Functionalization of dendritic polyethylene with cationic poly(p-phenylene ethynylene) enables efficient siRNA delivery for gene silencing

Article abstract of DOI:10.1039/c3tb00480e

A novel system based on a dendritic polyethylene-cationic poly(p-phenylene ethynylene) polyvalent nanocarrier was developed for siRNA delivery. By using the combination of a molecular wire and a dendritic effects strategy, this design provides the nanocarrier system with low cytotoxicity, cellular imaging and high siRNA delivery efficiency, allowing it to exhibit remarkable gene knockdown abilities as well as real-time monitoring of the siRNA delivery process. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3tb00480e

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Materials Chemistry B
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Functionalization of dendritic polyethylene with
cationic poly(p-phenylene ethynylene) enables efficient
Cite this: J. Mater. Chem. B, 2013, 1,
2245
siRNA delivery for gene silencing
*
Ling Zhang, Qi-He Yin, Jin-Ming Li, Hong-Ying Huang, Qing Wu and Zong-Wan Mao
A novel system based on a dendritic polyethylene–cationic poly(p-phenylene ethynylene) polyvalent
nanocarrier was developed for siRNA delivery. By using the combination of a molecular wire and a
“dendritic effects” strategy, this design provides the nanocarrier system with low cytotoxicity, cellular
imaging and high siRNA delivery efficiency, allowing it to exhibit remarkable gene knockdown abilities
as well as real-time monitoring of the siRNA delivery process.
Received 3rd December 2012
Accepted 21st February 2013
DOI: 10.1039/c3tb00480e
www.rsc.org/MaterialsB
water-soluble cationic conjugated polyelectrolytes provide an
excellent polyvalent scaffold for siRNA delivery, since cationic
Introduction
Recently, short interfering RNA (siRNA) has been considered as
a new class of nucleic acid therapeutic for treatment of various
infectious and genetic diseases including cancer via RNA
interference (RNAi).1 A successful approach used for siRNA
delivery in vitro and in vivo involves the formation of ionic
complexes (polyplexes) through noncovalent interaction
between the negatively charged phosphate groups in siRNA and
the cationic charges in the macromolecular vectors such as
lipids,2a dendrimers,2b,c and polymers.2d–h
Despite these intense studies, effective delivery of siRNA
to target cells under in vivo conditions still remains a
signicant challenge to realizing its full therapeutic potential
due to their low tolerability under physiological conditions
and poor cellular uptake.3 Furthermore, it would be difficult
to observe the siRNA delivery process, which is very useful
for RNAi therapy investigation, because these delivery
systems usually lack intrinsic uorescence. While quantum
dots (QDs) have been reported as siRNA carriers for high
efficiency and monitored siRNA delivery,4 the cytotoxicity of
the QDs must be considered if using this kind of siRNA
delivery system in vivo. Thus, the development of a low
cytotoxicity, high efficiency and monitored system for siRNA
delivery is essential.
conjugated polyelectrolyte chains with multiple recognition
elements can bind to the negatively charged siRNA through
electrostatic interactions. Moreover, water-soluble cationic
conjugated polyelectrolytes feature efficient optical signal
transduction and high resistance to photobleaching,7–9 allowing
them to monitor the siRNA delivery process. However, no
methods have been developed for utilizing uorescent conju-
gated polyelectrolyte-based polyvalent nanoparticles and their
unusual properties to load and transport siRNA across cell
membranes.
Among them, water-soluble poly(p-phenylene ethynylene)
(PPEs) with synthetic versatility are especially attractive uo-
rescence probes for sensing cells, imaging living cells and
cancer cells in vitro.10–13 In spite of these promising properties,
even charged PPEs have a strong tendency to aggregate which
results in subsequent uorescent quenching in aqueous
solution,14 which presents a major bottleneck for siRNA
delivery and intracellular imaging. We have developed a
method for synthesizing dendritic polyethylene–cationic
poly(p-phenylene ethynylene) (DPE–PPE+) core–shell nano-
particles with small size, biocompatibility and good cellular
permeability.15
In this work, polyvalent nanocarriers based on dendritic
polyethylene–cationic poly(p-phenylene ethynylene) (DPE–
PPE+) were investigated as siRNA carriers to target silencing of
VEGF protein in HeLa cells. In comparison with efficient
uptake of the target cells, folic acid (FA) ligands may be
introduced on the shell of DPE–PPE to obtain water-soluble
DPE–PPE–FA+. We expect that these polyvalent nanocarriers
will not only deliver siRNA much more effectively for gene
silencing, but also will be applicable to the observation of the
siRNA delivery process.
In Segura and Guo's review papers,5 they have highlighted
biocompatible polyvalent nanocarriers as delivery vehicles for
siRNA delivery. Meanwhile, some biocompatible polyvalent
nanocarriers based on the tunable surface charge of gold
nanorods have been applied in siRNA delivery.6 Most of all,
DSAPM Lab, PCFM Lab, OFCM Institute, MOE Key Laboratory of Bioinorganic and
Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen
University, Guangzhou 510275, China. E-mail: ceszl@mail.sysu.edu.cn
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conducted to obtain DPE–PPE–FA+ in the same way as the
previous description of DPE–PPE+.18,19
Experimental
Materials and general methods
The formation of DPE–PPE+/siRNA and DPE–PPE–FA+/siRNA
complexes
All chemicals and solvents were purchased from Sigma-Aldrich
or Acros Organics, and used without further purication unless
otherwise noted. HeLa cells were obtained from the Experi-
mental Animal Center of Sun Yat-sen University.15 The anti-
VEGF antibody and b-actin monoclonal antibody were obtained
from Santa Cruz (USA). VEGF siRNA (siVEGF) was purchased
from Dharmacon (USA) including Cy3-labeled siRNA (siR-
NACy3). siRNACy3 has the sequences of 50-CCUACGCCAAUUUC
GUdTdT-30 (sense) and 50-ACGAAAUUGGUGGCGUAGGdTdT
(antisense). The siRNA was labeled with Cy3 at the 50-end of its
sense strand. The sequences of siVEGF were 50-AUGUGAAU
GCAGACCAAAGAATT-30 (sense) and 30-TTUAACACUUACGUCU
GGUUUCUU-50 (antisense). All NMR spectra were recorded at
25 ꢀC on a Varian Mercury-Plus 300 M or a Varian Inova 500 M
spectrometer in D2O or DMSO-d6 with reference to solvent
residual peaks or TMS. Cytotoxicity assay was described in our
previous report.15
The DPE–PPE–FA+/siRNA complex was prepared by mixing
siRNA with the specied amount of DPE–PPE–FA+ solution
followed by incubation in serum-free medium at room
temperature for 15 min. The weight ratio of DPE–PPE–FA+ to
siRNA varied from 4 to 32. The complex formation was
conrmed by agarose gel electrophoresis. The siRNA and DPE–
PPE–FA+/siRNA complexes were loaded in the wells of 1.0 wt%
agarose gel containing ethidium bromide (EtBr) at a concen-
tration of 0.5 ug mLꢁ1, to which was applied 190 V electrodes in
1ꢂ TBE buffer (pH 9) for 10 min. The siRNA was visualized by
EtBr staining and the gel image was taken under UV. The gel
electrophoresis of DPE–PPE+/siRNA complex was conducted in
the same way as described above for the DPE–PPE–FA+/siRNA
complex.
siRNA protection of DPE–PPE+ and DPE–PPE–FA+
nanocarriers
Synthesis of DPE–PPE+ and DPE–PPE–FA+
The dendritic macromonomer bearing multiple iodine groups
(DPEI) was synthesized using the Brookhart palladium–a-dii-
mine chain walking catalyst. Based on DPEI, a neutral DPE–PPE
was synthesized by palladium/copper-catalyzed Sonogashira
cross-coupling. Moreover, quaternization of DPE–PPE was
conducted with bromoethane to obtain the water-soluble
cationic DPE–PPE (DPE–PPE+). The detailed synthesis of DPE–
PPE+ was described in our previous work.15 N-Hydrox-
ysuccinimide ester of folic acid (FA-NHS) was prepared in
accordance to a reported procedure.16,17 Vacuum-dried FA-NHS
was then dissolved in 1.5 mL of a mixture of DMSO and TEA
with a volume ratio of 2 : 1. An equal molar amount of 2-ami-
noethanebromine was added to the mixture, and the reaction
was conducted under anhydrous conditions overnight.17,18
Therefore, a bromine group was introduced into folic acid to
form FA–Br by nucleophilic substitution (Scheme 1). 1H NMR of
FA–Br (DMSO-d6) d 8.61 (s, 1H), 8.06 (d, 1H), 7.62 (d, 2H), 7.42
To study the ability of nanocarriers in protection of loaded
siRNA from being degraded by RNaseA, DPE–PPE+/siRNA and
DPE–PPE–FA+/siRNA complexes were formed following the
procedure described above. Next, 10 unit RNaseA was added to
the complexes, and the resulting mixtures were incubated at
37 C for 90 min. Aer incubation, 1 mL of 50 U mL RNaseA
inhibitor was added to stop the degradation reaction, and then
3 mL of 1% SDS was added to dissociate the siRNA from the
complexes. The gel electrophoresis images were taken under a
UV illuminator, and the results were analyzed to identify the
remains of siRNA aer the degradation of RNaseA, and
compared with naked siRNA in the presence of RNaseA. A
parallel experiment was conducted as a control for naked siRNA
in the absence of RNaseA.
ꢁ1
ꢀ
Flow cytometric analysis
(d, 1H), 7.21 (t, 1H), 6.91 (br), 6.60 (d, 2H), 5.56 (d, 1H), 4.48 (d, HeLa cells were grown in DMEM containing 10% FBS at 37 ꢀC in
2H), 4.20 (m, 1H), 3.06 (t, 1H), 2.99 (q, 1H), 2.53 (mm, 4H), 2.30 a 5% CO2 atmosphere. Six-well plates (Corning Inc., Corning,
(t, 1H), 2.10–1.85 (mm, 2H). Moreover, quaternization of the NY) were used with a well volume of 2.6 mL. The cells were
neutral DPE–PPE with a mixture of FA–Br and bromoethane was trypsinized, counted, and adjusted to 1 ꢂ 105 cells per mL and
Scheme 1 Synthesis of FA–Br.
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1 mL was added per plate. Stock solutions of DPE–PPE+, DPE–
PPE–FA+ and their siRNA complexes were diluted using DMEM
and added to the plate in 20 mL quantities (c ¼ 50 mg mLꢁ1).
Aer incubation for 6 h, cells were thoroughly washed with PBS
three times in order to eliminate DPE–PPE+, DPE–PPE–FA+ and
their siRNA complexes that were not internalized. The cells were
trypsinized and centrifuged in PBS buffer. Then the DPE–PPE+,
DPE–PPE–FA+ and their siRNA complex bound cells were har-
vested and a single cell suspension in 0.5 mL PBS buffer was
prepared and subjected to ow cytometric analysis. A ow
cytometer (Coulter Co. USA) was used to measure the uores-
cence intensity with excitation at 488 nm.
Western blotting
Transfected cells and untransfected controls were lysed using
M-PERꢀ protein extraction reagent (Pierce Inc, USA). The
lysates were separated by centrifugation at 12k rpm, 4 ꢀC for
10 min on an Eppendorf 5417R centrifuge (Eppendorf Inc,
Germany). Supernatants were then collected, and the protein
concentration was measured by a standard BCA protein assay
kit (Novagen Inc, USA). Equal amounts of protein were loaded
and separated on 12% sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and were then transferred for
1 h at 150 V using Bio-Rad Mini-PROTEAN4 (Bio-Rad Inc, USA)
to nitrocellulose membranes (Bio-Rad Inc, USA) in transfer
buffer (25 mM Tris–HCl, 200 mM glycine, 10% methanol) and
blocked with 5% milk blocking buffer for 2 h on a horizontal
shaker. The blocked membranes were incubated with 1 : 1000
rabbit antihuman VEGF antibody (Santa Cruz Inc, USA) diluted
in 5% milk blocking buffer. The membranes were washed in
Tween-Tris Buffered Saline (TTBS: 0.1% Tween-20 in 100 mM
Tris–HCL [pH 7.5], 0.9% NaCl) and probed with secondary
antibody (goat antirabbit IgG conjugate HRP) diluted at 1 : 5000
in 5% milk blocking buffer. The blots were developed by using
an ECL kit (Amersham Inc, USA). The membranes were exposed
to Kodak X-OMAT lm for 10–30 s for data acquisition and
developed using a conventional lm developing machine.
Laser confocal microscopy image analysis
For achieving confocal images, HeLa cells were seeded in a laser
confocal microscopy 35 mm2 Petri dish (MatTek, USA) at a
density of 1.0 ꢂ 105 cells and stored for 48 h. DPE–PPE–FA+/
siRNA was prepared in serum-free media and incubated for 15
min at room temperature. The media containing serum were
added into the solution, and cells were transfected with the
complex and incubated for different time intervals (2 h and 5 h).
Aer the transfection, the media were discarded and the cells
were washed 3 times with 1ꢂ PBS. Confocal images were
obtained at each time point using a Leica TCS SP5 confocal laser
scanning microscope (Leica Inc., USA). To acquire the uores-
cence signal, each sample was excited at 488 nm with an Ar
Statistical analysis
laser. A uorescence signal of siRNACy3 was detected at 570–600 Statistical analysis of data was performed with the one-factor
nm and the DPE–PPE–FA+ signal was obtained at 480–510 nm analysis of variance (SPSS soware, version 13.0, SPSS Inc). The
with excitation at 488 nm.
results were expressed as mean ꢃ SD (standard deviation), and
P < 0.05 was considered to be statistically signicant. All
statistical tests were two-sided.
Cell transfection procedures
Cellular transfection of siRNA was performed using DPE–PPE+
and DPE–PPE–FA+ and commercial transfection reagents
Results and discussion
(siPort NeoFX, Ambion Inc, USA; HiPerFect, Qiagen Inc, Ger- For this study, we utilized cationic ammonium PPE as
many). For siRNA transfection, cells were trypsinized with nonspecic binding element for siRNA, which was spatially
0.25% of trypsin solution (HyClone Inc, USA) for 3 min at 37 restricted by the DPE framework. As showed in Scheme 2, a
ꢀC. Next, 1 ꢂ 104 cells per well were plated into 24-well plates neutral DPE–PPE was synthesized by a tandem polymerization
(Corning Inc, USA) overnight to achieve 60–80% conuence. methodology, chain walking polymerization (CWP) followed by
On the day of transfection, cultured cells were washed with 1ꢂ palladium/copper-catalyzed Sonogashira cross-coupling. The
PBS and incubated for 30 min with DMEM without serum and water-soluble cationic DPE–PPE (DPE–PPE+) was obtained
antibiotics. siPort NeoFX (2 mL per well) and HiPerFect (2 mL through quaternization of its neutral polymer.15 The quaterni-
per well) were diluted in 500 mL of DMEM (manufacturer rec- zation degrees (QDs) of DPE–PPE+ could be estimated to be
ommended concentrations) and were incubated for 15 min at about 50% by 1H NMR spectroscopy (Fig. 1), suggesting that
room temperature. Then 100 nM siRNA against VEGF (Dhar- almost half of all side chains featured a tertiary amino group.20
macon Inc, USA) was added to the mixture of medium and In an attempt to develop a siRNA nanocarrier with more effi-
transfection agents and incubated for an additional 20 min at cient uptake of the target cells, FA ligands may be installed on
room temperature. Immediately before transfection, 500 mL of the periphery of the PPE palisade of DPE–PPE to obtain water-
DMEM were added to DPE–PPE+/siRNA, DPE–PPE–FA+/siRNA soluble DPE–PPE–FA+ through a FA–Br-mediated quaterniza-
complexes and were mixed by pipetting. The DPE–PPE+/siRNA, tion pathway. Coupling of the folate residue to the periphery of
DPE–PPE–FA+/siRNA complexes diluted in DMEM were then the PPE palisade of DPE–PPE+ was conrmed by the appearance
added to each well, and the cells were incubated at 37 ꢀC for 24 of signals at d 6.0–9.0 ppm in the magnied 1H NMR spectrum
h. DMEM with 10% FBS was then added to the cells and of DPE–PPE–FA+ (Fig. 1), which corresponded to the aromatic
ꢀ
incubated for 24 h at 37 C in a CO2 incubator. Aer 24 h, the protons of folic acid. This is consistent with the ref. 16 and 17.
protein was extracted from the transfected cells for Western The relevant signals of folate are much weaker than the broad
blotting.
and strong proton signals of PPE+.
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Scheme 2 Synthetic routes for DPE–PPE+ and DPE–PPE–FA+.
of their concentration by standard MTT assay in HeLa cells. Aer
48 h treatment, the viability of HeLa cells was more than 80% at
concentrations ranging from 0 to 200 mg mLꢁ1 for DPE–PPE+
(Fig. 2). For comparison with DPE–PPE+, a similar cytotoxicity
result of DPE–PPE–FA+ was obtained, suggesting that the intro-
duction of FA on the surface of the nanocarrier has little effect on
the cell viability for DPE–PPE–FA+. The results demonstrate that
both DPE–PPE+ and DPE–PPE–FA+ have signicantly low cyto-
toxicity and have potential applications for in vivo siRNA delivery.
Fig. 1 1H NMR spectra of DPE–PPE+ and DPE–PPE–FA+ in D2O.
Cytotoxicity of DPE–PPE+ and DPE–PPE–FA+
The formation of DPE–PPE+/siRNA and DPE–PPE–FA+/siRNA
To evaluate the applicability of DPE–PPE+ and DPE–PPE–FA+ in
live cell studies, we assessed their acute cytotoxicity as a function
In this study, DPE–PPE+ and DPE–PPE–FA+ were investigated as
siRNA carriers. The formation of DPE–PPE–FA+/siRNA complex by
Fig. 2 Cell cytotoxicity of DPE–PPE+ and DPE–PPE–FA+ in HeLa cells by MTT (left) and agarose gel electrophoresis of DPE–PPE–FA+/siRNA complex with various ratios of
DPE–PPE–FA+ conjugate to siRNA (right).
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the electrostatic interaction between negatively charged siRNA
and positively charged DPE–PPE–FA+ conjugate was assessed by
agarose gel electrophoresis (Fig. 2). The DPE–PPE–FA+/siRNA
complex appeared to be formed when the weight ratio of DPE–
PPE–FA+ conjugate to siRNA was higher than 20. A similar result
wasobtainedfortheformationofDPE–PPE+/siRNAcomplex. Thus
the optimized weight ratios of DPE–PPE+/siRNA and of DPE–PPE–
FA+/siRNA were determined to be 20 : 1 in the preparation exper-
iments, therefore these values were selected for further work.
siRNA protection of DPE–PPE+ and DPE–PPE–FA+
nanocarriers
Fig. 3 The protection effect of DPE–PPE+ and DPE–PPE–FA+ carriers on siRNA in
the presence of RNaseA. Protection of siRNA exposed to RNaseA for 90 min was
measured for DPE–PPE+/siRNA (lane 2) and DPE–PPE–FA+/siRNA (lane 5)
complexes, and compared with naked siRNA in the presence of RNaseA (lanes 1
and 4). Lane 3 corresponds to control naked siRNA in the absence of RNaseA.
As we know, siRNA can be easily degraded by nucleic acidase,
which has hindered the application of siRNA as tools in gene
silencing to some extent. Therefore, the capability to protect
siRNA is very important for the carrier. The siRNA protection
Fig. 4 Flow cytometry for transfection of DPE–PPE+/siRNA, DPE–PPE–FA+/siRNA and the control. Incubation time: 6 h. Dose: 100 nM siRNACy3
.
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Fig. 5 Time-dependent confocal microscopy images of DPE–PPE–FA+/siRNA complexes and their entry and transportation in HeLa cells. The red florescence is siRNACy3
and the green florescence is DPE–PPE–FA+. Scale bars: 25 mm.
experiment was performed to measure the capability of the rapid transport of siRNA into the cell. Like QD particles, the
DPE–PPE+ and DPE–PPE–FA+ carriers to protect siRNA from intrinsic uorescence of DPE–PPE+ and DPE–PPE–FA+ renders
being degraded by RNaseA. As shown in Fig. 3, naked siRNA was them well-suited for the observation of the siRNA delivery
completely degraded under the experimental conditions (Fig. 3, process, but they have lower cytotoxicity than QD particles.
lanes 1 and 4). However, siRNA bound within the carriers was
stable to RNaseA over a 90 min incubation at 37 ꢀC (Fig. 3, lanes
2 and 5), suggesting the conjugation of siRNA to DPE–PPE+ and
DPE–PPE–FA+ carriers could both effectively protect siRNA from
enzymatic degradation.
Gene silencing efficiency of DPE–PPE+/siRNA and DPE–PPE–
FA+/siRNA complexes
The gene silencing efficiency of DPE–PPE+ and DPE–PPE–FA+
were investigated by western blotting experiments, and
commercial transfection reagents, siPort NeoFX and HiPerFect
were kept as contrasts (Fig. 6). The results showed similar
silencing efficiencies of DPE–PPE+ and DPE–PPE–FA+ on VEGF
protein in HeLa cells aer transfection compared with the
commercial transfection reagents. Compared with the controls,
the levels of VEGF protein expression were reduced to 18 ꢃ 8.5%
Intracellular uptake of DPE–PPE+/siRNA and DPE–PPE–FA+/
siRNA complexes
Flow cytometry was utilized to evaluate transfer efficiency when
using DPE–PPE+ and DPE–PPE–FA+ as nanocarriers to deliver
siRNACy3 into HeLa cells (Fig. 4). According to the quantitative
ow cytometric analysis, DPE–PPE–FA+ showed greater uptake
than DPE–PPE+ in HeLa cells at the same concentrations, sug-
gesting the FA on the surface of nanocarrier could make the
entry of the nanocarrier into the targeted cells easier. It was also
shown that DPE–PPE+ and DPE–PPE–FA+ nanocarriers dis-
played siRNA transfer activity. Cationic conjugated polymer
chains with multiple recognition elements can bind to the
negatively charged siRNA through electrostatic interactions,
thereby increasing the delivery efficiency of siRNA. Moreover,
siRNA was more efficiently transferred into cells when using
DPE–PPE–FA+ rather than DPE–PPE+, as shown in Fig. 4.
Considering that DPE–PPE–FA+ as siRNA nanocarrier
exhibited better results than DPE–PPE+, we chose confocal
microscopy to monitor the time-dependent accumulation of
DPE–PPE–FA+/siRNA complex in HeLa cells (Fig. 5). Aer inter-
nalization, the DPE–PPE–FA+/siRNA complexes were observed to
become accumulated at a region outside the cell nucleus at 2 h.
Cellular uptake of more complexes was observed and more
siRNAs were transported into the cells at 5 h. It is shown that
Fig. 6 The suppression of VEGF protein expression was evaluated by western
DPE–PPE–FA+ nanocarrier exhibits efficient cellular uptake, and blotting. The siRNA carriers are DPE–PPE+ and DPE–PPE–FA+ (n ¼ 3; p < 0.05).
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and 23 ꢃ 5.8% for DPE–PPE–FA+ and DPE–PPE+ treated cells,
respectively. Using DPE–PPE+ and DPE–PPE–FA+ as siRNA
nanocarriers, we achieved gene silencing efficiency of almost
80% in HeLa cells. It is shown that an appreciable silencing of
the target gene at an extremely low siRNA concentration is
achieved through the assembly of siRNA into smart DPE–PPE+
and DPE–PPE–FA+ polyvalent nanoparticles.
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Conclusions
We report here an effective monitored siRNA delivery system
based on the water-soluble dendritic polyethylene–cationic
poly(p-phenylene ethynylene). For utilizing uorescent conju-
gated polyelectrolyte-based polyvalent nanocarriers and their
unusual properties to load, protect and transport siRNA across
cell membranes, the distinctive nanocarrier design achieved
low cytotoxicity, high transfer efficiency and monitored siRNA
delivery. Notably, the complex of the nanocarrier system and
siRNA revealed remarkable gene knockdown. These results
suggest that this newly designed system of polyvalent nano-
carriers will have great promise for in vivo gene therapeutics.
Acknowledgements
This work was nancially supported by the program of National
Natural Science Foundation of China (Grant no. 20804059.),
and the Fundamental Research Funds for the Central Univer-
sities (Grant no. 31000-3161711).
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