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N-Succinyl-chitosan grafted with low molecular weight polyethylenimine

as a serum-resistant gene vector
Bo Lu, Yun-Xia Sun, Yun-Qiu Li, Xian-Zheng Zhang* and Ren-Xi Zhuo*
Received 15th December 2008, Accepted 23rd March 2009
First published as an Advance Article on the web 17th April 2009
DOI: 10.1039/b822505b
Published on 17 April 2009. Downloaded by Dalhousie University on 26/05/2014 10:12:47.

Low transfection efficiency and inactivation by serum are the major drawbacks for cationic
polymers when used as non-viral gene vectors. Here, a series of N-succinyl-chitosan-graft-
polyethylenimine (NSC-g-PEI) copolymers with different compositions were synthesized
through grafting low molecular weight PEI (800 Da) to N-succinyl-chitosan. An agarose gel
electrophoresis assay showed NSC-g-PEIs had good binding capability with DNA and the
particle size of the NSC-g-PEI–DNA complexes was between 150 to 300 nm as determined by a
Zeta sizer. In vitro transfection of NSC-g-PEI–DNA complexes for 293T, HeLa and CHO cells
was investigated. It was found that the transfection efficiency of NSC-g-PEI–DNA complexes was
higher than that of DNA combined PEI (25 kDa) and the transfection efficiency increased with
the increasing GD of PEI. More importantly, the NSC-g-PEI–DNA complexes were stable and
the transfection efficiency was not affected obviously in the presence of serum with different
concentrations. In addition, NSC-g-PEIs had a lower cytotoxicity than PEI (25 kDa) and the
toxicity increased with increasing GD of PEI. The NSC-g-PEI copolymers will have a good
potential as efficient non-viral gene vectors in the presence of serum.

Introduction saline or acetic acid solution. Since the potential of chitosan to

Numerous non-viral gene vectors with several advantages over
their viral counterparts, including ease of production, low
immune response, the ability to transfer large DNA molecules
and potential cell targeting properties have been developed for
gene delivery.1,2 However, a major problem associated with
non-viral gene vectors is low gene transfection efficiency,
especially in the presence of serum in the transfecting
medium.3 For example, for polyethylenimine (PEI), a cationic
polymer which has been used widely as gene vector, it has been
reported that related gene expression is significantly inhibited
by serum.4 The inactivation by serum can be overcome in vitro
by replacing serum-containing medium with a serum-free
medium, but it cannot be solved in vivo. And this shortcoming
appears to be one of the major limitations for efficient
applications of non-viral vectors in vivo. Much effort has been
devoted to solve this problem and a serum-resistant non-viral
vector has been prepared.5 A method for preparing the
non-viral vector that would make vectors insensitive to serum
has been proposed,6 and increasing the net positive charge
of the complexes to overcome the serum effect was also
reported.7 However, the overall progress is inadequate
and the development of a non-viral vector with high
gene transfection in the presence of serum is still critically
important for practical applications of gene therapy in vivo.
Chitosan is the only natural cationic polysaccharide, and
has been shown to be capable of effectively binding DNA in
Key Laboratory of Biomedical Polymers of Ministry of Education &
Department of Chemistry, Wuhan University, Wuhan 430072,
P. R. China. E-mail: xz-zhang@whu.edu.cn, bmp@whu.edu.cn;
Fax: +86 27-68754509
deliver DNA into cells was found by Mumper et al. in 1995,8
chitosan-based carriers have been an important class of the
non-viral vectors that have attracted increasing interest in gene
therapy. As a gene vector, chitosan has advantageous qualities
such as good biocompatibility, low immunogenicity and low
cytotoxicity.9 Besides, chitosan can be degraded into N-acetyl
glucosamine by general lysozymes in the body, which is
subsequently excreted as carbon dioxide via the glycoprotein
way. It was widely reported that the chitosan based vector–
DNA nanoparticles could transfect cells.10–12 Importantly,
using chitosan as the gene vector, it was found that the
transfection efficiency of chitosan–DNA complexes was higher
in the presence of serum than of that without serum.13
However, the low gene transfection efficiency is still the major
disadvantage of chitosan-based gene delivery systems. It has
been suggested that the low transfection efficiency is attribu-
table to the strong interactions between chitosan and DNA,
resulting in highly stable particles, thereby preventing disso-
ciation within the cell and ultimately precluding translation of
the DNA.11 Another drawback for chitosan also affects the
transfection seriously because chitosan is insoluble in a neutral
or basic pH range. To overcome the above obstacles and
improve transfection efficiency, recent studies were focused on
oligomeric chitosan as well as the derivatives of chitosan for
gene vectors.14–16
In this work, N-succinyl-chitosan (NSC) was prepared first
by introducing succinyl groups into the N-terminus of
glucosamine units in chitosan, the succinylation degree of
NSC was adjusted by controlling the reaction conditions.17
Prepared NSC has been reported as an ideal matrix to load
hydrophilic and bioactive biomacromolecules.18 Thereafter,
low molecular weight polyethylenimine (PEI 800 Da) was

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grafted onto the NSC to obtain a novel gene vector. Table 1 Feed composition and characteristics of NSC-g-PEI
The synthesized N-succinyl-chitosan-graft-polyethylenimine
Sample ID
(NSC-g-PEI) copolymers as the potential non-viral gene
vectors were evaluated in vitro with respect to the physico- NSC-g-PEI1 NSC-g-PEI2
chemical characteristics, morphology, in vitro cytotoxicity etc. NSC/mmol a
1.0 1.0
The transfection activity of NSC-g-PEI–DNA complexes in PEI (800 Da)/mmol 2.5 2.5
the presence of serum with different concentrations was also EDAC/mmol 0.2 0.4
GDb 0.167 0.266
investigated. Mwc 19 700 (PDI 1.54) 23 800 (PDI 1.41)
a
Calculated based on the saccharide unit of chitosan. b The number
Results and discussion of PEI molecules grafted to the saccharide unit of NSC-g-PEI.
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c
Determined by GPC measurement.
Synthesis of NSC-g-PEI copolymers
In this study, PEI (800 Da) was introduced to NSC to obtain
NSC-g-PEI graft copolymers. Since the intermolecular
hydrogen bonding interactions of NSC were weakened as
compared with chitosan, the water solubility of NSC was
improved dramatically.19,20 As shown in Scheme 1, after being
activated by EDAC, which is known as a ‘zero-length’
crossing-linker because the amide linkages are formed without
leaving a spacer molecule,21 NSC reacted with PEI to form the
graft copolymer. We used excess PEI to avoid gelation and we
acquired different grafting degrees by changing the weight
ratio of NSC : EDAC. The feed composition and the grafting
degree (GD) of NSC-g-PEI with two different values are
shown in Table 1. The advantages of using this method to
synthesize NSC-g-PEI included ease of production because it
was very easy to adjust the GD by altering the molar ratio of
NSC to EDAC. Besides, the characteristic of the chitosan
chain could be kept for not breaking the glucosamine units.
The FTIR spectra of chitosan, NSC, and NSC-g-PEI are
shown in Fig. 1A. From the spectrum of chitosan, it was

Fig. 1 (A) FTIR spectra of (Aa) chitosan, (Ab) N-succinyl-chitosan

(NSC), and (Ac) NSC-g-PEI2. (B) 1H NMR spectra of (Ba) chitosan,
(Bb) NSC, and (Bc) NSC-g-PEI1 in D2O.

found that distinctive amide absorptions appear at (1) 1638 cm1

and 1561 cm1. From the spectrum of NSC, the amide peaks
became stronger and a new peak appeared at (2) 1728 cm1,
which was assigned to –COOH. In the spectrum of
NSC-g-PEI, it was found that the peak at 1728 cm1 disappeared,
Scheme 1 Schematic illustration of the synthesis of NSC-g-PEI. which indicated that –COOH had reacted with –NH2.

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The 1H NMR spectra of chitosan, NSC, and NSC-g-PEI are
given in Fig. 1B. The 1H NMR assignments of NSC are as
follows: 1H NMR (D2O) d = 2.28–2.33 ppm (multiplet,
CH2CH2 of succinyl); d = 2.70 ppm (broad, H-2 of
D-glucosamine unit); d = 3.46–3.71 ppm (multiplet, N-succinyl–
0
D-glucosamine unit, H-3, H-4, H-5, H-6, H-6 ), d = 4.38 ppm
(broad, H-1 of D-glucosamine unit). The chemical shifts of
NSC agree with the literature.22 According to the ratio of the
integral peak of H in NSC to H in the chitosan structure, it can
be known that the succinylation degree of NSC is 0.305. In the
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spectrum of NSC-g-PEI, the peaks of –CH2CH2 of succinyl
are overlapped with the methylene peaks of PEI.23 Based on
the 1H NMR spectra, the GD values of NSC-g-PEI1 and
NSC-g-PEI2 were determined to be 0.167 and 0.266, respec-
tively. GPC analyses are also shown in Fig. 2. The results
indicated that the molecular weights (Mw) of NSC-g-PEI1 and
NSC-g-PEI2 were ca. 19 700 (PDI 1.54) and 23 800 (PDI 1.41),
respectively.
Characterization of NSC-g-PEI–DNA complexes
The formation of polymer–DNA complexes is an important
prerequisite for polycations as gene vectors. Chitosan-based
carriers are attractive non-viral vectors as they can form
complexes with DNA based on electrostatic interaction
between the positive amino groups of chitosan and negative
phosphate groups of DNA. The condensation capability of
NSC-g-PEIs was evaluated by agarose gel electrophoresis
assay at various weight ratios ranging from 0 to 4. As shown
in Fig. 3, the movement of complexes at vector/DNA weight
ratios of 4 (Fig. 3a) and 3 (Fig. 3b) were retarded completely
for both NSC-g-PEI1 and NSC-g-PEI2. The results confirmed
that the vectors had strong binding capability to DNA. The
ability of NSC-g-PEI1 to bind DNA was slightly lower than
that of NSC-g-PEI2. It is clear that the binding ability of the
copolymer became stronger with the increasing GD of PEI
(800 Da).
The particle size of polymer–DNA complexes is an
important factor that affects the cellular uptake and trans-
fection efficiency. It was reported that cells typically uptake
particles ranging from about 50 to several hundred nano-
metres.24 It was reported that chitosan with molecular weight
of 10 kDa could complex DNA to form particles with the size
of 600–1000 nm.12 Fig. 4A shows the particle sizes of
NSC-g-PEI–DNA complexes with various weight ratios. The
ranges of particle sizes of the complexes were from 150 nm to
300 nm, and the size of the complexes decreased with the
Fig. 2 GPC profiles of (a) NSC-g-PEI1, (b) NSC-g-PEI2.
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Fig. 3 Agarose gel electrophoresis retardation assay of the copoly-
mers at various weight ratios of NSC-g-PEI–DNA complexes.
(a) NSC-g-PEI1, (b) NSC-g-PEI2.
increasing weight ratio. The results indicated that the weight
ratio of the complexes had an important effect on the compac-
tion of DNA. With increasing weight ratio , the size decreased
gradually. The representative morphologies of PEI–DNA and
NSC-g-PEI–DNA complexes are shown in Fig. 4C. The
typical images indicate that the complexes had a spherical
shape and a compacted structure. The particle size of
NSC-g-PEI–DNA complexes was about 250 nm, which was
in agreement with the particle sizes measured by Nano-ZS
ZEN3600. Therefore, we have confirmed that our copolymers
could form nanoparticles with DNA and the average
diameters of the complexes were within the size requirements
for efficient cellular endocytosis.12,24–26
The surface charge of the gene delivery systems is known to
be one of the major factors influencing their transfection
efficiency.27 Surface charge of the complexes plays an impor-
tant role in non-specific cellular uptake and physical stability
in blood. The zeta potential of the complexes at various weight
ratios is shown in Fig. 4B. When the weight ratio was below 2,
the NSC-g-PEI–DNA complexes had a negatively charged
surface, suggesting that the amount of NSC-g-PEI was not
enough to complex the DNA completely. When the weight
ratio increased to 2, the zeta potential of the complexes
became positive. The surface charge of the complexes
increased with the increasing weight ratio and gradually
approached a plateau due to the saturation of polycations
complexed with DNA. Compared with chitosan, the
NSC-g-PEI copolymers had an increased charge, and their
binding affinity to DNA had been improved greatly.
Cell viability
For eventual application in vivo, a very important required
characteristic of vectors is minimal cytotoxicity. To investigate
the cytotoxicity of NSC-g-PEI copolymers, cell viability was
determined by the MTT assay. The sample, without treatment
of polymer, was considered as a positive control with a cell
viability of 100%. As illustrated in Fig. 5, compared to PEI
(25 kDa), the NSC-g-PEI copolymers had low cytotoxicities
in 293T and HeLa cells. In addition, the cytotoxicity of
NSC-g-PEI1 was lower than that of NSC-g-PEI2 in both cell
lines, which showed that the cytotoxicity gradually increased
with the increasing GD of the copolymers. The cytotoxicity of
cationic polymers was probably caused by polymer aggrega-
tion on cell surfaces impairing the important membrane
functions.28 When the polymer complexes with DNA, the
positive charge of NSC-g-PEI is counterbalanced by the
negative charge of DNA minimizing the direct contact of
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Fig. 4 (A) The particle size and (B) zeta-potential of the NSC-g-PEI–DNA complexes at various weight ratios of NSC-g-PEI–DNA complexes.
(C) Typical SEM images of polymer–DNA complexes. (a) PEI (25 kDa)–DNA (N/P = 10), (b) NSC-g-PEI1–DNA (w/w = 5) and
(c) NSC-g-PEI2–DNA (w/w = 5).

Fig. 5 Cytotoxicity of the copolymers at various concentrations for (a) 293T, and (b) HeLa cells.

polycations with the cell membrane, and the complexes would
show less toxicity. Besides, chitosan as a biocompatible poly-
mer is helpful in reducing the cytotoxicity of the complexes.
Moreover, NSC-g-PEI may degrade in cells, become chitosan
units and non-toxic low molecular weight PEI.
Buffer capacity
It had been reported that the ability of polycation–DNA
complexes to escape the endolysosomal compartment could
be correlated to the buffer capacity of the polymer in the pH
range of 5 to 7.29 An appropriate buffer capacity of poly-
cations is important in the design of gene vectors. The buffer
capacities of the NSC-g-PEI copolymers, PEI (25 kDa), PEI
(800 Da) were determined by an acid–base titration method.
The polymer of high buffer ability would undergo a small
change in pH when the same amount of HCl was added into
the polymer solution during titration.30 As shown in Fig. 6,
PEI (25 kDa) and PEI (800 Da) had a similar buffer capacity.
NSC-g-PEI copolymers had reduced buffer capacities, and the
buffer capacity of NSC-g-PEI1 is lower than that of
NSC-g-PEI2. As previously mentioned, the buffer capability
of the polycation depends on the presence of primary, secondary,
and tertiary amine groups, the relatively lower buffer capacity
of the NSC-g-PEI1 might result from lower levels of amine
groups of NSC-g-PEI1 as compared with NSC-g-PEI2.
In vitro transfection
The gene transfection efficiencies of the NSC-g-PEIs were
evaluated by in vitro transfection of pEGFP in 293T, HeLa
and CHO cells. Typical fluorescence images of the transfected

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Fig. 6 Buffer capacity of PEI (25 kDa), PEI (800 Da) and the
NSC-g-PEIs copolymers in 150 mM NaCl solutions.
293T, HeLa and CHO cells are shown in Fig. 7. It can be seen
that NSC-g-PEI copolymers have similar gene transfection
ability to that of PEI (25 kDa) which has been considered to be
the most effective cationic gene vector. We also investigated
the gene transfection efficiency of the copolymers by luciferase
assay. The PEI (25 kDa) at an optimal ratio (N/P = 10)
was used as the control. As shown in Fig. 8, NSC-g-PEI
Fig. 7 Typical fluorescence images of (a) 293T, (b) HeLa and
(c) CHO cells transfected by NSC-g-PEI–DNA (pEGFP) complexes
at the optimized polymer–DNA weight ratios and PEI–DNA
complexes at optimized N : P ratio.
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Fig. 8 Transfection efficiencies of NSC-g-PEI–DNA (pGL-3) com-
plexes at various NSC-g-PEI–DNA weight ratios and for (a) 293T,
(b) HeLa and (c) CHO cells.
copolymers could effectively transfect all the three cell lines,
with the transfection efficiencies higher than that of PEI
(25 kDa) (N/P = 10). The higher transfection efficiency of
the NSC-g-PEI copolymers was ascribed to the following
reasons. NSC-g-PEI copolymers had a similar density of
amine groups to PEI but had much lower cytotoxicity. After
the grafting of PEI to NSC, the PEI moieties acted as a
sponge and enhanced the release of NSC-g-PEI–DNA com-
plexes, leading to improved transfection efficiency. Besides,
the transfection efficiency of NSC-g-PEI copolymers depended
on their GD, and the transfection efficiency of the copolymer
with a higher GD was increased. In addition, transfection
efficiency of the copolymers increased with the increasing
weight ratio of NSC-g-PEI–DNA when the weight ratio was
below 20.
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The development of gene vectors that are stable even in
serum after coagulation of blood is very important for
practical applications.31,32 In this study, the effect of serum
with different concentrations on the transfection efficiency of
NSC-g-PEI1–DNA complexes was further evaluated. As
shown in Fig. 9, the transfection efficiency of NSC-g-PEI1–DNA
complexes was not inhibited apparently at low concen-
tration of serum, and the presence of serum even led to
enhancement of gene transfection efficiency in all three kinds
of cell lines. However, in the presence of 40% serum, the
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transfection ability of complexes was decreased obviously in
the CHO cell line. The reason may be attributed to the fact
that the interference of serum depends on the cell type.33
Similar findings were also reported in the literature. For
Fig. 9 Effect of serum concentration on NSC-g-PEI–DNA complex
transfection efficiency at weight ratios of 10 and 20. (a) 293T, (b) HeLa
and (c) CHO cells.
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instance, Sato et al. reported that the transfection efficiency
of chitosan–DNA complexes in HeLa cells in the presence of
serum was higher than the one in the absence of serum.13
Jeong et al. reported that a hydrophobically modified glycol
chitosan showed increasing in vitro transfection efficiency in
the presence of serum.34 A similar effect was also shown by
stearic acid grafted chitosan oligosaccharide in the presence
of 10% serum-containing medium.35 The improved gene
transfection in the presence of serum may be presumed to be
due to the following reasons. First, the cell function was
raised by the addition of serum. Secondly, NSC-g-PEI forms
complexes with DNA in which the hydrophilic chitosan layer
is apt to locate on the complex surfaces, thus discouraging the
serum-mediated aggregation. Conventionally, large steric
barriers of carbohydrate polymer are often grafted on the
periphery of nanoparticles to prohibit flocculation in
biological media. Tseng et al. used dextran grafted branched
polyethylenimine to improve the stability of polycationic
vectors and promote gene delivery in serum. Srinivasachari
et al. used trehalose click polymers to inhibit nanoparticle
aggregation and promote DNA delivery in the presence of
serum. These polymers successfully prevent aggregation
through steric stabilization, and increased the gene trans-
fection in serum.36–38 Therefore, we speculate that the
saccharide ring chains of chitosan in the chitosan-based
polymers play an important role in these complexes to resist
the inhibition of serum.
Experimental
Materials
Chitosan (degree of deacetylation: 85%, Mw: B10 kDa) was
purchased from Haidebei Co. Ltd., Jinan, China. Branched
polyethylenimines with molecular weights of 25 kDa and
800 Da were purchased from Sigma-Aldrich. Dimethyl
sulfoxide (DMSO) was obtained from Shanghai Chemical
Reagent Co., China, and was dried by refluxing with
anhydrous MgSO4 overnight and then was distilled under
reduced pressure. Water-soluble 1-ethyl-3-(3-dimethylamino-
propyl) carbodiimide hydrochloride (EDAC) was obtained
from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium
(DMEM), penicillin-streptomycin, trypsin, fetal bovine serum
(FBS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT), and Dulbecco’s phosphate buffered saline
(PBS) were purchased from Invitrogen Corp. The reporter
plasmids, pEGFP-C1 and pGL-3, were purchased from
Invitrogen and Promega, respectively. Plasmids were amplified
in Escherichia coli and extracted and purified with an E.Z.N.A.
fast filter endo-free plasmid maxi kit (Omega). The plasmid
DNA was stored at 20 1C until the transfection experiments.
All other reagents were analytical grade and were used as
received.
Preparation of NSC-g-PEI
NSC-g-PEI was synthesized by grafting PEI onto NSC as
shown in Scheme 1. NSC was synthesized according to the
literature.39 In brief, 2 g (12 mmol) of chitosan, dipped in
20 mL of sodium hydroxide (20 wt%) for 12 h, was filtered and
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added to 40 mL of dimethyl sulfoxide, and 2 g (20 mmol) of
succinic anhydride was added at room temperature with
stirring, and then was placed in a 60 1C oil bath to carry out
the reaction for a certain time. Then the mixture was poured
into a 50 mL beaker followed by the addition of acetic acid
solution with stirring until the pH reached a value of 7.0.
Then, the precipitate was filtered, dissolved in distilled water,
and purified by acetone. The product was filtered and washed
with ethanol (70%) three times and anhydrous alcohol once
and then dried.
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To graft PEI onto NSC, 0.2 g (1.0 mmol) NSC was
dissolved in 40 mL distilled water with a proper weight of
EDAC at room temperature. The aqueous solution with 2.0 g
(2.5 mmol) PEI (800 Da) was then added into the above
solution, and followed by the addition of hydrochloric acid
solution with stirring until the pH reached a value of 5.6. The
mixture was stirred and incubated at room temperature for
24 h before dialysis (MWCO: 3500 Da) for 3 days and
lyophilized for 3 days.
Characterization of polymer
KBr pellets of chitosan, NSC, and NSC-g-PEI were prepared
for FTIR measurement via a Fourier transform infrared
(FTIR) spectroscope (a Lambda Bio40 UV-vis spectrometer,
PerkinElmer). 1H NMR of chitosan, NSC, and NSC-g-PEI
were recorded on a Mercury VX-300 spectrometer at 300 MHz
(Varian, USA) by using D2O as a solvent and TMS as an
internal standard. The molecular weight of the NSC-g-PEIs
copolymer was measured by a gel permeation chromatograph
(GPC) equipped with a Waters 26 900 separations module and
a Waters 2410 refractive index detector. Acetic acid–ammonium
acetate (pH 5.3) buffer solution was used as elute at a flow rate of
1.0 mL min1. The column temperature was maintained as 35 1C
NSC-g-PEI–DNA complex formation
NSC-g-PEI was dissolved in NaCl solution (150 mM) with a
concentration of 2 mg mL1. A plasmid DNA stock solution
(120 ng mL1) was prepared in Tris-HCl buffer solution.
NSC-g-PEI solution with a particular volume was added into
8.3 mL of DNA solution, vortexed for 15 s, and incubated
for 30 min at 37 1C prior to characterization. A series of
NSC-g-PEI–DNA complexes at various weight ratios were
prepared.
Agarose gel retardation assay
The NSC-g-PEI–DNA complexes with different weight ratios
ranging from 0 to 4 were formed according to the conditions
described above. Then 6 mL of complex suspensions
containing 0.1 mg DNA was electrophoresed on the 0.7%
(w/v) agarose gel containing GelRedTM and with Tris-acetate
(TAE) running buffer at 80 V for 80 min.
Measurement of particle sizes and zeta-potential
The particle size and zeta-potential were assessed by Nano-ZS
ZEN3600 (MALVERN Instrument) at 25 1C. The complexes
at various weight ratio ranges from 0.5 to 15 were prepared by
adding an appropriate volume of plasmid pGL-3 (in 40 mM
Tris-HCl buffer solution) to an appropriate volume of
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NSC-g-PEI solution (in 150 mM NaCl solution). Then the
complexes were incubated at 37 1C for 30 min. After that the
complexes were diluted with 150 mM NaCl solution (for zeta-
potential measurement, the solution was changed with distilled
water) to a final volume of 1 mL prior to measurement.
Determination of buffer capacity of copolymers
The buffer capacities of PEIs with Mw of 800 Da and 25 kDa,
and NSC-g-PEIs were determined by acid–base titration assay
over the pH range 10 to 2 as described by Benns et al.40,41
Briefly, 0.2 mg mL1 of each sample solution was prepared in
30 mL 150 mM NaCl solution. The sample solution was first
titrated with 0.1 M NaOH to a pH of 10, then 0.1 M HCl with
different volumes was added to the solution to obtain mixtures
with different pH values which were measured using a
microprocessor pH meter.
Scanning electron microscopy (SEM)
The polymer–DNA complexes were prepared according to the
conditions described above. 100 mL of complex suspension was
deposited onto a glass slide. After the solvent vaporized at
room temperature, the morphology of the sample was
observed using a scanning electron microscope (SEM,
FEI-QUANTA 200, Holland). Before the SEM observation,
the samples were fixed on an aluminium stub and coated with
gold for 7 min.
Cell culture
293T (epithelial human embryonic kidney cells), HeLa (human
cervix epithelial carcinoma cells) and CHO (Chinese hamster
ovary cells) were incubated, respectively in DMEM
supplemented with 10% FBS and 1% antibiotics (penicillin–
streptomycin, 10 000 U mL1) at 37 1C in a humidified
atmosphere containing 5% CO2.
Cytotoxicity assay
Cells were seeded in a 96-well plate at a density of 6000 cells
per well in 100 mL DMEM containing 10% FBS. After
incubation for 24 h, polymer solutions were added to the
culture medium. Cell viability was tested after the addition of
polymer for 48 h. The cell viability was measured using the
MTT method.42 The absorbance at 570 nm, which is
proportion to the cell viability, was measured using a micro-
plate reader (BIO-RAD, Model 550, USA). The relative
cell viability was calculated as: cell viability (%) =
(ODsample/ODcontrol)  100, where ODcontrol was obtained in
the absence of polymers and ODsample was obtained in the
presence of polymers. Each value was averaged from 4 parallel
experiments.
In vitro transfection
Cells were seeded at a density of 6  104 cells per well in a
24-well plate in triplicate with 1 mL DMEM containing 10%
FBS and incubated at 37 1C for 24 h in 5% CO2. The
polymer–DNA complexes were formed at different weight
ratios ranging from 5 to 30 according to the conditions
described above (containing 1 mg DNA in every weight ratio).
Before transfection, the cells were washed with PBS, and the
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cells were incubated with the complexes in serum-free or
different concentrations of serum-containing culture medium,
respectively for 4 h at 37 1C. Then the medium was replaced
with fresh medium containing 10% serum and incubated for
48 h. And the cells were analyzed for green fluorescence
protein (pEGFP-C1) expression with a fluorescence micro-
scope (OLYMPUS IX70, Japan).
To assay the expression of luciferase, the medium was
removed and the cells were rinsed gently with phosphate
buffered saline (PBS, 0.1 M, pH 7.4). After thorough lysis of
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the cells with reporter lysis buffer (Promega) (200 mL per well),
the luciferase activity was determined by detecting the light
emission from an aliquot of cell lysate incubated with 100 mL
of luciferin substrate (Promega) in a luminometer (Lumat
LB9507, Berthold). The protein content of the cell lysate was
determined by BCA protein assay kit (Pierce).43 All the
experiments were carried out in triplicate to ascertain the
reproducibility.
Conclusions
In this study, a series of NSC-g-PEIs with different GDs were
synthesized by coupling low molecular weight PEI to NSC.
The introduction of oligoethylenimine to NSC led to improved
DNA binding ability, buffer capacity, and solubility as
compared with that of chitosan. These copolymers when used
as gene vectors displayed comparable or higher transfection
efficiencies and lower cytotoxicities than that of PEI (25 kDa).
Importantly, the transfection efficiencies of the copolymer–
DNA complexes were not affected remarkably by the presence
of serum, indicating that resulting NSC-g-PEI copolymers will
have great potential as safe and efficient non-viral vectors in
the presence of serum.
Acknowledgements
This work was financially supported by the National Key
Basic Research Program of China (2005CB623903) and the
Ministry of Education of China (Cultivation Fund of Key
Scientific and Technical Innovation Project 707043).
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