ARTICLE
A Near-Infrared Triggered
Nanophotosensitizer Inducing Domino
Effect on Mitochondrial Reactive Oxygen
Species Burst for Cancer Therapy
Zhengze Yu, Qiaoqiao Sun, Wei Pan, Na Li,* and Bo Tang*
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities
of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals,
Shandong Normal University, Jinan 250014, PR China
ABSTRACT Photodynamic therapy (PDT) is a well-established modality for cancer
therapy, which locally kills cancer cells when light irradiates a photosensitizer. However,
conventional PDT is often limited by the extremely short lifespan and severely limited
diffusion distance of reactive oxygen species (ROS) generated by photosensitizer, as well as
the penetration depth of visible light activation. Here, we develop a near-infrared (NIR)
triggered nanophotosensitizer based on mitochondria targeted titanium dioxide-coated
upconversion nanoparticles for PDT against cancer. When irradiated by NIR laser, the
nanophotosensitizer could produce ROS in mitochondria, which induced the domino effect on
ROS burst. The overproduced ROS accumulated in mitochondria, resulting in mitochondrial
collapse and irreversible cell apoptosis. Confocal fluorescence imaging indicated that the
mitochondrial targeting and real-time imaging of ROS burst could be achieved in living cells.
The complete removal of tumor in vivo confirmed the excellent therapeutic effect of the nanophotosensitizer.
KEYWORDS: near-infrared . nanophotosensitizer . reactive oxygen species . domino effect . cancer therapy
P
hotodynamic therapy (PDT) has
emerged as one of significant therapies in the treatment of cancer.14
Compared to the conventional therapeutics,
PDT possesses several advantages owing to
its noninvasive nature, negligible drug resistance, and low systemic toxicity.58 Most modern PDT applications involve three key components: a photosensitizer, a light source and
tissue oxygen.9 Upon irradiation, the excited
photosensitizer transfers energy to the surrounding O2 to generate reactive oxygen species (ROS), which can be exploited to destroy
cancer cells for cancer therapy.1012 However,
the produced ROS exhibits the extremely short
lifespan and severely limited diffusion distance, so the damage of ROS to biomolecules
is strongly restricted to the immediate vicinity
of ROS generation.1315 Recently, most of the
photosensitizers were performed in cytoplasm to generate ROS, which greatly limits
the therapeutic effect of PDT.1618 Mitochondria are the primary source of cellular ROS
YU ET AL.
generation (approximately up to 90%),19,20
and mitochondrial dysfunctions are closely
correlated with the disruption in the balance
of mitochondrial ROS.2124 In addition, mitochondria are decisive regulators of the intrinsic pathway of apoptosis, which is regarded
as the major mode of cell death in cancer
therapy.2527 Therefore, a mitochondriatargeted photosensitizer can perturb ROS
homeostasis and further induce cell apoptosis,
which is beneficial to improve the effect of
PDT treatment.
Furthermore, another obstacle of conventional PDT is the limited penetration
depth of visible light activation.28,29 The
upconversion nanoparticles (UCNPs) can convert a near-infrared (NIR) excitation into
ultraviolet or visible emission through the
lanthanide doping.3034 Along with the
improved tissue penetration depth and reduced autofluorescence background in biological samples, the UCNPs have attracted
considerable interests.3537 Titanium dioxide
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* Address correspondence to
[email protected].
Received for review July 20, 2015
and accepted October 12, 2015.
Published online October 12, 2015
10.1021/acsnano.5b04501
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Scheme 1. Schematic illustration of (a) the structure of the naophotosensitizer (TPP anchored UCNPs@TiO2 nanoparticles)
and ROS generation; (b) the near-infrared triggered nanophotosensitizer inducing domino effect on mitochondrial ROS burst
for cancer therapy.
(TiO2) is considered to be an ideal candidate due
to high stability, nontoxicity and high efficiency.38
TiO2 with band gap energies of 3.2 eV requires activation under UV exposure conditions. However, UV is
cytotoxic to living cells and has low tissue penetration
capabilities, thus preventing its use in deep tissues
in a clinical setting.39 When modified on the surface
of UCNPs, TiO2 can be photoactivated by the NIR
excitation to generate harmful radicals.4042
Herein, we present a novel strategy to construct an
NIR-responsive nanophotosensitizer for PDT based on
mitochondria targeted TiO2-coated UCNPs. The Tm3þdoped UCNPs can emit UV light with 980 nm laser
excitation and activate TiO2 to produce a flux of ROS,
especially superoxide anion radicals(O2•). The triphenylphosphine (TPP), a mitochondria-targeted group, was
then anchored on the surface of TiO2 to selectively
trigger the localized ROS burst in the mitochondria,
resulting in initiation of mitochondria-mediated intrinsic
apoptotic pathway, which was associated with cascade
reactions, such as the activation of an inner membrane
anion channel (IMAC), the opening of mitochondrial
permeability transition pores, the decrease in mitochondrial membrane potential (ΔΨm), the release of cytochrome C to cytoplasm.2527 The released cytochrome
C will activate the caspase-3 and caspase-7 to induce
apoptosis. These caspases can, in return, disturb the mitochondrial electron transport chain, induce the domino
effect on ROS burst and cause irreversible cell death.43
The structure of the nanophotosensitizer (UCNPs@
TiO2-TPP) and details of this approach are illustrated in
Scheme 1.
RESULTS AND DISCUSSION
Synthesis and Characterization of the Nanophotosensitizer.
NaYF4:Yb3þ,Tm3þ nanocrystals (β-phase) were first prepared via a solvothermal method with some modifications.44 As given in Figure 1ac, high resolution
transmission electron microscopy (HRTEM) showed that
the oleic acid (OA)-capped UCNPs possessed uniform
morphology with sizes around 25 nm and the pattern
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Figure 1. Characterization of the nanophotosensitizer.
High resolution transmission electron microscopy images
of OA-capped NaYF4:Yb3þ,Tm3þ (a); OA free NaYF4:Yb3þ,
Tm3þ (b); UCNPs@TiO2 (c). Scale bars are 25 nm.
was hexagonal (β-) phase. After being treated with
hydrochloric acid, the OA free UCNPs were obtained
with good monodispersity in aqueous solution. Meanwhile, the size did not show an obvious change. Then a
layer of TiO2 was coated on the surface of UCNPs
(denoted as UCNPs@TiO2) through a versatile kineticscontrolled method.45 The TiO2 shell of UCNPs@TiO2
is estimated to have a homogeneous thickness of about
3 nm. The EDX spectra showed a KR energy peak of
titanium element appears at 4.51 Kev for UCNPs@TiO2
compared with UCNPs (Figure S1a, b).46 X-ray photoelectron spectroscopy (XPS) was also employed to confirm the TiO2 coating. As shown in Figure S1c, the XPS
pattern of titanium (Ti) was observed only for UCNPs@TiO2 and not for UCNPs. The results indicated that the
UCNPs were successfully modified with TiO2. Moreover,
the appearance of the strong absorbance in the UV
region of UCNPs@TiO2 further confirmed the coating
of TiO2 (Figure 2a). In order to further modify TPP groups
on the surface of UCNPs@TiO2, the surface of TiO2
was functionalized with amino groups. Zeta potential
experiments further verified the successful treatment,
i.e., 20.2 ( 0.3 mV (before functionalization) and
þ18.9 ( 0.7 mV (after functionalization). And the content of amino groups was calculated to be 6.87 μmol/mg
UCNPs@TiO2 by TGA analysis (Figure S2a).
Next, the ability of the nanophotosensitizer for the
UV emission of UCNPs to activate the TiO2 to generate
O2• was evaluated. The upconversion emission spectra
of UCNPs and UCNPs@TiO2 were shown in Figure 2b.
The emission peaks at 347 and 362 nm were assigned to
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Figure 2. Characterization of the FRET and O2• production. (a) Absorption spectra of the pure UCNPs and UCNPs@TiO2.
(b) Emission spectra of pure UCNPs and UCNPs@TiO2 under 980 nm laser. (c) Amplified images of black box shown in b.
(d) Fluorescence spectra of nanophotosensitizer UCNPs@TiO2-TPP-HE before and after irradiation with 980 nm laser for
15 min. λex/λem = 488/610 nm.
I63F4 and 1D23H6 transitions of Tm3þ ions doped
in NaYF4, respectively. Except emissions in UV region,
the two blue emission peaks at 452 nm, 476 nm were
contributed to the 1D23F4 and 1G43H6 transitions of
Tm3þ ions. As can be seen from Figure 2a, b, the absorption band of UCNPs@TiO2 overlaps with the UV emissions of the UCNPs, thereby enabling the generation
of FRET. Compared with UCNPs, the upconversion
emission peaks for UCNPs@TiO2 at 347 and 362 nm
decreases sharply, which indicated the efficient energy
transfer from UCNPs to TiO2 (Figure 2c). IR806 or hydroethidine (HE) probe on the surface of the nanophotosensitizer were employed to label the nanoparticle or
determine the O2• produced by TiO2. The content of
IR806 and HE was calculated to be 0.81 and 2.35 μmol/mg
UCNPs@TiO2 using fluorescence spectra, respectively
(Figure S2b, c). And TEM images showed that almost
no obvious morphology change and cross-linking were
found after the modifications (Figure S3). As shown
in Figure 2d, the fluorescence intensity at 610 nm of HE
probe obviously increased compared to that before
irradiation when the nanophotosensitizer was irradiated
continuously with 980 nm laser for 15 min. The results
indicated that the TiO2 shell of the nanophotosensitizer
can effectively absorb the UV emission of UCNPs to
generate O2•.
Colocalization and Cellular Internalization Pathways. To
evaluate the capability of the nanophotosensitizer
to selectively target in mitochondria, colocalization
imaging experiments in human breast cancer cell line
(MCF-7) were performed. Mito-Tracker Green (MTG), a
commercial mitochondrial dye, was employed to label
1
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mitochondria. First of all, the concentration of TPP
group was optimized through the colocalization ability
of the nanophotosensitizer. A series of nanoparticles
(UCNPs@TiO2-TPP-IR806) (Figure 3a) with different
concentrations of TPP were prepared (calculated to
be 0.94, 1.95, 2.94, 3.85, 4.87, 5.89 μmol/mg UCNPs@
TiO2 using UVvis spectra) (Figure S2d). MCF-7 cells
were incubated with UCNPs@TiO2-TPP-IR806 for 12 h,
and mitochondria were labeled with MTG before the
imaging experiment. As shown in Figure S4, the colocalization effect increased until the TPP concentration reached 3.85 μmol/mg, so it was chosen in this
study. Figure 3b indicated that the fluorescence of
nanophotosensitizer overlapped well with that of MTG
(Pearson's correlation coefficient, F = 0.653), which
was evidenced by the clear yellow signals. The above
observations are further verified by quantifying fluorescence intensity of the line scanning profiles
(Figure 3b). Bio-TEM images of MCF-7 cells incubated
with nanophotosensitizer were taken to demonstrate
the spatial localization of the nanoparticles. As shown
in Figure S5, the nanophotosensitizer were indeed
located inside mitochondria. The results confirmed
that the nanophotosensitizer could specifically localize
in mitochondria of living cells.
The intracellular trafficking profile was further evaluated by confocal laser scanning microscopy. MCF-7
cells were incubated with UCNPs@TiO2-TPP-IR806. As
shown in Figure S6, when the incubation time was
2 h, most of the nanoparticles were in endo/lysosome
(Pearson's correlation coefficient, F = 0.653). After MCF-7
cells were incubated with UCNPs@TiO2-TPP-IR806 for
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Figure 3. Mitochondrial targeting and cellular uptake pathways. (a) Schematic illustration of the structure of UCNPs@
TiO2-TPP-IR806 and UCNPs@TiO2-TPP-HE. Structure formulas of IR806 and HE are showed on the bottom. (b) Mitochondrial
targeting of nanophotosensitizer under confocal imaging. MCF-7 cells were incubated with UCNPs@TiO2-TPP-IR806 for 12 h
before measurement. Confocal images of nanophotosensitizer (excitation = 633 nm, emission = 750800 nm), Mito-Tracker
Green (MTG) stained mitochondria (excitation = 488 nm, emission = 500550 nm), the overlay channel of nanophotosensitizer and mitochondria(top); The quantification of fluorescent intensity of the line scanning profiles in the corresponding
confocal images in b(bottom). (c) Confocal imaging of MCF-7 cells treated without (control) with dynasore (inhibitor of
dynamin-mediated uptake, 100 μM), chlorpromazine (inhibitor of clathrin-mediated uptake, 10 μM), ethylisopropylamiloride
(EIPA, inhibitor of macropinocytosis, 50 μM) and filipin (inhibtor of caveolaemediated uptake, 5 μM) before incubated with
UCNPs@TiO2-TPP-IR806 (0.1 mg/mL).
4 h and the excess nanophotosensitizer was removed,
the cells were incubated with fresh culture media for
an additional 1, 2, 4, and 8 h. A continuously increased
Pearson's correlation coefficient was obtained in the
overlay channel. The increased colocalization ratio of
nanophotosensitizer within mitochondria from 34.8%
to 62.4% showed the transfer to mitochondria within
8 h in living cells (Figure S7). The efficient endosomal
escape of the designed nanophotosensitizer can be
attributed to their high buffering capacity caused by
the untreated amino groups on the surface, which was
caused as “proton sponges”.47,48 To confirm the uptake
of nanoparticles in MCF-7 cells, inductively coupled
plasma atomic emission spectroscopy (ICP-AES) was
employed. The analysis showed that the nanoparticles
were taken up into the cells and the content of Ti in
each group was almost the same. The similar result was
obtained by quantifying the fluorescence of IR806 in
each group (Figure S8).
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The cellular uptake and internalization pathways
were also investigated by applying various endocytosis
inhibitors including ethylisopropylamiloride (EIPA, inhibitor of macropinocytosis, 50 μM),49 dynasore (inhibitor of
both endocytotic pathways, 100 μM),49 chlorpromazine
(inhibitor of clathrin-mediated endocytsis 10 μM),50
and filipin (inhibitor of caveolae-mediated endocytosis, 5 μM).50 As shown in Figure 3c and Figure S9,
the fluorescence intensity did not show an obvious
change for EIPA-treated cells, indicating that the
pathway of endocytosis of the nanophotosensitizer
is not mediated by macropinocytosis. However, the
fluorescence intensity of cells treated with the other
three inhibitors significantly decreased. The results suggested that the nanophotosensitizer might be mainly
internalized via caveolae-mediated and clathrinmediated endocytic pathways, which generally plays
a key role for the internalization of nanoparticles into
cells.51
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Figure 4. Real-time imaging of intracellular O2• burst. (a) Confocal imaging of O2• in MCF-7 cells. After being incubated with
UCNPs@TiO2-TPP-HE (0.1 mg/mL) and irradiated for different time, the confocal images were captured at 12 h. (b) Real-time
monitoring the fluorescence of O2• for 12 h at 1 h interval. The increasing green pixels indicated the domino effect on O2•.
The power of the irradiation was 3W 3 cm2.
Real-Time Monitoring O2• Burst in Living Cells. The ability
of the nanophotosensitizer to trigger ROS burst in mitochondria and induce cell apoptosis was then evaluated.
In order to monitor the ROS change in real-time, hydroethidine (HE), a probe response to O2•, was employed
to anchor on the surface of the nanophotosensitizer
(UCNPs@TiO2-TPP-HE) (Figure 3a). After being incubated with UCNPs@TiO2-TPP-HE, 6 groups of MCF-7
cells were irradiated for different periods of time
(0, 30, 60, 90, 120, and 150 s), respectively. As shown
in Figure 4a and Figure S10, no obvious fluorescence
signal was observed when the irradiation time was less
than 90 s, while bright green fluorescence signals were
obtained for longer irradiation time (90, 120, 150 s). The
results suggested that the nanophotosensitizer could
trigger O2• burst in mitochondria as expected when
the irradiation time reached the threshold time, which
was consistent with the concept of “mitochondrial
criticality”.52,53
The unique O2•-responsive fluorescence of HE on
the nanophotosensitizer made it possible to monitor
the mitochondrial O2• change in real-time, which was
beneficial to evaluate the photoactivated cytotoxicity
arising from triggered apoptosis process. To verify
this capability, the fluorescent changes of UCNPs@
TiO2-TPP-HE in MCF-7 cells were tracked in real-time
by confocal fluorescence imaging. After being incubated with the UCNPs@TiO2-TPP-HE, the cells were
irradiated for 90 s to induce apoptosis and performed
on the CLSM immediately. The confocal images were
captured for 12 h at 1 h interval at the same region.
As shown in Figure 4b, a time-dependent increase
of fluorescence intensity suggested that ROS were
continuously produced without further irradiation.
The results confirmed that the domino effect of ROS
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Figure 5. Cell viability. Cell viability of MCF-7 cells incubated with the nanophotosensitizer (0.1 mg/mL) (a) and
UCNPs@TiO2NH2 (0.1 mg/mL) (b) upon irradiation for
different period of time. The power of the irradiation was
3W 3 cm2. Cell viability was measured 24 h after the
irradiation.
burst indeed happened. After 12 h, very strong
fluorescence signal and obvious change of cell morphology were observed, which further confirmed the
NIR triggered nanophotosensitizer could induce cell
apoptosis (Figure S11).
Therapeutic Effect of the Nanophotosensitizer in Living
Cells. An MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide) assay was then employed
to evaluate the ability of the nanophotosensitizer
to induce cell apoptosis. The absorbance of MTT at
490 nm is dependent on the degree of activation of the
cells. Then the cell viability was expressed by the ratio
of absorbance of the treated cells (incubated with
the nanophotosensitizer or irradiated with NIR laser)
to that of the untreated cells. After being incubated
with UCNPs@TiO2-TPP for 12 h, the MCF-7 cells were
irradiated for different periods of time. Figure 5a
showed that the cell viability was more than 90%
when the irradiation time was less than 60 s, while
the cell viability was less than 10% after administration
of longer irradiation time (>90 s). The results indicated
when the irradiation time reached the critical toxic
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time, that is, the initial concentration of O2• was large
enough to induce the domino effect on ROS burst
in mitochondria, which could induce cell apoptosis. To
evidence the pivotal role of the mitochondria targeting
for the nanophotosensitizer in inducing domino effect
on ROS burst to kill cells, the nanophotosensitizer
without TPP groups was also prepared as comparison.
After MCF-7 cells were incubated with UCNPs@TiO2
NH2 without TPP groups under the same condition
as mentioned above, the cell viability was more than
70% even though the irradiation time up to 150 s
(Figure 5b). It demonstrated that the initiation of
mitochondrial domino effect on O2• burst is the key
factor for cell apoptosis. As a control, the viability
of cells treated with only nanophotosensitizer or only
NIR laser irradiation were also investigated. The results
indicated that more than 90% cells were still alive in
both the samples, suggesting that the nanophotosensitizer possessed good biocompatibility and the
irradiation of NIR light showed negligible side effects
(Figure S12). We further investigated whether mitochondrial antioxidant (MitoQ10) had effect on the
apoptosis. As shown in Figure S13a, cell viability was
still less than 15% in the presence of MitoQ10, which
indicated that it could not reverse cell death.
Determination of Mitochondrial Membrane Potential. Previous report showed the loss of mitochondrial membrane potential (Δψm) was an early event in mitochondria triggered apoptosis.54 The change of Δψm
was monitored using rhodamine 123 staining. MCF-7
cells were incubated with UCNPs@TiO2-TPP and irradiated for different time as above. Confocal fluorescence
imaging indicated that the fluorescence of rhodamine
123 sharply reduced when the cells irradiated more than
90 s from confocal images and the intensity quantification, suggesting the decrease of Δψm (Figure S14). This
is because the ROS burst activates an inner membrane
anion channel (IMAC) and promotes the opening of
mitochondrial permeability transition pore via oxidation the matrix glutathione.25,55 Cyclosporine A (CsA),
a desensitizer of the mitochondrial permeability transition pore (PTP) was employed to clarify the apoptosis
mechanism of mitochondrial membrane potential depolarization. MTT results showed that the cell viability
was less than 20% in the presence of CsA, which
confirmed that the opening of MPTP was not the only
reason for Δψm reduction (Figure S13b).
Cell Death Pathways. Cell death pathways induced by
the nanophotosensitizer were examined by exploring
ann exin V-fluorescein isothiocyanate (annexin V-FITC)
and propidium iodide (PI) staining. Apoptosis initially
induces phosphatidylserine exposure outside the cell
membrane without permeabilization. This process enables annexin V-FITC to bind to phosphatidylserine,
but PI is unable to enter into cells owing to the integrity
of the cell membrane at the initial stage of apoptosis.
When the membrane is disrupted upon onset of
Figure 6. Cell apoptosis pathway. Confocal images of DAPI
and Annexin V-FITC/PI stained MCF-7 cells with different
treatments: (a) the nanophotosensitizer (0.1 mg/mL) with
laser irradiation for 90 s; (b) UCNPs@TiO2NH2 (0.1 mg/mL)
with laser irradiation for 90 s; (c) laser irradiation for 90 s
only; (d) control group without treatment. The power of the
irradiation was 3W 3 cm2. Confocal images were captured
4 h after irradiation.
necrosis, annexin V-FITC and PI interact with the surface and DNA inside the cell, respectively. As shown
in Figure 6, neither annexin V-FITC nor PI-stained cells
are detected before NIR light irradiation. After being
treated with NIR light for 4 h, only annexin V-FITC
stained cells are detected, indicating the inversion of
phosphatidylserine and no permeabilization of the cell
membrane, which confirmed the early apoptosis stage.
As controls, neither annexin V-FITC nor PI-stained cells
were observed when cells were treated with UCNPs@TiO2NH2 upon irradiation for 90 s or with only
irradiation for 90 s. When the incubation time extended
to 12 h after irradiation, both annexin V-FITC and PI
stained cells were observed, indicating that the membrane is disrupted at late apoptosis stage. (Figure S15)
These results suggested that O2• induced cell death
by the nanophotosensitizer occurred predominantly
through mitochondria.
Caspase 3 Activation. Apoptosis of MCF-7 cells through
the mitochondrial signaling pathways was also evidenced by the activity of caspase-3 using the method
of immunefluorescent staining. As shown in Figure 7,
increased activity of caspase-3 was detected when
MCF-7 cells were loaded the nanophotosensitizer and
irradiated for 90 s.
Therapeutic Effects of the Nanophotosensitizer In Vivo. We
next assessed the ability of the nanophotosensitizer
for PDT against cancer in a mouse model. A xenograft
mouse model (the mice were treated with MCF-7 cells)
was then applied to evaluate the therapeutic effect
of the nanophotosensitizer in tumor tissue. Figure 8a
illustrates the schematic diagram of the NIR light
triggered PDT for cancer therapy. MCF-7 cells are first
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Figure 7. Immunofluorescent staining images of caspase 3.
(a) MCF-7 cells without any treatment. (b) MCF-7 cells
incubated with the nanophotosensitizer (0.1 mg/mL) and
irradiated with NIR laser for 90 s (3W 3 cm2). Confocal
images were taken 12 h after irradiation.
NIR laser only, nanophotosensitizer only and UCNPs@
TiO2NH2 with irradiation for 90 s, respectively. The
changes of the tumor size were monitored in the same
way and the observed trend was similar to the control
group. As can be seen in Figure 8b, c, the tumor grew
rapidly as same as the control group. Body weight is an
important parameter to evaluate the systemic toxicity
of the material to the body. As shown in Figure 8d, the
body weight of all groups does not decrease with
the time prolonged in 14 days, implying that the
treatments did not show obvious toxicity. In addition,
the treatment efficacy in term of tumor cell death was
also evaluated by H&E staining on tissue sections from
the different treatment groups at 12 h after treatment.
The tumors treated with nanophotosensitizer upon
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xenografted to the flank of the mice. The nanophotosensitizer dispersed in PBS buffer (1 mg/mL, 50 μL),
was then directly injected into the tumor for each
mouse. The NIR laser with a parameter of 3 W 3 cm2
was conducted at the tumor region for 90 s. As can be
seen in Figure 8b, the tumor was completely removed
after 14 days when treated with the nanophotosensitizer upon NIR laser irradiation for 90 s. The change
in the tumor volume was monitored over a period of
14 days without extra irradiation. In the control group
of mice treated with PBS buffer, the tumor size
was found to increase about 5-fold over this period
(black line in Figure 8c). Notably, the tumors of mice for
the experimental group with nanophotosensitizer
mediated PDT were eliminated by day 6 (blue line in
Figure 8c). Another 3 groups of mice were treated with
Figure 9. H&E staining of tumor slides. The tumors were
treated differently: (a) PBS only; (b) PBS with laser irradiation
for 90 s; (c) nanophotosensitizer only; (d) UCNPs@TiO2NH2
with irradiation for 90 s; (e) nanophotosensitizer with irradiation for 90 s. (f) Amplified images of the black box shown in e.
The power of the irradiation was 3W 3 cm2.
Figure 8. In vivo application of nanophotosensitizer in a mouse model. (a) Schematic illustration of the PDT treatment setup.
(b) Photographs of the mice taken before treatment (0 day) and at 14 days with different treatments: i, PBS only; ii, PBS with
laser irradiation for 90 s; iii, nanophotosensitizer only; iv, UCNPs@TiO2NH2 with irradiation for 90 s; v, nanophotosensitizer
with irradiation for 90 s. A dosage of nanophotosensitizers in PBS (1 mg/mL, 50 μL) was administrated intratumorally for all
mice (n g 5). Tumor growth curves (c) and mice body weight curves (d) of different groups of tumor-bearing mice after PDT.
They were measured at 2 days interval for 14 days. The power of the irradiation was 3W 3 cm2.
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CONCLUSION
In conclusion, we have demonstrated a near-infrared
triggered nanophotosensitizer based on mitochondria
targeted titanium dioxide-coated upconversion nanoparticles, which can be used for photodynamic therapy
against cancer in living cells and in vivo. To the best of
our knowledge, this is the first time that cancer therapy
could be achieved using a photosensitizer to induce
mitochondrial ROS burst. The nanophotosensitizer
makes use of the advantages of UCNPs, such as remarkable light penetration depth, large anti-Stokes shifts and
MATERIALS AND METHODS
Materials and Reagents. Hydroethidine (HE), 1-octadecene
(ODE), rare earth oxides yttrium(III) oxide (Y2O3), ytterbium(III)
oxide (Yb2O3), thulium(III) oxide (Tm2O3) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Company; oleic acid (OA),
(4-carboxybutyl)triphenylphosphonium bromide, (3-aminopropyl)triethoxysilane (APTES), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS)
were purchased from Alfa Aesar Chemical Ltd. (tianjin, China);
tetrabutyl titanate (TBOT) was purchased from China National
Pharmaceutical Group Corporation (Shanghai, China). Annexin
V-FITC/propidium iodide (PI) Cell Apoptosis Kit and Cyclosporine
A (CsA) were obtained from Sangon Biotechnology Co., Ltd.
(Shanghai, China). Mito-Tracker Green was purchased from
Molecular Probes (Invitrogen, USA); MitoQ was purchased from
Vosun chemical Co., Ltd. (Suzhou, China). Oleic acid and 1-octadecene were of technical grade and the others were of analytical
grade. All the chemicals were used without further purification.
The human breast cancer cell line (MCF-7) was purchased from
KeyGEN biotechnology Company (Nanjing, China).
Synthesis of NaYF4:20%Yb3þ,0.2%Tm3þ Nanocrystals. The β-phase
NaYF4:Yb3þ,Tm3þ nanocrystals were prepared via a solvothermal
method with some modifications.44 To obtain rare earth chlorides, 1 mmol rare earth oxides Y2O3, Yb2O3, and Tm2O3 with a
stoichiometric ratio of 79.8:20:0.2 were dissolved in hydrochloric
acid, and then the solution was stirred and heated to evaporate
the water completely. In the typical synthesis procedure, YCl3
(0.798 mmol), YbCl3 (0.20 mmol), and TmCl3 (0.002 mmol) were
dispersed in oleic acid (OA, 8 mL) and 1-octadecene (ODE, 18 mL),
and then the mixture was heated to 160 °C for 30 min. After
a homogeneous solution was formed, the mixture was cooled
to room temperature, then followed by adding NaOH (0.1 g,
2.5 mmol) and NH4F (0.148 g, 4 mmol) in 10 mL of methanol
solution under vigorously stirring for 30 min. The temperature
was heated to 100 °C to evaporate methanol, then was raised to
295 °C in an argon atmosphere for 90 min and finally cooled
down to room temperature naturally. The resulting NaYF4:Yb,Tm
nanoparticles were precipitated by adding ethanol and then
centrifuged and washed with ethanol and cyclohexane for
YU ET AL.
high photochemical stability. The coated TiO2 can be
photoactivated by the emission of UCNPs with an NIR
laser, which could produce large amounts of ROS. The
triphenylphosphine modification can guide the nanophotosensitizer to specifically target mitochondria.
When irradiated with a 980 nm NIR laser, the nanophotosensitizer can selectively trigger the mitochondrial
ROS burst and initiate a series of cascade reaction,
leading to mitochondrial collapse and irreversible cell
apoptosis. Confocal fluorescence imaging indicated
that the nanophotosensitizer could target mitochondria
and induce domino effect on ROS burst above the
critical irradiation time in living cells. MTT assay confirmed the designed nanophotosensitizer could effectively destroy cancer cells with less than 10% of the cell
viability. Moreover, it revealed mitochondria targeting
of the nanophotosensitizer played a pivotal role in
inducing the domino effect on ROS burst and cell
apoptosis. In vivo study demonstrated that the tumor
could be completely removed owing to the excellent
therapeutic effect of the nanophotosensitizer. We anticipate that this novel approach can provide new insights
for cancer therapy.
ARTICLE
irradiation exhibited a wide range of tissue damage in
histological sections, while most tumor cells showed
no obvious change in the control groups (Figure 9e, f).
The histological effect of nanophotosensitizer on five
major organs (liver, lung, spleen, kidney, and heart) of
healthy mice was monitored at 7 days after intratumor
injection and no histopathological abnormalities were
found (Figure S16). These results indicated that the
nanophotosensitizer is highly effective for cancer therapy using PDT and has little side effects to normal issue
via intratumor administration.
several times. The precipitates were redispersed in 10 mL cyclohexane solution.
Synthesis of UCNPs@TiO2 CoreShell Nanoparticles. Before the
synthesis, the OA ligand on the NaYF4:Yb,Tm surface was
removed.44 0.5 mL of as-synthesized Oleate-capped UCNPs was
dispersed in a 10 mL aqueous solution, and the pH was adjusted
to 2 by adding a solution of HCl 0.5 M. The reaction was performed with vigorous stirring for 4 h. The nanoparticles were
centrifuged and washed with ethanol for three times. UCNPs@
TiO2 was synthesized according to a versatile kinetics-controlled
coating method with some modifications.45 The core particles
were dispersed in 25 mL absolute ethanol and mixed with concentrated ammonia solution (75 μL, 28 wt %) under ultrasound
for more than 15 min. Afterward, 120 μL of TBOT was added
dropwise in 5 min, and the reaction was allowed to proceed for
24 h at 45 °C under continuous mechanical stirring. The resultant
products were separated and collected, followed by washing
with deionized water and ethanol for 3 times, respectively.
Synthesis of UCNPs@TiO2-TPP and UCNPs@TiO2-TPP-IR806. UCNPs@
TiO2NH2 was synthesized first. As-prepared UCNPs@TiO2
(2 mg) was dispersed in a solution of ethanol (10 mL) and
deionized water (100 μL) under stirring for 15 min. Then, 10 μL
APTES (40 μmol) was added to the mixture and the reaction was
processed for another 12 h. The precipitates were washed and
redispersed in 4 mL of MES buffer (10 mM, pH 6.0). The content
of amino groups was measured by TGA analysis. UCNPs@
TiO2-TPP-IR806 was obtained by coupling the carboxl groups
of the (4-Carboxybutyl)triphenylphosphonium bromide and
infrared dye IR806 and the amino group on the surface of
UCNPs@TiO2NH2 to form the amido bonds. EDC (20 μmol),
NHS (20 μmol) and EDC (20, 40, 60, 80, 100, 120 μmol), NHS
(20, 40, 60, 80, 100, 120 μmol) were added to IR806 (2 μmol) and
(4-Carboxybutyl)triphenylphosphonium bromide (2, 4, 6, 8, 10,
12 μmol) solution, respectively. The reaction was performed for
30 min at room temperature in the dark to activate carboxylate
groups and then both of them were added to above UCNPs@
TiO2NH2 solution under gentle stirring for 12 h, which resulted
in the formation of the amido bonds. Following this, the
precipitates were centrifuged (10 000 rpm, 10 min) and washed
with methanol and PBS buffer (10 mM, pH 7.4) for three times
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YU ET AL.
quantified. The colocalization ratio of Lyso-Tracker DND-26 or
Mito-Tracker Green with IR806 of UCNPs@TiO2-TPP-IR806 was
quantified via Image-Pro Plus Imaging software.
ICP-AES. Five groups of MCF-7 cells (seeded at 1 105/mL)
were incubated with UCNPs@TiO2-TPP-IR806 (0.1 mg/mL) for
4 h. Then the cells were washed with PBS buffer to remove the
nanoparticles that were not uptake into the cells. Then, 2 mL
fresh DMEM culture medium was added, and the cells were
further cultured for 0, 1, 2, 4, and 8 h. At the end of incubation,
the cells were washed with PBS buffer and collected in centrifuge tubes. Next, the cells were treated with nitric acid (2 mL),
hydrofluoric acid (3 mL) and perchloric acid (0.5 mL), and then
heated to dissolve TiO2. The samples were finally solved in 4 mL
of hydrochloric acid, and then analyzed for total Ti content by
ICP-AES (Thermo, IRIS Advantage, 308.8 nm) and the measurement was repeated three times.
In Vitro Detecting O2• Burst. Six groups of MCF-7 cells were
seeded in a confocal dish and incubated at 37 °C in 5% CO2
for 24 h. UCNPs@TiO2-TPP-HE (0.1 mg/mL) was delivered into
the cells in DMEM culture medium for 12 h. Then, the cells were
washed with PBS buffer to remove the nanoparticles outside
the cells and fresh DMEM medium containing 10% fetal bovine
serum medium was added. Six groups of the cells were irradiated with 980 nm laser for 0, 30, 60, 90, 120, and 150 s,
respectively. Subsequently, the cells were examined with confocal laser scanning microscopy (CLSM) with 488 nm excitation.
Next, the cells were cultured for another 12 h and were also
examined with confocal laser scanning microscopy (CLSM) with
the same parameters.
Real-time monitoring O2• experiment was also carried out.
The cells were incubated with UCNPs@TiO2-TPP-HE (0.1 mg/mL)
for 12 h. Then, the cells were washed with PBS buffer. After
that, fresh DMEM medium containing 10% fetal bovine serum
medium was added followed with irradiation with 980 nm laser
for 90 s. Subsequently, the cells were examined with confocal
laser scanning microscopy (CLSM) with 488 nm excitation for 12 h.
Confocal images were obtained at 1 h interval.
In Vitro Cytotoxicity. (1) To inspect the applicability of UCNPs@
TiO2-TPP, 2 groups of MCF-7 cells were cultured in 96-well
microtiter plates and incubated at 37 °C in 5% CO2 for 24 h.
UCNPs@TiO2-TPP (0.1 mg/mL) and UCNPs@TiO2NH2 (0.1 mg/mL)
were delivered into the cells in DMEM culture medium for 12 h
followed by washing the cells with PBS buffer to remove the
nanoparticles that were not uptake into the cells. Then, each plate
of MCF-7 cells was divided into 6 groups and the laser irradiation of
980 nm (3W 3 cm2) was performed on each group for 0, 30, 60, 90,
120, and 150 s, respectively. The cells were further cultured for 24 h.
Next, 150 μL MTT solution (0.5 mg/mL) was added to each well.
After 4 h, the remaining MTT solution was removed, and 150 μL of
DMSO was added to each well to dissolve the formazan crystals.
The absorbance was measured at 490 nm with microplate reader
(Synergy 2, Biotek, USA). (2) MCF-7 cells were cultured in 96-well
microtiter plates and incubated at 37 °C in 5% CO2 for 24 h. Cells
were divided into 3 groups. UCNPs@TiO2-TPP (0.1 mg/mL) without
irradiation and only irradiation with 980 nm laser were performed,
respectively. And another groups of cells as the control without any
treatment. Next, 150 μL MTT solution (0.5 mg/mL) was added to
each well. After 4 h, the remaining MTT solution was removed, and
150 μL of DMSO was added to each well to dissolve the formazan
crystals. The absorbance was measured at 490 nm with microplate
reader (Synergy 2, Biotek, USA). (3) Two groups of MCF-7 cells
loaded with the nanophotosensitizer (0.1 mg/mL) were irradiated
with the CsA (1 μM) and MitoQ10 (20 μM) for 5 min, respectively.
Then the cells were irradiated with NIR laser for 90 s (3W 3 cm2).
After incubation for another 24 h, MTT assay was carried out as the
same procedure above.
Detection of Mitochondrial Membrane Potential (ΔΨm). MCF-7
cells were cultured in 96-well microtiter plates and incubated
at 37 °C in 5% CO2 for 24 h. UCNPs@TiO2-TPP (0.1 mg/mL) was
delivered into the cells in DMEM culture medium for 12 h. Then,
the cells were irradiated for 90 s and further incubated for 4 h.
Then, the cells were incubated with rhodamine 123 (5 μg/mL)
for 15 min in darkness at 37 °C. Confocal images of rhodamine
123-stained cells were obtained by excitation of the samples at
488 nm (emission = 500550 nm).
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and finally redispersed in PBS buffer (1 mg/mL). The content
of TPP groups and IR806 molecules was calculated according
to the standard linear calibration curve of each group using
subtraction through UVvis absorption spectra and fluorescence analysis, respectively.
Synthesis of UCNPs@TiO2-TPP-HE. UCNPs@TiO2NH2 and UCNPs@
TiO2-TPP were prepared successively with the method mentioned
above and further modified with carboxyl groups.56 UCNPs@
TiO2-TPP were collected by centrifugation and redispersed in
4 mL DMSO containing triethylamine (1 mg) and succinic anhydride (1 mg). The mixture was allowed to stir at 40 °C for 48 h. The
carboxylic acid-functionalized particles were centrifuged and
washed for three times and finally redispersed in MES buffer
(10 mM, pH 6.0). HE probe was anchored on the nanophotosensitizer by formation of amido bond.57 19.1 mg EDC (100 μmol)
and 11.5 mg NHS (100 μmol) were added to the above solution
with reaction for 30 min to activate carboxylate groups. Finally,
HE probe (10 μmol) in methanol was mixed under gentle stirring
for 12 h in the dark and the resulting coreshell nanoparticles
were centrifuged and washed with deionized water and redispersed in PBS buffer (1 mg/mL). The content of HE molecules was
calculated according to the standard linear calibration curve of
each group using subtraction through fluorescence analysis.
Cell Culture. MCF-7 cells were cultured in Dulbecco's modified
Eagles medium (DMEM) with 10% fetal bovine serum and 100 U/mL
1% antibiotics penicillin/streptomycin and maintained at 37 °C in a
100% humidified atmosphere containing 5% CO2.
Colocalization into Mitochondria. MCF-7 cells were seeded in a
confocal dish for 24 h. Then, UCNPs@TiO2-TPP-IR806 (0.1 mg/mL)
was delivered into the cells in DMEM culture medium. After
incubation for 12 h, Cells were then washed three times with PBS
buffer to remove the nanoparticles that were not uptake into the
cells. Then, 2 mL fresh DMEM culture medium was added and
the cells were stained by Mito-Tracker Green (25 nM) at 37 °C for
15 min. The cells were then washed by PBS twice and immediately
observed using confocal laser scanning microscopy (CLSM) and
confocal images of cells fluorescence were captured with 488 nm
excitation for Mito-Tracker Green (emission = 500550 nm) and
633 nm excitation for IR806 (emission = 750800 nm). The
colocalization ratio of Mito-Tracker Green with IR806 of UCNPs@
TiO2-TPP-IR806 was quantified using Image-Pro Plus Imaging
software.
Cellular Uptake and Internalization Pathways. MCF-7 cells were
cultured in 96-well microtiter plates and incubated at 37 °C in
5% CO2 for 24 h. Cells were incubated with different inhibitors
including dynasore (inhibitor of dynamin-mediated uptake,
100 μM), chlorpromazine (inhibitor of clathrin-mediated uptake,
10 μM), ethylisopropylamiloride (EIPA, inhibitor of macropinocytosis, 50 μM) and filipin (inhibitor of caveolae-mediated
uptake, 5 μM) in serum free DMEM medium for 30 min prior
to incubation with the nanoparticles UCNPs@TiO2-TPP-IR806
(0.1 mg/mL) for further 12 h. Subsequently, the medium was
removed and the cells were washed 3 times using PBS. Confocal
images were obtained by excitation of the samples at 633 nm.
Intracellular Trafficking UCNPs@TiO2-TPP-IR806. Six groups of
MCF-7 cells were seeded in confocal dishes for 24 h. Then,
UCNPs@TiO2-TPP-IR806 (0.1 mg/mL) was delivered into the cells
in DMEM culture medium at 37 °C in 5% CO2. One group of cells
were incubated for 2 h, washed three times and then stained with
Lyso-Tracker DND-26 at 37 °C for 15 min. The cells were then
washed by PBS twice and immediately observed using confocal
laser scanning microscopy (CLSM) and confocal images of cells
fluorescence were captured with 488 nm excitation for LysoTracker DND-26 and 633 nm excitation for IR806. Another five
groups of cells were incubated with UCNPs@TiO2-TPP-IR806
(0.1 mg/mL) for 4 h. Then the cells were washed with PBS buffer
to remove the nanoparticles that were not uptake into the cells.
Then, 2 mL of fresh DMEM culture medium was added and the
cells were further cultured for 0, 1, 2, 4, and 8 h. Subsequently,
the cells were stained by Mito-Tracker Green (25 nM) at 37 °C for
15 min. The cells were then washed by PBS twice and immediately
observed using confocal laser scanning microscopy (CLSM) and
confocal images of cells fluorescence were captured with 488 nm
excitation for Mito-Tracker Green and 633 nm excitation for IR806.
The IR806 fluorescence intensity of the cells in each group was also
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Conflict of Interest: The authors declare no competing
financial interest.
Acknowledgment. This work was supported by 973 Program
(2013CB933800), National Natural Science Foundation of China
(21535004, 21227005, 21390411, 21422505, 21375081, 21505087),
and Natural Science Foundation for Distinguished Young Scholars
of Shandong Province (JQ201503).
Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website
at DOI: 10.1021/acsnano.5b04501.
Instruments and supporting figures. (PDF)
YU ET AL.
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Cell Apoptosis. For monitoring cell death pathways, the Annexin V-FITC/PI Apoptosis Detection Kit is used. Here, MCF-7
cells were incubated with 0.1 mg/mL of UCNPs@TiO2-TPP at
37 °C in 5% CO2 for 12 h. After that, the cells were washed thrice
with DMEM medium and then subjected to the 980 nm NIR laser
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without any treatment were examined the same as above.
Another experiment of MCF-7 cells incubated with the nanophotosensitizer (0.1 mg/mL) was carried out at 12 h post irradiation (3W 3 cm2) for 90 s as the same procedure above.
Caspase 3 Activation. Briefly, MCF-7 cells were incubated with
0.1 mg/mL of UCNPs@TiO2-TPP at 37 °C in 5% CO2 for 12 h. Then,
the cells were washed thrice with DMEM medium and then
subjected to the 980 nm NIR laser irradiation for 90 s. After further
incubation for 12 h, the cells were fixed with paraformaldehyde
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control was performed as the same procedure above.
Animal Tumor Xenograft Models. All animal experiments were
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Care (People's Republic of China) and the Guidelines of the
Animal Investigation Committee, Biology Institute of Shandong
Academy of Science, China. Female nude mice (46 week old,
∼20 g) were housed under normal conditions with 12 h light
and dark cycles and given access to food and water ad libitum.
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were suspended and harvested after trypsinization and approximately 1 106 MCF-7 cells in 150 μL PBS were injected
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was determined by measuring length (L) and width (W), and
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