Radio-opaque theranostic nanoemulsions with synergistic anti-cancer activity of paclitaxel and Bcl-2 siRNA

Article abstract of DOI:10.1039/c3ra40883c

The integration of therapeutic and imaging functions in a nanoscale structure is gaining increasing attention to monitor drug and gene delivery in a non-invasive real-time manner. Here we introduce radio-opaque nanoemulsions incorporating paclitaxel and Bcl-2 small interfering RNA (siRNA) for synergistic anti-cancer theranostics. Water-insoluble paclitaxel is readily dissolved in iodinated poppy seed oil (Lipiodol) and emulsified in an aqueous solution using a mixture of PEGylated phospholipids, cholesterols, and linear polyethylenimine grafted with cholesterols. The prepared cationic nanoemulsions were electrostatically complexed with Bcl-2 siRNA for the co-delivery of paclitaxel and siRNA, inducing a dramatically higher level of apoptosis and cytotoxic activity in breast adenocarcinoma compared to individual treatments. The feasibility of the nanoemulsions for bio-imaging applications is preliminarily investigated using micro-computed tomography with a mouse model. This study suggests that the Lipiodol nanoemulsions can be used as a multifunctional nanocarrier platform for the co-delivery of anti-cancer drugs and siRNA with bio-imaging functionality to efficiently increase the modality of anti-cancer theranostics. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra40883c

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Radio-opaque theranostic nanoemulsions with
synergistic anti-cancer activity of paclitaxel and Bcl-2
Cite this: RSC Advances, 2013, 3,
1
4642
siRNA3
ac
b
b
a
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Mi Hwa Oh, Jee Seon Kim, Jeong Yu Lee, Tae Gwan Park and Yoon Sung Nam*
The integration of therapeutic and imaging functions in a nanoscale structure is gaining increasing
attention to monitor drug and gene delivery in a non-invasive real-time manner. Here we introduce radio-
opaque nanoemulsions incorporating paclitaxel and Bcl-2 small interfering RNA (siRNA) for synergistic
anti-cancer theranostics. Water-insoluble paclitaxel is readily dissolved in iodinated poppy seed oil
(
Lipiodol) and emulsified in an aqueous solution using a mixture of PEGylated phospholipids, cholesterols,
and linear polyethylenimine grafted with cholesterols. The prepared cationic nanoemulsions were
electrostatically complexed with Bcl-2 siRNA for the co-delivery of paclitaxel and siRNA, inducing a
dramatically higher level of apoptosis and cytotoxic activity in breast adenocarcinoma compared to
individual treatments. The feasibility of the nanoemulsions for bio-imaging applications is preliminarily
investigated using micro-computed tomography with a mouse model. This study suggests that the
Lipiodol nanoemulsions can be used as a multifunctional nanocarrier platform for the co-delivery of anti-
cancer drugs and siRNA with bio-imaging functionality to efficiently increase the modality of anti-cancer
theranostics.
Received 20th February 2013,
Accepted 24th May 2013
DOI: 10.1039/c3ra40883c
www.rsc.org/advances
cles for specific applications is determined by the physico-
chemical properties of payloads. For example, SLNs and
Introduction
There has been much attention on a variety of polymeric and
lipid nanoparticulate systems for the delivery of drugs, genes,
and other functional materials.
nanoemulsions have been used for the delivery of water-
insoluble and poorly-soluble molecules because of the
presence of a hydrophobic core. Liposomes can be used to
encapsulate hydrophilic biomacromolecules (e.g., proteins,
carbohydrates, genes).
Recently, a variety of nanostructures have been employed
for the co-delivery of therapeutic drugs and genes with the aim
to achieve their synergistic effects. In particular, the combina-
tion of therapeutic siRNA with anti-cancer drugs was proved to
be very effective for improving their therapeutic efficacy. For
instance, the co-delivery of paclitaxel with B-cell lymphoma-2
1
,2
Polymeric nanoparticles
exhibit relatively better structural stability than lipid-based
ones, but their biodegradability and biocompatibility need to
be carefully considered for practical applications. Various lipid
nanoparticles, including liposomes, solid lipid nanoparticles,
and lipid nanoemulsions, have been widely investigated for
clinical applications because they can improve the efficacy of
drugs without showing significant cytotoxicity. Excellent
biocompatibility is the most important reason why lipid-based
systems have been studied despite relatively poor physical
stability. The most appropriate type of lipid-based nanoparti-
(Bcl-2) siRNA using a cationic amphiphilic polymer or solid
lipid nanoparticles significantly increased the cytotoxicity of
3
,4
a
human breast cancer cells. Liposomes co-encapsulating Bcl-
or multidrug resistance-associated protein-1 (MRP-1) siRNA
Department of Biological Sciences, Korea Advanced Institute of Science and
2
Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
b
Department of Materials Science and Engineering, KAIST Institute for NanoCentury
with doxorubicin increased the efficacy of anti-cancer treat-
5
(
KINC) and BioCentury (KIB), Korea Advanced Institute of Science and Technology,
91 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
ments. Nanoemulsions have been also investigated as a
2
nanocarrier for drug and gene delivery, bio-imaging, and
personal care, although their use for the co-delivery of anti-
E-mail: yoonsung@kaist.ac.kr; Fax: 82 42 350 3310; Tel: 82 42 350 3311
c
Pharmaceutical Research Institute, CJ Corporation, 522-1, Icheon 467-812, Republic
6
–9
of Korea
cancer drugs and siRNA has not yet been reported. For anti-
cancer therapy, nanoemulsions can be very effective to
solubilize water-insoluble drugs and reduce the severe side
effects of anti-cancer drugs through passive and active
targeting to tumor sites. Nanoemulsions can also be prepared
3
Electronic supplementary information (ESI) available: The nanoemulsion
formulations prepared with different amounts of LPEI-g-chol, calculation of the
1
number of siRNA loaded on the surface of the nanoemulsions, H NMR analysis
of LPEI-g-chol, DLS results of the nanoemulsions, GFP gene silencing efficiency
and relative total protein level in MDA-MB-GFP cells and PC3-GFP cells
transfected with GFP sr-NE. See DOI: 10.1039/c3ra40883c
1
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only using biocompatible amphiphilic molecules, including
phospholipids, cholesterols, and their derivatives.
Results and discussion
To prepare nanoemulsions, a mixture of paclitaxel, choles-
terol, linear polyethylenimine grafted with cholesterols
Besides efficient drug and gene delivery to target sites, the
ability to monitor the fate of nanocarriers in systemic and
subcellular levels is highly required to achieve effective anti-
cancer therapy. Nanocarrier systems co-encapsulating imaging
and therapeutic agents have shown great potential for
(denoted ‘LPEI-g-chol’), and 1,2-distearoyl-sn-glycero-3-phos-
phoethanolamine–polyethylene glycol (DSPE–PEG, molecular
weight = 2 kDa) was dissolved in a mixture of chloroform and
methanol. Lipiodol was added to the above organic solution,
and then emulsified into water to generate oil-in-water
emulsions. After emulsification, the organic solvents were
removed by evaporation in vacuum, and Lipiodol remained
within the nanoemulsions while solubilizing paclitaxel.
Besides paclitaxel, Lipiodol can be used to solubilize a wide
range of lipophilic anti-cancer drugs, including, doxorubicin
and poly(styrene–maleic acid) copolymer conjugated with
4
,8,10,11
theranostic applications.
Multiple imaging techniques
have been used to non-invasively monitor the bio-distribution
1
2
and efficacy of drugs in a real-time manner. They include
computed tomography (CT), magnetic resonance imaging
(MRI), positron emission tomography (PET), single-photon
emission tomography (SPECT), and optical imaging (biolumi-
1
3
nescence and fluorescence).
In this study, we introduce radio-opaque nanoemulsions
for the co-delivery of anti-cancer drugs and therapeutic siRNA
for efficient theranostic applications. Lipiodol, an iodinated
derivative of poppy seed oil having a high content of iodine
1
7,30,31
neocarzinostatin (SMANCS).
In particular, SMANCS
dissolved in Lipiodol is a well-known tumor targeted che-
motherapy to treat hepatocellular carcinoma and renal cell
3
0,32
carcinoma.
Our previous study also demonstrated that
(38–40 wt%), was employed as a radio-opaque contrast agent
Pluronic/PEG cross-linked nanoemulsions with Lipiodol as a
hydrophobic core oil effectively solubilized paclitaxel.
that can be used to monitor the in vivo fate of delivery
1
9
1
4–16
carriers.
The important advantage of our system is that
Therefore, our Lipiodol-based nanoemulsions are not limited
to the selection of materials for the current study, but can be
readily extended to a wide range of other active and carrier
materials.
Lipiodol can effectively solubilize water-insoluble drugs as well
1
7–19
as working as a contrast agent.
The use of nanoemulsions
for theranostic applications has been reported by incorporat-
ing both the imaging agents (e.g., iron oxide nanocrystals and
fluorescent dyes) and water-soluble drugs into the nanoemul-
Cholesterol was added as an emulsion stabilizer to the
formulation containing paclitaxel-solubilized Lipiodol.
Cholesterol has been widely used for lipid-based nanoparticles
because it can mediate membrane fusion and cellular
internalization of lipid-based nanoparticles and improve their
dispersion stability in a serum-containing medium by increas-
5
sions. The novelty of our proposed nanoemulsion platform is
that a well-known CT contrast agent, Lipiodol, was employed
as an oil component, for the first time, to solubilize lipophilic
drugs. Triglycerides and soybean oil are generally used for
nanoemulsions. Note that Lipiodol has been proved to be an
effective radio-opaque contrast agent for CT imaging in clinics,
although it has never been studied in nanoemulsion systems.
We believe that our radio-opaque Lipiodol nanoemulsions can
be utilized as a unique theranostic platform for a wide range of
different drugs. In our work, paclitaxel was solubilized and
loaded into the Lipiodol core of nanoemulsions while Bcl-2
siRNA was also incorporated onto the surface of nanoemul-
3
3–36
ing the structural rigidity of nanoparticles.
In our work, all
of the formulations prepared without cholesterol exhibited
very poor dispersion stability. Apparent phase separation
appeared in 72 h when the samples were incubated at an
ambient temperature. DSPE–PEG was incorporated to inhibit
the adsorption of serum proteins to nanoemulsions. The anti-
fouling effect of the PEG chain is well established as it can
reduce in vivo clearance via the reticuloendothelial or mono-
2
0–22
sions for synergistic anti-cancer effects.
Paclitaxel is a
3
7
nuclear phagocytic systems. LPEI-g-chol was used as a
positively-charged shell stabilizer, making the surface of the
prepared nanoemulsions positively charged. LPEI-g-chol
enables the electrostatic complexation of negatively charged
siRNA with the nanoemulsions. It can also facilitate the
endosomal escape of the nanoemulsions via a proton sponge
chemotherapeutic agent widely used for the treatment of
various cancers, including lung, ovarian, breast, head and
2
3
neck cancer, and Kaposi’s sarcoma. Paclitaxel destabilizes
microtubules during cell cycles and inhibits cell divi-
2
0,24,25
sion.
However, its anti-cancer effects can be reduced
by the defence mechanism of cancer cells against cell death
mainly through the activation of an anti-apoptotic defence
3
8
effect, as previously demonstrated by PEI based complexs.
2
6–28
Although both branched and linear PEIs have been reported as
effective gene delivery carriers, PEI having a high molecular
weight is highly cytotoxic. Linear PEI with a low molecular
weight is much less toxic and less efficient for endosomal
escape. It has been reported that the endosomal release of
therapeutic genes can be enhanced when a low molecular
weight PEI conjugated with cholesteryl chloroformate was
used because the secondary and tertiary amines of PEI
mechanism, in which Bcl-2 proteins are involved.
The
selective knockdown of the Bcl-2 gene can make cancer cells
more susceptible to apoptosis induced by anti-cancer
2
0,29
drugs.
Here, the co-delivery and synergistic anti-cancer
effect of paclitaxel and Bcl-2 siRNA were evaluated using breast
adenocarcinoma cells, and the potential bio-imaging capabil-
ity of Lipiodol nanoemulsions was preliminarily investigated
using micro-CT with a mouse model.
3
9
provided an efficient buffering capacity. Hence, we employed
LPEI-g-chol, in which linear PEI of 2.5 kDa was conjugated
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Fig. 1 Schematic illustration of the formation of core-shell type Lipiodol
nanoemulsions. Paclitaxel is entrapped within the hydrophobic Lipiodol core,
while siRNA is electrostatically complexed with the positively charged outer
shell.
with cholesterols, to enable the electrostatic complexation of
siRNA with LPEI-g-chol and facilitate the endosomal release of
siRNA with no significant toxicity.
Fig. 2 Characterization of the prepared Lipiodol nanoemulsions. (A)
Hydrodynamic diameter and zeta potential of b-NE, ptx-NE, and their complexes
with siRNA. (B) Time-course change of the hydrodynamic diameter of sr-ptx-NE
in PBS. TEM images of ptx-NE (C) and sr-ptx-NE (D).
LPEI-g-chol was synthesized by chemically grafting choles-
teryl acrylates to the secondary amines of LPEI and used as a
positively charged shell stabilizer to attract negatively charged
siRNA to the surface of the prepared nanoemulsions (Fig. 1).
The grafting percentage of cholesterol to LPEI was 3.8%, as
calculated from the ratio of the number of the grafted
cholesterol to the number of the LPEI monomer units. It was
tively. The complexation of the nanoemulsions with siRNA
increased the hydrodynamic diameters to 162.1 ¡ 0.8 nm for
sr-NE and 178.8 ¡ 1.5 nm for sr-ptx-NE (Fig. 2A and S3, ESI ).
3
No significant change in the size of sr-ptx-NE was observed
during the incubation in PBS at pH 7.4 at 37 uC for 48 h (179.4
1
determined by H NMR analysis, indicating that about three to
¡
2.3 nm), indicating excellent dispersion stability (Fig. 2B).
four cholesterols were attached to each LPEI chain (Fig. S1,
The presence of the PEG chains in the shell is likely to
effectively prevent coalescence via steric stabilization. The
complexation of ptx-NE with siRNA decreased the zeta
potential from +59.7 ¡ 0.7 mV to +43.0 ¡ 0.3 mV in PBS
because the binding of siRNA to the nanoemulsions was
driven by electrostatic complexation (Fig. 2A). The spherical
ESI
3
). The nanoemulsion formulations with different amounts
. The
of LPEI-g-chol were prepared as listed in Table S1, ESI
3
smallest hydrodynamic diameter of the prepared Lipiodol
nanoemulsions was about 130 nm when 10 mg of LPEI-g-chol
per 37.55 mg of the total amount of the oil phase was used in
the total formulation described in Formula 4 of Table S1, ESI
as determined by dynamic light scattering (DLS) (Fig. S2, ESI
3
,
morphology of the nanoemulsions having
a core–shell
3
).
structure was observed using transmission electron micro-
scopy (TEM) without staining (Fig. 2C and D). The average
diameters of ptx-NE and sr-ptx-NE determined from the TEM
images were 87.9 ¡ 18.7 nm and 100.1 ¡ 13.9 nm,
respectively. Both of the values were much smaller than the
hydrodynamic diameters obtained by DLS. The difference
might be caused by the shrinkage of the nanoemulsion
In this condition, no significant loss of Lipiodol and paclitaxel
was found during the fabrication process. The loading
efficiencies of Lipiodol and paclitaxel within the resulting
nanoemulsions were 97.8% and 98.5%, respectively.
The incorporation of paclitaxel into the nanoemulsions
slightly increased their hydrodynamic size. The hydrodynamic
diameters of Lipiodol nanoemulsions (denoted ‘b-NE’) and
paclitaxel-loaded Lipiodol nanoemulsions (denoted ‘ptx-NE’)
were 132.3 ¡ 0.6 nm and 140.7 ¡ 0.9 nm, respectively, in
phosphate buffered saline (PBS) at pH 7.4. Despite the high
solubility of paclitaxel in Lipiodol, some paclitaxel molecules
might interact with the shell materials, altering the interfacial
properties and structure of the outer shell layer. The prepared
b-NE and ptx-NE were electrostatically complexed with Bcl-2-
targeted siRNA and denoted ‘sr-NE’ and ‘sr-ptx-NE’, respec-
2
2
structure during the drying process.
In addition, the
difference could originate from the differences between the
techniques. TEM only shows the size of the condensed solid
part. On the contrary, DLS determines the hydrodynamic
diameter of particles from the intensity of light scattered from
a particle suspension. The hydrodynamic size includes the
core size, the thickness of any additional layers of particles,
and the thickness of diffuse layers of counter-ions moving
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around the particles. Therefore, the particle size determined
from DLS is generally larger than that observed in TEM
analysis. The successful incorporation of Lipiodol into the core
of the nanoemulsions was indicated by the dark spot in the
interior of spherical nanoemulsions, which was attributed to
the high electron density of Lipiodol, as observed in previous
into two cell lines. After 48 h incubation, cell viability was
determined using CCK8. It did not show any significant cell
deaths, indicating that b-NE did not have a cytotoxic effect on
the cells (Fig. 3B).
We determined the weight ratio of the nanoemulsions to
siRNA effective for complexation and gene silencing. Agarose
gel analysis showed that the retardation of siRNA, which
represents the complexation of siRNA with the nanoemul-
sions, increased with the increased weight ratio of the
nanoemulsions to siRNA. At a weight ratio of 20, practically
all of the siRNA molecules were complexed with the
nanoemulsions as no detectable amount of free siRNA was
found (Fig. 3C). In this condition, on average, about 3857
siRNA molecules were loaded on each nanoemulsion (see
1
9,40
studies.
Note that the TEM images of conventional
nanoemulsions without staining have very low contrast, often
making it impossible to obtain recognizable images. The
siRNA-nanoemulsion complexation also caused a morpholo-
gical change to the surface of the nanoemulsions. A new shell-
like structure, which was not observed in the absence of
siRNA, was formed around the surface of nanoemulsions as a
result of complexation with siRNA (Fig. 2D). The siRNA–
nanoemulsion condensation could be sterically restricted by
the rigid structure of siRNA, generating a relatively thick but
loose layer-like structure on the nanoemulsions. Note that the
3
Supplementary Information ). Gene silencing efficiency of sr-
NE was determined by using green fluorescent protein (GFP)-
targeted siRNA, which has a molecular structure similar to Bcl-
2-targeted siRNA. The intracellular gene expression level was
visualized by using MDA-MB-435 human breast carcinoma
cells, which express a high level of GFP from a GFP-encoding
transgene integrated to the genomic DNA of the cell. The cells
are denoted ‘MDA-MB-GFP’ cells. The nanoemulsions com-
plexed with GFP-targeted siRNA induced dose-dependent gene
silencing in MDA-MB-GFP cells, whereas the total expression
level of cellular proteins, determined by the bicinchoninic acid
persistence length (l ) of double stranded RNA is 65–80 nm,
p
corresponding to about 150 base pairs, which is relatively large
4
1
compared to that of DNA (l = about 45–50 nm).
p
The chemical stability of Bcl-2 siRNA in sr-ptx-NE against
hydrolysis was examined in the presence of 50% FBS at 37 uC.
At pre-determined time intervals, the samples were collected
and analyzed using 1% agarose gel electrophoresis. Naked
siRNA degraded completely in 4 h, while sr-ptx-NE showed
excellent chemical stability of siRNA for 48 h with a marginally
decreased amount of intact siRNA (Fig. 3A). It is very likely that
the hydrophilic PEG chain of DSPE–PEG is exposed to the
outer shell layer of the nanoemulsions and acts as a steric
barrier that interrupts nuclease attack to siRNA. Our previous
work also showed that cationic SLNs formed polyelectrolyte
complexes with siRNA–PEG conjugates, where the PEG chain
was exposed outwards, and stabilized the bound siRNA in a
(BCA) assay, was not significantly altered (Fig. 3D). These
results indicate that the gene silencing effect was derived from
the effective cellular uptake of the nanoemulsions complexed
with siRNA rather than other side effects (e.g., the cytotoxicity
of carriers).
The co-delivery of paclitaxel and Bcl-2 siRNA incorporated
in the nanoemulsions was examined using confocal laser
scanning microscopy (CLSM) and fluorescence-activated cell
sorting (FACS). Bcl-2 siRNA and paclitaxel were labeled with
6
,42
serum-containing medium.
The cytotoxic test of b-NE was
1
fluorescein and BODIPY 564/570, respectively. Breast adeno-
examined in MCF7 and MDA-MB-435 cells before cellular
carcinoma MCF7 cells were treated with either sr-NE, ptx-NE,
or sr-ptx-NE, and then the intracellular distributions of
fluorescently-labeled paclitaxel and siRNA were visualized
after 3 h incubation. It is shown that paclitaxel (red) and
siRNA (green) were highly co-localized in the cytoplasm
experiments. Different concentrations of b-NE were treated
(Fig. 4A). It has been well known that the physicochemical
properties of nanocarriers influence the cellular uptake
mechanism. In particular, the surface charge of nanocarriers
plays a key role in their cellular uptake and internalization
4
3
pathway. The cationic nanoparticles can bind to sulfated
proteoglycan of cell surfaces through electrostatic interactions
and subsequently be internalized by pinocytosis and endocy-
3
8,43
tosis.
In addition, it has been reported that the mechan-
ism of cellular uptake of polyplex with PEI-cholesterol
conjugates may include pinocytosis and adsorptive endocyto-
4
4
sis. Our results showed that sr-NE and sr-ptx-NE were found
in the cytoplasm by efficient cellular uptake and endosomal
escape. FACS analysis was used to confirm the simultaneous
intracellular translocation of paclitaxel and Bcl-2 siRNA. The
cells incubated with either sr-NE or ptx-NE for 3 h were only
localized in the one-positive quadrant of the FACS diagram
Fig. 3 Stability and gene silencing effect of NE. (A) dispersion stability of naked
siRNA (a) and Bcl-2 sr-ptx-NE (b) incubated in 50% serum solution at 37 uC for
4
8 h. (B) Cytotoxic effect of b-NE against two cell lines. (C) Binding affinity of NE
to siRNA at different weight ratios. (D) GFP gene silencing efficiency and relative
total protein level in MDA-MB-GFP cells post-incubation with GFP sr-NE.
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Fig. 4 CLSM images and FACS diagrams of cellular uptake of sr-ptx-NE in MCF7
cells. (A) Confocal images for the intracellular distributions of sr-NE, ptx-NE, and
sr-ptx-NE. For fluorescence imaging, fluorescein-conjugated siRNA (green) and
1
BODIPY 564/570-labeled paclitaxel (red) were used. Cell nuclei were stained
Fig. 5 Inhibition of therapeutic target gene expression in MCF7 cells. (A)
with DAPI (blue). (B) FACS diagrams of cellular uptake of sr-NE, ptx-NE, and sr-
ptx-NE.
Representative Bcl-2 mRNA expression determined by RT-PCR. (B) Analysis of
light intensities of Bcl-2 mRNA expression as the ratio of Bcl-2 to b-actin from
RT-PCR results (*p , 0.01, **p , 0.5).
(Fig. 4B). Only the cells treated with sr-ptx-NE were observed in
the double-positive quadrant, indicating that the nanoemul-
sions effectively co-delivered siRNA and paclitaxel into the
cells.
due to forming a high density complex with DNA, thereby
4
9
enhancing the transfection efficiency in vitro. Moreover,
Lipiodol itself could function as a transfection agent to
enhance the uptake of siRNA into dendritic cells, and
effectively deliver caspase-related therapeutic siRNA to the
The efficacy of chemotherapy is often limited by the bypass
mechanism of cancer cells to escape the apoptosis. Highly
invasive cancer cells have survival mechanisms that overcome
apoptosis through the up-regulation of various anti-apoptotic
genes. For instance, Bcl-2 proteins are over-expressed and play
a key role in apoptotic activity in several types of cancer cells
5
0–52
liver in mice.
The cytotoxicity and apoptotic effects of the co-delivered
paclitaxel and Bcl-2 siRNA in MCF7 cells were determined
using the CCK8 and Annexin V-FITC apoptosis assays. The
viability of the cells treated with sr-ptx-NE was 42.8%, which
was much lower than that with sr-NE (80.6%) and ptx-NE
4
5,46
(e.g., colon, breast, and prostate).
They can protect invasive
cancer cells from apoptosis by inhibiting the efflux of
2
1
cytochrome c that triggers the cascade of caspase-activation
(62.4%). Note that the siRNA concentration was 0.5 mg mL
(38 nM) for sr-NE and sr-ptx-NE, and the paclitaxel concentra-
4
7,48
events.
Bcl-2 gene silencing in cancer cells makes them
2
1
more susceptible to apoptosis induced by anti-cancer
tion encapsulated in 10 mg mL of the nanoemulsion was 140
nM for ptx-NE and sr-ptx-NE. When the paclitaxel concentra-
2
0,29
agents.
Therefore, in this work, we evaluated the relevant
2
1
biological effects of the co-delivery of Bcl-2 siRNA and
paclitaxel by the nanoemulsions.
tion increased to 560 nM, which was equal to 40 mg mL of
the nanoemulsions, the viability of the cells treated with sr-ptx-
NE decreased to 14.7% (Fig. 6A). These results indicate that
the cytotoxic effect can be effectively increased by the
synergistic combination of Bcl-2 siRNA and paclitaxel. The
high cytotoxicity of sr-ptx-NE was derived from the enhanced
apoptotic activity of paclitaxel. A relatively high level of
apoptosis (about 60.1%) was induced by sr-ptx-NE in MCF7
cells, while sr-NE and ptx-NE led to only about 12.8% and
33.8% of apoptosis, respectively (Fig. 6B). In vitro release
profiles of paclitaxel from ptx-NE and sr-ptx-NE are shown in
MCF7 cells were incubated with sr-NE for 24 h, and the
mRNA expression level of Bcl-2 was measured using quanti-
tative reverse transcription polymerase chain reaction (RT-
PCR). It is shown that mRNA expression of Bcl-2 gene
significantly decreased in MCF7 cells transfected with sr-NE
(
Fig. 5A). In particular, sr-NE showed the most efficient
knockdown of Bcl-2 (47.9%) compared to Lipofectamine
61.3%), which is a widely used positive control, as determined
by the ratio of the intensities of Bcl-2 to b-actin mRNAs
Fig. 5B). The similar effects of gene silencing were also
observed in GFP-expressing cell lines through transfection of
sr-NE loaded with GFP-targeted siRNA (Fig. S4, ESI ). It has
(
(
3
Fig. S5, ESI , indicating that there was no significant effect of
the siRNA complexation on the drug release. This result
suggests that the increased apoptosis by sr-ptx-NE was not
caused by the facilitated release of paclitaxel from sr-ptx-NE.
Our results are comparable with the previous findings
3
been reported that the cationic lipid emulsion system contain-
ing Lipiodol increased the contact time with the adherent cells
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interesting to determine how the molar ratio of paclitaxel to
siRNA affects their cytotoxic activity, though one may need to
carefully consider the change in the structural and chemical
properties of the nanoemulsions. The synergistic effects of
siRNA–drug combination were also reported for other delivery
systems. For instance, the co-delivery of doxorubicin and Bcl-2
siRNA by diblock co-polymer micelles, comprising poly(e-ca-
prolactone) (PCL) and linear PEI conjugated with folic acid,
exhibited an enhanced cytotoxic effect in human hepatic
cancer cells Bel-7402. The cell apoptosis dramatically
increased to 84.2% by using the doxorubicin–Bcl-2 siRNA co-
2
1
delivery. We believe that our Lipiodol nanoemulsions can be
easily extended to other combinations of anti-cancer drugs
and siRNA and serves as a versatile platform for synergistic
anti-cancer therapy applications.
In recent years, various nanoscale carriers have been
employed for theranostic applications, where therapeutic
agents are combined with imaging agents for non-invasive
monitoring of bio-distribution and efficacy of drugs by using
8
,10–12
CT, MRI, PET, and other imaging techniques.
Although
nuclear imaging tools provide higher sensitivity compared to
other imaging tools, CT has a unique advantage in terms of
5
4
high resolution and contrast in soft tissues. The most
important advantage of our nanoemulsion system is that
Lipiodol, which solubilizes paclitaxel within the nanoemul-
sions, can be used as a good contrast agent for CT because of a
1
4
high iodine content. In addition, Lipiodol has been clinically
used as a chemo-embolizing agent for hepatocellular carci-
5
5
noma. In this work, the diagnostic capability of the radio-
opaque Lipiodol nanoemulsions was studied by using micro-
CT analysis. MCF7 cells were treated with PBS, Lipiodol-free
nanoparticles (denoted ‘sr-NP’), and sr-NE, followed by the
fixation in an agarose gel phantom. The Lipiodol content was
1
.39 mg per 2 mg nanoemulsions. The CT number of the cells
6
(
5 6 10 ) treated with sr-NE was 206.1 HU (Hounsfield Unit),
which was a much higher value than that of untreated cells
82.2 HU) and sr-NP treated cells (132.6 HU) (Fig. 7A). This
(
Fig. 6 Effect of the co-delivery of paclitaxel and Bcl-2 siRNA. (A) Cytotoxic effect
of co-delivery by Bcl-2 sr-ptx-NE in MCF7 cells. (B) Induction of apoptosis on
MCF7 cells by Bcl-2 sr-ptx-NE (*p , 0.05, **p , 0.01).
feasibility study suggests that Lipiodol nanoemulsions can be
used as a useful CT contrast material as well as the
hydrophobic oil core that solubilizes water-insoluble pacli-
taxel.
Preliminary CT imaging studies were also performed using
a mouse model to evaluate the Lipiodol nanoemulsions as a
potential in vivo contrast agent. MCF7 cells were treated with
PBS, sr-NP, and sr-NE, and then subcutaneously injected into
the dorsal side of nude mice. The whole mouse body was then
visualized on a micro-CT scanner. The cells transfected with
PBS and sr-NP were not found in the subcutaneous region of
nude mice, whereas a bright spot (indicated by a red circle)
was clearly detected in the mouse injected with the sr-NE
transfected cells (Fig. 7B). The 3D-rendered CT image of the
mouse treated with sr-NE transfected cells also confirmed the
visualization of the Lipiodol nanoemulsions (Fig. 7C). It
showed that Lipiodol nanoemulsions can be a promising
radio-opaque nanocarrier that can be used to trace and image
the delivery of drugs. More extensive in vivo CT imaging
reported by Yang and her colleagues, who developed
poly(N-methyldietheneamine sebacate) grafted with cholester-
ols for the simultaneous delivery of paclitaxel and Bcl-2 siRNA
3
to MDA-MB-231 human breast cancer cells. They showed that
the cell viability decreased from 78% to 59% by the co-delivery
of 100 nM paclitaxel with 20 nM siRNA. When they used 400
nM paclitaxel, the cell viability further decreased to 39%. Our
results showed a similar cytotoxic effect (about 40% cell
viability with 38 nM siRNA and 140 nM paclitaxel) presumably
because both of the studies employed breast cancer cell lines,
where paclitaxel can effectively cause endoplasmic reticulum
stress through the mitochondrial apoptotic pathway in various
breast cancer cell lines, including MCF7, MDA-MB, T47D, and
2
0,53
BT20.
For further investigation, it will be still very
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Experimental
Materials
Cholesterol, acryloyl chloride, triethylamine, and DAPI were
obtained from Sigma-Aldrich (St. Louis, MO), linear polyethy-
lenimine (LPEI, molecular weight
=
2.5 kDa) from
Polysciences, Inc. (Warrington, PA), DSPE–PEG from Avanti
1
Polar Lipids (Alabaster, AL), and Lipiodol (Lipiodol Ultra
fluid) from Guerbet (Aulnay-sous-Bois, France). Rosewell Park
Memorial Institute medium 1640 (RPMI-1640), Dulbecco’s
modified Eagle’s medium (DMEM), Phosphate Buffered Saline
1
(PBS), BODIPY
564/570-paclitaxel conjugates, and fetal
bovine serum (FBS) were purchased from Invitrogen
Carlsbad, CA). GFP-expressing MDA-MB-435 cell line was
(
kindly donated by Samyang Co. (Seoul, Republic of Korea), and
MCF7 cell line was obtained from Korean Cell Line Bank
1
(
Seoul, Republic of Korea). TRI Reagent , reverse transcrip-
tase, and Taq DNA polymerase were purchased from Ambion,
Inc. (Austin, Texas), Promega (Madison, WI), and Takara
TM
(Tokyo, Japan), respectively. ApoScan
Annexin V-FITC
Apoptosis Detection Kit and Cell-Counting Kit-8 (CCK-8) were
obtained from BioBud (Seoul, Republic of Korea) and Dojindo
laboratories (Kumamoto, Japan), respectively. Paclitaxel
(
.97%) was purchased from Wako (Osaka, Japan). GFP
siRNA and Bcl-2 siRNA were obtained from Genolution
Pharmaceuticals, Inc. (Seoul, Republic of Korea): GFP siRNA
sense, 59-GCAAGCUGACCCUGAAGUUdTdT-39; GFP siRNA
Fig. 7 (A) Feasibility test of NE as a CT contrast agent. CT images were obtained
from the cells by treating with PBS (a), sr-NP (b), and sr-NE (c). (B) In vivo CT
imaging by subcutaneous injection of the cells treated with each formulation.
anti-sense,
siRNA sense, 59-CCGGGAGAUAGUGAUGAAGdTdT-39; and Bcl-
siRNA anti-sense, 39-dTdTGGCCCUCUAUCACUACUUC-59.
39-dTdTCGUUCGACUGGGACUUCAA-59;
Bcl-2
(
C) 3D-rendered CT image of the mouse treated with sr-NE.
2
Bcl-2 siRNA modified with fluorescein at 39 end of sense and
antisense, and primers for reverse transcription-polymerase
chain reaction (RT-PCR) were purchased from Bioneer Co.
studies via systemic administration are currently underway
with the introduction of a tumor cell-targeting moiety to the
surface of the nanoemulsions, facilitating the accumulation of
the Lipiodol nanoemulsions in a localized tumor tissue.
(Daejeon, Republic of Korea): Bcl-2 primer forward,
59-GTCCAAGAATGCAAAGCACA-39; Bcl-2 primer reverse,
59-ACATCTCCCGCATCCCACT-39; b-actin primer forward,
59-GTGGGGCGCCCCAGGCACCAGGGC-39; and b-actin primer
reverse, 59-CTCCTTAATGTCACGCACGATTTC-39. BALB/c nude
mice were purchased from Orient Bio Inc. (Seongnam,
Republic of Korea). Mice were raised in a pathogen-free
environment at Korea Research Institute of Bioscience and
Biotechnology (KRIBB) according to the KRIBB’s Animal
Experiment Guidelines.
Conclusions
This study has demonstrated that the radio-opaque Lipiodol
nanoemulsions can be an effective, biocompatible carrier for
the intracellular co-delivery of paclitaxel and Bcl-2 siRNA.
Water-insoluble paclitaxel was readily solubilized into the
Lipiodol core of the nanoemulsions, while negatively charged
siRNA was electrostatically complexed with the cationic
nanoemulsions. The surface modification of the nanoemul-
sions with PEG was effective for maintaining the dispersion
stability in the presence of serum proteins. In addition,
Lipiodol was shown to work as an imaging contrast agent for
micro-CT. These results suggest that the Lipiodol nanoemul-
sions can be a versatile, multifunctional nanocarrier for drugs
and genes. We expect that further surface modification for
cell-specific targeting makes the nanoemulsions effective for
in vivo theranostic applications.
Synthesis of LPEI grafted with cholesterols
Cholesteryl acrylate was synthesized and grafted to the
backbone chain of LPEI, producing LPEI-g-chol. Briefly,
cholesterol (580 mg, 1.5 mmol) was dissolved in chloroform
(5 mL), and then triethylamine (209 mL, 1 mmol) and acryloyl
chloride (81 mL, 1 mmol) were added. The reaction mixture
was stirred for 3 h at ambient temperature, and then the
organic solvent was removed in vacuo. The oily yellow crude
product was washed with cold ethanol and recrystallized to
give a pale yellow solid. LPEI (62.5 mg) was dispersed in 1 mL
deionized water, and its pH was adjusted to 7.2 using 0.1 M
HCl. Water was removed under reduced pressure, and then the
dried LPEI was dissolved in a 4 : 1 mixture of methanol and
chloroform (5 mL) with argon purging for 5 min. The prepared
cholesteryl acrylate was added to the solution (as pre-
1
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determined equivalent) and reacted for 48 h at 50 uC. The
reaction mixture was evaporated under reduced pressure to
obtain a yellow sticky crude product, which was further
purified by recrystallization in cold diethyl ether.
sr-ptx-NE were also observed using transmission electron
microscopy (TEM, Omega912, Carl Zeiss, Germany). A drop
of the sample solution (20 mL) was deposited three times on a
300-mesh carbon-coated copper grid and dried in air at
ambient temperature. The TEM images were taken without
staining.
Preparation of paclitaxel-loaded Lipiodol nanoemulsions
Lipiodol nanoemulsions were prepared to encapsulate pacli-
taxel by an emulsification/solvent evaporation method.
Paclitaxel (0.5 mg) was dissolved in a 2 : 1 (v/v) mixture of
chloroform and methanol (1 mL) and mixed with Lipiodol
Gene silencing by GFP-targeted siRNA complexed with
nanoemulsions
The effect of the weight ratio of the nanoemulsions to siRNA
on gene silencing was determined from GFP-targeted siRNA
transfection with MDA-MB-GFP cells. The cells were seeded
(
22.5 mg). The mixture was added into a mixture of
cholesterol, LPEI-g-chol and DSPE–PEG in the same solvent
1 mL). Deionized water (8.0 mL) was then added and vortexed
5
(
onto a 24-well plate at a density of 1.0 6 10 cells per well and
for 1 min. The solution was sonicated in a bath-type sonicator
for 5 min with an output control of 3.5 at 20 kHz (Branson
Sonifier 450). The emulsified solution was quickly trans-
incubated for 24 h before transfection. The nanoemulsions
complexed with GFP siRNA formulated with different weight
ratios of the nanoemulsions to siRNA at a fixed weight of 0.5
mg siRNA were transfected into MDA-MB-GFP cells for 5 h
using a serum-free medium. The medium was replaced with
DMEM containing 10% FBS, and the cells were incubated for
1
ferred to
a
rotary evaporator (R-200, B u¨ chi, Flawil,
Switzerland), and the solvent was removed under reduced
pressure around 50 uC. The prepared solution was dialyzed
using a Spectra/Por dialysis membrane (molecular weight
cutoff = 10 kDa) against deionized water, and then filtered
through a 1.2 mm cellulose filter. Bcl-2 siRNA was electro-
statically complexed with the nanoemulsions. The complexa-
tion of siRNA with the nanoemulsions was performed with
various weight ratios (5/1, 10/1, 15/1, 20/1, and 40/1) of the
nanoemulsions to siRNA. The concentration of siRNA was
48 h. The cells were then lysed using 1% Triton X-100 solution
and centrifuged to separate GFP-containing supernatants from
cell debris. The supernatants were then carefully isolated and
measured using a spectrofluorometer (SLM-AMINCO 8100,
SLM Instruments Inc., IL) at excitation and emission
wavelengths of 480 nm and 520 nm, respectively.
2
1
fixed to 20 mg mL
.
Stability of siRNA in serum
To determine the structural stability of siRNA complexed with
Quantification of paclitaxel and Lipiodol within
nanoemulsions
ptx-NE in serum, samples were prepared as previously
5
7
reported. Naked siRNA and ptx-NE complexed with siRNA
were incubated using DMEM containing 50% FBS at 37 uC.
To determine the loading amount of paclitaxel, the prepared
ptx-NE were lyophilized and re-suspended in acetonitrile with
orbital shaking at about 60 rpm for 4 h. The solution was
filtered through a 0.45 mm filter and analyzed using reversed
phase high performance liquid chromatography (RP-HPLC).
The HPLC system HP 1100 series (Agilent 1100 series, Agilent
Technologies, Palo Alto, USA) was used with a Waters
Spherisorb ODS2 column (5 mm, 4.6 mm 6 250 mm).
Acetonitrile was used as an isocratic mobile phase at a rate
21
The final siRNA concentration was 10 mg mL . The samples
were collected at a pre-determined time interval (1, 2, 4, 8, 16,
24, and 48 h) and analyzed by 1% agarose gel electrophoresis.
Before gel electrophoresis, sr-ptx-NE were treated with 50 mg
21
mL heparin for 30 min to dissociate siRNA from the cationic
nanoemulsions. After ethidium bromide staining, gel images
TM
were visualized on a UV transilluminator (GelDoc-It TS
Imaging system, CA, USA).
2
1
of 1.0 mL min . The eluate was monitored by light
absorbance measured at 227 nm. To determine the amount
of Lipiodol loaded within the nanoemulsions, the nanoemul-
sions were lyophilized, dissolved in dichloromethane (100 mL)
with vigorous shaking, and diluted with ethanol. The Lipiodol
concentration was measured using the standard curve of
Lipiodol in ethanol by UV-visible spectrophotometry (UV-1601,
Targeting effect by Bcl-2-targeted siRNA complexed with
nanoemulsions
To demonstrate the inhibition of an endogenous target gene,
the expression level of target mRNA was examined using RT-
PCR. MCF7 cells were seeded onto a 6-well plate at a density of
5
5
6
2.0 6 10 cells per well for 24 h prior to transfection. Cells
SHIMADZU, Japan) at a wavelength of 260 nm.
were transfected with the nanoemulsions electrostatically
complexed with Bcl-2 siRNA for 5 h using a serum-free
medium, and then the medium was replaced with a fresh
medium containing 10% FBS. The concentration of siRNA in
Characterization of Lipiodol nanoemulsions
The hydrodynamic diameters and zeta potentials of the
prepared nanoemulsions in PBS (10 mM Na HPO , 2 mM
2
4
2
1
the transfection experiment was 38 nM (0.5 mg mL ). After 24
2 4
KH PO , and 156 mM NaCl) were measured using a Zetasizer
h incubation, the total RNA was isolated from the cells using
Nano ZS (Malvern Instruments Ltd., UK) equipped with a
vertically polarized He–Ne laser at a wavelength of 632.8 nm.
The scattering angle was fixed at 173u, and the temperature
was maintained at 25 uC. Size distribution was also calculated
using the CONTIN routine, which gives access to the
distribution of relaxation times in the experimental time
correlation functions. The morphology and size of ptx-NE and
1
TRI Reagent . The extracted RNA (2 mg) was reversely
transcribed and amplified with reverse transcriptase and Taq
DNA polymerase. The PCR products were examined using 1%
agarose gel electrophoresis. The gel images were obtained by a
UV transilluminator and then analyzed to measure the light
intensity using Image J software (NIH, Bethesda, MD).
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Cellular uptake of paclitaxel and Bcl-2 siRNA loaded
nanoemulsions
to determine CT numbers in the ROI (region of interest) of 10
mm using Image J software (NIH, Bethesda, MD). For mouse
2
CT imaging, cells were treated with PBS, sr-NP, or sr-NE at 37
uC for 5 h in a serum-free medium. The cells were harvested
and injected subcutaneously into the dorsal part of nude mice
and CT images were obtained using a micro-CT scanner with
the following parameters: voltage = 60 kVp, current = 60 mA,
exposure time = 500 ms, field of view (FOV) = 80 mm, matrix =
Simultaneous cellular uptake of paclitaxel and Bcl-2 siRNA was
examined using confocal microscopy and flow cytometry. For
1
confocal images, fluorescent BODIPY
564/570-paclitaxel
conjugates and FITC conjugated Bcl-2 siRNA were incorpo-
rated into NE. MCF7 cells seeded in a chamber slide (4-well
5
CultureSlides, BD Falcon, MA) at a density of 1.0 6 10 cells
512 6 512, and slice thickness = 0.16 mm.
per well were cultured in RPMI 1640 at 37 uC for 24 h. The cells
were transfected with the nanoemulsions containing the
Statistical analysis
2
1
fluorescently-labeled paclitaxel (1 mg mL ) and FITC con-
Statistical analysis was determined by a two-tailed Student’s
t-test. Statistical significance was determined using a con-
fidence level of 95%.
2
1
jugated Bcl-2 siRNA (2 mg mL ) at 37 uC for 3 h using a serum-
free medium. The cells were washed with PBS and fixed by
using 1 wt-% formaldehyde. The cell nuclei were stained with
2
1
4
,6-diamidino-2-phenylindole (DAPI, 1.5 mg mL ) for 10 min.
The specimens were visualized on a CLSM (LSM510, Carl
Zeiss, Germany). For fluorescence activated cell sorting (FACS)
Acknowledgements
analysis, the cells were cultured in a 6-well plate at a density of
This study was supported by a grant of the Korea Healthcare
Technology R&D Project, Ministry of Health and Welfare,
Republic of Korea (A111552). This work was also supported by
CJ Pharmaceutical Institute (Icheon, Republic of Korea). We
thank Hye-youn Jung and Dr Yoonkyung Kim at Korea
Research Institute of Bioscience and Biotechnology (KRIBB)
for technical assistance and analysis for micro-CT imaging.
5
2.0 6 10 cells per well and treated with the nanoemulsions
loaded with fluorescently-labeled paclitaxel and/or Bcl-2 siRNA
at 37 uC for 3 h using a serum-free medium. The cells were
harvested and analyzed using a flow cytometer (FACSCalibur,
BD Biosciences).
Determination of cytotoxicity and induced apoptosis
MCF7 cells were maintained in RPMI 1640 medium containing
1
0% FBS at 37 uC for 24 h prior to transfection. The cells were
4
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