Low molecular weight PEI-based polycationic gene vectors via Michael addition polymerization with improved serum-tolerance

Article abstract of DOI:10.1016/j.polymer.2015.03.070

A series of polycationic gene delivery vectors were synthesized via Michael addition from low molecular weight PEI and linking compounds with various heteroatom compositions. Agarose gel electrophoresis results reveal that these polymers can well condense plasmid DNA and can protect DNA from degradation by nuclease. The formed polyplexes, which are stable toward serum, have uniform spherical nanoparticles with appropriate sizes around 200-350 nm and zeta-potentials about +40 mV. In vitro experiments show that these polymers have lower cytotoxicity and higher transfection efficiency than 25 KDa PEI. Furthermore, the title materials exhibit excellent serum tolerance. With the present of 10% serum, up to 19 times higher transfection efficiency than PEI was obtained, and no obvious decrease of TE was observed even the serum concentration was raised to >40%. Flow cytometry and confocal microscopy studies also demonstrate the good serum tolerance of the materials.

Full text of DOI:10.1016/j.polymer.2015.03.070

Polymer 65 (2015) 45e54
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Low molecular weight PEI-based polycationic gene vectors via
Michael addition polymerization with improved serum-tolerance
,
Miao-Miao Xun a, Ya-Ping Xiao a, Ji Zhang a **, Yan-Hong Liu a, Qi Peng b, Qian Guo a,
,
*
Wan-Xia Wu a, Yong Xu c, Xiao-Qi Yu a
a Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, PR China
b West China School of Medicine, Sichuan University, Chengdu 610041, PR China
c Humanwell Healthcare (Group) Co., LTD., 666 Gaoxin Road, East High-Tech Development Zone, Wuhan, Hubei 430075, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of polycationic gene delivery vectors were synthesized via Michael addition from low molecular
weight PEI and linking compounds with various heteroatom compositions. Agarose gel electrophoresis
results reveal that these polymers can well condense plasmid DNA and can protect DNA from degra-
dation by nuclease. The formed polyplexes, which are stable toward serum, have uniform spherical
nanoparticles with appropriate sizes around 200e350 nm and zeta-potentials about þ40 mV. In vitro
experiments show that these polymers have lower cytotoxicity and higher transfection efficiency than
25 KDa PEI. Furthermore, the title materials exhibit excellent serum tolerance. With the present of 10%
serum, up to 19 times higher transfection efficiency than PEI was obtained, and no obvious decrease of TE
was observed even the serum concentration was raised to >40%. Flow cytometry and confocal micro-
scopy studies also demonstrate the good serum tolerance of the materials.
Received 26 December 2014
Received in revised form
20 March 2015
Accepted 25 March 2015
Available online 2 April 2015
Keywords:
Gene delivery
Low molecular weight PEI
Michael addition
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
material, has been used as a gene delivery vector since 1995 and
become one of the most promising and widely studied gene car-
Gene therapy represents a promising option for the treatment of
various diseases such as viral infections, inherited disorders and
cancers [1e3]. The safe and effective delivery of genes with various
types of vectors promises exceptional advancements in clinical
disease treatment, next-generation vaccines, and tissue engineer-
ing [4e6]. Current gene delivery vectors can be divided into viral
and non-viral. The use of non-viral vectors mitigates some issues
generally associated with viral gene therapy, such as limited gene
insertion size, immune response, mutagenesis and large scale
production limitations [7e9]. These non-viral vectors consist of a
diverse set of materials which are typically cationic, including
natural and synthetic polymers, lipids, peptides, dendrimers and
combinations of these structures [10,11]. Cationic polymers, which
can efficiently complex with negatively charged DNA, thereby
increasing DNA stability, were frequently studied. Among the pol-
riers [12,13] due in large part to efficient escape from the endocytic
pathway through the proton-sponge mechanism. However, the
gene transfection efficiency (TE) and cytotoxicity of PEI are heavily
correlated with their chain length and topology [14,15]. High mo-
lecular weight (HMW) PEIs are effective in condensing nucleic acids
but exhibit pronounced cytotoxicity and induce membrane damage
in the initial stages of treatment [16] and mitochondrial-mediated
apoptosis in the later stages. Meanwhile, low molecular weight
(LMW) PEIs, bearing buffering capacity equivalent to their longer
chain counterparts, are almost non-toxic but display poor TE owing
to inefficient pDNA condensation and low cellular uptake through
diminished charge-mediated interactions [17].
To achieve high TE as well as low toxicity, PEI might be modified
by many strategies such as covalent grafting [18], cross linking [19]
and electrostatic coating [20]. Crosslinked LMW PEIs have been
examined over the past decade, and various types of hydrolytically
or reductively degradable PEI polymers and networks have been
designed for in vitro transfection. Zhong et al. reported the
reversibly hydrophobilized 10 KDa PEIs based on rapidly acid-
degradable acetal-containing hydrophobe for nontoxic and
marked enhanced non-viral gene transfection [21]. Zhuo and co-
ycations, poly(ethyleneimine) (PEI),
a commercially available
* Corresponding author. Fax: þ86 28 85415886.
** Corresponding author.
E-mail addresses: jzhang@scu.edu.cn (J. Zhang), xqyu@scu.edu.cn (X.-Q. Yu).
http://dx.doi.org/10.1016/j.polymer.2015.03.070
0032-3861/© 2015 Elsevier Ltd. All rights reserved.

46
M.-M. Xun et al. / Polymer 65 (2015) 45e54
workers described the disulfide cross-linked PEI via click reaction
[22]. It was proved that these materials can maintain the TE of
25 KDa PEI with much less cytotoxicity.
(12.45 g, 0.057 mol) in anhydrous CH2Cl2 solution was added
dropwise to the above stirred solution under the ice bath. The
mixture was stirred overnight at room temperature, followed by
evaporation of the organic solvents. The residue was purified with
silica gel column chromatography (dichloromethane/methyl
alcohol ¼ 30: 1). The synthesis of naked two primary amine com-
pound according to the literature [29]. Then, acryloyl chloride
(6.95 g, 0.077 mol) in anhydrous dichloromethane (50 mL) was
added dropwise to a stirred solution of diol or diamine (0.038 mol)
and triethylamine (7.77 g, 0.077 mol) in anhydrous dichloro-
methane (50 mL) under the ice bath. The mixture was stirred
overnight at room temperature and then filtered off generated salt,
followed by evaporation of the volatile solvent. The residue was
purified with silica gel column chromatography (PE: EA ¼ 3: 1, v/v)
to give LC1eLC6.
For potential clinical applications, the interaction between
electropositive polycation/pDNA complexes (polyplexes) and the
negatively charged blood components cannot be ignored [23]. After
intravenous injection, some unwanted effects would arise including
the rapid clearance by the RES (Reticuloe Endothelin System) upon
polyplexes aggregation, and structure destabilization as well as the
premature DNA release and degradation [24]. Thereby it is highly
indispensable to make models about the serum-conditioned
transfection for the forecast evaluation on the in vivo TE of poly-
cationic vectors. For example, zwitterionic betaine species were
used for the functionalization of polymeric materials to enhance
their serum-tolerance [25,26]. Zhuo et al. also put forward that
branched PEI could be modified via the catalyst-free aminolysis
reaction with 5-ethyl-5-(hydroxymethyl)-1,3-dioxan-2-oxo (EHDO)
to promote the serum-tolerant capability [27]. Poly(ethylene glycol)
(PEG) was also used to enhance the biocompatibility of the polymer
vectors, and these modified PEIs maintained low cytotoxicity and
showed enhanced transfection activity [28]. Meanwhile, they pro-
tected the polyplexes from undesired interactions with the nega-
tively charged components in the bloodstream.
LC1 (Yield 41.2%): 1H NMR (400 MHz, CDCl3):
d
¼ 1.44 (s, 9H,
(CH3)3CO), 4.19e4.31 (m, 4H, OCH2CH(NH)CH2O), 4.84 (m, 1H,
OCH2CH(NH)CH2O), 5.85e5.88 (d, J ¼ 12 Hz, 2H, CH2CHCO),
6.09e6.15 (t, J ¼ 8 Hz, 2H, CH2CHCO), 6.40e6.45 (d, J ¼ 20 Hz, 2H,
CH2CHCO). 13C NMR (100 MHz, CDCl3):
d
¼ 165.92, 155.24, 131.80,
127.88, 80.24, 63.45, 48.61, 28.43. MALDI-HRMS: m/z 322.1265
([MþNa]þ), C14H21NO6Naþ, calc. 322.1267.
LC2 (Yield 55.3%): 1H NMR (400 MHz, CDCl3):
d
¼ 1.42e1.71 (m,
In the present study, we developed a series of polycations
(MP1eMP6) via Michael addition from LMW PEI 600 Da and linking
compounds (LC1eLC6). These materials showed good pH buffering
capacity and DNA binding ability. Improved TEs were achieved
compared to 25 KDa PEI, especially in the presence of serum.
6H, CH2(CH2)3CH2), 4.12e4.15 (m, 4H, OCH2(CH2)3CH2O), 5.77e5.80
(m, 2H, (CH2CHCO)2), 6.05e6.12 (m, 2H, (CH2CHCO)2), 6.34e6.39
(m, 2H, (CH2CHCO)2). 13C NMR (100 MHz, CDCl3):
d
¼ 166.27,130.67,
128.54, 64.32, 28.28, 22.52. MALDI-HRMS: m/z 235.0947
([MþNa]þ), C14H21NO6Naþ, calc. 235.0946.
LC3 (Yield 52.6%): 1H NMR (400 MHz, CDCl3):
d
¼ 3.71e3.73 (t,
2. Experimental details
J ¼ 4 Hz, 4H, CH2CH2OCH2CH2), 4.28e4.30 (t, J ¼ 4 Hz, 4H,
CH2CH2OCH2CH2), 5.80e5.83 (m, 2H, (CH2CHCO)2), 6.08e6.15 (m,
2H, (CH2CHCO)2), 6.37e6.42 (m, 2H, (CH2CHCO)2). 13C NMR
2.1. Materials and methods
(100 MHz, CDCl3):
d
¼ 166.13, 131.18, 128.22, 69.08, 63.58. MALDI-
All chemicals and reagents were obtained commercially and
were used as received. Anhydrous methanol and anhydrous chlo-
roform were dried and purified under nitrogen by using standard
methods and were distilled immediately before use. LMW PEI
(branched, average molecular weight 600 Da, 99%) was purchased
from Aladdin (Shanghai, China). 25 KDa PEI (branched, average
molecular weight 25 KDa) was purchased from SigmaeAldrich (St.
Louis, MO, USA) and nucleic acid labeling kit Label IT® Cy5™ was
obtained from Mirus Bio Corporation (USA). The plasmids used in
the study were pGL-3 (Promega, Madison, WI, USA, coding for
luciferase DNA) and pEGFP-N1 (Clontech, Palo Alto, CA, USA, coding
for EGFP DNA). The Dulbecco's modified Eagle's medium (DMEM)
and fetal bovine serum were purchased from Invitrogen Corp. The
MicroBCA protein assay kit was obtained from Pierce (Rockford, IL,
USA). The luciferase assay kit was purchased from Promega
(Madison, WI, USA). The endotoxin free plasmid purification kit
was purchased from TIANGEN (Beijing, China).
HRMS: m/z 237.0742 ([MþNa]þ), C14H21NO6Naþ, calc. 237.0739.
LC4 (Yield 45.4%): 1H NMR (400 MHz, CDCl3):
d
¼ 1.43 (s, 9H,
(CH3)3CO), 3.50e3.54 (t, J ¼ 16 Hz, 4H, CH2CH2N(CO)CH2CH2),
4.24e4.26 (t, J ¼ 8 Hz, 4H, OCH2CH2N(CO)CH2CH2O), 5.83 (m, 2H,
CH2CHCO), 6.06e6.13 (t, J ¼ 8 Hz, 2H, CH2CHCO), 6.37e6.41 (d,
J ¼ 16 Hz, 2H, CH2CHCO). 13C NMR (100 MHz, CDCl3):
d
¼ 166.02,
155.26,131.36,131.18,128.26,128.21, 80.43, 62.81, 62.68, 47.16, 46.93,
28.41. MALDI-HRMS: m/z 336.1426 ([MþNa]þ), C15H23NO6Naþ, calc.
336.1423.
LC5 (Yield 33.2%): 1H NMR (400 MHz, CDCl3):
d
¼ 1.44 (s, 18H,
{(CH3)3CO}2), 3.32e3.49 (t, J ¼ 20 Hz, 8H, CH2CH2N(CO) (CH2)2N(CO)
CH2CH2), 4.23e4.26 (t, J ¼ 4 Hz, 4H, OCH2CH2N(CO) (CH2)2N(CO)
CH2CH2O), 5.83e5.85 (d, J ¼ 8 Hz, 2H, CH2CHCO), 6.08e6.15 (t,
J ¼ 12 Hz, 2H, CH2CHCO), 6.39e6.43 (d, J ¼ 16 Hz, 2H, CH2CHCO). 13
C
NMR (100 MHz, CDCl3):
d
¼ 166.04, 155.42, 155.21, 131.46, 131.26,
131.09, 128.35, 80.34, 80.14, 62.75, 62.61, 46.71, 46.19, 45.50, 28.46.
MALDI-HRMS: m/z 479.2370 ([MþNa]þ), C22H36N2O8Naþ, calc.
479.2369.
1H NMR spectra were obtained on a Bruker AV400 spectrometer.
CDCl3 or D2O was used as the solvent and TMS as the internal
reference. The molecular weight of polymers were determined by
gel permeation chromatography (GPC) (Waters 515 pump, Waters
2410 Refractive Index Detector (25 ꢀC, incorporating Shodex col-
umns OHPAK KB-803). A filtered mixture of 0.5 mol Lꢁ1 HAc/NaAc
LC6 (Yield 19.5%): 1H NMR (400 MHz, CDCl3):
d
¼ 1.40 (s, 9H,
(CH3)3CO), 3.43e3.53 (m, 8H, NCH2CH2N(CO)CH2CH2N), 5.63e5.66
(d, J ¼ 12 Hz, 2H, CH2CHCO), 6.07e6.34 (m, 4H, {CH2CHCO}2). 13
C
NMR (100 MHz, CDCl3):
d
¼ 169.04, 157.12, 132.11, 131.06, 128.41,
126.61, 80.81, 49.59, 47.96, 40.04, 39.03, 28.41. MALDI-HRMS: m/z
buffer was used as the mobile phase with
a flow rate of
334.1740 ([MþNa]þ), C15H25N3O4Naþ, calc. 334.1743.
0.5 mL minꢁ1. Molecular weights were calculated against poly(-
ethylene glycol) standards of number average molecular weights
ranging from 900 to 80,000 Da.
2.3. Synthesis and characterization of target cationic polymers
MP1eMP6
2.2. Synthesis and characterization linker LC1eLC6
Polymers were successfully synthesized following modified
Michael addition reaction as reported previously. Briefly, PEI 600
(1.26 mmol) and linker LC1eLC6 (1.26 mmol) were separately
dissolved in 1.5 mL of anhydrous methanol and 1.5 mL anhydrous
Diols or diamine (0.048 mol) and triethylamine (4.81 g,
0.057 mol) were dissolved in anhydrous CH2Cl2 (50 mL). (Boc)2O

M.-M. Xun et al. / Polymer 65 (2015) 45e54
47
chloroform, they were mixed in a flask, under a nitrogen atmo-
sphere, the reaction mixtures were heated at 45 ꢀC with constant
stirring for 24 h in an oil bath. After the reaction, MeOHeHCl so-
lution was added to Boc-containing polymers and the reaction
mixture was stirred overnight at room temperature. The solvent
was removed under reduced pressure, and the residue was dis-
solved in a little water, strong alkaline ion-exchange resin was
added and then filtered off, the raw producs (MP1, MP4e6) were
obtained by lyophilization. The mixture of the six crude product
(MP1eMP6) was diluted with 3 mL of anhydrous methanol, and
then precipitated by the addition of anhydrous diethyl ether. The
precipitation was collected and dried in vacuum to get the product
as colorless oil. The molecular weights of compounds MP1eMP6
were measured by GPC.
10,000 U mLꢁ1) at 37 ꢀC in a humidified atmosphere containing 5%
CO2.
2.6. Amplification and purification of plasmid DNA
pGL-3 and pEGFP-N1 plasmids were used. The former one as the
luciferase reporter gene was transformed in Escherichia coli JM109
and the latter one as the enhanced green fluorescent protein gene
was transformed in E. coli DH5a. Both plasmids were amplified in
LuriaeBertani broth at 37 ꢀC overnight. The plasmids were purified
by an EndoFree Tiangen TM Plasmid Kit. Then the purified plasmids
were dissolved in TE buffer solution and stored at ꢁ20 ꢀC. The
integrity of plasmids was confirmed by agarose gel electrophoresis.
The purity and concentration of plasmids were determined by ul-
traviolet (UV) absorbance at 260 and 280 nm.
MP1 (Yield 45.16%): 1H NMR (400 MHz, D2O):
d
¼ 2.59e3.41 (m,
45H, (OCCH2CH2)2, (CH2CH2NH)n), 3.55e3.59 (m,1H, NH2CH(CH2)2),
3.67e3.83 (m, 4H, NH2CH(CH2O)2).
2.7. Agarose gel electrophoresis (binding capacity and stability of
polymers/pDNA complexes)
MP2 (Yield 56.08%): 1H NMR (400 MHz, D2O):
d
¼ 2.36e2.90 (m,
80H, (OCCH2CH2)2, CH2(CH2)3CH2, (CH2CH2NH)n), 3.13e3.21 (m, 2H,
(OCCH2CH2)2), 3.32 (m, 4H, (OCCH2CH2)2, CH2(CH2)3CH2), 3.47 (m,
2H, CH2(CH2)3CH2).
MP1eMP6/DNA complexes at different weight ratios ranging
from 0.2 to 6.4 were prepared by adding an appropriate volume of
MP3 (Yield 57.21%): 1H NMR (400 MHz, D2O):
d
¼ 2.36e2.99
MP1eMP6 to 5
mL of Puc-19 (0.025 mg/mL). The obtained complex
(m, 79H, (COCH2CH2)2, (CH2CH2NH)n), 3.13e3.19 (m, 2H,
CH2CH2OCH2CH2), 3.32 (m, 4H, (OCOCH2CH2)2, CH2CH2OCH2CH2),
3.47 (m, 2H, (OCOCH2CH2)2).
solution was diluted to a total volume of 15
mL, and then the
complexes were incubated at 37 ꢀC for 30 min. After that the
complexes were electrophoresed on a 1% (W/V) agarose gel con-
taining GelRed™ and with Triseacetate (TAE) running buffer at
150 V for 30 min. DNA was visualized under an ultraviolet lamp
using a Vilber Lourmat imaging system. In the experiments with
MP4 (Yield 42.37%): 1H NMR (400 MHz, D2O):
d
¼ 2.43e2.87 (m,
72H, (COCH2CH2)2, (CH2CH2NH)n), 3.11e3.17 (m, 2H, (OCH2CH2)2NH),
3.30e3.32 (m, 4H, (OCH2CH2)2NH, (COCH2CH2)2), 3.50e3.59 (m, 2H,
(COCH2CH2)2), 3.68e3.71 (m, 4H, (OCH2CH2)2NH).
serum, the complexes solution (20
mL contained 10% or 50% serum)
MP5 (Yield 40.32%): 1H NMR (400 MHz, D2O):
d
¼ 2.37e2.96 (m,
was obtained by adding PBS buffer and serum (2
m
L or 10 L) and
m
68H, NHCH2CH2NH, (OCCH2CH2)2, (CH2CH2NH)n), 3.08e3.25 (m, 2H,
(OCH2CH2NH)2), 3.34e3.51 (m, 4H, (OCH2CH2NH)2, (OCCH2CH2)2),
3.52e3.67 (m, 2H, (OCCH2CH2)2), 3.68e3.81 (m, 4H,
(OCH2CH2NH)2).
incubating for a certain time. Stabilities of MP4, MP6/DNA poly-
plexes were evaluated by testing the abilities of them to protect
pDNA against DNase degradation. Generally, MP4, MP6 and pDNA
(0.125
mg) were mixed at a weight ratio of 1.6 for 30 min at room
MP6 (Yield 35.11%): 1H NMR (400 MHz, D2O):
d
¼ 2.47e3.11 (m,
temperature. Then 2 mL DNase (2unit/uL) was added and the mix-
61H, (OCCH2CH2)2, (OCH2CH2)2NH, (CH2CH2NH)n), 3.34e3.55 (m,
4H, (OCH2CH2)2NH).
tures of MP4, MP6, pDNA, and DNase were incubated at 37 ꢀC for
2 h. The mixtures were then heated in a water bath at 65 ꢀC for
10 min to denature DNase I. Stability of the polyplex was further
analyzed by apolyanion competition assay, 5 mL heparin sodium
2.4. Acidebase titration
(1.6 mg/mL) was added to the MP4, MP6/DNA polyplex solution
and the mixture was incubated for 10 min at room temperature.
The samples were run at the same electrophoresis condition as
described above [30].
In this assay, briefly, MP1eMP6 (0.25 mmol of amino groups)
was dissolved in 5 mL of 150 mM NaCl aqueous solution, and 1 N
HCl was added to adjust the pH to 2.0. Aliquots (50 mL for each) of
0.1 M NaOH were added, and the solution pH was measured with a
pH meter (pHS-25) after each addition. For comparison, 25 KDa PEI
was used under same experimental conditions. The buffering ca-
pacity defined as the percentage of amine groups becoming pro-
tonated from pH 5.1 to 7.4, was calculated from the equation:
2.8. Ethidium bromide displacement assay
The ability of MP1eMP6 to condense DNA was studied using
ethidium bromide (EB) exclusion assays. Fluorescence spectra were
measured at room temperature in air by a Horiba Jobin Yvon
Fluoromax-4 spectrofluorometer and corrected for the system
m
L, 1 mg mLꢁ1) was put into quartz cuvette con-
buffercapacityð%Þ¼100½ðDV1NaOHꢁDV2NaOHÞꢂ0:1Mꢃ=Nmol
response. EB (5
taining 2.5 mL of 10 mM Hepes solution. After shaking, the fluo-
rescence intensity of EB was measured. Then CT DNA (10
mL,
wherein
to bring the PH value of the polymer solution from 5.1 to 7.4,
V2NaOH is the volume of NaOH solution (0.1 M) required to bring
the pH value of the NaCl solution from 5.1 to 7.4, and N is the total
moles (0.25 mmol) of protonable amine groups in the polymer.
DV1NaOH is the volume of NaOH solution (0.1 M) required
1 mg mLꢁ1) was added to the solution and mixed symmetrically,
and the measured fluorescence intensity is the result of the inter-
action between DNA and EB. Subsequently, the solutions of
D
MP1eMP6 (0.2 mg mLꢁ1, 5
mL for each addition) were added to the
above solution for further measurement. All the samples were
excited at 520 nm and the emission was measured at 600 nm. The
pure EB solution and DNA/EB solution without cationic polymer
were used as negative and positive controls, respectively. The
percent relative fluorescence (%F) was determined using the
equation %F ¼ (F ꢁ FEB)/(F0 ꢁ FEB), wherein FEB and F0 denote the
fluorescence intensities of pure EB solution and DNA/EB solution,
respectively.
2.5. Cell culture
HEK (human embryonic kidney) 293 cells, human osteosarcoma
(U2OS) cells, HeLa and HepG2 cells were incubated in Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal bovine
serum (FBS) and
1
‰
antibiotics (penicillinestreptomycin,

48
M.-M. Xun et al. / Polymer 65 (2015) 45e54
2.9. Particle size and
z-potential measurements in water
For luciferase assays, cells were transfected by complexes con-
taining pGL-3. For a typical assay in a 24-well plate, 24 h post
transfection as described above, cells were washed with cold PBS
Particle size and zeta potential measurements of polyplexes
were carried out using a Nano-ZS 3600 (Malvern Instruments, USA)
with a HeeNe Laser beam (633 nm, fixed scattering angle of 901) at
25 ꢀC. MP1eMP6/DNA polyplexes at weight ratios ranging from 0.2
and lysed with 100
m
L 1ꢂ Lysis reporter buffer (Promega). The
luciferase activity was measured by microplate reader (Model 550,
BioRad, USA). Protein content of the lysed cell was determined by
BCA protein assay. Gene TE was expressed as the relative fluores-
cence intensity per mg protein (RLU/mg protein). All the experi-
ments were done in triplicates.
to 12.8 were prepared by adding 1 mg of pUC-19 to the appropriate
volume of the polymer solution (in ultrapure water). Then the so-
lution of the polyplexes was incubated at 37 ꢀC for 30 min and then
diluted with deionized water to 1 mL prior to measurement. Data
were shown as mean standard deviation (SD) based on triplicate
independent experiments.
2.13. Cellular uptake of plasmid DNA (flow cytometry)
The cellular uptake of the polymer/fluorescein labeled-DNA
complexes was analyzed by flow cytometry. The Label IT Cy5 La-
beling Kit was used to label pDNA with Cy5 according to the
manufacturer's protocol. Briefly, HeLa and HepG2 cells were seeded
in 12-well plates (2.0 ꢂ 105 cells/well for HeLa, 2.4 ꢂ 105 cells/well
for HepG2) and allowed to attach and grow for 24 h. For trans-
fection in the absence of serum, the medium was exchange with
serum-free medium. As for transfection in the presence of serum,
the medium was exchanged with serum-containing medium. Cells
2.10. Transmission electron microscopy (TEM)
The morphologies of the polyplexes were observed by TEM
(JEM-100CXa) with an acceleration voltage of 100 kV. 2 mg of pUC-
19 was added to the appropriate volume of the polymer solution
(weight ratio of polymer relative to pDNA, w/w ¼ 6.4: 1), then
diluted to the total volume of 50 mL. The solution of the polyplexes
was incubated at 37 ꢀC for 0.5 h. 15 min before measurement, the
polyplex solution was diluted with deionized water or water con-
taining 10% fetal bovine serum (FBS) to 1 mL. A drop of DNA/
polymer polyplexes suspension was placed onto the copper grid.
After a few minutes, the excess solution was blotted away with
filter paper. Then, a drop of 0.5% (w/v) phosphotungstic acid was
placed on the above grid. The grid was dried at room temperature
at atmospheric pressure for several minutes before observation.
were incubated with Cy5 labeled DNA complexes (1.6 mg DNA/well,
optimal weight ratio of each sample) in media for 4 h at 37 ꢀC.
Subsequently, the cells were washed with 1ꢂ PBS and harvested
with 0.25% Trypsin/EDTA and resuspended in 1ꢂ PBS. Mean fluo-
rescence intensity was analyzed using flow cytometer (Becton
Dickinson and Company). Cy5-labeled plasmid DNA uptake was
measured in the FL4 channel using the red diode laser (633 nm).
The flow cytometer was calibrated for each run to obtain a back-
ground level of ~1% for control samples (i.e., untreated cells).
2.11. Cell viability assay
Toxicity of MP1eMP6 toward U2OS cells, HEK293 cells, HeLa
cells and HepG2 cells was determined by using a Cell Counting Kit-
8. The U2OS cells and HepG2 cells (9000 cells/well), HEK293 cells
and HeLa cells (8000 cells/well) were seeded into 96-well plates
and cultured overnight for 70e80% cell confluence. The cells were
then incubated in a culture medium containing MP1eMP6 with a
particular concentration for 24 h. After that, polymer solutions
2.14. Confocal laser scanning microscopy (CLSM) analysis
HepG2 and HeLa cells were seeded at a density of 1.8 ꢂ 104 cells
(HepG2 cells) and 1.5 ꢂ 104 cells (HeLa cells) per well in 35 mm
confocal dish (
F
¼ 15 mm), 24 h prior to transfection. For trans-
fection in the absence of serum, the medium was exchange with
serum-free medium. As for transfection in the presence of serum,
the medium was exchanged with serum-containing medium. Cells
were removed, 100 m
L of sterile filtered CCK-8 (0.1 mg mLꢁ1) stock
solution in PBS was added to each well for additional 1 h incubation
at 37 ꢀC. Then, the absorbance of each sample was measured using
an ELISA plate reader (model 680, BioRad) at a wavelength of
450 nm. The cell survival was expressed as follows: cell
viability ¼ (ODtreated/ODcontrol) ꢂ 100%. Besides, the cell viability of
25 KDa PEI was also performed.
were incubated with Cy5 labeled DNA complexes (0.8 mg DNA/well,
optimal weight ratio of each sample) in media for 2 h and 4 h at
37 ꢀC. Subsequently, cells were rinsed twice with PBS (pH 7.4) to
remove complexes that were not uptaken by cells, fixed with 4%
paraformaldehyde (dissolved with PBS buffer) for 10 min, nuclear
staining was done with DAPI. The CLSM observation was performed
using Leica TCS SP5 at excitation wavelengths of 405 nm for DAPI
(blue), 633 nm for Cy5 (red), respectively.
2.12. Transfection procedure
Gene transfection of a series of complexes was investigated in
U2OS cells, HEK293 cells, HeLa cells and HepG2 cells. Cells were
seeded in 24-well plates (1.0 ꢂ 105 cells/well for U2OS and HepG2
cells, 8 ꢂ 104 cells/well for HeLa and HEK293 cells) and grown to
reach 70e80% cell confluence at 37 ꢀC for 24 h in 5% CO2. Before
transfection, the medium was replaced with a serum-free or a 10%
serum-containing culture medium containing polymer/pDNA
3. Results and discussion
3.1. Synthesis and chemical properties target polymers MP1eMP6
The preparation method of polymers MP1eMP6 is shown in
Scheme 1. Cross linking compounds LC1e6 with different lengths
and carbon-heteroatoms distributions were first prepared by
relative diol or diamine and 2 equiv of acryloyl chloride. For com-
pounds LC1 and LC4e6, the amino groups in the middle of the
chain must be protected by (Boc)2O before the acylation. Subse-
quent Michael addition polymerization took place between the
linker LC1e6 and PEI 600 Da with mole ratio of 1: 1 in anhydrous
methanol and chloroform at 45 ꢀC for 24 h. After the reaction,
MeOHeHCl solution was added to remove the Boc group (if
needed). The crude products were recrystallized by methanol and
diethyl ether for 3 times to ensure their polydispersity. Gel
permeation chromatography (GPC) was used to measure the
(0.8 mg) complexes at various weight ratios. After 4 h under stan-
dard incubator conditions, the medium was replaced with fresh
medium containing serum and incubated for another 20 h.
For fluorescent microscopy assays, cells were transfected by
complexes containing pEGFP-N1. After 24 h incubation, GFP-
expressed cells were observed with an inverted fluorescence mi-
croscope (Nikon Eclipse TE 2000E) equipped with a cold Nikon
camera. Control transfection was performed in each case using a
commercially available transfection reagent 25 KDa PEI based on
the standard conditions specified by the manufacture.

M.-M. Xun et al. / Polymer 65 (2015) 45e54
49
Scheme 1. Synthetic route of cationic polymers.
Table 2
molecular weights of the polymers, and the results are listed in
The buffer capacity of synthesized polymers and 25 KDa PEI.
Table 1. Due to the lack of bulky Boc group, which would hinder the
polymerization process, MP2 and MP3 have distinctly higher mo-
lecular weights than others.
MP1
16.0
MP2
20.0
MP3
16.0
MP4
20.0
MP5
22.0
MP6
20.0
PEI
16.0
Buffering capacity (%)
Cationic polymers with various types of amino groups are
assumed to have high buffering capacity, which is also called as
“proton-sponge effect”. Such effect may lead to the disruption of
endosome in the transfection process, facilitating the escape of
polymer/DNA complexes (polyplexes) [31]. Therefore, the buffering
capacity of the polymers may be defined as the percentage of amine
groups that can become protonated during endosomal acidification
in the pH range from pH 7.4e5.1 (Table 2). It was shown that these
polymers have comparable or slightly higher buffering capacities
than 25 KDa PEI. The acid-base titration curves in Fig. S1 also shows
similar results. The structure of linking group has slight effect on
the buffering capacity, and the polymer with linking groups bearing
primary amine (MP1) has lower pH buffering ability than those
with secondary amine-contained linking groups (such as MP5).
benefit the interaction, and its higher molecular weight might also
contribute. Similar assay was also carried out with the presence of
serum, and results show that serum has little effect on the
condensation ability of the polymers (Fig. 1B). Besides, the binding
abilities of the polymers were also investigated by the ethidium
bromide (EB) dye replacement assay [33]. The fluorescence of EB
intercalating into the base pairs of DNA would be quenched by
another binding agent which may exclude EB molecules. It was
shown that the addition of polymers to EB pretreated DNA caused
considerable decrease of the fluorescent emission intensity along
with the increase of w/w ratio (Fig. S2), indicating that the EB was
gradually replaced by the polymers. Such results further illustrate
the strong DNA binding ability of the polymers.
3.2. The formation and properties of polymer/DNA polyplexes
Furthermore, the stability of polyplexes was studied via gel
electrophoresis. MP1eMP6/DNA polyplexes were incubated with
10% and 50% serum against different times. Results in Fig. 2A show
that no released or degraded DNA was found even after 2 h incu-
bation, indicating the stability of the polyplexes. The incubation
was further elongated with 50% serum. It was shown that DNA
would be degraded by serum in 24 h (Fig. S3, lane 7), however, the
polyplexes could remain stable after 48 h incubation (Fig. S3, lane
9). We also investigated the decomplexation of the polyplexes by
heparin, which is more negatively charged than DNA and can lead
to the release of DNA by its stronger interaction with cationic
polymers. As shown in Fig. 2B, DNA was found to be gradually
released with the increase of heparin. By comparing the polyplexes,
it was found that the heparin amount needed to release the DNA
from MP6/DNA complex was more than those from other poly-
plexes (MP2eMP4), indicating that MP6 has higher DNA affinity,
especially in the presence of negatively charged materials. On the
other hand, excessively high DNA binding ability (e.g. PEI) may
hinder the release of DNA in the cells [34], thus the balance be-
tween the DNA condensation and release abilities is essential for
non-viral vectors. Fig. 2B shows that although MP6 has higher DNA
binding ability than its analogs, such ability is also lower than that
of PEI.
The cationic property of the polymers facilitates their electro-
static interaction with negatively charged nucleic acids, which is
the cargo in the delivery process. The formation of polymer/DNA
complex (polyplex) can reduce the electrostatic repulsion between
DNA and the cell surface and can protect DNA against enzymatic
degradation by nucleases in cytoplasm or serum [32]. The DNA
condensation ability of MP1eMP6 was assessed by observing the
electrophoretic mobility of DNA band in agarose gel. As shown in
Fig. 1A, these polymers could effectively condense DNA from the
weight ratio (polymer/DNA, w/w) of 0.8. MP6 showed slightly
higher DNA binding ability, suggesting that amide groups might
Table 1
Molecular weights and polydispersities of target polymers.
Polymers
Mw (Da)
PDI
MP1
MP2
MP3
MP4
MP5
MP6
12,543
24,996
33,057
10,271
10,038
14,934
2.52
1.83
1.92
1.98
2.03
2.49

50
M.-M. Xun et al. / Polymer 65 (2015) 45e54
Fig. 1. DNA retardation assay by agarose gel electrophoresis in the absence (A) and presence (B) of 10% serum (þ). Lanes from left to right: pDNA control, MP1eMP6/DNA w/w ratios
of 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4, respectively.
Fig. 2. Polyplex stability studies via electrophoretic gel retardation assay. (A) polyplexes (w/w ¼ 1.6) against different incubation times (0, 30, 60, 120 min) in the presence of 10% or
50% serum (ꢁ: without serum, þ: with 10% serum, þþ: with 50% serum); (B) release of DNA with the addition of heparin at various concentrations (heparin/DNA: w/w ¼ 0, 0.025,
0.5, 1, 2, 4, 8, 16, 32, 64; polymer/DNA: w/w ¼ 0.8).
DNase I is an endonuclease that catalyzes the hydrolytic cleav-
age of phosphodiester linkages in the DNA backbone. Considering
the abundant amounts of DNase I in tissues and blood, DNA
degradation by DNase I is a major barrier for gene delivery in vitro
and in vivo [35,36]. The DNA protection by MP4 and MP6 against
DNase was studied, and results are shown in Fig. 3. Naked DNA was
degraded in the presence of DNase within 2 h (Lane 3), while MP4
and MP6 could protect DNA from degradation, and the protected
DNA could be released almost intactly by the addition of heparin
(Lanes 4 and 6).
The size of nanoparticles is important for its substantial effect on
their pharmacokinetics and pharmacodynamics [37]. The conden-
sation of DNA by MP4 and MP6 resulted in nanoscale particles,
which were directly visualized under transmission electron mi-
croscopy (TEM). As shown in Fig. 4A and B, the polyplexes in
deionized water at weight ratio of 6.4 were observed as regular
spherical shape with the diameters about 40 nm. Polyplex formed
from MP6 seemed to have smaller size and more regular shape than
the other. Besides, with the presence of 10% serum, the size of MP4/
DNA polyplex decreased, and the two polyplexes had similar
diameter and shape (Fig. 4C and D), and still well dispersed uni-
formly in the medium. These results also indicate their good serum
tolerance.
Dynamic light scattering (DLS) assay was subsequently carried
out to study the particle sizes and zeta potentials of all formed
polyplexes. Results in Fig. 5A reveal that with the increase of w/w
ratio, the particle sizes decreased and tended to be constant with
mean sizes of 200e350 nm. It is worth mentioning that the particle
sizes determined by DLS were much larger than those obtained by
TEM. This might be due to the experimental conditions: the particle
sizes determined by DLS were examined in the hydrated state in
solution, while those obtained by TEM had been dried after drop-
ping complexes solution onto C-coated Cu meshes [38,39]. MP1/
DNA shows smaller particle size than others, this might be due to
the primary amines in MP1 structure. Meanwhile, MP6/DNA also
shows small size which could be attributed to its higher DNA
binding ability. With respect to the surface charge, the excess of
cationic polymer over DNA molecules generally resulted in an
overall positive zeta-potentials. As shown in Fig. 5B, the zeta po-
tential of polyplexes gave an increasing trend along with the rise of
weight ratio. The values turned to be positive from w/w of 0.8,
which is in accord with the w/w for complete DNA retardation
observed in gel electrophoresis. Further increase of w/w led to
much slighter increase of zeta potentials, which finally reached
about þ40 mV. This positive charge might facilitate the interaction
between the polyplexes and cell membrane, leading to more effi-
cient cellular uptake.
3.3. Cytotoxicity
CCK8-based cell viability assays were performed in HEK293,
HeLa, U2OS and HepG2 cells at various concentrations which cover
the range used in the gene transfection assays. 25 KDa PEI was used
as control, and the results are shown in Fig. 6. In most cases, the cell
viabilities of studied polymers were higher than that of 25 KDa PEI
after 24 h incubation, suggesting that these polymers have better
biocompatibility. The cytotoxicity was found to be affected by
molecular weight and surface charge of the polymers. The poly-
mers with higher molecular weights (MP2, MP3, MP6) have higher
toxicity, and the cell viability were lower. MP5 seems to have the
lowest cytotoxicity, which might be attributed to its lower molec-
ular weight and zeta potential. We speculate that the presence of
ester or amide bonds in the polymer backbone may reduce their
toxicity.
Fig. 3. DNA protection by MP4 and MP6 against DNase. Lane 1: naked DNA; Lane 2:
polyplex (w/w ¼ 1.6); Lane 3: pDNA with DNase for 2 h; Lane 4: Polyplex (w/w ¼ 1.6)
with DNase for 2 h; Lane 5: Polyplex (w/w ¼ 1.6) treated with heparin; Lane 6: Pol-
yplex (w/w ¼ 1.6) with DNase for 2 h followed by heat-inactivation of DNase and then
treated with heparin.

M.-M. Xun et al. / Polymer 65 (2015) 45e54
51
Fig. 4. TEM images of MP4/DNA (A and C) and MP6/DNA (B and D) polyplexes at w/w of 6.4 in deionized water (A and B) and in water with 10% serum (C and D).
Fig. 5. Average particle size (A) and zeta-potential (B) of MP1eMP6/DNA polyplexes at weight ratio of 0.2, 0.4, 0.8, 6.4 and 12.8 (mean SD, n ¼ 3).
3.4. In vitro gene transfection
reporter gene. Results in Fig. 7A reveal that in the absence of serum,
only MP4 has slightly higher TE than PEI, which was used under its
optimal w/w ratio. However, to our delight, with the presence of
10% serum, all of the polymers exhibited higher TE than PEI.
Especially for MP4 and MP6, 12 and 19 times respectively higher
TEs were obtained at the optimized w/w ratio (12.8). Results clearly
Theoretically, satisfactory polycationic gene vectors should
possess both low cytotoxicity and high TE. To inspect the trans-
fection performance of the newly prepared polymers, gene delivery
experiments were first performed in HeLa cells using pGL-3 as
Fig. 6. Relative cell viabilities caused by MP1eMP6 toward HEK293 (A), HeLa (B), U2OS (C) and HepG2 cells (D), 25 KDa PEI was used as control. Data represent mean SD (n ¼ 3).

52
M.-M. Xun et al. / Polymer 65 (2015) 45e54
Fig. 7. Luciferase gene expression transfected by polyplexes MP1eMP6 at different weight ratios in comparison with 25 KDa PEI (w/w ¼ 1.4, N/P ¼ 10) in HeLa cells in the absence
(A) and presence (B) of serum. Data represent mean SD (n ¼ 3).
indicate that these materials have much higher serum tolerance
than 25 KDa PEI. Such biocompatibility might be ascribed to the
cross-linking LMW PEI structure, in which the oxygen atoms may
screen the positive charges on the polyplex, preventing their ag-
gregation with negatively charged serum proteins.
The transfections mediated by MP4 and MP6 in other cell lines
were subsequently processed. Normal cell HEK293 and tumor cells
U2OS/HepG2 were applied to study the TE without or with the
presence of serum. Although such two materials exhibited some
difference of TE between these cell lines, we could also find that
better TE could be obtained in the presence of serum, similar to the
results in HeLa cells. MP6 gave better TE than MP4 in HEK293 and
HepG2 cells, and 4.5 and 17 times higher TE than PEI were obtained
in the presence of serum, respectively (Fig. 8A and C). Meanwhile,
in U2OS cells, MP4 gave better results (Fig. 8B). Results further
demonstrate that this type of cationic polymers might act as
promising non-viral gene vectors toward various cell lines. In
addition, the effect of serum was further studied by varying its
concentration in MP6-mediated transfection in HepG2 cells. As
shown in Fig. 8D, the TE of PEI dramatically decreased with the rise
of serum concentration. On the contrary, no obvious TE decrease
was observed in MP6-mediated transfection until the serum con-
centration was raised to >40%, and up to 355 times higher TE than
PEI was obtained at its optimal w/w ratio (9.6) with 70% serum.
Results also indicate that with the increase of serum, higher poly-
mer dose (w/w) is needed for better TE. This might be attributed to
the more amount of negatively charged proteins in higher con-
centration of serum, thus more cationic polymers is necessary for
efficient DNA binding and protection. To directly visualize the
transfected cells, enhanced green fluorescent protein (eGFP)
Fig. 8. Luciferase gene expression transfected by MP4 and MP6 at various w/w in comparison with 25 KDa PEI (w/w ¼ 1.4, N/P ¼ 10) in HEK293 (A), U2OS (B) and HepG2 (C) cells in
the absence and presence of 10% serum. (D): TE of MP6 in HepG2 cells in the presence of different concentrations of serum, the weight ratios in each group were 3.2, 6.4, 9.6 and
12.8. Data represent mean SD (n ¼ 3).

M.-M. Xun et al. / Polymer 65 (2015) 45e54
53
advantage of MP6 may contribute to its better serum tolerance. In
HeLa cells, the cellular uptake results are quite consistent with the
transfection results shown in Fig. 7, indicating that cellular uptake
might be crucial element to affect the TE. In HepG2 cells, although
the TE of MP6 is much higher than PEI in the presence of serum
(Fig. 8C), the uptake of MP6/DNA is still lower. Such celluar uptake
was also observed by confocal microscopy (Fig. S5). Therefore, it
may be concluded that MP6 has much more efficient intracellular
DNA delivery ability in such cell line. To visually illuminate the
internalization and intracellular distribution of the polyplexes,
molecular probes Cy5 and DAPI were used to tag DNA and nucleus
in the transfection toward HeLa cells. As shown in Fig. 10, after 2 h
incubation, a small quantity of Cy5 labeled complexes were accu-
mulated in the perinuclear region of cells, and serum had no
obvious effect on the transfection. When the incubation time was
elongated to 4 h, the red fluorescence evidently increased. It could
also be found that serum has little effect on MP6-mediated trans-
fection but has large negative effect on that involving PEI, indi-
cating the uptake inhibition of PEI/DNA complex.
Fig. 9. Cellular uptake of MP6/DNA polyplexes at optimal transfection w/w ratio in
HeLa and HepG2 cells (in the absence and presence of serum) quantified by flow
cytomety analysis. 25 KDa PEI was used as control. Data represent mean SD (n ¼ 3).
expression in HeLa and U2OS cells was also observed by an inverted
fluorescent microscope, and the weight ratios were used according
to the optimal w/w obtained in luciferase assay. Results also show
that unlike PEI, the GFP expression mediated by these polymers
with the presence of 10% serum was ever higher than those without
serum (Fig. S4).
The TE of gene vector depends on a multiple factors, i.e. DNA
condensation and protection, serum stability, cellular uptake effi-
ciency, intracellular trafficking such as endosomal escape, DNA
unpacking, and nuclear entry [40]. To further study the transfection
mechanism by title polymers, we applied flow cytometry to
investigate the cellular uptake of the MP6/DNA polyplexes, and the
results in HeLa and HepG2 cells are shown in Fig. 9. After 4 h in-
cubation of polyplexes with the cells, the percentage of positive
cells for Cy5-labeled pDNA was calculated. It was shown that in
both cell lines, serum has less negative effect on the celluar uptake
of MP6/DNA than that of the polyplex formed from PEI. Such
4. Conclusion
In summary, a new series of polycationic gene delivery vectors
were synthesized via Michael addition from LMW PEI and linking
compounds. These materials can efficiently condense DNA into
stable nanoparticles with proper sizes and zeta-potentials.
Compared to 25 KDa PEI, these polymers exhibited lower cyto-
toxicity and higher TE. More importantly, these materials have
distinctly higher serum tolerance than PEI. In the presence of 10%
serum, MP6 may give 19 and 17 times higher TE than PEI in HeLa
and HepG2 cells, respectively. No obvious TE decrease was
observed even the serum concentration was raised to >40%, indi-
cating that these materials may act as promising candidates for
non-viral gene delivery in future in vivo application.
Fig. 10. Confocal microscopic images of HeLa cells treated with Cy5 labeled MP6/DNA polyplexes at optimal transfection w/w ratio for 2 h and 4 h in the absence and presence of
serum, 25 KDa PEI was used as control (w/w ¼ 1.4). For each triad image, left: cell nuclei stained by DAPI (blue); middle: Cy5-labeled pDNA (red); right: merged image. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

54
M.-M. Xun et al. / Polymer 65 (2015) 45e54
[16] Parhamifar L, Larsen AK, Hunter AC, Andresen TL, Moghimi SM. Soft Matter
Acknowledgments
2010;6:4001e9.
[17] Tripathi SK, Gupta S, Gupta KC, Kumar P. RSC Adv 2013;3:15687e97.
[18] Petersen H, Fechner PM, Martin AL, Kunath K, Stolnik S, Roberts CJ, et al.
Bioconjug Chem 2002;13:845e54.
[19] Deng J-Z, Sun Y-X, Wang H-Y, Li C, Huang F-W, Cheng S-X, et al. Acta Biomater
2011;7:2200e8.
[20] Ito T, Yoshihara C, Hamada K, Koyama Y. Biomaterials 2010;31:2912e8.
[21] Liu Z, Zheng M, Meng F, Zhong Z. Biomaterials 2011;32:9109e19.
[22] Liu J, Jiang X, Xu L, Wang X, Hennink WE, Zhuo R. Bioconjug Chem 2010;21:
1827e35.
[23] Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. Nat Rev
Genet 2014;15:541e55.
This work was financially supported by the National Program on
Key Basic Research Project of China (973 Program, 2012CB720603),
National Science Foundation of China (No. 21472131, 21232005 and
J1103315), and Specialized Research Fund for the Doctoral Program
of Higher Education (20120181130006).
Appendix A. Supplementary data
[24] Li S, Tseng WC, Stolz DB, Wu SP, Watkins SC, Huang L. Gene Ther 1999;6:
585e94.
[25] Xiu K-M, Zhao N-N, Yang W-T, Xu F-J. Acta Biomater 2013;9:7439e48.
[26] Sun J, Zeng F, Jian H, Wu S. Biomacromolecules 2013;14:728e36.
[27] Luo X-H, Huang F-W, Qin S-Y, Wang H-F, Feng J, Zhang X-Z, et al. Biomaterials
2011;32:9925e39.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.polymer.2015.03.070.
References
[28] Brus C, Petersen H, Aigner A, Czubayko F, Kissel T. Bioconjug Chem 2004;15:
677e84.
[29] Srinivasachari S, Liu Y, Zhang G, Prevette L, Reineke TM. J Am Chem Soc
2006;128:8176e84.
[30] Liu H, Wang H, Yang W, Cheng Y. J Am Chem Soc 2012;134:17680e7.
[31] Martello F, Piest M, Engbersen JFJ, Ferruti P. J Control Release 2012;164:
372e9.
[32] Farber IY, Domb AJ. Mater Sci Eng C 2007;27:595e8.
[33] Wang B, Yi W-J, Zhang J, Zhang Q-F, Xun M-M, Yu X-Q. Bioorg Med Chem Lett
2014;24:1771e5.
[34] Liu G, Xie J, Zhang F, Wang Z, Luo K, Zhu L, et al. Small 2011;7:2742e9.
[35] Wang J, Dou B, Bao Y. Mater Sci Eng C 2014;34:98e109.
[36] Ferguson EL, Duncan R. Biomacromolecules 2009;10:1358e64.
[37] van Asbeck AH, Beyerle A, McNeill H, Bovee-Geurts PHM, Lindberg S,
Verdurmen WPR, et al. ACS Nano 2013;7:3797e807.
[38] Xun M-M, Liu Y-H, Guo Q, Zhang J, Zhang Q-F, Wu W-X, et al. Eur J Med Chem
2014;78:118e25.
[1] Wang M, Cheng Y. Biomaterials 2014;35:6603e13.
[2] Kanasty R, Dorkin JR, Vegas A, Anderson D. Nat Mater 2013;12:967e77.
[3] Whitehead KA, Langer R, Anderson DG. Nat Rev Drug Discov 2009;8:129e38.
[4] Sinclair A, Bai T, Carr LR, Menye JRE, Zhang L, Jiang S. Biomacromolecules
2013;14:1587e93.
[5] Sheridan C. Nat Biotechnol 2011;29:121e8.
[6] Ferraro B, Morrow MP, Hutnick NA, Shin TH, Lucke CE, Weiner DB. Clin Infect
Dis 2011;53:296e302.
[7] Sun J, Zeng F, Jian H, Wu S. Polym Chem 2013;4:5810e8.
[8] Lu Y, Han Y, Liang J, Meng H, Han F, Wang X, et al. Polym Chem 2011;2:
1866e71.
[9] Hamilton SK, Sims AL, Donavan J, Harth E. Polym Chem 2011;2:441e6.
[10] Jia L, Li Z, Zhang D, Zhang Q, Shen J, Guo H, et al. Polym Chem 2013;4:156e65.
[11] Cheng C, Convertine AJ, Stayton PS, Bryers JD. Biomaterials 2012;33:6868e76.
[12] Liu L, Zheng M, Renette T, Kissel T. Bioconjug Chem 2012;23:1211e20.
[13] Park JW, Bae KH, Kim C, Park TG. Biomacromolecules 2011;12:457e65.
[14] Hu Y, Zhou D, Li C, Zhou H, Chen J, Zhang Z, et al. Acta Biomater 2013;9:
5003e12.
[39] Ma M, Li F, Yuan ZF, Zhuo RX. Acta Biomater 2010;6:2658e65.
[40] Wang X, Shao N, Zhang Q, Cheng Y. J Mater Chem B 2014;2:2546e53.
[15] Hu C, Peng Q, Chen F, Zhong Z, Zhuo R. Bioconjug Chem 2010;21:836e43.