Guanidinated multi-arm star polyornithines with a polyethylenimine core for gene delivery

Article abstract of DOI:10.1016/j.polymer.2014.07.037

Multi-arm star polyornithines PEI-P(Orn)_n are prepared by grafting polyornithine arms onto branched polyethylenimine (PEI) with an M _w of 600 via the ring-opening polymerization of N-carboxyanhydride of benzyloxycarbonyl ornithine. To enhance gene delivery efficiency and reduce cytotoxicity, the amino side groups on the polyornithine arms are partially guanidinated that transforms the ornithine units to arginine units. Thus, the guanidinated products G-PEI-P(Orn)_n contain multiple poly(ornithine-co-arginine) arms. PEI-P(Orn)_n and G-PEI-P(Orn) ₇₁ mediate the transfection of pGL3 plasmid to 293T cells almost as efficient as 25 kDa PEI in serum-free medium. Notably, in contrast to the dramatically lowered efficiency of 25 kDa PEI in the presence of serum, the efficiency of G-PEI-P(Orn)₇₁ can be retained or even enhanced in the medium containing 10% serum. The improved serum-compatibility and high efficiency of the guanidinium-modified star polyornithines make them promising for gene delivery.

Full text of DOI:10.1016/j.polymer.2014.07.037

Polymer 55 (2014) 4634e4640
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Guanidinated multi-arm star polyornithines with a polyethylenimine
core for gene delivery
Mengmeng Cai, Zhenguo Zhang, Xin Su, Hui Dong, Zhenlin Zhong*, Renxi Zhuo
Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Multi-arm star polyornithines PEI-P(Orn)n are prepared by grafting polyornithine arms onto branched
polyethylenimine (PEI) with an Mw of 600 via the ring-opening polymerization of N-carboxyanhydride of
benzyloxycarbonyl ornithine. To enhance gene delivery efficiency and reduce cytotoxicity, the amino side
groups on the polyornithine arms are partially guanidinated that transforms the ornithine units to
arginine units. Thus, the guanidinated products G-PEI-P(Orn)n contain multiple poly(ornithine-co-
arginine) arms. PEI-P(Orn)n and G-PEI-P(Orn)71 mediate the transfection of pGL3 plasmid to 293T cells
almost as efficient as 25 kDa PEI in serum-free medium. Notably, in contrast to the dramatically lowered
efficiency of 25 kDa PEI in the presence of serum, the efficiency of G-PEI-P(Orn)71 can be retained or even
enhanced in the medium containing 10% serum. The improved serum-compatibility and high efficiency
of the guanidinium-modified star polyornithines make them promising for gene delivery.
© 2014 Elsevier Ltd. All rights reserved.
Received 21 March 2014
Received in revised form
8 July 2014
Accepted 22 July 2014
Available online 1 August 2014
Keywords:
Poly(amino acid)s
Star polymers
Gene delivery
1. Introduction
DNA and swell in endosome when released from DNA. The partially
degraded dendrimers with a flexible structure mediate higher level
Gene therapy is a very promising way for treating congenital
diseases and acquired diseases [1e6]. One of the major factors for
effective gene therapy is an efficient and safe vector that delivers
genes into cells. Viral vectors are usually highly efficient but with
difficulty in large scale preparation and with safety issues of
immunogenicity and mutagenesis. The disadvantages of viral vec-
tors have stimulated the investigation of synthetic vectors.
Commonly used non-viral vectors include linear polymers such as
polylysine [7e10], branched polymers such as polyethylenimine
(PEI) [11e14], and dendritic polymers such as poly amidoamine
dendrimers [15e19].
Dendritic and hyperbranched polymers have attracted intensive
attentions in gene delivery because of their three-dimensional ar-
chitectures with abundant terminal functional groups and inner
cavities. Dendritic polyamidoamine polymers consist of primary
amines on the surface and tertiary amines in the interior. This
structure has high buffer capacity that helps the escape of DNA
from endosome [20e23]. Among these polyamidoamines, the
partially degraded are especially noticeable. Due to the random
solvolysis of amide bonds between polyamidoamine units, these
polymers can collapse into a compact form when complexed with
transfection than intact dendrimers [24e25]. Redox-responsive
hyperbranched polyamidoamines with a thiol-responsive core
and aminoethylpiperazine terminal groups exhibited high trans-
fection efficiency and low cytotoxicity, showing that both the
degradation of disulfide bonds in the core and the type of the ter-
minal groups play an important role [26]. Investigation on the in-
fluence of molecular weight and architecture of polylysine in gene
transfection efficiency showed that hyperbranched polylysine has
higher transfection efficiency than linear and dendritic polylysine
[27]. Hyperbranched PEI-methyl acrylate-PEI conjugate prepared
with low-molecular weight PEIs exhibited lower cytotoxicity and a
rather high gene transfection efficiency than 25 kDa PEI in various
cell lines [28].
Basic peptides such as arginine-rich peptides have been re-
ported to have a membrane permeability and a carrier function for
intracellular protein delivery [29e34]. These functions are related
to the guanidinium groups. Guanidinium groups displayed from
numerous non-proteinogenic scaffolds can facilitate internalization
[35]. Primary guanidinium groups bear a positive charge under
physiological conditions and have the potential to donate up to five
hydrogen bonds to electron-rich functional groups [36] such as the
carboxyl, phosphoryl, and sulfuryl groups of cell-surface carbohy-
drates and phospholipids which are essential for efficient cellular
uptake [37]. Arginine-rich peptides or poly(amino acid)s were
reported to be effective gene vectors [38e40]. The synthesis of
* Corresponding author. Tel.: þ86 27 6875 4061; fax: þ86 27 6875 4509.
E-mail address: zlzhong@whu.edu.cn (Z. Zhong).
http://dx.doi.org/10.1016/j.polymer.2014.07.037
0032-3861/© 2014 Elsevier Ltd. All rights reserved.

M. Cai et al. / Polymer 55 (2014) 4634e4640
4635
arginine-containing poly(amino acid)s, however, is comparatively
more complex because additional protection of the guanidine side
groups is required [41e43].
dissolved in DMF, and re-precipitated by adding diethyl ether. 1H
NMR (300 MHz, CDCl3, ): 7.30 (br s, C6H5CH2), 4.99 (s, C6H5CH2),
3.95 (br s, COCHNH), 3.11 ( br s, CH2CH2CH2NHCOO), 2.55e2.39 (m,
NCH2CH2N), 1.94e1.28 (m, CHCH2CH2CH2NH).
PEI-polyornithines (PEI-P(Orn)n) were synthesized by removing
the benzyloxycarbonyl (Z) groups on PEI-P(ZOrn)n. PEI-P(ZOrn)n
polymers weretreatedwith4 equivof HBr (33%in HOAc)respect toZ
groups in CF3COOH at 0 ꢁC for 1.5 h. The product was precipitated
with excess diethyl etherand dried in vacuo.1H NMR (300 MHz, D2O,
d
In this work, we design multi-arm star polyornithines con-
sisting of a hyperbranched PEI (Mw ¼ 600) core and multiple
positively charged polyornithine arms. The multi-arm structure
endows the polymer with a three-dimensional architecture while
the arms are yet not as crowded as the branches in typical den-
drimers or hyperbranched polymers. The star polyornithines are
transformed to ornithine-arginine copolymers by guanidination
of the amino side groups using O-methylisourea hemisulfate.
Compared to the original polyornithines, the guanidinated poly-
mers show reduced cytotoxicity and increased gene transfection
efficiency.
d): 4.24 (br s, COCHNH), 3.75e3.11 (m, NCH2CH2NHCO), 2.91 (br s,
CH2CH2CH2NH2), 2.65 (m, NCH2CH2N), 1.66 (br s, CH2CH2CH2NH2).
2.3. Guanidinium-modified PEI-poly(ornithine) (G-PEI-P(Orn)n)
PEI-P(Orn)71 (100 mg) was dissolved in 6 mL of ammonia solution
in a 15 mL centrifuge tube. To this tube a known amount of O-
methylisourea hemisulfate was added to the PEI-P(Orn)71 solution.
The solution was stirred at 65 ꢁC for 1.5 h. Then the mixture was
transferred to an MWCO 3500 Da dialysis bag and dialyzed against
deionized water for 2 days. The product was obtained by lyophili-
2. Experimental section
2.1. Materials
PEI with an Mw of 600 Da was purchased from Alfa Aesar, and
PEI with an Mw of 25 kDa was purchased from Aldrich. ε-Benzy-
loxycarbonyl ornithine (ZOrn) and 33 wt% solution of HBr in HOAc
were supplied by Chengdu Chengnuo New-Tech Co., Ltd. Tetra-
hydrofuran (THF) was distilled over NaeK alloy in the presence of
benzophenone before use. Dimethyl formamide (DMF) was dried
over CaH2 and distilled under vacuum before use. Plasmid pGL3
under the control of SV40 promoter and with enhancer sequences
encoding luciferase was obtained from Promega, Madison, WI,
USA. Plasmids were propagated in Escherichia coli in Lur-
zation to give a dry solid mass. 1H NMR (300 MHz, D2O,
d): 4.25 (br s,
COCHNH), 3.75e3.11(m,NCH2CH2NHCO),2.96(brs, CH2CH2CH2NH),
2.65 (m, NCH2CH2N), 1.74e1.59 (m, CHCH2CH2CH2NH). 13C NMR
(75 MHz, D2O,
d): 173.8 (COCHNH), 156.8 (NHC(NH)NH2), 53.4
(COCHNH), 40.6 (CHCH2CH2CH2NH), 28.3 (CHCH2CH2CH2NH), 24.5
(CHCH2CH2CH2NH).
2.4. Cytotoxicity assay
iaeBertani (LB) medium containing 60
mg
mLꢀ1 ampicillin
respectively at 37 ꢁC and purified using E. Z. N. A. Fastfilter
Endofree Plasmid Midi kits (Omega) according to the manufac-
turer's instruction. The purity of DNA was assessed spectropho-
tometrically by measuring absorbance at wavelengths of 260 and
280 nm (OD260/OD280 1.8 or greater) and confirmed using 0.7%
agarose gel electrophoresis containing GelRed. The DNA concen-
tration was determined by measuring the UV absorbance at
260 nm. DNA aliquots of pGL3 were stored at ꢀ20 ꢁC prior to use.
Dulbecco's Modified Eagle's Medium (DMEM) was purchased
from Invitrogen Corp. RPMI 1640 medium was purchased from
Biological Industries. Phosphate buffered saline (PBS) and
Trypsin-EDTA, Penicillin-streptomycin, fetal bovine serum (FBS)
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) were obtained from Bioind. Other commercially
available reagents were used as received.
The cytotoxicity was evaluated on the basis of an MTT assay on
293T cells. 293T cells were seeded in 96-well plates at an initial
density of 5000 cells per well in 100 mL of DMEM complete me-
dium. The cells were allowed to grow for 24 h. The PEI-P(Orn)n and
G-PEI-P(Orn)n solutions were added to the media. Each dosage was
replicated in 4 wells. Treated cells were incubated at 37 ꢁC under a
humidified atmosphere of 95% air and 5% CO2 for 24 h. MTT reagent
(20
were incubated for 4 h at 37 ꢁC. The liquid in each well was
removed and 150 L of DMSO was added to each well to dissolve
m
L in PBS, 5 mg mLꢀ1) was added to each well, and the cells
m
the crystals. The absorbance at 570 nm in each well was recorded
using a spectrophotometer Multiskan Go (Thermo Scientific). Cell
viability was calculated according to the following equation: Cell
viability (%) ¼ (ODsampleꢀODblank)/(ODcontrolꢀODblank) ꢂ 100, where
ODsample is the absorbance of the solution of the cells cultured with
the polymer; ODblank is the absorbance of the medium; ODcontrol is
the absorbance of the solution of the cells cultured with the me-
dium only.
2.2. Synthesis of PEI-polyornithine (PEI-P(Orn)n)
ε-Benzyloxycarbonyl ornithine N-Carboxyanhydride (ZOrn-
NCA) was synthesized by following a literature procedure [44].
Briefly, ZOrn (5.00 g, 18.7 mmol) and triphosgene (3.50 g,
11.8 mmol) was suspended in 100 mL of dry THF under argon. The
mixture was stirred in a 55 ꢁC oil bath until the cloudy solution
turned clear. The solution was precipitated by addition of excess
petroleum ether. The precipitate was collected by filtration and
purified by recrystallization from ethyl acetate and petroleum
MTT assay on L929 cells was carried out by the similar way,
except RPMI 1640 medium was used instead of DMEM medium.
2.5. Agarose gel retardation assay
Designed amounts of PEI-P(Orn)n and G-PEI-P(Orn)n aqueous
solutions were added slowly to 25 mL of pGL3 solutions (40 m
g mLꢀ1
ether. 1H NMR (300 MHz, CDCl3,
d): 7.35 (br s, C6H5), 5.09 (s,
in 40 mM TriseHCl buffer solution), and then the polyplexes were
diluted to a total volume of 1 mL with 150 mM NaCl and vortexed
for 15 s. The mixture was incubated at room temperature for 30 min
for the polyplex formation. The polyplexes at various polymer/DNA
C6H5CH2), 4.32 (s, COCHNHCOO), 3.23 (s, CH2CH2CH2NHCOO),
1.95e1.61 (m, CHCH2CH2CH2NH).
A representative procedure for the preparation of PEI-poly(ε-
benzyloxycarbonyl ornithine) (PEI-P(ZOrn)n) is as follows: To a
solution of ZOrn-NCA (1.00 g, 3.40 mmol) in 30 mL of dry DMF was
added a proportional PEI (600 Da) as an initiator under argon. The
reaction solution was stirred for 72 h at 30 ꢁC, and then precipitated
by addition of excess diethyl ether. The precipitate was collected,
(w/w) ratios mixed with 1
m
L of 6 ꢂ loading buffer were loaded on
0.7% (w/v) agarose gel containing GelRed and electrophoresed with
Tris-acetate (TAE) running buffer at 80 V for 80 min. DNA was
visualized with a UV lamp using a Vilber Lourmat imaging system
(France).

4636
M. Cai et al. / Polymer 55 (2014) 4634e4640
2.6. Particle size and zeta potential measurements
measured with a BCA protein assay kit (Pierce, USA). Luciferase
activity was expressed as RLU/mg protein.
The particle size and zeta potential were measured with a Nano-
ZSZEN3600 (Malvern) zetasizer. The polyplexes at various polymer/
DNA (w/w) ratios were prepared by adding appropriate volume of
3. Results and discussion
polymer to 1.0
mg of pGL3 DNA solution. The polyplexes were
incubated at room temperature for 30 min. Then the polyplexes
were diluted with pure water, DMEM without containing FBS, or
DMEM containing 10% FBS to 1 mL for size measurements and zeta
potential measurements.
3.1. Preparation and characterization of star polyornithines PEI-
P(Orn)n
The strategy for the preparation of star polyornithines PEI-
P(Orn)n is shown in Scheme 1. First, PEI-P(ZOrn)n was obtained by
grafting polyornithine arms on the branched PEI core through the
ROP of ZOrn-NCA initiated by the amino groups of PEI. Then, the
benzyloxycarbonyl protective groups were removed by acidolysis
with a solution of HBr in trifluoroacetic acid.
Molecular weights of PEI-P(ZOrn)n were measured by gel
permeation chromatography coupled with multi-angle laser light
scattering (GPC-MALLS) in DMF. The n values in PEI-P(ZOrn)n star
polymers were calculated according to their number average mo-
lecular weights (Mn), i.e., PEI-P(ZOrn)29, PEI-P(ZOrn)42, and PEI-
P(ZOrn)71 have an Mn of 7900, 11,000, and 18,200, respectively. As
shown by the data in Table 1, control of molecular weights was
achieved through the use of specific feed ratios of the NCA mono-
mer to PEI initiator. The deprotection of PEI-P(ZOrn)n was
confirmed by 1H NMR. The strong benzyl signal at 7.2 ppm dis-
appeared, and the signal at 3.95 ppm of the polyornithine chain
shifts to 4.24 ppm after deprotection because of the leaving of the
benzoxycarbonyl group from the amino group.
2.7. In vitro transfection efficiency
The in vitro transfection efficiency of the PEI-P(Orn)n and G-
PEI-P(Orn)n was evaluated in 293T cells, using pGL3 plasmid. Cells
were seeded at a density of 70,000 cells/well into 24-well plates.
They were incubated at 37 ꢁC under a humidified atmosphere of
95% air and 5% CO2 for 24 h. The medium in each well was replaced
with 1 mL of DMEM without containing FBS or 1 mL of DMEM
containing 10% FBS. The polyplexes containing 1.0 mg of pGL3 at
various polymer/DNA (w/w) ratios were added to each well and
incubated with cells for 4 h at 37 ꢁC. Then the medium was
replaced with fresh DMEM complete medium and the cells were
incubated for another 48 h. After incubation, cells were lysed with
200 mL of cell lysis buffer. The luciferase activity in cell extracts
was measured using a luciferase assay kit (Promega, USA). The
relative light units (RLU) were normalized by the total protein
concentration of the cell extracts, and the total protein was
Scheme 1. Schematic representation of the preparation of PEI-P(Orn)n and G-PEI-P(Orn)n.

M. Cai et al. / Polymer 55 (2014) 4634e4640
4637
Table 1
Synthesis and molecular weights of PEI-P(ZOrn)n.
PEI-P(ZOrn)n Feed
Molecular weights
Mn (103) Mw (103) Mw/Mn
a
a
PEI (g) ZOrn-
NCA/PEI
NCA (g) (molar ratio)
PEI-P(ZOrn)29 0.1280 1.9615 31.5
PEI-P(ZOrn)42 0.0788 2.0123 52.5
PEI-P(ZOrn)71 0.0430 1.5368 73.4
7.9
11.0
18.2
8.6
12.7
20.2
1.09
1.15
1.11
a
Molecular weights were determined by GPC-MALLS in DMF.
3.2. Guanidination of PEI-poly(ornithine)
Star polymer PEI-P(Orn)71, which demonstrated the highest
transfection efficiency among the PEI-P(Orn)n series, was guanidi-
nated with O-methylisourea hemisulfate to give the guanidinium-
modified G-PEI-P(Orn)71. The content of guanidine groups was
determined by elemental analysis, on the basis that the guanidi-
nated polymers have higher N/C ratios than the originals. The
amino groups of ornithine were transformed to guanidino groups
at two different percentages (34% and 60%), and the resulted
guanidinium-modified polymers are represented as 34% G-PEI-
P(Orn)71 and 60% G-PEI-P(Orn)71, respectively. These polymers
were used to investigate the possible effect of amino/guanidino
ratio on DNA condensation, cytotoxicity and transfection efficiency.
3.3. Cytotoxicity of PEI-P(Orn)n and G-PEI-P(Orn)n
MTTassay was carried out to evaluate the cytotoxicity of the PEI-
P(Orn)n and G-PEI-P(Orn)71 polymers in 293T cells. The results are
shown in Fig. 1A. The IC50 values of PEI-P(Orn)29, PEI-P(Orn)42, and
PEI-P(Orn)71 were 110, 45, and 17 m
g mLꢀ1, respectively, indicating
that the cytotoxicity increases with the increase of molecular
weight. Under the same conditions, the IC50 value of 25 kDa PEI was
detected to be 7 m
g mLꢀ1. Therefore, the cytotoxicity of PEI-P(Orn)n
was only slightly lower than that of PEI with a similar molecular
weight. Noticeably, the cytotoxicity of PEI-P(Orn)n could be
remarkably lowered by guinidination of the amino groups. The IC50
of 34% G-PEI-P(Orn)71, and 60% G-PEI-P(Orn)71 was elevated to
Fig. 1. Relative cell viability of 293T cells (A) and L929 cells (B) at 24 h after the
addition of the polymers.
26
P(Orn)71
m m m
g mLꢀ1 and 51 g mLꢀ1, respectively, from 17 g mLꢀ1 of PEI-
3.4. Characterization of PEI-P(Orn)n/pDNA and G-PEI-P(Orn)71
/
.
pDNA polyplexes
Cytotoxicity of the polymers in L929 mouse fibroblasts was also
evaluated by MTT assay. The results are shown in Fig. 1B. The
cytotoxicity of the PEI-P(Orn)n and G-PEI-P(Orn)71 polymers in
L929 cells was obviously higher than that in 293T cells. Similar
trend was observed in L929 cells that the cytotoxicity increases
with the increase of molecular weight and the cytotoxicity could be
lowered by guinidination.
The polyplexes of PEI-P(Orn)n and G-PEI-P(Orn)71 with plasmid
pGL3 were prepared by mixing their aqueous solutions at chosen
polymer/DNA weight ratios (w/w). The PEI-P(Orn)n/pGL3 and G-
PEI-P(Orn)71/pGL3 polyplexes were characterized by agarose gel
retardation, particle size, and zeta potential.
The ability of PEI-P(Orn)n series to interact with DNA was
characterized by agarose gel retardation. As shown in Fig. 2, the
concentration of the polymer to retard DNA reduced with the
molecular weight increasing. The required polymer/DNA weight
ratios to retard DNA were 0.3, 0.2 and 0.15 for PEI-P(Orn)29, PEI-
P(Orn)42, and PEI-P(Orn)71, respectively. The star polymer could
bind DNA at a very low polymer/DNA ratio in contrast to the linear
PLL (Mn ¼ 4 kDa) that needs a high weight ratio of 1 [48]. 25 kDa PEI
as a very efficient polymer could completely retard DNA at weight
ratio of 0.39, or equivalently at N/P ratio of 3 [49]. This suggests that
the star PEI-P(Orn)n polymers bind the plasmid DNA as efficiently
as PEI of similar molecular weights.
The cytotoxicity of cationic polymers was probably caused by
polymer aggregation on cell surfaces impairing the important
membrane function. The contents of amino groups increased with
the increase of molecular weight, resulting to increased positive
charges. The polymers with higher molecular weights more seri-
ously impaired cell membranes that are negatively charged. Gua-
nidination of amino groups results in delocalization of the positive
charge which leads to reduction in toxicity [45]. Further, guanidino
groups form bidentate hydrogen bonding with the constituents
present in cell membrane rather than electrostatic interactions
alone which could cause cell damage [46e47]. After amino groups
in PEI-P(Orn)71 were converted to guanidino groups, the electro-
static force was weakened due to partial role of hydrogen bonding
of guanidine in binding cell membrane. It is deemed that the
depressed electrostatic interaction is beneficial to the improvement
of biocompatibility of vector.
Guanidinated G-PEI-P(Orn)71 polymers exhibit lower DNA-
binding strength relative to their non-guanidinated counterparts.
The required weight ratios for PEI-P(Orn)71, 34% G-PEI-P(Orn)71,
and 60% G-PEI-P(Orn)71 to completely retard the DNA were 0.15,

4638
M. Cai et al. / Polymer 55 (2014) 4634e4640
Fig. 2. Agarose gel electrophoresis assay of the polyplexes of pGL3 with (a) PEI-P(Orn)29, (b) PEI-P(Orn)42, (c) PEI-P(Orn)71, (d) 34% G-PEI-P(Orn)71, and (e) 60% G-PEI-P(Orn)71
.
0.3, and 0.6, respectively. Guanidination results in delocalization of
the polymer charge which leads to weaker interaction between
polymer and DNA [45]. As reported previously, guanidine has an
attenuated interaction with DNA than amine does [47,50].
The particle sizes of the polyplexes in pure water were deter-
mined by DLS and shown in Fig. 3. The particle size decreased with
the increase of the polymer/DNA ratio and was from 400 to 160 nm
in the polyplexes formed by PEI-P(Orn)n and DNA. G-PEI-P(Orn)71
formed polyplexes with a size in the range of 150e300 nm.
Surface charge of DNA polyplexes is another important param-
eter affecting the efficiency of cellular uptake. Zeta potentials of the
polyplexes in various media were shown in Fig. 4. For PEI-P(Orn)n
series and G-PEI-P(Orn)71 in pure water (Fig. 4A), the polyplexes
were positively charged and the zeta potentials of increased with
the polymer/DNA ratio increase. At polymer/DNA weight ratio of
20, zeta potentials trended to reach a plateau of 34 mV. In DMEM
medium (Fig. 4B), the zeta potentials were much lower than those
in pure water, probably because the surface charge was partially
neutralized by the amino acids and other electrolytes in the me-
dium. In the 10% FBS-containing DMEM medium, zeta potential was
found to become negative. This is possibly due to negatively
charged serum proteins in FBS that adsorb to the positively charged
polyplexes and made them a net negative surface charge.
The effect of media type on particle size and stability over time
was shown in Fig. 5. The results demonstrated that PEI-P(Orn)71
/
DNA and 60% G-PEI-P(Orn)71/DNA particles in DMEM without
containing FBS were significantly larger than those in DMEM con-
taining 10% FBS. It was also demonstrated that in DMEM without
containing FBS, these particles were unstable and particle sizes
increased over time. In contrast, particles were stable over time in
DMEM containing 10% FBS serum, presumably, from negative
electrostatic repulsions. The sizes of 60% G-PEI-P(Orn)71/DNA par-
ticles were significantly smaller than the sizes of PEI-P(Orn)71/DNA
particles in both DMEM without containing FBS and DMEM con-
taining 10% FBS serum. The size of the gene delivery complex is
known to dramatically affect transfection efficacy. Small particles
that are stable in serum are typically preferred in gene transfection.
3.5. In vitro transfection efficiency
The gene delivery efficiency of PEI-P(Orn)n series and G-PEI-
P(Orn)71 was evaluated by luciferase expression of pGL3 plasmid
DNA in 293T cells. The amount of pGL3 plasmid DNA was 1.0 mg
in vitro transfection assay. Polyplexes were prepared at polymer/
DNA weight ratios ranging from 1 to 20. Branched 25 kDa PEI at its
Fig. 3. Particle size of the polymer/pGL3 polyplexes in water measured by DLS.

M. Cai et al. / Polymer 55 (2014) 4634e4640
4639
optimal N/P ratio of 10 (equivalently at weight ratio of 1.3) was used
for comparison.
Fig. 6 shows the luciferase expression of 293T cells in the pGL3
transfection mediated by PEI-P(Orn)n series and G-PEI-P(Orn)71 in
the serum-free medium. The PEI-P(Orn)71/pGL3 polyplexes at w/w
polymer/DNA weight ratio of 2 exhibited the highest efficiency,
while PEI-P(Orn)29/pGL3 and PEI-P(Orn)42/pGL3 reached their
maxima at weight ratios of 5 and 2, respectively. At its optimal ratio
of 2, PEI-P(Orn)71 achieved nearly the same luciferase expression of
PEI 25 kDa. The polyplexes prepared at higher ratios led to reduced
efficiency, possibly ascribed to the increased cytotoxicity and tight
interaction with DNA as revealed by gel agarose gel electrophoresis
that the polymer retarded DNA at very low ratios. 34% G-PEI-
P(Orn)71 and 60% G-PEI-P(Orn)71 reached their highest efficiency at
ratio of 2. In contrast to the other polymers, 60% G-PEI-P(Orn)71 did
not significantly reduce efficiency at polymer/DNA weight ratios
higher than the optimal value of 2. The P-values calculated by T-test
by comparing the data of 60% G-PEI-P(Orn)71 with PEI-P(Orn)29
,
PEI-P(Orn)42, PEI-P(Orn)71, and 34% G-PEI-P(Orn)71 are 0.011, 0.012,
0.015, and 0.052, respectively. The statistical calculation reveals
that the efficiency changing trend of 60% G-PEI-P(Orn)71 along with
polymer/DNA ratio changes is significantly different from that of
the non-guanidinated polymers. These results are consistent with
the tendency of cytotoxicity difference of the polymers, suggesting
that cytotoxicity may be an important cause for the efficiency
decrease at higher ratios.
It is important to investigate the influence of serum on the
transfection efficiency since therapeutic gene transfection is
in vivo. Fig. 7 shows luciferase expression of the pGL3-transfected
293T cells mediated by PEI-P(Orn)n series and G-PEI-P(Orn)71 in
the medium containing 10% serum. Compared to the results in
serum-free medium, the maximum transfection efficiency achieved
by PEI-P(Orn)n series as well as 25 kDa PEI was reduced by
approximate 10 times. In the case of G-PEI-P(Orn)71, though the
efficiency was also significantly reduced at lower polymer/DNA
ratios, it was retained or even enhanced at higher polymer/DNA
ratios of 10 and 20.
These multi-arm star polymers have a flexible structure in some
extent similar to the degraded polyamidoamine dendrimers. At
neutral pH, electrostatic repulsion between protonated primary
amines at branch chains cause the star polymer an extended
conformation, and when the amino groups are charge neutralized
Fig. 4. Surface charge of polymer/pGL3 polyplexes at various weight ratios with 1 mg of
pGL3 in pure water (A) and in DMEM medium with or without FBS (B).
Fig. 6. In vitro transfection efficiency of the polyplexes of pGL3 with the polymers in
293T cells in serum-free medium. The transfection efficiency of 25 kDa PEI was ob-
tained at an optimal weight ratio of 1.3.
Fig. 5. Polymer/pGL3 polyplexes (20 w/w) particle size and stability in DMEM with or
without containing FBS.

4640
M. Cai et al. / Polymer 55 (2014) 4634e4640
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This research was supported by the National Natural Science
Foundation of China (21374084) and National Basic Research Pro-
gram of China (2011CB606202).