Non-peptidic guanidinium-functionalized silica nanoparticles as selective mitochondria-targeting drug nanocarriers

Article abstract of DOI:10.1039/c8tb01358f

We report on the design and fabrication of a Fe₃O₄ core-mesoporous silica nanoparticle shell (Fe₃O₄@MSNs)-based mitochondria-targeting drug nanocarrier. A guanidinium derivative (GA) was conjugated onto the Fe₃O₄@MSNs as the mitochondria-targeting ligand. The fabrication of the Fe₃O₄@MSNs and their functionalization with GA were carried out by the sol-gel polymerization of alkoxysilane groups. Doxorubicin (DOX), an anti-cancer drug, was loaded into the pores of a GA-attached Fe₃O₄@MSNs due to both its anti-cancer properties and to allow for the fluorescent visualization of the nanocarriers. The selective and efficient mitochondria-targeting ability of a DOX-loaded GA-Fe₃O₄@MSNs (DOX/GA-Fe₃O₄@MSNs) was demonstrated by a co-localization study, transmission electron microscopy, and a fluorometric analysis on isolated mitochondria. It was found that the DOX/GA-Fe₃O₄@MSNs selectively accumulated into mitochondria within only five minutes; to the best of our knowledge, this is the shortest accumulation time reported for mitochondria targeting systems. Moreover, 2.6 times higher amount of DOX was accumulated in mitochondria by DOX/GA-Fe₃O₄@MSNs than by DOX/TPP-Fe₃O₄@MSNs. A cell viability assay indicated that the DOX/GA-Fe₃O₄@MSNs have high cytotoxicity to cancer cells, whereas the GA-Fe₃O₄@MSNs without DOX are non-cytotoxic; this indicates that the DOX/GA-Fe₃O₄@MSNs have great potential for use as biocompatible and effective mitochondria-targeting nanocarriers for cancer therapy.

Full text of DOI:10.1039/c8tb01358f

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Page 1 of 11
J Po lue ran sael od fo Mn aot te rai ad l jsu Cs ht emm ai rs gt ri ny sB
Journal of
View Article Online
DOI: 10.1039/C8TB01358F
Materials Chemistry B
Paper
Non-peptidic guanidinium-functionalized silica nanoparticles as
selective mitochondria-targeting drug nanocarriers
Junho Ahn, Boeun Lee,b,† Yeonweon Choi,a,† Hanyong Jin, Na Young Lim, Jaehyeon Park, Ju
a,†
b
a
a
Received 00th January 20xx,
Accepted 00th January 20xx
a
,b
,a
Hyun Kim, Jeehyeon Bae,* and Jong Hwa Jung*
DOI: 10.1039/x0xx00000x
We report on the design and fabrication of a Fe
chondria-targeting drug nanocarrier. A guanidinium derivative (GA) was conjugated onto the Fe
chondria-targeting ligand. The fabrication of the Fe @MSNs and their functionalization with GA were carried out by the
sol-gel polymerization of alkoxysilane groups. Doxorubicin (DOX), an anti-cancer drug, was loaded into the pores of a GA-
attached Fe @MSNs due to both its anti-cancer properties and to allow for the fluorescent visualization of the nanocarri-
ers. The selective and efficient mitochondria-targeting ability of a DOX-loaded GA-Fe @MSNs (DOX/GA-Fe @MSNs)
was demonstrated by a co-localization study, transmission electron microscopy, and a fluorometric analysis on isolated
mitochondria. It was found that the DOX/GA-Fe @MSNs selectively accumulated into mitochondria within only five
minutes; to the best of our knowledge, this is the shortest accumulation time reported for mitochondria targeting systems.
Moreover, 2.6 times higher amount of DOX was accumulated in mitochondria by DOX/GA-Fe @MSNs than by DOX/TPP-
Fe @MSNs. A cell viability assay indicated that the DOX/GA-Fe @MSNs have high cytotoxicity to cancer cells, whereas
the GA-Fe @MSNs without DOX are non-cytotoxic; this indicates that the DOX/GA-Fe @MSNs have great potential for
use as biocompatible and effective mitochondria-targeting nanocarriers for cancer therapy.
3
O
4
core–mesoporous silica nanoparticle shell (Fe
3
O
4
@MSNs)-based mito-
3
O
4
@MSNs as the mito-
www.rsc.org/
3
O
4
3
O
4
O
3 4
O
3 4
3
O
4
O
3 4
O
3 4
O
3 4
3
O
4
O
3 4
mitochondrial targeting is highly desirable, as it would allow for
these compounds to be tracked and their therapeutic effects to
be directly visualized.
Introduction
22–24
Using fluorescence image-guided therapy for the site-selective
delivery of diagnostic and therapeutic agents to cancer cells is a
Various methods have been developed to target mitochondria;
these include the use of targeting ligands, such as peptides
composed of guanidine or guanidinium, and chemical
1–3
great challenge. One of the reasons as to why this is so
challenging is the lack of specificity for specific organelles, as it
25–26
4
,
compounds like triphenoylphosphonium (TPP).
TPP, which
results in both high cytotoxicity and high dosage requirements.
5
consists of three phenyl moieties, is a cation that has a
delocalized positive charge and sufficient lipophilicity; this
enables it to facilitate the transportation process when a
The precise delivery of chemotherapy agents at the sub-
cellular level has gained a lot of attention as an approach for
addressing this challenge, as it can improve therapy efficiency,
2
7
6
,
7
mitochondrial membrane is crossed. For example, small
molecules that use TPP as a targeting ligand for mitochondria
decrease side-effects, and combat multidrug resistance.
Mitochondria are vital subcellular organelles in eukaryotic cells,
and mitochondrial dysfunctions have been linked to
2
8
have been developed by Murphy et al. In contrast,
guanidinium-rich scaffolds (such as peptides, spaced peptides,
oligocarbamates, dendrimers, and oligocarbonates) have also
8, 9
tumorigenesis and tumor progression.
The most common
method for developing mitochondria-targeting therapy agents
is to conjugate a mitochondria-targeting moiety with model
drugs or to direct drugs into surface-functionalized
29–30
been used to efficiently enter cells.
These transporters have
been shown to enable and enhance the passage of a large
number of substances, including small molecules, peptides,
proteins, and oligonucleotides, across multiple biological
1
0–21
aggregates.
Furthermore, a fluorescent tag is required if
one wishes to monitor a mitochondria-targeting drug delivery
system. Such modifications have the potential to alter the
metabolic pathways of drug molecules, and the fluorescence is
not associated with the drugs once they are released. As a
result, developing fluorescent therapeutic agents with
31–34
barriers (such as the cell wall of algae).
Although many
research groups have attempted to deliver small molecules
using guanidinium-rich modified peptides, cyclic peptides, and
peptide nucleic acids, guanidinium without a polymeric
backbone as a selective mitochondria targeting ligand has rarely
been reported upon; there are only two papers that have in fact
3
5, 36
done so.
In one of these, Mao et al. reported on the use of
iridium–guanidinium complexes as
a.Department of Chemistry and Research Institute of Natural Sciences Gyeongsang
National University, Jinju, 52828, Korea.
cyclometalated
b.
School of Pharmacy, Chung-Ang University, Seoul 06974, Korea.
mitochondria-targeting anticancer agents; these exhibited
higher cytotoxicity than cisplatin against a cisplatin-resistant
†
These authors contributed equally. *Email - jonghwa@gnu.ac.kr
Electronic Supplementary Information (ESI) available: [synthetic procedures of
DOX/GA-Fe @MSNs and DOX/TPP-Fe @MSNs, IR, TGA and its analysis, PL
35
3
O
4
O
3 4
cell. They also found that these complexes could be effectively
spectra, TEM-EDS, Zeta potential]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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Journal Name
accumulated into HeLa cells and used to specifically target ligand-immobilized MSNs were accumulated aVniedw ArutiscleedOnlitnoe
mitochondria. Giraft et al., meanwhile, investigated a new deliver anticancer drugs to selected mitoDchOoI:n1d0.r1i0a3.9T/Co8tThBe01b35e8sFt
family
of
powerful
non-peptidic
cell-penetrating of our knowledge, this is the first approach for selective
oligocyclicguanidinium systems; these were found to be more mitochondria accumulation that uses guanidinium without
efficient translocation through HeLa membrane than either further chemical functionalization as a mitochondria-targeting
antennapedia (Antp) or TAT peptides at low concentrations in ligand attached to silica nanoparticles. The accumulation rate of
36
mitochondria.
guanidinium-immobilized silica nanoparticles in the
Developing a new class of mitochondria targeting ligands using mitochondria was found to be faster than that of TPP-
only guanidinium ions (i.e., no other chemical functional immobilized silica nanoparticles.39–43 We also found that the
groups) would be valuable, as it would be easy to synthesize, it guanidinium-immobilized silica nanoparticles exhibited no
would be simple to immobilize the surfaces of the nanocarriers, significant cytotoxicity.
and there would be a high amount of transportation by
intermolecular hydrogen-bonding interactions with phosphate
17, 29
Experimental
membranes in the cells.
Mesoporous silica nanoparticles
(
MSNs) have been used as drug carriers in the design of drug
Materials and instruments
delivery systems, as they can be easily synthesized and their size
and pore volume are controllable; furthermore, they exhibit
good biocompatibility, which allows for them to be used in in
All chemical reagents were purchased from Sigma-Aldrich.
Solvents were purchased from Samchun Pure Chemicals Co. and
used without further purifications. An ultraviolet–visible light
37
vivo applications.
(UV–Vis) spectrophotometer (Thermo Evolution 600, Thermo
In this report, we describe a new class of mitochondria-
targeting ligand; namely a guanidinium derivative that does not
contain any other functional groups. We found that it exhibited
high delivery efficacy and offered several advantages over
Fisher Scientific) was used to obtain the absorption spectra. The
fluorescence spectra were recorded with a Shimadzu RF-5301
PC to confirm the doxorubicin loading on the nanocarriers. TEM
8– and TEM-EDS, which were both operated at 300 kV, were used
to obtain the morphologies and compositions of nanoparticles
3
previously reported systems, including that of a TPP moiety.
4
3
The guanidinium ligand was attached to the surfaces of the
MSNs by a sol-gel reaction in which a covalent bond was used
as a model carrier material. Fe core has a potential to be
applied to hyperthermia, and its magnetic properties allowed
(JEM2010, Jeol) carbon/formvar-covered Cu-supported grid
was used. Fourier-transform infrared (FTIR)
A
3 4
O
spectrophotometer was used to confirm the functional groups
in the nanoparticles (FTIR 8400S, Shimadzu). BET was used to
confirm the surface areas and pore distributions of the
nanoparticles (Belsorp II mini, BEL Japan). Before the ad-
sorption experiment, the materials were vacuum-dried at 130
11, 16
for the easy separation and removal of particles.
used in this approach as platform materials to support the
conjugation of the guanidinium ligands. The guanidinium-
MSNs were
3
-
DOX/R-Fe O @MSNs
Cell membrane (R-PO )
Guanidinium derivative
3
4
Model drug:
Cell membrane
Cytoplasm
Cytoplasm
“
Direct targeting and penetration”
+
+
+
-
-
-
-
Mitochondrial Outer membrane
+
+
-
-
-
H+
H+
H+
H+
+
-
H+
H
+
H+
H+
H+
-
+
-
-
+
+
-
-
Mitochondrial inner membrane
Matrix
-
GA
-
-
-
-
-
-
-
Continuous imaging
and
Mitochondria
Biodegradation-induced release
Mitochondrial-membrane-potential-driven accumulation
TPP
3 4
Scheme 1. Schematic illustration of the mitochondria targeting of the DOX/GA-Fe O @MSNs.
2
| J. Name., 2012, 00, 1-3
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ARTICLE
°
C for 10 h. The ligand concentrations of the surfaces of the twice with ethanol, the Fe
3
O
µ
µ
4
@MSNs were dried. A mixture
mol) and 3-(triethoxysilyl)propyl
View Article Online
DOI: 10.1039/C8TB01358F
nanoparticles were determined using thermogravimetric solution of DOX (1 mg, 1.72
analysis (TA Q50, TA Instruments) over a temperature range of isocyanate (0.38 mg, 1.55
mol) stirred 4 h at 45
@MSNs (150 mg) solution
performed by NANO ZS (Malvern). A Nikon Microscope ECLIPSE dispersed in EtOH 30 ml for 4 h at 50 ℃. Impurities were
0i was used to record images with a 420 nm UV light excitation removed by centrifugation and dried at 60 ℃ . This
℃ was
1
3 4
00–800 °C. A zeta potential analysis of the nanoparticles was prepared and reacted with Fe O
8
source. The emission spectra between 400 and 650 nm were particles were used to TPP or GA modifications on silica
obtained using a 100× magnification. The samples used for surfaces.
microscopy were made by drop-casting onto a glass slide and
allowing for slow evaporation to occur.
Synthesis of 246 and 1
The compound
reported previously.
2
was synthesized by adapting procedures
Fabrications of Fe
Iron oxide (Fe
3
O
4
NPs44
46
A
mixed solution of 1,2-bis(2-
3
O
4
) nanoparticles were fabricated using two aminoethoxy)ethane and diisopropylethylamine (DIPEA, 1 mL)
steps. Firstly, The metal–oleate complexes were prepared by a was stirred at −20 °C for an hour. N,N’-bis(tertbutoxycarbonyl)-
reaction of iron(III) chloride with sodium oleate. In a typical 1H-pyrazol-1-carboxamide (0.5 g, 1.61 mmol) in
synthesis, 10.8 g of iron(III) chloride hexahydrate (98%, CH
3
CN/Tetrahydrofuran (20 mL, v/v = 2/1) was added dropwise
−
1
FeCl •6H O, 40 mmol, Sigma-Aldrich) and 36.5 g of sodium by a syringe pump at a rate of 0.15 mL min , and the solution
3
2
oleate (120 mmol, TCI, 95%) were dissolved in a mixture of 80 was stirred for 2 h at −20 °C. The product was confirmed by TLC
mL ethanol, 60 mL distilled water, and 140 mL hexane. monitoring. After the reactions were complete, the reaction
Following this, the mixture was heated at 70 °C for 4 h. Upon mixture was evaporated until dry, and the residue was dissolved
completion of the reaction, organic layer was washed three in MC (100 mL) and washed with brine (100 mL). The organic
times with 30 mL distilled water; this was followed by a extract was dried over Na
concentration in vacuo, which produced a waxy solid. residue was subjected to recycling preparative high
In the second step, the general procedure for synthesizing performance liquid chromatography with MeOH/MC (v/v
mono-disperse iron oxide (Fe ) nanocrystals with a size of 13 =1:10) as an eluent; to allow isolation of in 37% yield (0.234
nm initially involved dissolving the prepared iron–oleate g). Nuclear magnetic resonance (NMR, 300 MHz, DMSO-d ) δ
complex (36 g, 40 mmol) and oleic acid (5.7 g, 20 mmol, Sigma- ppm 11.47 (s, 1H), 8.63 (s, 1H), 3.67–3.61 (m, 8H), 3.51 (t, 2H),
2 4
SO , evaporated until dry, and the
3
O
4
2
6
1
3
6
Aldrich, 90%) in 1-octadecene (200 g, Sigma-Aldrich, 90%) at 2.89 (t, 2H), 1.50 (m, 18H); C NMR (75 MHz DMSO-d ) δ ppm
room temperature. Following this, the reaction mixture was 162, 157, 153, 86, 77, 72, 68, 43, 32, 29; ESI-MS: calculated for
+
heated to 100 °C for 2 h so that excess water in the solution was
17
C H
34
N
4
O
6
[M] : 390.25, Found 390.92; Anal. Calculated for C,
removed. The reaction mixture was heated to 320 °C at a 52.29; H, 8.78; N, 14.35; O, 24.58, Found: C, 52.34; H, 8.79; N,
−
1
constant heating rate of 2.3 °C min under a nitrogen flow; this 14.33; O, 24.54.
temperature, 320 °C, was then maintained for an hour without In order to synthesize compound 1, a mixed solution of com-
gas flow. A noticeable reaction occurred when the reaction pound 2 (0.1 g, 0.256 mmol) and 3-(triethoxysilyl)propyl isocya-
temperature reached 320 °C, and the initially transparent nate (0.63 g, 0.254 mmol) was stirred in dry N,N-
solution became turbid and a brown-black color. The reaction Dimethylformamide (4 mL) at 40 °C for 6 h. Following this, the
mixture was then cooled to room temperature, and 500 mL solution was cooled to room temperature and stored at 0 °C.
ethanol was added so as to precipitate iron oxide particles; This solution was used without any further purification.
these were subsequently separated by centrifugation.
Fabrication of 1-Fe
Fe @MSNs (150 mg) were dispersed in EtOH (20 mL).
nanocrystals (34 mg mL ) Compound (0.065 g, 0.1 mmol) was added to a solution
in chloroform was poured into 10 mL of 0.55 M aqueous cetyl containing Fe @MSNs and the reaction mixture was stirred
3 4
O @MSNs
3 4
O
@MSNs45
Fabrications of Fe
In a typical procedure, 1 mL of Fe
3 4
O
−
1
3
O
4
1
3 4
O
trimethylammonium bromide (CTAB) solution, and the resulting at 60 °C for 3 h. The unreacted species were separated by
solution was stirred vigorously for 30 min. An oil-in-water centrifugation, and the collected product was washed three
microemulsion then formed that was a turbid brown color. The times with water.
mixture was then heated to 60 °C and maintained at that
Fabrication of doxorubicin-loaded GA-Fe
-immobilized Fe @MSNs (100 mg) were
The solution was then added to a mixture of 110 mL water and dispersed in MC (10 ml). TFA (0.2 ml) was slowly added under 0
3 4
O @MSNs
temperature under stirring so as to remove the chloroform; this
resulted in a transparent black Fe /CTAB solution forming. Compound
3
O
4
1
3 4
O
0
7
.62 mL of 2 M NaOH solution, and the mixture was heated to °C. Thirty minutes later, mixture solution was stirred for 4 hours
0 °C under stirring. 1 mL TEOS was then slowly added to the at room temperature. Water was added to extract the GA-
reaction solution. The as-synthesized Fe
washed three times with ethanol to remove any unreacted nanoparticles were washed
species and was dispersed in 20 mL ethanol. In order to extract centrifugation. As a final step, further doxorubicin was loaded
CTAB from the NPs, 40 μL HCl was added to the dispersion (pH to the pores of GA Fe @MSNs. GA-Fe @MSNs (10 mg)-
level of ca. 1.4) and stirred for 3 h at 60 °C. After being washed dispersed aqueous solution containing doxorubicin (1 mg/ml, 5
3
O
4
@MSNs was then immobilized Fe
3
O
4
@MSNs from MC phase. Collected
3
times with water by
-
O
3 4
3 4
O
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Page 4 of 11
ARTICLE
Journal Name
ml) was stirred for 12 hours at room temperature. Nanoparticle supplemented with a 10% fetal bovine serum (FBS; Caisson) and
View Article Online
DOI: 10.1039/C8TB01358F
2
were collected by centrifugation and washed with HEPES Buffer 1% penicillin-streptomycin (Caisson) at 37 °C/5% CO .
(
0.01 M, pH 7.4) three times. Final product was dispersed in 10
Confocal fluorescence microscopy
mL of HEPES buffer (0.01 M, pH 7.4).
4
HeLa (2 × 10 ) cells were treated with the indicated DOX-loaded
nanoparticles for 2 h. The cells were washed with phosphate-
In vitro DOX release test
3 4
The DOX/GA-Fe O @MSNs (15 mg) were dispersed in 5 mL of
HEPES Buffer solution (0.01 M, pH 7.4) by stirring under dark
condition. The temperature of HEPES Buffer was maintained at
37 ℃. After a predetermined time interval, an aliquot (0.2 mL)
of the supernatant solution was taken out for UV-Vis analysis to
determine the DOX release amount.
Synthesis of 1A47 and its triethoxysilyl group functionalization.
The compound 1A was synthesized by adapting procedures
47
reported previously.
bromoethy1)amine hydrobromide (7 g, 34.16 mmol) and
triphenylphosphine (10 g, 1.12 equiv.) in CH CN (50 mL) was
refluxed at 95 °C for 15 h. The precipitate was then dissolved in
.5 equivalent of KOH aqueous solution and evaporated before
being treated with ethanol. The product was filtered and
triturated with ether; white precipitate which was
A
mixture solution of (2-
3
Figure 1. (A) TEM image of GA-Fe
3 4 2
O @MSNs, (B) its N adsorption–
1
desorption isotherm, and (C) BJH plot of B.
a
recrystallized from ethanol/ether was produced. (3.0 g, 19%): buffered saline (PBS) twice and incubated with 100 nM Mito-
1
H NMR (CDCl
3
) δ ppm 1.86 (s, 2 H), 3.1 (m, 2 H), 4.1 (m, 2 H), Tracker Green FM (M7514; Life Technologies, Gaithersburg,
7
D
1
.9 (m, 15 H); the resonance at 1.90 ppm fully exchanged with MD, USA) for 30 min. The cells were fixed with 4%
1
3
2
O. C NMR (CDCl
3
) δ ppm 135.1 (d), 135.0, 130.5 (d), 130.3, paraformaldehyde (Sigma, St. Louis, MO, USA) and analyzed
21BrNP: C, 62.19; using LSCM (LSM700; Carl Zeiss, Jena, Germany).
16.8 (d), 116.0, 31.5, 26.8. Anal. Calcd for C20
H
H, 5.48; Br, 20.69. Found: C, 61.89; H, 5.22; Br, 20.5.
A mixed solution of 3-(triethoxysilyl)propyl isocyanate (0.1 g,
TEM imaging
7
0
(
.4 mmol) and (2-aminoethyl)triphenylphosphonium bromide HeLa cells (1 × 10 ) were treated with DOX/GA-Fe
3
O
4
@MSNs
−
1
2A, 0.16 g, 0.41 mmol) was dissolved in dry DMF. The reaction (500 μg mL ) for 12 h before being washed three times with
mix-ture was stirred for 6 h at 40 °C. Following this, the solution PBS; they were subsequently fixed in PBS containing 2.5%
was cooled to room temperature and stored at 0 °C. This glutaraldehyde (Sigma-Aldrich). The cell pellets were further
solution was used without further purification.
4
fixed with a PBS buffer solution containing 1% OsO before
being washed with ethanol for dehydration, treated with
propylene oxide, and embedded in Epon. For the TEM
Fabrication of TPP-Fe
The Fe
triethoxysilyl)propyl isocyanate-modified compound 1A (0.063 using uranyl acetate and lead citrate. TEM imaging was carried
g, 0.1 mmol) was added to the Fe @MSNs solution, and the out using a Technai G2 Spirit Twin TEM fitted with an anti-
3 4
O @MSNs
3 4
O @MSNs (150 mg) were dispersed in EtOH (20 mL). 3- observations, the cells were cut into ca. 80 nm slices and stained
(
3 4
O
reaction mixture was stirred at 60 °C for 3 h. Any unreacted contaminator (FEI Company, Hillsboro, OR, USA) operated at
species were separated by centrifugation, and the collected 120 kV.
product was washed three times with water.
Mitochondria isolation
Fabrication of doxorubicin-loaded TPP-Fe
Doxorubicin was loaded into the pores of the TPP-immobilized nanoparticles for 2 h before mitochondria were isolated by a
Fe @MSNs. A TPP-Fe @MSNs (10 mg) dispersed aqueous mitochondria isolation kit (Thermo Fisher Scientific, Rockford,
3
O
4
@MSNs
7
HeLa cells (1 × 10 ) that had been treated with the indicated
3
O
4
3 4
O
−
1
solution containing doxorubicin (1 mg mL , 5 mL) was stirred IL, USA) as per the manufacturer's instructions. The
for 12 h at room temperature. Nanoparticles were collected by fluorescence of the mitochondria isolates was measured by a
centrifugation and washed with a HEPES buffer solution (0.01 FlexStation 3 microplate reader (480 nm excitation, 550 nm
M, pH 7.4) three times. The final product was dispersed in a 10 emission; Molecular Devices, Sunnyvale, CA, USA).
mL HEPES buffer solution (0.01 M, pH 7.4).
Cell viability assay
Cell culture
HeLa and 293T cells purchased from the Korean Cell Line Bank plates. After 24 h, the cells were incubated with the indicated
Seoul, Korea) and American Type Culture Collection (Manassas, concentrations of Fe @MSNs, GA-Fe @MSNs,
VA, USA), respectively, cultured in Dulbecco's Modified Eagle DOX/Fe @MSNs or DOX/GA-Fe @MSNs for 24 h. The cell
Medium (DMEM) (Caisson, North Logan, UT, USA) viability assay was performed according to the process reported
4
4
HeLa (1 × 10 ) and 293T (2 × 10 ) cells were seeded in 96-well
(
3
O
4
3 4
O
3
O
4
3 4
O
4
| J. Name., 2012, 00, 1-3
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J Po lue ran sael od fo Mn aot te rai ad l jsu Cs ht emm ai rs gt ri ny sB
Journal Name
in a previous paper using a CellTiter-Glo Assay kit (Promega, Fe
ARTICLE
3
O
4
@MSNs were characterized using
a
N
2
adsorp-
View Article Online
66
DOI: 10.1039/C8TB01358F
Madison, WI, USA).
tion/desorption measurement. The N
2
adsorption/desorption
curves of the GA-Fe @MSNs exhibited a shape typical of
3 4
O
type-4 mesoporous materials (Fig. 1(B)). Of the type 4
mesoporous materials compared against, the curves were
found to be quite similar to those of MCM-41; this indicated
Results and discussion
3 4
Preparation and characterization of GA-Fe O @ MSNs (Scheme S1)
3 4
that the GA-Fe O @MSNs formed with a mesoporous structure
Fe
decomposition of iron-oleate. The mesoporous silica shell of the
Fe @MSNs was fabricated by a cetrimonium bromide (CTAB)-
templated sol-gel polymerization of tetraethylorthosilicate
TEOS). A trace of DOX was treated on the silica surface to give
3
O
4
nanoparticles were synthesized by the thermal
of the type MCM-41. The Brunauer–Emmett–Teller (BET)
surface area and Barrett–Joyner–Halenda (BJH) pore diameter
3
O
4
2
−1
of the GA-Fe
and 3.33 nm, respectively.
The immobilization of the guanidinium derivative on the outer
surface of the Fe @MSNs was determined using infrared (IR)
3 4
O @MSNs were determined to be 545.44 m g
(
partial hydrophobic natures. Either a Boc-protected guanidine
derivative or (2-aminoethyl)triphenylphosphonium bromide
was reacted with 3-(triethoxysilyl)propyl isocyanate. Following
3 4
O
spectroscopy (Fig. S1). When compared to the IR spectrum of
−1
3 4
the Fe O @MSNs, four new peaks were found at 1685 cm
this, the Fe
3
O
4
@MSNs were functionalized by a sol-gel reaction
−1
−1
(
1
C=N), 1645 cm (C=O, urea), 1577–1565 cm (N-H), and
of the silica shell with either an alkoxysiliyl-appended guanidine
derivative or a TPP derivative. In order to remove the Boc
groups from the guanidine derivative, trifluoroacetic acid (TFA)
−1
477~1368 cm (C–H); these were felt to originate from the
@MSNs, which suggests
that the guanidinium derivative was attached onto the surface
of the Fe @MSNs. Energy dispersive spectroscopy (EDS) also
3 4
guanidinium derivative in the GA-Fe O
was treated on
GA-Fe @MSNs and TPP-Fe
cation exchange reaction using an acidic aqueous solution.
The prepared GA-Fe @MSNs were characterized by trans-
mission electron microscopy (TEM). The average diameter of
the GA-Fe @MSNs was ca. 54 nm, and these consisted of a
Fe core diameter of ca. 13 nm and silica shell thickness of
0.5 nm (Fig. 1(A)). The surface area and pore size of the GA-
1
-Fe
3
O
4
@MSNs. The CTAB in the silica pores of
3 4
O
3
O
4
3 4
O
@MSNs was removed by a
pro-vided evidence of the conjugation of the guanidinium
derivative. The presence of nitrogen in the guanidinium moiety
3 4
O
was confirmed by the TEM-EDS of the GA-Fe
did not exist in the Fe @MSNs (Fig. S2).
In order to further investigate the grafting of the guanidinium
derivative, the zeta potentials of the Fe @MSNs without the
guanidinium derivative and the GA-Fe @MSNs were
measured (Fig. S3). The zeta potential of the Fe @MSNs was
determined to be −12.5 mV; however, the zeta potential of the
GA-Fe @MSNs was 11.27 mV; this was due to the positive
3 4
O @MSNs; these
3 4
O
3 4
O
3 4
O
3 4
O
2
3 4
O
3 4
O
DOX
red)
Mitotracker
(orange)
DAPI
Merged
(
(blue)
3 4
O
charge of the guanidinium. These results strongly indicated that
the guanidinium derivative had been successfully grafted onto
(
A)
the surface of the Fe
3 4
O @MSNs; it was also found by a
thermogravimetric analysis (TGA) to account for ~6.51 wt%
−
1
(
~0.16 mmol g ) of the GA-Fe
3 4
O @MSNs (Fig. S4).
DOX loading into GA-Fe @MSNs
3
O
4
(
B)
We utilized DOX, a well-known chemotherapeutic anticancer
agent that acts on deoxyribonucleic acid (DNA), as a model drug
for demonstrating the effectiveness of the GA-Fe O @MSNs as
3 4
a mitochondrial targeting material. DOX disrupts the DNA
replication process in mitochondria, which leads to dysfunction
(
(
C)
48, 49
of organelles.
Furthermore, it exhibits a characteristic red
florescence emission, which allows for the imaging and tracking
of the location of DOX-including materials without any
fluorescent labels.
D)
In order to load DOX, the GA-Fe
DOX-saturated aqueous solution. After 24 h, DOX-loaded GA-
Fe @MSNs (DOX/GA-Fe @MSNs) were collected from the
3 4
O @MSNs were dispersed in a
3
O
4
3 4
O
solution by centrifugation and washed with a pH 7.4 HEPES
buffer solution. In order to confirm the loading of the DOX, the
fluorescence spectra of both DOX and the DOX/GA-
(
E)
Fe
3
O
4
@MSNs were obtained (Fig. S5). The fluorescence
@MSNs exhibited fluorescence
spectrum of the DOX/GA-Fe
3 4
O
Figure 2. Confocal laser scanning microscopy images of HeLa cells
treated with nanoparticles. ((A) control, (B) DOX/Fe @MSNs, (C)
DOX/NH -Fe @MSNs, (D) DOX/TPP-Fe @MSNs and (E)
DOX/GA-Fe @MSNs) (100 μg/mL and scale bar: 20 μm)
emissions at both 560 and 590 nm, which were both found for
DOX; this suggested that the DOX had been successfully loaded
into the GA-Fe O @MSNs.
3 4
3 4
O
2
3
O
O
4
4
3 4
O
3
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J. Name., 2013, 00, 1-3 | 5
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ARTICLE
Additional evidence of DOX loading was obtained using IR demonstrated that the DOX/GA-Fe
Journal Name
3 4
O @MSNs selectively
View Article Online
DOI: 10.1039/C8TB01358F
spectroscopy (Fig. S1). In the IR spectrum of the DOX/GA- accumulated in the mitochondria rather than in the nuclei. The
−
1
−1
3 4 3 4
Fe O @MSNs, three peaks were found at 1724 cm , 1673 cm , intracellular distribution of the DOX/Fe O @MSNs that did not
−
1
and 1623–1635 cm ; these were not found for the GA- contain guanidinium were used as a control under the same
@MSNs. The peaks at 1724 cm−1 and 1673 cm were de- conditions. As expected, the tango fluorescence due to the
−1
3
Fe O
4
termined to be due to the C=O bonds and the peak at 1623– overlap of the fluorescence of the MitoTracker and DOX could
−
1
1
635 cm was determined to be due to the aromatic C=C bonds not be seen, which indicated that the DOX/Fe
3 4
O @MSNs could
of DOX. As shown in Fig. S4, the capacity of DOX loading was not target the mitochondria (Fig. 2(B)). The blue fluorescence of
−
1
estimated to be 9.7 wt% (0.167 mmol g ) by TGA.
DAPI remained unchanged; as such, it could be concluded that
the DOX/Fe @MSNs moved to an unspecified area in the
cytoplasm of the HeLa cell without any selectivity.
DOX release behavior in HEPES buffer solution (0.01 M, pH 7.4) Positively charged amine-conjugated Fe @MSNs (NH
@MSNs (TPP-
@MSNs) were also prepared (Figs. S7 and S8). DOX was
-Fe @MSNs and TPP-Fe @MSNs in order
@MSNs. The release of DOX reached an to compare their efficiencies for the selective accumulation into
equilibrium in 2.5 h. At the equilibrium point, the accumulated mitochondria with that of the DOX/GA-Fe @MSNs. Amine-
percentage of the released DOX was only ca. 3.8 %. This result modified Fe @MSNs loaded with DOX have positive charge
implies that tracking the distribution of GA-Fe @MSNs based values in acidic environment such as inside of cancer cells due
3 4
O
3 4
In vitro DOX release from DOX/GA-Fe O @MSNs
3
O
4
2
-
was evaluated using UV-Vis spectroscopy (Fig. S6). For the test, Fe
the DOX/GA-Fe @MSNs were dispersed in the release Fe
medium at 37
from DOX/GA-Fe
3
O
O
4
3 4
@MSNs) and TPP-conjugated Fe O
3 4
O
℃
O
3 4
3
4
. Fig. S6 shows the cumulative DOX release loaded into NH
2
3
O
4
3 4
O
3 4
O
3 4
O
3 4
O
4
3
on the fluorescence of DOX can be performed and it is reliable. to the amine groups on nanoparticles. TPP is known to
selectively and effectively accumulate into mitochondria due to
3 4
Mitochondria targeting of DOX/GA-Fe O @MSNs
the negative potential gradient of the organelle, and it does not
In order to evaluate the mitochondria targeting of the DOX/GA- alter any of the genes or protein structures; it is the most
Fe @MSNs, their intracellular distribution was investigated. commonly used moiety for preparing mitochondria-targeting
The DOX/GA-Fe
2 h before their intracellular localization was observed using the punctiform distribution of DOX/NH
3 4
O
2
5, 39–43
3
O
4
@MSNs were incubated with HeLa cells for drug delivery systems.
In the merged image in Fig. 2(C),
-Fe @MSNs was
1
2
3 4
O
confocal laser scanning microscopy (CLSM) (Fig. 2). Two shown, and no significant tango fluorescence was observed as
markers, DAPI and MitoTracker Orange, were used to stain the compared to that in Fig. 2(E). The clear appearance of isolated
nuclei and mitochondria, respectively. As shown in Fig. 2(E), the original orange and red fluorescences implies that the DOX was
orange fluorescence of the MitoTracker and the red mainly localized in cytoplasm. Thus, it could be concluded that
fluorescence of DOX in the DOX/GA-Fe
overlapped in the merged image, which shows a clear tango mitochondria. This result would be related with the targeting
fluorescence due to co-coloration, whereas the blue behaviors of DOX-loaded NH -MSNs on HeLa cells, which was
fluorescence of DAPI remained unchanged. This result reported that the amine-modified silica nanoparticles were not
3 4 2 3 4
O @MSNs almost DOX/NH -Fe O @MSNs could not be selectively accumulated in
2
DOX/GA-Fe
Mitotracker DAPI
(orange) (blue)
3 4
O @MSNs
DOX/TPP-Fe
Mitotracker DAPI
(orange) (blue)
3 4
O @MSNs
(
A)
DOX
red)
Merged
DOX
(red)
Merged
(
0
5
N
10
200 nm
2
μm
(
B)
30
60
DOX/GA-Fe O @MSN
3
4
N
120
Mitochondrial
membrane
180
(min)
2
μm
200 nm
Figure 3. (A) Bio-TEM images of free HeLa cell and (B) those of HeLa
Figure 4. Accumulation time measurement by CLSM images of the
HeLa cells treated with DOX/TPP-Fe @MSNs and DOX/GA-
Fe @MSNs, respectively (100 μg/mL, scale bar: 20 μm).
−
1
cell treated with DOX/GA-Fe
nucleus).
3
O
4
@MSNs (500 μg mL ) for 12 h (N:
3 4
O
3 4
O
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 11
J Po lue ran sael od fo Mn aot te rai ad l jsu Cs ht emm ai rs gt ri ny sB
Journal Name
ARTICLE
localized in nucleus and mitochondria.43,50 These results bonding interactions. This association leads toVietwhAerticclehOanrlginee
indicate that the charge effect of amine-modified nanoparticles neutralization and subsequently hydDrOoI:p1h0i.l1i0c3it9y/C8ToBf013t5h8eF
could not induce mitochondrial targeting ability. The merged guanidinium groups are reduced. The neutralized non-polar
29, 30
image in Fig 2(D) showed that somewhat constant distribution complex crosses the bilayer of the cell.
of red dots from DOX in DOX/TPP-Fe @MSNs, however, co-
localization level was lower than that in Fig 2(E), suggesting that
the mitochondria-targeting ability of GA-Fe @MSNs is higher
than that of TPP-Fe @MSNs. These CLSM analyses indicated
3 4
O
3 4
O
3 4
O
that the guanidinium moiety played a critical role in the
selective targeting of mitochondria.
In order to provide further visual evidence of the selective
targeting of mitochondria of the DOX/GA-Fe @MSNs, TEM
3
O
4
images of free HeLa cell and DOX/GA-Fe O @MSNs-treated
3 4
HeLa cell slices were observed after cross section with
ultramicrotome to observe the insides of the mitochondria in
detail. HeLa cells (10,000,000) were incubated with DOX/GA-
−
1
Fe
so as to obtain cell slices. As shown in Fig. 3B, the DOX/GA-
Fe @MSNs selectively accumulated inside the mitochondria,
which exhibited an inherent double membrane; this was
consistent with the results of the CLSM analyses, which showed Figure 5. Fluorescence intensity of DOX of isolated mitochondria
selective accumulation of DOX/GA-Fe
@MSNs in in HeLa cells treated with free DOX, DOX/Fe @MSNs, DOX/GA-
Fe @MSNs, and DOX/TPP-Fe @MSNs (100 μg mL ) for 2 h.
3 4
O @MSNs (0.5 mg mL ) for 24 h, and then the cells were cut
3 4
O
3
O
4
3 4
O
−
1
3
O
4
3 4
O
mitochondria. A few particles that were found to exist outside
of the mitochondria could have leaked out due to a cracking of
the double membrane of the mitochondria after they have been
damaged by the DOX. The rattle-type DOX/GA-Fe
the mitochondria were observed to have an average diameter In order to further prove the mitochondria-targeted
of ca. 60 nm; these may have become somewhat distorted and accumulation of the DOX/GA-Fe @MSNs, a fluorescence
Fluorescence spectroscopic analysis of isolated mitochondria
3 4
O @MSNs in
3 4
O
swollen by the biodegradation of the silica shells via the spectroscopic analysis of the isolated mitochondria was
hydrolysis and subsequent ion exchange mechanism in the performed based on the fluorescence of the DOX. Free
52–54
cells.
Typically, the biodegradation of MSNs is very slow, mitochondria, free DOX-treated mitochondria, and
@MSNs without
occurred relatively fast. Furthermore, several damaged areas guanidinium were used as control groups. TPP-conjugated
could be seen in the image of the DOX/GA-Fe @MSNs- Fe @MSNs (TPP-Fe @MSNs) were also prepared (Fig. S7).
treated HeLa cell; these could not be found in those of the free DOX was loaded into the TPP-Fe @MSNs in order to compare
HeLa cell (Fig. 3). This cell damage was considered to be due to its efficiency for the selective accumulation into mitochondria
however, that of MSNs which were not calcinated can be mitochondria treated with the DOX/Fe
3 4
O
5
5
3
O
4
3
O
4
3 4
O
3 4
O
the cell dysfunction caused by the DOX.
3 4
with that of the DOX/GA-Fe O @MSNs. TPP is known to
The accumulation times of the DOX/GA-Fe O @MSNs and selectively and effectively accumulate into mitochondria due to
3 4
DOX/TPP-Fe
investigated by a CLSM analysis (Fig. 4). In the CLSM images, the alter any of the genes or protein structures; it is the most
DOX/GA-Fe @MSNs in the mitochondria were visualized commonly used moiety for preparing mitochondria-targeting
over a period of 5 min. To the best of our knowledge, this is the drug delivery systems.
3 4
O @MSNs in the mitochondria were also the negative potential gradient of the organelle, and it does not
3 4
O
25, 39–43
HeLa cells (10,000,000) were
-1
shortest accumulation time used for mitochondria-targeting incubated with each of the materials (100 μg mL ) for 2 h, and
nanoparticles (Table S1).3
9–43, 56–66
However, the red then mitochondria isolated from the HeLa cells according to the
4
2
3 4
fluorescence of DOX in DOX/TPP-Fe O @MSNs was shown after previously reported process.
3
0 min. The gap of accumulation time between DOX/GA- As shown in Fig. 5, the DOX/GA-Fe
Fe @MSNs and DOX/TPP-Fe @MSNs would be due to the chondria exhibited remarkably higher fluorescence intensity
delivery pathway of TPP-attached nanoparticles. A previously than the control groups; this indicated that the DOX/GA-
reported literature revealed that TPP-attached nanoparticles Fe @MSNs had successfully accumulated into the
are not directly delivered to mitochondria, but they are mitochondria. Furthermore, the gap between the fluorescence
3 4
O @MSNs-treated mito-
3
O
4
3 4
O
3 4
O
51
delivered to mitochondria by way of endosome. The intensity of the free mitochondria and that of the mitochondria
introduction of the guanidinium moiety may have effectively treated with the free DOX or the DOX/Fe @MSNs was
accelerated the accumulation of the particles into the negligible; this suggested that the free DOX and
mitochondria. A hypothesis for the acceleration, called DOX/Fe @MSNs were unable to selectively target the
adaptive translocation”, is that the polycationic guanidinium- mitochondria. We therefore confirmed that guanidinium is
3 4
O
3 4
O
“
rich nanoparticle forms a complex with negatively charged important for mitochondria targeting in this system. We also
phosphate groups on the surface of the cell using hydrogen noted that the fluorescence intensity of the DOX/GA-
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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ARTICLE
Journal Name
Fe @MSNs-treated mitochondria was about 2.6 times higher HeLa cells; more than 80% of them were killed after being
3 4
O
View Article Online
@MSNs (DOX/TPP- treated with 1000 μg mL− of DOX/GA-Fe
Fig. 6(A)). However, the treatment of cells with DOX-loaded
nanocarriers without mitochondrial targeting GA
DOX/Fe @MSNs) showed much less efficient cell death
1
DOI: 10.1039/C8TB01358F
O @MSNs for 24 h
than that of the DOX-loaded TPP-Fe
3
O
4
3 4
(
HeLa
(
3 4
O
(A)
activity as only 50% of HeLa cells were killed (Fig. 6(A)).
Moreover, DOX/GA-Fe3O4@MSNs elicited dramatically
reduced cytotoxicity to the 293T normal cells (Fig. 6(B)). This
3 4
occurred because the DOX/GA-Fe O @MSNs were able to
selectively accumulate into the mitochondria through an
effective endocytosis process utilizing electrostatic interactions.
After the mitochondria transmembrane process induced by the
guanidinium moiety, the DOX/GA-Fe
accumulated into the mitochondria, and DOX in the DOX/GA-
Fe @MSNs were able to interact with the adenine-thymine
3 4
O @MSNs successfully
3 4
O
base pair of the mitochondrial DNA (mtDNA); this led to the
dysfunction of the mitochondria and subsequent death of the
67
2
93T
HeLa cells. These cell viability assays demonstrated that the
GA-Fe @MSNs have the potential to be used as
(
B)
3 4
O
biocompatible, non-lethal, and effective mitochondria-
targeting nanocarriers for cancer therapy.
Conclusions
Fe
targeting nanocarriers could be developed. A guanidinium
derivative was grafted onto the surface of Fe @MSNs so as
3 4
O @MSNs were prepared so that novel mitochondria-
3 4
O
to act as a mitochondria-targeting functional group, and DOX
was loaded into the nanoparticles as anti-cancer agent. The
selective targeting property of the DOX/GA-Fe O @MSNs for
3 4
mitochondria in HeLa cells was successfully demonstrated by a
co-localization investigation using CLSM, TEM, and fluorescence
spectroscopic analyses of isolated mitochondria. The CLSM
study showed that the accumulation of the DOX/GA-
Figure 6. Cell viability tests of the HeLa (A) and 293T (B) cells
treated
DOX/Fe
with
Fe
3
O
4
@MSNs,
3 4
GA-Fe O @MSNs,
3 4
Fe O @MSNs in the mitochondria was visualized within 5 min,
3
O
4
@MSNs, or DOX/GA-Fe O
3 4
@MSNs for 24 h. Data are
which was considered to be the shortest accumulation time
reported to date. Furthermore, in the fluorescence
spectroscopic analyses of the isolated mitochondria, the
presented as the mean ± SEM of two independent experiments
performed in triplicate. Asterisks indicate statistically significant
values (* P < 0.05; ** P < 0.001).
DOX/GA-Fe
mitochondria far more efficiently than the DOX/TPP-
Fe @MSNs could. The DOX/GA-Fe @MSNs exhibited high
cytotoxicity for the HeLa cells, whereas the GA-Fe @MSNs
without DOX did not exhibit cytotoxicity; this indicated that the
GA-Fe @MSNs are biocompatible and could acted as a drug
carrier that would avoid cell death. As a result, we believe that
the proposed system shows promise as a platform for future
clinical medication, especially for specific suborganelle-targeted
drug delivery systems.
3 4
O @MSNs were found to be able to target
3 4
Fe O @MSNs)-treated mitochondria; this indicated that
guanidinium was much more efficient than TPP for
mitochondria targeting.
3
O
4
3 4
O
3 4
O
Evaluation of cytotoxic effect of DOX/GA-Fe
3 4
O @ MSNs
3 4
O
In order to evaluate the cytotoxicity of DOX/GA-Fe
3 4
O @MSNs
in both cancer and normal cells, a CellTiter-Glo® cell viability
assay was performed. For the assay, HeLa cervical carcinoma
and 293T human kidney normal cells were incubated with the
Fe
3
O
4
@MSNs, GA-Fe
@MSNs under concentrations of 0.1–1000 μg
mL for 24 h. For the GA-Fe @MSNs without the DOX, no
3 4 3 4
O @MSNs, DOX/Fe O @MSNs, or
DOX/GA-Fe
3 4
O
−
1
3 4
O
Conflicts of interest
There are no conflicts to declare.
obvious cytotoxic effects were observed in both HeLa and 293T
cells; over 95% of these cells maintained viability over the entire
range of tested concentrations, which indicates the non-
cytotoxicity and good biocompatibility of the GA-Fe
3
O
O
3 4
4
@MSNs.
@MSNs
3 4
O @MSNs) exhibited high cytotoxic activity to the
Acknowledgements
(Fig. 6(A) and (B)). In contrast, DOX-loaded GA-Fe
(DOX/GA-Fe
8
| J. Name., 2012, 00, 1-3
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2
3
3
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0 E. G. Stanzl, B. M. Trantow, J. R. Vargas and P. A. Wender, Acc.
Chem. Res., 2013, 46, 2944 2954.
This work was supported by
a
grant from NRF
2
DOI: 10.1039/C8TB01358F
(2017R1A4A1014595 and 2015R1A2A2A05001400).
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Journal of Materials Chemistry B
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DOI: 10.1039/C8TB01358F
Non-peptidic guanidinium derivative-functionalized nanoparticle as a drug nanocarrier possessing
unprecedentedly high mitochondria-targeting efficiency in HeLa cell was developed.