A Mitochondria-Targeted Cryptocyanine-Based Photothermogenic Photosensitizer

Article abstract of DOI:10.1021/jacs.7b04263

Cryptocyanine-based probes exhibit highly efficient photothermal conversion and represent a new class of photothermal agents for use in photothermal therapy (PTT). With the thermal susceptibility of mitochondria in mind, we have prepared a mitochondria-targeted, NIR-absorbing cryptocyanine probe (Mito-CCy) and evaluated its photophysical properties, photothermal conversion efficiency, biological compatibility, cytotoxicity, and mitochondrial localization in HeLa cells. Upon subjecting 0.5 mL of a PBS buffer solution (10 mM, pH 7.4, containing 50% DMSO) of Mito-CCy (0.5 mM) to 730 nm laser irradiation at 2.3 W/cm², the temperature of the solution increased by 13.5 °C within 5 min. In contrast, the corresponding cryptocyanine (CCy) lacking the triarylphosphonium group gave rise to only an 3.4 °C increase in solution temperature under otherwise identical conditions. Mito-CCy also exhibited high cytotoxicity in HeLa cells when subject to photoirradiation. This light-induced cytotoxicity is attributed to the endogenous production of reactive oxygen species (ROS) induced under conditions of local heating. ROS are known to interfere with the mitochondrial defense system and to trigger apoptosis. By targeting the mitochondria, the present sensitizer-based photothermogenic approach is rendered more effective. As such, the system reported here represents the vanguard of what might be a new generation of organelle-targeted photothermal therapeutics.

Full text of DOI:10.1021/jacs.7b04263

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Article
A Mitochondria-targeted Cryptocyanine-
Based Photothermogenic Photosensitizer
Hyo Sung Jung, Jae Hong Lee, Kyutae Kim, Seyoung Koo, Peter
Verwilst, Jonathan L. Sessler, Chulhun Kang, and Jong Seung Kim
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 23 Jun 2017
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A Mitochondria-Targeted Cryptocyanine-Based Photothermo-
genic Photosensitizer
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Hyo Sung Jung,†,§ Jae-Hong Lee,‡ Kyutae Kim,‡ Seyoung Koo,† Peter Verwilst,† Jonathan L. Sessler,*,§
Chulhun Kang,*,‡ and Jong Seung Kim*,†
†Department of Chemistry, Korea University, Seoul 02841, Korea
‡The Graduate School of East-West Medical Science, Kyung Hee University, Yongin 17104, Korea
§Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712-1224, United States
ABSTRACT: Cryptocyanine-based probes exhibit highly efficient photothermal conversion and represent a new class of photother-
mal agents for use in photothermal therapy (PTT). With the thermal susceptibility of mitochondria in mind, we have prepared a
mitochondria-targeted, NIR-absorbing cryptocyanine probe (Mito-CCy) and evaluated its photophysical properties, photothermal
conversion efficiency, biological compatibility, cytotoxicity, and mitochondrial localization in HeLa cells. Upon subjecting 0.5 mL
of a PBS buffer solution (10 mM, pH 7.4, containing 50% DMSO) of Mito-CCy (0.5 mM) to 730 nm laser irradiation at 2.3 W/cm2,
the temperature of the solution increased by 13.5°C within 5 min. In contrast, the corresponding cryptocyanine (CCy) lacking the
triarylphosphonium group gave rise to only a ~3.4°C increase in solution temperature under otherwise identical conditions. Mito-
CCy also exhibited high cytotoxicity in HeLa cells when subject to photoirradiation. This light-induced cytotoxicity is attributed to
the endogenous production of reactive oxygen species (ROS) induced under conditions of local heating. ROS are known to interfere
with the mitochondrial defense system and to trigger apoptosis. By targeting the mitochondria, the present sensitizer-based photo-
thermogenic approach is rendered more effective. As such, the system reported here represents the vanguard of what might be a new
generation of organelle-targeted photothermal therapeutics.
To date, nanomaterials, including transition metal oxides,8
sulfide nanoparticles,9 gold nanoparticles,10 platinum nanopar-
ticles11 and nano-carbon species,12,13 have been studied as pho-
tosensitizers in PTT. Their compatibility with NIR irradiation,
excellent photothermal conversion efficiency combined with
high absorption and superior photostability, are appealing fea-
tures. However, the poor biodegradability and potential long-
term toxicity of many nanomaterials represent potential limita-
tions to clinical translation efforts.14 Organic photosensitizers,
such as cyanines,15,16 formulated in micelles or liposomes17,18 or
conjugated to polymers,19,20 have thus been studied as potential
alternative PTT photosensitizers. Their safety is reflected by the
US FDA’s decision to approve indocyanine green (ICG) as a
medical imaging agent. ICG has since been used off-label as a
PTT agent.18 However, PTT is not devoid of issues. In particu-
lar, the relatively high temperatures needed to produce a robust
cancer effect via PTT can increase damage to surrounding nor-
mal cells.21 In addition, many of the existing agents suffer from
poor inherent bio-localization features, which can limit their
utility. The agents that combine singlet oxygen generation with
thermal production could obviate some of these concerns.22
Also attractive would be PTT agents that localize specifically
to particularly susceptible organelles. With these considerations
in mind, we have developed a new, mitochondria-targeted PTT
sensitizer based on cryptocyanine. This system, referred to as
Mito-CCy, displays a high photothermal conversion efficiency,
■ INTRODUCTION
Phototherapy is a light-induced therapeutic modality, which tra-
ditionally includes photodynamic therapy (PDT) and photother-
mal therapy (PTT). In both, chemical agents are used as photo-
sensitizers that then produce either reactive oxygen species
(ROS) or induce heat shock, or a combination thereof.1,2 These
methods continue to attract the attention of researchers engaged
in cancer therapy, as a number of cancers have been found to be
particularly sensitive to these types of treatment.3,4 To date,
more attention has focused on PDT than on PTT; indeed, sev-
eral PDT photosensitizers have received the US Food and Drug
Administration (FDA) approval for specific indications. How-
ever, PTT has emerged as a potentially safer option. This is be-
cause off-target sensitizing effects and irreversible oxidative
damage beyond the targeted cancer locus is often seen in the
course of PDT.1,3,5 Additionally, PDT as practiced clinically, re-
quires an adequate tumor oxygen supply.5 This can limit its ef-
ficiency in hypoxic environments, whereas PTT does not suffer
from this drawback. In many instances, PTT effects can be pro-
duced using dyes in the near-infrared (NIR) spectral region,
which further lowers damage to tissues surrounding the tumor
targets of interest. It is thus suggested that PTT could emerge as
a less invasive treatment option than not only PDT, but also cur-
rent standards of care, such as, e.g., radiation or chemother-
aphy.2,6,7 Not surprisingly, therefore, considerable effort is be-
ing devoted to the study of new potential PTT sensitizers.
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good biological compatibility in vitro, and photo-induced cyto-
toxicity in HeLa cells that is ascribed in part to organelle-spe-
cific endogenous ROS production.
Synthesis and Characterization of Cryptocyanines.
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The putative mitochondria-targeted cryptocyanine (Mito-CCy)
and a non-targeted cryptocyanine (CCy) control were synthe-
sized as outlined in Figure 2. Briefly, the synthesis entailed the
alkylation of 4-methylquinoline with 4-bromomethylbenzoic
acid to yield 3, which was further reacted with N,N′-diphenyl-
formamidine and acetic anhydride to produce 2. Extension of
the π-conjugated system 2 with 3 and triethylamine (TEA) re-
sulted in cryptocyanine 1. The amide-linked target compound,
Mitochondria are indispensable cellular organelles responsi-
ble for the production of the majority of the cell’s energy sup-
ply. They also play a key role in programed cell death such as
apoptosis. These organelles are particularly sensitive to changes
in cardiolipin metabolism23 or heat shock24,25 and are easily
damaged by ROS formation.26,27 Therefore, mitochondrial heat
delivery mediated through various forms of nanostructures28-30
has been attempted by adopting mitochondrial-guiding agents
(e.g., triarylphosphonium (TPP) cations,31 mitochondrial phos-
pholipid cardiolipin,32 mitochondria-penetrating peptides,33 an
oligomeric carbohydrate scaffold,34 mitochondrial targeting se-
quences,34 as well as a vesicle-based transporter system34). Re-
cently, we reported a mitochondrial targeting hyperthermia-in-
ducing agent using TPP-conjugated nanoparticles, that induced
mitochondrial dysfunction, resulting in apoptosis.8 However,
small organic molecules offer advantages relative to other na-
nomaterials, including in many instances improved safety, ease
of preparation and modification. We therefore suggest that mi-
tochondria-targeting PTT agents based on organic molecules
might provide significant benefits. However, to our knowledge,
such systems have yet to be reported.
Mito-CCy, was obtained from
1
by reacting with
(PPh3 (CH2)2NH2)Br− in the presence of 2-(1H-9-azobenzo-tri-
azole-1-yl)-1,1,3,3-tetramethylammonium hexafluorophos-
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phate (HATU), diisopropylethylamine (DIPEA) and TEA in
N,N-dimethylformamide (DMF). CCy, an analogue lacking the
TPP unit, was prepared using an analogous procedure. Both
Mito-CCy and CCy and gave 1H NMR and 13C NMR spectral
and ESI-MS analytical data consistent with their proposed
structures (Figures S26-S40).
In this study, we set out to explore the potential benefits as-
sociated with a small molecule-based approach to mitochon-
dria-localized PTT. Towards this end, the putative mitochon-
dria targeting NIR-thermogenic cryptocyanine dye (Mito-CCy)
was prepared (cf. Figure 1). Mito-CCy was designed to fulfill
two critical requirements expected to maximize the PTT effi-
ciency: i) efficient light absorption but a low fluorescence quan-
tum yield (QY), as well as a low singlet oxygen QY, so as to
maximize the conversion of absorbed photo-energy into ther-
mal energy35 and ii) mitochondrial localization of the photosen-
sitizer to enhance the presumed photo-cytotoxicity.8,28-30 As de-
tailed below, the absorption spectrum of Mito-CCy is charac-
terized by an intense NIR absorption band and a low fluores-
cence QY (< 0.01), as well as a low singlet oxygen QY (< 0.02).
It also possesses lipophilic cationic parts to enhance mitochon-
drial targeting.36 Despite the low QY of Mito-CCy, it still
proved possible to visualize its fluorescence in vitro by means
of confocal microscopy. This allowed the real-time monitoring
of its uptake by the mitochondria. Subsequent NIR light irradi-
ation generates heat and, in conjunction with endogenous ROS
production, results in mitochondrial dysfunction and cell apop-
tosis as outlined in Figure 1.
Figure 2. Synthesis of Mito-CCy and CCy. (i) Tri-
phenylphosphine, acetonitrile, reflux, 24 h, 88%; (ii) 4-(bromome-
thyl)benzoic acid, acetonitrile, 110°C, 6 h, 72%; (iii) N,N′-diphe-
nylformamidine, acetic anhydride, 150°C, 1 h, 87%; (iv) 3, TEA,
dichloromethane, 25°C, 24h, 48%; (v) propylamine, HATU, TEA,
DIPEA, DMF, r.t., 24 h, 32%; (vi) 4, HATU, TEA, DIPEA, DMF,
r.t., 24 h, 35%.
Spectroscopy. The absorption and emission features of
Mito-CCy and CCy were probed in dimethyl sulfoxide
(DMSO) using UV-Vis and fluorescence spectroscopy, respec-
tively (Figure 3). The absorption spectra exhibited an intense
peak at 713 nm with large molar extinction coefficients of 15.5
× 104 M-1 cm-1 and 17.0 × 104 M-1 cm-1 for Mito-CCy and CCy,
respectively (Figure S1). In the case of both probes, the fluores-
cence maximum was observed at 734 nm (Figure 3). The fluo-
rescence QYs (Φf) of Mito-CCy and CCy were calculated ac-
cording to equation (S1) and found to be 0.007 and 0.011, re-
spectively. These values are in agreement with what has been
reported for other dyes sharing the same basic cryptocyanine
scaffold.37 As shown in Figure S2, the two cryptocyanines,
Mito-CCy and CCy, also exhibited large molar extinction co-
efficients in the NIR region in other common solvents such as
CH2Cl2, CH3CN, MeOH, and water. The emission maxima of
these probes showed slight variations as a function of solvent,
with modest bathochromic shifts (< 20 nm) being seen upon in-
creasing solvent polarity from CH2Cl2 to H2O.
Figure 1. Schematic representation of enhanced photothermogen-
esis by mitochondria-targeting cryptocyanine (Mito-CCy).
Whereas the UV-Vis absorption features of either Mito-CCy
or CCy were not appreciable affected by the choice of organic
solvents, in aqueous media variations were seen in the optical
■ RESULTS AND DISCUSSION
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signature of CCy as a function of the ionic strength. This latter
finding was ascribed to their limited solubility in aqueous envi-
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ronments. As shown in Figure 3d-e, the spectra of both Mito-
CCy and CCy exhibit an intense peak at 702 nm in aqueous
media containing 10% DMSO. However, in a solution of higher
ionic strength (10 mM PBS various pH conditions) an hypso-
chromic shift in the absorption maximum (~200 nm) with an
associated color change from green to purple (Figure 3f-g and
Figure S3) was seen for CCy. The absorption bands of CCy
show similarly hypsochromic shifts in various buffer solutions
(e.g., HEPES, MES, monosodium phosphate and Tris) (Figure
S4). Similar spectral behavior was seen for more biomimetic
CCy solutions, including those containing CT-DNA (50
μg/mL) or HeLa cell cytosol extract (20 μg/mL) (Figure S5).
These results lead us to suggest that the observed spectral shifts
are not caused by the choice of buffering agent. Rather, the hyp-
sochromic shifts in the absorption maxima of CCy are ascribed
to aggregation effects that are likely to be relatively enhanced
in aqueous environments. Support for this latter conclusion
comes from an observation of similar shifts in dye molecules
that interact in a face-to-face manner to form so-called H-ag-
gregates.38-40 However, aggregation is not a desirable feature for
PTT agents since it can lead to reduced heat transfer. Fortu-
nately, no evidence of aggregation was seen for Mito-CCy (as
opposed to CCy) under any of these conditions (Figures 3 and
S3-S5). This desirable lack of aggregation is rationalized in
terms of the presence of the cationic phosphonium substituents,
which provide for electrostatic repulsion between the individual
monomers.
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Photothermal Conversion Efficiency. The fluores-
cence QYs (Φf) of Mito-CCy (0.007) and CCy (0.011) tabu-
lated in Figure 3a are much lower than that of the FDA-
approved agent ICG (0.078) (cf. Table S1).41 To obtain insights
into the relative photothermal effect of the present cryptocya-
nines as compared to ICG, their conversion efficiencies were
compared under conditions of 730 nm laser irradiation, using
DMSO as the solvent to ensure complete solubilization of both
photosensitizers. The temperature of the Mito-CCy (0.5 mM)
solution was raised by 24.5 °C upon irradiation for 5 min at 730
nm laser irradiation (3.0 W/cm2). Moreover, the extent of tem-
perature increase was found to correlate well with the laser
power (Figure 4a-b) as well as Mito-CCy’s concentration (Fig-
ure 4c-d). Similar photothermal efficiencies were observed for
CCy (Figure S6).
Figure 3. UV-Vis-NIR absorption and emission spectral features
of Mito-CCy and CCy. (a) Photophysical data for Mito-CCy and
CCy. All values were determined in DMSO. ε: molar extinction
coefficient (×104 M-1 cm-1). λabs: absorption maximum wavelength
(nm). λem: emission maximum wavelength (nm). Φf: fluorescence
quantum yield. Normalized absorption (black) and emission spec-
tra (red) of (b) Mito-CCy and (c) CCy. Absorption spectra of var-
ious concentrations (i.e., 2.5, 5.0 and 10 μM) of (d) Mito-CCy and
(e) CCy in 10% DMSO-water solution and (f) Mito-CCy and (g)
CCy in 10% DMSO-buffer solution (pH 7.4, 10 mM PBS).
Photochemical Properties of Mito-CCy and CCy.
Prior to application of the cryptocyanines to in vitro based PTT
studies, we investigated their basic chemical and photochemical
features. First, the ROS generating ability of the cryptocyanines
was estimated by quantifying the singlet oxygen co-production
under conditions of photoirradiation. The co-production of ROS
during PTT has been advanced as being both advantageous in
terms of increased cytotoxicity at lower temperatures22 and
problematic due triggering undesired side-effects.43 The singlet
oxygen QYs (ΦΔ) of Mito-CCy and CCy were determined ac-
cording to equation (S6) and were found to be 0.015 and 0.018,
respectively, upon irradiation at 730 nm (Figure S7). These
QYs (ΦΔ) are small compared to those of dyes that are used
clinically, such as methylene blue (MB) (0.52 in DMSO, Figure
S8).44 Moreover, the singlet oxygen QY of Mito-CCy is ~4.3
times lower than that of ICG (ΦΔ = 0.065, Figure S9), a potential
photosensitizer with a negligible singlet oxygen QY.45 Low
QYs for Mito-CCy and CCy were also found in more biomi-
metic media produced by the addition of calf thymus DNA (50
μg/mL) to PBS or in HeLa cell cytosol extract (20 μg/mL) (Fig-
ures S12-15). Given the low singlet oxygen QYs seen for Mito-
To evaluate the heat transfer efficiency of the cryptocya-
nines, their photothermal conversion efficiencies (η) were
measured following a reported method.42 Briefly, under condi-
tions of 730 nm laser irradiation (2.3 W/cm2), the temperature
of the solution including the cryptocyanines (0.5 mM) was al-
lowed to increase to the point of steady state. After switching
the laser off, the temperature decrease was recorded so as to es-
timate the heat transfer rate from the solution to the environ-
ment (Figures 4e-f and S6e-f). As defined by Equation S2, the
efficiencies (η) of Mito-CCy and CCy were found to be ap-
proximately 9.5 and 8.4%, respectively, and thus approximately
3-fold higher than the value reported for ICG (~3.1%; cf. Table
S1). The enhanced photothermal efficiencies seen for Mito-
CCy and CCy relative to ICG are consistent with their lower
fluorescence quantum yields, which is expected to allow a
greater non-emissive conversion of NIR light into heat.
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CCy and CCy and the fact that Mito-CCy is less effective at
producing singlet oxygen than ICG, we rule out the direct pro-
duction of significant quantities of ROS when our probes are
subject to photoactivation.
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Figure 5. Photostability of Mito-CCy and CCy in DMSO. IR ther-
mal images of (a) Mito-CCy and (b) CCy. Curves showing the
temperature change of (c) Mito-CCy and (d) CCy over several
ON/OFF cycles involving irradiation with a 730 nm laser (2.3
W/cm2) for 1 h followed by passive cooling. A Ti:Sa femtosecond-
pulsed laser (Chameleon XR by Coherent, 200 fs pulse width, 90
MHz repetition rate) was used.
Consistent with the relatively non-aggregated nature of Mito-
CCy in aqueous media (cf. Figure 3f-g), a buffer solution of
Mito-CCy leads to a higher temperature elevation than a corre-
sponding one containing CCy when studied under identical
conditions (~13.5°C vs. ~3.4°C for Mito-CCy and for CCy)
(Figure 6a-b). Similar temperature elevation effects were ob-
served for Mito-CCy and CCy in more biomimetic media con-
taining calf thymus DNA (50 μg/mL) (~11.1°C vs. ~3.1°C for
Mito-CCy and for CCy) or HeLa cell cytosolic extract (20
μg/mL) (~11.1°C vs. ~2.4°C for Mito-CCy and for CCy) over
the course of 5 min (Figure S16). On the basis of these findings,
we considered it likely that Mito-CCy would prove effective as
a PTT agent in vitro.
Figure 4. Photothermal conversion efficiency of Mito-CCy. IR
thermal images of DMSO solutions of Mito-CCy (a and c). Mito-
CCy concentration = 0.5 mM and irradiation intensities of 0, 1.0,
2.3, and 3.0 W/cm2 at 730 nm (a) and 2.3 W/cm2 irradiation (730
nm) at various Mito-CCy concentrations (0, 0.25, 0.50, and 1.0
mM) (c) as a function of irradiation time. Photothermal heating
curves for DMSO solutions of Mito-CCy at constant 0.5 mM con-
centrations of Mito-CCy and various laser irradiation intensities
(0, 1.0, 2.3, and 3.0 W/cm2, 730 nm) (b) and constant irradiation at
2.3 W/cm2 with varying Mito-CCy concentrations (0, 0.25, 0.50,
and 1.0 mM, 730 nm) (d) as a function of irradiation time. Photo-
thermal effects observed upon irradiating Mito-CCy with 730 nm
laser light (2.3 W/cm2) for 600 s and then stopping the irradiation
(e). Time versus −ln(θ) plot (with θ being the driving force temper-
ature, cf. equation (S5)) (f) obtained using the data recorded during
the cooling period of the experiment outlined in (e). A Ti:Sa femto-
second-pulsed laser (Chameleon XR by Coherent, 200 fs pulse
width, 90 MHz repetition rate) was used.
The photostability of Mito-CCy and CCy was also investi-
gated. Towards this end, these two cryptocyanines were subject
to repeated irradiation-cooling cycles using 730 nm laser irradi-
ation (2.3 W/cm2) for 1 h followed by passive cooling to room
temperature. As shown in Figure 5a-d, the variation in the max-
imal temperature increase during each cycle proved to be less
than 2.0%, leading us to conclude that Mito-CCy and CCy are
stable under conditions of 730 nm laser irradiation.
Figure 6. Photothermal heating curves of Mito-CCy and CCy.
Mito-CCy (a) and CCy (b) (0.5 mM, respectively) dispersed in
50% DMSO-buffer solution (pH 7.4, 10 mM PBS) with 730 nm
laser irradiation (2.3 W/cm2) as a function of irradiation time. A
Ti:Sa femtosecond-pulsed laser (Chameleon XR by Coherent, 200
fs pulse width, 90 MHz repetition rate) was used.
Cellular Localization of Mito-CCy. Prior to investigating
the presumed photothermal cytotoxicity of Mito-CCy in vitro,
the intracellular location of Mito-CCy was determined in HeLa
cells using fluorescence confocal microscopy and various com-
mercially available organelle-selective trackers. As can be seen
from an inspection of Figure 7, the fluorescence image of Mito-
CCy overlaps well with that of a commercially available mito-
chondrial tracking dye, MitoTracker Green FM. However,
poorer overlap was seen in the case of ER- or Lyso-Tracker.
The corresponding Pearson Correlation (PC) coefficients are
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0.95, 0.64, and 0.44 for these three probes, respectively. These the photoirradiated region as shown in Figure 8c. In this Figure,
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results provide support for the notion that Mito-CCy localizes
in mitochondria, presumably as the result of the cationic ap-
pended triarylphosphonium moieties that help direct the dye to
the mitochondrion.36
many red PI-fluorescence spots were found in the photoirradi-
ated region whereas cells not subject to photoirradiation re-
mained viable. We thus conclude that Mito-CCy showed no
significant cytotoxicity in the dark but gives rise to cytotoxicity
upon photoirradiation.
We further confirmed mitochondrial localization of Mito-
CCy at various post-incubation times (1, 2, 4, and 20 h). At 1 h
post-incubation of Mito-CCy, the PC value for mitochondrial
localization was 0.80, presumably reflecting some residual ad-
herence of the agent at or near the cell plasma membrane (Fig-
ure S17). At later times, the fluorescent image overlapped well
with that of MitoTracker (PCs: 0.91, 0.95, and 0.92 for 2, 4, and
20 h incubation, respectively) (Figure S17). From these results,
we infer no significant relocalization behavior up to 20 h and
thus conclude that Mito-CCy may be used to achieve mitochon-
dria targeting under our experimental conditions.
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Figure 8. Effect of NIR irradiation following Mito-CCy admin-
istration on the viability of HeLa cells. (a) Confocal fluorescence
images of propidium iodide-treated HeLa cells after incubation
with various concentration of Mito-CCy for 4 h, following by 730
nm irradiation (2.3 W/cm2) for 10 min. (b) The proportion of PI-
positive cells was determined from the ratio of PI-positive cells to
the total number of cells as determined by Hoechst 33342 staining.
(c) Confocal fluorescence images of HeLa cells obtained after in-
cubation with Mito-CCy (20 μM) for 4 h following 730 nm laser
irradiation (2.3 W/cm2) for 10 min. Dead cells are labeled in red by
PI staining, whereas all cells were visualized using Hoechst 33342
(green). A Ti:Sa femtosecond-pulsed laser (Chameleon XR by Co-
herent, 200 fs pulse width, 90 MHz repetition rate) was used. The
energy density (fluence) was 1380 J/cm2.
Figure 7. Confocal microscopic images of Mito-CCy co-localized
with various commercially available organelle trackers in HeLa
cells (a, e, and i). HeLa cells were incubated with Mito-CCy (2.0
μM) for 4 h and then with (b) MitoTracker Green FM (0.05 μM),
(f) ER Tracker Green (1.0 μM) or (j) Lysotracker Green DND-26
(0.05 μM) for 15 min. (c) Merged image of (a) and (b). (g) Merged
image of (e) and (f). (k) Merged image of (i) and (j). (d) Co-locali-
zation scatterplots of (c). PC: 0.95. (h) Co-localization scatterplots
of (g). PC: 0.64. (l) Co-localization scatterplots of (k). PC: 0.44.
The fluorescence images of the organelle trackers and Mito-CCy
were collected with 488 nm and 633 nm light being used as the
excitation and detection wavelengths, respectively, with 505−530
nm band pass (green) and 650 nm long pass (red) filters being em-
ployed as warranted.
Photo-induced ROS Formation by Mito-CCy and Its
Mechanism. To investigate the mechanism of the cytotoxicity
by photoirradiation of Mito-CCy in Figure 8, the mitochondrial
ROS levels were measured using MitoSOX, a mitochondrial
ROS sensor. Indeed, the fluorescent intensity of MitoSOX is
elevated upon 730 nm NIR irradiation (Figure 9b-c) whereas N-
acetylcysteine (NAC), a ROS scavenger,46 dramatically de-
creased the emission (Figure S21). These results are taken as an
evidence that ROS play a role in mediating the cytotoxicity pro-
duced by Mito-CCy under photothermal activation. On the
other hand, considering that Mito-CCy is an ineffective ROS
photosensitizer (see Figures S7, 10, 12 and 14) but an efficient
photothermal agent (see also Figures 6 and S16), we suggest
that the cytotoxic ROS production seen in the case of Mito-
CCy (cf. Figure 9b-c) is not the result of direct ROS produced
by photoirradiation; rather, it comes from the generated heat.
Photothermal Cytotoxicity of Mito-CCy in Cells. The
cytotoxicity of Mito-CCy was tested by incubating HeLa cells
with the presumed PTT sensitizer and subjecting to NIR irradi-
ation. Propidium iodide (PI), an indicator of dead cells, was
used to judge cell killing efficacy. As can be seen from an in-
spection of Figure 8a, the number of PI-positive cells observed
upon photoirradiation gradually increases as a function of Mito-
CCy concentration in the cell medium, whereas few PI-positive
red signals were seen in the case of the dark studies (Figure
S19). A quantitative analysis of the proportion of PI-positive
cells is shown in Figure 8b. Here, the dead cells give rise to
positive PI staining. The total number of cells was determined
by staining with Hoechst 33342, a dye that allows nuclei to be
visualized regardless of their viability status (cf. Figure S20).
The cytotoxic effect produced by Mito-CCy is localized within
One concern associated with relying on thermal effects to
produce ROS is that the associated heat might damage enzymes,
such as superoxide dismutase (SOD), that provide protection
against ROS. To the extent this occurs, it may complicate ef-
forts to ascertain the mechanistic determinants of the putative
cytotoxicity. To address this concern, ROS formation was
measured using MitoSOX in the presence of a potent SOD in-
hibitor, 2-methoxyestradiol.47 The results, shown in Figure S22,
reveal that upon irradiation in the absence of Mito-CCy, addi-
tion of the estradiol inhibitor served to increase slightly the
emission intensity of MitoSOX relative to control. However,
the addition of the inhibitor enhanced dramatically the ROS in-
duced by Mito-CCy under identical conditions of irradiation.
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MHz repetition rate) was used. The energy density (fluence) was
We interpret these results in terms of the estradiol SOD inhibi-
1380 J/cm2.
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tor contributing to mitochondrial ROS generation in a manner
that is independent from the action of Mito-CCy. We thus con-
sider it unlikely that Mito-CCy mediates its action by deactivat-
ing SOD or other protective enzymes present in the mitochon-
dria.
■ CONCLUSIONS
To the best of our knowledge, this work is the first report
wherein the mitochondria is targeted as the primary site of ac-
tion for a NIR irradiation-activated cryptocyanine dye. The new
PTT agent reported here, Mito-CCy, contains two functional
components, namely a triarylphosphonium mitochondrial local-
izing unit and a cryptocyanine photosensitizer. Based on fluo-
rescence microscopy, it was determined that Mito-CCy local-
izes preferentially in the mitochondria of HeLa cells. When sub-
ject to 730 nm photoirradiation, Mito-CCy produces an in-
crease in temperature in test systems. This increase is dependent
on both the laser power and probe concentration. It is much
more effective in terms of photothermal conversion than a con-
generic control system, CCy, that, in contrast to Mito-CCy, is
aggregated in aqueous media. When HeLa cells are incubated
with Mito-CCy and subject to photoirradiation, cytotoxicity is
observed. On the basis of ROS detection experiments and con-
trol studies involving inhibition of the mitochondrial respiratory
chain, it is concluded that the Mito-CCy-induced PTT cytotox-
icity is due to endogenous ROS generation, rather than direct
ROS production. The present study thus leads us to suggest that
systems such as Mito-CCy, which take advantage of thermally-
induced mitochondrial-based ROS production to mediate cell
killing, could represent promising PTT photosensitizers. We
believe this work provides an important starting point for creat-
ing cryptocyanine-based PTT agents. More broadly, we believe
the present studies provide important mechanistic insights that
could help drive further advances in the area of sensitizer-based
anticancer research and treatments.
We next sought to determine whether Mito-CCy-mediated
PTT perturbs the respiratory electron transfer chain. Toward
this end, the ROS levels were measured when Mito-CCy was
photoirradiated in the absence and presence of rotenone, anti-
mycin A or both. These agents are potent inhibitors of the mi-
tochondrial respiratory chain complexes I and III,48 respectively.
As shown in Figure 9c(iv), in the presence of both inhibitors,
the ROS levels in the cells decreased dramatically under condi-
tions of photoirradiation. In light of this result, we conclude that
PTT-mediated ROS generation in mitochondria requires an
electron transport chain that functions in whole or in part and
that the photo-action of Mito-CCy originates from its photo-
thermal effect.
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We also found that the fluorescence intensity of Mito-CCy
was not altered after being incubated for 30 min in the presence
of rotenone and antimycin A (Figure S24) and that the fluores-
cence signal ascribed to Mito-CCy was still localized in the mi-
tochondria after treatment with both inhibitors (Figure S25), as
well as in their absence. We thus conclude that Mito-CCy is
subject to negligible efflux under the conditions of these exper-
iments, in spite of the reduced membrane potential caused by
the inhibitors. This, in turn, leads us to rule out the possibility
that the variations in the ROS levels induced by the inhibitors
rotenone and antimycin A are caused by reductions in effective
photosensitizer concentration.
Many other photochemical therapeutic agents are reported to
involve ROS generation that could also originate in the mito-
chondrion.8,30 As such, the results in this study lead us to suggest
that it may be necessary to reconsider whether the ROS gener-
ating capabilities of other photosensitizers are direct (i.e., orig-
inate from their photoactivation and ROS production) or are in-
direct and involve perturbations of mitochondrial respiration
events stimulated through a photothermal effect.
■ ASSOCIATED CONTENT
Supporting Information. Detailed experimental conditions and
methods, synthesis, structural characterization, spectroscopic anal-
yses, functional characterization of cryptocyanines, and biological
evaluations. This material is available free of charge via the Internet
at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
* sessler@cm.utexas.edu
* kangch@khu.ac.kr
* jongskim@korea.ac.kr
ORCID
Hyo Sung Jung -- 0000-0003-2018-8226
Jae-Hong Lee -- 0000-0001-6590-0841
Kyutae Kim -- 0000-0001-8049-0588
Seyoung Koo -- 0000-0002-0620-5395
Peter Verwilst -- 0000-0002-4673-2050
Jonathan L. Sessler -- 0000-0002-9576-1325
Chulhun Kang -- 0000-0002-8695-3392
Jong Seung Kim -- 0000-0003-3477-1172
Figure 9. Use of a ROS indicator to evaluate the production of ROS
in mitochondria after incubation of HeLa cells with MitoSOX. (a)
Matrix showing experimental design. (b) Fluorescence intensity
per cell. *P < 0.05. (c) Confocal fluorescence microscopic images
of HeLa cells treated with MitoSOX (1.0 μM) according to the
treatment variables, including with or without co-incubation with
Mito-CCy (10 μM), 730 nm NIR irradiation (2.3 W/cm2) for 10
min, antimycin A (0.5 μM) and rotenone (0.5 μM). Fluorescence
images were recorded using 488 nm excitation and 550−650 nm
emission (red) wavelengths, respectively. A Ti:Sa femtosecond-
pulsed laser (Chameleon XR by Coherent, 200 fs pulse width, 90
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by CRI project (No. 2009-0081566, JSK)
and
the
Basic
Science
Research
Programs
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