A new strategy for intracellular delivery of enzyme using mesoporous silica nanoparticles: Superoxide dismutase

Article abstract of DOI:10.1021/ja3105208

We developed mesoporous silica nanoparticle (MSN) as a multifunctional vehicle for enzyme delivery. Enhanced transmembrane delivery of a superoxide dismutase (SOD) enzyme embedded in MSN was demonstrated. Conjugation of the cell-penetrating peptide derive

Full text of DOI:10.1021/ja3105208

Article
pubs.acs.org/JACS
A New Strategy for Intracellular Delivery of Enzyme Using
Mesoporous Silica Nanoparticles: Superoxide Dismutase
Yi-Ping Chen,† Chien-Tsu Chen,‡ Yann Hung,† Chih-Ming Chou,‡ Tsang-Pai Liu,§,∥ Ming-Ren Liang,†
Chao-Tsen Chen,† and Chung-Yuan Mou*,†
†Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
‡Department of Biochemistry, Taipei Medical University, Taipei 106, Taiwan
§Division of General Surgery, Department of Surgery, Mackay Memorial Hospital, Taipei 106, Taiwan
∥Mackay Medicine, Nursing and Management College, Taipei 106, Taiwan
S
* Supporting Information
ABSTRACT: We developed mesoporous silica nanoparticle
(MSN) as a multifunctional vehicle for enzyme delivery.
Enhanced transmembrane delivery of a superoxide dismutase
(SOD) enzyme embedded in MSN was demonstrated.
Conjugation of the cell-penetrating peptide derived from the
human immunodeficiency virus 1 (HIV) transactivator protein
(TAT) to mesoporous silica nanoparticle is shown to be an
effective way to enhance transmembrane delivery of nano-
particles for intracellular and molecular therapy. Cu,Zn-
superoxide dismutase (SOD) is a key antioxidant enzyme
that detoxifies intracellular reactive oxygen species, ROS, thereby protecting cells from oxidative damage. In this study, we fused a
human Cu,Zn-SOD gene with TAT in a bacterial expression vector to produce a genetic in-frame His-tagged TAT-SOD fusion
protein. The His-tagged TAT-SOD fusion protein was expressed in E. coli using IPTG induction and purified using FMSN-Ni-
NTA. The purified TAT-SOD was conjugated to FITC-MSN forming FMSN-TAT-SOD. The effectiveness of FMSN-TAT-SOD
as an agent against ROS was investigated, which included the level of ROS and apoptosis after free radicals induction and
functional recovery after ROS damage. Confocal microscopy on live unfixed cells and flow cytometry analysis showed
characteristic nonendosomal distribution of FMSN-TAT-SOD. Results suggested that FMSN-TAT-SOD may provide a strategy
for the therapeutic delivery of antioxidant enzymes that protect cells from ROS damage.
carrier for delivery of protein; engineered multifunctional
nanoparticles with targeting specific cells, cell-tracking signal,
and cargo-delivery capacity have been extensively developed,
and its extension to carry protein as cargo would certainly be
very interesting; and (b) fusogenic peptides (TAT) to increase
cell uptake and avoid endosome trapping; the human
immunodeficiency virus 1 (HIV) TAT peptide containing a
small region of residues YGRKKRRQRRR (TAT peptide) had
been reported as one of the most effective carrier peptides,
which has been proven to effectively translocate proteins across
cell membrane in an apparently energy-independent manner.
The TAT peptide is able to mediate nanoparticles across the
blood−brain barrier (BBB) and locate them around the cell
nucleus of neurons.8
INTRODUCTION
■
Intracellular protein delivery is receiving increasing attention
for treatment of protein deficiency, mutation, and misfolding
diseases such as amyotrophic lateral sclerosis (ALS) and
Parkinson’s Disease.1−3 To restore biological function of
proteins relevant to protein abnormal diseases, protein-based
therapy has been developed.4 The idea is to repair the protein-
mediated diseases through the delivery of normal protein in
vitro or in vivo. Some strategies have been used to manipulate
cell functions by influencing signaling pathways.5,6 For example,
we can regulate the biomarkers of p53 and caspase to turn on/
off the cell death for the diseases of apoptosis pathways.
Delivering antigens to a specific site to stimulate a desired
immune response has been explored.7 Protein therapy offers a
new direction for the next generation of medicine.
However, applications of protein-based therapies remain rare,
partly due to the intrinsic properties of most proteins: poor
stability and membrane permeability. In addition, endosome
trapping and digestion, which often occur during intracellular
protein delivery, significantly limit the treatment efficiency. In
recent years, there have been two main strategies for
overcoming these problems: (a) nanoparticles (NPs) as a
Our strategy is to combine a nanoparticle carrier and TAT
fusogenic peptide by surface functionalization of nanoparticle
and recombinant gene expression of protein (enzyme).
Mesoporous silica nanoparticle (MSN) is one of the promising
nanoparticles to serve as a multifunctional vehicle due to its
Received: October 30, 2012
Published: January 5, 2013
© 2013 American Chemical Society
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high surface area, uniform pore size, easy functionalization, and
biocompatibility. Therefore, it becomes highly suitable for
biological applications.9−12 Here, we demonstrate the con-
jugated complex with a nonpermeable antioxidant enzyme,
Cu,Zn-superoxide dismutase (SOD), will cross the cell
membrane efficiently. MSNs (with diameters between 50 and
100 nm) readily undergo an endocytosis in vitro with much
higher cellular uptake efficiency than the passive transfer or
simple diffusion of molecules across cell membranes.13 We
prepared the TAT-SOD fusion protein by a recombinant DNA
technique with built-in 6xHis codes to produce His-tag protein.
The TAT-SOD was expressed in E. coli using IPTG induction
and purified using FMSN-Ni-NTA by a one-step metal affinity
binding. Herein, we demonstrate a simple method for
preparation in aqueous solution of His-tag protein without
using organic solvents. In addition, the interactions by metal
affinity between TAT-SOD proteins and MSN nanoparticles
afford an advantage to isolate the His-tag TAT-SOD from the
whole cell lysate without any further purification step. At the
same time, the successful interactions of FMSN-Ni-NTA and
His-tag TAT-SOD enable the conjugate complex, which can be
selectively isolated as sediment, to be directly formed.
chose the 50 nm diameter MSN with functionalization of a
fluorescent organic dye, fluorescein isothiocyanate (FITC), to
form FITC-MSN (FMSN). For further conjugation of protein
to the MSN surface, we introduced Ni-NTA surface
functionality to interact with the His-tagged protein. We
modified the FMSN surface with a nitrilotriacetic acid tether,
NTA silane, which thereafter reacted with nickel metal ion
(Scheme 1).
Scheme 1. Schematic Illustration for the Ni-NTA-Modified
FMSN That Interacts Selectively with Histidine-Tagged
TAT-SOD Protein and Subsequently Gives a Protective
a
Effect on Cells against Oxidative Stress
In addition to traditional applications of mesoporous silica
such as catalysis14 and chromatography,15 biomedical applica-
tions of MSN such as enzyme immobilization,16 gene
transfection,17,18 and drug delivery agents19 have recently
gained much attention.20,21 We previously reported that MSNs
function in cell labeling13,22 and stem cell tracking.23 In this
work, a new method of conjugating protein to the surface of
MSN for delivery has been designed. We choose to study the
enzyme, Cu,Zn-superoxide dismutase (SOD),24 which is a key
antioxidant enzyme that detoxifies intracellular reactive oxygen
species (ROS), thereby protecting cells from oxidative damage.
Reactive oxygen species (ROS), such as free radicals and
peroxides, lead to oxidative damage in cells or tissue. There are
many different endogenous and exogenous sources by which
the reactive oxygen species are generated. The level of oxidative
stress is determined by the balance between the rate at which
oxidative damage is induced and the rate at which it is
efficiently repaired and removed. Antioxidant enzymes, such as
superoxide dismutase, have been found to exert a beneficial
effect against various diseases that are mediated by the reactive
oxygen species.
a
(a) First, we modified the FMSN with Ni-NTA to form FMSN-Ni-
NTA. Next, 6xHis-TAT-SOD was cloned and overexpressed by E. coli
using molecular biology method. The FMSN-Ni-NTA selectively
bound to histidine-tagged TAT-SOD protein in an E. coli cell lysate. 8
M urea was used to denature the TAT-SOD protein. (b) After the cell
delivery, the denatured TAT-SOD protein could be refolded to
catalyze with full activity.
In this study, a recombinant gene of human Cu,Zn-
superoxide dismutase (hSOD) fused with human immunode-
ficiency virus 1 (HIV) transducing peptide (TAT) was
constructed in a bacterial expression vector to overproduce a
genetic in-frame TAT-SOD fusion protein. The TAT-SOD was
conjugated to FMSN with a tether in a nickel-oligo histidine
interaction forming FMSN-TAT-SOD. The biological charac-
terization of FMSN-TAT-SOD as an agent against ROS was
investigated, which included the level of ROS and apoptosis
after free radicals induction, functional recovery after ROS
damage, and activation of caspase-9 and p21 in culture cells. We
will show that FMSN-TAT-SOD may provide a useful strategy
for the therapeutic delivery of antioxidant enzymes that protect
cells from ROS damage.
For the subsequent experiment of FMSN-SOD conjugation,
we overexpressed and purified the His-tag human Cu,Zn-
superoxide dismutase (SOD) protein containing a human
immunodeficiency virus 1 (HIV) transducing domain (Tat 49−
57; TAT) from a human TAT-SOD gene in-frame constructed
in pQE-30 vector (pQE-TAT-SOD) as described in Chou et al.
(2005) (Figure 1a).26 The construct was transformed into
JM109 E. coli cells and cultured in LB broth with IPTG
induction. The harvested bacterial cells were lysed at 4 °C in a
10% glycerol/ddH2O buffer. Protein content in the crude cell
extract (supernatant) obtained from the lysate was determined,
and then the overexpression of TAT-SOD was confirmed with
electrophoresis in 12% SDS-PAGE. The protein bands were
visualized by staining with Coomassie brilliant blue G250
showing a major 23 kD band (Figure 1b). The band marked by
an arrow in lane 2 (1 h), lane 3 (2 h), and lane 4 (3 h), in
comparison with that of the lane 1 (0 h), suggested that the
SOD protein was expressed at increasingly higher level with
increasing induction time. A major problem for efficient
delivery of a MSN-mediated agent into the cytoplasm of living
RESULTS AND DISCUSSION
■
Previously, we have reported the synthesis of size-controlled
multifunctional mesoporous silica nanoparticle (MSN) and
found that MSN of 50 nm gives the highest cell-uptake.25 For
the purpose of tracking the uptaken MSN in the cells, we thus
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immobilized TAT-SOD via formation of Nickel-6xHis
interaction on the FMSN surface was isolated by centrifugation
at 7000 rpm for 20 min and subsequently purified by removing
the supernatant. This procedure affords an easy and clean
separation of the FMSN-TAT-SOD protein conjugates. The
Western blotting assay was used to identify the SOD molecule
by a polyclonal antibody against human SOD and confirm the
protein associated with FMSN. Results (Figure 1c) indicated
that the SOD protein dissociated from FMSN by the imidazole
elution was clean, and the immunological recognition suggested
the SOD protein attached to FMSN through metal affinity
interaction. This conjugated complex has the following
advantages: (a) high affinity resulting in quantitative binding
and stability, (b) specificity for His-tag engineered proteins, and
(c) reversible binding, which could be dissociated by imidazole.
After the one-step purification, the TAT-SOD molecule was
denatured by treating FMSN-TAT-SOD with 8 M urea. Here,
we would like to mention that the SOD in as-synthesized
FMSN-TAT-SOD, FMSN-SOD, and TAT-SOD were all in
denatured form for subsequent experiments.
To further investigate the amount of TAT-SOD and Ni
attached on the MSN, the Bradford method and ICP-MS
elemental analysis were used, respectively. The amount of SOD
protein attached on FMSN was determined by measuring the
SOD molecules dissociated in the supernatant through the
imidazole buffer elution. We determined an average weight
percent of TAT-SOD in FMSN-TAT-SOD at 16.7 wt % and Ni
at 0.3 wt %. Thus, the Ni to SOD ratio is about 7:1. The
transmission electron microscopic (TEM) image in Figure 1d
shows the FMSN-TAT-SOD possesses an average diameter of
50 nm and well-ordered mesopores. The zeta potential is in the
range from −30 to −35 mV. The surface functionalizations of
TAT, NTA, and SOD had very little influence on the total
charge of the FMSN (see Figure S5).
We then proceed to evaluate the delivery efficiency of
FMSN-TAT-SOD into cells in vitro. FMSN-TAT-SOD was
added to the culture media of HeLa cells and incubated for 4 h.
Harvested cells were lysed and treated with 500 mM imidazole.
Western blotting of cell lysate was carried out to analyze the
content of cellular SOD (Figure 2a). The control cells without
any external loading of SOD show a weak presence of intrinsic
SOD. In comparison, cells with treatments of FMSN-TAT-
SOD give a very strong SOD signal, which indicated the TAT-
SOD protein has been transduced into the HeLa cells. To
determine the cellular localization of the transduced TAT-SOD
proteins, FMSN-TAT-SOD treated HeLa cells were fixed for
the immuno-cytostaining with DNA-specific DAPI (blue) and
anti-SOD antibody (red). Confocal microscopy observations
(Figure 2b) show colocalization image (yellow) of FMSN
(green) and SOD protein (red) in the FMSN-TAT-SOD-
delivered cells, whereas the cells with FMSN alone show only
green signal from the FITC fluorescence. In the cells treated
with FMSN only (upper row of Figure 2b), the fluorescence
signal of endogenous SOD protein was apparently low.
Figure 1. Bioconjugation of TAT-SOD with MSN nanoparticles. (a)
Construct of the pQE-TAT-SOD. The synthetic TAT oligomer was
cloned into the Bam HI site, and human Cu,Zn SOD cDNA was
cloned into Sph I and Pst I sites of vector pQE. (b) Electrophoretic
analysis of IPTG induced overexpression of Human Cu,Zn-SOD
protein. The SOD protein was induced and overexpressed from pQE-
TAT-SOD within 1, 2, and 3 h by IPTG in E. coli and analyzed by
electrophoresis in a 12% SDS-PAGE. (c) Western blotting analysis of
the purified SOD. The FMSN-Ni-NTA purified SOD shown as lane 2
as compared to E. coli total lysate (lane 1) by using Western blot
analysis with a polyclonal antibody to human SOD. (d) TEM image of
FMSN-TAT-SOD. The average diameter of MSN is 50 nm.
cells is that the nanoparticles may often be trapped in
organelles such as endosomes and lysosomes. Previously, it
has been reported that TAT-mediated delivery would result in a
nonendosomal mechanism for cell-uptake.27 The design of
FMSN-TAT-SOD was to avoid the endosomal trap inside
living cells.
The second important consideration in design is to ensure a
binding of protein to the external surface of nanoparticles
(NPs), which is convenient and specific. In this work, we take
advantage of the affinity interactions28,29 between Ni-NTA-
bearing MSN with His-tagged proteins. The affinity between
Ni-NTA and oligohistidine (Kd ≈ 10−13) has been shown to be
comparable to or superior than that of the protein−protein
interaction (Kd ≈ 10−5−10−12) such as carbohydrate−lectin or
streptavidin−biotin interactions.30−32 Therefore, the use of
such an interaction can provide a simple and sensitive method
that would overcome the limitation of conventional antibody-
based protein labeling. Particularly, we designed a novel one-
step method achieving the isolation and conjugation at the
same time. The design is based on the interaction between Ni-
NTA-NPs and His-tagged proteins.33 In the nickel bead
purification procedure, the His-tag TAT-SOD proteins are
associated with Ni(II) to form Nickel-6xHis complex, and the
metal affinity could be dissociated by adding excessive
imidazole. For one-step isolation and conjugation, the
The restoration of enzymatic activities of TAT-SOD protein
in cells is critical for the application of protein transduction for
therapeutic use. In our strategy, the denatured form of SOD
would be refolded back into the native form of SOD, which is
regulated by intracellular chaperons34 such as Hsp90.35
Therefore, we examined the SOD activities in the HeLa cells,
which had been delivered with denatured FMSN-TAT-SOD
proteins as mentioned before (Figure 2c). Here, we show that
denatured FMSN-TAT-SOD could successfully enter the HeLa
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arm. This avoids a large restriction from steric hindrance for
modification, conformational change, and interactions of metal
affinity. It also shows the delivered MSN as a multifunctional
vehicle without interfering with the biological function of
attached SOD molecules.
Previously, SOD has been reported with the biological
functions of inhibiting cell apoptosis and ROS protection in
mammalian cells.36,37 Because FMSN-TAT-SOD has been
successfully delivered and exhibits the antioxidant activity in
HeLa cells, we then determined whether FMSN-TAT-SOD
protects the cells from lipopolysaccharide (LPS) (50 μg/mL)-
induced oxidative stress and paraquet-induced free radicals.
First, we tested the effect of FMSN-TAT-SOD on cell viability
of a MTT colorimetric assay under oxidative stress induced by
LPS.38 When pretreated with FMSN-TAT-SOD, the viability of
LPS-treated cells was significantly increased in a dose-
dependent manner. As shown in Figure 3a, when the cells
Figure 2. Delivery of FMSN-TAT-SOD into HeLa cells. FMSN-TAT-
SOD and bare FMSN were added to the serum free cultured media for
4 h. (a) Cellular delivery of SOD by FMSN-TAT-SOD. The cells were
sonicated with lysis buffer containing 500 mM imidazole and analyzed
by Western blotting. (b) Immunocytochemical staining. Harvested
cells were fixed and immunocytochemical stained with DAPI (blue),
Cy3-labeled anti-SOD antibody (red), and FITC-MSN (green). HeLa
cells were first pretreated with various concentrations of MSN-TAT-
SOD, bare MSN, and TAT-SOD for 4 h. (c) The enzymatic assay of
SOD. SOD specific activity was analyzed by pyrogallol method.
(Details are in the Materials and Methods.)
Figure 3. Inhibition of apoptosis, ROS protection, and activation of
cleaved caspase-9 and p21 in HeLa cells by FMSN-TAT-SOD. HeLa
cells were first pretreated with various concentrations of FMSN-TAT-
SOD and control MSN for 4 h. (a) MTT assay. FMSN-TAT-SOD
treated cells were exposed with LPS (50 μg/mL) for 18 h and
determined the cell viability by a MTT colorimetric assay. (b)
Detection of ROS generation. FMSN-TAT-SOD treated cells
stimulated with paraquat (250 μM) and then oxidized dihydroethidine
(DHE) dye (2 μM) were determined by flow cytometry analysis. (c)
Western blotting analysis of cleaved caspase-9 and p21 protein.
FMSN-TAT-SOD treated cells exposed to LPS (100 ng/mL) for 24 h,
and the cellular levels of cleaved caspase-9 and p21 protein were
analyzed by Western blotting. The quantitative data of Western blots
were indicated by densitometric method.
cells and exhibit good cellular SOD activity. Results of the
enzyme activity of SOD show an increase in a dose-dependent
manner, which suggests that the denatured proteins were
refolded to active form through chaperon assembly. We further
examined whether the FMSN particles interfere with TAT-
SOD in its activity. As compared to the activity of a positive
control TAT-SOD (100 μg/mL), there is apparent similarity in
activity when TAT-SOD formed the conjugates with FMSN
matrix. Although there was no difference in SOD activity
between FMSN-TAT-SOD and TAT-SOD, the MSN could
serve as a multifunctional vehicle to load fluorescent organic
dye, drugs, targeting peptides, and other biomoleculars such as
siRNA. Evidently, these data confirm that the FMSN-TAT-
SOD were efficiently delivered with full activity in HeLa cells.
This is the first work reporting the MSN carrying denatured
enzyme could refold in the cell with good activity. The MSN
conjugated TAT-SOD was mediated by interactions of metal
affinity. The Ni-NTA is extended from a tether with flexible
were exposed to LPS with FMSN or without FMSN-TAT-
SOD, only ∼15% of cells stayed viable. However, the viability
of the cells doubled when pretreated with FMSN-TAT-SOD at
a high concentration. FMSN-TAT-SOD increases the cell
viability from LPS-induced oxidative stress in a dose-dependent
trend, suggesting that these fusion proteins on FMSN have a
critical protective effect on cells against oxidative stress.
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To address the protective role of FMSN-TAT-SOD against
the ROS-mediated apoptosis in HeLa cells, the cells were
stimulated with paraquat (methyl viologen), which is well-
known as an intracellular superoxide anion generator.39,40 The
amount of ROS was detected by reacting with dihydroethidine
(DHE; Invitrogen)41 and quantified by the red fluorescence
detected in flow cytometric method. As shown in Figure 3b, the
group with FMSN-TAT-SOD delivery (red) decreased 20%
mean fluorescence values as compared to the control group
with no additional treatment in paraquat stimulated cells
(black), whereas FMSN delivered cells (blue) retain the same
as the paraquet control. The nonparaquet stimulated cells
(green) as a negative control have little oxidation of DHE. This
suggested that the FMSN-TAT-SOD catalyze the superoxide
dismutation to reduce the amount of free radicals. Thus, the
risk of free radicals attacking the cells was reduced.
Most of the LPS-stimulated cell apoptosis involves activation
of the cleaved caspase-942 and p2143 protein signaling cascades
mechanism. To examine the protective effect of FMSN-TAT-
SOD on LPS-induced cleaved caspase-9 and p21 activation,
cells were treated with the FMSN-TAT-SOD (5−100 μg/mL)
for 4 h and subsequently exposed to LPS (100 ng/mL) for 24
h, and then the cellular cleaved caspase-9 and p21 was analyzed
by Western blot analysis (Figure 3c). Western blotting results
revealed that FMSN-TAT-SOD suppressed LPS-induced
cleaved caspase-9 and p21 activation in a dose-dependent
manner for HeLa cells. It indicates that FMSN-TAT-SOD was
able to remove the free radicals (or reduces oxidative stress),
which led to cell apoptosis in either caspase-9 signaling cascade
or p21 activation.
Taken together, we have shown MSN is a good nanocarrier
for the enzyme SOD. The MSN served as an inert carrier,
which does not interfere much with normal biological function.
From our experiments, we demonstrated FMSN-TAT-SOD
still retains the antioxidant activities and protection of cell from
apoptosis; ROS scavenger activity and p21 activation, which the
native SOD performs, were illustrated.
As mentioned above, the full SOD activity of a denatured
FMSN-TAT-SOD was restored through a refolding machinery.
Because the Cu,Zn-SOD is a metalloenzyme containing copper
and zinc ions, the molecular refolding to constitute the intact
structure may also need the incorporation of metal ions, which
is important for conformation and catalysis. In principle,
through the supply of the essential metal ions, the metal-
loenzymes would gain more activities if the ions are the limiting
factor in the SOD activity in cells. With the addition of those
metal ions, we examined whether the activity of FMSN-TAT-
SOD would increase after the transduction in cell and
molecular refolding. Copper ion (50 μM) and zinc ion (50
μM) were added into the HeLa cells culture and incubated for
24 h; FMSN-TAT-SOD and control FMSN were then added
for 4 h, which is usually enough for performing the enzyme
activity. The specific activity of SOD was determined by
pyrogallol auto-oxidation method.43 No significant enhance-
ment of the activity was observed in those cells treated with
additional metal ions in our experimental conditions. These
results suggest that the metal ion for full activity of SOD may
have been supplied by an endogenous source. Also in Figure 4a,
results of an SOD specific kinetic analysis revealed that the
SOD activity was markedly increased by the addition of FMSN-
TAT-SOD as compared to the FMSN-only case. Also, it is
moderately higher (40%) than the case of FMSN-SOD. That
the FMSN-SOD still retains some minor activity indicated
Figure 4. Effect of the TAT, Cu2+, Zn2+, and temperature on the
delivery activity of SOD. HeLa cells were first pretreated with FMSN-
TAT-SOD and control FMSN for 4 h. (a) Metal effect on the activity
of FMSN-TAT-SOD. Copper ion (0.05 mM) and zinc ion (0.05 mM)
were added into the culture medium and incubated for 24 h before
FMSN-TAT-SOD and control FMSN treatment. The specific activity
of SOD was determined by pyrogallol method. (b) Confocal
microscopy analysis of MSN in endosome. The living unfixed cells
were cotreated with endosome-specific marker FM 4−64 (5 μg/mL)
and analyzed by confocal microscopy for an endosomal colocalization
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mediated nonendocytosis delivery at 4 °C as compared to
FMSN-SOD.
Figure 4. continued
TAT is the signaling peptide to help the delivery and avoid
MSN-mediated SOD from the endocytosis. However, we found
the FMSN-SOD without any TAT sequence still has some
SOD activity. As shown in Figure 4a, FMSN-SOD had less
activity as compared to FMSN-TAT-SOD. FMSN-SOD and
FMSN alone would tanslocate through the endocytosis
mechanism. Therefore, possibly most FMSN-SOD were
trapped in endosomes, which resulted in loss of the activities.
TAT played an important role for the nonendocytosis
mechanism and enhanced the SOD activity significantly. We
have demonstrated the TAT-SOD-mediated translocation is
nonendosomal in contrast to FMSN alone. We further
measured the amount of delivered FMSN-SOD and FMSN-
TAT-SOD in cells, which may answer some of the questions
about why FMSN-TAT-SOD retains high cellular activity.
FMSN-SOD and FMSN-TAT-SOD were added to the serum
free culture for 4 h. The relative quantities of delivered protein
were then analyzed by Western blotting and densitometry. As
shown in Figure 4d, high density of SOD band was determined
in FMSN-TAT-SOD treated cells, indicating that FMSN-TAT-
SOD was more efficiently delivered into cells than FMSN-
SOD. The results support the FMSN carrying a TAT sequence
will perform an efficient delivery of enzyme or other cargo,
which has low endosomal trapping.
In conclusion, we have demonstrated that the MSN
conjugated with TAT-SOD gave good dismutation activity
toward superoxides in vitro. As a multifunctional vehicle, MSNs
were modified by a Ni-NTA tether for interactions of metal
affinity and by FITC for tracing the nanoparticle images. The
conjugation of TAT mediates the nonendocytosis pathway,
which MSN alone does not. To our best knowledge, this is the
first report that the SOD conjugated on nanoparticles in
denatured form was refolded after translocating in the cell. This
success in protein delivery lays the groundwork for future
complementation experiments and for eventual delivery of
therapeutic proteins into patients in the form of protein
treatment of ROS-mediated diseases.
image. The fluorescent images show the MSN (green, FITC) and FM
4−64-labeled endosomes (red). (c) Temperature effect on MSN
endocytosis. The cells were incubated at 37 or 4 °C individually, and
the cellular uptake of nanoparticles was determined by FACS analysis
for cell endocytosis activity. (d) Delivery efficiency by Western blot
analysis. Various concentrations of FMSN-TAT-SOD and FMSN-
SOD were added into the cells, and the cellular level of SOD protein
was measured in the cells by Western blotting.
some FMSN-SOD could perform nonendocytosis translocation
without the TAT sequence.
While the cellular uptake of MSN has been shown to follow
an endocytosis process,22,23 the cellular uptake of the cationic
TAT cell-penetrating peptide has been shown to follow a
nonendocytosis mechanism.44−46 In our experiment, we
constructed a combinational FMSN-TAT cargo and success-
fully delivered the SOD protein into the HeLa cells. We then
investigated possible mechanism for the uptake of the FMSN-
TAT-SOD protein. FM4−64 dyes have been widely used to
study endocytosis, vesicle trafficking, and organelle organization
in living eukaryotic cells.47 The FMSN or FMSN-TAT-SOD
(green) was cocultured with FM4−64 (red) (Molecular
Probes) to trace the distributions of certain specific molecule
in living HeLa cells. As shown in the confocal microscopic
results (Figure 4b), a punctate cytoplasmic distribution pattern
for both FMSN and FMSN-TAT-SOD was observed. For the
FMSN alone, the FMSN (green) predominantly co- localized
with FM 4−64 (red, the marker of endosome) in living cells as
indicated by the resulting yellow coloration, but the minority of
green dots still remains. However, FMSN-TAT-SOD (green)
shows a different distribution pattern from that of FM4−64,
indicating they were not colocalized. These data suggest that
the TAT peptide on FMSN-TAT-SOD allows the cargo
delivery into cells through a nonendocytosis mechanism and
avoids subsequent endosomal trapping, while FMSN alone
would remain in endosomes. Many groups used chemically
synthesized TAT peptides to conjugate on nanoparticles to
reach the nonendocytosis mechanism approach. This work is
the first to develop a protein expression system by molecular
cloning method combined with nanoparticles for TAT-
conjugation and delivery.
The efficiency of endocytosis is limited by the low lipid
fluidity and available energy. Further evidence from cellular
uptake at low temperature also illustrated some part of the
FMSN-TAT-SOD delivery is an energy-independent process to
drive a nonendocytosis translocation. The cellular uptake of
FMSN-SOD and FMSN-TAT-SOD was measured by flow
cytometry at 4 and 37 °C for 1 and 4 h. As shown in Figure 4c,
the delivery efficiency of FMSN-TAT-SOD (90−92%) was
better than FMSN-SOD (78−80%) at 37 °C for 1 and 4 h,
which suggests that the TAT enhances the delivery more
effectively. Experiments on both nanoparticles at 4 °C for 1 and
4 h showed a similar result (FMSN-TAT-SOD of 38−40% vs
FMSN-SOD of 18−21%). However, the delivery efficiency was
reduced at 4 °C because the low temperature decreased the
endocytosis pathway. The 20% increase of uptake of FMSN-
TAT-SOD as compared to FMSN-SOD at 4 °C indicates that
the TAT provides a nonendocytosis delivery. Furthermore, our
results from the cellular uptake experiment illustrated that the
FMSN-TAT-SOD delivery at low temperature is still active.
FMSN-TAT-SOD enhanced the uptake of cells through TAT-
METHODS
■
Materials and Methods. All chemicals were purchased from
commercial suppliers (Acros, Aldrich, Sigma, and Merck) and were
used without further purification.
Synthesis of Green Fluorescent Mesoporous Silica Nano-
particles. Following the literature method, C16TABr (0.58 g, 1.64 ×
10−3 mol) and 5 mL of 0.226 M ethanol solution of TEOS (1 mL of
TEOS in 20 mL of 99.5% ethanol) were dissolved in 300 g of 0.51 M
aqueous ammonia solution. The stock solution was stirred at 50 °C for
5 h. Five milliliters of 1.13 M ethanol solution of TEOS (5 mL of
TEOS in 20 mL of 99.5% ethanol) and FITC-APTMS was added with
vigorous stirring for 1 h and then aged statically at 50 °C for 24 h.
FITC-APTMS, N-1-(3-trimethoxysilyl propyl)-N′-fluoreceyl thiourea),
was prepared in advance by stirring fluorescein isothiocyanate (FITC,
1 mg) and 3-aminopropyltrimethoxysilane (APTMS, 5 μL) in 5 mL of
ethanol (99%) at room temperature for 24 h. As-synthesized samples
were then collected by centrifugation at 18 000 rpm for 10 min and
washed five times with 99% ethanol. 200 mg of as-synthesized samples
was redispersed in 25 mL of 95% ethanol with 0.5 g of 37 wt % HCl.
Surfactant was extracted by heating the ethanol suspension at 60 °C
for 24 h. The product, called FITC-MSN (FMSN), was collected by
centrifugation, washed with ethanol several times, and stored in sterile
water or ethanol.
Conjugation of NTA-Silane and Ni(II) with MSNs (FMSN-Ni-
NTA). As-synthesized FMSN with template surfactants (50 mg)
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suspended in toluene (50 mL) containing NTA-silane (25 mg) were
stirred under reflux for 18 h. The products were collected by
centrifugation and washed with ethanol three times to eliminate
unreacted silane. To remove the surfactants, the products were
suspended in acidic ethanol (1 g of HCl in 50 mL of EtOH) and
refluxed for 24 h. Subsequently, hydrolysis of methoxycarbonyl on
NTA linker was achieved in the presence of aqueous ρ-TsOH (0.133
g, pH = 2) under stirring at 65 °C for 6 h. The products, called FMSN-
NTA, were collected following the same procedure described above.
Next, FMSN-NTA suspended in 50 mL of aqueous NiCl2 (50 mM)
was stirred at 25 °C for 6 h. Finally, the nickel coordinated FMSN-
NTA (FMSN-Ni-NTA) was obtained after excess NiCl2 was removed
by ethanol washing.
Immobilization of His-TAT-SOD Proteins with FMSN-Ni-NTA.
A crude cell lysate of E. coli containing His-TAT-SOD proteins was
mixed and incubated with FMSN-Ni-NTA at 4 °C overnight. After
that, the His-TAT-SOD conjugated with FMSN-Ni-NTA (FMSN-
TAT-SOD) was isolated by centrifugation and washed several times
with water as well as ethanol. Afterward, the FMSN-TAT-SOD was
suspended and stored in ethanol at 4 °C.
Cell Lines and Reagents. The HeLa cell line was obtained from
the American Type Culture Collection (Manassas, VA). Restriction
endonucleases and T4 DNA ligase were purchased from Promega
(Madison, WI). Oligonucleotides were synthesized from Gibco BRL
(Gaithersburg, MD) customized primers. Isopropyl-L-D-thiogalacto-
pyranoside (IPTG) was obtained from Promega. Escherichia coli strain
JM109 (DE3) was obtained from Stratagene (La Jolla, CA). Human
Cu,Zn-SOD cDNA fragment was isolated using the polymerase chain
reaction (PCR) technique using the human liver cDNA library.
Polyclonal antibodies raised against human Cu,Zn-SOD were
produced in our laboratory.
Isolation of the Full-Length Human Cu,Zn-SOD. Total RNA
was isolated from human liver using the RNAzol reagent (Tel-Test,
Friendswood, TX) according to the instructions of the manufacturer.
To isolate the cDNA covering the complete open reading frame
(ORF) of human Cu,Zn-SOD according to the sequences of one
human expressed sequence tag (accession number K00065, Genbank),
the full-length human Cu,Zn-SOD was amplified with a Cu,Zn-SOD
sense primer, CuZn-SOD-F, 5′-ATG GCG ACG AAG GCC GTG
TGC GTG CTG-3′, and a Cu,Zn-SOD antisense primer, CuZn-SOD-
R, 5′-TTA TTG GGC GAT CCC AAT TAC ACC ACA-3′. PCR
amplification was performed in a 50 mL reaction mixture containing 2
mL of first-strand cDNA, 0.5 mg of primers, 1.5 mM MgCl2, 0.2 mM
dNTP, and 2.5 U of HiFi-DNA polymerase (Yeastern Biotech, Taipei,
Taiwan). Samples were incubated in a thermal cycler (Hybaid
MultiBlock System; Hybaid Limited, Franklin, MA). The cDNA
from brain template was used for PCR amplification using the program
of 94 °C for 3 min; 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72
°C for 60 s; and a final extension step at 72 °C for 15 min. All products
were ligated into pGEM-T easy vector (Promega) and subjected to
sequence analysis.
its optical density at 600 nm reached 1.2. To induce expression of
these recombinant proteins, IPTG was added to a final concentration
of 1.0 mM, and the incubation was continued for 1−3 h. All
recombinant proteins were collected and purified with FMSN-Ni-NTA
by metal affinity binding.
Cell Culture. HeLa cells, a human epithelial cervical cancer cell
line, were maintained in Dulbecco’s modified eagles medium
supplemented (DMEM; GIBCO) with 10% fetal bovine serum
(FBS; GIBCO), 100 U/mL penicillin, and 100 μg/mL streptomycin
(GIBCO) at 37 °C in a humidified and 5% CO2 atmosphere. When
adherent cells reached ∼60−70% confluence, they were detached with
0.25% trypsin-EDTA growth medium to allow for continued
passaging.
Western Blotting Analysis. Cell lysates were separated on a 10%
SDS-PAGE, and the proteins were then electrophoretically transferred
to a polyvinylidene difluoride (PVDF) membrane and blocked 1 h at
toom temperature in blocking buffer [1xTris-buffered saline (TBS)−
0.1% Tween 20, 5% w/v nonfat dry milk]. Membranes were incubated
overnight at 4 °C with primary antibodies:cleaved-caspase 9 and p21
from Santa Cruz Biotechnology (Santa Cruz, CA), along with α-
tubulin from Oncogene Science. All primary antibodies were diluted in
dilution buffer [1xTris-buffered saline (TBS)−0.1% Tween 20, 5% w/
v BSA]. The PVDF membranes were extensively washed and
incubated with a horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulin G antibody (1:2000 dilution, Rockland, Inc.) for 1 h
at room temperature. Immunoreactive bands were visualized with the
enhanced chemiluminescence substrate kit (Amersham Pharmacia
Biotech, GE Healthcare UK Ltd., Bucks, UK) according to the
manufacturer’s protocol.
Immunocytochemical Staining for TAT-SOD Expression.
HeLa cells were fixed with 4% paprformaldehyde, and permeabilized
with 0.1% Triton X-100. After being washed in PBS, cells were blocked
with 3% skim milk for 1 h and then incubated with a polyclonal anti-
human Cu,Zn-SOD antibody for 16 h at 4 °C. The cells were
extensively washed and incubated with rabbit antihuman Cu,Zn-SOD
antibody and visualized with antirabbit Cy3-labeled secondary antibody
at a 1/1000 dilution for 2 h, and counterstained for nuclei with DAPI
(DNA marker) for 5 min. The fluorescent images were obtained on a
confocal microscope (TCS SP5, Leica).
Determination of Superoxide Dismutase Activity. Superoxide
dismutase (SOD) activity of cell lysates was measured according to the
method of Marklund.48 Superoxide anion radical is involved in the
auto oxidation of pyrogallol. At alkaline pH, SOD dismutates
superoxide, thereby inhibiting the auto-oxidation of pyrogallol. The
stock pyrogallol solution contained 1.0 mL of 0.5 M HCl and was
stored at 30 °C. The assay mixture, containing 500 μL of buffer (50
mM Tris−cacodylic acid buffer, pH 8.4, 1 mM diethylenetriamine
pentaacetic acid, 1 mM potassium phosphate buffer, pH = 7.0), 50 μL
of sample, and 400 μL of deionized water, was put into a cuvette. The
control sample contained 500 μL of assay buffer and 450 μL of water.
The optical density of each sample was measured at 420 nm before the
addition of pyrogallol. The increase in absorbance was measured at 10
s intervals and lasted for 3 min. SOD specific activity was expressed as
units per milligram (U/mg) of protein.
Construction of Expression Clones. The expression plasmid
pQE-SOD was constructed by inserting the full-length human Cu,Zn-
SOD cDNA into pQE30 at the SphI and PstI sites, which allows
generation of the Cu,Zn-SOD protein with in-framed His-tag at the N-
terminal end. The pQE-TAT-SOD expression vector was constructed
to express the basic domain (amino acids 49−57) of HIV-1 TAT as a
fusion with Cu,Zn-SOD as follows. In brief, two oligonucleotides were
synthesized and annealed to generate a double-stranded oligonucleo-
tide encoding nine amino acids from the basic domain of HIV-1 TAT.
Their sequences are (BamHI-TAT-F) 5′-GAT CCG GAA GAA GCG
GAG ACA GCG ACG AAG ACG-3′ and (BamHI-TAT-R) 5′-GAT
CCG TCT TCG TCG CTG TCT CCG CTT CTT CCG-3′. The
double-stranded oligonucleotide was directly ligated into the BamHI-
digested pQE-SOD to generate the His-TAT-SOD expression plasmid
pQE-TAT-SOD.
Cell Viability Assay. The 1 × 105 cells per well were seeded in 24-
well plates for proliferation assays. After incubation with different
amounts of nanoparticles suspended in serum-free medium for 4 h,
respectively, then the lipopolysaccharide (LPS; 50 μg/mL; Sigma) was
added to the culture medium for 18 h. Cell viability was estimated by a
colormetric assay using MTT [ 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (0.5 mg/mL)]. The dark blue formazan
dye generated by the live cells was proportional to the number of live
cells, and the absorbance at 570 nm was measured using a microplate
reader (Bio-Rad, model 680). Cell numbers were determined from a
standard plot of known cell numbers versus the corresponding optical
density.
Expression and Purification of Recombinant Proteins. pQE-
TAT-SOD was expressed in E. coli JM109. A colony of E. coli cells was
separately inoculated into Luria−Bertani broth in the presence of 100
mg/mL ampicillin, and the culture was grown overnight at 37 °C until
Superoxide Detection. The production of superoxide anion was
fluorometrically estimated using a fluorescent probe, dihydroethidine
(DHE), which is oxidized to a fluorescent intercalator, ethidium, by
cellular oxidants, in particular, superoxide radicals. To measure
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superoxide anion generation, dihydroethidine (DHE; 2 μM;
Invitrogen) was used. Increase in DHE fluorescence upon paraquat
(superoxide anion generator) stimulation indicated an increase in
superoxide anion levels. After various experimental treatments, cells
were incubated with DHE for 20 min and trypsinized, followed by
cytofluorimetric analysis with a FACS Calibur flow cytometry (BD
Biosciences).
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental methods for obtaining powder X-ray diffraction,
nitrogen adsorption−desorption isotherms, TEM micrographs,
flow cytometry analysis, and endosome staining and results in
Figures S1−S5. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
cymou@ntu.du.tw
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Science Council of
Taiwan through the National Nanotechnology Program. We
thank Mr. Si-Han Wu, Dr. Fan-Ching Chien, and Prof. Peilin
Chen for technical help.
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