A mitochondrial-targeting and NO-based anticancer nanosystem with enhanced photo-controllability and low dark-toxicity

Article abstract of DOI:10.1039/c5tb00522a

Compared to the generation of singlet oxygen in photodynamic therapy, photo-generation of nitric oxide (NO) would not be limited by the concentration of molecular oxygen. However, therapeutic applications of exogenous nitric oxide are usually limited by i

Full text of DOI:10.1039/c5tb00522a

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DOI: 10.1039/C5TB00522A
Journal Name
RSCPublishing
ARTICLE
A Mitochondria-targeted and NO-based Anticancer
Nanosystem with Enhanced Photo-controllability and
Low Dark-toxicity
Cite this: DOI: 10.1039/x0xx00000x
Jiangsheng Xu, Fang Zeng*, Hao Wu, Shuizhu Wu*
Received 00th January 2012,
Accepted 00th January 2012
Compared to the generation of singlet oxygen in photodynamic therapy, photoꢀgeneration of
nitric oxide (NO) would not be limited by the concentration of molecular oxygen. However,
the therapeutic applications of exogenous nitric oxide are usually limited by its short halfꢀlife
and its vulnerability to many biological substances, thus straightforward and precise control of
NO delivery may be critical to its therapeutic effects. Herein, we demonstrate a mitochondriaꢀ
targeted and photoactive NOꢀreleasing system as an anticancer drug. Fabricated by covalently
incorporating a photoꢀresponsive NOꢀdonor and a mitochondria targeting ligand onto carbon
dot, this nanosystem exhibits a multiꢀfunctional nature which combines mitochondriaꢀtargeting,
DOI: 10.1039/x0xx00000x
www.rsc.org/
photocontrollable NOꢀreleasing and cell imaging
. Upon cellular internalization, the
nanosystem could target mitochondria effectively. Furthermore, the system displays little
dark toxicity under physilogical temperature; but upon light irradiation, it could release NO,
efficiently damage the mitochondria and consequently cause prominent apoptosis of cancer
cells. Moreover, evaluated by using MTT assay, this nanosystem shows high cytotoxicity
towards two cancer cell lines. These observations provide new insights for exploiting NO in
disease therapy.
Schoenfisch et al. have fabricated a number of NOꢀreleasing
scaffolds featuring enhanced NO storage and some other
Introduction
1
3ꢀ15
advantageous properties;
Ford and coworkers used several
Nitric oxide (NO), one of the most appealing and studied
molecules in the fascinating realm of biomedical sciences,
plays important roles in various physiological as well as
metal complexes as the photochemical NO donor in cancer
treatment; and this photochemical strategy allows for good
1
6ꢀ18
1
ꢀ
control of the timing and dosage for the NO delivery.
On
pathological processes, such as tumor growth and suppression.
3
the other hand, Sortino’s group developed a variety of lightꢀ
controlled NO delivering molecular assemblies (such as
nanostructured films, polymers, nanoparticles and etc.) through
Endogenous NO mediates multiple processes in various
physiological systems including cardiovascular system,
immune system, and the central and peripheral nervous system.
1
9ꢀ24
different approaches,
and these assemblies exhibit
On the other hand,
a number of potential therapeutic
promising potential in NOꢀinvolved therapeutic applications.
However, severe limitations do exist for NOꢀinvolved
therapeutic applications. This ephemeral species has a very
short lifetime and exhibits high reactivity with many biological
substances, which makes NOꢀbased therapies highly site,
applications of exogenous NO delivery in cardiovascular
diseases, respiratory diseases, cancer treatment, antimicrobial
therapy, wound healing, and functional biomedical devices
4
ꢀ7
have so far been proposed and explored. In particular, human
solid tumors contain hypoxic regions that have considerably
2
5ꢀ29
6
,8
concentration and dosage dependent.
And the solutions,
lower oxygen tension than normal tissues.
This imparts
which enable us to precisely determine where, when, and to
what extent a process is started or stopped, are highly desirable.
Among the possible solutions able to harness the release of
therapeutics, the use of light is definitely one of the most
elegant ones. Light has an easy manipulation with an
exquisite spatiotemporal control in terms of intensity,
wavelength, duration and location. It can be generated and
manipulated with wellꢀestablished techniques and also harmless
if applied correctly.
resistance to radiotherapy and anticancer chemotherapy. For
instance, photodynamic therapy involves three main
components: a photosensitizer (PS), molecular oxygen and
9,10
light,
which means the lack of molecular oxygen in tumor
2
2
tissue or cells would reduce the efficacy of the photodynamic
treatment. Compared to singlet oxygen generation, photoꢀ
generation of NO involves a neat photochemical reaction
without the dependence on oxygen.
In response to the need for NO delivery, researchers have
1
1,12
On the other hand, the mitochondria are energy factories of
the cell, playing important roles in energy metabolism of
paid great attention to the development of NO donors.
For
example, to improve NO’s pharmacological potential,
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DOI: 10.1039/C5TB0052
Journal Na2Ame
various biochemical processes. They exert a variety of
To gain enhanced control over the NO release, we herein
important cellular functions including the production of the prepared a new mitochondriaꢀtargeting and photoactive NOꢀ
largest part of cellular ATP needed for endergonic releasing system as an anticancer drug, as shown in Scheme 1.
3
0,31
processes.
They are also the central regulators of cellular For this system, a triphenylphosphonium moiety which was
metabolism and the executioners of programmed cell death used as a mitochondriaꢀtargeting ligand and a photoꢀresponsive
2
5,32ꢀ35
(
apoptosis).
Thus, mitochondriaꢀtargeting ligands have NO donor 4ꢀnitroꢀ3ꢀ(trifluoromethyl) aniline derivative were
been used as effective vectors for some therapeutic drugs that conjugated onto the carbon dots. Multiꢀfunctionality (including
affect mitochondrion function, the apoptotic pathway, or other mitochondriaꢀtargeting, efficient lightꢀtriggered NOꢀreleasing
signal transduction factors localized in mitochondria. Some and cell imaging) was achieved for the nanosystem. In this
ligands such as phosphonium derivatives have been employed study, we evaluated the action of the nanosystem on two cancer
by Murphy, Chang and others to target mitochondria as these cell lines; and our results indicate that, the mitochondriaꢀ
lipophilic cations selectively accumulate in mitochondria due to targeted and NOꢀreleasing system can cause high toxic effect
3
6ꢀ39
proton gradient considerations.
towards the cancer cells by specifically damaging their
It also has been found that many of the regulatory tasks mitochondria upon light irradiation. Our study demonstrates
undertaken by NO in the cell are associated with that the combination of mitochondriaꢀtargeting and highly
4
0,41
mitochondria.
endogenous NO is synthesized by inducible NO synthase apoptosis action, thereby providing new insights for exploiting
iNOS) in mammalian cells and plays a critical role in NO in cancer therapy with the ultimate goal of maximizing the
Mitochondrion is the main area where controlled NOꢀreleasing can enhance the system’s proꢀ
(
regulating cell functions. Increasing amount of evidences therapeutic action while minimizing side effects.
suggests that small amounts of NO stimulate mitochondrial
biogenesis and boost the supply of oxygen and respiratory
substrates to mitochondria. In contrast, high level of NO
endogenously produced by inducible NO synthase (iNOS)
4
2ꢀ44
block mitochondrial respiration, and can be cytotoxic.
NO
is cytotoxic partly because mitochondrial NO can inactivate the
respiratory chain enzymes by binding to the heme group of
cytochrome c oxidase or by controlling mitochondrial pH, and
−
partly because NO can be further oxidized into ONOO , N O ,
2
3
−
−
NO , and NO by excess reactive oxygen species and induces
2
3
nitrosative damage to proteins, ultimately stimulates the
4
5
mitochondrial pathway of apoptosis. Despite of the fact that
the mitochondria maintain homeostasis in the cell by interacting
with NO and other reactive nitrogen species (RNS) and there is
a growing consensus of the vital role mitochondria play in
medicine (e.g. mitochondrion has recently emerged as a
4
6,47
promising therapeutic target in cancer treatment),
the
investigation on the interaction of NOꢀreleasing
pharmacological agents with mitochondria is too often
disregarded, and to date only a few reseaches involving both
the mitochondriaꢀtargeting and NOꢀreleasing have been
4
8ꢀ51
reported.
Nakagawa, Miyata and coworkers, for the first
time, demonstrated effective NOꢀreleasing systems for timeꢀ
controlled, mitochondriaꢀspecific NO treatment, which were
synthesized by linking a NO donor, a mitochondriaꢀtargeted
moiety and a fluorophore. And the intracellular localization of
these molecular NOꢀreleasing systems could be clearly
observed under confocal microscope due to their fluorescent
Scheme 1. Schematic illustration for the action of the Cdot-based multi-
functional system on the mitochondrion of a cancer cell.
Experimental
4
9,50
Materials.
nature.
On the other hand, our group has developed a
mitochondria targeting and NOꢀreleasing nanosystem and used
Citric acid, triphenylphosphine, ethylenediamine, 3ꢀ
bromopropionic acid, 3ꢀbromopropylamine hydrobromide, 5ꢀ
5
1
it as a proꢀapoptosis agent in cancer theraphy. However, we
could not precisely control the release of NO by using light,
since the nanosystem was also heatꢀresponsive and slowly
chloroꢀ2ꢀnitrobenzotrifluoride,
trifluoromethyl)aniline,
dibromobutane,
nanoparticles, have attracted growing interest due to their dihydrochloride (NEED) and sulfanilamide (SULF) were
4ꢀnitroꢀ3ꢀ
(
1,3ꢀpropanediamine 1,4ꢀ
released NO at 37
°C in the dark.
Nꢀ(1ꢀnaphythyl)ꢀethylenediamine
Recently, carbon dots (Cdots), as a new class of fluorescent
alluring optical and biological properties, such as water supplied
by
Aladdin
Industrial
Inc.
1ꢀEthylꢀ3ꢀ(3ꢀ
solubility, biocompatibility and excellent cell membrane
dimethylaminopropyl) carbodiimide hydrochloride (EDCꢀHCl),
sulfoꢀNꢀhydroxysuccinimide (sulfoꢀNHS) and rhodamine 123
permeability, tunable surface functionalities and good
5
2ꢀ57
photostability.
0 nm) makes them very suitable as the carriers for subcellular
organelles targeting; and their photoluminescence allows for
visualization of the intracellular trafficking of the drug delivery obtained from Invitrogen. Triethylamine (TEA), N,Nꢀ
More importantly, their small size (less than
(
RhB123) were purchased from SigmaꢀAldrich. The culture
1
medium RPMI1640 and the bovine serum albumin (BSA) were
system.
dimethylformamide (DMF) and dimethyl sulfoxide (DMSO)
were distilled before use. JCꢀ1 (5,5',6,6'ꢀtetrachloroꢀ1,1',3,3'ꢀ
2
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ART22ICLE
DOI: 10.1039/C5TB005 A
tetrethyl benzimidazolylcarbocyanine iodide), LysoTracker Red to the solution of NFꢀNH (130 mg, 0.5 mmol) in THF (10 mL)
2
and annexin VꢀFITC kit were purchased from the Beyotime and 35 ꢁL TEA. The mixture was stirred at room temperature
Institute of Biotechnology. All other chemicals used were of overnight, then the solvent was removed, and the resulting
analytical reagent grade. The water used in this study was the yellow solid was purified by silica gel flash chromatography
1
tripleꢀdistilled water which was further treated by ion exchange (MeOH:CH Cl =10:1) to produce the yellow solid MitoNF. H
2
2
columns and then by a MilliꢀQ water purification system.
NMR (400 MHz, CDCl , δ) 8.86 (t, 1H), 7.87 – 7.69 (m, 15H),
3
7
3
5
.14 (t, 1H), 6.98 (s, 1H), 6.77 (d, 1H), 3.60 – 3.51 (m, 2H),
Synthesis of (3-Carboxyethyl)triphenylphosphonium Bromide
+
.31 (d, 4H), 1.88 – 1.78 (m, 2H). ESIꢀMS m/z [MꢀBr]
79.38.
=
(TPP-COOH). A solution of 3ꢀbromopropionic acid (1.53 g,
1
0mmol) and triphenylphosphine (2.62 g, 10 mmol) in CH CN
3
(
25 mL) was stirred at reflux for 15 h. Then the solvent was Synthesis of Cdots with targeting ligand (MitoCdot). Cdots were
5
1
removed, and the resulting white solid was purified by silica gel prepared according to our previously reported procedure. Cdots
flash chromatography (MeOH:CH Cl =10:1) to produce the (200 mg) was treated with EDC HCl (345 mg, 1.8 mmol) and
white solid (2.6 g, 64% yield). H NMR (400 MHz, CDCl , δ) sulfoꢀNHS (345 mg, 1.59 mmol ) in 25.0 mL DMSO with
·
2
2
1
3
7
3
.86 – 7.75 (m, 15H), 3.64 (m, 2H), 3.76 (t, J = 7.2 Hz, 2H), stirring at room temperature for 1 h before being added to the
+
.08 (m, 2H). ESIꢀMS m/z [MꢀBr] = 334.14.
solution of (3ꢀaminopropyl)triphenylphosphonium bromide
TPPꢀNH2) (120 mg, 0.3 mmol) in 10.0 mL DMSO and 150 ꢁL
(
Synthesis of (3-aminopropyl)triphenylphosphonium Bromide
TPP-NH ). An aminoꢀcontaining triphenylphosphonium (TPPꢀ
of TEA. The mixture was stirred at room temperature for
another 48 h. The resultant solid was precipitated out by
addition of sufficient amount of diethyl ether/acetone (7:3) and
washed repeatedly with methanol and ethanol respectively.
Thereafter, the redꢀbrown solid was dissolved in purified water
and dialyzed against purified water using a dialysis tubing
(
2
NH2) was synthesized as described below: briefly, a solution of
ꢀbromopropylamine hydrobromide (2.19 g, 10 mmol) and
3
triphenylphosphine (2.62 g, 10 mmol) in CH CN (25 mL) was
stirred at reflux for 15 h. The resultant precipitate was collected
by filtration and then dissolved in H O and treated with
saturated aqueous K CO until the pH reached 11. The product
was extracted into 2 vol of CH Cl , dried (by MgSO ), filtered;
then the solvent was removed, and the resulting white solid was
purified by silica gel flash chromatography (MeOH: CH Cl
3
2
(
molecular weight cutꢀoff 100 Da) for 48 h. The solid was
2
3
collected by freezeꢀdrying.
2
2
4
Synthesis of mitochondrial-targeting and NO-releasing
nanosystem (MitoCdot-NF). MitoCdot (100 mg), EDC
·HCl
2
2
1
(192 mg, 1.0 mmol) and sulfoꢀNHS (212 mg, 1.0 mmol) were
dispersed in 10 ml DMSO, and placed into a 50 ml roundꢀ
=
10:1) to produce the white solid (1.2 g, 24% yield). H NMR
(
400 MHz, MeOD, δ) 7.96 – 7.75 (m, 15H), 3.64 (m, 2H), 3.26
+
bottomed flask under N
atmosphere at room temperature. After
2
–
3
3.17 (t, J = 7.2 Hz, 2H), 2.05 (m, 2H). ESIꢀMS m/z [MꢀBr] =
19.9.
1
h of stirring, Nꢀ(3ꢀaminopropyl)ꢀ4ꢀnitroꢀ3ꢀ(trifluoromethyl)
aniline NFꢀNH (130 mg, 0.5 mmol) and TEA (100 ꢁL) were
2
Synthesis
of
N-(3-aminopropyl)-4-nitro-3-(trifluoromethyl) added to the dispersion. The mixture was stirred at room
aniline (NF-NH2). 5ꢀchloroꢀ2ꢀnitrobenzotrifluoride (225 mg, temperature for 24 h in dark. The resultant solid was
.0mmol) and 1,3ꢀpropanediamine (158 mg, 2.0 mmol) were precipitated out by addition of sufficient amount of diethyl
1
refluxed in 25 mL MeCN for three days. The obtained solution was ether/acetone (7:3) and washed repeatedly with methanol and
concentrated under reduced pressure and purified by column ethanol respectively, and then dialyzed against purified water
chromatography (CH Cl :MeOH, 2:1) to give Nꢀ(3ꢀaminopropyl)ꢀ4ꢀ for 4 days in dark. The solid was collected by freezeꢀdrying to
2
2
1
nitroꢀ3ꢀ(trifluoromethyl)aniline (yield 60%) as a yellow solid.
H
give 78 mg of MitoCdotꢀNF.
NMR (600 MHz, CDCl , δ) 8.01 (t, 1H), 6.93 – 6.79 (m, 1H), 6.63
3
Synthesis of NO-releasing nanosystem without targeting ligand
(Cdot-NF). The control, CdotꢀNF, was prepared with a similar
procedure as that of MitoCdotꢀNF by using pristine Cdots
(
d, 1H), 6.51 – 6.34 (m, 1H), 3.37 – 3.25 (m, 2H), 2.93 (d, 2H), 1.85
+
–
1.77 (m, 2H). ESIꢀMS m/z [M+H] = 264.18.
Synthesis of N-(4-bromobutyl)-4-nitro-3-(trifluoromethyl)aniline instead of MitoCdot.
(Br-NF). 4ꢀNitroꢀ3ꢀ(trifluoromethyl)aniline (206 mg, 1.0 mmol)
1
Characterization. H NMR spectra were recorded on a Bruker
and 1,4ꢀdibromobutane (928mg, 2.0 mmol) were refluxed in 25
mL MeCN for three days. The resultant solution was
concentrated under reduced pressure and purified by column
chromatography (CH Cl : MeOH, 50:1) to give Nꢀ(4ꢀ
Avance 400 MHz NMR spectrometer. UVꢀvis spectra were
recorded on a Hitachi Uꢀ3010 UVꢀvis spectrophotometer.
Fluorescence spectra were recorded on a Hitachi Fꢀ4600
fluorescence spectrophotometer. Confocal Laser Scanning
Microscopy (CLSM) images were collected using a Leica TCSꢀ
SP5 confocal microscope. Atomicꢀforce microscopy (AFM)
was performed on a DI Veeko Multimode V atomic force
2
2
bromobutyl)ꢀ4ꢀnitroꢀ3ꢀ(trifluoromethyl)aniline (yield 70%) as a
1
yellow solid. H NMR (400 MHz, CDCl , δ) 8.09 (d, 1H), 6.84
3
(
s, 1H), 6.59 (d, 1H), 3.44 (t, 4H), 2.18 – 2.06 (m, 4H).
Synthesis of mitochondrial-targeting and NO-releasing microscope operated in the tapping mode. Highꢀresolution
Molecule (MitoNF). TPPꢀCOOH (167 mg, 0.5 mmol) was transmission electron microscopy (HRꢀTEM) was performed on
treated with EDC·HCl (115 mg, 0.6 mmol) in 10 mL CH Cl2 a JEMꢀ2100HR electron microscope. Dynamic light scattering
2
with stirring at room temperature for 30 min before being added measurement was carried out on a Malvern Nanosizer. Flow
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5TB0052
DOI: 10.1039Jo/Curnal Na2Ame
cytometry were performed on a Beckman Coulter Epics XL Determination of Change in Mitochondrial Membrane
flow cytometer.
Potential. Two different methods were employed to detect
mitochondrial membrane potential changes using the
fluorescent probe JCꢀ1.
For flow cytometry, cells were treated for 4 h with the
nanosystem (during which the cells were also subject to 15 or
Photo-triggered variation in absorption spectrum of MitoCdot-
NF. The variation in absorption spectrum for nanosystem upon
light irradiation at different time periods were determined using
UVꢀvis spectra method, MitoCdotꢀNF samples were incubated
at a simulated physiological condition (PBS, pH 7.4) at 37
3
0 min of light irradiation) in 6ꢀwell plates and were then
°C
washed three times with warm PBS. The cells were then
detached with trypsin –EDTA solution. Collected cells were
in dark or under a 12 W LED lamp (spectral peak position at
2
4
00 nm) with light intensity at 10 mW/cm . The absorbance
incubated for 30 min with 1
ꢀg/mL of JCꢀ1 in culture medium
spectra were recorded.
at 37 °C in the dark. Stained cells were collected in 37 °C
NO Release Assay: The production of NO by the nanosystem PBS/2% BSA and washed twice by centrifugation. Finally,
was determined using Griess assay. MitoCdotꢀNF samples were cells were resuspended in 0.3 mL of PBS with 2% BSA and
incubated at a simulated physiological condition (PBS, pH 7.4) used for data acquisition. A Beckman Coulter Epics XL
at 37 °C in dark or under light irradiation, and the release of NO equipped with a 488 nm argon laser was used in the study.
ꢀ
from nanosystem was monitored as nitrite (NO ) formation by After the flow cytometer was set for green (FLꢀ1) and red (FLꢀ
2
using the Griess reaction. Typically, 1mL solution of 2) fluorescence, the data were collected and analyzed with
nanosystem was immersed in a quartz cuvette containing Griess Beckman Coulter EXPO 32 software.
ꢀ
1
ꢀ1
ꢀ
solution (SULF 0.1 mol L , NEED 0.1 mol L , HCl 4.0 mol L
)
For imaging (mitochondria damage), cells were passaged and
. The absorbance at 550 nm, which provides a quantitative plated on polylysineꢀcoated cell culture glass slides inside 30ꢀ
measurement of NO released to the solution after its conversion mm glass culture dishes and allowed to grow to 50ꢀ70%
1
ꢀ
to NO , was kinetically monitored at 37 °C. The relationship of confluence. Fresh staining solution (1
absorbance and concentrations of nitrite was constructed by immediately before use by diluting a 100
ꢀ
×
g/mL) was prepared
stock solution of JCꢀ
2
drawing a standard curve from known concentrations of 1 in warm (37 °C) culture medium supplemented with 10%
NaNO2.
FBS. Cells treated with NOꢀreleasing nanosystems for 4 hours
during which the cells were also subject to 30 min of light
irradiation) were incubated with JCꢀ1 for 30 min in a 37 °C
(
Cell Culture. Two cancer cell lines, HeLa (human cervical
cancer cell) and HepG2 (human liver carcinoma cell) were
incubated in RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS) at 37 °C with 5% CO2. The cells were
regularly subcultured to maintain them in a logarithmic phase
CO incubator. Cells were then washed twice with warm PBS
2
and examined immediately under a Leica TCSꢀSP2 confocal
microscope. The JCꢀ1 was excited at 488 nm, and light
emissions were collected between 515 and 545 nm (green) and
at 570ꢀ600 nm (red). All image acquisitions and analyses were
performed using Leica LAS AF software. The intensity of the
laser beam and the photodetector sensitivity were kept constant
in order to compare the relative fluorescence intensities
between experiments.
of growth.
Co-localization Imaging. One day before imaging, cells were
passaged and plated on polylysineꢀcoated cell culture glass
slides inside 30ꢀmm glass culture dishes and allowed to grow to
5
0ꢀ70% confluence. Afterwards, cells (on glass slides) were
washed with RPMI1640, and reꢀincubated in RPMI1640
medium containing the Cꢀdotꢀbased sample (0.30 mg/mL) and
Cell Viability Assay. The viability of HeLa and HepG2 cells
treated by BrꢀNF, MitoꢀNF, MitoCdotꢀNF, MitoCdot or Cdotꢀ
NF subject to light irradiation were assessed by MTT assay.
The cells were cultured in RPMI 1640 medium supplemented
with 10% FBS. The cells were seeded in 96ꢀwell plates at the
cell population of ca. 5000 cells/well. After 24 h of incubation
in 96ꢀwell plates at 37 °C, cells were washed with prewarmed
PBS buffer, then the PBS was replaced with fresh medium
containing MitoCdot, MitoCdotꢀNF or CdotꢀNF (corresponding
a fluorescent dye (2
ꢀ
M rhodamine 123, 1 ꢀg/mL JCꢀ1 or 500
C under 5 % CO for a certain
ꢀ
M LysoTracker Red) at 37
°
2
period of time. Afterwards, the culture dishes were washed with
PBS, and the cells (on glass slides) were taken out and washed
with PBS for three times and then placed on a Leica TCS SP5
laser confocal scanning microscope for imaging (using a
1
00×1.4 NA objective). The laser excitation wavelength for the
samples was 405 nm and their fluorescent signals were
collected from 415 to 510 nm (blue channel). Rhodamine 123
was excited at 488 nm, and its emission was collected from 515
to 550 nm (green). Lyso Tracker Red was excited at 543 nm,
and its emission was collected from 580 to 630 nm (red). All
image acquisitions and analyses were performed using Leica
LAS AF software. The intensity of the laser beam and the
photodetector sensitivity were kept constant in order to
compare the relative fluorescence intensities among
experiments.
to 0 ~ 100 ꢀM of NF moiety) and the samples were incubated
for 4 h, and then the cells were exposed to 400 nm light (using
2
a 12 W LED lamp with light intensity at 10 mW/cm ) for 30
min. The samples were then incubated for another 19.5 h.
Thereafter, the wells were washed with PBS buffer and
incubated for another 4 h with RPMI 1640 medium containing
0
.5 mg/mL MTT. After discarding the culture medium, 150 ꢀL
of DMSO was added to dissolve the precipitates and the
absorbance was read with a Thermo MK3 ELISA reader at 570
nm. As for cell viability without light illumination (dark
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DOI: 10.1039/C5TB005 A
cytotoxicity), similar procedures were adopted only without the
light illumination. As for all the assays, three independent
experiments were performed for each concentration, and for
each independent experiment, the assays were performed in
eight replicates. And the statistical mean and standard deviation
were used to estimate the cell viability.
Statistical Analysis. All data are reported as mean ± SD from
three independent experiments, each performed in eight
replicates; differences between groups were assessed by oneꢀ
way ANOVA with Tukey’s post hoc test.
Figure 1. Structures of four NO-releasing systems (A). The HR-TEM (B), DLS (C)
and AFM (D) analysis results for Mito-Cdot-NF. The lower panel of (D) gives the
height profile along the line in the topographic image in the upper panel.
Results and discussion
Preparation of mitochondrial-targeting and NO-releasing
systems.
The spectral properties for some NOꢀreleasing systems are
given in Figure 2. It can be seen from Figure 2(A), with the NO
In this study, a 4ꢀnitroꢀ3ꢀ(trifluoromethyl) aniline derivative donor (4ꢀnitroꢀ3ꢀ(trifluoromethyl)aniline, NF) in their structure,
was synthesized as the nitric oxide donor; and
both the nanosystem MitoCdotꢀNF and the molecular MitoꢀNF
a
triphenylphosphonium (TPP) derivative was used as the ligand exhibit an absorption bond at 402 nm. On the other hand,
to target mitochondria. For comparison, we prepared two MitoCdotꢀNF displays a strong emission band at 460 nm when
molecular NOꢀreleasing systems and two Cdotꢀbased NOꢀ excited at 405 nm, and an extremely weak emission when
releasing systems (BrꢀNF, MitoꢀNF, CdotꢀNF and MitoCdotꢀ excited at 488 nm (Figure 2B). This is important for subsequent
NF, their structures are shown in Figure 1A and the synthesis cell imaging studies because the emission behavior of the Cdot
routes are shown in Scheme S1). Among them, BrꢀNF and allows us to trace the cell uptake of the nanosystem, but does
CdotꢀNF, which have no triphenylphosphonium in their not interfere with other imaging experiments.
structures, were used as control systems to verify the
Light induced NO release.
mitochondria targeting capability of MitoꢀNF and MitoCdotꢀNF.
The structures of the intermediates and final products were well
The lightꢀinduced spectrum changes for the NOꢀreleasing
confirmed and characterized (Figures S1ꢀ S11 in the systems are demonstrated in Figure 2C, 2D and in Figure S12.
Supplementary Information). The content of carboxyl groups One can see from the figures that, with the increasing
on asꢀprepared Cdot surface was determined as 318.87 ꢁM/g irradiation time, the absorbance for MitoCdotꢀNF or MitoꢀNF
carbon dots (or about 12 carboxyl groups per Cdot), as decreases, this is due to the photoꢀbleaching of NO donor (NF),
2
0,21
described in Figure S8 and S9. And the content for the targeting which is accompanied by the release of NO.
ligand (TPP) and the NO donor (NF) on MitoCdotꢀNF were NOꢀreleasing systems exhibit almost no decrease in absorbance
determined as 9.96 mg/g (39.8 M/g) and 39.12 mg/g (146.2 in the absence of light irradiation (Figure 2D, Figure S13). In
M/g), respectively, as described in Figure S7, S10 and S11. addition, the Griess assay was adopted to confirm the
In contrast, the
ꢀ
5
8
ꢀ
The high resolution transmission electronic microscope (HRꢀ generation of NO by the nanosystem upon photoꢀirradiation
TEM), dynamic light scattering (DLS) and atomic force (Figure S14ꢀS16). First, a standard curve (Figure S14) for the
microscope (AFM) analysis results for MitoCdotꢀNF are given assay was constructed using NaNO (the reaction product of
2
in Figure 1B, 1C and 1D. The average diameter for MitoCdotꢀ NO and Griess agent) of known concentrations. The absorption
NF determined from dynamic light scattering measurement is spectra for MitoCdotꢀNF treated by Griess agent over different
4
.5 nm (Figure 1 C), and the nanoparticles generally exhibit periods of time under irradiation in pH 7.4 PBS buffer at 37
°C
spherical shape (Figure 1B and 1D).
are given in Figure S15; and the concentrations of NO released
from the sample over time by Griess assay with or without
irradiation are presented in Figure S16. It is clear that without
the light irradiation, there is almost no nitric oxide released
from the nanosystem, while continuous light exposure results in
NO production, as evidenced by the enhancement of the
absorption at 540 nm. This indicates that the release of NO for
the systems at 37
°C is strictly dependent on the external light
inputs, and the systems do not exhibit heat response.
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Figure 3. Confocal microscopic images for HeLa cells respectively co-stained with
MitoCdot-NF/JC-1 (A – C), MitoCdot-NF/rhodamine 123 (E – G) or MitoCdot-
NF/LysoTracker Red (I –K). (D), (H) and (L): Intensity profile of ROIs across the
cells. For Figure 3B, signals only from red-channel were collected. Concentration
Figure 2. (A) Absorption spectra for three TPP-containing systems. Among them,
MitoCdot is
a
control sample which was prepared by linking
triphenylphosphonium onto carbon dot; (B) Emission spectra for MitoCdot-NF
excited at 405 nm or 488 nm, respectively; (C): Absorption spectra variation for
MitoCdot-NF over different time periods under 400 nm light irradiation, the
arrows indicate the spectral evolution with irradiation time from 0 to 45 min; (D):
Absorbance values at 402 nm for Mito-NF and MitoCdot-NF plotted against
of MitoCdot-NF: 300 ꢀg/mL; JC-1: 1 ꢀg/mL; Rh123: 2 ꢀM; LysoRed: 500 ꢀM.
On the other hand, the use of nanosystems to target
mitochondria is often limited by the fact that nanosystems is
taken up by the endocytic pathway, and in this pathway
endosomes/lysosomes serve as the barrier to mitochondrial
trafficking. Thus, to rule out the possibility that the current
nanosystems mainly distribute in endosomes/lysosomes upon
cell uptake, we performed a coꢀlocalization experiment using
the MitoCdotꢀNF and a lysosome marker LysoTracker Red, and
the results are also presented in Figure 3 (IꢀK). It is clear that
irradiation time in pH=7.4 buffers at 37
ꢀ
°
MitoCdot-NF is 133 g/mL, equivalent to 19.5
C. In (C) and (D), the concentration for
M of NF.
ꢀ
Mitochondria Targeting of the Nanosystem.
there is
a poor match between the intracellular blue
To verify the triphenylphosphonium (TPP)ꢀmodified fluorescence (from MitoCdotꢀNF) and the red fluorescence
nanosystem can target and specifically stain the mitochondria, (from the lysosome marker), suggesting the good lysosomal
two mitochondriaꢀspecific staining dyes, rhodamine 123 escape ability of the nanosystem.
(
Rh123) and JCꢀ1, were employed to coꢀstain HeLa cells with
MitoCdotꢀNF. Figure 3 (A, B, E and F) shows the confocal
fluorescence images for the cells coꢀstained by MitoCdotꢀNF
and rhodamine 123 (or JCꢀ1). The Cdots show blue
fluorescence; while Rh123 shows green fluorescence, and the Jꢀ
aggregates of JCꢀ1 dye exhibit red fluorescence in cells. The
Mitochondria damage induced by NO.
The exact molecular mechanism of cell apoptosis is not
completely clear, but it appears that a variety of key events are
5
9
focused on mitochondria;
and loss of mitochondrial
6
0
merged images (Figure 3C and 3G) indicate effective overlap membrane potential (ꢁΨ) is the one of these events. The
for the Rh123/MitoCdotꢀNF pair or JCꢀ1/MitoCdotꢀNF pair. damaging of mitochondria will lead to the depolarization of
6
1
These fluorescence images all indicate the selective mitochondria with a drop in the membrane potential. In this
accumulation of the nanosystem in mitochondria. In addition, study, a membraneꢀpermeable JCꢀ1 dye was used to monitor
as shown in Figures 3D and 3H, the intensity profiles of linear mitochondrial status upon NO treatment. The loss of
regions of interest (ROIs) also prove that the nanosystem mitochondrial membrane potential
(ꢁΨ, a hallmark of
exhibits greater overlap with Rh123 or JCꢀ1. These results cytochrome c translocation and the start of the apoptotic
suggest that, with the targeting ligand on its surface, MitoCdotꢀ process) is an important indicator to evaluate the dysfunction of
NF specifically stains the mitochondrial region in live cells.
mitochondria, since the damaging of mitochondria will lead to
the depolarization of mitochondria with a drop in the membrane
6
2
potential. The JCꢀ1 dye undergoes a reversible change in
fluorescence emission, cells with high mitochondrial membrane
potential promote the formation of dye aggregates, which
fluoresce red; cells with low potential will contain monomeric
JCꢀ1 and fluoresce green. Therefore, the red/green ratio can
help to identify the status of mitochondria and cells; and we
could distinguish live cells from damaged ones following the
NO treatment by recording the fluorescence change of JCꢀ1
using confocal microscopy and flow cytometry.
6
| J. Name., 2012, 00, 1-3
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ART22ICLE
DOI: 10.1039/C5TB005 A
Figure 5. Representative flow cytometric analyses of mitochondrial membrane
potential (using JC-1 as indicator) for HeLa cells incubated with Mito-NF (B, C) or
Mito-Cdot-NF (D, E) (equivalent to 50
39 g/mL of Cdot-NF) for 4 hours, and then exposed to light irradiation for 15
min (B, D) and 30 min (C, E) respectively. The shift of JC-1 fluorescence from red
FL2) to green (FL1) indicates a collapse of mitochondrial membrane potential.
Red lines separate populations with high (live cells) and low membrane potential
ꢀM of NF, or 342 ꢀg/mL of MitoCdot-NF or
3
ꢀ
(
(
apoptotic cells) according to the untreated HeLa cells (A).
Figure 4. Confocal microscopic images for HeLa cells stained with 1
ꢀ
g/mL JC-1.
Moreover, we investigated the change in population of cells
Cells were first cultured in 35 mm dishes and incubated respectively with of different fluorescence following the nitric oxide treatment
different NO donors (all equivalent to 50
ꢀ
2 W LED lamp with light intensity at 10 mW/cm ) for 30 min, and then stained
ꢀM of NF, or 342 ꢀg/mL of MitoCdot-
NF or 339 g/mL of Cdot-NF) for 4 h and then exposed to light (using a 400 nm
using flow cytometry with JCꢀ1 as the indicator. Analysis of the
cells by flow cytometry reveals a decrease in red fluorescence
2
1
(
FL2 channel) after the treatment with NO donors (NOꢀ
with 1 ꢀg/mL JC-1. The cells without being treated with any of the NO donors
were used as the control.
releasing systems) and light irradiation (Figure 5). As shown in
the figure, without being treated by the NOꢀreleasing system,
the cells with high ꢁΨ predominate in the cell population.
However, following the treatment and irradiation, significant
increases in the percentage of the cells with low ꢁΨ are
recorded with the increasing time of irradiation, further proving
the action of the NO on mitochondria damage. It can also be
found in the figure that, compared to molecular NO donor
Figure 4 shows the representative results of JCꢀ1 assays for
HeLa cells as a result of nitric oxide treatment. Confocal
microscopy reveals that in untreated HeLa cells, wellꢀpolarized
mitochondria are marked by both punctate red and green
fluorescent staining. However, treatment with different forms
of NO donors (BrꢀNF, MitoꢀNF, CdotꢀNF, and MitoCdotꢀNF)
respectively followed by light irradiation results in changes in
cell staining: the red fluorescence regions gradually decrease,
while green fluorescence intensity in the cytoplasm increases. It
is noteworthy that the increase of green fluorescence in the
cytoplasm is more pronounced when cells are treated with the
mitochondriaꢀtargeted systems (MitoꢀNF and MitoCdotꢀNF),
and a big portion of the red dotꢀlike fluorescent images turn
into the diffuse green colors (Figure 4). Observed changes in
JCꢀ1’s fluorescence suggest the decrease of dye aggregate
accumulation in mitochondria and the diffusion of JCꢀ1
molecules into cytosol following the increase in the
permeability of the mitochondrial membrane with concomitant
collapse of mitochondrial membrane potential. The results
strongly suggest that, the incorporation of a mitochondriaꢀ
targeting ligand onto the photoactive NOꢀreleasing system
could significantly enhance its mitochondria damaging
capability after light irradiation. On the other hand, the carbon
dotꢀbased NOꢀreleasing system with mitochondria targeting
ligand on its surface (MitoCdotꢀNF) can damage the
mitochondria as efficiently as the molecular mitochondriaꢀ
targeting system (MitoꢀNF).
(
MitoꢀNF) (Figure 5 B, C), the Cdotꢀbased system (MitoCdotꢀ
NF) induces similarly degree of change in fluorescence upon a
certain time of treatment (Figure 5 D, E), suggesting the
treatment with the either nanoꢀ or molecularꢀbased system can
efficiently cause the mitochondrial damage.
To evaluate the efficacy of our NOꢀreleasing systems in cell
death, we performed MTT assay for two cancer cell lines
(
HeLa, HepG2) upon treatment by the targeted (MitoꢀNF or
MitoCdotꢀNF) system or the nonꢀtargeted NOꢀreleasing system
CdotꢀNF), and the result is presented in Figure 6. No
significant dark toxicity is observed for cells treated with up to
00 M (concentration of NF, equivalent to 685 g/mL of
MitoCdotꢀNF or 678 g/mL of CdotꢀNF) of MitoꢀNF,
(
1
ꢀ
ꢀ
ꢀ
MitoCdotꢀNF or CdotꢀNF as determined by the MTT assay.
This is due to their inability to generate nitric oxide in the dark.
After 4 h incubation with the NOꢀreleasing systems followed
by 30 min light irradiation, both cell lines treated by
mitochondriaꢀtargeting systems (MitoCdotꢀNF, MitoꢀNF) cause
much higher phototoxicity than the system with no
mitochondriaꢀtargeting ability (CdotꢀNF). To rule out the
possibility that the reduced viabilities are solely induced by
light irradiation or by the enhanced light absorption by the Cdot
and/or TPP moieties, we irradiated HeLa cells in the absence of
the NO donor, and the results suggest that irradiation alone or
the existence of Cdot and/or TPP could not reduce the viability
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Journal Na2Ame
of the cells (Figure S17 and S18). These results clearly indicate mitochondria by releasing NO upon light irradiation, affording
that the preferential localization in the target organelle a higher cytotoxicity towards cancer cells. We suppose,
(
mitochondrion) accounts for the enhanced cytotoxicity of the compared to the previously reported small molecular NOꢀ
4
9,50
targeted NOꢀreleasing nanosystem.
releasing system,
this nanosized system may exhibit some
advantageous properties such as ease of fabrication (due to the
existence of many reaction cites on carbon dot’s surface),
higher water solubility (no need for using DMSO in cell
experiments) and etc.; but it also has inherent drawbacks such
as relatively illꢀdefined structure. This strategy may provide
useful insights for rational design of NOꢀrelated therapies.
Acknowledgements
We gratefully acknowledge the financial support by NSFC
(
21474031, 21174040 and 21025415), and the National Key
Basic Research Program of China (Project No. 2013CB834702).
Notes and references
College of Materials Science and Engineering, State Key Laboratory of
Luminescent Materials and Devices, South China University of
Technology, 510640, China
Eꢀmail: mcfzeng@scut.edu.cn, shzhwu@scut.edu.cn
†
Electronic Supplementary Information (ESI) available. See
DOI: 10.1039/b000000x/
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DOI: 10.1039/C5TB00522A
A spatiotemporally controllable NO-releasing nanosystem for killing cancer cells with
high efficiency based on carbon dots has been developed, which exhibits
mitochondria targeting, light-responsive NO-releasing and cell imaging capability.