Photoresponsive cross-linked polymeric particles for phototriggered burst release

Article abstract of DOI:10.1111/php.12038

We synthesized a series of cross-linked photoresponsive polymeric particles with photolabile monomers and cross-linkers through miniemulsion polymerization. These particles are quite stable in dark, while light irradiation caused the breakage of particles and the efficient release of encapsulated contents up to 95% based on Nile red fluorescence. Photoswitches of particle systems were confirmed by fluorescence spectroscopy, SEM and colorimetry. Particle uptake and triggered release in RAW264.7 cells were confirmed by fluorescein diacetate loaded particles.

Full text of DOI:10.1111/php.12038

Photochemistry and Photobiology, 2013, 89: 552–559
Photoresponsive Cross-linked Polymeric Particles for Phototriggered
Burst Release
Zhen Wang1, Lili Yu2, Cong Lv1, Peng Wang1, Yedong Chen1 and Xinjing Tang*1
1State Key Laboratory of Natural & Biomimetic drugs, The School of Pharmaceutical Sciences, Peking University, No. 38,
Xueyuan Rd., Beijing, 100191, China
2School of Pharmaceutical Sciences, Xi′an Medical University, No. 1, Xinwang Rd., Xi′an, 710021, Shanxi, China
Received 28 September 2012, accepted 2 January 2013, DOI: 10.1111/php.12038
ABSTRACT
deliver target molecules which can be released from nanoparticles
upon light triggering(22,23). For nanoparticles formed by organic
substances, most researches are currently limited to photolabile
diblock copolymers which self-assemble into photoresponsive
micellar particles. Azobenzene moieties (change of dipole moment)
(24,25), spiropyran moieties (formation of zwitterionic species)
(26–28), stilbene(29), pyrenyl(30), coumarinyl(31) and nitrobenzyl
esters (change hydrophobic to hydrophilic block)(32–35) are the
typical chromophores incorporated into amphiphilic copolymers to
render them susceptible to light triggering. All these copolymers
contain PEG as hydrophilic block and poly-methacrylate (PMA)
derivatives as hydrophobic block. After UV irradiation, the PMA
block turned into water-soluble polymethacrylic acid and the
encapsulated targets were released. Even though great improve-
ments have been achieved with photolabile copolymers for micelle
formation, more photoresponsive particle systems are in need.
Miniemulsion polymerization has been widely used to synthesize
polymeric particles which target molecules can be encapsulated
during sonication-assisted miniemusification(36). We are inter-
ested in synthesizing cross-linked photoresponsive polymeric par-
ticles for target incorporation through mimiemulsion
polymerization and phototriggered target releasing.
Here, we report the syntheses of photoresponsive polymeric
particles, their photoswitches from hydrophobic to hydrophilic
internal environments, and the controllable release of model dyes
from these particles. Different from previous works by other labs
and ours which focused on self-assembly of photolabile linear
polymers or photolabile PEG/PMMA block copolymers using
photocleavable cross-linkers, this study illustrated cross-linked
particles formed by miniemulsion polymerization of photolabile
monomers with nonphotolabile cross-linkers. With cross-linkers
inside emulsions, more rigid solid particles will be formed. Inter-
nal hydrophobic cores of particles are filled with hydrophobic
molecules in their initial state. Upon light irradiation, the trans-
formation from a hydrophobic environment to hydrophilic one
allowed the penetration of water and the release of encapsulated
molecules, as shown in Fig. 1. To develop such photoresponsive
particles, we prepared hydrophobic methacrylate monomers in
which the hydrophilic acid groups were temporally protected
with photolabile groups.
We synthesized a series of cross-linked photoresponsive poly-
meric particles with photolabile monomers and cross-linkers
through miniemulsion polymerization. These particles are
quite stable in dark, while light irradiation caused the break-
age of particles and the efficient release of encapsulated con-
tents up to 95% based on Nile red fluorescence. Photoswitches
of particle systems were confirmed by fluorescence spectros-
copy, SEM and colorimetry. Particle uptake and triggered
release in RAW264.7 cells were confirmed by fluorescein diac-
etate loaded particles.
INTRODUCTION
Lipids, carbohydrates, hydroxyl acid polymers, dendrimers,
diblock copolymers etc. have been applied to form micro- and
nanoparticles and have been widely used in biological applica-
tions, such as biomolecular imaging and drug delivery(1,2).
Although great improvements for delivering targets into cells
using micro- and nanoparticles have been achieved, it is highly
desirable to be able to control the release of encapsulated targets
from capsules and reach high enough target concentrations to
mediate the effective responses via an external stimulus. The
design and syntheses of intelligent drug delivery systems which
respond to the environmental stimuli are promising research
areas(3,4).
Currently, intelligent drug delivery systems are mostly based
on stimuli-responsive polymers which sense a change of specific
variables, such as temperature, pH, redox-potentials, the level of
enzymes, ultrasound, light and specific donor molecules(4–14).
Among these intelligent stimuli, light stands out as a clean and
noninvasive one and has been widely used as an external trigger
in biological systems(7,8,10,14–19), especially with the develop-
ment of LitxTM technology for internal irradiation of tissues(20).
Unlike the pH and oxidation responsive systems, light does not
require any chemical environmental change(21). For photorespon-
sive particles, the release of entrapped drugs can be accelerated at
a specific location and time upon light activation, which promotes
maximum efficacy. Inorganic nanoparticles, such as Au and Fe2O3
nanoparticles, have been used as carriers with surface modification
through photolabile linkages. These nanoparticles can assist to
MATERIALS AND METHODS
*Corresponding author email: xinjingt@bjmu.edu.cn (XinJing Tang)
© 2013 The Authors
Photochemistry and Photobiology © 2013 The American Society of Photobiology 0031-8655/13
All commercial solvents and reagents were used without further purifica-
tion except as noted below. Tetrahydrofuran (THF), dichloromethane
552

Photochemistry and Photobiology, 2013, 89 553
Figure 1. Strategy of controllable release from photoresponsive particles, and the structures of photolabile (MA and MB), nonphotolabile (MC) mono-
mers and cross-linkers (CL1 and CL2) for particles synthesized through miniemulsion polymerization.
(DCM) and triethylamine (TEA) were distilled over CaH2. Nile Red
(NR) was purchased from J&K Chemicals. Fluorescein diacetate (FDA),
Triton B (40% in methanol) and cross-linkers (Ethylene glycol dimethac-
rylate [CL1] and hexamethylene glycol dimethacrylate [CL2]) were pur-
chased from Alfa Aesar. Methacryloyl chloride was purchased from TCI
Shanghai. Hexadecane was purchased from Haltermann in Germany.
sodium dodecyl sulfate (SDS) was purchased from Beijing Dingguo Bio-
technology Co., Ltd. Azobisisobutyronitrile (AIBN) was purchased from
Shanghai Trial of Four Hervey Chemical Co., Ltd.
nitroethyl benzene 3 (10 g, 66 mmol). Paraformaldehyde (2 g, 67 mmol)
was added to above mixture and purged with nitrogen. The solution was
then heated to 60°C for 6 h. The solution was concentrated in vacuo and
neutralized with 5% aqueous HCl followed by the extraction with ethyl
acetate, dried over anhydrous sodium sulfate and concentrated. The resi-
due was purified by column chromatography on silica gel (ethyl acetate/
petroleum ether = 2:8) to afford 4 (10 g, 84.4%). Compound 4. 1H NMR
(400 MHz, CDCl3) d 7.74 (d, J = 8.1 Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H),
7.49 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 3.82 – 3.71 (m, 2H),
3.50 (dd, J = 13.5, 6.8 Hz, 1H), 1.88 (s, 1H), 1.32 (d, J = 6.9 Hz, 3H).
13C NMR (100 MHz, CDCl3): d150.7 (C(NO2)), 138.1 (Ar, C), 132.6
(Ar, C), 128.2 (Ar, C), 127.2 (Ar, C), 124.1 (Ar, C), 67.8 (CH2), 36.4
(CH), 17.5 (CH3).
1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz
NMR spectrometer. Reported chemical shifts (ppm) are relative to CDCl3
and coupling constants are reported in Hz. The size of nanoparticles in
diameter was determined through dynamic light scattering (DLS) (Cumu-
lant method) using the Zetasizer Nana-ZS from Malvern Instruments at
25°C. UV/Vis spectra were performed on DU800 UV/Vis Spectropho-
tometer (Beckman Coulter, Inc.). Fluorescence spectra were obtained
using Cary Eclipse spectrofluorometer (Varian). An excitation wavelength
of 550 nm was used for NR and the emission spectra were recorded from
570 and 750 nm. Scanning electron microscope (SEM, Hitachi S-4300)
and transmission electron microscope (TEM) (JEM-2010; JEOL Ltd)
were used to observe the particles. Typically, one drop of diluted nano-
particle solution was cast on either silicon wafer (for SEM) or carbon-
coated copper grid (for TEM), and dried at 40°C. Nile red-loaded parti-
cles were visualized with Zeiss Upright Fluorescence Microscope (Axio
Imager A2). And toxicity of particles was evaluated by MTT assay after
incubation with blank particles for 48 h. FDA release in Raw 264.7 was
detected with FlexStation 3 (Molecular Devices). Particles uptake and
fluorescence images of cells were obtained using Invert Fluorescence
Microscope (OLYMPUS, IX81).
Synthesis of 1-(2-nitrophenyl)ethanol (2) (Scheme S1). Compound 2
was synthesized according to a literature procedure (37) with minor
modification. Aqueous solution of sodium borohydride (NaBH4)
(34.4 mL 10%) was added dropwise to the solution of 1-(2-nitrophenyl)
ethanone (1) (5 g, 30 mmol) in 30 mL 1, 4-dioxane at 0°C. The mix-
ture was stirred in an ice bath for 1 h and at room temperature for an
additional 30 min. Then, excess NaBH4 was quenched with 20 mL ace-
tone. After the removal of the solvents, the residue was dissolved in
water and extracted with ethyl acetate, and dried over anhydrous
sodium sulfate. After the removal of solvents, the obtained light yellow
oil was used in next step without further purification. Compound 2.
Yield 98%. 1H NMR (400 MHz, CDCl3) d 7.91 (d, J = 8.2 Hz, 1H),
7.85 (d, J = 7.8 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.43 (t, J = 7.7 Hz,
1H), 5.44 (q, J = 6.3 Hz, 1H), 1.59 (d, J = 6.4 Hz, 3H). 13C NMR d
(100 MHz, CDCl3): 147.9 (C(NO2)), 140.9 (Ar, C), 133.6 (Ar, C),
128.1 (Ar, C), 127.6 (Ar, C), 124.3 (Ar, C), 65.5 (CH), 24.2 (CH3).
Synthesis of (2 -nitrophenyl)propanol (4) (Scheme S1). Compound 4
was synthesized according to a literature procedure with minor modifica-
tion. (38) Triton B (40% in methanol, 27 g, 66 mmol) was added to
Synthesis of 1-(2-nitrophenyl)ethyl methacrylate (MA), 2-(2-nitrophe-
nyl)propyl methacrylate (MB), phenethyl methacrylate (MC) (Scheme S1).
Compounds 2, 4, 5 (phenethyl alcohol) (60 mmol) was dissolved in dry
DCM (30 ml), dry triethylamine (10 mL, 71.8 mmol) was added, then
purged with nitrogen. The mixture was allowed to stir in an ice bath for
5 min. Methacryloyl chloride (7.2 mL, 73.7 mmol) was slowly added over
a period of 10 min. The reaction mixture was then warmed to room temper-
ature while stirring overnight. The reaction mixture was then washed three
times with saturated NaHCO3 and once with brine. The resulting organic
phase was dried over anhydrous sodium sulfate and concentrated. The resi-
due was purified by column chromatography on silica gel (petroleum ether/
ethyl acetate = 15:1) to afford MA, MB, MC as light yellow oil.
Compound MA. Yield: 71.4%. 1H NMR (400 MHz, CDCl3) d 7.94
(d, J = 8.3 Hz, 1H), 7.65-7.59 (m, 2H), 7.45-7.39 (m, 1H), 6.38 (q,
J = 6.4 Hz, 1H), 6.17 (s, 1H), 5.60 (s, 1H), 1.94 (s, 3H), 1.69 (d,
J = 6.5 Hz, 3H). 13C NMR d (100 MHz, CDCl3): d=166.2 (C=O), 147.7
(C(NO2)), 136.1 (C=), 126.0 (CH2=), 138.1 (Ar, C), 133.6 (Ar, C), 128.3
(Ar, C), 127.1 (Ar, C), 124.5 (Ar, C), 68.4 (CH), 22.0 (CH3), 18.2(CH3).
MS [M + Na]: calculated: 258.08, found: 258.10.
Compound MB. Yield: 96%. 1H NMR (400 MHz, CDCl3) d 7.76 (d,
J = 8.1 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H),
7.40-7.33 (m, 1H), 6.00 (s, 1H), 5.51 (s, 1H), 4.31 (d, J = 6.8 Hz, 2H),
3.75 (dd, J = 13.7, 6.9 Hz, 1H), 1.87 (s, 3H), 1.39 (d, J = 7.0 Hz, 3H).
13C NMR (100 MHz, CDCl3): d 167.1 (C=O), 150.2 (C(NO2)), 135.9
(C=), 125.8 (CH2=), 137.4 (Ar, C), 132.6 (Ar, C), 128.2 (Ar, C), 127.4
(Ar, C), 124.1 (Ar, C), 68.5 (CH2), 33.1 (CH), 18.1 (CH3), 17.9 (CH3).
MS [M + Na]: calculated: 272.10, found: 272.15.
Compound MC. Yield: 93.2%. 1H NMR (400 MHz, CDCl3) d 7.28
(d, J = 6.6 Hz, 2H), 7.22 (d, J = 7.2 Hz, 3H), 6.07 (s, 1H), 5.52 (s, 1H),
4.35 (s, 2H), 2.97 (t, J = 7.0 Hz, 2H), 1.92 (s, 3H). 13C NMR
(100 MHz, CDCl3): d 167.3 (C=O), 136.4 (C=), 126.5 (CH2=), 138.0
(Ar, C), 128.9 (Ar, 2C), 128.5 (Ar, 2C), 125.4 (Ar, C), 65.2 (CH2), 35.2
(CH2), 18.3 (CH3).
Preparation of Nile red/FDA-loaded particles and blank particles.
Particles were prepared through miniemulsion polymerization method.

554 Zhen Wang et al.
In a typical run, 50 mg of monomers, varied amounts of cross-linkers
(1%, 5%, 10%, 15%, 25% of the molecular of the monomers), 0.5 mg
Nile red or 4 mg FDA, 10 lL 5% (w/w) AIBN in CH2Cl2 and 3.1 mg
costabilizer Hexadecane (for some cases) were dissolved in 500 lL
CH2Cl2. A quantity of 5 mL distilled water (for FDA loading, sodium
acetate buffer, pH = 4, was used due to the stability of FDA at 80°C)
containing 5 mg SDS was purged with bubbling nitrogen for 10 min,
and was then added to oil phase. This mixture was then sonicated in an
ice bath for 5 min (3 s pulses with 1 s delay) with 80 W of the power.
Following sonication, the mixture was transferred to 5 mL sealed con-
tainer having been purged with nitrogen. The polymerization was per-
formed at 80°C oil bath for 18 h. TLC confirmed the disappearance of
monomers. After polymerization, the suspension was stirred overnight,
which allowing CH2Cl2 to evaporate. The aqueous solutions were then
dialyzed with a dialysis membrane (8000–14000 MWCO) against dialysis
buffer (water with 1% DMSO) for 2 days with the exchange dialysis buf-
fer every 4 h to remove excess surfactants and other residues, then were
further dialyzed against pure water for 1 day with the exchange of water
every 4 h to remove DMSO.
Nanoparticle Characterization and Degradation Study. Nanoparticles
were characterized by SEM using Nanoscope IIIa. The particles were dis-
persed in water and then 5 lL solution was dripped onto a silicon wafer.
After drying 2 h at 35°C, particles were sputter coated with aurum. Size
distribution of particles was determined by DLS using Zetasizer Nano
ZS.
Cell culture. Macrophage cell line, RAW 264.7 was grown in Dul-
becco’s modified Eagle cell culture medium (DMEM) containing 10%
heat-inactivated fetal bovine serum, 100 IU/mL Penicillin-Streptomycin
in an atmosphere of 5% CO2 and 95% relative humidity. The cells were
routinely at 80–95% coverage.
FDA-loaded particle uptake and release in cell study. FDA is a non-
fluorescent hydrophobic fluorescein derivative that can pass through cell
membrane, where intracellular esterases hydrolyze ester groups, produc-
ing the highly fluorescent product, fluorescein. In our study, to investi-
gate particle uptake by macrophages and triggered release in cells, FDA
was mixed with monomers during microemulsion polymerization. These
particles were then dialyzed for 2 days with 1% DMSO water solution
and then with dialyzed with water.
Raw264.7 cells were incubated with 31.3 lg/mL FDA-loaded particles
of MA, MB, MC (MA-CL110%-FDA, MB-CL110%-FDA, MC-CL110%-
FDA) for 5 h. After incubation, cells were thoroughly washed with PBS
buffer and were irradiated with 365 nm UV light for 10 min. Fluores-
cence images were taken using inverted fluorescence microscope after 2 h
incubation of irradiated or nonirradiated cells. Fluorescence images of the
cells incubated with FDA-loaded particles either with or without light irra-
diation were then taken with reverted fluorescence microscope.
according to reported methods.(39,40) The particles were synthe-
sized with the initiation of AIBN and were further purified by dial-
ysis against pure water for 2 days with alternative water
replacement to remove excess surfactants. The precipitates were
collected after high speed centrifuge and stored for further uses.
The synthesized polymeric particles were characterized by IR as
showed in Fig. 2. Strong peaks for the N-C bond at 1529 or
1527 cmꢀ1 were observed in particles synthesized from MA and
MB but not from MC. These specific peaks are observed in their
corresponding monomers. Particle size and shape were then deter-
mined by DLS, SEM and TEM techniques. Typical results are
shown in Fig. 3a and b. DLS results show that the sizes of the
particles formed from monomer A and B are approximately 150–
200 nm in diameter for all compositions with the cross-linker CL1
(Table S1 and Figure S1). The size of particles with the nonphot-
olabile monomer C and 10% cross-linker CL1 was 168 nm in
diameter which is also similar to above photoresponsive particles.
With the addition of hexadecane in miniemulsion, the more com-
pact particles were formed with smaller sizes around 135–150 nm
in diameter by DLS (Table S1). SEM images also show the spheri-
cal shape with particle diameter around 100–250 nm in diameter
for particles synthesized from monomer A, monomer B and mono-
mer C with different compositions (Figure S2). To further confirm
the formation of particles instead of vesicles, results from TEM
show that particles instead of micelles were formed from both
monomer A and monomer B. (Figure S3) Nile red, a model hydro-
phobic guest molecule, was encapsulated inside hydrophobic par-
ticles during miniemulsion and following polymerization. The dye
has higher solubility and displays stronger fluorescence in the
hydrophobic environment, while its fluorescence is much weaker
when it is released into water.(32) Since Nile red was encapsulated
into the particles, we also characterized Nile red encapsulation by
imaging particles (MA-CL1-10%-NR) with fluorescence micro-
scope. As shown in Fig. 3c, red fluorescence spots from the parti-
cles encapsulated Nile red were clearly visible.
Cellular toxicity of particles. Blank nanoparticles synthesized from
MA, MB and MC were thoroughly dried under vacuum to remove meth-
ylene chloride inside the core of particles and resuspended in PBS buffer.
Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay was used for the assess of cell viability. Briefly,
5 9 104 RAW 264.7 cells were seeded in 96-well flat bottom plates (Co-
star, Corning, NY) and allowed to grow for 8 h. The cells were incu-
bated with increasing concentrations of MA and MB particles or
irradiated residues for 48 h. Then, 10 lL MTT (5 mg/ml in PBS) was
added and incubated for another 4 h. MTT-formazan crystals formed
were then dissolved in 150 lL three linked lysis solution (10% SDS/5%
isobutyl alcohol/0.01 M HCl) overnight. The absorbance was measured
on microplate reader at 570 nm. The cell viability (%) was determined
by comparing the absorbance at 570 nm with control wells containing
only cell culture media.
RESULTS AND DISCUSSION
Synthesis and Characterization of Photolabile Polymeric
Nanoparticles
Photolabile monomers MA, MB and MC were synthesized by
methacrylation of the corresponding 2-nitrobenzyl alcohol deriva-
tive moieties using methacryloyl chloride in dry THF and TEA, as
shown in Figure S1. The emulsification were carried out with the
cross-linkers (CL1 or CL2) and monomers MA, MB and MC
Figure 2. IR spectra of nanoparticle MA-CL1-10%, MB-CL1-10% and
MC-CL1-10%. Strong peaks for C-N at 1529 cmꢀ1 or 1527 cmꢀ1 were
observed in particles MA-CL1-10% and MB-CL1-10%, but not MC-
CL1-10%.

Photochemistry and Photobiology, 2013, 89 555
Figure 3. Characterization of photoresponsive particle MB-CL1-10%. (a) Image of scanning electron micrograph (SEM); (b) Particle size distribution
by dynamic light scattering (DLS); (c) Fluorescence microscopy of Nile red-encapsulated particles MB-CL1-10%-NR.
The stability of synthetic particles in water was verified by
fluorescence intensity of Nile red in particle aqueous solutions.
We monitored fluorescence intensity at 609 nm of Nile red
encapsulated particle solutions (0.5 mg/mL) with MB and 1%,
4% and 25% of a cross-linker (CL1) at 0, 2 and 4 days. Varia-
tions in fluorescence intensity were below 15% of initial intensi-
ties. With gentle vortexing, fluorescence intensity of the particle
solutions was almost recovered. For particles (MB-CL1-25%-
NR) with high percentages of the cross-linker (25% CL1), fluo-
rescence intensity of the particle solution almost did not change
in 4 days (Figure S4). These results showed that no obvious leak
of the encapsulated Nile red happened.
Photoresponsive Behaviors of Nanoparticles
An essential feature of these particle delivery vehicles is the trig-
gered release in response to external light. We tested the possi-
bility of uncaging these photoresponsive particles under our
photolysis conditions (365 nm, 11 mW cmꢀ2), as shown in
Fig. 4 and Figure S5. Figure 4 demonstrated the absorption spec-
trum changes in particles (MA-CL1-10% and MB–CL1-10%)
formed with MA or MB monomers with 10% CL1 after the irra-
diation of these particle aqueous solutions (0.5 mg/mL). The
presence of isosbestic points at 332 and 275 nm indicates a clean
conversion from esters to carbonyl moieties for MA and
MB. The process of the photolysis was followed by noting the
appearance of 1-(2-nitrosophenyl) ethanone and 2-(2-nitrosophe-
nyl)propanal, as indicated by absorption peaks at 290 and
310 nm for particles MA-CL1-10% and 290 and 318 nm for par-
ticles MB–CL1-10%. The spectral changes in particle solutions
were consistent to the changes of their monomers, respectively,
and the literature report of similar photolabile moieties,(41)
which verified the uncaging efficacy of particles as monomers
under our photolysis conditions. IR spectra also indicated that
light irradiation caused disappearance of C-NO2 vibration signal
Figure 4. Absorbance spectra variation in blank polymeric particles
at 1529.75 cmꢀ1 for particle MA-CL1-10% (Figure S6). These
MA-CL1-10% and MB-CL1-10% upon photolysis at 365 nm (11 mW/
cm2). The concentrations of particle solutions were 0.5 mg/mL.
observations confirmed the transformation of hydrophobic esters
to hydrophilic acids inside the polymers, as indicated in the
uncaging mechanism in previous report.
nonphotolabile MC. As we can see, there was only slightly fluo-
rescence intensity variation in 15 min light irradiation for parti-
cles MC-CL1-10%-NR (Figure S7). However, dramatic decrease
in fluorescence intensity was observed for photoresponsive parti-
cles MB-CL1-10%-NR, and the remaining fluorescence intensity
of the solution reached as low as 5% of the initial fluorescence
intensity. The fluorescence intensity was not recovered in couple
of hours due to the precipitation of Nile red. These results dem-
onstrated that the efficient transformation of hydrophobic to
hydrophilic structure of photoresponsive particles under mild UV
irradiation. Particles synthesized with different percentages of the
To further evaluate the photocontrolled release, light-triggered
transformation of hydrophobic to hydrophilic environment inside
particles was investigated by Nile red fluorescence upon expo-
sure to light (365 nm). Fluorescence emission is used to charac-
terize the efficiency of the triggered release due to its dramatic
change in fluorescence intensity during hydrophobic and hydro-
philic transformation. Figure 5 shows light triggered the changes
in Nile red fluorescence spectra of aqueous particle (MB-CL1-
10%-NR and MC-CL1-10%-NR) solutions (0.5 mg/mL) synthe-
sized with 10% cross-linker CL1 and the photolabile MB or

556 Zhen Wang et al.
Figure 6. Plots of normalized fluorescence intensity of Nile red at
609 nm vs irradiation time (365 nm, 11 mW/cm2) for aqueous solutions
of photoresponsive particles MB-CL1 with different percentages of the
cross-linker (1%, 5%, 10%, 15% and 25%). The concentrations of parti-
cle solutions were 0.5 mg/mL.
was efficient and the encapsulated contents were efficiently
released from these photoresponsive polymeric particle systems
under our photolysis conditions.
The effect of hydrophobic cores of MA-CL1-10%-NR and
MB-CL1-10%-NR on kinetics of triggered release of Nile red
was also studied. Under the same photolysis conditions, NR fluo-
rescence intensity was recorded with the increase of UV irradia-
tion time. Figure S10 shows that the normalized fluorescence
intensity for particle solutions of MA-CL1-10%-NR and MB-
CL1-10%-NR was an exponential change with irradiation time.
To illuminate the difference, the characteristics time (τ) was
introduced by fitting the data through the following equation(32):
Figure 5. Fluorescence spectra of particles encapsulated with Nile red
upon light irradiation (365 nm, 11 mW/cm2) with the excitation wave-
length at 550 nm. (a) particle MC-CL1-10%; (b) particle MB-CL1-10%.
The concentrations of particle solutions were 0.5 mg/mL.
I=I0 ¼ ðI=I0Þm þ ½1 ꢀ ðI=I0Þmꢁexpðꢀt=sÞ
cross-linker CL1 (1%, 5%, 10%, 15% and 25%) to MA and
MB, and their effects on photocontrolled release were also inves-
tigated. As shown in Fig. 6, normalized fluorescence intensity
variation in particles (MB-CL1-1/5/10/15/25%) was recorded as
a function of different irradiation time in aqueous solutions
(0.5 mg/mL). For all the compositions of particles MB-CL1, the
phototriggered fluorescence decrease was clearly observed and
the release efficiency was around 85–95% in less than 8 min.
Similar observations also happened for the photoresponsive parti-
cles MA-CL1 (Figure S8). When 10% cross-linker CL2 was
used instead of CL1 to miniemulsion polymerization of MB,
Nile red fluorescence intensity of aqueous particle (MB-CL2-
10%) solution also greatly decreased and release efficiencies
could also reach up to 95% in 15 min UV irradiation (Figure
S9). The initial rate of decrease in Nile red fluorescence for parti-
cle MB-CL1-10% solution was large than that of particle MB-
CL2-10% solution. This difference may be due to some small
size of particle MB-CL1-10% or longer hydrophibic linker of
CL2. If hexadecane was added during miniemulsion, it did not
influence the sensitivity of photolabile particles, and the encapsu-
lated Nile red could also be efficiently released upon light irradi-
ation. In 10 min, release efficiencies were also around 90–95%
for nanoparticle formed with MB and CL1. These experimental
data demonstrated that the photocleavage of photolabile moieties
Here, (I/I0)m is the achievable minimum of the normalized fluo-
rescence, and τ is the characteristic time at which the fluores-
cence intensity decreases to 36.8% (1/e). The curve fitting results
displayed τ for polymeric particle MB-CL1-10%-NR (τMB-Nile
= 72 s) were much shorter than that for particle MA-CL1-
red
10%-NR (τMA-Nile red = 273 s), which shows that the photoclea-
vage rate of MB was faster than that of MA. These results are
consistent to the releasing efficiencies of Nile red and the previ-
ous report for photo-uncaging of these two photolabile moie-
ties.(42)
The uncaging and crash of photolabile particles were also
clearly observed by SEM, fluorescence microscopy and colorime-
try. Figure 7a and b show typical SEM images of nanoparticle
MB-CL1-1%. Before light irradiation, particles were in spherical
shapes in SEM images. However, most spherical particles crashed
or melted due to light irradiation. Similar observations also
happened for the other photoresponsive particles synthesized from
MA and MB, but not from MC. The color variation in aqueous
particle (MA-CL1-10%) solutions was also observed before and
after light irradiation, as shown in Fig. 7c–e. These aqueous parti-
cle (MB-CL1-1/4/25%) solutions remained as a red opaque sus-
pension under ambient conditions even after 4-day standing if no
light irradiation was applied (Fig. 7c). In comparison, the red

Photochemistry and Photobiology, 2013, 89 557
Figure 7. SEM images of photoresponsive particles MB-CL1-1% before light irradiation (a) and 15 min light irradiation with 365 nm (b); (c) a typical
photo image of photoresponsive particle (MB-CL1) aqueous solutions with different percentages of cross-linker CL1 stored in the dark for 4 days; (d)
photo images of photoresponsive particle (MA-CL1-10%) aqueous solution before and after 15 min irradiation with 365 nm; (e) a typical photo image
of irradiated photoresponsive particle (MB-CL1) aqueous solutions with different percentages of cross-linker CL1 after 4 days. The concentrations of
particle solutions were 0.5 mg/mL.
opaque suspension turned to purple red suspension with 15 min
light irradiation (Fig. 7d), and purple Nile red solid gradually pre-
cipitated at the bottom of cuvettes. Figure 7e clearly shows that
most Nile red solid was at the bottom and aqueous solution became
transparent and almost colorless. Similar phenomena were also
observed using fluorescence microscopy (Figure S11). Light irradi-
ation could greatly reduce the fluorescence spots in the images for
particles MA-CL1-10%-NR and MB-CL1-10%-NR, while lots of
Nile red fluorescence spots still exist for particles MC-CL1-10%-
NR after light irradiation. These results further confirmed the
release of the encapsulated contents from internal particle cores
upon light activation due to the breakage of these photoresponsive
particles.
light activation. As we expected, these nanoparticles were stable
in cells and there are almost no fluorescence emission. However,
upon light activation, the cells clearly lighted up with the hydro-
lyzed FDA, as shown in Fig. 8g and h. In comparison to
MC-CL1-10%-FDA, MA-CL1-10%-FDA must have been photo-
degraded and the breakage of polymers triggers the release of
FDA, followed by the hydrolysis of FDA with intracellular ester-
ase.
To evaluate the toxicity of these particles themselves, macro-
phages were incubated with varying concentrations (0–1000 lg/
mL) of blank particles prepared from monomer A, B and C
with 10% CL1 for 48 h. As seen in Fig. 9, the results of cell
viabilities showed low toxicity at the concentration as high as
1000 lg/mL of aqueous particle (MA-CL1-10%, MB-CL1-10%
and MC-CL1-10%) solutions. Upon irradiation, cell viabilities
did not decrease at low concentration of particle solutions.
However, cell toxicity increased greatly at high concentration
for particle MA-CL1-10% and MB-CL1-10%, but not for MC-
CL1-10%. We then tested cell viability of a micelle self-assem-
Nanoparticle Uptake and Photocontrolled Release In Cells
We further studied cell uptake and cell viability of photorespon-
sive polymeric particles synthesized from MA, MB and MC
using RAW 264.7 macrophages. We used FDA-encapsulated
particles (MA-CL1-10%-FDA and MB-CL1-10%-FDA) to evalu-
ate particle cell uptake and phototriggered release in cells. FDA
is nonfluorescent, upon uptake by the cells, intracellular esterases
hydrolyze diacetate groups, producing the highly fluorescent
product, fluorescein. Uptake and release of FDA from above par-
ticles were confirmed by fluorescence of the hydrolyzed fluores-
cein (Fig. 8 and Figure S12). Figure 8a–b and c–d are the
images of cells incubated with MC-CL1-10%-FDA before and
after light activation. No obvious fluorescence was observed
which indicated FDA could not be released from the nonphotola-
bile nanoparticles even with light activation. Figure 8e and f are
images of cells with the uptake of MA-CL1-10%-FDA without
bled with
a
diblock copolymer (PEG5000-(MA)33). We
observed low toxicity of the above micelle before light irradia-
tion, while their toxicity increased at high concentration
(1000 lg/mL) upon light irradiation, which is similar to the
observation for nanoparticle MA-CL1-10% and MB-CL1-10%.
(Figure S13) We postulated that the cleavaged nitrosobenzyl by-
products lead to the high toxicity. We then evaluated cell viabil-
ity of these small organic compounds to RAW246.7 according
to the same experimental procedure as particle solutions. High
level of toxicity of these small molecules was observed before
or after light irradiation even at relative low concentrations, as
shown in Figure S13.

558 Zhen Wang et al.
Figure 8. Fluorescence microscopy image of RAW264.7 cells incubated with MA-CL1-10% and MC-CL1-10% before or after 10 min light irradiation.
(a,b) Particle MC-CL1-10%-FDA without light irradiation; (c,d) Particle MC-CL1-10%-FDA with 10 min light irradiation; (e,f) Particle MA-CL1-10%-
FDA without light irradiation; (g,h) Particle MA-CL1-10%-FDA with 10 min light irradiation. The concentration of nanoparticles was 31.3 lg/mL. After
particle incubation for 5 h at 37°C, the cells were thoroughly washed with PBS to remove the free nanoparticles. The cells were irradiated with UV lamp
(365 nm, 11 mW/cm2) for 10 min, then observed by inverted fluorescence microscope. All fluorescence images were taken under the same conditions.
Figure 9. Toxicity of particles (MA-CL1-10%, MB-CL1-10% and MC-CL1-10% and irradiated residues) measured by incubation with macrophages for
48 h. The viability of cells incubated with particles was normalized to cells cultured without particles.
macrophages at high particle concentration after 48 h incubation.
Increased toxicity was observed at high concentration of irradiated
CONCLUSIONS
particles MA-CL1-10% and MB-CL1-10%, but not for MC-CL1-
10%, which is due to the toxicity of the cleaved by-products. Fur-
ther studies will focus on using less toxic photolabile monomer,
such as coumarin derivatives, or using copolymers with methyl
methacrylate monomer for syntheses of photoresponsive particles
and their photocontrolled release.
In summary, a series of cross-linked photoresponsive polymeric
particles with different photolabile monomers and cross-linkers
have been synthesized through miniemulsion polymerization and
have been characterized through DLS, SEM, TEM and fluorescence
microscopy. The synthesized particles were quite stable in aqueous
solutions, while light irradiation caused the crash of these particles
and the efficient release of encapsulated contents. Releasing effi-
ciency could reach up to 95% based on fluorescence intensity of
Nile red. Photoswitches of photoresponsive particle systems were
confirmed by fluorescence spectroscopy, SEM and colorimetry.
Particle cell uptake and triggered release in RAW264.7 cells were
confirmed by Nile red-loaded particles and FDA-loaded particles.
The cell viability of particles shows low toxicity of blank particle
and particles MA-CL1-10%, MB-CL1-10% and MC-CL1-10% for
Acknowledgements—This work is supported by Innovation Team of
Ministry of Education (no. BMU20110263) and Higher Education
Doctoral Fund (20100001120051) of Ministry of Education of China, the
National Natural Science Foundation of China (grant no. 21072015) and
Program for New Century Excellent Talents in University (NCET-10-
0203). We thank Profs. ChenHo Tung and LiZhu Wu (Technical Institute
of Physics and Chemistry, CAS) for help with SEM measurements and
Prof. JingRong Cui (Peking University) for providing RAW264.7 cells.

Photochemistry and Photobiology, 2013, 89 559
20. LitxTM is developed by Light Sciences Oncology for innovative
light-activated drug treatment for solid tumors. Available at: www.
lsoncology.com.
21. Li, M.-H. and P. Keller (2009) Stimuli-responsive polymer vesicles.
Soft Matter 5, 927–937.
22. Banerjee, S. S. and D.-H. Chen (2009) A Multifunctional Magnetic
Nanocarrier Bearing Fluorescent dye for Targeted Drug Delivery by
Enhanced two-Photon Triggered Release Nanotechnology 20, 185103.
23. Rai, P., S. Mallidi, X. Zheng, R. Rahmanzadeh, Y. Mir, S. Elring-
ton, A. Khurshid and T. Hasan (2010) Development and Applica-
tions of Photo-triggered Theranostic Agents. Adv. Drug Deliv. Rev.
62, 1094–1124.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Particle characterization, NMR, DLS, TEM, SEM, photolysis,
particle stability, FDA release in cells and toxicity were all
included in supplementary materials.
24. Hwang, G. T., Y. J. Seo and B. H. Kim (2004) A Highly Discrimi-
nating Quencher-Free Molecular Beacon for Probing DNA. J. Am.
Chem. Soc. 126, 6528–6529.
25. Jochum, F. D., L. Z. Borg, P. J. Roth and P. Theato (2009) Thermo-
and Light-Responsive Polymers Containing Photoswitchable Azoben-
zene End Groups. Macromolecules 42, 7854–7862.
REFERENCES
1. Sahoo, S. K. and V. Labhasetwar (2003) Nanotech approaches to
drug delivery and imaging. DDT 8, 1112–1120.
2. Mohanraj, V. and Y. Chen (2006) Nanoparticles – A Review. Trop.
J. Pharmaceut. Res. 5, 561–573.
3. Alvarez-Lorenzo, C. and A. Concheiro (2008) Intelligent Drug
Delivery Systems:Polymeric Micelles and Hydrogels. Mini-Reviews
in Medicinal Chemistry 8, 1065–1074.
4. Esser-Kahn, A. P., S. A. Odom, N. R. Sottos, S. R. White and J. S.
Moore (2011) Triggered Release from Polymer Capsules. Macromol-
ecules 44, 5539–5553.
5. Fomina, N., J. Sankaranarayanan and A. Almutairi (2012a) Photo-
chemical mechanisms of light-triggered release from nanocarriers.
Adv. Drug Deliv. Rev. 64, 1005–1020.
6. Meng, F., Z. Zhong and F. Jan (2009) Stimuli-Responsive Polymer-
somes for Programmed Drug Delivery. Biomacromolecules 10, 197–
209.
7. Lux, C.d.G., C.L. McFearin, S. Joshi-Barr, J. Sankaranarayanan, N.
Fomina and A. Almutairi (2012) Single UV or Near IR Triggering
Event Leads to Polymer Degradation into Small Molecules. ACS
Macro Lett., 1, 922–926.
8. Lin, Y.-L., H.-Y. Chang and Y.-J. Sheng (2012) Photoresponsive
Polymersomes Formed by Amphiphilic Linearꢀ Dendritic Block
Copolymers: Generation-Dependent Aggregation Behavior. Macro-
molecules 45, 7143–7156.
9. Li, G., G. Fei, H. Xia, J. Han and Y. Zhao (2012) Spatial and tem-
poral control of shape memory polymers and simultaneous drug
release using high intensity focused ultrasound. J. Mater. Chem. 22,
7692–7696.
10. Kumar, S., J.-F. Allard, D. Morris, Y. L. Dory, M. Lepage and Y.
Zhao (2012) Near-infrared light sensitive polypeptide block copoly-
mer micelles for drug delivery. J. Mater. Chem. 22, 7252–7257.
11. Han, D., X. Tong and Y. Zhao (2012) Block Copolymer Micelles
with a Dual-Stimuli-Responsive Core for Fast or Slow Degradation.
Langmuir 28, 2327–2331.
26. Jin, Q., G. Liu and J. Ji (2010) Micelles and Reverse Micelles with
a Photo and Thermo Double-responsive Block Copolymer J. Polym.
Sci. Pol. Chem. 48, 2855–2861.
27. Lee, H., W. Wu, J. K. Oh, L. Mueller, G. Sherwood, L. Petean, T.
Kowalewski and K. Matyjaszewski (2007) Light-Induced Reversible
Formation of Polymeric Micelles. Angew. Chem. Int. Ed. 119, 2505–
2509.
28. Yan, B., X. Tong, P. Ayotte and Y. Zhao (2011b) Light-responsive
block copolymer vesicles based on a photo-softening effect. Soft
Matter 7, 100001–100009.
29. Menon, S., R. Thekkayil, S. Varghese and S. Das (2011) Photore-
sponsive Soft Materials: Synthesis and Photophysical Studies of a
Stilbene-Based Diblock Copolymer. J. Polym. Sci. Pol. Chem. 49,
5063–5073.
30. Jiang, J., X. Tong and Y. Zhao (2005) A New Design for Light-
Breakable Polymer Micelles. J. Am. Chem. Soc. 127, 8290–8291.
31. Babin, J., M. Pelletier, M. Lepage, J. F. Allard, D. Morris and Y.
Zhao (2009) A New Two-Photon-Sensitive Block Copolymer Nano-
carrier. Angew. Chem. Int. Ed. 121, 3379–3382.
32. Jiang, J., X. Tong, D. Morris and Y. Zhao (2006) Toward Photocon-
trolled Release Using Light-Dissociable Block Copolymer Micelles.
Macromolecules 39, 4633–4640.
33. Doh, J. and D. J. Irvine (2004) Photogenerated Polyelectrolyte Bilay-
ers from an Aqueous-Processible Photoresist for Multicomponent
Protein Patterning. J. Am. Chem. Soc. 126, 9170–9171.
34. Johnson, J. A., M. G. Finn, J. T. Koberstein and N. J. Turro (2007)
Synthesis of Photocleavable Linear Macromonomers by ATRP and
Star Macromonomers by a Tandem ATRP-Click Reaction: Precur-
sors to Photodegradable Model Networks. Macromolecules 40, 3589
–3598.
12. Han, D., X. Tong, O. Boissie re, and Y. Zhao (2012) General
Strategy for Making CO2-Switchable Polymers. ACS Macro Lett. 1,
57–61.
13. Han, D., O. Boissiere, S. Kumar, X. Tong, L. Tremblay and Y. Zhao
(2012) Two-Way CO2-Switchable Triblock Copolymer Hydrogels.
Macromolecules 45, 7440–7445.
35. Xie, Z., X. Hu, X. Chen, G. Mo, J. Sun and X. Jing (2009) A Novel
Biodegradable and Light-Breakable Diblock opolymer Micelle for
Drug Delivery. Adv. Eng. Mater. 11, B7–B11.
36. Schork, F. J., Y. Luo, W. Smulders, J. P. Russum, A. Butte and K.
Fontenot (2005) Miniemulsion Polymerization. Adv. Polym. Sci. 175,
129–255.
14. Fomina, N., C. L. McFearin and A. Almutairi (2012b) Increasing
materials’ response to two-photon NIR light via self-immolative den-
dritic scaffolds. Chem. Commun. 48, 9138–9140.
15. Yesilyurt, V., R. Ramireddy and S. Thayumanavan (2011) Photoreg-
ulated release of noncovalent guests from dendritic amphiphilic na-
nocontainers. Angew. Chem. Int. Ed. 50, 3038–3042.
16. Yan, B., J.-C. Boyer, N. R. Branda and Y. Zhao (2011a) Near-
infrared light-triggered dissociation of block copolymer micelles
using upconverting nanoparticles. J. Am. Chem. Soc. 133, 19714–
19717.
17. Shi, D., M. Matsusaki and M. Akashi (2011) Photo-tunable protein
release from biodegradable nanoparticles composed of cinnamic acid
derivatives. J. Control. Release 149, 182–189.
18. Alvarez-Lorenzo, C., L. Bromberg and A. Concheiro (2009) Light-
sensitive Intelligent Drug Delivery Systems. Photochem. Photobiol.
85, 848–860.
37. Tang, X., M. Su, L. Yu, C. Lv, J. Wang and Z. Li (2010) Photo-
modulating RNA cleavage using photolabile circular antisense oligo-
deoxynucleotides. Nucleic Acids Res. 38, 3848–3855.
38. Bhushan, K. R., C. DeLisi and R. A. Laursen (2003) Tetrahedron
44, 8585–8588.
39. Griset, A. P., J. Walpole, R. Liu, A. Gaffey, Y. L. Colson and M.
W. Grinstaff (2009) Expansile Nanoparticles: Synthesis, Character-
ization, and in Vivo Efficacy of an Acid-Responsive Polymeric Drug
Delivery System. J. Am. Chem. Soc. 131, 2469–2471.
40. Landfester, K., N. Bechthold, F. Tiarks and M. Antonietti (1999)
Macromolecules 32, 5222–5228.
41. Yan, F., L. Chen, Q. Tang and R. Wang (2004) Synthesis and Char-
acterization of a Photocleavable Cross-Linker and Its Application on
Tunable Surface Modification and Protein Photodelivery. Bioconju-
gate Chem. 15, 1030–1036.
42. Buler, S., I. Lagoja, H. Giegricha, K.-P. Stengele and W. Pfleiderer
(2004) New Types of Very Efficient Photolabile Protecting Groups
Based upon the [2-(2-Nitrophenyl)propoxy]carbonyl (NPPOC) Moi-
ety. Helv. Chim. Acta 87, 620–659.
19. Lv, C., Z. Wang, P. Wang and X. Tang (2012) Photodegradable
polyurethane self-assembled nanoparticles for photocontrollable
release. Langmuir 28, 9387–9394.