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, wurenan@dicp.ac.cn; Hanfa Zou, hanfazou@dicp.ac.cn

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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