dye exhibits very low solubility in
pure water, it can still be transferred to the spiropyran-
doped nanoparticles from its solid state in water by stirring
at 508C, as described in the Experimental Section. The am-
phiphilic nanoparticles acted like surfactant molecules and
incorporated the hydrophobic dye molecules, thereby drasti-
cally increasing the solubility of the dye in the aqueous dis-
Figure 2. Absorption spectra of A) the spiropyran-linked methacrylate
(
(
SPMA) monomer in toluene and B) spiropyran-containing nanoparticles
sample NP-A3) in water with a solid content of 5.0 wt% upon UV or
visible light irradiation. In panel B, the decrease in the absorption curves
from short to long wavelength is due to the light scattering effect of parti-
cles in water.
4854
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Chem. Eur. J. 2008, 14, 4851 – 4860

Photoreversible Fluorescence Modulation of a Dye
FULL PAPER
cence intensity of NBD by spiropyran moieties for three
nanoparticle samples. After a NBD–nanoparticle complex
dispersion was irradiated with UV light (300 nm, 15 W) for
1
5 minutes, it was immediately transferred to the fluores-
cence spectrometer to measure the emission (at an excita-
tion wavelength of 490 nm). The characteristic fluorescence
emission from NBD at 533 nm was significantly quenched
and a new emission band at 635 nm appeared, which is the
emission from the open-ring state of the spiropyran moiety.
After irradiation with visible light (525 nm, 15 W LED
lamp) for 20 minutes, the fluorescence intensity at 533 nm
was restored (Figure 5).
Figure 4. Fluorescence emission of the NBD dye in pure water and in
water dispersions of different nanoparticles (excited at 490 nm). The fluo-
rescence spectrum of NBD in pure water was recorded by using a satu-
rated water solution of NBD. The solid content of the nanoparticle dis-
persions was 5.0 wt%. The NBD dye was added to these nanoparticle
dispersions until the complexes reached their maximum emission intensi-
ꢁ
5
ꢁ5
ty and the NBD concentrations (in water) were 1.07”10 , 1.89”10
,
ꢁ
5
and 4.23”10 m in the NP-A3, NP-B3, and NP-C3 dispersions, respec-
tively.
persion. As a result of the introduction of the dye into the
nanoparticle, the NBD–nanoparticle dispersion exhibited a
prominent absorption at around 470 nm (Figure 3). More-
over, the fluorescence intensity of the NBD–nanoparticle
dispersion increased greatly (by ꢀ40–180 times) and a mod-
erate blue shift of 8 nm was observed relative to that in pure
water, as shown in Figure 4. The results indicate that the
dye molecules were actually incorporated into the nanopar-
ticles, and the strong fluorescence intensity of the dye sug-
gests that the dye molecules reside in a more hydrophobic
environment, in this case probably at the interface between
the hydrophilic PEI shell and the hydrophobic PMMA/
SPMA core.
In this study, we used three different nanoparticle samples
to accommodate the hydrophobic dye. To test the accommo-
dating capacity of the different nanoparticles, we gradually
increased the amount of NBD dye during complex forma-
tion until the emission intensity of the complexes reached a
maximum. It can be seen in Figure 4 that the fluorescence
emissions of the NBD dye in the three nanoparticle disper-
sions have different intensities. For the three nanoparticle
samples (NP-A3, NP-B3, and NP-C3), the one with PEI
chains of the highest molecular weight could incorporate
more NBD molecules and the higher intensity at the maxi-
mum emission wavelength was due to the higher amount of
NBD incorporated into the core–shell interface of the nano-
particles as the fluorescence intensities were not normalized
to the corresponding optical density at the excitation wave-
length of the solutions. However, the reason for this fluores-
cence dependence on the PEI molecular weight is presently
unclear.
Figure 5. Fluorescence emission (excited at 490 nm, 258C) of NBD–nano-
particle complexes modulated by UV irradiation (300 nm) or by visible-
ꢁ
5
light (525 nm) irradiation. The concentration of NBD is 1.07”10 m in
ꢁ
3
aqueous dispersions and that of the spiropyran moiety is A) 1.2”10
ꢁ
4
(
sample NP-A1, spiropyran feed: 0.10 g), B) 3.0”10 (sample NP-A3,
spiropyran feed: 0.025 g), and C) 1.5”10 m (sample NP-A4, spiropyran
Photoreversible modulation (switching) of fluorescence of
ꢁ4
the NBD dye: Figure 5 shows the modulation of the fluores-
feed: 0.0125 g), respectively.
Chem. Eur. J. 2008, 14, 4851 – 4860
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4855

S. Wu et al.
[31]
For photoreversible fluorescence modulation using the
range of R ꢂ0.5R . For NP-A1, this distance is 22ꢃrꢃ
0
0
[28–30]
dyad strategy,
the energy transfer process can be ex-
65 Š.
ploited to modulate the emissive behavior of the fluoro-
phore–spiropyran (or other photochromes) dyads or triads
as long as the emission band of the fluorophore and the ab-
sorption bands of the photochrome overlap significantly in
one of the two states of the spiropyran fragment and the dis-
tance between the fluorophore (donor) and the spiropyran
moiety (acceptor) is within the Fçrster radius (generally 1–
6
0
R
E ¼
ð1Þ
6
6
R þ r
0
We used the sample NP-A1 to compare the effective energy
transfer distance with the nanoparticle size. The measured
average diameter of the core–shell nanoparticle is 82 nm,
and according to the amount of shell (PEI) and core materi-
al (mainly PMMA) fed into the reaction medium, the aver-
age diameter of the hydrophobic core of this sample of
nanoparticles can roughly be calculated to be 76 nm. In the
core–shell nanoparticles, the NBD molecules (donor) reside
in the core–shell interface, whereas the spiropyran moieties
(acceptor) reside in the core. As the upper limit of the effec-
tive energy transfer distance in this sample is 65 Š (6.5 nm),
an outer sphere of about 6.5 nm thickness, which represents
about 43% of the overall volume of the hydrophobic core
of the nanoparticle system, serves as the effective area in
which the McH form of the spiropyran can quench the fluo-
rescence emission of NBD. In this regard, preparation of
smaller nanoparticles can further increase the ratio of the
effective volume in a nanoparticle.
Figure 5 also shows that, for the three samples (NP-A1,
NP-A3, and NP-A4, with their average diameter of hydro-
phobic cores ranging from ca. 76 to ca. 90 nm), the amount
of spiropyran moieties in the nanoparticle core can affect
the energy transfer efficiency. We calculated the experimen-
tal energy transfer efficiency E between NBD and the McH
form of spiropyran, the concentration of spiropyran in the
[31]
10 nm). Under these conditions, the photochemical trans-
formation of the spiropyran can activate or suppress the
transfer of the excitation energy from a fluorophore to a spi-
ropyran moiety. For the dyad or triad systems, the distance
between the energy donor and the acceptor can be con-
trolled by inserting a spacer between them. These dyad or
triad systems have the well-defined structure needed for ef-
fective energy transfer as well as good fluorescence modula-
tion. However, a complicated synthetic process is required
to prepare them. In this case, the emission band (500–
650 nm) of the fluorophore NBD overlaps the absorption
[
32]
band (500–650 nm) of the McH state of the spiropyran in
the nanoparticles. However, the SP state does not exhibit
any absorption band in the emission band of NBD (500–
650 nm), as shown in Figure 2. Thus, energy transfer from
the excited state of NBD to the SP form of spiropyran is im-
possible, whereas energy transfer to the McH form is possi-
ble. From the perspective of an energy-level match, the
energy of the first-excited singlet state of NBD was estimat-
ed to be 2.50 eV from the average frequencies of the ab-
sorption and emission bands (lmax =460 nm, lem =533 nm).
Similar calculations based upon absorption and emission
[12]
maxima
.65 eV (lmax =340 nm) for the SP form of the spiropyran
moiety in nanoparticles, and 2.05 eV (lmax =575 nm, lem
30 nm) for the McH form of the spiropyran moiety. These
gave a first-excited singlet-state energy of
3
dispersion (C ), the number of spiropyran moieties in one
nanoparticle (NSP/NP), and the average distance between the
spiropyran moieties (D ) for the three samples (see Sup-
SP
=
6
SP
data also indicate that energy transfer from NBD to the SP
form of spiropyran is impossible, whereas that from NBD to
the McH form is possible. Therefore, before ring-opening of
the spiropyran moiety, no fluorescence quenching of NBD
could be observed, whereas upon UV irradiation, the spiro-
pyran moiety converts into the open-ring McH form and
then the energy transfer from NBD to the McH form of spi-
ropyran became efficient.
The energy-level match between the excited state of NBD
and the McH form of spiropyran clearly indicates that the
observed quenching of NBD fluorescence by the McH form
arises through an energy transfer process. Fçrsterꢁs critical
porting Information) and listed them in Table 2.
Table 2. Characteristics of the three NBD–nanoparticle complexes sam-
ples.
Sample
C
SP
ꢁ4
D
[nm]
SP
N
SP/NP
E
R
0
[Š]
A
H
R
U
G
NP-A1
NP-A3
NP-A4
3.56
5.42
6.84
13340
3470
1720
0.926
0.917
0.592
43
42
42
Based on the monomer feed ratio, for the three samples,
each particle roughly contains on average 1720–13340 spiro-
pyran moieties, which corresponds to a spiropyran-linked
methacrylate (SPMA) feed of 0.0125–0.1 g (see the Experi-
mental Section). The corresponding volume occupied by
each spiropyran moiety is in the range of roughly 23–
distances R for this donor–acceptor pair in samples NP-A1,
0
NP-A3, and NP-A4 have been calculated to be 43, 42, and
42 Š, respectively (see the Supporting Information). The
slight differences in the R values between the three samples
0
may be due to different microenvironments in the different
[31]
3
nanoparticle samples. According to Fçrsterꢁs nonradiative
165 nm , or, on average, two spiropyran moieties are sepa-
[31,32]
energy transfer theory,
the energy transfer efficiency E,
rated by a distance of around 3.56–6.84 nm. In this study,
the NBD molecules were loaded into the nanoparticles at
ambient temperature, which is far below the glass transition
temperature of the hydrophobic core. Thus, most of the
expressed by Equation (1), depends on R and on the dis-
0
tance (r) between the donor (NBD) and the acceptor
(
McH). The energy transfer is effective over distances in the
4856
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Chem. Eur. J. 2008, 14, 4851 – 4860

Photoreversible Fluorescence Modulation of a Dye
FULL PAPER
NBD molecules cannot penetrate the inside of the cores and
are located at the interface between the hydrophilic shell
and the hydrophobic core. Based on the above estimation of
the inter-spiropyran distance, at the higher spiropyran feed
The switching time is comparable to that of a polymer film
[33]
incorporating spiropyran molecules, but longer than that
of spiropyran molecules in toluene or mineral oil solutions
[34]
(tens of seconds to a few minutes). The longer switching
time may be due to the solid-state environment of the nano-
particles in which configurational switching must overcome
energy barriers imposed by the closely packed organic mole-
cules. In nonpolar or low-polar solutions, however, the barri-
ers posed by fluidic solvent molecules may be much lower
than those in the solid-phase core of the nanoparticles.
Nonetheless, for some water-soluble spiropyran derivatives,
the conversion from the McH form to the SP form is slow in
water because high-polar solvent molecules tend to stabilize
(
for sample NP-A1 and NP-A3), there are plenty of spiro-
pyran moieties located within the Fçrster radius of the NBD
molecules at the interface between the hydrophilic shell and
hydrophobic core. Therefore, upon UV irradiation, intrapar-
ticle energy transfer from the excited NBD dye to the McH
state of the spiropyran moieties inside the nanoparticle core
can take place. Thus the fluorescence emission of NBD was
efficiently quenched (Figure 5). With a lower spiropyran
feed (sample NP-A4), the fluorescence emission of NBD
cannot be efficiently quenched, as illustrated in Figure 5C.
For this NBD–nanoparticle sample, the measured energy
transfer efficiency is only 0.59, and the calculated number of
spiropyrans in the effective outer sphere is about 720, still
more than that of NBD in one nanoparticle (ca. 120). We
suppose one possible reason for the absence of quenching is
that the spiropyran moieties are not homogeneously distrib-
uted in the hydrophobic cores of the nanoparticles.
[35]
the zwitterionic McH form.
Recently, we developed a
strategy for photoreversible fluorescence modulation by
modification of b-cyclodextrin with spiropyran, and we used
light to switch the fluorescence of rhodamine B (RhB) in-
[36]
cluded in b-cyclodextrin.
However, the reversibility was
only efficient in nonaqueous solution; the photochemical
conversion from the McH form to the SP form took several
hours in water. In this case, however, the spiropyran groups
reside in the hydrophobic cores of the nanoparticles, in
which the zwitterionic McH form cannot be stabilized by
water, and the fluorescence intensity of NBD can be recov-
ered much more quickly.
The reversible nature of the fluorescence modulation of
the NBD–nanoparticle complexes upon exposure to alter-
nating cycles of UV (300 nm) and visible-light (525 nm) illu-
mination is illustrated in Figure 7 and in Table S1 of the
Supporting Information. The visible light or UV can be ap-
plied to reversibly “turn on” and “turn off” the fluorescence
of NBD. The appearance of an aqueous dispersion of NBD–
nanoparticle complexes also reversibly changes on irradia-
tion with UV or visible light, as shown in Figure S4 in the
Supporting Information.
The above results and discussions suggest that the photo-
physical theory can be applied to explain FRET in this core/
shell nanoparticle system. However, the theory cannot accu-
rately work for this system because its structure is much
more complicated than those of unimolecule dyad systems,
for example, the particle sizes of the different samples are
not identical, the spiropyran moieties may not be homoge-
neously distributed in the particle cores, and short-range
[31b]
(
less than 1 nm) interactions
between donors (NBD) and
acceptors (spiropyran) may exist.
Figure 6 shows a typical photoresponse of a NBD–nano-
particle complex in a water dispersion. Upon irradiation
with 300 nm light, the fluorescence intensity gradually de-
creased over 20 min and then increased upon irradiation
with 525 nm light, taking about 20 min for the complex to
recover its fluorescence intensity. The switching of the fluo-
rescence intensity of NBD is due to the photochemical con-
version between the two states of the spiropyran moieties.
In Figure 7 and Table S1 in the Supporting Information,
with repeated cycles of visible and UV irradiation, one can
observe an increase in the fluorescence intensity of the “off
state” (upon UV irradiation) and a decrease in the fluores-
Figure 6. Fluorescence intensity (at 533 nm) changes of the NBD–nano-
particle (sample NP-A3) complex upon alternate irradiation with 300 nm
UV and 525 nm visible light. The concentration of NBD in the dispersion
Figure 7. Fluorescence emission intensities for the NBD–nanoparticle
ꢁ
5
complex in an aqueous medium (NBD concentration: 1.07”10 m, lex
=
490 nm) recorded at 533 nm and 258C upon cycles of UV and visible-
light irradiation.
ꢁ
5
is 1.07”10 m.
Chem. Eur. J. 2008, 14, 4851 – 4860
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4857

S. Wu et al.
cence intensity of the “on state” (upon visible-light irradia-
tion), which indicates that some NBD molecules and spiro-
with chloroform. Evaporation of water gave l-(b-carboxyethyl)-2,3,3-tri-
methylindolenine iodide (73% yield). This iodide (0.04 mol), 5-nitrosali-
cylaldehyde (0.04 mol), and piperidine (3.8 mL, 0.04 mol) were dissolved
in ethyl methyl ketone, and the red solution was refluxed for 3 h. On
standing overnight, the product precipitated as a yellow crystalline
powder. This was filtered and washed with methanol to give the product
[37]
pyran moieties undergo irreversible photodamage.
As a
result of photodamage, the amount of intact NBD dye in
the nanoparticles decreases. Thus the fluorescence intensity
upon repeated visible-light irradiation is lower than the “on
state” value observed upon the first visible-light irradiation.
On the other hand, the amount of intact spiropyran moieties
also decreases as a result of photodamage, and the remain-
ing intact spiropyran (McH form) is not capable of quench-
ing the fluorescence intensity of NBD to the “off state”
value observed upon the first UV irradiation.
1
SPCOOH (75% yield). The H NMR spectrum of SPCOOH is shown in
1
Figure S1 in the Supporting Information. H NMR (400 MHz, deuterated
DMSO, 258C, TMS): d=1.0–1.3 (6H; CH
2H; CH N), 5.9–6.0 (2H; olefinic protons), 6.6–8.2 (aromatic protons),
2.0 ppm (COOH, hydrogen-bonding).
3 2
), 2.6 (2H; CH COO), 3.4–3.5
(
2
1
For the synthesis of SPMA, SPCOOH (3.8 g, 10 mmol), HEMA (2.6 g,
20 mmol), and DMAP (0.272 g, 2 mmol) were added to a 100 mL round-
bottomed flask equipped with a pressure-equalized dropping funnel,
magnetic stirrer, and a nitrogen inlet. Dry THF (80 mL) was added to
the flask and the solution was cooled to 08C; a red-brown solution result-
ed. DCC (2.06 g, 10 mmol) was dissolved in dry THF (20 mL), and the
solution was added to the SPCOOH/HEMA solution through the pres-
sure-equalized dropping funnel over 45 min. The flask was maintained at
Conclusion
0
2
8C for 2 h and then the temperature was raised gradually to 258C over
4 h. The product was filtered with cold (08C), dry THF (3”50 mL) to
Amphiphilic core–shell nanoparticles containing spiropyran
moieties have been prepared and they not only greatly en-
hance the fluorescence emission of the hydrophobic dye
NBD in aqueous media, probably by accommodating the
dye molecules in the interfaces between the hydrophilic
shells and the hydrophobic cores, but also photoreversibly
modulate the fluorescence of the dye through energy trans-
fer. The hydrophobic core of the nanoparticles contains and
protects the spiropyran moieties, whereas the hydrophilic
shell allows the nanoparticles to be dispersed in water and
forms an interface with the hydrophobic core to accommo-
date the hydrophobic dyes. This approach can be readily ap-
plied to other hydrophobic fluorophores (chromophores),
thus opening up possibilities for their fluorescence modula-
tion in aqueous media.
give a red filtrate. Then most of the solvent was evaporated from the fil-
trate under vacuum and the residue was washed with distilled water to
give a red-purple precipitate. The precipitate was dissolved in benzene
and filtered again. Afterwards, most of the solvent was evaporated, the
solution was precipitated in a large amount of petroleum ether, and final-
ly a fine red-purple precipitate of purified 2-[3-(3’,3’-dimethyl-6-nitro-
A
H
R
U
G
methacrylate
(
SPMA) was obtained. The target product was dried in a vacuum oven
1
overnight at room temperature. The H NMR spectrum of this product is
shown in Figure S2 in the Supporting Information. H NMR (400 MHz,
1
CDCl
HEMA, connected to olefinic carbon), 2.6–2.7 (2H; CH
pyran), 3.5–3.6 (2H; CH N of spiropyran), 4.2 (4H; CH
.5–6.0 (4H; olefinic protons, CH and 2 CH), 6.6–8.1 ppm (aromatic pro-
tons).
Synthesis of the spiropyran-based amphiphilic core/shell particles:
Branched poly(ethyleneimine), with M of 1800, 10000, or 70000, respec-
3
, 258C): d=1.0–1.3 (6H; CH
3
of spiropyran), 1.8–1.9 (3H; CH
COO- of spiro-
O of HEMA),
3
of
2
2
2
5
2
w
tively, was dissolved in water (75 mL) and then mixed with a mixture of
purified MMA (3.0 g), ethylene dimethacrylate (EDMA, 0.03 g), and var-
ious amounts of SPMA (0.0125–0.1 g) in a three-necked flask equipped
with a thermometer, a condenser, a magnetic stirrer, and a nitrogen inlet.
The stirred mixture was purged with nitrogen for 30 min. The appropriate
amount of TBHP was added and the mixture was heated at 808C for 6 h
under nitrogen. The product was filtered through a G2 sintered glass
funnel. Then the colloid was centrifuged to remove residual water-soluble
molecules, and the precipitate was recovered and dialyzed against water
for 48 h. Finally an aqueous dispersion was obtained.
Experimental Section
Materials: Branched poly(ethyleneimines) (Acros), tert-butyl hydroper-
oxide (TBHP, Sigma), N,N’-dicyclohexylcarbodiimide (DCC, 99%, Alfa
Aesar), 4-dimethylaminopyridine (DMAP, 99%, Alfa Aesar), 2,3,3-tri-
methylindolenine (Aldrich), 3-iodopropanoic acid (Aldrich), ethylene di-
methacrylate (EDMA, Acros), and 5-nitrosalicylaldehyde (Aldrich) were
used as received. Dichloromethane (DCM, A.R.) was washed with sulfu-
Synthesis and introduction of the NBD dye into nanoparticles in aqueous
ric acid and then distilled from CaH
HEMA, 97%, Aldrich) was dissolved in water (25 vol%) and washed
four times with an equal volume of hexane, then dried with MgSO and
2
. 2-Hydroxyethyl methacrylate
media: The NBD dye (NBD-C8) was synthesized and purified according
(
[
38]
to a literature procedure. The prepared NBD dye was dissolved in di-
4
ꢁ
4
distilled under vacuum prior to use. The phenolic inhibitor in methyl
methacrylate (MMA, Aldrich) was removed by washing three times with
0% sodium hydroxide solution and then with deionized water until the
pH of the water layer was 7, and then it was further purified by vacuum
distillation. Double-distilled water further purified with a Milli-Q system
was used in this work. Tetrahydrofuran (THF, A.R.) was distilled over
chloromethane to give a 10 m of solution. A given amount of the solu-
tion was transferred to a 25 mL flask and then the solvent was evaporat-
ed under vacuum. The same flask with the dried NBD dye was then
filled with the aqueous dispersion of nanoparticles and the mixture was
stirred for 48 h at 508C to allow the dye molecules to move into the
nanoparticles. Subsequently the mixture was cooled to room tempera-
ture.
1
2
CaH . Petroleum ether, benzene, and other reagents were analytical re-
agents and used without further purification.
1
Characterization: H NMR spectra were recorded on a Bruker Avance
Synthesis of the carboxy-containing spiropyran (SPCOOH) and the spi-
ropyran-linked methacrylate monomer (SPMA): During the synthesis, all
the reaction vessels were wrapped in aluminum foil to ensure the reac-
tion was performed in the dark. The carboxy-containing spiropyran l’-(b-
400 MHz NMR spectrometer. UV/Vis spectra were recorded on a Hita-
chi U-3010 UV/Vis spectrophotometer at room temperature. Fluores-
cence spectra were recorded on a Hitachi F-4500 Fluorescence spectro-
photometer. Nanoparticle morphology was observed with a Seiko SII
atomic force microscope (AFM) in the tapping mode at room tempera-
ture. The diameters of the nanoparticles were analyzed with a MAL-
VERN Nano-ZS90 instrument. The diameters of the samples are the
averaged values with a systematic (instrumental) error of around 5%.
For the core–shell nanoparticle samples, the weight ratio of the shell
carboxyethyl)-3’,3’-dimethyl-6–nitrospiro[indoline-2’,2-chromane]
(re-
ferred to as SPCOOH) was synthesized as follows. 2,3,3-Trimethylindole-
nine (0.06 mol), 3-iodopropanoic acid (0.06 mol), and ethyl methyl
ketone (5 mL) were heated under nitrogen at 1008C for 3 h. The result-
ing solid material was dissolved in water and the solution was washed
4858
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 4851 – 4860

Photoreversible Fluorescence Modulation of a Dye
FULL PAPER
(
PEI) to core material (PMMA and spiropyran) are known and thus, by
P. E. Dawson, B. R. Fisher, B. OꢁRourke, H. Mattoussi, Adv. Mater.
2007, 19, 1921.
assuming all the materials underwent reaction and were incorporated
into the core–shell particles, the thickness of the shells and the diameters
of the cores could be calculated with errors of about 5%.
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Acknowledgement
[
This work was supported by the NSFC (Project Nos. 50573023, 50773022,
2
NCET.
0534020 and 50473035), the GDNSF (Project No. 07006497), and the
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