Jin 2019

Theranostics 2019, Vol.
9, Issue 1 246
Ivyspring
International Publisher
Theranostics
2019; 9(1): 246-264. doi: 10.7150/thno.30174
Research Paper
Near-infrared light-regulated cancer theranostic

nanoplatform based on aggregation-induced emission
luminogen encapsulated upconversion nanoparticles
Guorui Jin1,2*, Rongyan He1,2*, Qian Liu1,2, Min Lin1,2, Yuqing Dong2,3, Kai Li4, Ben Zhong Tang5,6, Bin Liu7,
Feng Xu1,2
1. The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Shaanxi, 710049,
China
2. Bioinspired Engineering & Biomechanics Center (BEBC), Xi’an Jiaotong University, Shaanxi, 710049, China
3. State Key Laboratory for Mechanical Behavior of Materials, School of Materials Science and Engineering, Xi’an Jiaotong University, Xi’an 710049, People’s Republic of
China
4. Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 510855, China.
5. Department of Chemistry, Institute for Advanced Study, Institute of Molecular Functional Materials, Division of Biomedical Engineering, Division of Life Science, and
State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China
6. Guangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University
of Technology, Guangzhou, 510640, China
7. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117585, Singapore.
Authors contributed equally

*
 Corresponding author: [email protected]; [email protected]
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Received: 2018.09.25; Accepted: 2018.11.13; Published: 2019.01.01
Abstract
Photodynamic therapy (PDT) has been widely applied in the clinic for the treatment of various types of
cancer due to its precise controllability, minimally invasive approach and high spatiotemporal accuracy as
compared with conventional chemotherapy. However, the porphyrin-based photosensitizers (PSs) used
in clinics generally suffer from aggregation-caused reductions in the generation of reactive oxygen species
(ROS) and limited tissue penetration because of visible light activation, which greatly hampers their
applications for the treatment of deep-seated tumors.
Methods: We present a facile strategy for constructing a NIR-regulated cancer theranostic
nanoplatform by encapsulating upconversion nanoparticles (UCNPs) and a luminogen
(2-(2,6-bis((E)-4-(phenyl(40-(1,2,2-triphenylvinyl)-[1,10-biphenyl]-4-yl)amino)styryl)-4H-pyran-4-ylidene
)malononitrile, TTD) with aggregation-induced emission (AIEgen) characteristics using an amphiphilic
polymer, and further conjugating cyclic arginine-glycine-aspartic acid (cRGD) peptide to yield
UCNP@TTD-cRGD NPs. We then evaluated the bioimaging and anti-tumor capability of the
UCNP@TTD-cRGD NPs under NIR light illumination in an in vitro three-dimensional (3D) cancer
spheroid and in a murine tumor model, respectively.
Results: With a close match between the UCNP emission and absorption of the AIEgen, the synthesized
NPs could efficiently generate ROS, even under excitation through thick tissues. The NIR-regulated
UCNP@TTD-cRGD NPs that were developed could selectively light up the targeted cancer cells and
significantly inhibit tumor growth during the NIR-regulated PDT treatment as compared with the cells
under white light excitation.
Conclusion: In summary, the synthesized UCNP@TTD-cRGD NPs showed great potential in NIR
light-regulated photodynamic therapy of deep-seated tumors. Our study will inspire further exploration
of novel theranostic nanoplatforms that combine UCNPs and various AIEgen PSs for the advancement of
deep-seated tumor treatments with potential clinical translations.
Key words: aggregation-induced emission (AIE), photodynamic therapy, near infrared light, active targeting,
tumor imaging
http://www.thno.org
Theranostics 2019, Vol. 9, Issue 1 247
Introduction as activatable PSs via triggered aggregation to further

optimize the therapeutic outcome of PDTs and to
While numerous advances have been made in
increase the signal-to-background ratio in bioimaging
cancer diagnosis and chemotherapy, cancer patients
[24-27]. However, most AIEgen PSs are regulated by
still suffer from severe side effects, drug resistance
visible light (400–700 nm) which suffers from limited
and frequent recurrence of cancer [1]. This serves to
tissue-penetration depth, thus restricting their
emphasize the importance of developing new
applications to only therapy for superficial cancers
antitumor therapy strategies that have new
(e.g., melanoma) [28, 29]. Recently, AIEgen PSs with
mechanisms of action. Nanoparticle-based theranostic
two-photon absorption (TPA) capability have been
platforms that incorporate both imaging and
developed to realize NIR light-regulated PDTs using
therapeutic reagents into one single probe are
expensive femtosecond pulsar lasers [29]. However,
strongly desired for cancer diagnosis and treatment
this requires complicated synthesis routines which are
because they facilitate image-guided diagnosis and
not feasible for obtaining AIEgen PSs with usable TPA
therapy concurrently [2-4]. Among the various
efficiency [30]. In addition, two photon-PDT
theranostic platforms, photodynamic therapy (PDT)
treatment is restricted to a tiny tissue volume
has been increasingly recognized as an attractive
resulting in prolonged treatment time, thus limiting
approach for cancer treatment because of the
its clinical use [31]. Therefore, there is still an unmet
advantages of precise controllability, its minimally
need for a facile and efficient approach to solve these
invasive nature and high spatiotemporal accuracy
issues to further extend the applications of AIEgen
[5-11]. PDT relies on photosensitizers (PSs) to
PSs across a broad range of cancer therapies.
generate toxic reactive oxygen species (ROS) to induce
Upconversion nanoparticles (UCNPs), which can
cell death upon light illumination, while the
harness energy from NIR light and convert it to higher
fluorescence from the photosensitizers can also
energy (e.g., visible or UV light) [32, 33], have
facilitate the image-guided theranostic process.
emerged as a promising NIR light “transducer” to
However, traditional fluorescent photosensitizers,
engineer NIR light-regulated theranostic
especially the clinically used porphyrin derivatives,
nanoplatforms for PDTs to solve the issue of limited
generally suffer from π-π stacking due to the intrinsic
tissue-penetration depth [34-36]. Therefore, by
hydrophobic and rigid planar molecular structures,
choosing UCNPs whose emission spectra match the
which further lead to aggregation caused quenching
absorption spectrum of AIEgen PSs, a robust NIR
(ACQ) and a significant decrease in ROS generation
light-regulated theranostic platform can be
[12, 13]. Additionally, the requirement of visible light
engineered for the treatment of deep-seated tumors.
activation for most of the conventional PSs greatly
This facile and efficient strategy thus greatly expands
hampers their clinical applications in treating solid or
the potential applications of AIEgen PSs in treating a
deep-seated tumors, due to the limited
broad range of cancers. To the best of our knowledge,
tissue-penetration depth of visible light [14].
the in vivo PDT of deep-seated tumors based on the
Therefore, there is an unmet demand for developing
combination of UCNPs with an AIEgen PS has not
effective PDT PSs with good ROS yield and stable
been reported yet.
fluorescence in an aggregated state, which can be
In this research work, we developed a
activated by near-infrared (NIR) light that has greater
monodisperse nanoplatform by direct encapsulation
tissue-penetration depth and less tissue scattering and
of UCNPs with a spectrally matchable AIEgen PS,
blood absorption [15, 16].
which offers targeted cell imaging and PDT under
Fluorogens with aggregation-induced emission
single NIR laser illumination. By encapsulating the
characteristics (AIEgens) have emerged as effective
hydrophobic AIEgen, 2-(2,6-bis((E)-4-(phenyl(40-
fluorescent materials for theranostic applications
(1,2,2-triphenylvinyl)-[1,10-biphenyl]-4-yl)amino)styr
[17-19]. In contrast to ACQ materials, AIEgens are
yl)-4H-pyran-4-ylidene)malononitrile (TTD) and
generally non-emissive in good solutions but can
UCNPs using a biocompatible poly(ethylene
become highly emissive upon aggregation, due to the
glycol)-lipid and further decoration of the particle
restriction of intramolecular rotations, which
surface with cyclic arginine-glycine-aspartic acid
prohibits energy dissipation through nonradiative
(cRGD), we obtained NIR-regulated multifunctional
channels [20]. As such, AIEgen PSs have attracted
probes (i.e., UCNP@TTD-cRGD) for the PDT
significantly increasing interest as excellent key
treatment of cancer. The synthesized
components in PDTs with the expectation of yielding
UCNP@TTD-cRGD can still efficiently generate ROS
bright emission and high ROS-induced phototoxicity
in the presence of a 6 mm thick tissue when excited by
upon loading into nanocarriers in an aggregated state
a 980 nm laser. In addition, the UCNP@TTD-cRGD
[21-23]. Additionally, by taking advantage of the AIE
could selectively and efficiently kill the targeted triple
phenomenon, AIEgens have been developed further
negative breast cancer cells (MDA-MB-231) as sonicated immediately for 1.5 min using a probe
demonstrated in both two-dimensional (2D) and sonicator at 60 W output to form UCNP@TTD NPs.
three-dimensional (3D) in vitro tumor models. The The THF was evaporated by stirring the clear red
therapeutic efficacy of UCNP@TTD-cRGD NPs as an solution in the fume hood for 8 h. The cRGD peptides
in vivo PDT agent was further evaluated in a mouse (5×10−2 M) were then added to the solution containing
model. After 980 nm laser illumination, the growth of UCNP@TTD NPs with further stirring at room
tumors intratumorally injected with the NPs was temperature for 12 h. Then we removed the excess
significantly inhibited despite the different initial cRGD peptides from the particle solution by dialysis
sizes (60, 120 and 240 mm3), indicating an excellent against MilliQ water for 1.5 d and changed it with
PDT efficacy of the UCNP@TTD-cRGD NPs in fresh MilliQ water four times. The UCNP@TTD-cRGD
deep-seated tumor treatments. Additionally, the NPs that were generated were collected for further
UCNP@TTD-cRGD NPs could accumulate at the use.
tumor site for tumor imaging and significant
restriction of tumor growth after intravenous Characterizations
administration. These results demonstrate that the To verify the aggregation-induced luminescence
NIR light-regulated cancer theranostic nanoplatform phenomenon, the same amount of molecular AIEgens
with AIEgen PSs will open up new opportunities for were dissolved in THF and a water mixture solution
targeted and image-guided PDT of deep-seated with different water fractions (fw) at the same total
tumors allowing for great clinical translation volume (the water fraction was from 0% to 100% with
potential. 10% intervals). The change in fluorescence intensity
was measured with a fluorometer (Photon
Materials and methods Technology International). The average size of the
NPs was measured using a Brookhaven Zeta Plus zeta
Synthesis of upconversion nanoparticles potential analyzer (Brookhaven Instruments,
(UCNPs) Holtsville, NY). The morphology of the NPs was
The NaYF4:Yb,Er UCNPs were synthesized studied by transmission electron microscopy (Nippon
according to reports in the literature [2, 37]. Briefly, Tekno Co., Ltd). The emission spectra (200–900 nm) of
deionized water (2 mL) containing YCl3·6H2O (242.69 the UCNP@TTD-cRGD NPs and UCNPs were
mg, 0.8 mmol), YbCl3·6H2O (69.75 mg, 0.18 mmol), recorded by excitation with a 980 nm laser. To study
and ErCl3·6H2O (7.64 mg, 0.02 mmol) was added to a the photostability of the synthesized NPs,
100 mL flask that was pre-loaded with oleic acid (7.5 UCNP@TTD-cRGD NPs (25 μg/mL or 5 μg mL-1 of
mL) and 1-octadecene (15 mL). The solution was TTD) were dispersed in DMEM supplemented with
stirred for 0.5 h at room temperature, followed by 10% FBS and 1% PS, and the emission intensity was
stirring for 1 h at 120 °C, and for another 1.5 h at 166 recorded every day up to 10 days using a fluorometer
°C to remove water under an argon atmosphere. After (Photon Technology International). A Qtracker® 585 (a
the solution was cooled down to room temperature, a commercial fluorescent probe) with the same
methanol solution (5 mL) with NH4F (148.15 mg, 4 concentration was used as a control.
mmol) and NaOH (100 mg, 2.5 mmol) was added, and
it was further stirred for 2 h. Then, the methanol was Detection of ROS in solution
removed by heating the solution to 120 °C for 1 h. The The 9,10-anthracenediyl-bis(methylene)
solution was further heated to 296 °C for 2 h, followed dimalonic acid (ABDA) probe was sensitive to the
by a cooling down to room temperature. The resulting singlet oxygen (1O2) which was used to measure 1O2
product was washed with ethanol and cyclohexane generation from the NPs irradiated by a 980 nm laser.
three times and then finally dissolved in The absorbance at 378 nm of ABDA would decrease in
tetrahydrofuran (THF). the presence of 1O2 because of the oxidized ABDA
[38]. Rose Bengal (RB) with a 75% 1O2 quantum yield
Synthesis of UCNP@TTD-cRGD NPs in water was used as the standard photosensitizer
To synthesize UCNP@TTD-cRGD NPs, 2 mL [39]. The ABDA solid was dissolved in the mixture of
THF was used to dissolve DSPE-PEG2000-Mal (2 mg), UCNP@TTD-cRGD NPs (5 μg mL-1 of TTD) with a
DSPE-PEG2000 (1 mg) and TTD molecules (0.75 mg). concentration of 40 µM and RB (5 μg/mL) and then
The UCNPs dissolved in THF (70 µL, 0.4 mg/mL) irradiated for 0, 30, 60, 90 and 120 seconds using a 980
were added into the formed solution and mixed nm laser (for UCNP@TTD-cRGD) and white light (for
under sonication for 5 min followed by standing for RB), both at a power density of 200 mW cm-2. The
30 min. Then the formed mixture solution was quickly power output of the white light and 980 nm laser used
injected into 18 mL of deionized (DI) water, and in this study was measured at 500 nm (the absorbance
maximum of TTD) and at 980 nm using an optical cm-2) illumination, followed by fixation with 4%
power and energy meter (Thorlabs, PM100D), paraformaldehyde. The fixed cells were imaged using
respectively. confocal microscopy at 644 nm excitation and a
The 1O2 quantum yield of the UCNP@TTD- 665/20 nm bandpass filter.
cRGD was calculated based on the following equation
[39]. Cytotoxicity of the UCNP@TTD-cRGD NPs
𝐾𝐾NPs 𝐼𝐼RB The cytotoxicity of the UCNP@TTD-cRGD NPs
ΦNPs = ΦRB was studied using a
𝐾𝐾RB 𝐼𝐼𝑁𝑁𝑁𝑁𝑁𝑁
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazol
where ΦNPs and ΦRB were the 1O2 quantum yield for
ium bromide (MTT) assay. MDA-MB-231, MCF-7 and
UCNP@TTD-cRGD and RB, respectively. KNPs and KRB
NIH 3T3 cells (5×103 per well) were co-cultured with
represent the slope of plot ln(A0/A) versus
UCNP@TTD-cRGD NPs at a concentration of 0, 12.5,
illumination time from the UCNP@TTD-cRGD and
25 and 50 µg/mL in 96-well plates, respectively. After
RB, respectively, where A and A0 stands for the
48 h of incubation, a 100 µL MTT solution (0.5
absorbance of ABDA at 378 nm with and without light
mg/mL) was added into each well, and incubation
illumination, respectively. INPs and IRB were calculated
continued for a further 3 h. Then, 100 µL DMSO was
from the areas of their absorption spectra, from
used to dissolve all the formed precipitates with the
400–800 nm, respectively.
help of gentle shaking. The absorbance of MTT at 570
The tissue-penetration ability of UCNP@TTD- nm was monitored with a microplate reader and the
cRGD NPs viability of the cells was reflected by comparing the
The UCNP@TTD-cRGD NPs in the cuvettes were absorbance of the nanoparticle-treated cells to that of
covered by chicken tissues of different thicknesses the cells incubated with nanoparticle-free medium
(i.e., 0, 3 and 6 mm), and further illuminated by a 980 only.
nm laser and white light at a power density of 200 Photocytotoxicity studies
mW cm-2 for 2 min, respectively. Then we used a ROS
The photocytotoxicity of UCNP@TTD-cRGD
probe, ABDA to detect the ROS generation.
NPs was studied by evaluating the metabolic activity
Targeting capability of UCNP@TTD-cRGD of NP-treated cells after illumination with a 980 nm
NPs laser. For this, MDA-MB-231, MCF-7 and NIH 3T3
Coverslips with a diameter of 15 mm were cells at a density of 5000 cells/well were cultured in
located in a 24-well plate. Three kinds of cells 96-well plates, and each type of cells was treated with
(MDA-MB-231, MCF-7 and NIH 3T3) at a density of NPs (25 µg/mL) alone, 2 min of 980 nm laser
3×104 cells per well were seeded on the coverslips, illumination alone, and also 2 min of 980 nm laser
respectively. After 12 h of incubation, the culture illumination with NPs, respectively. After 48 h, cell
medium was replaced by fresh medium viability was evaluated by MTT assay. For live/dead
supplemented with UCNP@TTD-cRGD NPs (5 μg staining assay, the cells seeded in 24-well plates at a
mL-1 of TTD) and the cells were further incubated for density of 5×104 cells per well were subjected to the
4 h. The cells treated by NPs were fixed by 4% same treatments, and then live/dead staining was
paraformadehyde for 15 min and washed with 1×PBS performed to characterize the viability of cells.
buffer twice followed by flow cytometry detection. Cell apoptosis assay
The NP-treated cells were used for fluorescence
The APC Annexin V/Dead Cell Apoptosis Kit
imaging study after being stained with
(Life Technologies) was used to assess cell apoptosis
4',6-diamidino-2-phenylindole (DAPI).
after the PDT treatment according to the
Intracellular ROS detection manufacturer's instructions. Briefly, UCNP@TTD-
The CellROX® Deep Red Reagent (Thermo cRGD NP-treated MDA-MB-231 cells (3×105
Fisher Scientific) was used to detect intracellular ROS, cells/well) in 6-well plates were exposed to a 980 nm
following the manufacturer's instructions. Briefly, The laser (200 mW cm-2) for 2 min and cultured further for
MDA-MB-231 cells treated with UCNP@TTD-cRGD 12, 24 and 48 h, respectively. At each time interval, the
NPs (5 μg mL-1 of TTD) were further incubated with cells were collected from the 6-well plate and washed
CellROX® Deep Red Reagent that was diluted to 5 µM three times with 1× annexin-binding buffer, followed
with pure DMEM medium for 30 min. Then the cell by centrifugation and resuspension in 1×
culture medium was replaced with fresh medium annexin-binding buffer at a concentration of 1×106
after three times washing with 1×PBS. After that, the cells/mL. Then, APC annexin V (5 μL) and SYTOX®
cells were subjected to 2 min of 980 nm laser (200 m W Green solution (1 μL, 1 μM) were added to each 100
μL cell suspension, respectively. After 15 min
incubation, an additional 1× annexin-binding buffer mm3, the PDT experiments were then conducted,
(400 μL) was added into each sample by gentle mixing respectively.
and the samples were kept on ice before the flow
cytometry test. The stained cells were analyzed by In vivo antitumor efficacy of UCNP@TTD-
recording the fluorescence emission at 530 nm cRGD NPs with intratumoral injection
(excited by 488 nm) and 660 nm (excited by 633 nm). For in vivo imaging studies, the tumor bearing
Confocal microscopy was applied to verify the flow mice having a tumor size of 120 mm3 were
cytometry results. intratumorally injected with 20 µL
UCNP@TTD-cRGD NPs (2 mg/mL of TTD). Then, in
In vitro 3D cell spheroids for PDT treatment
vivo fluorescence images were obtained using an
The 3D cell spheroids were fabricated using a IVIS® Lumina Series III with a 500 nm excitation and a
microwell approach as reported in our previous study bandpass filter from 550 nm to 750 nm at the
[40]. Briefly, 15% polyethylene glycol-dimethacrylate designated time intervals (1, 2, and 7 D post-injection).
(PEG-DMA, MW=1000; Polysciences, Inc., The NP-treated mice were sacrificed after 7 days and
Warrington, PA, USA) and 0.5% (v/v) of a the collected organs were immediately imaged ex vivo
photoinitiator (PI), using a IVIS® Lumina Series III with a 500 nm
2-hydroxy-2-methylpropiophenone (TCI, Shanghai excitation and a bandpass filter from 550 nm to 750
Development Co., Ltd., Shanghai, China) were used nm.
to fabricate microwells facilitated by a photomask. For in vivo PDT treatment using intratumorally
After UV cross-linking, microwells of 800 μm in injected UCNP@TTD-cRGD NPs, we divided the
diameter and 1 mm in depth were formed. The tumor-bearing mice into three groups based on their
hydrogel microwell array located in the 24-well plates initial tumor volumes (60 mm3 120 mm3 and 240
was sterilized by placing the plate in the biosafety mm3). Each group included five sub-groups that were
cabinet with ultraviolet light illumination for 4 h. treated as follows: control group, without any
MDA-MB-231 cells at a concentration of 1×106 treatment; nanoparticle-only group, tumors
cells/mL were seeded on the microwells. The cell intratumorally injected with nanoparticles only;
spheroids were formed after three days’ culture. Then laser-only group, tumors intratumorally injected with
the spheroids were fed with UCNP@TTD-cRGD NPs saline with NIR laser illumination (200 mW cm-2) for
(5 μg mL-1 of TTD) according to the above-mentioned 20 min; white light PDT group, tumors intratumorally
protocol with extended co-culture duration of 12 h. injected with UCNP@TTD-cRGD NPs (2 mg/mL, 20
After replacement with fresh medium, the NP-treated µL), and at 4 h injection illuminated with white light
spheroids were illuminated under a 980 nm laser (200 (200 mW cm-2) for 20 min; laser PDT group, tumors
mW cm-2) for 5, 10, 20 and 30 min, respectively. MTT intratumorally injected with UCNP@TTD-cRGD NPs
assay and live/dead staining were used to study the (2 mg/mL, 20 µL), and a 4 h injection illuminated with
viability of the 3D cell spheroids after 48 h further a 980 nm laser (200 mW cm-2) for 20 min. The tumor
incubation. The cell spheroids treated with NP-free volume and mice body weight were measured every
medium with or without NIR laser illumination were two days.
used as the control, respectively. We took three All the mice were euthanized after 10 days’
fluorescence images in the Z-direction of each treatment, and the tumors were collected and fixed in
spheroid and analyzed the fluorescence intensity of 10% neutral-buffered formalin for further evaluation.
each with ImageJ software to obtain the ratio of live For the hematoxylin and eosin (H&E) staining, the
and dead cells within each spheroid after PDT formalin-fixed tumor sections were stained and
treatment. visualized by fluorescence microscope (Olympus
Animal study IX51, Japan). For the TUNEL staining, the fixed tumor
sections were stained by the One Step TUNEL
All the animal studies conformed to the
Apoptosis Assay Kit (Beyotime Biotechnology,
guidelines set by the Institutional Animal Care
Beijing, China) according to the manufacturer’s
Committee of Xi'an Jiaotong University. The tumors
protocol and visualized by fluorescence microscope
were developed by subcutaneously implanting 5×106
(Olympus IX51, Japan).
MDA-MB-231 cells into the right front flanks of
female BALB/c nude mice having an average weight In vivo targeted PDT treatment with
of 20 ± 2 g. Tumor volume was monitored using a intravenous injection
caliper and calculated using the following formula: For investigating the biodistribution of NPs, the
volume = (tumor length) × (tumor width)2/2. When tumor bearing mice having a tumor size of 120 mm3
the tumor volume reached 60 mm3, 120 mm3 and 240
were intravenously injected with 125 µL
UCNP@TTD-cRGD NPs (2 mg/mL of TTD). Then, in toxicity of UCNP@TTD-cRGD NPs by collection of the
vivo fluorescence images were obtained using an major organs (such as heart, liver, spleen, lung and
IVIS® Lumina Series III with a 500 nm excitation light kidney) from the mice intravenously injected with
and a bandpass filter from 550 nm to 750 nm at UCNP@TTD-cRGD NPs after 5 days. Then the organs
different time intervals (0, 4, 8, 24, 48 and 96 h were fixed, embedded and sectioned for H&E
post-injection). The NP-treated mice were sacrificed staining. Photos were taken to evaluate the toxicity of
after 24 h and the collected organs were immediately the UCNP@TTD-cRGD NPs in vivo by comparing the
imaged ex vivo by IVIS® Lumina Series III with a 500 morphology and structure of the major organs with
nm excitation and a bandpass filter from 550 nm to tumor-free mice without nanoparticle injection.
750 nm. Intravenous injection of free cRGD (15 mg
kg-1) was conducted to block the overexpressed Statistical analysis
integrin of the MDA-MB-231 tumors. After injection All quantitative results were obtained from at
of free cRGD for 30 min, 125 µL of UCNP@TTD-cRGD least three samples for analysis. Data were expressed
NPs (2 mg/mL of TTD) were then intravenously as the mean ± standard error of the mean. A
injected into the tumor-bearing mice. The two-tailed paired Student's t-test was used to compare
tumor-bearing mice intravenously injected with the differences. Difference with p < 0.05 was
125 µL of UCNP@TTD-cRGD NPs (2 mg/mL of TTD) considered to be statistically significant.
were used as positive control. The mice were then
Results and discussion
imaged using the IVIS® Lumina Series III following
the same protocol. Preparation of UCNP@TTD-cRGD NPs
We chose the tumor-bearing mice with an initial In this study, we developed a NIR light-
tumor volume of 120 mm3 for further evaluation of regulated theranostic platform based on
the in vivo targeted PDT treatment of deep-seated AIE-luminogen-encapsulated UCNPs (Figure 1). This
tumors using UCNP@TTD-cRGD NPs administered platform possesses capabilities for enhanced
by intravenous injection. The tumor-bearing mice bioimaging and deep-seated tumor treatment,
were randomly divided into five groups: control because of the AIEgen PS that can yield bright
group, nanoparticle-only group, laser-only group, emission and high ROS-induced phototoxicity in an
white light PDT group, and laser PDT group. Both aggregated state, as well as UCNPs that can convert
NPs and saline were administered intravenously to NIR light to shorter wavelength light for the
tumor-bearing mice in the corresponding groups. The activation of the AIEgen PS [41, 42]. To achieve high
tumor volume and mice body weight were measured ROS yield, we chose the NaYF4:Yb,Er UCNP as the
every two days. NIR light transducer, as its emission matches well
Hemolysis assay and in vivo toxicity assay with the excitation of TTD at around 540 nm (Figure
S1). Briefly, we used amphiphilic polymers (i.e.,
Red blood cells (RBCs) were isolated by
DSPE-PEG2000 and DSPE-PEG2000-MAL) to
centrifugation of mice blood at 3000 rmp for 10 min
encapsulate the hydrophobic UCNPs and TTD that
and thrice washed with saline. Then the RBCs were
were synthesized according to our previous protocol
co-cultured with distilled water, saline and the
[43, 44] and synthetic route [17], respectively. The
UCNP@TTD-cRGD NP solution (20 µg/mL, 40
synthesized TTD molecules showed typical twisted
µg/mL and 80 µg/mL of TTD), respectively. After
intra-molecular charge transfer and AIE
culture for the designated time intervals (1, 2 and 3 h),
characteristics (Figure 2A). The DSPE segments along
all suspensions were subjected to 5 min centrifugation
with the hydrophobic UCNP and TTD tended to be
at 3000 rpm, followed by transfer of the supernatant
entangled into the hydrophobic core, while the
liquid to a 96-well plate for absorbance reading at 577
hydrophilic PEG chains spread out into the aqueous
nm by a microplate reader (Thermo Scientific, USA).
phase [45]. The hydrophobic interaction between TTD
The same concentration of UCNP@TTD-cRGD NPs
and the NaYF4:Yb,Er UCNPs provides a simple
was suspended in saline as a blank control. The
method to keep the PS molecule TTD close to the
precipitations at the bottom of the tubes were used to
UCNPs, favoring energy transfer between the two
make the cell smear to observe the morphologies of
components. The formed PEG sheath has the potential
the RBCs. The hemolysis ratio of the RBCs was
to enhance the circulation time of the synthesized
calculated using the following formula:
NPs, therefore improving nanoparticle accumulation
Hemolysis (%) = (OD sample − OD negative control )/(OD at the tumor site for in vivo applications [46]. No
positive control − OD blank ) ×100%. obvious precipitation formed in the UCNP@TTD NP
Tumor-free mice were used to study the in vivo suspensions after removing THF, indicating the high
encapsulation efficiency of TTD and the UCNPs. The
Mal-decorated UCNP@TTD NPs were further fluorescence of Qtracker® 585 was dramatically
conjugated with cyclic arginine-glycine-aspartic acid decreased to ~20% of the initial fluorescence after only
(cRGD) peptide to yield UCNP@TTD-cRGD NPs, 3 days (Figure 2D). The dramatically decreased
which have the ability to target integrin- fluorescence of Qtracker® 585 in a cell culture medium
overexpressed cancer cells. The UCNP@TTD-cRGD has been reported previously [49, 50]. The probable
NPs that were obtained have excellent colloidal reason is that the quantum dot-based Qtracker® 585
stability in a water suspension as demonstrated by the suffers from potential degradation caused by reactive
fact that no obvious precipitation was observed even oxygen species, resulting in a fluorescence decrease
after being stored at 4 ℃ for 3 months. The [51, 52]. The results indicate that the synthesized
bioimaging and anti-tumor capability of UCNP@TTD-cRGD NPs are suitable for long-term
UCNP@TTD-cRGD NPs upon NIR light illumination imaging applications.
was further evaluated in an in vitro three-dimensional Considering that the generation of cytotoxic
(3D) cancer spheroid and in a murine tumor model, singlet oxygen (1O2) plays a major role in determining
respectively. PDT efficacy, we evaluated the ability of the
UCNP@TTD-cRGD NPs to generate 1O2 under direct
Characterization of the UCNP@TTD-cRGD NIR light and white light illumination by monitoring
NPs the absorbance decrease of 9,10-anthracenediyl-
The ideal NP for image-guided PDT applications bis(methylene) dimalonic acid (ABDA) at 378 nm
should have characteristics of monodispersity, (Figure 2E), respectively. Rose Bengal (RB) with a 1O2
photostability, targeting, and a high quantum yield of 75% in water was used as the
signal-to-background ratio, as well as negligible dark standard photosensitizer [39]. We observed that 980
cytotoxicity and acute cytotoxicity under light nm laser illumination of UCNP@TTD-cRGD NPs
illumination [47]. In particular, PS-loaded NPs with resulted in an ABDA absorbance decrease at 378 nm,
excellent monodispersity favor the diffusion of ROS corresponding to 1O2 generation of TTD encapsulated
considering the fact that ROS have a small active on the UCNPs’ surface (Figure S3A). The initial
range (<20 nm) in a PDT protocol [48]. We therefore absorbance of ABDA at 378 nm before illumination is
used dynamic light scattering analysis and defined as A0 and the photosensitizer
transmission electron microscopy to study the (UCNP@TTD-cRGD NPs or RB) induced ABDA
dispersity and morphology of the synthesized absorbance which decreases at 378 nm after 2 min
UCNP@TTD-cRGD NPs. We observed that the illumination is defined as A. We observed a linear
monodisperse NPs have a corona morphology and a increase of ln(A0/A) over time (Figure 2E). The
narrow size distribution of 90 ± 4 nm (Figure 2B and decomposition rate constants of the
Figure S2). Since a close match between the UCNP UCNP@TTD-cRGD NPs (KNPs) and RB (KRB) were
emission and absorption of the AIEgen PS is calculated according to the slopes of the linear curves,
necessary for efficient upconversion to occur, we and determined to be 0.0076 and 0.0176, respectively.
therefore examined the spectral match between the The integration ratio of the absorption from 400 to 800
UCNP emission and absorption of TTD (Figure 2C). nm between RB (IRB) and UCNP@TTD-cRGD NPs
We observed that the emission of UCNP upon NIR (INPs), IRB/INPs, was calculated to be 1.13. Therefore, the
excitation matches well with the absorption of TTD. 1O quantum yield of UCNP@TTD-cRGD NPs (Φ
2 NPs)
As compared with UCNP alone, nearly all the green under 980 nm laser illumination was calculated to be
peaks (~540 nm) of the UCNP were absorbed by the 36.4%, which is higher than the 1O2 quantum yield of
TTD within the UCNP@TTD-cRGD NPs. Moreover, the FDA-approved photosensitizer meso-tetra-
the green color of UCNPs changes to a red color for hydroxyphenyl-chlorin (mTHPC, ΦmTHPC = 31%) [53].
the UCNP@TTD-cRGD NPs upon 980 nm laser The 1O2 generation of UCNP@TTD-cRGD NPs under
illumination (Figure 2C, insert), further confirming white light illumination was also monitored in Figure
efficient energy transfer between the UCNP and TTD. S3B and the 1O2 quantum yield (ΦNPs) was calculated
The enhanced red fluorescence of UCNP@TTD-cRGD to be 63.7%. However, the higher 1O2 quantum yield
NPs favors in vivo bioimaging. Since photostability is of UCNP@TTD-cRGD NPs under white light
important for long-term tumor imaging, we checked illumination as compared to that under 980 nm laser
the photostability of UCNP@TTD-cRGD NPs illumination was greatly attenuated after coverage
dispersed in a cell culture medium, using the with chicken tissues (Figure S4), as demonstrated in
commercial cell imaging probe Qtracker® 585 as a the following study. We further investigated the
control (Figure 2D). We observed that penetration of a 980 nm laser and white light
UCNP@TTD-cRGD NPs could still maintain 75% of illumination, using 3 mm and 6 mm thick chicken
their initial fluorescence after 10 days, while the tissues to mimic clinical skin tissue (Figure S4),
respectively. We observed that the efficiency of 1O2 generation of 1O2 could still be detected when
generation is significantly decreased (5.1-fold illuminated at 980 nm. These results indicate a better
decrease) with white light illumination due to the 3 penetration depth of 980 nm excitation compared
mm thick chicken tissue coverage, while there is only with white light illumination, which makes
a 1.0-fold decrease in 1O2 generation when UCNP@TTD-cRGD NP-based PDT suitable for
illuminated at 980 nm (Figure 2F). When the tissue effective tumor treatment in living systems, especially
thickness increases to 6 mm, there is barely any 1O2 for deep-seated tumors.
generation with white light illumination, while the
Figure 1. A rational design for a NIR light-regulated theranostic nanoplatform based on AIE luminogen encapsulated upconversion
nanoparticles. Schematic illustration of preparation of UCNP@TTD-cRGD nanoparticles and their applications in bioimaging and PDT of deep-seated tumors upon
NIR laser illumination, in an in vitro three-dimensional (3D) cancer cell spheroid and in a murine tumor model, respectively.
Figure 2. Characterization of the theranostic nanoplatform based on UCNP@TTD-cRGD NPs. (A) Change of PL intensity of 5 µM of TTD in
THF-water mixtures with different water fractions (fw %) at the same total volume. (B) Particle size distribution of UCNP@TTD-cRGD NPs studied by dynamic light
scattering. Insert image represents the morphology of the UCNP@TTD-cRGD nanoparticle under transmission electron microscopy (the enlarged TEM image can
be found in Figure S2). (C) Fluorescence spectra of UCNPs and UCNP@TTD-cRGD NPs. (D) Time courses for fluorescence intensity change of
UCNP@TTD-cRGD in DMEM supplemented with 10% FBS after continuous incubation at 37 °C. (E) Relative ROS generation with Rose Bengal (RB) as the standard
photosensitizer (the 1O2 quantum yield for RB is 75% in water). (F) Relative ROS generation of UCNP@TTD-cRGD excited by NIR or visible light with the coverage
of tissues of different thicknesses.
Figure 3. Targeting study of UCNP@TTD-cRGD NPs. (A) Fluorescent images of MDA-MB-231, MCF-7, NIH 3T3 cells after incubation with
UCNP@TTD-cRGD NPs (5 μg mL-1 of TTD) for 4 h. Red fluorescence is from the UCNP@TTD-cRGD NPs. Blue fluorescence labels the nuclei. (B) Quantitative
analysis of fluorescence intensity of UCNP@TTD-cRGD nanoparticle signals from confocal images shown in (A). Flow cytometry results of UCNP@TTD-cRGD
nanoparticle-treated MDA-MB-231 cells against MCF-7 cells (C) and NIH 3T3 cells (D), respectively. (E) Confocal images of MDA-MB-231 cells pre-treated with free
cRGD before co-incubation of UCNP@TTD-cRGD NPs. (F) Confocal images of MDA-MB-231 cells treated with UCNP@TTD nanoparticles without cRGD
conjugation. (G) Confocal images of MDA-MB-231 cells without any treatment. Scale bars: 20 μm.
intensity of MCF-7 and NIH 3T3 cells was only 36.3%

Targeting and intracellular ROS detection and 34.4%, respectively. We also performed a flow
Active targeting of NPs to cancer cell-specific cytometry study to further evaluate the targeting
receptors could promote rapid intracellular uptake of capability of UCNP@TTD-cRGD NPs on
the enhanced permeability and retention (EPR) MDA-MB-231 cells. We observed that the mean
accumulated nanoformulations. To examine the fluorescence intensity of MDA-MB-231 cells was
targeting ability of UCNP@TTD-cRGD NPs, the ανβЗ significantly higher than that of the MCF-7 and NIH
integrin overexpressed MDA-MB-231 cells were used 3T3 cells (Figure 3C-D). The results indicate that
as integrin-positive cancer cells, using MCF-7 and UCNP@TTD-cRGD NPs can be selectively
NIH 3T3 cells as the negative controls. The internalized by MDA-MB-231 cells in significant
MDA-MB-231 cells, MCF-7 and NIH 3T3 cells were quantities, demonstrating the targeting capability of
incubated with UCNP@TTD-cRGD NPs (5 μg mL-1 of UCNP@TTD-cRGD NPs. Besides, the uptake of
TTD) for 4 h and then imaged using a confocal UCNP@TTD-cRGD NPs by cRGD pre-treated
microscope (Figure 3A). We observed clear red MDA-MB-231 cells was significantly suppressed as
fluorescence from the UCNP@TTD-cRGD NPs in the shown in Figure 3E. Moreover, the UCNP@TTD NPs
cytoplasm of the MDA-MB-231 cells, but much lower without cRGD conjugation could not be effectively
signals in the MCF-7 and NIH 3T3 cells (Figure 3A). internalized by the MDA-MB-231 cells (Figure 3F).
We also quantified the fluorescence intensity using These results clearly suggest that the targeting
the ImageJ program from confocal images and set the capability of UCNP@TTD-cRGD NPs is mainly due to
fluorescence intensity of the MDA-MB-231 cells at the cRGD conjugation on the particle surface.
100% (Figure 3B). We observed that the fluorescence
It has been well accepted that the loss of cell viability of the MCF-7 and NIH 3T3 cells was not
viability induced by PDT is associated with excessive significantly affected by the NP treatment,
intracellular ROS production. To evaluate the demonstrating the robust capability of UCNP@TTD-
intracellular ROS production of UCNP@TTD-cRGD cRGD NPs in selectively killing MDA-MB-231 cells
NPs under NIR laser illumination, we used the under both 980 nm laser and white light illumination
CellROX® Deep Red Reagent as the intracellular ROS (Figure 5B). In addition, we found that 2 min 980 nm
indicator. After oxidation by ROS, CellROX® Deep laser illumination does not affect the cell viability of
Red reagent becomes fluorescent at 633 nm laser all the cells without NP treatment, further confirming
illumination. As shown in Figure 4, excessive that the acute cytotoxicity is only induced by the
intracellular ROS generation was observed in the internalized UCNP@TTD-cRGD NPs under light
cytoplasm of MDA-MB-231 cells treated with illumination. The live/dead staining results
UCNP@TTD-cRGD NPs and 980 nm laser demonstrate that most MDA-MB-231, MCF-7 and
illumination (Figure 4A) as compared to the NIH 3T3 cells treated with NPs or 2 min 980 nm laser
MDA-MB-231 cells with only 980 nm laser illumination alone are still alive, as revealed by the
illumination (Figure 4B). The results demonstrate the green fluorescence in Figure 5C. However, nearly all
robust capability of UCNP@TTD-cRGD NPs in the UCNP@TTD-cRGD NP-incubated MDA-MB-231
producing intracellular ROS under NIR light cells were dead under 980 nm laser illumination or
regulation. white light illumination, as indicated by the red
fluorescence in Figure 5C, demonstrating the
In vitro PDT efficiency of UCNP@TTD-cRGD effectiveness of UCNP@TTD-cRGD NPs in selectively
NPs killing MDA-MB-231 cells. Furthermore, the laser
The potential photodynamic cytotoxicity of output power and exposure time to obtain a
UCNP@TTD-cRGD NPs was further evaluated significant antitumor efficacy in our technique (200
through an in vitro cytotoxicity assay (Figure 5A). We mW cm-2, 2 min) is much lower and shorter than that
observed that without 980 nm laser illumination, needed in the previous study (3 W cm-2, 10 min)
there is negligible cytotoxicity in the UCNP@TTD- which also used AIE-encapsulated UCNPs for cancer
cRGD NP-treated MDA-MB-231, MCF-7 and NIH 3T3 PDT [54]. Collectively, the synthesized UCNP@TTD-
cells in a 50 μg/mL concentration, suggesting a cRGD NPs can selectively light up the
minimum cytotoxicity caused by the NPs. However, MDA-MB-231cells and then kill the MDA-MB-231
with 980 nm laser illumination (200 mW cm-2, 2 min) cells by elevating intracellular ROS generated from
and white light illumination (200 mW cm-2, 2 min), the UCNP@TTD-cRGD NPs under NIR light
only 28.3% and 16.6% of the MDA-MB-231 cells illumination.
treated with UCNP@TTD-cRGD NPs (25 μg/mL, or 5
μg mL-1 of TTD) remained alive, respectively. Yet the
Figure 4. Intracellular ROS generation of UCNP@TTD-cRGD NPs. (A) Detection of intracellular ROS generation in living MDA-MB-231 cells after
co-culture with UCNP@TTD-cRGD NPs (5 μg mL-1 of TTD) and CellROX® Deep Red reagent, followed by light illumination (λex = 980 nm; 530/25 nm bandpass
filter). The red fluorescent signals represent the intracellular ROS. (B) Detection of intracellular ROS generation in living MDA-MB-231 cells only with NIR laser (980
nm) illumination.
Figure 5. Cytotoxicity study of UCNP@TTD-cRGD NPs with or without light illumination. (A) Cell viabilities of UCNP@TTD-cRGD
nanoparticle-treated MDA-MB-231 cells, MCF-7 cells and NIH-3T3 cells without light or NIR laser irradiation. (B) Viability of MDA-MB-231, MCF-7 and NIH 3T3
cells after incubation with UCNP@TTD-cRGD NPs (5 μg mL-1 of TTD) for 4 h followed by 980 nm laser or light irradiation (100 mW cm-2, 2 min). (C) Live/dead
staining after NIR laser PDT treatment.
Since 2D in vitro tumor models usually cannot staining of the 3D cell spheroids demonstrate the clear
predicate the in vivo outcomes because of their disruption of the 3D cell spheroid under NIR
oversimplification and non-representation of 3D light-regulated PDT (Figure S5).
natural tissues [55], we further investigated the To study the mechanism of PDT using
therapeutic effect of UCNP@TTD-cRGD NPs as a PDT UCNP@TTD-cRGD NPs in killing MDA-MB-231 cells,
agent for tumor therapy in a 3D in vitro tumor model we used APC Annexin V/Dead Cell Apoptosis Kit to
(Figure 6A). We observed that contrary to the PDT stain the PDT-treated cells for flow cytometry
outcomes observed in the 2D model, prolonged evaluation (Figure 7). We observed that after 12, 24
illumination durations up to 30 min are required to and 48 h incubation, the apoptosis rate was 20.5%,
kill ~70% of the cells from the 3D cell spheroids 54.5% and 12.2% (Figure 7A), respectively and the
(Figure 6B). The metabolic activity of the cell corresponding necrosis rate was 12.5%, 24.2% and
spheroids after PDT treatment was evaluated by MTT 78.9%, respectively. The result demonstrates a clear
assay, after further culture for 5 days. We observed apoptosis-to-necrosis process for cells with PDT
that the viability of the 3D cell spheroid declined to treatment. The confocal images showed that the
60.5% after 30 min of 980 nm laser illumination intracellular NPs (yellow) are encapsulated by a
(Figure 6C). The bright field images and F-actin damaged cell membrane as revealed by the red
fluorescence (Figure 7B), while no fluorescence is cell apoptosis under different illumination durations
observed in the untreated MDA-MB-231 cells (Figure (such as 0, 2, 5, and 10 min) and found that a
S6A), confirming that cell apoptosis is the mechanism prolonged illumination time can promote cell
in NIR light-regulated PDT. We further studied the apoptosis to necrosis (Figure S6B).
Figure 6. The evaluation of UCNP@TTD-cRGD NPs in cancer therapy using an in vitro 3D cancer model. (A). Live/dead staining of cell spheroids
irradiated with a 980 nm laser. (B) Quantitative analysis of live/dead fluorescence signals in each cell spheroid shown in (A). (C) The viability of cell spheroids after
NIR light PDT treatment revealed by MTT study.
Figure 7. Apoptosis and necrosis staining of MDA-MB-231 cells after PDT based on UCNP@TTD-cRGD NPs. (A) Flow cytometry results for the
MDA-MB-231 cells after co-culture with UCNP@TTD-cRGD NPs (5 μg mL-1 of TTD) for 4 h at 37 °C and then exposed to 2 min of 980 nm laser illumination (100
mW cm-2), following by further incubation for 12, 24 and 48 hours, respectively. Then, the apoptosis/necrosis was accessed with APC Annexin V/Dead Cell Apoptosis
Kit staining by flow cytometry. (B) Confocal images of MDA-MB-231 cells treated with PDT and continuously cultured for 12 h are shown. The green fluorescence
signal in the left image from the SYTOX® Green (λex = 488 nm; 520/20 nm bandpass filter) was used to stain the nucleus of the necrotic cells. The yellow fluorescence
signal was from UCNP@TTD-cRGD NPs (λex = 488 nm; 585/20 nm bandpass filter). The red fluorescence signal was from APC annexin V (λex = 633 nm; 660/20 nm
bandpass filter).
In vivo antitumor efficacy of UCNP@TTD- the tumors for the 980 nm laser illumination. The
cRGD NPs tumor sizes in the different groups were measured to
assess the PDT efficacy over a period of 10 days.
Inspired by the promising imaging and PDT In the group with an initial tumor volume of 60
results in both in vitro 2D and 3D cancer models, we mm , both NIR-regulated PDT treatment (P ≤ 0.001)
3
then examined the in vivo theranostic efficacy of and white light-regulated PDT treatment (P ≤ 0.009)
UCNP@TTD-cRGD NPs for treatment of deep-seated could significantly inhibit tumor growth as compared
tumors via intratumoral injection of with that observed in other groups, thus confirming
UCNP@TTD-cRGD NPs into living female Balb/c the capability of UCNP@TTD-cRGD NPs in killing
nude mice (Figure 8). Instead of covering a piece of cancer cells (Figure 8C). Importantly, the growth of
tissue over the subcutaneous tumor to represent tumors in the laser PDT group was significantly
deep-seated tumors, we grew MDA-MB-231 tumors restricted as compared to that in the white light PDT
with different initial therapeutic tumor volumes group, indicating the superior capability of NIR
including 60 mm3 (widely used as the initial light-regulated PDT in enhancing therapeutic
therapeutic tumor size), 120 mm3 and 240 mm3, outcomes, due to the deep tissue penetration of NIR
respectively. We used the enlarged initial therapeutic light and the successful energy transfer between the
tumor volumes (120 mm3 and 240 mm3) for the UCNPs and TTD (Figure 8C). On the other hand, the
deep-seated tumors which are more relevant to the tumors in the laser-only group, nanoparticle-only
tumors seen in clinical practice as compared to group and control group grew in an exponential
covering a piece of tissue over the subcutaneous manner over the period of time, suggesting that the
tumor. We found that the intratumorally injected treatment with 980 nm laser only or
UCNP@TTD-cRGD NPs with bright fluorescence can UCNP@TTD-cRGD NPs without light illumination
clearly reveal the boundary of the tumors for at least 7 induces minimum inhibition of tumor growth.
days (Figure 8A). After 7 days, we obtained the tumor In the group with an initial tumor volume of 120
and major organs of the mice with intratumorally mm3, the therapeutic outcomes of white
injected UCNP@TTD-cRGD NPs. We observed that light-regulated PDT treatment (P ≤ 0.016) were
most NPs were still within the tumors, while only a sub-optimal as revealed by the limited tumor
few NPs were metabolized by the liver (Figure 8B). shrinkage ratio (30%, reduced tumor volume in PDT
The results indicate the excellent internalization and group/tumor volume in control group). However, the
retention of UCNP@TTD-cRGD NPs at the tumor shrinkage ratio of tumors with an initial tumor
sites, which favors long-term tumor imaging and volume of 60 mm3 after white light-regulated PDT
tumor treatment by PDT. Furthermore, we treatment (Figure 8D) was around 60%. However,
investigated the therapeutic effect of NIR-regulated PDT treatment (P ≤ 0.001) could still
UCNP@TTD-cRGD NPs as a PDT agent for obviously inhibit tumor growth as compared with
deep-seated tumor therapy in living mice by dividing other groups, demonstrating the robust capability of
the tumor-bearing mice into three groups based on UCNP@TTD-cRGD NPs in killing cancer cells under
their initial tumor volumes (60 mm3 120 mm3 and 240 NIR light illumination (Figure 8D). When the initial
mm3). In each group, there were five sub-groups tumor volume increased to 240 mm3, white light PDT
including the laser PDT group, white light PDT treatment could not inhibit tumor growth anymore, as
group, laser-only group, nanoparticle-only group and indicated by the same tumor growth rate in the white
control group. In the laser PDT group (n=6), light PDT group, laser-only group, nanoparticle-only
UCNP@TTD-cRGD NPs were injected into the group and control group (Figure 8E). However,
implanted tumor, and followed by a 980 nm laser tumor growth in the 980 nm laser PDT group could
illumination on the tumors after intratumoral still be obviously restricted, confirming the great
injection for 4 h. The tumor-bearing mice in the white potential of UCNP@TTD-cRGD NPs in treating
light PDT group (n = 6) were irradiated with white deep-seated tumors under NIR light illumination
light under identical conditions. Tumor-bearing mice (Figure 8E).
with injection of only UCNP@TTD-cRGD NPs but no To further evaluate the therapeutic efficacy
NIR or white light illumination were assigned to be against deep-seated tumors, we also performed
the nanoparticle-only group (n = 6). In the laser-only TUNEL and H&E staining using the tissue sections
group (n = 6), the tumor-bearing mice were only from the laser PDT group and white light PDT group
irradiated with a 980 nm laser. The mice in the control after 10 days (Figure 8F and Figure S7), respectively.
group (n = 6) were injected with saline only. To These tissue sections from each isolated tumor were
minimize the overheating effect of 980 nm laser further divided based on their layers (upper, middle
illumination, we used a transparent ice bag to cover and bottom) within the tumor. We observed that in
the group comprising initial tumor volumes of 60 the same superior therapeutic outcomes of NIR
mm3, both laser PDT and white light PDT can induce light-regulated PDT as compared to white
cancer cell apoptosis in the bottom layers of the light-regulated PDT in tumors from the different
tumors (Figure 8F). However, when the initial tumor groups (60 mm3, 120 mm3 and 240 mm3) (Figure S7).
volume becomes 120 and 240 mm3, white light PDT In addition, the weight of all mice could be
can only induce apoptosis of cancer cells in the middle maintained within a healthy range (Figure S8A) and
and upper layers, respectively, due to the limited the tumors under NIR laser-regulated PDT treatment
tissue penetration of the white light. In contrast, NIR had a small tumor weight (Figure S8B). These results
light-regulated PDT treatment can still cause clearly imply that the UCNP@TTD-cRGD NPs
apoptosis of cells in the corresponding deeper layers, accompanied by laser illumination at 980 nm could
further confirming the potential of UCNP@TTD- serve as a reliable platform for effective in vivo
cRGD NPs in treating deep-seated tumors under NIR deep-seated tumor treatment.
light illumination. H&E staining results demonstrate
Figure 8. In vivo fluorescence imaging and PDT efficiency by intratumoral injection of UCNP@TTD-cRGD NPs. (A). The in vivo fluorescence images
of the tumor after being intratumorally injected with UCNP@TTD-cRGD NPs for 1, 2 and 7 days. (B) Ex vivo fluorescence imaging of the tumor and organs harvested
from the euthanized mice with intratumoral injection of nanoparticles after administration for 7 days (1 Tumor, 2 Kidney, 3 Spleen, 4 Liver, 5 Heart and 6 Lung).
Growth curves of tumors in laser PDT, white light PDT, nanoparticle-only, laser-only and control groups with initial tumor volumes of 60 (C), 120 (D) and 240 mm3
(E), respectively. (F) TUNEL staining results of tumor slices collected from mice that were divided by their initial tumor volumes of 60, 120 and 240 mm3, respectively.
*P < 0.05, **P < 0.01.
To study whether cRGD conjugation can aid in receptors of Kupffer cells which are responsible for
tumor uptake in living mice, we used fluorescence the majority of phagocytic activity in the liver, with
imaging to monitor in real time the targeting effect of decoy and nontoxic nanoparticles prior to
UCNP@TTD-cRGD NPs in vivo (Figure 9A). The administration of nanotherapeutics [59], and coating
fluorescent images were recorded at different time the membrane of cells (such as red blood cells,
intervals after the first intravenous injection of leukocytes, and monocytes) have been designed to
UCNP@TTD-cRGD NPs. We observed that the NPs reduce nanoparticle entrapment by the liver [60-62],
reveal a time-dependent biodistribution and tumor thus enhancing the delivery of therapeutic NPs to the
preferential profile in the mice. At 0 h, the intense diseased tissues. The quantity of NPs accumulating at
fluorescence near the tail reveals the presence of NPs the tumor site is still much higher than that in other
at the tail vein after intravenous injection. We organs such as the kidney, heart, lung, and spleen
observed that there was a significant increase in (Figure 9B and Figure S9), indicating the in vivo
fluorescence in the tumor of living mice at 4 h targeting capability of UCNP@TTD-cRGD NPs. To
post-injection of UCNP@TTD-cRGD NPs (Figure 9A) further evaluate the in vivo targeting specificity of the
and then the fluorescence reached the highest level in UCNP@TTD-cRGD NPs, we first intravenously
the tumor at 8 h post-injection. Then the fluorescent injected free cRGD (15 mg kg-1) into MDA-MB-231
signals gradually decreased at 24 h post-injection in tumor bearing mice. At 30 min post-injection, we then
the circulation process. The weak fluorescence at the intravenously injected the same dose of
neck reveals the potential presence of NPs at the UCNP@TTD-cRGD NPs. We found that the uptake of
lymph nodes [56]. The biodistribution result UCNP@TTD-cRGD NPs at 24 h post-injection by the
demonstrates that the NPs are mainly accumulated in tumor pretreated with cRGD was significantly
the liver (Figure 9B), which agrees with previous inhibited (Figure 9C), demonstrating the in vivo
findings [57, 58]. Methods such as saturating the targeting specificity of UCNP@TTD-cRGD NPs.
Figure 9. In vivo targeted tumor imaging and PDT therapy by intravenous injection of UCNP@TTD-cRGD NPs. (A) Biodistribution of
UCNP@TTD-cRGD NPs in tumor-bearing mice after intravenous injection of UCNP@TTD-cRGD NPs (30 mg/kg) at different times. Black circles indicate the
tumor. (B) Ex vivo fluorescence imaging of various organs and tumor tissues from mice intravenously injected with UCNP@TTD-cRGD NPs. The mice were
sacrificed at 12 h post-injection. (C) Biodistribution of UCNP@TTD-cRGD NPs in tumor-bearing mice 8 h after intravenous injection of UCNP@TTD-cRGD NPs
(30 mg/kg) without or with blocking the receptors. Red circles indicate the tumor. (D) Growth curves of tumors in laser PDT, white light PDT, nanoparticle-only,
laser-only and control groups, respectively, and nanoparticles were intravenously injected into tumors with an initial tumor volume of 120 mm3. (E) H&E staining
results of tumor slices collected from different groups of mice. **P < 0.01.
Encouraged by the imaging data that revealed targeting moieties could further enhance the selective
the effective tumor uptake of UCNP@TTD-cRGD accumulation of the PS loaded NPs at the target site.
NPs, we continued the light-mediated PDT therapy However, NP-based PDT still suffers from
via intravenous injection of UCNP@TTD-cRGD NPs drawbacks such as the severe aggregation of
or saline into five different groups of mice with an hydrophobic PSs, which have an ACQ property, and
initial tumor volume of 120 mm3. As shown in Figure the therapeutic efficiency of ACQ PSs is dependent on
9D, after 980 nm laser illumination, the growth rate of their aggregation state. To solve the aggregation-
the tumors in the laser PDT group (n = 6, P ≤ 0.01) was caused fluorescence and ROS reduction of ACQ PSs
found to be restricted significantly over the [14], AIEgen PSs with the remarkable feature of
therapeutic period, whereas the tumors in the white generating ROS and fluorescent signals even in the
PDT group, laser-only group, nanoparticle-only aggregated state have been developed as a better
group and control group (n = 6) revealed a fast choice for image-guided PDT with improved
growth rate. Further increase in the NP injection times therapeutic outcomes [26, 65]. In particular, inspired
and light irradiation times could improve the by exactly the opposite photophysical properties of
therapeutic outcome to realize complete cancer AIEgen PSs and ACQ PSs in the aggregated and
ablation. These data suggest that the facilitated tumor monomeric states, the ACQ PS (pheophorbide-a) and
uptake by cRGD and effective tumor reduction is AIEgen PS (tetraphenylsilole) have been encapsulated
mainly due to the production of 1O2 from the TTD into a pH-responsive nanoprobe that can realize
encapsulated UCNPs under 980 nm laser activated photodynamic ablation of cancer cells with
illumination. limited side effects, and in situ prediction of the
In addition to the promising antitumor therapy therapeutic response [66]. Various AIEgen PSs with
in vivo, the histological tumor tissue analysis also NIR emission have been developed for cancer
reveals the efficacy of the PDT treatment. After 10 theranostics [28, 67-69]. However, these reported
days of light-mediated PDT at 980 nm, H&E and AIEgen PSs are excited in a UV-visible wavelength
TUNEL staining of the tumor tissues displayed more ranging from 378 to 600 nm, which unfortunately
significant damage in the mice with intravenous shows limited penetration depth in biological tissues
injection of UCNP@TTD-cRGD NPs than those [70]. Efforts have been devoted to developing AIEgen
tumors treated in the other groups (Figure 9E). PSs that can be excited by red or NIR light [17, 71].
Moreover, there was no obvious hemolysis and organ However, the preparation of red/NIR fluorescent
damage observed during the treatment (Figure S10). molecules is extremely complicated regardless of
These results clearly indicate that UCNP@TTD-cRGD either the AIEgens or conventional dyes [72, 73],
NPs hold great potential for improving the which usually require several-step reactions and
therapeutic efficacy of in vivo deep-seated tumor inconvenient purifications. Recently, AIEgen PSs with
treatment with a desirable PDT outcome. high two-photon absorption (TPA) have been
developed for PDT of deep-seated tumors. However,
Discussion the TPA protocol requires the use of a tightly focused
Photodynamic antitumor therapy has been femtosecond laser beam as a light source to excite a
widely applied in the clinical treatment of various small focused area and obtain sufficient instant
types of cancers such as lung cancer, gastrointestinal energy for two-photon excitation [74, 75]. Moreover,
cancer, head and neck tumor and prostate tumors TPA materials often suffer from severely decreased
[14], due to its distinct advantages which include quantum yields after transfer into water through
precise controllability, a minimally invasive nature molecular engineering or nanoparticle formulation
and high spatiotemporal accuracy compared with [74]. Therefore, there is an urgent need to develop a
conventional chemotherapy [63, 64]. However, its facile and effective method for fabrication of a NIR
application in treating deep-seated tumors is light activated PDT platform based on AIEgens.
restricted due to the shortcomings of classical PSs Lanthanide-doped UCNPs with an intrinsic
such as poor solubility and aggregation under photon-converting nature upon exposure to
physiological conditions, undesirable long-wavelength light illumination have received
pharmacokinetics, and low tumor selectivity. In considerable attention in cancer imaging and therapy
contrast, NP-based PDT has emerged as an extremely [5, 63]. These UCNPs are excited by NIR light and can
promising avenue for future technological emit fluorescence across UV, vis, or even the NIR
breakthroughs. NPs can transport hydrophobic PSs in range in accordance with the absorption
a high PS “payload” to the tumor site via an enhanced characteristics of PSs. Recently, various UCNPs have
permeability and retention (EPR) effect. Additional been loaded with PSs (such as chlorin e6 and
surface modification and functionalization with β-carboxyphthalocyanine zinc) to engineer a
NIR-triggered cancer theranostic nanoplatform for under white light. With the rapid development of
cancer treatment [63, 76-78]. However, these PSs synthetic and theoretical approaches, the emission
generally suffer from ACQ, which restricts their range of UCNPs can be easily tuned by the type and
therapeutic efficiency. Therefore, the combination of amount of ions doped, which is advantageous in the
UCNPs and AIEgen PSs [70] which usually require selection of available AIEgen PSs as photosensitizing
visible light excitation provides a facile and effective agents in this protocol [64]. Thus, a therapeutic
way to fabricate a NIR light activated PDT nanoplatform based on AIEgen PS-encapsulated
nanoplatform based on AIEgen PSs. Although, the UCNPs is emerging as a versatile tool for deep-seated
combination of UCNPs with AIEgen PS for in vitro tumor therapy. It is known that the upconversion
PDT treatment of cancer cells was attempted once efficiency from NIR light to UV/vis light of UCNP is
[54], the ultra-small UCNPs that were used (< 12 nm) low, which reduces the singlet oxygen generation
as the NIR light “transducer” suffered from a quantum yield of UCNP@TTD-cRGD NPs. Methods
significant decrease in upconversion emission such as the homogeneous doping based on a
intensity and luminescence lifetime as compared with successive layer-by-layer approach [82], suppressing
UCNPs with a larger particle size (> 40 nm), due to surface quenching effects via heteroepitaxial growth
the severe surface quenching [79, 80]. Moreover, the of a biocompatible CaF2 shell [83], and coupling
severe aggregation of the synthesized particles UCNPs with an organic NIR dye molecule [84] have
hampers their blood circulation and diffusion of ROS been developed to increase the upconversion
generated in the inner part of the aggregates because efficiency of UCNPs. A 100-time enhancement in the
of the severely small active radius of the ROS (< 20 upconversion efficiency of NaYbF4:Tm@NaYF4:Nd
nm) [48], which leads to the dramatic decrease in both UCNPs has been achieved by their coupling with a
in vitro and in vivo PDT efficiency. Therefore, a NIR NIR sensitive organic dye [84]. Therefore, a
light-regulated cancer theranostic nanoplatform with NIR-regulated cancer theranostic nanoplatform
bright emission, high ROS production and good engineered by encapsulating UCNPs and AIEgen PSs
monodispersity is highly desired to successfully treat with improved singlet oxygen generation quantum
deep-seated tumors. yield can be realized to achieve excellent PDT efficacy.
In this research work, we encapsulated AIEgen It should be noted that the generation of ROS is highly
PS TTD and NaYF4:Yb,Er UCNPs using amphiphilic oxygen-dependent, which will limit the therapeutic
polymers and then conjugated cRGD on the particle effect of PDTs in treating hypoxic tumors [85].
surface to yield UCNP@TTD-cRGD NPs. The TTD Integration of an oxygen supplier (perfluorohexane)
and UCNP within the NP core could be densely [86, 87] or oxygen-independent radical source [88],
packed together because of the hydrophobic within the NIR light-regulated cancer theranostic
interactions between TTD and the UCNPs, which nanoplatform could be the solution for expanding its
favor energy transfer between these two components, application in treating hypoxic tumors.
approved by experimental results. Therefore, the
higher singlet oxygen production with 980 nm Conclusion
illumination in the presence of tissue coverage In summary, the synthesized UCNP@TTD-cRGD
compared with the white light illumination makes NPs showed great potential for NIR light-regulated
UCNP@TTD-cRGD NP-based PDT suitable for photodynamic therapy of deep-seated tumors. With
deep-seated tumor treatment in living systems. high photostability, the UCNP@TTD-cRGD NPs
Compared with two-photo excitation which also could maintain their fluorescent intensity above 70%
possesses deep tissue-penetration ability, after 30 days, and under NIR light illumination, a
upconversion processes are more efficient, and they significant amount of ROS could still be detected even
can be realized using inexpensive continuous-wave with coverage with a 6 mm tissue. The
(CW) lasers, unlike two-photon excitation, which UCNP@TTD-cRGD NPs could specifically target αvβ3
requires pulsed lasers [81]. Our extensive in vitro and integrin overexpressed MDA-MB-231 cells and
in vivo fluorescence imaging studies validated the selectively kill these cells in both 2D and 3D cancer
successful active targeting, which can be easily models upon NIR light illumination, without obvious
recognized by the enhanced fluorescence intensity dark cytotoxicity. The results of in vivo antitumor
within the targeted cancer cells and the in vivo tumors. evaluation demonstrated that with NIR light
Both in vitro and in vivo theranostic studies illumination, the intravenously injected UCNP@TTD-
successfully demonstrated that such AIEgen cRGD NPs could light up the tumors and significantly
PS-encapsulated UCNPs with NIR laser activation induce apoptosis of tumor cells, and thus inhibit the
could significantly inhibit tumor growth in PDT growth of large tumors in a mouse model compared
treatment compared to those AIEgen PSs excited to white light illumination. The potential for
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Competing Interests 27. Liang J, Tang BZ, Liu B. Specific light-up bioprobes based on AIEgen
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The authors have declared that no competing 28. Wu W, Mao D, Hu F, Xu S, Chen C, Zhang C-J, et al. A Highly Efficient and
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Theranostics 2019, Vol.

Near-infrared light-regulated cancer theranostic

Authors contributed equally

 Corresponding author: [email protected]; [email protected]

Received: 2018.09.25; Accepted: 2018.11.13; Published: 2019.01.01

Introduction as activatable PSs via triggered aggregation to further

intensity of MCF-7 and NIH 3T3 cells was only 36.3%

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