Enhanced NIR radiation-triggered hyperthermia by mitochondrial targeting

Article abstract of DOI:10.1021/ja5122809

Mitochondria are organelles that are readily susceptible to temperature elevation. We selectively delivered a coumarin-based fluorescent iron oxide nanoparticle, Mito-CIO, to the mitochondria. Upon 740 nm laser irradiation, the intracellular temperature of HeLa cells was elevated by 2.1 °C within 5 min when using Mito-CIO, and the treatment resulted in better hyperthermia and a more elevated cytotoxicity than HeLa cells treated with coumarin iron oxide (CIO), which was missing the mitochondrial targeting unit. We further confirmed these results in a tumor xenograft mouse model. To our knowledge, this is the first report of a near-infrared laser irradiation-induced hyperthermic particle targeted to mitochondria, enhancing the cytotoxicity in cancer cells. Our present work therefore may open a new direction in the development of photothermal therapeutics.

Full text of DOI:10.1021/ja5122809

Article
pubs.acs.org/JACS
Enhanced NIR Radiation-Triggered Hyperthermia by Mitochondrial
Targeting
Hyo Sung Jung,†,⊥ Jiyou Han,‡,⊥ Jae-Hong Lee,† Ji Ha Lee,§ Jong-Min Choi,∥ Hee-Seok Kweon,#
Ji Hye Han,▽ Jong-Hoon Kim,*,‡ Kyung Min Byun,*,∥ Jong Hwa Jung,*,§ Chulhun Kang,*,▽
and Jong Seung Kim*,†
†Department of Chemistry, Korea University, Seoul 136-701, Korea
‡Division of Biotechnology, Laboratory of Stem Cells and Tissue Regeneration, College of Life Sciences and Biotechnology, Korea
University, Seoul 136-713, Korea
§Department of Chemistry and Research Institute of Natural Science, Gyeongsang National University, Jinju 660-701, Korea
∥Department of Biomedical Engineering, Kyung Hee University, Yongin 446-701, Korea
#Division of Electron Microscopic Research, Korea Basic Science Institute (KBSI), Daejeon 305-806, Korea
▽The Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea
S
* Supporting Information
ABSTRACT: Mitochondria are organelles that are readily
susceptible to temperature elevation. We selectively delivered a
coumarin-based fluorescent iron oxide nanoparticle, Mito-
CIO, to the mitochondria. Upon 740 nm laser irradiation, the
intracellular temperature of HeLa cells was elevated by 2.1 °C
within 5 min when using Mito-CIO, and the treatment
resulted in better hyperthermia and a more elevated
cytotoxicity than HeLa cells treated with coumarin iron
oxide (CIO), which was missing the mitochondrial targeting
unit. We further confirmed these results in a tumor xenograft
mouse model. To our knowledge, this is the first report of a
near-infrared laser irradiation-induced hyperthermic particle targeted to mitochondria, enhancing the cytotoxicity in cancer cells.
Our present work therefore may open a new direction in the development of photothermal therapeutics.
been pursued; however, this method can increase damage to
normal cells too.17−19 In this study, we demonstrate a novel
method to enhance cytotoxicity for a given amount of heat by
selectively delivering MPs to a thermally susceptible subcellular
organelle with minimal side effects.
INTRODUCTION
■
Hyperthermia, a therapeutic procedure to induce apoptosis in
tumor tissues, is an efficient complement to standard cancer
treatments (radiotherapy and chemotherapy) that artificially
elevates tissue temperatures1,2 to take advantage of cells with a
weak defense against heat, causing minimal side effects.3 Several
heat delivery materials, including nanoparticles (NPs), have
been developed for applications in anticancer therapy.4,5 Many
studies have investigated methods to enhance the hyperthermic
efficacy of the NPs by varying their size, shape, surface charge,
and coating material (e.g., protein, polymer, or cell-penetrating
peptides).6−11
Recently, heat-generation systems using near-infrared (NIR)
laser irradiation have attracted attention because they are less
invasive and less harmful to normal tissues than other
methods.12,13 Among the NIR-sensitive NPs, magnetic particles
(MPs) such as iron oxide particles (IOPs) are excellent
candidates for hyperthermic anticancer therapy, because they
are nontoxic, biocompatible, and, in addition, compatible with
magnetic resonance imaging technology.4a,14−16 To achieve
higher radiation-to-heat conversion in hyperthermia with the
NIR-sensitive MPs, the delivery of large amounts of MPs has
Mitochondria are critical energy-producing cellular organelles
and are highly sensitive to heat shock,20−22 frequently causing
apoptotic cell death through the production of reactive oxygen
species (ROS).23,24 Recently, the synergy of mitochondrial
targeting and magnetic field-induced hyperthermia was
exploited by Shah et al. to utilize an enhanced effect on
mitochondrial dysfunction, resulting in cell apoptosis.25 There-
fore, MP delivery to mitochondria may enhance hyperthermic
cytotoxicity. Herein, we describe the design and synthesis of
mitochondria-targeting iron oxide NPs (Figure 1) composed of
a lipophilic triphenylphosphonium cation (TPP) for efficient
mitochondrial targeting26−29 and a coumarin fluorophore for
fluorescence-based real-time tracking of its uptake and
distribution into various subcellular compartments. We show
Received: December 2, 2014
© XXXX American Chemical Society
A
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iodoethane in the presence of potassium carbonate (K2CO3)
in dimethylformamide (DMF) yielded 8,30 which was further
reacted with tert-butyl bromoacetate, K2CO3, and potassium
iodide to produce 7. Through a Vilsmeier−Haack reaction, 7
was converted to 6, which then underwent a piperidine-
catalyzed Knoevenagel condensation with diethyl malonate to
produce the coumarin derivative 5. Compound 3 was obtained
from 5 via a deprotection with sodium hydroxide in ethanol
and the subsequent coupling with dopamine hydrochloride by
using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide
(EDCI) and 4-dimethylaminopyridine (DMAP). Compound
Figure 1. Schematic representation of enhanced hyperthermia by
using mitochondria-targeting iron oxide NPs.
2 was obtained from 3 by reaction with (PPh3 (CH2)2NH2)Cl−
+
in the presence of isobutyl chloroformate in DMF. To
demonstrate that the TPP unit in 2 was responsible for
mitochondrial targeting,26−29 an analogue lacking the TPP unit,
10, was also prepared as a comparison. Coupling of dopamine
hydrochloride with 11, prepared according to previously
reported methods,31 yielded compound 10. The chemical
structures of 2 and 10 were confirmed by 1H NMR, 13C NMR,
FT-IR, and ESI-MS analyses (see Supporting Information).
To prepare Mito-CIO and CIO, IOPs were added to an
anhydrous toluene solution of compounds 2 or 10. The
mixtures were refluxed with continuous stirring under N2
atmosphere for 24 h. The collected products were washed
three times with dichloromethane and acetone to remove
excess 2 or 10 and dried under vacuum. Functionalization of
Mito-CIO and CIO was characterized by transmission electron
microscopy (TEM), thermogravimetric analysis (TGA), infra-
red spectroscopy (IR), and X-ray photoelectron spectroscopy
(XPS). TEM images of iron oxide-based Mito-CIO revealed
spherical particles with a diameter of ∼15 nm and a narrow size
distribution (Figure 2a). Their distinct lattice fringe patterns
that the specially designed NPs localize preferentially to
mitochondria and induce significant cell hyperthermia upon
NIR irradiation.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Coumarin Iron
Oxides. Mitochondrial-targeted coumarin iron oxide (Mito-
CIO) and coumarin iron oxide (CIO) were synthesized as
outlined in Scheme 1. Coupling of 3-aminophenol and
a
Scheme 1. Synthetic Routes to Mito-CIO and CIO
Figure 2. Characterization of Mito-CIO. (a) TEM image of Mito-
CIO. Inset: high-resolution TEM image. (b) UV−vis absorption
spectra of Mito-CIO and compound 2. (c) TEM mapping image of
Mito-CIO: (i) bright-field image, (ii) iron, (iii) oxygen, (iv) nitrogen,
and (v) phosphorus components.
were evident in a high-resolution TEM image (Figure 2a,
inset), indicative of a highly crystalline nature. The IR spectra
of Mito-CIO showed strong new bands at 3300, 2922, 2848,
1692, 1614, 1576, 1511, 1432, 1260, 1224, and 1015 cm−1 that
originated from 2 (Figure S1). The XPS spectrum of Mito-CIO
(Figure S2) revealed an increase in nitrogen atoms, thereby
confirming the presence of 2 on the surface of the iron oxide
NPs. Mito-CIO is composed of 67.5% carbon, 5.6% nitrogen,
22.7% oxygen, and 4.1% phosphorus, and TEM mapping
a
DMF, N,N-dimethylformanide; EDCI, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide; DMAP, 4-dimethylaminopyri-
dine.
B
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results showed that Mito-CIO contains carbon, oxygen,
nitrogen, phosphorus, and iron (Figure 2c). These findings
confirmed that compound 2 was securely attached to the
surface of NPs, accounting for ∼52.0 wt % of Mito-CIO as
determined by TGA (Figure S3). The reference material (CIO)
without the targeting unit was characterized as a comparison
(see Supporting Information). Aqueous dispersions of Mito-
CIO and CIO displayed a broad and continuous absorption
and an increased NIR absorption (Figures 2b and S5b).
Photothermal Conversion Efficiency of Mito-CIO. The
photothermal conversion efficiency of Mito-CIO dispersed in a
phosphate-buffered saline (PBS, pH 7.4) solution was
investigated as a function of concentration and NIR irradiation
power. Upon irradiation with a 740 nm NIR laser at 2.0 W/
cm2, we observed that the temperature of the dispersed solution
gradually increased with the concentration (Figure 3a). The
Figure 4. Photothermal conversion efficiency of Mito-CIO inside
cells. (a) Schematic diagram of HeLa cells in a delta T dish loaded with
Mito-CIO upon NIR laser irradiation. The red circle shows the area of
laser illumination. The local temperature change was monitored using
an IR thermal camera. (b) Hyperthermia heating curves. (c) IR
thermal images of HeLa cell media with and without Mito-CIO at
various irradiation intensities (2.0, 6.0, and 10 W/cm2) as a function of
irradiation time.
demarcation between the green calcein AM- and red PI-
fluorescent regions (Figure 5a), indicating that the irradiation is
Figure 3. Photothermal conversion efficiency of Mito-CIO. Hyper-
thermia heating curves of Mito-CIO dispersed in PBS solution (pH
7.4) at (a) various Mito-CIO concentrations (0, 0.3, 1.4, and 7.0 mg/
mL) with irradiation at 2.0 W/cm2 and (b) 1.4 mg/mL Mito-CIO
with irradiation at various intensities (0.3, 2.0, 6.0, and 10 W/cm2) as a
function of irradiation time.
solution at 7.0 mg/mL showed a ∼13 °C increase in
temperature within 2 min, whereas no such change was
observed with the particle-free solution. Furthermore, the
temperature of the dispersed solution at 1.4 mg/mL markedly
increased as the laser power increased from 0.3 to 10 W/cm2
(Figure 3b). These results demonstrate that the heat is
generated by NIR irradiation of the iron oxide-based Mito-
CIO, with an excellent photothermal conversion efficiency.
Photothermal Conversion Efficiency of Mito-CIO
Inside HeLa Cells. To characterize the photothermal
conversion efficiency of the MPs inside cells, the temperature
of HeLa cells loaded with Mito-CIO was monitored under
constant NIR irradiation by using an infrared thermal camera
(Figure 4a). The local temperature at the irradiation point
increased with increasing laser power (Figure 4b,c). In
particular, 10 W/cm2 irradiation with a 740 nm NIR laser
rapidly elevated the temperature of Mito-CIO loaded cells by
Figure 5. Cytotoxicity of HeLa cells loaded with Mito-CIO and CIO.
(a) Confocal fluorescence microscopic images of HeLa cells treated
with Mito-CIO following NIR irradiation (740 nm, 0.04 W/cm2) for
20 min. (b) Dead cell ratio (cytotoxicity is defined as a number of PI
positive cells of the number of the total cells) and (c) confocal
fluorescence microscopic images of HeLa cells treated with PBS, Mito-
CIO and CIO following laser irradiation (740 nm, 0.04 W/cm2) for 20
min as a function of irradiation time. Live/dead cells are green/red
(Calcein AM/PI), respectively. *P < 0.05.
2.14
0.25 °C within 5 min, whereas such a change was
negligible without Mito-CIO (approximately 0.3 °C). These
results demonstrate that Mito-CIO is capable of elevating the
cell temperature upon NIR irradiation. We next tested whether
this NP-based hyperthermia is sufficient to induce cell death.
Cytotoxicity of CIO-Loaded HeLa Cells. To investigate
and fluorescently visualize the cytotoxicity upon NIR
irradiation, Mito-CIO- or CIO-loaded HeLa cells were
prestained with propidium iodide (PI) and calcein AM.
Irradiation of the Mito-CIO-loaded cells reveals a clear
indeed a critical factor in cell cytotoxicity as determined by the
percentage of PI-stained cells. The fluorescence microscopic
results in Figure 5c revealed more rapid and extensive cell death
for Mito-CIO than for CIO. The cytotoxicity induced by
irradiated Mito-CIO reached 89.90
whereas that by CIO was 42.58
7.72% within 20 min,
5.09% under similar
experimental conditions. Consequently, Mito-CIO showed no
C
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significant cytotoxicity when the cells were not irradiated
(Figure S6) but induced the superior cytotoxicity than CIO
upon NIR irradiation. The fluorescence intensities (FI) of
HeLa cells treated with Mito-CIO and CIO were approx-
(PC 0.833) (Figure S10). We further confirmed the
mitochondrial localization of Mito-CIO by TEM (Figure
S11), revealing that the dark iron oxide particles, which were
identified by their diameters, are located in the mitochondria.
These results confirm that the TPP unit of Mito-CIO causes
preferential localization to the mitochondria.26−29 Therefore,
intracellular hyperthermia induced by NIR-sensitive CIOs in
the mitochondria exerts cytotoxic effects superior to CIOs in
other subcellular locations.
imately equal (FI = 14.15
0.18 and 14.98
0.27,
respectively) (Figure S7), indicating that differential amounts
of Mito-CIO or CIO delivered to the cells are not responsible
for the superior cytotoxicity of Mito-CIO. Moreover, the
coumarin moiety in the chemical structure is not responsible
for the superior cytotoxicity of Mito-CIO, compared to CIO
(Figure S8). The enhanced cytotoxic effect of Mito-CIO could,
therefore, be attributed to its TPP moiety, which is a well-
known mitochondrial targeting unit and absent in CIO. Thus,
we propose that the hyperthermic cytotoxicity of Mito-CIO
may depend on its intracellular localization. The mode of cell
death by the hyperthermic cytotoxicity of Mito-CIO was
examined by using annexin V-fluorescein isothiocyanate
(annexin V-FITC) and PI stainings (Figure S9). Before NIR
irradiation, neither annexin V-FITC nor PI-stained cells were
observed, but at 6 h after NIR laser irradiation, both annexin V-
FITC and PI stained cells were observed simultaneously. These
results thus indicate that hyperthermic cytotoxicity by the
Mito-CIO induces mixed types of cell death, necrosis, and
apoptosis.
Co-Localization of Mito-CIO and CIO with Fluorescent
Organelle Trackers. To identify the intracellular localizations
of the CIOs, we performed TEM imaging and fluorescence co-
localization experiments employing organelle trackers (Mito-,
ER-, and LysoTracker Red). The fluorescence localization of
Mito-CIO overlapped with that of MitoTracker (Pearson’s
coefficient (PC) 0.871) but only partially overlapped with
Lyso- (PC 0.550) or ER-Tracker (PC 0.664) (Figure 6). This
strongly indicates that Mito-CIO localizes to the mitochondria
(Figure 6c). In comparison, CIO primarily localized to the ER
In Vivo Xenografted Tumor Imaging and Therapy by
Using MPs. To gain insight into the cytotoxicity of CIOs in an
animal model, we examined the in vivo tumor target specificity
and therapeutic potential of Mito-CIO in A549 cell xenografted
BALB/c nude mice. The A549 cells are human adenocarci-
nomic alveolar basal epithelial cells, which reported a relatively
slow vascular formation in a xenograft mouse model.32
Generally, fast growing tumor vasculature in immunocompro-
mised mouse models can be a bias for evaluating tumor
targeting efficiency. Therefore, to avoid this undesirable bias,
we used A549 cells as our xenograft model. We intravenously
injected mice tails with either PBS (control group) or Mito-
CIO and observed gradually increased fluorescence signals in
the Mito-CIO group. The maximum signal intensity at the
tumor site was observed 6 h after injection, confirming that
Mito-CIO can target tumors in vivo (Figure 7a). Examination
of the ex vivo fluorescence biodistribution revealed that Mito-
CIO and CIO preferentially accumulated at the tumor site
(>10-fold higher than the normal liver, kidney, heart, lung, and
spleen tissues; P < 0.05) (Figure 7b,c). We measured the tumor
levels of CIOs by inductively coupled plasma atomic emission
spectroscopy (ICP-AES) and found that the amount of
accumulated iron in the tumor tissues (w/w) was similar
among the IO, Mito-CIO and CIO-injected groups (8.288
0.919, 11.2552
1.774, and 8.939
0.982, respectively (×
10−5)) (Figure 7d). However, the iron was not detectable in the
tumor tissues of the control group (P < 0.05, compared to all
CIO-injected groups). Accumulation of NPs inside tumors is
assumed to be mediated by an enhanced permeability and
retention effect.33,34
We examined the therapeutic effects of IO, Mito-CIO, and
CIO upon NIR irradiation in the tumor xenograft model
(Figure 7e). After six doses with NIR irradiation on one side of
the grafted tumor region, the irradiated tumor volume in the
Mito-CIO group was significantly reduced compared to the
nonirradiated tumor volume (Figure 7f, P < 0.05). In particular,
the Mito-CIO treatment induced a more dramatic decrease in
the tumor volume after day 28 (Figure 7g). Histochemical
analysis revealed several regions of increased cell death in the
tumor tissue of the Mito-CIO-injected group after photo-
thermal therapy (Figure 7h, inside area of black dashes in lower
left panel) compared to the PBS control group. Furthermore,
proliferating cell nuclear antigen (PCNA) staining of tumor
cryosections also showed that proliferation was significantly
decreased in the Mito-CIO-treated group, and Prussian blue
staining detected increased iron in the tumor from the Mito-
CIO-treated group, compared to the PBS group (Figure 7h).
Considering the similar amounts of accumulated iron oxide
localized to the tumor sites (Figure 7d), the superior antitumor
efficacy of Mito-CIO may be attributed to its TPP moiety. The
agreement between the therapeutic effects of CIOs in the
animal model and the in vitro cytotoxicity results (Figure 5)
suggests that mitochondria-directed photothermal therapy may
be a more effective cancer treatment.
Figure 6. Confocal fluorescence microscopic images of HeLa cells
treated with Mito-CIO and MitoTracker Red, ER-Tracker Red, or
LysoTracker DND-99. (a, d, and g) Fluorescent images of Mito-CIO
in HeLa cells excited at 740 nm (two-photon excitation) and collected
at 450−550 nm. (b) Fluorescent image of MitoTracker Red (0.2 μM)
collected using a 650−700 nm filter with excitation at 633 nm. (c)
Merged image of (a) and (b). PC: 0.871. (e) Fluorescent image of ER-
Tracker Red (0.5 μM) collected using a 600−650 nm filter with
excitation at 543 nm (two-photon excitation). (f) Merged image of (d)
and (e). PC: 0.550. (h) Fluorescent image of LysoTracker DND-99
(0.5 μM) collected using 600−650 nm filter with excitation at 543 nm
(two-photon excitation). (i) Merged image of (g) and (h). PC: 0.664.
D
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generating system can enhance the therapeutic efficacy of
hyperthermia in cancer treatment and may allow the develop-
ment of new photothermal therapeutics.
EXPERIMENTAL SECTION
■
Synthetic Materials and Methods. UV−vis and ESI-MS spectra
were obtained using Shinco S-3100 spectrophotometers and Shimadzu
LCMS-2020, respectively. NMR spectra were collected on either
Varian 300 or Varian 400 MHz spectrometers. IR spectra were
recorded on a Shimadzu FTIR 8400S instrument. TEM images of the
NPs were taken on a JEOL JEM-2010 transmission electron
microscope operating at 200 kV (293 K) using an accelerating voltage
of 100 kV and a 16 mm working distance. XPS analysis of the NPs was
measured by XPS (X-ray photoelectron spectroscopy, Sigma Probe,
Thermo-VG, England) with monochromatic Al Kα (1486.7 eV) X-ray
source. TGA data were obtained with the TA Instruments Q-50.
Synthesis of Iron Oxide NPs. These NPs were synthesized by the
previously reported procedure.35 A mixture of iron(III) chloride
hexahydrate (5.4 g, 20 mmol) and sodium oleate (18.3 g, 60 mmol) in
a solvent mixture (40 mL EtOH, 30 mL distilled water and 70 mL
hexane) was stirred at 70 °C. After a 4 h reaction, the organic layer was
washed with distilled water, and the organic layer was collected. After
removal of the solvents, the iron−oleate complex was obtained as a
solid. For preparation of the monodisperse iron oxide NPs (12 nm),
oleic acid (2.9 g, 10 mmol) and 1-octadecene (100 g) were added to
the iron−oleate complex (18.0 g, 20 mmol) at 25 °C, and the solution
was stirred at 320 °C and maintained constant heating rate (3.3 °C
min−1). After 30 min reaction, the reaction mixture including the NPs
was cooled to 25 °C, and the precipitates were washed with ethanol
(200 mL × 3) to remove the excess oleate. The NPs were prepared by
separation using a centrifuge.
Cell Culture and Confocal Microscopy Study. HeLa cells were
cultured in DMEM (Invitrogen) applied with 10% FCS (Invitrogen).
The cells were seeded onto 24-well flat-bottomed plates 1 day prior to
the confocal imaging experiments. After 1 day later, Mito-CIO (1.40
mg/mL) and CIO (1.22 mg/mL) were added to the cells to confirm
their cellular localization and cytotoxicity as mentioned in this
manuscript. To check cellular localization of CIOs, the cells were
pretreated with media containing organelle trackers (Mito- (0.2 μM),
Lyso- (0.5 μM), and ER (0.5 μM) tracker, respectively, Invitrogen)
prior to incubation for 2 h with CIOs and washed with PBS buffer
three times. HeLa cells incubated with organelle trackers and CIOs
were measured by a Leica TCS-SP2 confocal fluorescence multiphoton
microscope. For monitoring cell cytotoxicities, propidium iodide (PI,
50 μg/mL in PBS buffer, Aldrich) and calcein AM (1 μg/mL in PBS
buffer, Aldrich) were used. The HeLa cells were incubated 2 h with or
without CIOs prior to incubation with media containing PI and
calcein AM and then subjected to the 740 nm pulsed laser exposure
(1.3 mW, 200 fs, 90 MHz) with 0.04 W/cm2 power densities for 20
min. The cytotoxicity results in HeLa cells were monitored under a
Leica TCS-SP2 confocal fluorescence multiphoton microscope.
MTT Assay. HeLa Cells were washed with 1 mL of PBS twice and
the medium was changed into FBS-free DMEM culture media prior to
assay. The cell viability was tested by the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide, Life Technol-
ogies, Carlsbad, CA, USA) assay following the manufacture’s
instruction. Briefly, HeLa cells (1.5 × 104 cells in 100 μL of media)
were seeded onto a 96-well plate. On the following day, the culture
medium was changed, and cells were incubated with different
concentrations (0.70, 1.40, 7.00, 14.0, 21.0, or 28.0 mg/mL) of
CIOs in 100 μL of fresh medium at 37 °C for 6 h. After 6 h, the
medium was discarded, and prepared culture medium containing 12
mM MTT solution was added into each well, including a negative
control of culture media alone. After 1 h incubation, the medium was
removed from the wells. Then 30 μL of DMSO was added to each well
and mixed thoroughly for 15 min. The supernatant of each well was
transferred to a 96-well plate, and the absorbance was measured at 540
nm using a microplate spectrophotometer (PowerWave XS, Bio-Tek,
Winooski, VT, USA).
Figure 7. In vivo xenograft tumor imaging and therapy using MPs. (a)
In vivo NIR image of xenografted BALB/c nude mice 1, 3, and 6 h
after tail intravenous injection of PBS or Mito-CIO. (b) Ex vivo
fluorescence in various tissues (i: liver, ii: kidney, iii: tumor, iv: heart, v:
lung, and vi: spleen) from xenografted BALB/c nude mice 6 h after
injection with PBS, IO, Mito-CIO, or CIO. (c) Quantification of ex
vivo fluorescence [regions of interest (ROI) as individual pixels]. (d)
Quantification of accumulated iron in tumor tissues by ICP-AES. (e)
Representative images of xenografted BALB/c nude mice on day 0 and
21 days after 740 nm photothermal therapy of the right tumor (red
circle). The left tumor (blue circle) was used as nonirradiated control.
(f) Ratio of irradiated tumor volume to nonirradiated tumor volume.
*P < 0.05. (g) Dissected tumor tissues from each group after
photothermal therapy. (h) Histochemical analysis of snap-frozen
tumor cryosection from PBS and Mito-CIO mice after photothermal
therapy. Photothermal cell death is indicated by the black dashed area
in the H&E-stained panel. PCNA with DAPI staining indicates cellular
proliferation. Prussian blue indicates intracellular iron accumulation.
Magnification: 200×.
CONCLUSIONS
■
We prepared a new mitochondria-targeting iron oxide NP
fabricated with two functional groups: TPP as the mitochon-
drial targeting unit and coumarin as a fluorescent signaling unit.
Upon irradiation with a 740 nm NIR laser at 2.0 W/cm2, the
solution temperature of Mito-CIO was increased by 13 °C,
whereas no such changes occurred in a NP-free solution. Laser
irradiation of HeLa cells incorporating Mito-CIO increased the
cellular temperature by 2.1 °C within 5 min. In HeLa cells,
Mito-CIO and CIO were localized to the mitochondria and the
ER, respectively. Mito-CIO induced significantly more cell
death after 20 min NIR irradiation than did CIO, and the
differential cytotoxicity can be attributed to the mitochondrial
localization of NPs. Furthermore, in vivo therapy using
mitochondria-directed hyperthermia clearly displayed tumor
suppression, which is in agreement with the in vitro cytotoxicity
results. Together, these results strongly support our hypothesis
that mitochondria are more susceptible to hyperthermia than
the ER. From this study of both the in vitro and in vivo effects of
mitochondria-targeted MPs, we conclude that this heat
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Monitoring the Mode of Cell Death by the Hyperthermic
Cytotoxicity of Mito-CIO. HeLa cells (2.0 × 106 per dish) were
seeded on 35 mm confocal dishes (glass bottom dish, SPL, Kyong-Gi,
Republic of Korea) and stabilized for 48 h. Then the cells were treated
with 1.40 mg/mL Mito-CIO in 2 mL culture medium. After 2 h
incubation, the cells were irradiated with a 740 nm pulsed laser (0.04
W/cm2 power densities) for 10 min to induce hyperthermic
cytotoxicity. Six h later, the mode of cell death was examined by
using an annexin V-FITC and PI staining kit according to
manufacturer’s instructions (BD, San Jose, California, USA). The
results in HeLa cells were monitored under a confocal fluorescence
microscope.
Photothermal Conversion Efficacy. To investigate the photo-
thermal conversion effect, a series of solutions of Mito-CIO (0, 0.3,
1.4, and 7.0 mg/mL, respectively) in pH 7.4 PBS solution were
irradiated by the 740 nm pulsed laser exposure (1.3 mW, 200 fs, 90
MHz) with 2.0 W/cm2 power densities during 5 min. To verify the
function of NIR laser power density, the solutions of Mito-CIO (1.4
mg/mL) in pH 7.4 PBS were irradiated with different NIR power
densities (0.3, 2.0, 6.0, and 10 W/cm2). The temperature changes were
measured with an IR thermal camera (A300, FLIR, Boston, MA) over
5 min. The camera captured a time series of images that display
solution temperatures by color. All the experiments were conducted at
room temperature.
In Vitro Photothermal Conversion Efficacy. HeLa cells were
incubated with Mito-CIO (1.4 mg/mL) and then subjected to the 740
nm pulsed laser exposure (1.3 mW, 200 fs, 90 MHz) with different
power densities (0.3, 2.0, 6.0, and 10 W/cm2) over 5 min. During
exposure to a NIR laser, thermographic images were recorded with a
FLIR thermal camera at a time interval of 10 s over a course of 5 min,
and the temperature of the HeLa cells was calculated from the data
using FLIR R&D software. All the experiments were conducted at
room temperature.
Bio-TEM Image. The HeLa cells were grown on the MatTek Petri
dish with CELLocate coverslips and incubated for 2 h with Mito-CIO
(1.40 mg/mL) and washed with PBS buffer three times. Mito-tracker
Red (0.2 μM) was used for mitochondrial staining. Imaging was done
with a Leica TCS-SP2 confocal fluorescence multiphoton microscope.
After this observation, the cells were then fixed with 2.5%
glutaraldehyde (pH 7.3, 0.1 M phosphate buffer) at 25 °C for 1 h.
Then, the cells were treated with 1% OsO4 plus 1.5% potassium
ferrocyanide (pH 7.3, 0.1 M phosphate buffer) at 4 °C in the dark for
1 h and embedded in Epon 812 after dehydration with an ethanol and
propylene oxide series. After 2 days polymerization at 70 °C with pure
resin, 70 nm ultrathin sections were collected with an ultramicrotome
(UltraCut-UCT, Leica, Austria). Then, the sections were collected on
copper grids (100-mesh). After staining (2% uranyl acetate (15 min)
and lead citrate (5 min)), bio-TEM images of the sections were taken
on a JEOL JEM-1400Plus transmission electron microcopy operating
at 120 kV.
provided with sterilized food and water ad libitum. Animal facility was
kept 12 h light/dark cycles at room temperature 21 2 °C with 30−
40% humidity. Approximately 5.0 × 106 cells of A549 were mixed with
354234-matrigel (BD) and subcutaneously injected in the right and
left flanks.
In and Ex Vivo Imaging. In vivo and ex vivo investigations were
examined to evaluate the tumor target specificity and organ
distribution of CIOs using A549 inoculated xenograft mice. After
4−5 weeks of inoculation, xenograft mice were assigned into four
groups, and tail intravenous injection of 3.36 mg/mL IO, 7.00 mg/mL
Mito-CIO, and 7.47 mg/mL CIO in 70 μL PBS was performed. As a
negative control, 70 μL PBS was injected. After tail intravenous
injection, in vivo spectral fluorescence images were measured by using
an in vivo imaging system (IVIS) spectrum (PerkinElmer, Waltham,
MA, USA). For ex vivo images, the tumor tissues and other organs
(lung, heart, liver, kidney, and spleen) of all animals were dissected 6 h
after injection with PBS, IO, Mito-CIO, or CIO and observed by using
an IVIS spectrum. The filter set for both in vivo and ex vivo images was
Blue: excitation and emission; 430 to 500 nm. The fluorescence
images consisting of spectra and autofluorescence were then unmixed
with commercial software (Living Image software for IVIS Spectrum/
200, ver. 4.1, Waltham, MA, USA).
In Vivo Therapy. To validate the therapeutic effect, the initiated
tumor volume and size were weekly measured with a digital caliper.
CIOs were tail intravenously injected 6 times every other day as
described above. Six h after injection, 740 nm pulsed laser irradiation
(1.3 mW, 200 fs, 90 MHz) with 2.0 W/cm2 power densities for 10 min
was performed on the tumors on the right side in each of the xenograft
mice of the four different groups. Then final tumor volume, size, and
weight on both sides were measured at 3 weeks subsequent to the last
injection and irradiation treatment.
Histochemistry Analysis. After taking ex vivo images, the grafted
tumor tissues and other organs (lung, heart, liver, kidney, and spleen)
were dissected and embedded in Tissue-Tek 100% optimal cutting
temperature compound (O.C.T., Sakura Finetek, USA) for cryosec-
tion. Then the fresh tissue was rapidly snap frozen by placing it into
liquid nitrogen, and the tissues were stored in a −80 °C freezer until
further analysis. Freshly cryopreserved tissues were cut (6 μM thick)
using a −25 °C ultramicrotome (Leica CM 3050 S, Wetzlar,
Germany). Each section was picked up on an adhesion microscope
glass slide (Paul Mariendeld GmbH & Co.KG, Lauda-Konigshofen,
̈
Germany), and then the O.C.T compound was carefully washed out
with PBS (twice). The tissue cryosections were stained with
hematoxylin (Merck, Readington Township, NJ, USA) and eosin
(H&E) (BBC Biochemical, Mt. Venom, WA, USA).
For PCNA immunohishtochemistry of tumor cryosection, snap
frozen tumor sections were washed with PBS and blocked with 10%
goat or donkey serum in PBS. Then, the tissue sections were incubated
for 24 h at 4 °C with anti-PCNA antibodies (Abcam, Cambridge, MA,
USA) diluted with 10% goat or donkey serum in PBS. On the
following day, the tissue sections were washed with PBS and incubated
at room temperature for 1 h with appropriate secondary antibodies
(Life Technologies, Carlsbad, CA, USA). Nuclei were labeled with 4,6-
diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO,
USA) for 5 min. After rinsing in 70% ethanol for 5 min, tissue
sections were treated with an autofluorescence eliminator reagent
(Millipore, Billerica, MA, USA) for 5 min to avoid false positive
signals. Finally, tissue sections were washed and mounted with Immu-
mount (Thermo, Waltham, MA, USA) and examined with an Axiovert
200 M fluorescence microscope (Zeiss, Oberkochen, Germany).
The Retention of the Iron Oxide NPs Inside the Tumor
Tissues. The tumor-bearing mice were tail-intravenously injected with
PBS, 3.36 mg/mL IO, 7.00 mg/mL Mito-CIO, and 7.47 mg/mL CIO
in 70 μL PBS. After 6 h, the injected mice were terminated. The tumor
tissues (0.1 g) were dissected and immersed in 0.5 mL of water. The
immersions were ground into a mash with a tissue grinder, and 0.5 g of
aqua regia was added. After 10 days, the tissue samples were
centrifuged, the supernatants were collected and diluted with 9 mL of
water. The concentrations of iron in these solutions were measured in
triplicate by ICP-AES (Perkin−Elmer Optima 4300 DV, USA) at the
Korean Basic Science Institute (KBSI). The calculated average iron
concentration as determined from three independent samples.
Mouse Xenograft Model. To examine the therapeutic effect and
imaging of Mito-CIO and CIO in the tumor tissue, 6- to 8-week-old
BALB/c nude mice were purchased from RaonBio (Kayonggido,
Yonginsi, South Korea) (control xenograft, n = 8; treatment injected
xenograft, n = 8). All animals were acclimatized to the animal facility
for at least 48 h prior to experimentation and maintained according to
the Guide for the Care and Use of Laboratory Animals published by
the NIH. They were housed in a barrier under HEPA filtration and
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis, structural characterization, subcellular localization,
functional characterization of NPs, and biological evaluations.
This material is available free of charge via the Internet at
http://pubs.acs.org.
F
DOI: 10.1021/ja5122809
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
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2010, 62, 339−345.
AUTHOR INFORMATION
■
Corresponding Authors
*jhkim@korea.ac.kr
*kmbyun@khu.ac.kr
*jonghwa@gnu.ac.kr
*kangch@khu.ac.kr
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1998, 431, 285−286.
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*jongskim@korea.ac.kr
Author Contributions
⊥These authors contributed equally.
Funding
The authors declare no competing financial interest.
Notes
(23) Yuen, W. F.; Fung, K. P.; Lee, C. Y.; Choy, Y. M.; Kong, S. K.;
Ko, S.; Kwok, T. T. Life Sci. 2000, 67, 725−732.
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(25) Shah, B. P.; Pasquale, N.; De, G.; Tan, T.; Ma, J.; Lee, K.-B. ACS
This work was supported by CRI project (no. 2009-0081566 to
J.S.K.), Basic Science Research Program (2012R1A1A2006259
to C.K.) from the National Research Foundation of the
Ministry of Science, ICT & Future Planning in Korea, the Next-
Generation BioGreen 21 Program (SSAC) (PJ009041022012
to J.H.J) from the Rural development Administration in Korea,
and the National Research Foundation of Korea (NRF) grant
funded by the Korea government (MEST) (NSF-
2013R1A1A1A05011990 to K.M.B.) and the National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MEST) (no. 2012M3A9B4028636 to J.-H.K.).
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