A highly emissive conjugated polyelectrolyte vector for gene delivery and transfection

Article abstract of DOI:10.1002/adma.201202145

An intrinsically fluorescent cationic polyfluorene (CCP) has been designed, synthesized, characterized, and examined as a plasmid DNA (pDNA) delivery vector. This material facilitates nucleic acid binding, encapsulation and efficient cellular uptake. CCP can effectively protect pDNA against nuclease degradation, which is necessary for gene carriers. Green fluorescent protein (GFP) expression experiments reveal that CCP can achieve efficient delivery and transfection of pDNA encoding GFP gene with 92% efficiency, which surpasses that of commercial transfection agents, lipofectamine 2000 (Lipo) and polyethylenimine (PEI). CCP is also highly fluorescent, with 43% quantum yield in water, and exhibits excellent photostability, which allows for real-time tracking the location of gene delivery and transfection. These features and capabilities represent a major step toward designing and applying conjugated polymers that function in both imaging and therapeutic applications. An intrinsically fluorescent cationic polyfluorene (CCP) has been designed, synthesized, characterized, and examined as a plasmid DNA (pDNA) delivery vector. CCP can effectively protect pDNA against nuclease degradation. CCP can achieve highly efficient delivery and transfection of gene with 92% efficiency, which surpasses that of commercial transfection agents. Copyright

Full text of DOI:10.1002/adma.201202145

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A Highly Emissive Conjugated Polyelectrolyte Vector
for Gene Delivery and Transfection
Xuli Feng, Fengting Lv, Libing Liu,* Qiong Yang, Shu Wang,* and Guillermo C. Bazan*
toxicity.[18] These challenges prompt the
design of new safe and efficient trans-
fection agents.[19] New materials such as
aptamer-siRNA conjugates,[20] minicells,[7]
carbon nanotubes,[21] and inorganic nano-
particles including silica,[22] iron oxide,[23]
and gold[24] have been exploited recently
as alternatives of nonviral vectors. All
of these delivery technologies lack an
intrinsic signal for long-term and real-
time imaging of gene delivery and release.
In this context, by attaching fluorescent
dyes onto gene vectors, gene delivery can
be tracked via fluorescence microscopy.
Despite these advantages fluorescent dyes
often suffer from photobleaching and cel-
luar toxicity. Quantum dots (QDs) can be
considered to their unique optical proper-
ties (e.g., tunable emission, photostability,
and brightness),[25,26] but their cytotoxicity
An intrinsically fluorescent cationic polyfluorene (CCP) has been designed,
synthesized, characterized, and examined as a plasmid DNA (pDNA) delivery
vector. This material facilitates nucleic acid binding, encapsulation and
efficient cellular uptake. CCP can effectively protect pDNA against nuclease
degradation, which is necessary for gene carriers. Green fluorescent protein
(GFP) expression experiments reveal that CCP can achieve efficient delivery
and transfection of pDNA encoding GFP gene with 92% efficiency, which
surpasses that of commercial transfection agents, lipofectamine 2000
(Lipo) and polyethylenimine (PEI). CCP is also highly fluorescent, with 43%
quantum yield in water, and exhibits excellent photostability, which allows
for real-time tracking the location of gene delivery and transfection. These
features and capabilities represent a major step toward designing and
applying conjugated polymers that function in both imaging and therapeutic
applications.
is severe owing to leaching of harmful metals and the genera-
tion of reactive oxygen species. Developing novel materials with
efficient luminescence and high transfection efficiencies thus
remains challenging.
Gene-based therapies are relevant to pharmaceutical
research.[1–3] Antisense DNA and small interfering RNA
(siRNA) are widely studied due to their relevance to cancer
therapy.[4–9] Important obstacles to overcome include inade-
quate delivery and transfection, due to the membrane perme-
ability inhibition by the negative charges of the gene, and rapid
degradation by serum nucleases. Therefore, there is a need for
gene carriers.[10] Recombinant viruses have proven to be effec-
tive transfection vectors, but suffer from problems of immuno-
genicity, carcinogenicity and inflammation that hinder clinical
implementation.[11–13] Nonviral vectors have been recently devel-
oped in response, such as cationic polymers,[14] lipid or lipid-
like materials,[15] dendrimers[16] and peptides.[17] These systems,
however, exhibit lower delivery efficiency or higher celluar
Conjugated polymers have established themselves as useful
optical platforms to sensitively detect chemical and biological
molecules due to the signal amplification by a collective system
response.[27–32] Several conjugated polymers have been recently
prepared for live-cell imaging because of their high fluores-
cence brightness, good photostability, and lower toxicity.[33–37]
Conjugated polymer nanoparticles have also been used for
siRNA delivery in plants.[38] Herein, we describe the prepara-
tion of a new cationic water-soluble conjugated polymer (CCP,
see Scheme 1 for its chemical structure). This material has ver-
satile features to serve as an imaging and gene delivery vehicle,
including protection of pDNA against nuclease degradation,
high delivery and transfection efficiency, high fluorescent
quantum yield in water and good photostability. These new fea-
tures and capabilities provide unique guidelines for the design
of synthetic polycationic vectors for both imaging and thera-
peutic applications.
Prof. S. Wang, Dr. X. Feng, Dr. F. Lv,
Dr. L. Liu, Dr. Q. Yang
Beijing National Laboratory for Molecular Sciences
Key Laboratory of Organic Solids
Institute of Chemistry
Chinese Academy of Sciences
CCP was synthesized by the route depicted in Scheme 1.
Reaction of 3,5-dihydroxyl-benzyl alcohol (1) with 1,3-dibromo-
propane in acetone affords compound 2 in 64% yield. Reaction
of compound 2 with N-bromosuccinimide (NBS) gives com-
pound 3 in 93% yield. Compound 4 is obtained in 63% yield
by reacting 2,5-dibromohydroquinone and compound 2 in the
presence of K2CO3 for 2 days. Compound 6 is prepared in 98%
yield by reaction of 2,7-dibromofluorene with bromoethane
in the presence of KOH and (n-Bu)4NBr. Treatment of 6 with
Beijing 100190, P. R. China
E-mail: liulibing@iccas.ac.cn; wangshu@iccas.ac.cn
Prof. G. C. Bazan
Departments of Chemistry & Biochemistry and Materials
Center for Polymers and Organic Solids
University of California
Santa Barbara, CA 93106-9510, USA
E-mail: bazan@chem.ucsb.edu
DOI: 10.1002/adma.201202145
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Adv. Mater. 2012,
DOI: 10.1002/adma.201202145
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Br
Br
Br
Br
HO
OH
O
O
O
O
Br
Br
NBS
CH2OH
PPh3
K2CO3
CH2Br
CH2OH
1
3
2
Br
OH
Br
Br
Br
O
O
O
Br
OH
O
O
O
K2CO3
Br
Br
4
Br
B
O
O
Br
O
O
Br
O
O
O B
Br
Br
Br
KOH
B
n-BuLi
5
7
6
Br
Br
N
O
Br
O
O
Br
N
O
O
O
4
Me3N
n
Pd(dppf)Cl2
K2CO3
n
O
O
O
O
Br
O
N
O
N
Br
Br
Br
8
CCP
Scheme 1. Schematic preparation and molecular structure of the cationic conjugated polyelectrolyte CCP.
n-BuLi in anhydrous THF at –78 ºC, followed by treatment with
isopropoxyboronic acid pinacol ester gives compound 7 in 67%
yield. Polymerization of 4 with 7 can be carried out by Suzuki
coupling reaction in the presence of 2.0 M aqueous K2CO3 and
PdCl2(dppf) in toluene to afford polymer precursor 8 in 45%
yield. The molecular weight of 8 was determined by gel per-
meation chromatography (GPC) with polystyrene standards
and THF as eluent. The number-average molecular weight (Mn)
of 8 is 13360 with a polydispersity index (PDI) of 2. Quaterni-
zation of 8 by 33% trimethylamine alcoholic solution in THF
gives the desired positively charged CCP, which is soluble in
polar solvents, such as DMSO, DMF and water. CCP exhibits
an absorption maximum at 372 nm in water, corresponding to
the π–π∗ transition of the conjugated backbone. It emits blue
fluorescence in water with a maximum at 422 nm. The fluores-
cence quantum yield of CCP in water was measured to be 43%
by using quinine sulfate in 0.1 M H2SO4 as the standard. These
results demonstrate that CCP has good water solubility and
optical performance. Furthermore, CCP is positively charged
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The photostability of CCP (important for real time cell
imaging experiments) was evaluated by comparison against
the commonly used fluorescein dye. As shown in Figure 2a,
the fluorescence intensity of fluorescein quickly drops below
a detectable level after only 10 seconds irradiation with a mer-
cury lamp (100 W) at 480 nm, while CCP retains 80% of the
original fluorescence upon continuously irradiating at 380 nm
for 60 seconds. Based on the good optical property and photo-
stability, CCP was tested for use in cell imaging. As shown
in Figure 2b-c, blue fluorescence from the cellular cytoplasm
is visible for all CCP stained cells. The cellular nuclei were
co-stained with propidium iodide (PI) that exhibits red emis-
sion color. The absence of fluorescence overlap between CCP
and PI indicates that CCP is efficiently internalized by these
cells and is mainly accumulated around the perinuclear region.
This cationic conjugated polyelectrolyte therefore provides an
optical tool for real-time tracking of gene delivery and transfec-
tion, as described in more detail below.
CCP was studied as a vector to deliver pDNA-encoding green
fluorescent protein (GFP) into Hela cells. Figure 3a shows the
phase contrast (left) and fluorescence images of Hela cells
treated with CCP/pDNA complex. Both the blue fluorescence
of CCP (middle) and the green fluorescence of GFP (right)
were observed. Thus, we can conclude that CCP has success-
fully delivered plasmid into cells for further transcription and
translation. As shown in Figure 3b, CCP can achieve approxi-
mately 92% GFP expression, which surpasses the efficiency
of commonly used transfection reagents, Lipo (83%) and PEI
(88%) (based on the same pDNA concentration and the opti-
mized amount for each transfection reagent). Thus, CCP can
a
Hela cell
MCF7/ADR cell
100
90
80
70
60
50
40
0
5
10
[CCP] (
µ
M)
b100
Hela cell
MCF7/ADR cell
80
60
40
20
0.0
0.2
0.4
0.6
0.8
1.0
Lipo Amount (µL)
Figure 1. (a) Cell viability as a function of CCP concentration after incuba-
tion with Hela and MCF7/ADR cells. (b) The effect of commercial trans-
fection agent Lipo2000 on cell viability as comparison experiment.
1.0
a
0.8
0.6
which lays a foundation for its electrostatic interactions with
oppositely charged pDNA and siRNA macromolecules.
CCP
0.4
0.2
0.0
To study CCP for gene delivery and transfection, the plasmid
DNA that can express green fluorescent protein (GFP) in Hela
cells was used as model pDNA. To demonstrate in vitro bio-
compatibility of CCP, its cytotoxicity on Hela and human breast
adenocarcinoma (MCF7/ADR) cells was assayed using the
methylthiazolyldiphenyl-tetrazolium bromide (MTT) method,
in which the conversion of soluble MTT into formazan is
directly related to mitochondrial activity and thereby to cell via-
bility. Hela and MCF7/ADR cells were grown in 96-well culture
plates, and were then treated with CCP. Figure 1a shows the
effect of CCP with varying concentrations (0 ∼ 10 μM, relative
to repeat unit) on the viability of Hela and MCF7/ADR cells.
Although the cell viability decreases after 24 h, incubation upon
increasing concentration of CCP, the viability is still larger
than 70%. For Lipo2000, a commercial product for gene trans-
fection, the cell viability was reduced to lower than 20% upon
increasing its amount from 0 to ∼ 1.0 μL, a concentration nec-
essary to obtain high transfection efficiency (Figure 1b). Thus,
the CCP shows lower toxicity than Lipo2000, which is critical
for allowing subsequent cell experiments.
Fluorescein
0
10
20
30
40
50
60
Irradiation time (s)
Figure 2. (a) Photostability of CCP with fluorescein as comparison. Irra-
diation at 480 nm for fluorescein and 380 nm for CCP using a mercury
lamp (100 W). (b,c) Fluorescence images of Hela and MCF7/ADR cells
treated with CCP. The cellular nuclei are stained by propidium iodide (PI)
that exhibits red emission color.
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uptake. CCP can effectively protect pDNA against nuclease
degradation, which is a desirable attribute of gene carriers.
CCP exhibits a transfection efficiency of 92%, which surpasses
the performance of commercial transfection agents Lipo and
PEI. CCP is also highly fluorescent with 43% quantum yield
in water and exhibits excellent photostability, thereby enabling
real-time tracking the location of the gene during delivery. To
the best of our knowledge, this is the first reported conjugated
polymer that can be employed both for imaging and highly
efficient gene delivery. These new features and capabilities
represent an innovative step toward multifunctional synthetic
polycationic vectors both for imaging and therapeutic applica-
tions and highlight the multiple functions that can be achieved
by suitably tailoring the molecular structures of conjugated
polyelectrolytes.
b100
80
60
40
20
0
PEI
CCP
Lipo
Figure 3. (a) Phase contrast (left) and fluorescence microscopy images
of Hela cells incubated with CCP/pDNA complex. Fluorescence image
of CCP (middle) was recorded on fluorescence microscopy (Olympus
1 × 71) using a 380/30 nm excitation filter and that of GFP (right) was
recorded using a 455/70 nm excitation filter. (b) Quantifying the expres-
sion of GFP using flow cytometry. Each bar represents the mean GFP
expression in three independent experiments. In this and all other fig-
ures, error bars represent standard deviations. (c) Protection of pDNA
from nuclease degradation. lane 1: 1 kb marker; lane 2: naked pDNA;
lane 3: naked pDNA with FBS; lane 4: CCP/pDNA; lane 5: CCP/pDNA
digested with FBS; lane 6: CCP/pDNA with addition of heparin solution
after digested with FBS.
Experimental Section
Materials and Measurements: All chemicals were purchased from
Acros, Aldrich or Alfa-Aesar Chemical Company and used as received. All
organic solvents were purchased from Beijing Chemical Works and used
as received. The water was purified using a Millipore filtration system.
Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was obtained from
Xinjingke Biotechnology Co., Ltd (Beijing, China). Goldview DNA dye
was purchased from Beijing SBS Genetech Co. Ltd. LipofectamineTM
2000 was purchased from Invitrogen. TIANprep Mini Plasmid Kit was
purchased from Tiangen Biotech (Beijing) Co., Ltd. Hela (human cervical
cancer) cell was purchased from cell culture center of Institute of Basic
Medical Sciences, Chinese Academy of Medical Sciences (Beijing,
China). MCF7/ADR cells were purchased from KeyGEN Biotech. The
1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance
400 MHz spectrometer. Mass spectra were recorded on Bruker Biflex
MALDI-TOF spectrometer. Elemental analyses were carried out with a
Flash EA1112 instrument. The gel permeation chromatography (GPC)
measurement was performed on a Water-410 system against polystyrene
standards with tetrahydrofuran (THF) as eluent. UV-Vis absorption
spectra were taken on a JASCO V-550 spectrophotometer and the
fluorescence spectra were measured using Hitachi F-4500 fluorometer
with a Xe lamp as excitation source. Zeta potentials and size analysis
were measured on a Nano ZS (ZEN3600) system. PCR was conducted
on a Bio-Rad Mycycler Thermocycler. The image of the gel was taken
by a Bio-Rad Molecular Imager ChemiDox XRS system. Phase contrast
bright-field and fluorescence images were taken with fluorescence
microscopy (Olympus 1 × 71) with a mercury lamp (100 W) as the light
source. The absorbance for MTT analysis was recorded on a microplate
reader (BIO-TEK Synergy HT, USA) at a wavelength of 520 nm.
Synthesis of conjugated polymer (8): A mixture of the monomer 4
(1 equiv) and 7 (1 equiv) in toluene and 2.0 M aqueous K2CO3 solution
was degassed, and then catalytic quantity of Pd(dppf)Cl2 was added
under nitrogen atmosphere. The reaction mixture was stirred at 100 ºC
under nitrogen for 48 h. After cooling down to room temperature,
50 mL water was added and was extracted with chloroform. After the
organic solvent was removed, the residue was precipitated in acetone
or methanol. The crude polymers were purified by precipitation from
chloroform into acetone or methanol again and dried under vacuum to
give a yellow solid (yield 45%). 1H-NMR (400MHz, CDCl3): δ (ppm) 0.46
(br, 6H) 2.13-2.31 (br, 12H), 3.58 (br, 8H), 4.12 (br, 8H), 5.01 (br, 4H),
6.38 (br, 2H), 6.52 (br, 4H), 7.13 (br, 2H), 7.64 (br, 2H), 7.77-7.82 (br, 4H).
13C-NMR (100MHz, CDCl3): δ (ppm) 8.4, 29.3, 32.1, 55.6, 66.8, 68.2,
71.6, 100.7, 106.2, 116.3, 118.9, 123.6, 127.8, 128.7, 131.2, 136.6, 130.8,
139.9, 149.3, 149.8, 152.4, 159.8.
successfully deliver nucleic acid into Hela cells for efficient
transcription and translation. We surmise that the ability of
CCP to protect pDNA against nuclease degradation is a likely
contributor to the high transfection efficiency. As demonstrated
by the gel electrophoresis images in Figure 3c (lane 4), pDNA
forms a tight complex with CCP, due to electrostatic comple-
mentarity, and the migration of pDNA on agarose gel is thereby
retarded because of charge neutralization and the increase in
molecular size of the complexes.
The effect of protection from DNase degradation was exam-
ined by using foetal bovine serum (FBS) as a model. Naked
pDNA shows complete degradation, while intact pDNA can
be recovered from the CCP/pDNA complex after sequential
incubation with FBS, followed by dissociating the complexes
with heparin solution (lane 6). These results indicate that CCP
renders a significant protection to the plasmid against nuclease
digestion. The compaction of the pDNA into stable nanoparti-
cles is also important for efficient cellular uptake. Dynamic light
scattering (DLS) and zeta potential (ζ) experiments were con-
ducted to examine the size and surface charge of CCP/pDNA
complex. The mean particle size of CCP/pDNA is 125 nm from
SLD experiment, which is in favor of the endocytosis.[33] The ζ
value of the CCP/pDNA complex is +42.7 mV. Since the cellular
membranes are negatively charged, the positive ζ is helpful for
interaction with cells. All these data ensure the high transfec-
tion efficiency achieved by the above experiments.
In conclusion, the new intrinsically fluorescent cationic con-
jugated polyelectrolyte CCP has been synthesized and charac-
terized. This material was designed with the intention to facili-
tate nucleic acid binding, encapsulation and efficient cellular
Synthesis of cationic conjugated polymer (CCP): To a solution of
polymer 8 in 10 mL THF was added 33% trimethylamine alcoholic
solution. After stirring for 24 h at room temperature, the solvent and
excessive trimethylamine was removed and 10 mL of acetone was added
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to precipitate the product as a solid (yield 95%). 1H-NMR (400MHz,
DMSO-d6): δ (ppm) 0.38 (br, 6H) 1.88-1.96 (br, 12H), 3.04 (br, 36H,
NCH3), 3.29 (br, 8H, NCH2), 3.99 (br, 8H), 5.06 (br, 4H), 6.42 (br, 2H),
6.62 (br, 4H), 7.44 (br, 2H), 7.54 (br, 2H), 7.74-7.84 (br, 4H).
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Transfection experiment: In a typical transfection experiment, Hela cells
were first seeded onto 24-well plates (6 × 104 cells per well, in 0.6 mL
growth medium) 24 h prior to the experiment. 1 μg of plasmid DNA
encoding GFP was diluted with 50 μL serum-free medium, and a certain
amount of CCP were also diluted with 50 μL serum-free media, then
the diluted CCP (50 μL) was added to the 50 μL diluted DNA solution,
mixing gently and incubating for 20 ∼ 30 minutes at room temperature.
The 100 μL of the complex was then added to a well containing cells and
600 μL serum-free medium, mixing gently by rocking the plate back and
forth. Four hours after transfection, 500 μL of the supplemented DMEM
was added to each well. Twenty-four hours after transfection, the media
was replaced with fresh supplemented DMEM (1 mL). Forty eight hours
after initial transfection, the medium was removed and the cells were
washed with phosphate buffered saline (PBS, pH 7.4) three times for
gene expression analysis and image studies. The cells were evaluated
for expression of the green fluorescent protein by flow cytometry. To
compare the transfection efficiency of CCP with other commercially
available transfection reagents, Lipo and PEI were used to transfect the
Hela cells under the similar experimental condition.
Cell imaging: In order to study the endocytosis and location of CCP
mediated transfection, Hela and MCF7/ADR cells were seeded onto
35 mm plate, after 24 h incubation, the medium was replaced with fresh
supplemented DMEM (1 mL), and then CCP was added to the fresh
medium. After incubation for 8 h, the cells were washed three times with
1 × PBS buffer and then fixed by 75% DMSO for 20 minutes and further
washed twice with 1×PBS buffer. The nuclei were stained with PI for
40 minutes. The cell monolayer was washed twice with 1×PBS buffer and
imaged by fluorescence microscopy (Olympus 1 × 71). The fluorescence
images of CCP were recorded using a 380/30 nm excitation filter, and
that of PI were recorded using a 532/50 nm excitation filter.
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Acknowledgements
The work described in this manuscript was supported by the National
Natural Science Foundation of China (Nos. 21033010, 21021091),
the National Basic Research Program of China (Nos. 2011CB932302,
2012CB932600) and the National Science Foundation (DMR-1005546).
Received: May 29, 2012
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Adv. Mater. 2012,
DOI: 10.1002/adma.201202145
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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