Hybrid of baculovirus and galactosylated PEI for efficient gene carrier
You-Kyoung Kima, Jae Young Choia,c, Hu-Lin Jianga,c, Rohidas Arotea, Dhananjay Jerea, Myung-Haing Chob,
Yeon Ho Jea,c,⁎, Chong-Su Choa,c,⁎
aDepartment of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea
bLaboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea
cResearch Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea
a b s t r a c t a r t i c l ei n f o
Received 13 October 2008
Returned to author for revision
29 January 2009
Accepted 2 February 2009
Available online 9 March 2009
Baculovirus, containing an appropriate eukaryotic promoter, is considered an attractive approach for an
efficient and safe gene delivery vehicle. However, the drawbacks of baculovirus, such as the lack of specificity
and the inactivation of baculovirus by the complement system in human serum, negatively affect efficient
gene delivery. Therefore, a hybrid system utilizing the positive aspects of both viral and non-viral vector
systems would be useful to overcome the obstacles of either system alone. In this study, we constructed a
hybrid system composed of baculovirus (B) and galactosylated polyethylenimine (GP)/DNA complexes
through electrostatic interaction. The resulting GP/B hybrid had suitable physicochemical properties and low
cytotoxicity for use in gene therapy. Furthermore, the GP/B significantly enhanced transduction efficiency
and showed good cell-specificity compared to either viral or non-viral vector systems. These results suggest
that the GP/B hybrid system can be used in gene therapy to enhance transduction efficiency and hepatocyte
© 2009 Elsevier Inc. All rights reserved.
Gene therapy has the potential to treat devastating inherited
diseases and can lead to the prevention, correction, or modulation of
genetic and acquired diseases by introducing genes coding for
therapeutic proteins (Cavazzana-Calvo et al., 2004; Mahato et al.,
1999). The primary challenge for vector-based gene therapy treat-
ments for cancer or any other disease is the ability to develop vectors
that enable a dose of the therapeutic genes to be delivered to and
expressed in the diseased tissues with high efficiency, specificity, and
safety (Yangetal.,2007a,2007b). Viralvectorshave greatpotential for
both gene delivery and expression, and virus-mediated gene therapy
has been well studied as a strategy for correcting a variety of genetic
disorders (Smith, 1995). Among the many types of viral vectors,
animal viral vectors have many advantageous attributes including
their high infectivity of most mammalian cell types, high expression
level of transgenes (lentivirus and adenovirus), relatively large
packaging capacity (HSV-1 and adenovirus), and relatively low
induction of inflammation (AAV) (Yang et al., 2007a, 2007b; Thomas
et al., 2005). However, many critical obstacles remain which prevent
the safe use of animal viral vectors in gene therapy such as the
potential to induce strong host immune responses, the non-specificity
of transgene delivery, and the capability to cause insertional
mutagenesis inducing oncogenesis, especially for integrating viral
vectors (Thomas et al., 2005; Douglas, 2007).
Recently, Autographa californica multiple nuclear polyhedrosis
virus (AcMNPV), a prototype of the Baculoviridae family, has emerged
as an attractive candidate for gene therapy applications (Makela et al.,
2006). Interestingly, baculovirus containing an appropriate eukaryotic
promoter has been shown to transfer and express target genes
efficiently in several mammalian cell types both in vitro and in vivo
(Tani et al., 2003; Airenne et al., 2000). Furthermore, the lack of
toxicityand replication, large cloning capacity, ease of production, and
lack of preexisting immunity have made baculovirus a promising tool
for gene therapy (Hu, 2006). However, manychallenges remain before
the baculoviral vector system can be applied to human gene therapy.
Inparticular, baculovirus vectors exhibit transient expression, a lack of
cell target specificity, and are inactivated in human serum and whole
blood, all of which prevent efficient gene expression of baculoviral
vectors (Matilainen et al., 2005).
Recent studies have reported that the merging of viral and non-
viral vector research efforts could provide a promising strategy for the
future optimization of both vector classes because the currently used
classes of viral and non-viral vectors have complementary strengths,
such as the high intracellular efficiency of viral vectors, and the high
systemic potential and low immunogenicity of shielded non-viral
vectors (Boeckle and Wagner, 2006). To merge viral and non-viral
vectors, hybrid vector systems have been generated by combining
Virology 387 (2009) 89–97
⁎ Corresponding authors. C.-S. Cho is to be contacted at the Department of
Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea.
Fax: +82 2 875 2494. Y.H. Je, Department of Agricultural Biotechnology, Seoul National
University, Seoul 151-921, South Korea. Fax: +82 2 878 4706.
E-mail addresses: firstname.lastname@example.org (Y.H. Je), email@example.com (C.-S. Cho).
0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
viral and non-viral elements. The viral vector genome and the
separate non-viral gene expression cassette may synergize in some
applications (Boeckle and Wagner, 2006). In addition, the non-viral
component may be modified with targeting ligands to allow
retargeting of the hybrid vector, whereas the virus component may
confer greatly improved intracellular efficiency (Diebold et al., 1999).
Wagner et al. reported that the coupling of adenovirus (as a viral
component) to transferrin-polylysine/DNA complexes (as a non-viral
component) greatly enhanced receptor-mediated gene delivery and
expression of transfected genes (Wagner et al., 1992).
In this study, we constructed a hybrid system composed of viral
and non-viral vector components to combine their complementary
strengths. Galactosylated polyethylenimine (GP) as a non-viral vector
system was predicted to provide hepatocyte specificity to the hybrid
system via asialoglycoprotein receptor (ASGPR)-mediated endocyto-
sis and additionally prevent the inactivation of baculovirus (B) in
human serum byshielding the viral surface.The hybrid system (GP/B)
was formed by electrostatic interaction between the positively
charged GP/DNA complexes and the negatively charged baculovirus.
The transduction efficiency of GP/B was compared with non-hybrid
Fig.1. (A) Synthesis scheme for galactosylated PEI (GP). (B) Representative1H NMR spectra of PEI and galactosylated PEI (GP) in D2O. (C) Schematic representation of hybridization
between GP/DNA complexes and baculovirus (GP/B).
Y.-K. Kim et al. / Virology 387 (2009) 89–97
systems and the hepatocyte specificity via ASGPR-mediated endocy-
tosis was evaluated.
Results and discussion
Preparation of GP and GP/B
GP was successfully synthesized through an amide linkage
between the carboxyl group of lactobionic acid and the primary
amine of PEI (MW: 1800) (Fig.1A). The resultant GP was analyzed by
1H nuclear magnetic resonance (1H NMR) (Fig. 1B). The composition
of the galactose group in the GP was approximately 4 mol-%. Hybrid
(GP/B) can be easily formed by electrostatic interactions between the
GP/DNA complexes and the baculovirus (Fig. 1C) because naked
baculovirus is highly negatively charged (about −33 mV) (Kim et al.,
2007) and GP/DNAcomplexes have a positivesurface charge(Fig. 2C).
Characterization of GP/DNA complexes and GP/B
The DNA condensation ability of GP was evaluated by gel
retardation assay. As shown in Fig. 2A, the GP/DNA complexes were
completely formed when the GP/DNA charge ratio (N/P) was
approximately 3. Protection and release of the DNA were also
evaluated because the DNA in the gene vehicle needs to be protected
2005). As shown in Fig. 2B, DNA in the complexes was protected from
DNase I, while DNA alone treated with DNase I was completely
Surface characteristics such as surface charge, particle size, and
morphology, are important parameters for efficient uptake of the
complexes by cells. For example, a positive surface charge is required
for binding to anionic cell surfaces, thereby facilitating uptake by the
cell (Kunath et al., 2003). As shown in Fig. 2C, the surface charge of
GP/DNAcomplexes increased with increasing N/P ratios. Inparticular,
the high negative charge of the baculoviral surface was reversed to a
positive charge up to +12 mV following hybridization with GP/DNA
complexes. Raty et al. reported that avidin-displaying baculovirus
enhanced the transduction efficiency due to the high positive charge
of avidin (Raty et al., 2004). Therefore, the positive surface charge of
the hybrid baculoviral system was expected to enhance the transduc-
tion efficiency than native baculovirus.
Morphologies of GP/DNA complexes, naked baculovirus, and GP/B
hybrid were observed by energy-filetring transmission electron
microscope (EF-TEM) (Fig. 2D). GP/DNA complexes had well-formed
spherical and compacted structure less than 100 nm (Fig. 2D (I)),
while naked baculovirus had a cigar-shaped nucleocapsid 25 by
260 nm (Fig. 2D (II)), as seen previously (Yang et al., 2007a, 2007b).
Importantly, the morphologies of GP/B observed by EF-TEM as shown
in Fig. 2D (III) were consistent with our schematic representation of
hybridization (Fig. 1C).
Cytotoxicity of GP/B
was determined at various concentrations of GP or GP/B using the Cell
B exhibited high cell viability (greater than 80% of control) even at high
concentrations (50 μg/ml), while the cell viability of PEI 25K and low
molecular weight PEI (PEI 1800) was drastically decreased with
increasing concentrations (Fig. 3). The cytotoxicity of cationic polymers
is most likely caused by polymer aggregation on the cell surface that
impairs important membrane functions (Bottega and Epand, 1992;
Wong et al., 2006) and typically occurs in a molecular weight-
dependent manner (Kunath et al., 2003). In contrast, galactosylation
of cationic polymers has been reported to decrease cytotoxicitybecause
of the reduction of charge density of cationic polymers (Jiang et al.,
2007b; Carreno-Gomez and Duncan,1997). Therefore, GP showed high
cell viability due to the properties of low molecular weight PEI and the
shielding of the primary amines of PEI 1800 via galactosylation.
Moreover, Makela et al. reported that the cytotoxic effects of
baculovirus in mammalian cells appear to be comparatively small
owing to the baculovirus' high species specificityand lack of replication
in mammalian cells (Makela et al., 2006). Because of these character-
istics of the baculovirus, GP/B showed high cell viability regardless of
multiplicity of transduction (MOT) as shown in Fig. 3.
For further experiments, various concentrations from 0.52 μg/ml to
15.6 μg/ml of GP and GP/B were used according to various N/P ratios
from1 to30.As showninFig. 3,thecellviabilityintheseconcentrations
was higher than 90% of control, indicating that cytotoxicity of used
carriers was not affected on further studies.
Transduction efficiency of GP/B
To optimize the transduction conditions of the hybrid system,
luciferase activity was measured according to various parameters,
such as N/P ratio, MOT, and sample treatment conditions. The
transduction efficiency of GP/B was first evaluated in relation to the
N/P ratio (Fig. 4A). The transduction efficiency of GP/B was higher
than PEI 25K, PEI 1800, GP, and naked baculovirus. However, the
transduction efficiency of GP/B decreased with an N/P ratio greater
than 10, indicating that there is an optimum N/P ratio for use of the
hybrid system in gene therapy. Therefore, N/P ratios of 3, 5,10, and 20
were selected for further studies. In addition, to investigate the
transduction efficiency of GP/B with regards to the MOT of
baculovirus, luciferase activity was measured with various MOTs
(50, 100, and 300) as shown in Fig. 4B. The transduction efficiency of
GP/B was MOT-dependent at every N/P ratio tested, indicating that
the virus remained functional even after formation of the hybrid
system which contains non-viral components.
Various sample treatment conditions were compared to optimize
the efficiency of the hybrid system because traditional sample
treatment conditions of viral and non-viral vector systems substan-
tially differ. In a previous study, baculovirus was transduced into
seeded cells for 1 h in PBS, resulting in enhanced transduction
Fig. 1 (continued).
Y.-K. Kim et al. / Virology 387 (2009) 89–97
efficiency (Kim et al., 2006, 2007). Hsu et al. also reported that
baculovirus showed high transduction efficiency and gene expression
levels in D-PBS as the surrounding solution (Hsu et al., 2004), while
non-viral vectors were usually transfected into seeded cells for 4–6 h
in serum-free growth medium (Jiang et al., 2007a; Arote et al., 2007).
Therefore, to compare the transduction efficiency of GP/B in different
conditions, luciferase activities weremeasured aftersampletreatment
for 1 h in PBS (Protocol A) or for 4 h in serum-free growth medium
(Protocol B), respectively. As shown in Fig. 4C, the transduction
efficiency of the non-viral vector systems (PEI 25K, PEI 1800, and GP)
was greater for protocol B than A. In contrast, the transduction
efficiency of the hybrid system was higher than that of the non-viral
vector systems under all conditions tested. Furthermore, although the
transduction efficiency of GP/B using protocol B could be increased by
increasing the N/P ratio, the transduction efficiency of GP/B was more
efficient using protocol A, indicating that the optimum treatment
condition for the hybrid system is protocol A. Therefore, all further
studies were performed using protocol A.
There is no way to compare the exact amount of genetic materials
between non-viral and viral vector systems and a hybrid system is
more complicated. Therefore, to evaluate the transduction efficiency
Fig. 3. CellviabilityassayofGPandGP/BinHepG2cellsatvariousconcentrationsofGPand
Fig. 2. (A) Agarose gel electrophoresis was performed to confirm DNA condensation of GP at various N/P ratios. (B) Protection and release assay of DNA. (I) DNA alone and (II) GP/DNA
complexes at an N/P ratio of 5 were treated with PBS, while (III) DNA alone and (IV) GP/DNA complexes at an N/P ratio of 5 were treated with DNase I. (C) Surface charges of GP/DNA
Y.-K. Kim et al. / Virology 387 (2009) 89–97
of a hybrid system on the fair ground, we indirectly compared the
transduction efficiency of Bac 100 and GPGFP/B (a hybrid system
composed of GP/DNA complexes containing GFP genes and recombi-
nant baculovirus with luciferase genes). Both of them had same
amount of genetic materials by non-viral compartment (GPGFP)
containing GFP genes instead of luciferase genes of the hybrid system.
The transduction efficiency of GPGFP/B 100 was similar to that of GP/B
100 and was significantly higher than that of Bac 100 (Fig. 5),
indicating that enhanced transduction efficiency of a hybrid system
was due to the combination of the complementary strengths of viral
and non-viral vector systems regardless of gene copies. In addition,
the transduction efficiency of GP and GP/BW (a hybrid system
composed of GP/DNA complexes containing luciferase genes and
wild-type baculovirus) was compared. The amount of their genetic
materials was also same as viral compartment (BW) without any
recombinant genetic materials of the hybrid system. The transduction
efficiency of GP/BW was about 10 times higher than that of GP
although it was not higher than that of Bac 100 (data not shown). This
result suggested that a hybrid system dominantly transduced into
cells with viral vector-centered transduction manner.
To elucidate the mechanism of transduction, transduction effi-
ciency was evaluated after treatment with bafilomycin A1, a specific
inhibitor of vacuolar type H+ATPase. As shown in Fig. 6, the
Fig. 4. Optimization of luciferase activity of GP and GP/B in HepG2 cells under various conditions. Transduction efficiency of GP/DNA complexes and GP/B at (A) various N/P ratios,
(B) various MOTs, and (C) various sample treatment conditions (Protocol A: samples treated for 1 h in PBS and Protocol B: samples treated for 4 h in serum free growth medium)
(n=3, error bars represent standard deviations, ⁎pb0.05, ⁎⁎pb0.01).
Fig. 5. Comparison of transduction efficiency of Bac, GP/B, and GPGFP/B (GPGFP/B: a
hybrid system composed of GP/DNA complexes containing GFP genes and recombinant
baculovirus with luciferase genes) (n=3, error bars represent standard deviations,
Y.-K. Kim et al. / Virology 387 (2009) 89–97
transduction efficiency of GP/B was drastically decreased after
bafilomycin A1 treatment. Furthermore, the reduction in transduction
efficiency of GP/B after bafilomycin A1 treatment (GP/B 50: 597 times
reductionatanN/Pratio of5; GP/B100: 1158timesreductionatanN/
PEI 1800 (4 times reduction), Bac 50 (280 times reduction), and Bac
100 (335 times reduction). Several studies have reported that the
transduction efficiency of PEI-containing carrier was abruptly
decreased after treatment with bafilomycin A1 due to blocking of the
proton sponge effect by PEI (Jiang et al., 2007a, 2007b; Arote et al.,
2007). In addition, Long et al. reported that reduction in transduction
efficiency of baculovirus caused by treatment of bafilomycin A1 was
due to the entry mechanism of baculovirus through a low-pH-
dependent endocytic pathway (Long et al., 2006). Therefore, it is
reasonable to assume that the transduction mechanism of GP/B
utilizes both the proton sponge effect in PEI-mediated transduction
from a non-viral element (GP) and the low-pH-dependent endocytic
entry from the viral element (baculovirus).
It has been reported that inactivation of baculovirus by comple-
ment system is a great hurdle for in vivo application (Sandig et al.,
1996). To investigate the serum effect of hybrid system, transduction
efficiency with or without serum was evaluated. As shown in Fig. 7,
transduction efficiency of hybrid systems (GP/B 50 or GP/B 100) was
slightly decreased, whereas transduction efficiency of baculovirus and
PEI was drastically decreased. Even though serum affected both
baculovirus and PEI on their transduction efficiency, transduction
efficiencyof hybridsystemwas alittle affectedunder serumcondition.
It was already reported that hydrophilic group inhibits the interaction
between serum component and polymer/DNA complexes (Kuo,
2003). Therefore, it is thought that hydrophilic galactose group in
the GP prevents aggregation of GP/DNA complexes and protects
baculovirus in the GP/B from inactivation under serum condition
owing to steric interference of serum by galactose group. This result
indicated that the transduction efficiency of the GP/B was not affected
in the presence of serum.
Receptor-mediated endocytosis studies
To confirm ASGPR-mediated transduction of GP/B, luciferase
activity of GP/B was compared to that of P/B in the ASGPR-positive
HepG2 cell line (Fig. 8A). The transduction efficiency of GP/B was
significantly higher than that of P/B at the same N/P ratio and MOT. In
addition, the transduction efficiency was greater in the ASGPR-
positive HepG2 cell line as compared to the ASGPR-negative A549 cell
line (Fig. 8B). These results are consistent with previous reports that
galactosylated cationic polymers show hepatocyte specificity because
ASGPR, highly presented on the hepatocyte cell surface, can recognize
the galactose moiety as a specific ligand (Steer and Ashwell, 1980;
Sagara and Kim, 2002; Kim et al., 2005).
Finally, a competition assay was performed by pre-treatment of
HepG2 cellsor A549 cellswithvarious concentrationsof freegalactose
(1,10, and 100 mM) as a competitor. As shown in Fig. 8C, the luciferase
activity of GP/B was decreased by pre-treated free galactose in HepG2
cells. Especially, inhibition of transduction efficiency of GP/B in
presence of free galactose depended on concentration of pre-treated
galactose whereas the effect was not observed on the transduction
efficiency of P/B, suggesting that pre-treatment of free galactose as a
competitor prevents GP/B from being transported into HepG2 cells by
competitive binding to ASGPRs on the cell surface although the
inhibition of transduction was incomplete in the competition assay
because the GP/B still entered into HepG2 cells via non-specific
endocytosis as well as ASGPR-mediated endocytosis.
In contrast to HepG2 cells, both GP/B and P/B had similar
transduction efficiency regardless of presence of free galactose and
Fig. 6. Effect of bafilomycin A1 on transduction efficiency. Bafilomycin A1 (200 nM) solution dissolved in DMSO was added into wells. After a 10 min incubation, the bafilomycin A1
solution was removed and PBS containing GP or GP/B was added and incubated for 1 h and analyzed 24 h after transduction (n=3, error bars represent standard deviations,
Fig. 7. Effect of serum on transduction efficiency. HepG2 cells were incubated in the
absence or presence of 10% serum with PEI/DNA complexes, Bac, and GP/B at various
MOTs and N/P ratios. (n=3, error bars represent standard deviation, ⁎pb0.05,
Y.-K. Kim et al. / Virology 387 (2009) 89–97
therewere not muchdifferencesof thetransduction efficiencybetween
GP/B and P/B in A549 cells. This result indicated that transduction of
GP/B showed independence of concentration of galactose in A549 cells
owing to the lack of ASGPR on A549 cells.
In this study, a hybrid system composed of viral and non-viral
vectors was successfullyconstructed and its potential as a hepatocyte-
targeting gene delivery system was evaluated in vitro. The GP/B had
suitable physicochemical properties, and transduction conditions
were optimized for efficient gene delivery. In addition, the GP/B
showed low cytotoxicity and significantly enhanced transduction
efficiency compared to other viral or non-viral vector systems.
Furthermore, the targeted delivery of GP/B by ASGPR-mediated
endocytosis was confirmed via a competition assay. This hybrid
system, therefore, has potentials for use as a safe and efficient gene
delivery vehicle for hepatocyte-targeting gene therapy.
Materials and methods
Polyethylenimine (PEI; Mw 1800 Da) and 1-ethyl-3-(3-dimethy-
laminopropyl)-carbodiimide hydrochloride (EDC) were purchased
from Wako (Osaka, Japan). Lactobionic acid (LA) and N-hydroxysucci-
nimide (NHS) were obtained from Sigma-Aldrich (St. Louis, Mo, USA).
A 5.3 kb expression vector, pGL3-control, containing a luciferase gene
driven by an SV40 promoter was obtained from Promega (Madison,
WI, USA) and a gene coding for enhanced green fluorescent protein
purchased from Clontech Laboratories (San Jose, CA, USA). pBacPAK8
was also purchased from Clontech Laboratories (San Jose, CA, USA).
Synthesis of GP
PEI was coupled with LA as previously described by Jiang et al.
(2007b), with modifications. Briefly, PEI (1.72 g; 10 mM) and LA
Fig. 8. (A) Comparison of transduction efficiencies between GP/B and hybrid of PEI/DNA complexes and baculovirus (P/B) at various MOTs and N/P ratios in HepG2 cells. (B)
Comparison of relative luciferase activity of GP/B normalized to P/B in asialoglycoprotein receptors (ASGPR)-positive HepG2 cells and ASGPR-negative A549 cells. Competition assay
of GP/B by adding free galactose with various concentrations (1, 10, and 100 mM) as a competitor to (C) HepG2 cells and (D) A549 cells (n=3, error bars represent standard
deviations, ⁎pb0.05, ⁎⁎pb0.01, ⁎⁎⁎pb0.001).
Y.-K. Kim et al. / Virology 387 (2009) 89–97
(0.537 g; 1.5 mM) were each dissolved in 2-morpholinoethanesulfo-
nic acid buffer (0.1 M, pH 6.5). NHS and EDC were added toLAsolution
and stirred for 20 min at 4 °C. PEI was coupled with LA by mixing PEI
solution and activated LA solution with stirring for 72 h at room
temperature. Hydroxylamine (10% v/v) was added to stop the
reaction and GP solution was adjusted to pH 8 by adding NaOH. The
reactants were dialyzed for 3 days against distilled water (Spectra/
Por®, MW cut-off 1000) and then freeze-dried. The composition of the
prepared GP was estimated by measuring1H NMR (Avance™ 600,
Hybridization of GP/B
Recombinant baculovirus was constructed as previously reported
(Kim et al., 2007). The prepared baculoviral stocks were stored at 4 °C
GP/DNA complexes were formed by adding GP solution to equal
volumes of pGL3-control solution with gentle vortexing and incubated
for 30 min at room temperature. The desired plaque forming units (pfu)
of baculovirus was concentrated from cell culture medium by sedimen-
reported (Kim et al., 2006). GP/DNA complexes and baculovirus were
hybridized by adding GP/DNA complexes to baculovirus suspension
solution with gentle vortexing and incubated for 30 min at room
temperature. The hybrid system was freshly formed before every use.
Characterization of GP and GP/B
Complex formation with GP and DNA at various N/P ratios was
confirmed by electrophoresis. The DNA retardation was observed by
irradiation with UV light and assayed with Cam2com software.
Protection and release of DNA from GP/DNA complexes were
performed by electrophoresis as previously reported (Park et al.,
2005). DNase I or PBS in DNase/Mg2+digestion buffer (50 mM Tris–
Cl, pH 7.6 and 10 mM MgCl2) was added to GP/DNA complexes. After
incubation at 37 °C with shaking for 30 min, EDTA (250 mM) was
treated for 10 min for DNase inactivation. Sodium dodecyl sulfate
(SDS) was added to the sample for DNA release for 2 h at room
temperature. DNA protectionand releasewere observed byirradiation
with UV light and assayed with Cam2com software.
The surface charges of GP/DNA complexes and GP/B were
measured with an electrophoretic light scattering spectrophotometer
(ELS 8000, Otsuka Electronics, Osaka, Japan) with 20 scattering angle
at room temperature. The volume of sample containing GP/DNA
complexes and (or) baculovirus (1.5×107pfu) was 2 ml.
B on a copper grid and stained with 1% uranyl acetate solution for 5 s.
After drying for 10 min, these specimens were observed under the
Cell lines and cell culture
HepG2 (human hepatoblastoma) and A549 (human lung carci-
noma) cells were cultured in DMEM medium (HyClone, Logan, UT,
USA). Each cell culture medium was supplemented with 10% FBS,
streptomycin at 100 μg/ml, and penicillin at 100 U/ml. All cells were
incubated at 37 °C in a humidified 5% CO2atmosphere.
Cell viability assay
Cell viability was evaluated by the Cell Titer 96®Aqueous One
Solution Cell Proliferation Assay (Promega, Madison, WI, USA) as
previously described (Jianget al., 2007a). Cells wereseeded in 96-well
plates at an initial density of 2×104cells/well. After incubation for
18 h, polymers and GP/B with various concentrations were added to
the 96-well plates. After an additional incubation for 24 h, the media
were changed with growth media containing 20 μl of Cell Titer
96®Aqueous One Solution Reagent. Finally, after further incubation for
3 h, the metabolic activity of the cells was measured at 570 nm by an
ELISA plate reader (GLR 1000, Genelabs Diagnostics, Singapore). Cell
viability (%) was calculated according to the following equation: cell
Luciferase activity assay for transduction efficiency
GP/DNA complexes or GP/B at various N/P ratios and MOTs were
transduced into seeded cells as previously described (Kim et al.,
2007). Briefly, cells were seeded in 24-well plates at an initial density
of 1×105(A549) or 2×105(HepG2) cells/well. After incubation for
18 h, GP/DNA complexes or GP/B with various N/P ratios and MOTs
wereadded tothe 24-well plates. The transduced cells were incubated
for 1 or 4 h, and then PBS solution or DMEM containing GP/DNA
complexes or GP/B were replaced with fresh media containing serum
and 10 mM sodium butyrate (a histone deacetylase inhibitor) and
incubated for an additional 24 h. The luciferase assay was performed
according to the manufacturer's protocol (Promega, Madison, WI,
USA, Cat. E1501). Relative light units (RLUs) were measured with a
chemiluminometer (Autolumat LB953, EG&G Berthold, Germany).
RLUswere normalized tothe protein concentration of cell extracts and
protein quantification was determined by the BCA method.
To confirm receptor-mediated transduction, a competition assay
was performed by treatment of free galactose as a competitor in
ASGPR-positive HepG2 cells and ASGPR-negative A549 cells. Various
concentrations of free galactose (1,10, and 100 mM) were added into
seeded HepG2 or A549 cells for 10 min before GP/B or P/B (hybrid
between PEI/DNA complexes and baculovirus) were transduced.
Transduced cells were measured by luciferase activity following the
same method as described above.
Statisticalanalysiswas performedusing Student's t-test (GraphPad
Software, San Diego, CA). All results are given as the mean±standard
deviation (SD). Statistical significance is indicated by ⁎ for p-values
less than 0.05, ⁎⁎ for p-values less than 0.01, and ⁎⁎⁎ for p-values less
This work was supported by the grants from the Ministry of
Science and Technology in Korea (F104AA010002-08A0101-00211).
We also acknowledge the National Instrumentation Center for
Environmental Management (NICEM) for permission to take NMR,
ELS, and EF-TEM measurements. Y. K. Kim was supported by the BK21
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