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Nano Res
1
Elevating Mitochondrial Reactive Oxygen Species by
Mitochondria-Targeted Inhibition of Superoxide
Dismutase with a Mesoporous Silica Nanocarrier for
Cancer Therapy
Yi Zhang1,2, Zhengyan Hu1,2, Guiju Xu1,2, Chuanzhou Gao3, Ren’an Wu1(),and Hanfa Zou1()
Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-014-0473-4
http://www.thenanoresearch.com on April 10, 2014
© Tsinghua University Press 2014
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Nano Research
DOI 10.1007/s12274-014-0473-4
1
TABLE OF CONTENTS (TOC)
Elevating Mitochondrial Reactive Oxygen Species by
Mitochondria-Targeted Inhibition of Superoxide
Dismutase with a Mesoporous Silica Nanocarrier for
Cancer Therapy
Yi Zhang1,2, Zhengyan Hu1,2, Guiju Xu1,2, Chuanzhou
Gao3, Ren’an Wu1* and Hanfa Zou1 *
1 Key Lab of Separation Sciences for Analytical
Chemistry, National Chromatographic R&A Center,
Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, China.
2 University of Chinese Academy of Sciences, Beijing
100049, China
3 Institute of Cancer Stem Cell, Dalian Medical University,
Dalian 116044, China
Page Numbers. The font is
ArialMT 16 (automatically
inserted by the publisher)
Since mitochondrial superoxide dismutase (SOD2) is vital in
maintaining the intracellular ROS levels, a general strategy in
killing cancer cells is reported by targeted inhibiting SOD2
using 2-methoxyestradiol (2-ME, an inhibitor for SOD family)
via an elaborately designed mitochondria-targeted mesoporous
silica nanocarrier (mtMSN). The elevation of mitochondrial
oxidative stress is demonstrated to be powerful in cancer
therapy.
Hanfa Zou, http://www.bioanalysis.dicp.ac.cn/
2
Elevating Mitochondrial Reactive Oxygen Species by Mitochondria-Targeted Inhibition of Superoxide Dismutase with a Mesoporous Silica Nanocarrier for Cancer Therapy
Yi Zhang
1,2, Zhengyan Hu
1,2, Guiju Xu
1,2, Chuanzhou Gao
3, Ren’an Wu
1(),and Hanfa Zou
1()
1 Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian 116023, China. 2 University of Chinese Academy of Sciences, Beijing 100049, China. 3 Institute of Cancer Stem Cell, Dalian Medical University, Dalian 116044, China.
Received: day month year / Revised: day month year / Accepted: day month year (automatically inserted by the publisher)
© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2011
ABSTRACT In the intrinsic pathway of apoptosis, stresses of mitochondrial reactive oxygen species (mitoROS) might be
sensed as more effective signals than those in cytosol, as mitochondria are the major sources of ROS and pivotal
components during cell apoptosis. Mitochondrial superoxide dismutase (SOD2) takes the leading role in
eliminating mitoROS, and inhibition of SOD2 might induce severe disturbances overwhelming the
mitochondrial oxidative equilibrium, which would elevate the intracellular oxidative stresses and drive cells to
death. Herein, we report a general strategy to kill cancer cells by targeted inhibiting SOD2 using
2-methoxyestradiol (2-ME, an inhibitor for SOD family) via a robust mitochondria-targeted mesoporous silica
nanocarrier (mtMSN), with the expected elevation of mitoROS and activation of apoptosis in HeLa cells. It was
the first report that Fe3O4@MSN was applied in the mitochondria-targeted drug delivery and selective
inhibition of mitochondrial enzymes, which was stable and had good biocompatibility and high loading
capacity. Due to the selective inhibition of SOD2 by 2-ME/mtMSN, enhanced elevation of mitoROS (132% of
that with free 2-ME) was obtained, coupled with higher efficiency in initiating cell apoptosis (395% of that with
free 2-ME in 4 hours). Finally, the 2-ME/mtMSN exhibited powerful efficacy in targeted killing HeLa cells by
both biological recognization and magnetic guiding, which caused 97.0% cell death with only 2 μg/mL
2-ME/mtMSN, hinting great potentials in cancer therapy through manipulation of the delicate mitochondrial
oxidative balance.
KEYWORDS Mitochondria, reactive oxygen species, apoptosis, mesoporous silica nanoparticles, drug delivery
1 Introduction Reactive oxygen species (ROS) are responsible for
most of intracellular oxidative stresses, which serve
in signaling pathways including immunity,
differentiation, aging and apoptosis etc., as natural
Nano Res DOI (automatically inserted by the publisher)
Research Article
————————————
Address correspondence to Ren’an Wu, [email protected]; Hanfa Zou, [email protected]
3
byproducts of oxygen metabolism [1]. Under
physical conditions, intracellular ROS are strictly
maintained at low levels and are usually harmless
for cells [2, 3]. While under intense stimuli, such as
heating or UV irradiation, intracellular ROS would
rise intensely and induce risks for cells due to
causing the irreversible damages of phospholipids,
proteins and DNA etc. or the direct transmission of
apoptosis signals [4, 5]. The active or inactive
elevation of intracellular ROS levels in cancer cells
has been demonstrated during the treatments using
chemotherapeutic drugs such as alkaloids and
doxorubicin, followed by the initiation of the cell
apoptosis and death of cancer cells [6-8]. However,
slight enhancements of ROS levels would be adapted
by the cellular oxidative clearance system to
maintain the normal cellular homeostasis, and in
some cases, moderate stimuli such as starvation
caused autophagy would introduce mild increases of
ROS, which might induce positive feedbacks to the
stressors and promote cell viability [1, 9, 10].
Mitochondria are major sources of ROS via the
respiration chain, with the levels of mitochondrial
ROS (mitoROS) tightly suppressed by effective
clearance [11]. Disturbances of the balance in the
production and elimination of mitoROS, such as the
slight changes in the mitochondrial membrane
potential, might induce highly intense enhancement
and accumulation of mitoROS against the
self-adaption [12]. The efforts associating with the
elevated oxidant states in mitochondria would
provide the maximum therapeutic benefits for the
pharmacological manipulation of mitochondrial
functions as well as the destinies of cancer cells. Up
to date, oxidative stress inducers, such as cisplatin,
As2O3 and elesclomol, have been applied to enhance
the production of mitoROS, which implies novel and
general strategies in anti-cancer treatments [13-15].
Meanwhile, the applications of antioxidants during
cancer therapy have been considered to be
antagonized in killing cancer cells, for which would
lower intracellular oxidative stresses and promote
cell survival [8], hinting that weakening the ROS
clearance in mitochondria would be in favor of
terminating lives of cancer cells [13, 16].
Mitochondrial superoxide dismutase (SOD2) takes
the essential role in the clearance of mitoROS to keep
cells from the oxidative risks [17]. It has been
reported that the mitoROS would induce more
powerful treatments for SOD2-deficient cells than
wild-type cells by the oxidation of c-Jun N-terminal
kinase (JNK) phosphatase catalytic cysteines and
subsequent cell death [18]. Thus, the selective
inhibition of SOD2 might be an efficient strategy in
enhancing the levels of mitoROS and activating
apoptosis, which could be fulfilled by the targeted
delivery of inhibitors for SOD families to
mitochondria.
Generally, mitochondria could be recognized and
targeted by the highly negative mitochondrial
membrane potential or the receptors of
mitochondrial locating signals (MLS, such as
peptides, nucleic acid sequences, etc.) [19-22].
However, it would possibly change the conformation
and reduce the treating efficacy of drugs, if the drug
molecules are functionalized with the locating
signals to target cancer cells or subcellular organelles
[23]. Nanocarriers are practicable for the
mitochondria-targeted delivery of SOD inhibitors,
for which could manage the subcellular targeting
and tumor internalization along with the
maintenance of the structures of drug molecules [21,
24, 25]. Nevertheless, the nanocarriers towards
mitochondria are mostly based on liposomes and
metallic oxide [20, 23], which might suffer either less
stability or poor loading capacity for intracellular
delivery, respectively. Thus, the construction of a
nanosystem with good biocompatibility, high specific
area and good mechanical strength would be favored
in the mitochondria-targeted drug delivery, which
has seldom been reported. Mesoporous silica
nanoparticles (MSNs) are easy to be synthesized and
modified, highly biocompatible, have large specific
surface area, and therefore have been widely used in
intracellular delivery [26]. The functionalized MSNs
have recently been utilized in the delivery of
doxorubicin to cell nuclei, which exhibit the
feasibility of subcellular targeting with MSN based
nanomaterials [27].
Meanwhile, the targeted treatments for cancer cells
also draw great concerns, where severe damages for
healthy cells might happen during the
non-discriminatory elevation of mitoROS levels.
Selective cancer therapy could be achieved by
nanocarriers through passive targeting (such as
enhanced permeability and retention effect, EPR
4
effect) [28], active targeting (such as the involvement
of ligands or antibodies for recognization of the over
expressed receptors on cancer cells) [29] and
magnetic targeting (using external magnetic field to
guide the nanocarriers towards tumor tissues) [30].
The Fe3O4 nanoparticles had been involved in the
composite materials, such as Fe3O4@MWCNT, to
provide the magnetic guiding ability, which
exhibited enhanced efficacy [31]. In this way, the
Fe3O4@MSN is of great promise in the
mitochondria-targeted delivery of 2-ME for targeted
and highly efficient cancer therapy.
In this work, a mitochondria-targeted mesoporous
silica nanocarrier (mtMSN) was constructed to
elevate the mitoROS levels via the targeted delivery
of inhibitors of SODs into mitochondria of HeLa cells
(represent cancer cells). 2-Methoxyestradiol (2-ME), a
metabolite of estradiol, is capable of inhibiting both
SOD1 (mostly localized in cytosol) and SOD2
(localized in mitochondria) [16]. The selective
delivery of 2-ME to mitochondria would be favorable
in the targeted inhibition of SOD2, and the artificial
elevation of mitoROS might present efficient
treatments in cancer chemotherapies, which has
never been utilized in the mitochondria-targeted
treatments. Also, it should be noticed that healthy
cells would be set into serious risks by using 2-ME,
since which could hardly present discriminatory
treatments for cancer cells. The mtMSN was
developed by the decoration of monodisperse
magnetic core/shell mesoporous silica nanoparticles
(Fe3O4@MSN) with MLS peptides and folic acids (FA)
(Figure 1). To minimize the injuries for healthy cells
during the treatments with 2-ME/mtMSN, the FA
ligands and the paramagnetic Fe3O4 core in the
mtMSN were assigned for active recognization of
HeLa cells, where the FA receptors were highly
overexpressed [32], and magnetic directing,
respectively. After internalization by HeLa cells, the
MLS peptides would direct the 2-ME/mtMSN to
mitochondria, which made 2-ME dominantly
released in the mitochondria for the inhibition of
SOD2; later, the accumulation of mitoROS would be
fulfilled as the selective inhibition of SOD2, and
subsequent oxidative damages and apoptotic signals
would drive the treated HeLa cells to death (Figure
1). Consist with our hypothesis, the mtMSN
successfully targeted at mitochondria, which offered
the selective inhibition of SOD2 by the relatively
more confined mitochondrial distribution of 2-ME.
The killing of HeLa cells owing to subsequent
apoptosis induced by the 2-ME/mtMSN was proven
to be highly effective. Furthermore, both FA based
active targeting and magnetic guiding exhibited
feasibility in diminishing side-effects for healthy cells
during treatments, implying great potentials in the
future in vivo applications. To the best of our
knowledge, it was the first time that Fe3O4@MSN was
utilized in mitochondria-targeted delivery and
regulation of mitochondrial enzyme activies. As a
proof of concept, the manipulation of endogenous
mitoROS exhibited as a general, powerful and
efficient strategy in the cancer therapy, which would
broad sights in clinical therapy by the regulation of
physical processes in subcellular components using
nanomaterials to kill or repair the cancer cells or cells
with defects.
Figure 1. Schematic illustration of the construction of mtMSN (upper)
and elevating mitoROS by targeted inhibition of SOD2 via loaded
2-ME in the mtMSN (lower).
2 Experimental Section
2.1 The construction and decoration of Fe3O4@MSN
At first, the Fe3O4 nanoparticles were synthesized
through a solvothermal method by heating the
solution of 1.5 mmol Fe(acac)3 in a mixed solvent
containing 12 mL octylamine and 36 mL octanol at
240 ˚C for 120 minutes [33]. The Fe3O4 nanoparticles
were later dispersed in chloroform (5 mg/mL) with
5
the assistance of ultrasonic treatment (scientz
biotechnology, China) at 400 W for 20 minutes. Then,
the Fe3O4@MSN was synthesis by the hydrolysis of
tetraethyl orthosilicate (TEOS) outside the Fe3O4
nanoparticles [34]. 6 mL Fe3O4 dispersion (5 mg/mL)
was added into the cetyltrimethyl ammonium
bromide (CTAB) solution (0.135 M, 60 mL) under
intense stirring, and the mixture was heated to 60 ˚C
for 30 minutes to evaporate the chloroform
completely. Then, 240 mL H2O and 1.8 mL NaOH (2
M) were added in the solution, followed by the
dropwise addition of TEOS (1 mL) and the ethyl
acetate (9 mL) 10 minutes later, and the mixtures
were kept at 70 ˚C for 3 hours with stirring. To
remove CTAB, the products were dispersed in 30 mL
ethanol solution containing ammonium nitrate (2
mg/mL) at 70 ˚C for 3 hours. The Fe3O4@MSN was
washed with ethanol and water for three times, dried
at 60 ˚C overnight and kept under dry condition.
For further functionalization, amino-groups were
first involved on the Fe3O4@MSN. 200 mg
Fe3O4@MSN was dispersed in 50 mL HCl (0.1 M) and
stirred at 50 ˚C for 3 hours to increase the activated
Si-OH. Then, Fe3O4@MSN was collected by
centrifugation at 20 000×g for 10 minutes and washed
with 30 mL isopropyl alcohol for 5 times to totally
remove the HCl solution. The Fe3O4@MSN was
subcequently dispersed in 50 mL isopropyl alcohol,
followed by the addition of 3 mL
(3-Aminopropyl)triethoxysilane (APTES). The
mixtures were stirred at room temperature for 24
hours under nitrogen atmosphere. The
as-synthesized Fe3O4@MSN-NH2 was collected by
centrifugation at 20 000×g for 30 minutes, washed
with ethanol for 3 times, and dried at 60 ˚C
overnight.
Both FA and MLS peptides went through similar
processes to link with the amino groups on the
Fe3O4@MSN-NH2. 2 μmol MLS (or FA) was first
dissolved in 1 mL 2-morpholinoethanesulfonic acid
(MES) buffer (0.1 M MES, 0.5M NaCl, pH=6.0), and
20 μmol
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) and 50 μmol sulfo-NHS were
added in the solution, which was mixed at room
temperature for 15 minutes. Then, 100 mg
Fe3O4@MSN-NH2 was dispersed in 10 mL PBS buffer
(pH=7.5) and mixed with the activated MLS (or FA)
at room temperature for 2 hours. Finally, the
products were collected by centrifugation at 20 000×g
for 30 minutes, washed with PBS buffer for 3 times,
and then stored at -20 ˚C before further applications.
2.2 Loading of 2-ME in the mtMSN
2-ME (50 μg/mL) was dispersed in the PBS buffer
(40 mL) containing 10% DMSO (v/v) as co-solvent
and was then loaded in the mtMSN (40 μg/mL) by
incubation at 37 ˚C for 2 hours. The 2-ME/mtMSN
was collected by centrifugation at 20 000×g for 10
minutes, washed with PBS for 3 times, and then
stored at -20 ˚C before further applications.
2.3 Cell culture and staining
HeLa cells were cultured with RPMI 1640 culture
medium containing 10% bovine serum (BS) and
penicillin/streptomycin (1×). The HEK 293 cells were
cultured with DMEM culture medium containing
15% fetal bovine serum (FBS) and
penicillin/streptomycin (1×). All the cells grew under
5% CO2 atmosphere at 37 ˚C, and they were split as
reaching a 90 % confluency.
For mitochondria staining, cells were incubated
with the MitoTracker Red CMXROS (100 nM,
Invitrogen, Carlsbad, CA) at 37 ˚C for 15 minutes,
and the redundant dyes were cleaned with PBS
buffer. The cells were fixed with PBS buffer
containing 4% formaldehyde at 37 ˚C for 15 minutes
before observation.
2.4 Confocal laser scanning microscopy imaging
The confocal laser scanning microscopy (CLSM)
imaging was performed by a FluoView™ FV1000
confocal laser scanning microscope (Olympus, Japan)
with an 100×objective. The cells were pre-planted in
the glass-bottom dishes (NEST, China) with the
concentration around 100 000 cells/mL, and then
florescent labeled materials or 2-ME were added and
incubated with cells as required. For FITC labeled
materials, the excitation wavelength was set at 488
nm, and the emission wavelength was set at 500-550
nm; for DNS-2-ME, the excitation wavelength was
set at 405 nm, and the emission wavelength was set
at 415-450 nm; and for stained mitochondria, the
excitation wavelength was set at 543 nm, and the
emission wavelength was set at 580-680 nm.
2.5 Subcellular location of mtMSN by TEM imaging
The HeLa cells (ca. 1 000 000 cells) were treated
with FA-Fe3O4@MSN and mtMSN (20 μg/mL) for 12
hours and washed with PBS buffer for 3 times, which
6
were later fixed in PBS buffer containing 2.5%
glutaraldehyde. The cell pellets were further fixed
with PBS buffer containing 1% OsO4, washed with
ethanol for dehydration, treated with propylene
oxide and embedded in Epon. For TEM observation,
slices (ca. 80 nm) of cells were cut, and stained by
uranyl acetate and lead citrate. TEM imaging was
carried out using a JEM-2000 EX (JEOL, Japan)
electronic microscope with an accelerating voltage at
120 keV.
2.6 Evaluation of mitochondrial ROS after treated with
2-ME, mtMSN and 2-ME/mtMSN
The mitochondrial ROS (mitoROS) were reported
by the MitoSOXTM Red Mitochondrial Superoxide
Indicator (Invitrogen, Carlsbad, CA) for the selective
detection of mitochondrial superoxides, which just
followed the protocol. Briefly, after treated with the
free 2-ME, mtMSN or 2-ME/mtMSN (20 μg/mL) for 4
hours, HeLa cells were incubated with fresh culture
medium (without BS) containing 5 μM MitoSOX™ at
37 ˚C for 10 minutes. Then the cells were washed
with PBS buffer for 3 times and collected. The
measurement of intracellular ROS levels was fulfilled
by a FACS Vantage SE flow cytometer from BD
(Franklin Lakes, NJ), where the excitation
wavelength was set at 510 nm, and the emission
wavelength was set at 580 nm. The data were
technically repeated for 3 times.
2.7 Cell lysis and western blotting assay
The HeLa cells (ca. 10 000 000 cells) were treated
with 2-ME, mtMSN and 2-ME/mtMSN (20 μg/mL)
for 12 hours, and then the cells were cracked with the
Cell Lysis Buffer for Western and IP (Beyotime
Institute of Bitechnology, China) according to the
protocol. Proteins in the cell lysates were quantified
with the bicinchoninic acid (BCA) protein assay kit
(Beyotime Institute of Biotechnology, China).
The proteins (ca. 45 μg) from cell lysates were first
separated by SDS-PAGE, and the caspase-8,
caspase-9 and β-actin were transferred from the gel
to nitrocellulose (NC) membranes. The NC
membranes containing the proteins were treated
with the block buffer (5% milk powders in TBST
buffer; TBST buffer: TBS buffer, 0.05% Tween20; TBS
buffer: 0.15 M NaCl, 0.01 M Tris-HCl, pH 7.5), and
then were incubated with the primary antibodies
(against caspase-8, 50 μM; caspase-9, 10 μM; β-actin,
5 μM) at 4 ˚C overnight. The NC membranes were
washed with TBS buffer for 3 times, and treated with
secondary antibodies for 2 hours at 25 ˚C, which
followed by 3-times washing with TBS buffer. Finally,
the proteins were visualized with the ECL Western
Blotting Substrate (Pierce).
2.8 Evaluation of the apoptosis of HeLa cells after treated
with 2-ME, mtMSN and 2-ME/mtMSN
At first, HeLa cells (ca. 500 000 cells per test) were
treated with 2-ME, mtMSN and 2-ME/mtMSN (20
μg/mL) for 4, 8 and 12 hours, which were washed
with PBS buffer for 3 times and collected. The
apoptotic HeLa cells were stained using Annexin V:
FITC Apoptosis Detection Kit (BD Biosciences, Inc.,
San Diego, CA, USA) according to the protocol. 5 μL
of both FITC Annexin V and Propidium Iodide were
added to incubate with HeLa cells at 25 ˚C for 15
minutes. The evaluation of apoptosis was fulfilled by
a FACS Vantage SE flow cytometer from BD
(Franklin Lakes, NJ), with the excitation wavelength
was set at 488 nm and the emission wavelength was
set at 525 nm. The data were technically repeated for
3 times, and the ratios of apoptotic cells were
obtained by the sum of both early and late stages of
apoptotic cells.
2.9 Cell viability assay
The viability of HeLa cells and HEK 293 cells under
treatments was evaluated using the Cell Counting
Kit-8 (CCK-8, DOJINDO, Japan) assay. According to
the protocol, 5 000 cells were pre-planted in the
96-well plates and incubated with the nanocarriers or
2-ME as required. In the meantime, different
amounts of cells (1 000, 2 000, 3 000, 4 000, 5 000 cells)
were also pre-grown in the plates to serve as the
standard value during incubation. After the cells
were treated, the materials or the 2-ME were cleared
with PBS buffer, and the CCK-8 solution, which was
eluted with the culture medium, was added and
applied to incubate with cells for 2 hours. The value
of absorbance was measured using a microplate
reader (BioRek, USA) at 450 nm. All the data were
repeated for 6 times technically.
3 Results and discussion
3.1 Construction and characterization of the mtMSN
For mitochondria targeting, the nanocarriers should
be controlled under ca. 50 nm for effective
intracellular moving and locating [20, 22]. And also,
high loading capacity of the nanosystem need to be
7
satisfied to carry drugs efficiently. Mesoporous silica
nanoparticles (MSN) are easy to be synthesized and
modified, highly biocompatible, have large specific
surface area, and therefore have been widely used in
drug delivery [35], and was applied in the
construction of mtMSN in this work.
The mtMSN was consisted of a Fe3O4 core for
magnetic targeting, a mesoporous silica shell for
2-ME loading/release, MLS peptides for
mitochondria internalization [19] and FA for HeLa
cell recognization (since FA receptors were
overexpressed in HeLa cells [32]). Fe3O4
nanoparticles (6-8 nm) were prepared with Fe(acac)3
in mixed solvents of octanol and octylamine using
solvothermal method [33] (Figure 2a) with the
saturation magnetization value of ca. 60.2 emu∙g-1
(Figure S1a in the ESM). The coating of mesoporous
silica shell on Fe3O4 nanoparticles was accomplished
via the hydrolysis of TEOS to yield the Fe3O4@MSN
(with the diameter of 40-60 nm) (Figure 2b) [34],
with the saturation magnetization value of 6.10
emu∙g-1 (Figure S1b in the ESM), specific area of 735.4
m2/g and pore diameter of 3.0 nm (BET, nitrogen
adsorption, Figure S2 in the ESM). After the
decoration with (3-aminopropyl) triethoxysilane
(APTES), MLS peptides and FA for the demands of
targeting mitochondria and recognizing HeLa cells
were coupled on the surface of Fe3O4@MSN using the
EDC/NHS method [31].
Figure 2. TEM images of a) Fe3O4 nanoparticles and b) Fe3O4@MSN;
and c) viability of HeLa cells under the incubation of 24, 48 and 72
hours with the mtMSN (20, 40, 60, 80, 100 μg/mL).
3.2 Evaluation of the loading capacity and
biocompatibility of mtMSN
The mesoporous silica shell of mtMSN with large
specific area was assigned to load 2-ME with high
capacity. The loading of 2-ME in mtMSN was
evaluated with UV-vis spectrometry at 286 nm
(Figure S3 in the ESM), and the maximum adsorption
of 2-ME could be reached within 60 minutes, with an
excellent capacity of 936.7±6.0 mg/g in the mtMSN.
Meanwhile, the mtMSN should be of good
compatibility for cells, which was analyzed by
measuring the viability of HeLa cells via the Cell
Counting Kit-8 (CCK8) assay after incubation with
the mtMSN (20, 40, 60, 80, 100 μg/mL) (Figure 2c).
The viability after treating with as-synthesized
mtMSN was maintained above 80% for HeLa cells
during 72h incubation, exhibiting acceptable
compatibility of mtMSN in further applications.
Figure 3. LSCM images of the intracellular location of fluorescent
labeled a-c) FA-Fe3O4@MSN, d-f) mtMSN and g-i) 2-ME (20 μg/mL)
by incubation with HeLa cells for 12 hours. The mitochondria were
dyed with Mitotracker (Invitrogen, USA).
3.3 Mitochondria targeting of mtMSN with the
decoration of MLS peptides
To evaluate the mitochondria targeting of loaded
2-ME in mtMSN (2-ME/mtMSN), the intracellular
distribution of mtMSN was profiled. The FITC
labeled fluorescent FA-Fe3O4@MSN and mtMSN (20
μg/mL) were used to track intracellular distribution
of the nanocarriers in HeLa cells by the 12-hour
incubation, which was observed with the laser
scanning confocal microscopy (LSCM). The LSCM
images showed that mtMSN could selectively target
at mitochondria, yet not the pristine FA-Fe3O4@MSN
without the MLS peptide functionalization (Figure
3a-3f). In the meantime, the intracellular distribution
of 2-ME in HeLa cells was also analyzed with the
dansyl chloride functionalized 2-ME (20 μg/mL,
DNS-2-ME, Figure S4 in the ESM). After incubation
with HeLa cells for 12 hours, DNS-2-ME exhibited
8
the poor targeting ability towards mitochondria
(Figure 3g-3i).
Moreover, to provide more direct evidences for the
selective targeting of mtMSN to mitochondria, the
HeLa cells (ca. 1 000 000) were treated with mtMSN
or FA-Fe3O4@MSN (20 μg/mL) for 12, 24 and 48
hours, and the stained cell slices were observed with
TEM imaging (Figure 4). Consistant with results
from CLSM imaging, mtMSN was confirmed to
target and localize in the mitochondria of HeLa cells
during a 12-hour incubation (Figure 4g-4i), and more
mtMSN could be observed inside mitochondria with
longer incubation (24 hours, Figure S5d-S5f in the
ESM; and 48 hours, Figure S5j-S5l in the ESM).
However, the FA-Fe3O4@MSN could only be found in
the vasicles with single layer of lipid membranes
(probably endocytotic vesicles) even for 48 hours
(Figure 4d-4f; Figure S5a-S5c and S5g-S5i in the
ESM). Thus, rather than the average distribution of
free 2-ME in the cytosol, 2-ME/mtMSN would tend
to internalize in the mitochondria as the
functionalization of MLS peptides, which would be
qualified to exert the selective inhibition of SOD2.
Figure 4. TEM images of a-c) HeLa cells and the intracellular
localization of d-f) FA-Fe3O4@MSN and g-i) mtMSN (20 μg/mL) in
HeLa cells with the 12-hour incubation. The rectangles in the figures
indicated the selected regions with higher magnification. (N: nucleus;
M:mitochondrion.)
3.4 Enhancing the mitochondrial ROS via the
2-ME/mtMSN
The inhibition of SOD family has been utilized for
the promotion of ROS in cancer therapy [16].
Superoxide dismutase (SOD2) possesses the
anti-oxidative roles inside mitochondria, and the
selective inhibition of SOD2 via the targeted delivery
of 2-ME into mitochondria is expected to elevate the
mitoROS levels effectively. After the incubation of
free 2-ME and 2-ME/mtMSN (20 μg/mL) with HeLa
cells for 4 hours, the mitochondrial superoxides
(represent the mitoROS levels) were reported by the
MitoSOXTM Red Mitochondrial Superoxide
Indicator (Invitrogen) and measured with the flow
cytometry (Figure 5a). A distinct increase of
mitoROS levels was detected in HeLa cells after
treated with 2-ME/mtMSN, while which was not
efficient with free 2-ME. To be noticed, the levels of
mitoROS were barely increased with the raw mtMSN,
further suggesting good biocompatibility of mtMSN.
The elevation of mitoROS levels with both free 2-ME
and 2-ME/mtMSN would be accounted for the
inhibition of proteins in the SOD family, while the
extra increase of mitoROS with 2-ME/mtMSN might
be due to the targeted inhibition of SOD2 via
mtMSN.
Figure 5. a) The levels of mitoROS and b) ratios of apoptotic cells after
the cells were treated with free 2-ME, mtMSN and 2-ME/mtMSN (20
μg/mL); c) western blotting analysis of the activated caspase-9 from
HeLa cells treated with mtMSN, 2-ME and 2-ME/mtMSN (20 μg/mL)
for 12 hours; d) viability of HeLa cells after treated with 2-ME, mtMSN
and 2-ME/mtMSN (5 μg/mL) for 24, 48 and 72 hours.
3.5 Induced apoptosis and cell death as the intense
accumulation of mitochondrial ROS
Later, investigations were carried out to examine
the hypothesis that the elevation of mitoROS by
2-ME/mtMSN would induce apoptosis efficiently.
The ratios of apoptotic HeLa cells after the
treatments of free 2-ME, pristine mtMSN and
2-ME/mtMSN (20 μg/mL) for 4, 8 and 12 hours were
9
measured with flow cytometry. As shown in Figure
5b, the mtMSN per se was inefficient in inducing
apoptosis of HeLa cells. In contrast, the
2-ME/mtMSN exhibited the remarkable
improvements (395% of that with the free 2-ME with
only the 4-hour treatment) in the initiation of
apoptosis, and during the 12-hour treatment, the
ratio of apoptotic cells (induced by both 2-ME and
2-ME/mtMSN) increased gradually Moreover, since
very few necrotic cells were detected (Figure S6 in
the ESM), the main reason for the final death of the
treated HeLa cells would be 2-ME/mtMSN induced
apoptosis.
To further verify that the involvement of
2-ME/mtMSN and elevation of mitoROS finally lead
to cell apoptosis, the activated caspase-9, which was
a marker in the mitochondrial apoptosis pathway
[36], was examined from HeLa cells treated with
mtMSN, 2-ME and 2-ME/mtMSN by western
blotting assay (Figure 5c), and significant increase of
activated caspase-9 fragments was observed from the
12-hour treated HeLa cells with 2-ME/mtMSN. Yet,
the activated caspase-8, which was associated with
the death receptor pathway of apoptosis, was not
detected during the treatments (data not shown). In
this case, the 2-ME/mtMSN was demonstrated to be
effective in the initiation of apoptosis via the selective
inhibition of SOD2 and promotion of mitoROS.
To evaluate the therapeutic efficacy of
2-ME/mtMSN, HeLa cells were treated with 2-ME,
2-ME/FA-Fe3O4@MSN and 2-ME/mtMSN (5 μg/mL)
for 12 hours, which was then replaced with fresh
culture mediums to get rid of the unused drugs, and
the cell viability was measured after another 24
hours, 48 hours and 72 hours, respectively. In Figure
5d, the 2-ME/mtMSN showed the highest treating
efficacy, while free 2-ME and 2-ME/FA-Fe3O4@MSN
presented less toxic. Thus, the 2-ME/mtMSN
demonstrated powerful efficacy in killing cancer
cells via the selective elevation of mitoROS.
3.6 Targeted treatments for cancer cells by using the
loaded 2-ME in the mtMSN combining both biological
active recognization and magnetic guiding
Free 2-ME exhibits poor ability in targeting at
tumor sites, which might put normal tissues in
severe risks. Efforts were made in this work to
provide selective administrations of 2-ME for cancer
cells, which might expand the in vivo applications of
2-ME/mtMSN in the future. Generally, selective
cancer therapy could be fulfilled with the
nanomaterial based drug delivery by passive
targeting, active targeting, and magnetic targeting
[28-30]. In this work, we tested the utility of both
active targeting and magnetic guiding as the
involvement of FA and Fe3O4 nanoparticles in the
mtMSN.
FA receptors on the membrane of HeLa cells are
overexpressed, which make FA a favorable and
widely-used modification in targeting HeLa cells [32,
37]. To test the feasibility of FA decoration, the
uptake of mtMSN (labeled with FITC, 20 μg/mL) by
both HeLa cells (represent cancer cells, FA receptors
over expressed) and HEK 293 cells (represent normal
cells, with FA receptors normally expressed) for 12
hours were quantified with flow cytometry (Figure
6a), and almost 2 folds of mtMSN could be ingested
by HeLa cells than by HEK 293 cells. As another
proof, we exploited the uptake amounts of mtMSN
(10 μg/mL) in HeLa cells with the involvement of
free FA (100 μg/mL) as competitors, and the
ingestion of mtMSN by HeLa cells was successfully
suppressed in this case (Figure S7 in the ESM). Later,
treating effects of 2-ME/mtMSN (5 μg/mL) were
evaluated for both HeLa cells and HEK 293 cells after
24, 48 and 72 hours treatments (Figure 6b). Only
19.78±1.42% HeLa cells could survive while
65.38±3.94% HEK 293 cells were still viable with the
24-hour administration, and after the 72-hour
treatments, almost all HeLa cells were killed while
there were still 34.15±1.65% HEK 293 cells alive.
Figure 6. a) Amounts of FITC-mtMSN (20 μg/mL) ingested by HeLa
cells and HEK 293 cells for 12 hours; b) viability of HeLa cells and
HEK 293 cells for the treatments with 2-ME/mtMSN (5 μg/mL) for 24,
48 and 72 hours.
In the meantime, external magnetic field has been
applied in guiding magnetic nanoparticles in vivo [31,
38, 39]. Thus, the paramagnetic Fe3O4 particle was
employed in the mtMSN to provide the site directed
treatments towards HeLa cells (Figure 7a). After 12
10
hours incubation with 2-ME/mtMSN (2 μg/mL),
more debris of HeLa cells were found around the
magnet as observed under an Olympus CKX 41
microscope (Olympus, Japan), which was failed with
the free 2-ME (Figure S8 in the ESM). By cell viability
assay, 96.97±0.12% HeLa cells were killed under the
assistant of a magnet, contrast with 50.08±8.56%
HeLa cells killed without the magnet (Figure 7b). In
this case, both FA ligands and paramagnetic Fe3O4
nanoparticles assisted the accomplishment of the
targeted treatments for cancer cells, and
consequently the 2-ME/mtMSN was qualified as a
potential chemotherapeutic agent for the in vivo
treatments.
Figure 7. a) Illustration of magnetic guided treatments with
2-ME/mtMSN for HeLa cells; d) viability of HeLa cells after treated
with free 2-ME, 2-ME/ FA-Fe3O4@MSN, 2-ME/mtMSN (2 μg/mL)
under the external magnetic field.
Uplifting of the intracellular oxidative stresses has
long been considered practicable in cancer therapy
[13-15]. Though mitoROS are responsible for most of
intracellular oxidative stresses, the targeted
manipulation of mitoROS has seldom been reported.
We proposed that mitochondria-targeted drug
delivery could be efficient in the elevation and
accumulation of oxidative stresses, which would
cause cell apoptosis and death with great efficiency.
Firstly, mitochondria are the major sources of
intracellular ROS via the electron transport
respiratory chain, and disturbances in the production
and clearance of mitoROS would be more effective in
destroying the mitochondrial oxidative equilibrium;
secondly, mitochondria are core subcellular
components in sensing the oxidative stresses during
cellular physical procedures such as apoptosis and
aging [3]; thirdly, accumulation of mitoROS at high
levels would present intense injuries for
mitochondria through the direct oxidation of
mitochondrial components. Since mitochondrial
SODs (SOD2) take the leading charge in the
clearance of mitoROS, targeted inhibition of SOD2
would be an efficient approach in enhancing the
mitochondrial oxidative stresses and activating
subsequent apoptosis, yet where the inhibitors for
SOD family could hardly present discriminatory
interactions for SOD1 or SOD2. As a proof of concept,
we developed a general strategy for mitochondria
targeted delivery of the inhibitor of SODs (2-ME)
into cancer cells (HeLa cells) via a multifunctional
mesoporous silica nanocarrier (mtMSN). The
mtMSN was composed by a Fe3O4 core for magnetic
targeting, a MSN shell for drug delivery, FA for HeLa
cell recognization and MLS peptides for
mitochondria internalization, which owned high
loading capacity, good biocompatibility and dual
targeting ability for mitochondria and cancer cells.
Utilizing the mtMSN, the 2-ME was delivered to the
mitochondria for the first time, with the elevation of
mitoROS and distinct initiation of cell apoptosis.
Interestingly, the 2-ME/mtMSN was more powerful
than free 2-ME in both enhancing the mitoROS,
activating the apoptosis, and killing cancer cells
(HeLa cells). As reported, free 2-ME would interact
with both SOD1 and SOD2 in intact cells without
discrimination in selecting the substrates [16]; via the
mitochondria targeted delivery using mtMSN, 2-ME
was supposed to mainly inhibit the SOD2 due to the
confined intracellular distribution, exhibiting the
more efficient promotion of mitoROS, and
subsequent activation of apoptosis of HeLa cells.
Therefore, the selective inhibition of SOD2 would
provide more intense promotion of mitoROS levels
than the nondiscriminatory inhibition of SODs,
which would be favorable in overwhelming the
cellular oxidative adaption system and further
induced more treated cells into the death program,
and moreover, manual manipulation of
mitochondrial physical progresses would be of great
promise in cancer therapy.
4 Conclusions
In summary, a mesoporous magnetic mtMSN was
constructed to target and deliver the inhibitor of
SOD2 (2-ME) to mitochondria of HeLa cells, and
11
provide selective elevation of mitoROS levels. The
enhancement of mitoROS was more effective in the
initiation of apoptosis than cytosol ROS, which lead
to powerful curative effects for HeLa cells.
Meanwhile, 2-ME/mtMSN exhibited targeted
treatments for cancer cells (HeLa cells), hinting
potential in vivo applications. The Fe3O4@MSN based
nanocarriers were of good biocompatibility and good
mechanical strength, and were utilized in
mitochondria-targeted delivery of anti-cancer agents
for the first time. Moving forward, the
nanomaterials-based subcellular delivery would be
applied for the manipulation of physical processes in
different organelles, implying potentials in clinical
medication and molecular biology.
Acknowledgements Financial support from the Creative Research Group
Project of NSFC (21021004), the National Natural
Science Foundation of China (Nos. 21235006,
21175134, 21375125, 81161120540), the China State
Key Basic Research Program Grant (2012CB910601),
National Key Special Program on Infection Diseases
(2012ZX10002009-011), the Analytical Method
Innovation Program of MOST (2012IM030900) are
grateful acknowledged.
Electronic Supplementary Material: Supplementary
material (detailed experimental procedures;
characterization of Fe3O4 and Fe3O4@MSN;
adsorption of 2-ME by the mtMSN; purity evaluation
of synthesized DNS-2-ME; intake inhibition of
FITC-mtMSN by free FA; and observation of HeLa
cells after treated with free 2-ME, mtMSN,
2-ME/mtMSN using the external magnetic field) is
available in the online version of this article at
http://dx.doi.org/10.1007/s12274-***-****-*
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