Amphiphilic BODIPY-Based Photoswitchable Fluorescent Polymeric Nanoparticles for Rewritable Patterning and Dual-Color Cell Imaging

Article abstract of DOI:10.1021/acs.macromol.5b00667

Photoswitchable fluorescent polymeric nanoparticles (PFPNs) with controllable molecular weight, high contrast, biocompatibility, and prominent photostability are highly desirable but still scarce for rewritable printing, super-resolution bioimaging, and r

Full text of DOI:10.1021/acs.macromol.5b00667

Article
pubs.acs.org/Macromolecules
Amphiphilic BODIPY-Based Photoswitchable Fluorescent Polymeric
Nanoparticles for Rewritable Patterning and Dual-Color Cell Imaging
Jian Chen,*,†,‡ Weibang Zhong,† Ying Tang,‡ Zhan Wu,‡ Ya Li,† Pinggui Yi,*,† and Jianhui Jiang*,‡
†Key Laboratory of Theoretical Organic Chemistry and Functional Molecule of Ministry of Education, Hunan University of Science
and Technology, Xiangtan 411201, P. R. China
‡State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, P. R. China
S
* Supporting Information
ABSTRACT: Photoswitchable fluorescent polymeric nanoparticles (PFPNs) with
controllable molecular weight, high contrast, biocompatibility, and prominent
photostability are highly desirable but still scarce for rewritable printing, super-
resolution bioimaging, and rewritable data storage. In this study, novel amphiphilic
BODIPY-based PFPNs with considerable merits are first synthesized by a facile one-
pot RAFT-mediated miniemulsion polymerization method. The polymerization is
performed by adopting polymerizable BODIPY and spiropyran derivatives, together
with MMA as monomer, and mediated by utilizing biocompatible PEO macro-RAFT
agent as both control agent and reactive stabilizer. The amphiphilic BODIPY-based
PFPNs not only exhibit reversibly photoswitchable fluorescence properties under the
alternative UV and visible light illumination through induced intraparticle fluorescence
resonance energy transfer (FRET) but also display controllable molecular weight with
narrow polydispersity index (PDI), high contrast of fluorescence, tunable energy
transfer efficiency, good biocompatibility, excellent photostability, favorable photo-
reversibility, etc. The as-prepared PFPNs are successfully demonstrated for rewritable fluorescence patterning and high-contrast
dual-color fluorescence imaging of living cells, implying its potential for rewritable data storage and broad biological applications
in cell biology and diagnostics.
different diffusion coefficients between the selected dyes with
monomer or matrix in water (or another media). In addition,
many reported PFNs often introduced fluorescent dyes via
doping or physical adsorption, which implies potential dye
leakage and aggregation, significantly limiting their advanced
biological applications.2d,8c Notably, although traditional
miniemulsion polymerization has displayed some advantages
for preparing PFNs, such as facile and versatile preparation
route, improved photostability, and tunable amount of
incorporated dyes,8 there are still some problems like surfactant
migration or desorption need to be resolved, when adopting
general ionic surfactant like sodium dodecyl sulfate (SDS).10 In
addition, the radical polymerization process in conventional
miniemulsion strategy also cannot control the polymerization
rate and the polydispersity index (PDI) of formed polymer,
which will be bring undesirable effects on the architecture of
prepared polymers or the morphology of particles.10 To
overcome these drawbacks, a facile and elegant way is to
adopt reversible addition−fragmentation chain transfer
(RAFT)-mediated miniemulsion polymerization strategy and
use amphiphilic macromolecular (macro-) RAFT agents as
both stabilizer and control agent.11 These macro-RAFT agents
INTRODUCTION
■
The successful preparation of photoswitchable fluorescent
nanoparticles (PFNs) has been extensively studied for their
potential applications as chemical sensing, rewritable data
storage, and ultrahigh-resolution biological imaging.1 In general,
PFNs often utilize photochromic compounds, such as
spiropyrans,2 diarylethenes,3 and spirooxazines,4 to bring
photoswitchable fluorescence property. In a popular fluores-
cence resonance energy transfer (FRET) strategy, these
photochromes, like spiropyrans,2 usually act as switchable
energy acceptors under the irradiation of UV or visible light to
quench or recover the fluorescence of another nearby energy-
matched fluorophores (energy donor). Obviously, these are the
desired features which help to overcome the autofluorescence
interference of cells in bioimaging or surpass the limitation of
spatial resolution of ∼250 nm for routine fluorescence
microscopy.1a,5
To date, various strategies such as surface modification,6 self-
assembly method,7 and microemulsion2a,b or miniemulsion
polymerization,8 etc., have been employed to prepare various
PFNs. However, it is noteworthy that the surface modification
of fluorescent nanoparticles like quantum dots has potential
defects such as blinking and cytotoxicity,9 and the self-assembly
strategy often involves a complicated synthesis,7 while the
adoption of microemulsion polymerization is hard to modulate
the amount of embedded dyes,2a,b which is ascribed to the
Received: March 30, 2015
Revised: May 4, 2015
© XXXX American Chemical Society
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Synthesis of BODIPY Methacrylate (BDPMA). BDPMA was
synthesized according to modified literature procedures.12 First, an
anhydrous CH2Cl2 solution (50 mL) of BODIPY phenol (475 mg, 1.2
mmol), methacrylic acid (309 mg, 3.6 mmol), and DMAP (29 mg,
0.24 mmol) was cooled to 0 °C under a N2 atmosphere; then, DCC
(370 mg, 1.8 mmol) in anhydrous CH2Cl2 (5 mL) was slowly added
to the opaque solution. The mixture was stirred at room temperature
for 24 h. After that the mixture was concentrated and purified by silica
gel column chromatography (CH2Cl2/petroleum ether = 7:3 v/v),
can induce the in situ generation of block copolymers with
controllable molecular weight and a predictably narrow PDI11
as well as form the final stable amphiphilic particles. However,
to the best of our knowledge, there is little information about
using macro-RAFT agents and RAFT-mediated miniemulsion
polymerization to fabricate PFNs.
On the other hand, as a well-known fluorescent dye,
BODIPY derivatives have exhibited high brightness and tunable
emission spectrum.12 Thus, they can be selected as ideal
candidates to fabricate various photoswitchable fluorescence
systems.4b,13 However, there is no report about introducing
BODIPY derivatives into nanoparticles to prepared novel
PFNs; most of the reported BODIPY-based photoswitchable
fluorescence systems are based on organic compounds which
often involve complicate synthetic pathway and cannot be
directly used in water due to their hydrophobic nature.4b,13
Obviously, these drawbacks strongly limit their potential
applications in the super-resolution imaging of biological
samples.
Herein, we reported the first preparation of novel
amphiphilic BODIPY-based photoswitchable fluorescent poly-
meric nanoparticles (PFPNs) in aqueous media via an one-pot
RAFT-mediated miniemulsion polymerization, with a biocom-
patible poly(ethylene oxide) (PEO) macro-RAFT agent (PEO-
TTC, Scheme S1) as both control agent and reactive stabilizer.
This novel strategy undoubtedly endows as-prepared PFPNs
with controllable molecular weight and narrow PDI, improved
stabilization, and biocompatibility. Moreover, in this study, two
hydrophobic dyes as BODIPY methacrylate (BDPMA) and
spiropyran-linked methacrylate (SPMA) were covalently linked
to the polymer backbone with tunable amount and ratio, which
can not only greatly increase the photostability of as-prepared
PFPNs but also significantly improve their brightness and
photoswitch contrast in water. In addition, these novel
amphiphilic PFPNs also reveal other advantages, such as
controllable FRET efficiency, improved long-term photo-
stability, and favorable photoreversibility, etc. More impor-
tantly, the novel PFPNs can be directly applied in rewritable
fluorescence patterning and high-contrast dual-color fluores-
cence imaging of living cells, which implies the great potential
in rewritable data storage and biological diagnosis.
1
affording 456 mg of BDPMA product (82% yield). H NMR (500
MHz, CDCl3, δ): 0.98 (t, 6H, J = 1.0 Hz, CH3), 1.34 (s, 6H, CH3),
2.09 (s, 3H, CH3), 2.30 (q, 4H, CH2), 2.53 (s, 6H, CH3), 5.81 (s, 1H,
CH), 6.40 (s, 1H, CH), 7.28 (d, 2H, Ar H), 7.33 (d, 2H, Ar H)
(Figure S3 of Supporting Information).
Synthesis of Spiropyran-Linked Methacrylate (SPMA). SPMA
was synthesized according to modified literature procedures.2c An
anhydrous CH2Cl2 solution (45 mL) of SP-OH (1.00 g, 2.84 mmol),
methacrylic acid (1.22 g, 14.20 mmol), and DMAP (0.069 g, 0.57
mmol) was cooled to 0 °C under a N2 atmosphere and protection
against sunlight exposure. Then DCC (0.877g, 4.26 mmol) in
anhydrous CH2Cl2 (5 mL) was slowly added to the solution over
0.5 h. The mixture was stirred for 24 h at room temperature, and then
the mixture was concentrated and purified by silica gel column
chromatography (CH2Cl2/petroleum ether = 2:1 v/v), affording 950
1
mg of SPMA product (80% yield). H NMR (500 MHz, CDCl3, δ):
1.17 (s, 3H, CH3), 1.28 (s, 3H, CH3), 1.92 (s, 3H, CH3), 3.45−3.54
(m, 2H, CH2), 4.3 (t, 2H, CH2), 5.57 and 6.07 (d, 2H, CH2), 5.87 (d,
1H, CH), 6.71 (q, 2H, Ar H) 6.92 (q, 2H, Ar H), 7.09 (d, 1H, Ar H),
7.17−7.23 (m, 1H, Ar H), 7.96−8.05 (m, 2H, Ar H) (Figure S4 of
Supporting Information).
Synthesis of Amphiphilic PFPNs. The organic phase containing
MMA, BDPMA, SPMA, hexadecane, and initiator AIBN was added to
the aqueous phase (water and PEO-TTC) and stirred for 15 min; then
the mixture was ultrasonicated (650 W, JY92-IIN, power 15%) for 15
min. The as-prepared miniemulsion was transferred to a three-necked
flask and purged with N2 for 30 min. The polymerization was
performed at 75 °C for 7 h to obtain a stable amphiphilic PFPNs
aqueous dispersion. The unreacted monomer or other impurities were
removed by dialysis three times through a porous cellulose membrane
(MWCO 3500). The detailed experimental parameters for this
polymerization are shown in Table 1.
Table 1. Summary of Some Data for BDPMA and SPMA
Contained Nanoparticle Samples
b
c
BDPMA feed
[mg]
SPMA feed
[mg]
diameter
[nm]
FL intensity
[au]
a
sample
NP-N0
NP-N1
NP-N2
NP-N3
NP-N4
NP-S
0
3
3
3
3
0
0
2.5
5
85
87
76
77
79
91
EXPERIMENTAL SECTION
■
616
600
595
377
9
Materials. Poly(ethylene glycol) methyl ether (PEO-OH, Mw
=
1900, Alfa), N,N′-dicyclohexylcarbodiimide (DCC, 99%, Alfa), 4-
(dimethylamino)pyridine (DMAP, 99%, Alfa), n-hexadecane (HD,
99%, Aldrich), 2,4-dimethyl-3-ethylpyrrole (97%, Aldrich), boron
trifluoride diethyl etherate (2 M in diethyl ether, Aldrich), 4-
hydroxybenzaldehyde (98%, Aldrich), tetrachloro-1,4-benzoquinone
(Chloranil, 99%, Aldrich), N,N-diisopropylethylamine (DIPEA, 99.5%,
Aldrich), trifluoroacetic acid (TFA, 99%, Aldrich), and 2-(3′,3′-
dimethyl-6-nitrospiro[chromene-2,2′-indolin]-1′-yl)ethanol (SP-OH,
93%, TCI) were used as received. Methyl methacrylate (MMA, 99%,
Aldrich) was washed with 10% sodium hydroxide solution and
deionized water for three times and then purified by vacuum
distillation. 2,2′-Azobis(isobutyronitrile) (AIBN, 99.99%, Aldrich)
was recrystallized from ethanol. The double-distilled water, which
was used throughout this work, was further purified with a Milli-Q
system. Tetrahydrofuran (THF, A.R.), dichloromethane (A.R.), and
acetonitrile (A.R.) were distilled over CaH2. Petroleum ether, benzene,
and other reagents were analytical reagents and used without further
purification. The PEO macro-RAFT agent (PEO-TTC, end-function-
ality >95%, Scheme S1 and Figure S1)11 and BODIPY phenol
(Scheme S2 and Figure S2)12 were synthesized as described elsewhere.
10
15
10
a
The MMA/HD/PEO-TTC/AIBN feed is 0.5/0.05/0.075/0.006 g,
respectively. Average nanoparticle diameter, determined by DLS.
Excitation at 500 nm and emission at 550 nm, 25 °C, CPFPNs = 0.3 wt
%.
b
c
Photorewritable Fluorescence Patterning. Quadrate filter
papers were soaked in a MFPNs dispersion (NP-N3, 3 wt %) for 24
h at room temperature and dried via natural seasoning to keep the
filter papers cover with MFPNs equably. Photomasks with hollowed
out letter “A” or “B” (Figure S8) were designed and fabricated by local
advertising design company. Photorewritable fluorescence patterning
were achieved by circularly covering/removing masks (A and B) on
the filter paper under alternating 302 nm UV and 525 nm visible light
illumination.
Cell Culture. CellTiter 96 AQueous One Solution Cell
Proliferation Assay was purchased from Promega (Madison, WI).
B
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Scheme 1. Schematic Illustration of Amphiphilic PFPNs via Covalently Combining BODIPY Monomer (BDPMA) and
Photochromic Derivative (SPMA)
Cell culture media was purchased from Thermo Scientific HyClone
(Waltham, MA). A549 cells, which were obtained from the cell bank at
Xiangya Hospital (Changsha, China), were cultured in RPMI-1640
medium containing 100 U/mL of penicillin−streptomycin and 10%
fetal bovine serum (FBS). The cells were grown at 37 °C in an
incubator with a 5% CO2 humidified atmosphere.
In Vitro Cytotoxicity Measurement. The effect of PFPNs on cell
proliferation in A549 cells was performed via the CellTiter 96
AQueous One Solution Cell Proliferation Assay. First, cells were
seeded at a density of 5.0 × 103 cells per well in 96-well plates in three
replicates and incubated overnight in 200 μL of complete medium.
Then, cells were dealt with various diluted samples of PFPNs in the
presence of 10% serum. After 24 h of incubation, 96-well plates were
washed with cold phosphate buffered saline (PBS) for three times
(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4,
pH 7.4), then 120 μL of fresh medium containing 20 μL of CellTiter
96 AQueous One Solution was added to every well, and the plate was
incubated again for 1 h at 37 °C. Thereafter, the absorbance of each
plate was measured via a Thermo Scientific Multiskan MK3Microplate
Reader (Thermo Fisher, USA) at 490 nm.
Confocal Imaging. A549 cells were seeded in confocal dishes at a
density of ca. 20% per plate and cultured for 24 h at 37 °C. Then, 500
μL of fresh cell growth medium supplemented with PFPNs (100 μg/
mL) was added to each dish. After incubation for 3 h, the dishes were
washed with cold PBS for three times. Then, A549 cells were
incubated with 1 mL of fresh medium for fluorescence imaging. All
fluorescence images were acquired using an oil immersion objective
(100×, NA 1.3) on an Olympus FV1000 confocal laser scanning
fluorescence microscope containing an Olympus IX81 inverted
microscope. First, an Ar+ laser (488 nm) was used as green irradiation
source, and a 505−560 nm bandpass filter was used for green
fluorescence detection. Then, cell dishes were exposed to a ZF-1 type
three ultraviolet analyzer UV lamp (365 nm) for 30 s and used Ar+
laser (405 nm) as red irradiation source, and a 575−650 nm bandpass
filter was used for red fluorescence detection.
Other Measurements. 1H NMR spectra was measured on a
Bruker Avance 500 MHz NMR spectrometer. The number-average
molecular weight (Mn) and PDI were determined by a Waters 2410
gel permeation chromatograph (GPC) at 30 °C and using THF as the
eluent (1.0 mL/min). The calibration curve was obtained by using
polystyrene (PS) as the standard. The diameter of nanoparticles was
determined by a Malvern Nano-ZS90 instrument, and their
morphology was recorded on a Bruker Dimension Icon atomic force
microscope (AFM) in the tapping mode. The UV−vis absorption
spectrum was measured on a Shimadzu UV-2501PC spectropho-
tometer at room temperature. The fluorescence spectrum was
determined by a Shimadzu RF-5301PC fluorescence spectrophotom-
eter at room temperature. The solid content of nanoparticles was
estimated by gravimetric analysis. Fluorescence lifetime (τ) was
recorded on a time-correlated single photon counting (TCSPC)
nanosecond fluorescence spectrometer (Edinburgh FLS920) at room
temperature. For PFPNs, data analysis was carried out by using a
simple tail fit method and fitting with a monoexponential decay
function. The goodness of fit was estimated by using χ2 values
(between 1.0 and 1.2).
RESULTS AND DISCUSSION
■
Synthesis of Amphiphilic PFPNs. In this study, novel
amphiphilic PFPNs were prepared by an one-pot RAFT-
mediated miniemulsion polymerization.11 In detail, two
polymerizable dyes as BDPMA (Figure S3) and SPMA (Figure
S4) were copolymerized with MMA in the presence of a PEO
macro-RAFT agent (PEO-TTC, Scheme S1 and Figure S1) as
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both control agent and reactive nonionic surfactants in
miniemulsion. The well-defined amphiphilic nanoparticles
with a hydrophobic photoswitchable fluorescent core and a
hydrophilic PEO shell were gradually achieved via the
generation of PEO-b-P(MMA-co-BDPMA-co-SPMA) diblock
copolymers from the hydrophilic PEO-TTC and the in situ self-
assembly process during the polymerization. Obviously, the
copolymerization of fluorescent monomer and photochromic
derivative can successfully avoid the potential dye leakage, so as
to greatly enhance the structural stability and photostability of
the obtained PFPNs. The as-prepared amphiphilic PFPNs
dispersion in aqueous media displayed reversibly distinct dual-
color fluorescence via alternate illumination of UV and visible
light, as illustrated in Scheme 1.
concentration (CMC),15 the micelle nucleation could take
place during the polymerization process. However, the adopted
hydrophobe (hexadecane) could provide enough osmotic
pressure to prevent Ostwald ripening, thus forming a stable
miniemulsion.8b
Figure 2 shows the AFM image of an amphiphilic PFPN
sample NP-N3 (Table 1). The AFM result exhibited the
Figure 1 shows the GPC traces of macro-RAFT agent PEO-
TTC and PEO-b-PMMA copolymer from obtained amphiphilic
Figure 2. AFM image of amphiphilic PFPNs (sample NP-N3).
presence of well-dispersed global particles with an average
diameter of 64 nm. For comparison, the diameter for this
sample (NP-N3) measured by DLS is 78 nm (Table 1 and
Figure S5). The difference of sizes obtained from AFM and
DLS was possibly due to that the AFM scanning was performed
on dry solid sample, while the DLS was measured on the
unrestricted extension of amphiphilic nanoparticles in aqueous
solution. These results indicated that RAFT-mediated mini-
emulsion polymerization can be used to prepare amphiphilic
PFPNs with tunable size.
Figure 1. GPC curves of macro-RAFT agent PEO-TTC and the
obtained PEO-b-PMMA copolymer via one-pot RAFT-miniemulsion
polymerization.
polymeric nanoparticles without incorporation of BDPMA and
SPMA at different reaction time via typical one-pot RAFT-
miniemulsion polymerization. For this experiment, a detailed
description is shown in the Supporting Information. As
exhibited in Figure 1, with the reaction time increased, GPC
trace of the obtained PEO-b-PMMA copolymer was gradually
shifted to the region of high molecular weight. The Mn and PDI
of the synthesized PEO-b-PMMA are also displayed in Figure 1.
The reaction time increased from 3 to 9 h. As shown in Figure
1, the Mn of PEO-b-PMMA gradually increased from 7970 to
12 850 g/mol; meanwhile, the PDI always kept a low value
(<1.25), which indicated the typical “living” property of
selected RAFT-miniemulsion polymerization technique. More-
over, in current experimental conditions, compared with other
reaction times, 7 h seems to be more helpful for us to prepare
the well-defined amphiphilic diblock copolymer with relatively
high molecular weight and narrow PDI.
Generally, particles with smaller size are affected little by light
scattering and display more potential for additional biomodi-
fication and biological applications.14 Herein, the averaged
particle diameter ranging from ca. 85 to ca. 129 nm was
achieved by modulating some experimental parameters as
shown in Table S1, which strongly relied on the concentration
of used surfactant, PEO-TTC. For example, as for sample NP-
S3, by using 7.5 mg/mL PEO-TTC, a stable particle with
diameter down to approximately 85 nm was obtained via
measuring by DLS (Table S1). However, if the concentration of
surfactant was added to 10 mg/mL, some big particles formed,
which induced the increase of both the average diameter and
the PDI of particles (NP-S4, Table S1). As the concentration
(7.5 mg/mL) of PEO-TTC exceeded its critical micelle
Spectroscopic Characterization of Amphiphilic
PFPNs. Figures 3A and 3B compare the absorption spectra
and the fluorescence emission spectra of BDPMA dye in
different conditions, including pure water, dichloromethane,
and nanoparticles (NP-N3). As a known hydrophobic dye,
BDPMA exhibited low absorption (Figure 3A) and negligible
fluorescence in water due to its low solubility (Figure 3B).12
However, it showed intense fluorescence intensity and ultrahigh
brightness after it was encapsulated in polymeric nano-
particles.12 Additionally, the maximum absorption wavelength
for BDPMA in nanoparticles sample (NP-N3) and dichloro-
methane were identical, revealing that the fluorescent dye was
located in the hydrophobic polymer matrix.8c Notably, inspired
by recent G. Clavier’s report about copolymerization of
BDPMA in polymer nanoparticles, it could be estimated that
more than 95% of the feed dyes were covalently linked to the
polymer backbone.12 Furthermore, it is certain that spiropyran
derivatives adopt the ring-opened merocyanine (MC) state
under UV irradiation, while changing to the ring-closed spiro
(SP) state after visible light illumination (Scheme 1).2,8 As
shown in Figure 3A, compared with the visible light irradiation
of SPMA-contained samples (NP-N3 and NP-S), a new
absorbance band around 570 nm appeared after UV irradiation,
which was due to the photoisomerization of the ring-closed SP
state to the ring-opened MC state. It demonstrated that the
SPMA dyes have also been wonderfully encapsulated in the
particles, which is highly consistent with our previously
reported results.8
D
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Figure 3. (A) Absorption spectra of BDPMA dye in pure water and dichloromethane, together with a PFPNs sample (NP-N3) and a SPMA
contained nanoparticle sample (NP-S) upon UV or visible light. (B) Fluorescence emission spectra of BDPMA dye in pure water and
dichloromethane and the PFPNs sample NP-N3 upon UV or visible light (λex = 500 nm).
The relationship between the fluorescence intensity and the
content of introduced fluorescent dye in nanoparticles is also
investigated in this study. For this experiment (Table S1),
without changing other experimental conditions, four nano-
particles with different feed amount of BDPMA were designed
to compare their fluorescence intensity at the same
concentration of particles as shown in Figure S6A,B. With
the BDPMA content increasing from 1 to 4 mg in
nanoparticles, their maximum absorbance improved corre-
spondingly (Figure S6A), while their fluorescent intensity first
increased and then decreased (Figure S6B). Specifically, for the
nanoparticle (NP-B4) possesses the highest BDPMA content,
its maximum fluorescence intensity is obviously lower than NP-
S3, and its fluorescence emission band exhibited a broadened
profile with a slightly red-shift (∼4 nm) of maximum emission.
It means that the possible self-quenching will be induced when
the content of fluorophores inside the nanoparticles gets to a
certain value.8 Thus, the proper content of fluorophores in
nanoparticles is crucial to provide the nanoparticles with high
fluorescence intensity as well as effectively to avoid self-
quenching.
moieties in PFPNs at 550 nm, the emission intensity of the MC
moieties in PFPNs at 640 nm is relatively weak (Figure 3B and
Figure S8) but still can be easily detected via changing the
measure condition (Figure S7). We are not sure if the low
emission is related to the PMMA matrix.8 Presumably, the
relatively high content of SPMA molecules in nanoparticles was
apt to induce fluorescence quenching. After the sample was
irradiated again by 10 min of visible light (525 nm), the
fluorescence emission at 550 nm was basically recovered, while
the emission band around 640 nm disappeared at the same time
(Figure 3 and Figure S8). Correspondingly, the luminescence
of the PFPNs dispersion in the dark was transparently varied
between red and yellow-green after illuminating by UV or
visible light, as illustrated in Scheme 1.
According to the principle of FRET,16 once the emission
spectrum of the donor overlaps remarkably with the acceptor’s
absorption spectrum, and the distance of donor−acceptor
resides in the proper radius (ca. 1−10 nm), the FRET between
the donor and acceptor is efficient.16 In the current work, as
shown in Figure S9, the emission spectrum of the BDPMA
(donor) overlaps wonderfully with the absorption spectrum of
the MC state of the SPMA (acceptor) in the PFPNs. In
contrast, the absorption spectrum of the SP state of SPMA and
the emission spectrum of the BDPMA overlap hardly.
Therefore, the FRET from the BDPMA to the SP state of
the SPMA is difficult, while the FRET to the MC state is
feasible.
Reversibly Photoswitching of PFPNs by UV and
Visible Light. Figure 4 compares the change of the
As can be seen in Table 2 and Figure 4, for four PFPNs
samples (NP-N1 to NP-N4), the estimated average donor−
acceptor distances (r) ranged from 3.7 to 5.7 nm, which are
similar to some previous reports.8 And there were multiple
acceptors (MC state of SPMA) within the effective energy
transfer distance from a donor, which ensured the presence of
intraparticle FRET mechanism as well as the photoswitching
fluorescence after the irradiation of UV light. As for the sample
NP-N1, it has the lowest ratio of acceptor/donor (0.9), and its
E is 68.0%; thus, the BDPMA fluorescence cannot be availably
decreased after illumination of UV light, while for the higher
ratio of acceptor/donor (3.7, NP-N3), there was more MC
state of SPMA units around a BDPMA molecule (donor), and
quite high E (93.4%) can be obtained. However, E cannot be
improved evidently and the initial fluorescence intensity
reduced much (Table 1 and Figure S7C), when continue to
increase the ratio of acceptor/donor (5.5, NP-N4). Therefore,
the optimization of the ratio of acceptor/donor is crucial to
achieve ideal PFPNs with sufficient energy transfer efficiency as
well as high photoswitch contrast.
Figure 4. Fluorescence intensity of four PFPNs samples (Table 1) at
550 nm upon UV and visible light irradiation.
fluorescence intensity of four PFPNs samples (Table 1) at
550 nm after irradiating by visible and UV light. As shown in
Figure 3B as well as Figures S7 and S8, after illumination of UV
light (302 nm) for 3 min, fluorescence emission of all PFPNs
samples at 550 nm was dramatically decreased; meanwhile, an
emission band around 640 nm appeared (Figure 3B and Figure
S7), which remained with the emission band of the MC form of
the SPMA units and was consistent with our previous reports.8
Compared with the intense fluorescence emission of BDPMA
E
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Table 2. Characteristics Data of Four PFPNs Samples
a
b
b
c
c
d
e
e
f
sample
DNP [nm]
Dcore [nm]
NBD
NSP
NSP/NBD
NA
E [%]
r [nm]
NP-N1
NP-N2
NP-N3
NP-N4
71
62
64
67
68
59
61
64
1358
887
1253
1637
3618
6268
0.9
1.8
3.7
5.5
3.6
7.3
68.0
85.7
93.4
96.8
5.7
4.8
4.2
3.7
981
14.5
21.8
1132
a
b
The MMA/HD/PEO-TTC/AIBN feed is 0.5/0.05/0.075/0.006 g, respectively. DNP and Dcore: the average nanoparticle diameter and core
c
diameter (Supporting Information), obtained by AFM. NBD and NSP: the average number of BDPMA and SPMA in a nanoparticle core,
respectively, estimated by presuming more than 90% of dyes are copolymerized. The average number of SPMA residing around one BDPMA
within the effective energy transfer distance (Supporting Information). The experimental energy transfer efficiency (Supporting Information). The
estimated average donor−acceptor distance (Supporting Information).
d
e
f
To further confirm the presence of intraparticle FRET in
current systems, fluorescence decay curves of typical PFPNs
sample (NP-N3, Figure 5) after irradiating UV/vis light were
and visible light (2 W), the fluorescence intensity of BDPMA-
containing nanoparticles at 550 nm changed little, which
indicated the undetectable photodegradation in such short-time
irradiation. In contrast, for the PFPNs sample NP-N3, the
fluorescence intensity at 550 nm dramatically decreased within
a very short time (3 min) UV irradiation, revealing the FRET
between donor and acceptor happened. While its fluorescence
intensity can basically recover after irradiation by visible light
for 10 min, which can be due to the photoisomerization from
MC state to SP state of SPMA units. On the other hand, the
fluorescence intensity of as-prepared MFPNs at 550 nm can be
reversibly turned on/off via cyclically illuminating UV and
visible light (Figure 6B). As revealed in Figure 6B, the
photoswitching of fluorescence can be performed for five cycles
with a negligible “fatigue” effect. Undoubtedly, the prepared
PFPNs with the fluorescent dye and the photochromic
incorporated by covalent bonding and protected by a polymer
matrix hold superior resistance to the possible “fatigue” effect,
as compared to other analogous dyad systems.13 Moreover, it is
worth pointing out that the present PFPNs exhibit a much
shorter photoresponse time than some previous systems,2a,f
which indicated a valuable potential for e.g. rewritable data
storage or selective highlighted biological labeling.2a,e
In the present study, the fluorescence long-term stability and
the thermal stability of MC state of the typical PFPNs sample
(NP-N3) were also explored. For the long-term stability test,
the diluted dispersion was sealed and stored in the dark under
ambient temperature. Then an aliquot was drawn out for
fluorescence test after every week. As displayed in Figure 7A,
not only the fluorescence intensity but also the photoswitching
efficiency of the diluted PFPNs sample changed seldom even
after storing for 6 weeks, implying remarkable long-term
photostability and the tiny leakage of dyes in PFPNs sample.
Figure 5. Fluorescence decay curves of NP-N3 upon UV/vis light
irradiation (λex = 405 nm, detection wavelength = 550 nm).
obtained by using the TCSPC technique.16 As shown in Figure
5, after illumination of visible light for 10 min (switching on),
the selected sample NP-3 displayed a mean lifetime of 2.49 ns,
which was similar to previously reported BDPMA-contained
polymeric nanoparticles,12 indicating there is no FRET process
between donor and SP state of the SPMA units. After
irradiation of UV light for 3 min (switching off), the average
lifetime of the PFPNs was significantly reduced to 0.83 ns,
which was mainly ascribed to the photoinduced FRET process
between donor and acceptor.
Figure 6A displays fluorescence response behavior of the
BDPMA-containing nanoparticles sample (NP-S3) and the
novel PFPNs sample (NP-N3) under illumination of UV and
visible light. During the whole irradiation period of UV (12 W)
Figure 6. (A) Fluorescence response of BDPMA-contained nanoparticles sample (NP-S3) and a PFPNs sample (NP-N3) via irradiating with UV
(302 nm) and visible light (525 nm). (B) Photoinduced switching cycles of a PFPNs sample (NP-3) under alternative illumination of UV for 3 min
and visible light for 10 min (CPFPNs = 0.3 wt %, λex = 500 nm, λem = 550 nm).
F
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Figure 7. (A) Fluorescence long-term stability of PFPN sample (NP-N3, CPFPNs = 0.3 wt %, λex = 500 nm, λem = 550 nm). (B) Thermal stability of
MC state in PFPNs dispersion (NP-N3, CPFPNs = 0.3 wt %, λex = 500 nm, λem = 550 nm) under physiological conditions (PBS, pH 7.4).
Presumably, it should be mainly attributed to the stable
hydrophilic PEO shell and the protection of hydrophobic
PMMA matrix as well as the covalent binding of dyes in the
particles. The results demonstrated that the PFPNs could be
used for long-term imaging or tracking in complex biological
application. On the other hand, it is noteworthy that the ring-
opened MC state of spiropyran units can return to the ring-
closed SP state through the thermal isomerization pathway.17
Thus, thermal stability of PFPNs sample (NP-N3) after UV
irradiation at physiological conditions was investigated at three
temperatures: 25, 37, and 42 °C. In contrast, as exhibited in
Figure 7B, the fluorescence intensity of PFPNs sample at 550
nm recovered slowly at lower temperature, i.e., 25 °C, and it
only added to 2.6-fold after being stored in the dark for 60 min,
which was mainly attributed to the weak thermal isomerization
of spiropyran moieties at room temperature. Therefore, the
photoswitchable fluorescence property of PFPNs at room
temperature should be more propitious to practical application.
Photorewritable Fluorescence Patterning. To take full
advantage of the high contrast of photoswitchable fluorescence
and favorable water-dispersibility nature, the prepared PFPNs
Figure 8. Photorewritable fluorescence patterning, prepared by PFPN
aqueous dispersion (NP-N3) was used as luminescent paint for
preparing photorewritable fluorescence patterning. The pat-
terning on filter paper covered with PFPNs sample (NP-N3)
was examined by modulated illumination through designed
photomasks (Figure S10). As shown in Figure 8, the
fluorescence pattern of letter “A” was recorded as a positive
image upon 302 nm UV excitation by covering with mask A,
which was subsequently erased by UV light. Then, another
fluorescence patterning image of letter “B” was recorded upon
525 nm visible light excitation via covering with mask B (Figure
8) and then erased via illumination of visible light for ca. 20
min. The results indicated that the reversibly photoswitchable
fluorescence property of PFPNs can be instantly used to
fabricate photorewritable fluorescence patterning under the
help of photomask.18
dispersion (NP-N3), via using two photomasks (masks A and B,
Figure S9) and irradiation of alternating 302 nm UV and 525 nm
visible light. Scale bar: 1 cm.
MTT Assay and Confocal Imaging of PFPNs in Living
Cells. The cytotoxicity of PFPNs (NP-N3) was estimated via
MTT assay by using A549 cells. For this test, the A549 cells
were incubated with various concentrations of PFPNs for 24 h.
The results in Figure 9 clearly indicate that no noticeable
reduction in cell viability is observed for cells treated with
PFPNs. It is noteworthy that over 85% of cell viability was
reserved even at a high concentration of PFPNs (600 μg/mL),
demonstrating that the PFPNs produced in this study is not
obviously toxic in vitro.
Figure 9. Cytotoxic effects of PFPNs sample (NP-N3) against A549
cells upon 24 h of incubation. Control: A549 cells in the absence of
the PFPNs.
We also investigated the photoswitchable capability of the
PFPNs in the living A549 (human lung carcinoma) cell lines
(Figure 10). Under visible light irradiation, the green
fluorescence from cells can be found as shown in Figure
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Figure 10. Confocal images of A549 cells treated with an amphiphilic PFPNs dispersion (NP-N3) at 37 °C for 3 h under visible light (A−C) or UV
illumination (D−F). The overlay images confirm the signals are from nanoparticles in cell, not interference. Scale bar: 10 μm.
10A,C. It was mainly ascribed to the SPMA moieties in
nanoparticles preserved as the nonfluorescence SP state, which
cannot quench the fluorescence of BDPMA; therefore, the
BDPMA in PFPNs emitted strong green fluorescence after
excitation. After illumination of these A549 cells with UV light
(30 s), the green fluorescence from cells has disappeared
(Figure 10D), while the red fluorescence emitted from cells as
exhibited in Figure 10E,F was clearly displayed. Because the
SPMA moiety in nanoparticles has changed to its fluorescent
MC state after irradiation of UV light, and the FRET process
between MC state of SPMA and BDPMA occurred, which led
to dramatic quenching of the green fluorescence of BDPMA
(Figure 10D) and the remarkable appearance of the red
fluorescence of the MC state of SPMA (Figure 10E). The
results significantly demonstrated that the photoswitchable
fluorescence property can also be realized in living cells, which
revealed the potential advantage of PFPNs as smart fluorescent
labels in complex biological environment with relatively high
autofluorescence or another interfering fluorescence in the
same wavelength region. Hence, the PFPNs may find broad
applications in selectively highlighted complex biological
systems with photoswitchable dual-color fluorescence.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed description of synthesis of PEO-TTC, BODIPY
phenol, and the amphiphilic polymeric nanoparticles; 1H
NMR spectrum of PEO-TTC, BODIPY phenol, BDPMA,
and SPMA; detailed description of calculation of the R0, E, r,
and estimation of NA, etc. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acs.macromol.5b00667.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: cj0066@gmail.com (J.C.).
*E-mail: pgyi@hnust.cn (P.G.Y.).
*E-mail: jianhuijiang@hnu.edu.cn (J.H.J.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support of the present
work by NSFC (Projects 51373002 and 21172066), Scientific
Research Foundation for the Returned Overseas Chinese
Scholars, Scientific Research Fund of Hunan Provincial
Education Department (Project 12B041), Project funded by
China Postdoctoral Science Foundation (Project
2014M550418), and Open Project Program of State Key
Laboratory of Chemo/Biosensing and Chemometrics (Project
2013008).
CONCLUSIONS
■
In summary, novel amphiphilic BODIPY-based PFPNs have
been successfully synthesized by a facile one-pot RAFT-
mediated miniemulsion method, in which the macro-RAFT
reagent was used both as reactive stabilizer and control agent.
Meanwhile, the BDPMA dye and the photochromic SPMA can
be covalently incorporated into the polymer backbone with
controllable amount and ratio, which can significantly increase
the structural stability and photostability of as-prepared PFPNs.
Notably, the novel PFPNs displayed reversibly distinct dual-
color (green-red) fluorescence not only in aqueous solution but
also in the living cell by irradiation of UV or visible light via
occurring switchable intraparticle FRET. In addition, the
tunable FRET efficiency, prominent long-term stability,
relatively fast photoresponsibility, and excellent photoreversi-
bility together with their instantaneous application in rewritable
fluorescence patterning further indicated that they are especially
suited to applications in rewritable optical data storage and
super-resolution biological imaging and labeling.
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