Effect of peptides and their introduction methods on target gene transfer of gene vector based on disulfide-containing polyethyleneimine

Article abstract of DOI:10.1016/j.ijpharm.2012.08.039

To evaluate the effect of different peptides as well as their introduction methods on target gene transfer of gene vectors based on disulfide-containing polyethyleneimine (SS-PEI), a series of peptides including N₃-GRGDSF, GRGDSF, and EEEEEEEEGRGDSF (E₈GRGDSF) were prepared. N ₃-GRGDSF was conjugated to SS-PEI by click chemistry and SS-PEI-GRGDSF was obtained. GRGDSF was non-covalently introduced into SS-PEI/DNA mainly through hydrogen bonding to obtain SS-PEI/DNA/GRGDSF complexes, whereas E₈GRGDSF was further non-covalently introduced to SS-PEI/DNA through electrostatic force to obtain SS-PEI/DNA/E₈GRGDSF complexes. Transfection efficiency of all complexes with peptides was lower than that of SS-PEI/DNA in COS-7 cells due to the fact that nonspecific endocytosis was prohibited after peptide introduction. Whereas in HeLa cells, transfection activity of SS-PEI-GRGDSF/DNA and SS-PEI/DNA/E₈GRGDSF at certain w/w ratios was higher than that of SS-PEI/DNA. But the transfection efficiency of SS-PEI/DNA/E₈GRGDSF at peptide/DNA w/w ratios higher than 30 dropped due to targeted binding interactions between surplus E₈GRGDSF and the integrins in HeLa cells, which would prohibit specific endocytosis of E ₈GRGDSF in complexes. Transfection activity of SS-PEI/DNA/GRGDSF was lower than or comparable to that of SS-PEI/DNA because of loose complexes constructed by hydrogen bonding between GRGDSF and SS-PEI/DNA.

Full text of DOI:10.1016/j.ijpharm.2012.08.039

International Journal of Pharmaceutics 438 (2012) 191–201
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Effect of peptides and their introduction methods on target gene transfer of gene
vector based on disulfide-containing polyethyleneimine
Hui-Yuan Wang, Yun-Xia Sun∗, Ji-Zhe Deng, Juan Yang, Ren-Xi Zhuo, Xian-Zheng Zhang∗
Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 May 2012
Received in revised form 19 July 2012
Accepted 21 August 2012
Available online 28 August 2012
To evaluate the effect of different peptides as well as their introduction methods on target gene transfer
of gene vectors based on disulfide-containing polyethyleneimine (SS-PEI), a series of peptides including
N3-GRGDSF, GRGDSF, and EEEEEEEEGRGDSF (E8GRGDSF) were prepared. N3-GRGDSF was conjugated
to SS-PEI by click chemistry and SS-PEI-GRGDSF was obtained. GRGDSF was non-covalently introduced
into SS-PEI/DNA mainly through hydrogen bonding to obtain SS-PEI/DNA/GRGDSF complexes, whereas
E8GRGDSF was further non-covalently introduced to SS-PEI/DNA through electrostatic force to obtain
SS-PEI/DNA/E8GRGDSF complexes. Transfection efficiency of all complexes with peptides was lower
than that of SS-PEI/DNA in COS-7 cells due to the fact that nonspecific endocytosis was prohibited
after peptide introduction. Whereas in HeLa cells, transfection activity of SS-PEI-GRGDSF/DNA and SS-
PEI/DNA/E8GRGDSF at certain w/w ratios was higher than that of SS-PEI/DNA. But the transfection
efficiency of SS-PEI/DNA/E8GRGDSF at peptide/DNA w/w ratios higher than 30 dropped due to targeted
binding interactions between surplus E8GRGDSF and the integrins in HeLa cells, which would prohibit
specific endocytosis of E8GRGDSF in complexes. Transfection activity of SS-PEI/DNA/GRGDSF was lower
than or comparable to that of SS-PEI/DNA because of loose complexes constructed by hydrogen bonding
between GRGDSF and SS-PEI/DNA.
Keywords:
Inclusion complexes
RGD peptide
Targeted gene delivery
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
and low cytotoxicity (Kim et al., 2005a,b; Breunig et al., 2007; Wen
et al., 2009; Deng et al., 2011).
Among various non-viral gene vectors, polycations have
received much attention due to the advantages of non-
immunogenic, easy to manufacture and adjustable structures
(Kima et al., 2005; Zhai et al., 2011). As the “gold standard” of
polycations (Lungwitz et al., 2005; Neu et al., 2005), cationic
polyethyleneimine (PEI) exhibits unique merits including substan-
tial buffering capacity and high transfection efficiency in many cell
lines (Godbey et al., 1999). However, its transfection efficiency was
strongly associated with its molecular weight and cytotoxicity. In
general, PEIs with high molecular weight (MW) of 25,000 Da or
greater demonstrated high transfection efficiency (Moghimi et al.,
2005), whereas low molecular weight PEIs (<2000 Da) exhibited
much lower cytotoxicity but showed poor transfection activity
(Fischer et al., 1999). One possible avenue to overcome this puzzle
was to crosslink low molecular weight PEI and then obtain degrad-
able high molecular weight PEI with high transfection efficiency
However, a series of barriers including poor cellular uptake,
endosomal escape, cytoplasmic mobility, and nuclear entry may
weak the gene expression during polycation-mediated in vitro gene
transfer (Funhoff et al., 2005; Zhai et al., 2011). Introduction of
active target ligand is a widely used method for promoting cellular
uptake, in which active ligand is able to specifically interact with
receptor on the surface of target cells (Cheng and Saltzman, 2011).
The widely used active ligands involve such as transferrin (Huang
et al., 2008; Kakimoto et al., 2007), asialoglycoprotein (ASGP) (Park
et al., 2000; Cho et al., 2006; Mi et al., 2007), and functional pep-
tides (Kim et al., 2005a,b; Oba et al., 2007). Peptides containing
arginine-glycine-aspartic acid (RGD) sequence as a targeting ligand
have been widely used in gene delivery system to selectively rec-
ognize and bind ␣ ␤ as well as ␣ ␤ integrin receptors that are
expressed on cell surfaces of certain cell types such as endothelial
cells, osteoclast, macrophage, platelets, and melanomas (Kim et al.,
2005a,b; Renigunta et al., 2006; Oba et al., 2007). RGD contain-
ing peptides were often chemically conjugated to non-viral gene
vectors for improving the receptor-mediated endocytosis (Alexis
et al., 2006; Suk et al., 2006; Chen et al., 2011). Unfortunately, these
chemical attempts might reduce or change the biophysical activ-
ities of peptides (Zeng et al., 2007) as well as the corresponding
␯
␯
3
5
∗
Corresponding authors. Tel.: +86 27 6875 5993; fax: +86 27 6875 4509.
E-mail addresses: yx-sun@whu.edu.cn (Y.-X. Sun), xz-zhang@whu.edu.cn
(X.-Z. Zhang).
0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2012.08.039

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H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
polymer. Therefore, it would be of more interest if we could pre-
pare similar or even more advanced gene delivery systems by the
use of a non-covalent approach to introduce desired peptides.
Previously, we reported targeted gene vectors based
on low molecular weight PEI including biotinylated
polyethyleneimine/avidin bioconjugates (Zeng et al., 2009)
and disulphide-containing polyethyleneimine (SS-PEI) with non-
covalently introduced RGD (Sun et al., 2008). However, the RGD
introduction reduced the transfection efficiency of SS-PEI in HeLa
cells due to the shorter amino acid sequence and zwitterionic
character of RGD. The interaction between short RGD and SS-
PEI/DNA complexes is weaker than the one of targeted binding
interaction between RGD and the integrins on the surface of HeLa
cells. Hence, when the complexes with RGD was added into the
cultured HeLa cells, the surplus RGD peptide is preferential for
cellular uptake by receptor-mediated endocytosis, but the binding
between integrin receptor and a spot of RGD in complexes is
inhibited.
2.2. Characterization
Fourier transformed infrared spectroscopy (FTIR) of GRGDSF,
SS-PEI, alkynyl-SS-PEI, and SS-PEI-GRGDSF were measured in a
Fourier transforms infrared spectroscope (a Lambda Bio40 UV–Vis
spectrometer, Perkin-Elmer) using KBr disks. 1H nuclear magnetic
resonance spectroscopy (1H NMR) was performed on a Varian
Mercury-VX 300 spectrometer using D2O as solvent. The molecu-
lar weights and polydispersity (Mw/Wn) of SS-PEI were determined
by Gel Permeation Chromatographic (GPC) relative to PEG stan-
dards using a Waters-2690D HPLC equipped with Ultrahydrogel
120 and 250 columns. HAc–NaAc buffer solution (0.03 M, pH 2.8)
was used as an eluent at a flow rate of 1.0 mL/min. The molecu-
lar weights of GRGDSF, N3GRGDSF, and E8GRGDSF were analyzed
by electrospray ionization mass spectrometry (ESI/MS, LCQ Advan-
tage, Finigan, USA).
2.3. Synthesis
In this study, a series of peptides including GRGDSF, N3GRGDSF,
and E8GRGDSF were synthesized to further investigate the effect
of peptide length, peptide charge character as well as peptide
introduction method (covalent as well as non-covalent method)
on gene transfer of SS-PEI gene vector. N3GRGDS was covalently
conjugated to SS-PEI to prepare SS-PEI-GRGDSF, whereas GRGDSF
was introduced to SS-PEI/DNA complex through hydrogen bonding
(Patwardhan et al., 2012) and anionic E8GRGDSF was introduced
into the surface of positively charged SS-PEI/DNA binary com-
plexes by electrostatic forces (Yang et al., 2012). The transfection
efficiency of SS-PEI-GRGDSF/DNA, SS-PEI/DNA/GRGDSF, and SS-
PEI/DNA/E8GRGDSF complexes was evaluated and compared in
COS-7 and HeLa cells.
2.3.1. Synthesis of disulfide-containing polyethyleneimine
(SS-PEI)
The synthesis of cystamine bisacrylamide (CBA) was according
to the literature (Emilitri et al., 2005). The SS-PEI was synthesized
by Michael addition between CBA and branched PEI with molecu-
lar weight of 1800 Da. Briefly, 0.86 g of PEI was dissolved in 10 ml
10% aqueous methanol and then added to the three-necked flask
equipped with a condenser at 45 ◦C under nitrogen atmosphere.
To obtain the SS-PEI, 0.162 g CBA was dissolved in 3 ml methanol
and added dropwise to the PEI solution. The reaction mixture was
stirred in the dark under nitrogen atmosphere for at least 24 h at
45 ◦C. Subsequently, 10% excess PEI (0.086 g) in 2 ml methanol was
added to consume any unreacted acrylamide groups for another 6 h
with stirring. After that the transparent solution was diluted with
water to 20 ml and acidified with 6 M HCl to pH of 4, and then dia-
lyzed against distilled water (MWCO: 3500) for 2 days to remove
the unreacted 1800 Da PEI. Lastly, the solution was lyophilized for 2
days to obtain the product. 1H NMR (300 MHz, D2O, ı ppm): 2.5–3.5
(m, mH, (NCH2CH2)n ).
2. Materials and methods
2.1. Materials
QIAfilterTM plasmid purification Giga Kit (5) was pur-
chased from Qiagen (Hilden, Germany). GelRedTM was purchased
from Biotium (CA, USA). Dulbecco’s Modified Eagle’s Medium
(DMEM), fetal bovine serum (FBS), 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazoliumbromide (MTT), penicillin-streptomycin,
and phosphate buffered saline (PBS) were purchased from
Invitrogen Corp. The Micro BCA protein assay kit was pur-
chased from Pierce. Acetate and methanol were of ana-
lytical grade and used as received. Hydroxyurea was pur-
chased from Alfa Aesar (USA). N-Fluorenyl-9-methoxycarbonyl
(FMOC) protected l-amino acids: Fmoc-Gly-OH, FMOC-Arg(Pbf)-
OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp-OH, Fmoc-Asp-OtBu and 2-
chlorotrityl chloride resin (100–200 mesh, loading: 1.32 mmol/g)
were purchased from GL Biochem Ltd. (Shanghai, China)
and used as received. N-Hydroxybenzotriazole (HOBt), tri-
fluoroacetic acid (TFA), O-Benzotriazole-N,N,N,N-tetramethyl-
uronium-hexafluorophosphate (HBTU), 1,4-Dithiothreitol (DTT),
piperidine, acryloyl chloride of analytical grade were provided
by Shanghai Reagent Chemical Co. (Shanghai, China) and used
directly. Dimethylformamide (DMF) and diisopropylethylamine
(DiEA) were obtained from Shanghai Reagent Chemical Co. (Shang-
hai, China) and distilled prior to use. Triisopropylsilane (TIS) was
purchased from ACROS (USA) and used without further purifi-
cation. Cystamine dihydrochloride (98%), branched PEI (bPEI,
water-free) with molecular weights of 25 and 1.8 kDa, propargyl
bromide, bromoacetic acid, sodium azide, sodium ascorbate were
purchased from Sigma–Aldrich (Taufkirchen, Germany) and used as
received. All other reagents and solvents were of analytical grade
and used directly.
2.3.2. Synthesis of peptide derivatives containing RGD
(Arg-Gly-Asp) sequence
All the peptide derivatives containing RGD sequence includ-
ing GRGDSF, N3GRGDSF, E8GRGDSF were prepared manually using
standard solid-phase peptide synthesis based on classical Fmoc (1-
(9H-fluoren-9-yl)-methoxycarbonyl)/tert-Butyl chemistry (Fields
and Noble, 1990).
Briefly, the peptide sequence was assembled on a 2-chlorotrityl
chloride resin (1.32 mmol/g), using DiEA/HBTU/HOBt in DMF to
promote coupling of the Fmoc-protected amino acid-OH, and 20%
piperidine in DMF for Fmoc-removal cycles. The coupling effi-
cacy was monitored via the ninhydrin assay. The peptide was
fully deprotected and cleaved from resin by trifluoroacetic acid
(TFA, 28.5 ml), water (1.5 ml), triisopropylsilane (1.5 ml) for 2 h at
room temperature. The resin was removed by filtration, and then
the cleavage mixture and three subsequent TFA washings were
collected. The combined solution was concentrated to a viscous
solution by rotary evaporation. Cold ether was added to precip-
itate the product. After washing with cold ether (five times) to
remove TFA residue, the precipitate was dissolved in distilled
water and then freeze-dried under vacuum for 3 days and stored
at −20 ◦C until used. The molecular weights were confirmed by
an electrospray ionization mass spectrometry (ESI/MS). ESI/MS
m/z: calculated for GRGDSF, 637, found 638 [M+H]+, calculated
for N3GRGDSF, 720, found, 721 [M+H]+, calculated for E8GRGDSF,
1669, found 1670 [M+H]+. IR of GRGDSF (KBr, cm−1):
CONH (1668, 1539). 1H NMR of GRGDSF (300 MHz, D2O, ı ppm):
N H (3347),

H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
193
1.64–1.80 (Arg 4H), 2.83–3.18 (Asp 2H, Arg 2H, and Phe 2H),
2.6. Agarose gel electrophoresis assay
3.62–4.03 and 4.30–4.43 (Gly 4H, Ser 2H), 4.60–4.73 (Arg H, Asp
H, Ser H, and Phe H), 7.26–7.32 (Phe 5H).
The SS-PEI/DNA at w/w ratios ranging from 0.1 to 2.0 and SS-
PEI-GRGDSF/DNA complexes at 0.5 to 4.0 were prepared by adding
appropriate volumes of polymer solution to l ␮g of pGL-3 DNA. The
SS-PEI/DNA at w/w ratio of 0.6 and SS-PEI-GRGDSF/DNA at w/w
ratio of 3.0 with DTT were prepared by adding different content
of DTT to SS-PEI/DNA and SS-PEI-GRGDSF/DNA complexes. And
the SS-PEI/DNA complexes at w/w ratio of 10 with GRGDSF and
E8GRGDSF were prepared by adding different content of peptides to
SS-PEI/DNA complexes. We first mixed SS-PEI with 1 ␮g of DNA at a
w/w ratio of 10 for 30 min, and added various amounts (0, 5, 10, 20,
30, 40, 50 ␮g) of GRGDSF or E8GRGDSF peptides for 30 min, then the
complexes were diluted to 10 ␮L with PBS and further incubated
at 37 ◦C for 30 min. Hence, the concentrations of two peptides are
0, 0.5, 1, 2, 3, 4, 5 ␮g/␮L respectively.
2.3.3. Synthesis of peptide modified SS-PEI (SS-PEI-GRGDSF)
2.3.3.1. Synthesis of azidoacetic acid. Briefly, bromoacetic acid was
dissolved in 50 ml water in a single-necked flask. After the solution
was cooled to 0 ◦C, 6.5 g sodium azide dissolved in 15 ml water was
added dropwise. The reaction mixture was stirred continuously for
16 h at 0 ◦C. After that the flavescent solution was acidified with 6 M
HCl to pH of 3.0. The final product was extracted by 200 ml diethyl
ether and then obtained by removing the solvent under vacuum.
2.3.3.2. Synthesis of N-terminal azidoacetylated GRGDSF (N3-
GRGDSF). The linear peptide analogue (N-terminal azidoacetylated
GRGDSF, N3-GRGDSF) was synthesized by standard solid-phase
peptide synthesis (SPPS) strategy on resin. Azidoacetic acid was
coupled to the last amino acids.
After that the complexes were loaded on the 0.7% (w/v) agarose
gel containing GelRedTM and with Tris–acetate (TAE) running buffer
at 80 V for 60 min. DNA was visualized with a UV lamp using a Vilber
Lourmat imaging system (France).
2.3.3.3. Synthesis of peptide modified SS-PEI (SS-PEI-GRGDSF). The
alkyne-modified SS-PEI was synthesized firstly. In brief, 1.0 g SS-
PEI and 0.060 g propargyl bromide were dissolved in 10 ml DMSO
and added into a single-necked flask. Then 0.053 g Na2CO3 was dis-
solved in 3 ml water and added into above solution. The reaction
was carried out for 24 h at room temperature. The alkyne-modified
SS-PEI was obtained by dialyzing and freeze-drying for 3 days. IR
(KBr, cm−1): CH (2096).
2.7. Particle size and ꢀ-potential measurement
The particle size and ꢀ-potential were measured by Nano-ZS
ZEN3600 (Malvern Instruments, MA) at 25 ◦C. The vector/DNA
binary complexes were prepared as 2.4 descriptions and diluted
with distilled water to 1 ml volume for the size and ꢀ-potential
measure respectively.
The SS-PEI-GRGDSF was synthesized through click chem-
istry reaction between alkyne-modified SS-PEI and N-terminal
azidoacetylated peptide derivatives containing RGD sequence
(N3GRGDSF) (Parrish et al., 2005; Robert et al., 2010; Moore et al.,
2010). Briefly, 0.5 g alkyne-modified SS-PEI and 0.18 g N3GRGDSF
were dissolved in 15 ml methanol and added into a single-necked
flask. Copper (II) sulfate and sodium ascorbate with appropriate
amount were added as catalyst. The reaction was carried out under
nitrogen atmosphere for 24 h at room temperature. The final prod-
uct SS-PEI-GRGDSF was obtained by dialyzing and freeze-drying for
3 days. 1H NMR (300 MHz, D2O, ı ppm): 7.1–7.3 (H, =CH).
2.8. Cytotoxicity assay in vitro
The cytotoxicity assay was performed with HeLa and COS-7
cells by MTT assay. Briefly, the cells were seeded respectively
in a 96-well plate at a density of 6000 cells/well, and then
cells were incubated in 100 ␮L DMEM containing 10% FBS for 1
day prior to adding the vectors. After the GRGDSF, E8GRGDSF,
PEI 25 kDa, SS-PEI, and SS-PEI-GRGDSF were added for 2 days,
the medium was replaced with 200 ␮L of fresh medium. Then
20 ␮L MTT (5 mg mL−1) solutions was added to each well and
further incubated for 4 h. After that, the medium was removed
and 200 ␮L DMSO was added. The absorbance was measured
at 570 nm using
a microplate reader (Bio-Rad, Model 550,
2.4. Cell culture and amplification of plasmid DNA
USA). The relative cell viability was calculated as: cell viabil-
ity (%) = (ODsamples − ODDMSO)/(ODcontrol − ODDMSO) × 100, where
ODcontrol was obtained in the absence of vectors and ODsamples was
obtained in the presence of vectors.
HeLa and COS-7 cell lines were used for transfection and were
incubated in DMEM medium with 10% FBS and 1% antibiotics
(penicillin–streptomycin, 10,000 U/mL) at 37 ◦C in a humidified
atmosphere containing 5% CO2.
pGL-3 plasmid was transformed in Escherichia coli JM109 and
was amplified in terrific broth media at 37 ◦C overnight at 250 rpm.
The plasmid was purified by an EndoFree QiAfilter Plasmid Giga Kit
(5) (QIAGEN, Germany). Then the purified plasmid was dissolved
in Tris–HCl buffer solution and stored at −20 ◦C.
2.9. In vitro transfection
The transfection efficiency of vector/DNA complexes were eval-
uated at various w/w ratios. The cells were seeded in the 24-well
plate at a density of 6 × 104 cells/well and cultured with 1 ml DMEM
containing 10% FBS for 1 day at 37 ◦C to give 50% confluence at the
time of transfection. Then the complexes were added into the plate
with DMEM containing 10% FBS for 4 h at 37 ◦C. After this period,
the medium was replaced with fresh DMEM containing 10% FBS
respectively and the cells were further incubated for 2 days to allow
gene expression. For luciferase assay, the medium was removed
and cells were washed with 200 ␮L PBS, then the cells were lysed
using 200 ␮L reporter lysis buffer.
The luciferase activity was measured with chemiluminometer
(Lumat LB9507, EG&G Berthold, Germany). Luciferase activity was
normalized to the amount of total protein in the sample, which was
determined using a BCA protein assay kit.
In order to further demonstrate the effect of peptides and their
introduction methods on target gene transfer of gene vector, the
competitive inhibition of peptides was further evaluated through
2.5. Preparation of vector/DNA complexes
To prepare DNA binary complexes, SS-PEI and SS-PEI-GRGDSF
stock solutions (in 0.01 M PBS solution) were added dropwise to
a 40 mmol/L Tris–HCl buffer solution containing a plasmid vector
encoding pGL-3 while vortexing at various w/w ratios. The com-
plexes were incubated at 37 ◦C for 30 min to allow formation of the
complexes.
For preparation of inclusion or electrostatic ternary complex,
DNA was first mixed with SS-PEI at a w/w ratio of 10 for 30 min,
and then added slowly various amounts of GRGDSF or E8GRGDSF
peptides. All complexes were used immediately after their
preparation.

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H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
Fig. 1. Molecular structures of synthesized peptides (A) and scheme for synthesis of SS-PEI-GRGDSF (B).
prior-addition peptides method. Different amount of GRGDSF or
E8GRGDSF (0, 10, 30, 50, 100 ␮g) were first incubated in COS-7
and HeLa cells with DMEM containing 10% FBS for 30 min, after
that the peptides were removed and then SS-PEI/DNA/GRGDSF
or SS-PEI/DNA/E8GRGDSF (peptide/DNA = 30) complexes were fur-
ther incubated in cells with DMEM containing 10% FBS for 4 h. And
then, the following procedure was accord with the above luciferase
assay.
E8GRGDSF was first incubated in cells for 30 min, after that the
peptide was removed and SS-PEI/DNA/E8GRGDSF complex was
added. The amount of YOYO-1 used was 1 dye molecule to 60 bp.
After transfection for 4 h, the complexes were removed and the cells
were washed with PBS three times. Then the nucleus was stained
with 20 ␮L (2 ␮g ␮L−1) of Hoechst 33258 for 15 min at 37 ◦C, after
which the cells were further washed with PBS three times and incu-
bated with 200 ␮L DMEM. The fluorescence was observed with a
confocal laser scanning microscope (C1-Si, Nikon, Japan) equipped
with a 405 nm diode for Hoechst 33258 and a 488 nm argon laser
for YOYO-1.
2.10. In vitro cellular uptake of complexes
The COS-7 and HeLa cells were respectively seeded at
3. Results and discussion
a
density of 1.0 × 105 cells on cover-glass slides and cul-
tured for 24 h. To prepare labeled DNA complexes, 1 ␮g
of pGL-3 DNA was intercalated with 2.5 ␮L of 10 mmol/L
YOYO-1 for 15 min at 37 ◦C. Then SS-PEI/DNA (w/w = 10) and
SS-PEI/DNA/E8GRGDSF (peptide/DNA = 30) complexes were incu-
bated with DMEM containing 10% FBS for 30 min at 37 ◦C.
To evaluate the competitive inhibition of peptides, 100 ␮g of
3.1. Synthesis and characterization
3.1.1. Synthesis and characterization of peptides derivatives
containing RGD sequence
As illustrated in Fig. 1, three different RGD peptide derivatives
(GRGDSF, N3GRGDSF, and E8GRGDSF) were designed and prepared

H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
195
Fig. 2. (A) FT-IR spectra of GRGDSF (a), SS-PEI (b), alkynyl-SS-PEI (c), and SS-PEI-GRGDSF (d). (B) 1H NMR spectra of GRGDSF (e), SS-PEI (f), and SS-PEI-GRGDSF (g).
by standard solid-phase peptide synthesis (SPPS) technique. The
molecular weights were determined by an electrospray ionization
mass spectrometry (ESI/MS).
condensable ability, and DNA mobility is significantly reduced
when the w/w ratio of the binary complex reached 3.0. And in
Fig. 3B, the two binary complexes were incubated with various
amounts of DTT at the w/w ratio of 0.6 (SS-PEI/DNA) and 3.0 (SS-
PEI-GRGDSF/DNA) respectively. The migration of DNA is observed
obviously at the DTT amounts ranging from 5 to 50 mmol/mL. It
is inferred that the presence of reducing agent of DTT leads to the
release of condensed DNA. That is to say, under the reducing con-
dition of cytoplasm, the condensed DNA would be released from
the complexes easily, allowing the increasing uptake of DNA and
enhancing transgene expression inside the nucleus. In Fig. 3C, we
mixed DNA with SS-PEI first at a w/w ratio of 10 for 30 min, and
then added slowly various amounts (0, 5, 10, 20, 30, 40, 50 ␮g) of
GRGDSF and E8GRGDSF peptides further for 30 min. It was found
that the ability of SS-PEI to condense DNA is not visibly affected by
the addition of different peptides contents.
3.1.2. Synthesis and characterization of SS-PEI and
SS-PEI-GRGDSF
From the chemical structures of three peptide derivatives
(Fig. 1A), it can be calculated that after the introduction of peptides
into the gene delivery system, the interaction between zwitteri-
onic GRGDSF and SS-PEI was mainly hydrogen bonding, while the
interaction between anionic E8GRGDSF and SS-PEI was electro-
static force as well as hydrogen bonding. In addition, the N3GRGDSF
was chemically conjugated to SS-PEI through click chemistry reac-
tion as shown in Fig. 1B. The SS-PEI was synthesized from the
Michael addition between CBA and branched 1800 Da PEI, and then
the SS-PEI-GRGDSF was synthesized from click chemistry between
alkyne-modified SS-PEI and N-terminal azidoacetylated peptide
derivatives of N3GRGDSF.
3.3. Particle size and ꢀ-potential measurement
The chemical structures of GRGDSF, SS-PEI, and SS-PEI-GRGDSF
were confirmed by FTIR (Fig. 2A) and 1H NMR spectroscopies
(Fig. 2B). As shown in Fig. 2A(c), the typical peaks of alkyne group
in alkyne-modified SS-PEI is clearly visible at 2096 cm−1, indicating
successful alkyne-modifying of SS-PEI. And in the spectrum of SS-
PEI-GRGDSF, the typical peaks of alkyne group were disappeared,
suggesting the consumption of the alkyne functional groups. As
shown in Fig. 2B, the peaks between ı 2.5 and 3.5 ppm in 1H NMR
spectra of SS-PEI (Fig. 2B(f)) in D2O solvent belong to the special
shift of branched PEI, but the characteristic acrylamide peaks in
CBA between ı 5.8 and 6.5 ppm disappeared, which confirm that
the SS-PEIs are prepared with terminal amino groups. The peaks at
ı 7.1–7.3 ppm in 1H NMR spectra of SS-PEI-GRGDSF (Fig. 2B(g)) are
characteristic peaks of imidazole ring, indicating that N3GRGDSF
was successfully conjugated to SS-PEI. The number average molec-
ular weights (Mn) of SS-PEI is 10.4 kDa (PDI = 1.1) measured by GPC.
Fig. 4 shows the mean particle diameter and ꢀ-potential of
SS-PEI/DNA, SS-PEI-GRGDSF/DNA, SS-PEI/DNA/GRGDSF, and SS-
PEI/DNA/E8GRGDSF. Fig. 4A shows that the surface charge of
SS-PEI/DNA complexes at all w/w ratios is positive. On the other
hand, the potential of SS-PEI-GRGDSF/DNA complexes is negative at
w/w ratios of 0.5 and 1, but at higher w/w ratios (>2), potential turns
to positive and is comparable to that of SS-PEI/DNA complexes. The
reason is that chemical conjugation of GRGDSF slightly decreases
the density of positive charge on the surface of SS-PEI. Hence,
SS-PEI-GRGDSF/DNA shows negative potential at lower ratios, but
with increasing of w/w ratios, SS-PEI-GRGDSF could condense DNA
compactly.
As shown in Fig. 4B, the particle size of SS-PEI/DNA com-
plexes drops with increasing of w/w ratios and becomes stable
at w/w ratios higher than 4 due to the net positive electrostatic
repulsion between complexes. However, the particle size of SS-PEI-
GRGDSF/DNA complexes increases first and then decreases at w/w
ratios ranging from 0.5 to 40. It is highly possible that the com-
plex get aggregates when SS-PEI-GRGDSF concentration is slightly
increased; however, further increasing concentration might induce
precipitation of polycations.
3.2. Gel retardation assay
The DNA binding capability of polycation is a prerequisite for
the gene vectors and Fig. 3 shows the results of agarose gel elec-
trophoresis of vector/DNA complexes with different w/w ratios. As
shown in Fig. 3A, the SS-PEI could condense DNA compactly at
w/w ratio of 0.6. In contrast, SS-PEI-GRGDSF shows visible lower
We further investigated the effect of non-covalent introduc-
tion of peptides on ꢀ-potential and particle size of SS-PEI/DNA

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H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
Fig. 3. (A) Gel retardation assay of SS-PEI/DNA (a) and SS-PEI-GRGDSF/DNA (b) complexes at different w/w ratio. (B) Reductibility of SS-PEI/DNA complexes at w/w ratio of
0.6 (c) and SS-PEI-GRGDSF/DNA complexes at w/w ratio of 3.0 with DTT (d). (C) Gel retardation assay of SS-PEI/DNA complexes at w/w ratio of 10 with different amounts of
GRGDSF (e) and E8GRGDSF (f).
complexes. The w/w ratio of 10 for SS-PEI/DNA complexes was cho-
sen as control. As presented in Fig. 4C, the ꢀ-potential of complexes
with addition of GRGDSF changes randomly. The possible reason
is that the main interaction between SS-PEI/DNA and GRGDSF is
hydrogen bonding, which is often influenced by subjective fac-
tors, such as environmental or working conditions. Whilst, the
ꢀ-potential of complexes with addition of E8GRGDSF decreases
with increasing content of peptide due to the anionic character of
E8GRGDSF peptide, and negative ꢀ-potential is observed at pep-
tides/DNA w/w ratios higher than 16.
The particle size of complexes was also altered with different
contents of added peptide. As shown in Fig. 4D, the particle size
of SS-PEI/DNA/GRGDSF complexes also changes randomly due to
loose complexes constructed by hydrogen bonding. In addition, the
particle size of SS-PEI/DNA/E8GRGDSF complexes increases with
increasing of peptides/DNA w/w ratios due to the particle aggrega-
tion with increase of E8GRGDSF.
3.4. In vitro cytotoxicity
The cytotoxicity of materials was monitored in COS-7 and HeLa
cells by MTT assay, and PEI 25 kDa was used as the control. As shown
in Fig. 5, the cell viability indicates that GRGDSF and E8GRGDSF
display no cytotoxicity in two cell lines even at the concentration
up to 200 mg/L.
Generally, the cytotoxicity of cationic gene vectors depends
on the molecular weight, the cell binding ability, and the mem-
brane permeability (Rea et al., 2008). Fig. 5 shows that SS-PEI has
lower cytotoxicity than PEI 25 kDa in both two cell lines, which is
attributed to the fact that the SS-PEI has lower molecular weight
than PEI 25 kDa. In addition, the rapid degradation of SS-PEI by
disulfide bond cleavage in the cytoplasm favorably contributes to
the lower cytotoxicity (Bauhuber et al., 2009). Furthermore, SS-PEI-
GRGDSF has shown significantly lower cytotoxicity as compared
with SS-PEI, and the viability of cells is always more than 75%.

H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
197
Fig. 4. Zeta potential (A) and (C) and particle size (B) and (D) of SS-PEI/DNA, SS-PEI-GRGDSF/DNA, SS-PEI/DNA/GRGDSF, and SS-PEI/DNA/E8GRGDSF complexes.
3.5. In vitro transfection
the transfection efficiency of SS-PEI/DNA complexes at optimal
w/w ratio of 10 (2.25 × 109 RLU/mg protein) is higher or com-
parable to that of PEI 25 kDa/DNA at optimal w/w ratio of 1.25
(1.97 × 109 RLU/mg protein). While that of SS-PEI-GRGDSF/DNA
complexes at optimal w/w ratio of 14 (2.40 × 108 RLU/mg pro-
tein) is much lower than that of SS-PEI/DNA and PEI 25 kDa/DNA
complexes at their optimal w/w ratios. This is attributed to the
fact that COS-7 cells do not possess integrin receptors and the
complex is uptaken by nonspecific endocytosis through electro-
static interactions. The covalent attachment of N3GRGDSF to SS-PEI
results in decreased positive charge and enlarged particle size,
and then decreased nonspecific uptake as well as transfection
efficiency. While in HeLa cells, the transfection efficiency of SS-
PEI-GRGDSF/DNA complexes at w/w ratio of 14 (1.62 × 109 RLU/mg
protein) is higher than that of SS-PEI/DNA complexes at w/w ratio
of 10 (6.20 × 108 RLU/mg protein) as well as PEI 25 kDa/DNA at
the optimal w/w ratio (5.18 × 108 RLU/mg protein). The results
3.5.1. Transfection of SS-PEI-GRGDSF/DNA complexes (covalent
interaction)
The transfection efficiency of SS-PEI/DNA and SS-PEI-
GRGDSF/DNA complexes at w/w ratios ranging from 4 to 40
was compared as shown in Fig. 6. In COS-7 and HeLa cell lines,
the highest luciferase ability of SS-PEI/DNA complexes is both
obtained at a w/w ratio of 10. The highest transfection efficiency
in COS-7 cells is 2.25 × 109 RLU/mg protein and that in HeLa cells
is 6.20 × 108 RLU/mg protein, respectively, hence, the SS-PEI/DNA
complexes at w/w ratio of 10 is chosen as the optimal point in the
following experiments. Correspondingly, the highest transfection
efficiency of SS-PEI-GRGDSF/DNA complex is obtained at w/w ratio
of 14.
However, the transfection ability mediated by SS-PEI and SS-
PEI-GRGDSFis opposite in two cell lines. For example, in COS-7 cells,
Fig. 5. Cytotoxicity induced by SS-PEI, SS-PEI-GRGDSF, GRGDSF, and E8GRGDSF in COS-7 (A) and HeLa (B) cells. PEI (25 kDa) were used as the control. The relative cell viability
was compared with the untreated cells. Data were shown as mean SD (n = 3).

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H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
Fig. 6. Transfection efficiency of SS-PEI/DNA and SS-PEI-GRGDSF/DNA complexes at w/w ratios ranging from 4 to 40 in (A) COS-7 and (B) HeLa cells. Data were shown as
mean SD (n = 3).
indicate that chemical conjugation of N3GRGDSF could increase
SS-PEI transfection activity and lead to more than 2-fold
in transfection can be explained by the changed zeta potential,
which is more affected by anionic E8GRGDSF.
a
increase of luciferase expression in HeLa cells. The reason is that
HeLa cells possess integrin receptor and complex is uptaken by
receptor-mediated endocytosis. Hence, although the introduction
of N3GRGDSF decreases the positive charge of SS-PEI, the receptor-
mediated endocytosis is primary and a weakly positively charged
surface is preferable for the specific binding by receptor-mediated
endocytosis (Zeng et al., 2007).
While in HeLa cells (Fig. 7B), the transfection activity of SS-
PEI/DNA/E8GRGDSF complexes at peptide/DNA w/w ratios ranging
from 0.5 to 30 is better than that of SS-PEI/DNA, but transfec-
tion activity does not show obvious alteration with increasing
of peptides. The introduction of anionic E8GRGDSF peptide could
reduce the positive charge of gene delivery complexes, which
would reduce nonspecific uptake but enhance receptor-mediated
specific uptake (Thomas and Klibanov, 2003). However, when the
amount of added E8GRGDSF is high enough (above w/w ratio 30 of
peptide/DNA), a considerable decrease of transfection efficiency is
observed, mainly due to the fact that excess E8GRGDSF may com-
pete with the peptide in ternary complexes for targeted binding
integrin receptors, leading to reduced specific cellular uptake of
complexes and plasmid DNA.
3.5.2. Transfection of SS-PEI/DNA/GRGDSF inclusion complex
(hydrogen bonding interaction)
As shown in Fig. 7A, the addition of GRGDSF decreases the trans-
fection activity of SS-PEI in COS-7 cells, due to the alteration of zeta
potential and particle size of original SS-PEI/DNA complexes. As
gene delivery nanoparticles are usually positively charged, which
is important for the nonspecific cellular uptake through electro-
static interaction in cells containing no integrin receptor, so the
nonspecific uptake of SS-PEI/DNA/GRGDSF complexes is reduced
(Trubetskoy et al., 2003). However, it seems that the amount of
GRGDSF does not correlate to the final gene expression level com-
pletely. This is attributed to the loose complexes constructed by
hydrogen bonding and SS-PEI/DNA as well as GRGDSF was uptaken
respectively by nonspecific endocytosis. The transfection efficiency
of complexes increases slightly at w/w ratios below 30 and then
drops gradually. It was also due to the loose complexes constructed
by hydrogen bondings. At low w/w ratios from 0.5 to 5, the main
cellular uptake is from nonspecific endocytosis. With increasing
of w/w ratios from 10 to 30, the complexes tend to be stable and
specific endocytosis becomes primary. But with further increasing
of ratios, the excess, free GRGDSF would compete with the pep-
tides in ternary complexes for binding interaction with the integrin
receptor (Sun et al., 2008), and the worst result (1.40 × 108 RLU/mg
protein) was achieved with a GRGDSF/DNA ratio of 60.
3.5.4. Comparison of transfection efficiency of complexes in
different cell lines
In order to further concisely compare the effect of different
introduction methods of peptides on the transfection activity of
SS-PEI gene vector, the SS-PEI/DNA and SS-PEI-GRGDSF/DNA at
optimal w/w ratios (10, 14) as well as SS-PEI/DNA/GRGDSF and
SS-PEI/DNA/E8GRGDSF at optimal peptide/DNA w/w ratios (30, 30)
were chosen for gene expression evaluation in COS-7 and HeLa
cells.
As shown in Fig. 8, in COS-7 cells, transfection activity of SS-PEI-
GRGDSF/DNA, SS-PEI/DNA/GRGDSF, and SS-PEI/DNA/E8GRGDSF
is lower than that of SS-PEI/DNA. The results demonstrate
that all the three introduced peptides decrease the transfection
efficiency of SS-PEI/DNA complexes due to the fact that the intro-
duction of peptides ruins the nonspecific endocytosis through
electrostatic interaction in COS-7 cells. While the transfection
efficiency of SS-PEI-GRGDSF/DNA and SS-PEI/DNA/E8GRGDSF in
HeLa cells is higher than that of SS-PEI/DNA, but efficiency of SS-
PEI/DNA/GRGDSF is only comparable to that of SS-PEI/DNA. The
reason is that the receptor-mediated endocytosis is primary in HeLa
cells and complexes with peptides constructed by chemical conju-
gation or electrostatic force show more stability as compared with
that by hydrogen bonding.
3.5.3. Transfection of SS-PEI/DNA/E8GRGDSF electrostatic
complexes (electrostatic force interaction)
As presented in Fig. 7A, addition of E8GRGDSF has significantly
negative effects on transfection activity of SS-PEI/DNA in COS-7
cells. With increasing content of E8GRGDSF peptide, gene expres-
sion decreases presumably due to decreased surface positive charge
of SS-PEI/DNA complexes and nonspecific uptake. Besides, the gene
expression decrease in COS-7 cells is more evident by E8GRGDSF-
mediated transfection than that by GRGDSF-mediated transfection,
and the luciferase expression level of peptide/DNA at w/w ratio of
60 is decreased by 250-fold and 24-fold, respectively. The changes
Moreover, the gene expression level of SS-PEI/DNA/E8GRGDSF
at the same w/w ratio in HeLa cells is up to 103-fold higher than
that in COS-7 cells, and the one of SS-PEI-GRGDSF/DNA as well as
SS-PEI/DNA/GRGDSF in HeLa cells is up to 7-fold higher than that in
COS-7 cells. The results demonstrate that the non-covalent intro-
duction of E8GRGDSF peptide with longer amino acid sequence and

H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
199
Fig. 7. Transfection efficiency of SS-PEI/DNA/E8GRGDSF and SS-PEI/DNA/GRGDSF complexes in (A) COS-7 and (B) HeLa cells. Data were shown as mean SD (n = 3).
transfection efficiency of ternary complexes in COS-7 cells alters
slightly with increasing amount of prior-addition of GRGDSF or
E8GRGDSF peptides from 0 to 100 ␮g. This is ascribed to that part
of prior-addition of peptides is uptaken by nonspecific endocytosis
after prior-incubated for 30 min and then the non-uptaken peptides
is removed, which has little effect on the nonspecific endocytosis of
SS-PEI/DNA/GRGDSF and SS-PEI/DNA/E8GRGDSF complexes. How-
ever, transfection activity of ternary complexes with prior-addition
of peptides in HeLa cells (Fig. 9B) is reduced gradually with increas-
ing amount of GRGDSF or E8GRGDSF peptides from 0 to 100 ␮g.
This is due to the fact that the prior-addition peptides first interact
with ␣ ␤ and ␣ ␤5 integrins on the surface of HeLa cells, and then
␯
␯
3
the specific interaction between peptides in ternary complexes and
integrin on surface of HeLa cells is inhibited. These results revealed
that prior-addition peptides cultured in cells induce inhibition of
target gene transfer in HeLa cells but have little inhibition effect on
gene transfer in COS-7 cells.
3.6. Cellular uptake of complexes
The cellular uptake of complexes was visualized by a con-
focal laser scanning microscope. YOYO-1 labeled DNA is green
while Hoechst 33258 stained nuclei is blue. As presented in
Fig. 10, YOYO-1 labeled DNA is internalized and mainly located
around the nuclear membranes. However, the amount of internal-
ized DNA through SS-PEI/DNA, SS-PEI/DNA/E8GRGDSF complexes,
and SS-PEI/DNA/E8GRGDSF with prior-addition of E8GRGDSF is
different. In COS-7 cells, fewer internalized DNA through SS-
PEI/DNA/E8GRGDSF (Fig. 10A(b)) and SS-PEI/DNA/E8GRGDSF with
prior-addition of E8GRGDSF (Fig. 10A(c)) is visible as compared
Fig. 8. Comparison of transfection efficiency in COS-7 and HeLa cells of SS-
PEI/DNA, SS-PEI-GRGDSF/DNA, SS-PEI/DNA/GRGDSF, and SS-PEI/DNA/E8GRGDSF at
w/w ratios of 10, 14, 30, 30, respectively.
anionic character through electrostatic force method would induce
enhancement of gene transfer in HeLa cells selectively.
3.5.5. Competitive inhibition of peptides (prior-addition peptides)
Competitive inhibition of GRGDSF or E8GRGDSF peptides was
investigated using luciferase assay. As shown in Fig. 9A, the
Fig. 9. Competitive inhibition of peptides was evaluated through the transfection efficiency of SS-PEI/DNA/GRGDSF and SS-PEI/DNA/E8GRGDSF with prior-addition different
amount of GRGDSF or E8GRGDSF (0, 10, 30, 50, 100 ␮g) in COS-7 (A) and HeLa (B) cells.

200
H.-Y. Wang et al. / International Journal of Pharmaceutics 438 (2012) 191–201
Fig. 10. Cellular uptake of SS-PEI/DNA (w/w = 10 (a) and (d)), SS-PEI/DNA/E8GRGDSF complexes (b) and (e) as well as SS-PEI/DNA/E8GRGDSF with prior-addition 100 ␮g of
E8GRGDSF (c) and (f) in COS-7 and HeLa cells cultured in DMEM with 10% FBS. Images were obtained 400×.
to that through SS-PEI/DNA complexes (Fig. 10(a)). In HeLa cells,
the amount of internalized DNA through SS-PEI/DNA/E8GRGDSF
with prior-addition 100 ␮g of E8GRGDSF (Fig. 10B(f)) is lower than
that through SS-PEI/DNA (Fig. 10B(d)) and SS-PEI/DNA/E8GRGDSF
complexes (Fig. 10B(e)). It was confirmed that the introduc-
tion of peptides into SS-PEI/DNA has important role on transfer
DNA into cells and further results in different transfection
efficiency.
Acknowledgments
We acknowledge the financial support from the Min-
istry of Science and Technology of China (2009CB930300,
2011CB606202), Natural Science Foundation of Hubei Province,
China (2009CDA024), National Natural Science Foundation of China
(21004046), and the Fundamental Research Funds for the Central
Universities (1101014).
4. Conclusions
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