Conjugation with betaine: A facile and effective approach to significant improvement of gene delivery properties of PEI

Article abstract of DOI:10.1021/bm301826m

Herein, we developed a new gene delivery vector by grafting a betaine monomer (N,N-dimethyl(acrylamidopropyl)ammonium propane sulfonate, DMAAPS) onto 25 KDa polyethylenimine (PEI 25K) via the Michael addition reaction. The graft ratio for betaine on PEI polymer could be readily controlled, and in this study three PEI-betaine conjugates PEI-DMAAPS_23%, PEI-DMAAPS_55%, and PEI-DMAAPS_95% were prepared with their graft ratios of 23, 55, and 95%, respectively. The PEI-betaine conjugates exhibited much lower protein adsorption and cytotoxicities compared with PEI 25K, and they also showed little or no hemolytic effect. Moreover, the PEI-betaine conjugates display satisfactory DNA condensation capability; and in the absence and presence of serum, PEI-DMAAPS_23%/pEGFP and PEI-DMAAPS_55%/pEGFP complexes exhibited remarkable gene transfection efficiencies determined by flow cytometry, which are in general several times higher than that of PEI 25K. With these favorable properties, the PEI-betaine conjugates hold great potential for use as efficient gene delivery vectors. This study suggests that the betaine monomer may serve as a biocompatible modifying agent and this facile strategy may provide a facile and effective way for constructing some other biocompatible materials.

Full text of DOI:10.1021/bm301826m

Article
pubs.acs.org/Biomac
Conjugation with Betaine: A Facile and Effective Approach to
Significant Improvement of Gene Delivery Properties of PEI
†
,†
†
,‡
Jun Sun, Fang Zeng,* Haoliang Jian, and Shuizhu Wu*
†Department of Biomedical Engineering, College of Materials Science and Engineering, South China University of Technology,
Guangzhou 510640, P. R. China
‡
State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, P. R.
China
*
S Supporting Information
ABSTRACT: Herein, we developed a new gene delivery
vector by grafting a betaine monomer (N,N-dimethyl-
(
acrylamidopropyl)ammonium propane sulfonate, DMAAPS)
onto 25 KDa polyethylenimine (PEI 25K) via the Michael
addition reaction. The graft ratio for betaine on PEI polymer
could be readily controlled, and in this study three PEI-betaine
conjugates PEI-DMAAPS23%, PEI-DMAAPS55%, and PEI-
DMAAPS95% were prepared with their graft ratios of 23, 55,
and 95%, respectively. The PEI-betaine conjugates exhibited
much lower protein adsorption and cytotoxicities compared
with PEI 25K, and they also showed little or no hemolytic effect. Moreover, the PEI-betaine conjugates display satisfactory DNA
condensation capability; and in the absence and presence of serum, PEI-DMAAPS23%/pEGFP and PEI-DMAAPS55%/pEGFP
complexes exhibited remarkable gene transfection efficiencies determined by flow cytometry, which are in general several times
higher than that of PEI 25K. With these favorable properties, the PEI-betaine conjugates hold great potential for use as efficient
gene delivery vectors. This study suggests that the betaine monomer may serve as a biocompatible modifying agent and this facile
strategy may provide a facile and effective way for constructing some other biocompatible materials.
INTRODUCTION
polyplexes with stealth behavior, because PEG takes on a
ballooning-type conformation that increases solution viscosity
and acts as a water barrier that prevents proteins from
■
Polyethylenimine (PEI), an off-the-shelf material, has been used
as a gene-delivery vector since 1995 and become one of the
most promising and widely studied gene carriers due in large
part to efficient escape from the endocytic pathway through the
proton-sponge mechanism. When PEIs are used as the gene
carriers, the high cationic charge densities in the polymeric
chains result in tight DNA condensation, leading to highly
positively charged polyplexes that show generally high cellular
uptake in vitro; however, high cationic charge densities also
bring about some severe problems, for example, the membrane-
disruptive properties that frequently cause high cytotoxicities,
the strong binding with negatively charged proteins which
25
1
−7
approaching the surface. Furthermore, PEG reduces the
tendency of particles to aggregate by steric stabilization, thereby
producing formulations with increased stability during storage
8
,9
2
6,27
and application.
Currently, PEG remains the most used
polymer and the gold standard in biomedical applications. On
the other hand, the search for potential alternatives with
comparable or even better properties is attracting attentions
due to some limitations of PEG such as compromised
28
bioactivity in some applications.
In recent years, zwitterionic polymers, such as phospholipid-
9
−11
based, polymer-like poly(2-methacryloyloxyethyl phosphoryl-
deteriorate serum stability of the polyplexes, and so on.
2
9−36
choline) (PMPC)
(
and betaine-based, polymer-like poly-
Hence, PEI has been chemically modified in different ways so
that additional functions or enhanced performances were
achieved for a better gene delivery, and the modification
methods include the incorporation of short hydrophobic
37,38
carboxybetaine) (PCB) and poly(sulfobetaine) (PSB),
have been applied to a broad range of biomedical and
engineering materials. Due to the electrostatically induced
hydration, surfaces modified by (via conjugation or coating)
zwitterionic polymers are highly resistant to nonspecific protein
adsorption, bacterial adhesion, and biofilm formation. For
examples, the pioneering studies performed by Ishihara et al.
1
2−14
substituents to yield more effective carriers,
PEGylation
or hydroxylation to improve the serum stability during
1
5−17
circulation,
biocompatibility,
PEI to reduce cytotoxicities,
functional molecules to target specific cells or tissues.
linking to polysaccharides to enhance
1
8,19
cross-linking of low molecular weight
2
0,21
and attachment of some
2
2−24
Received: November 27, 2012
Revised: February 1, 2013
Published: February 5, 2013
Among the PEI modification approaches, PEGylation is a
commonly applied conjugation method that affords the
©
2013 American Chemical Society
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have shown that the MPC polymers exhibit excellent
biocompatibility based on inhibition of protein adsorption
and activation on the surface, and the MPC-based polymers can
been applied for surface coating of many medical devices
ring-opening reaction of 1,3-propanesultone with N,N-dimethylami-
nopropylacrylamide (DMAPAA) in anhydrous acetone at ambient
5
1
temperature. DMAPAA (4.0 g, 25.6 mmol) and acetone (15 mL)
were charged into a 50 mL dried round-bottom flask. 1,3-Propane-
sultone (3.0 g, 24.6 mmol) solution in 15 mL of anhydrous acetone
was added dropwise into the flask in 0.5 h and stirring was proceeded
for 16 h at the same temperature. The resulting white precipitate was
39−41
including implantable artificial organs.
On the other hand,
42−47
Jiang and co-workers
have ingeniously developed a series
of betaine-based zwitterionic polymers that are more resistant
to nonspecific adsorption of proteins and can provide stability,
prolonged circulation, reduced degradation, and increased
water solubility when covalently attached to a protein. As for
filtered, washed with acetone, and dried under vacuum, yielding a
1
white powder, 92% (6.29 g, 22.6 mmol). H NMR (δ ppm, in D O,
2
Figure S1): 2.07 (m, 2H), 2.22 (m, 2H), 2.99 (t, J = 8 Hz, 2H), 3.12
(m, 6H), 3.40 (m, 4H), 3.49 (t, J = 8 Hz, 2H), 5.80 (d, J = 12.0 Hz,
1H), 6.19−6.52 (m, 2H). In mass spectrum (Figure S2), mass peaks at
48
the application of poly(zwitterion) in gene delivery, Liu et al.
+
synthesized a novel polysulfobetaine-methacrylate copolymer,
they found the incorporation of polysulfobetaine into the
polymer chain retained a good DNA condensation capability
and a high gene transfection efficacy comparable to PEI with
serum.
m/z 278.9 corresponds to [DMAAPS + H] .
Synthesis of DMAAPS-Modified PEI (PEI-DMAAPS). In a
typical run, to a reaction flask containing 25 kDa PEI (450 mg, 0.018
mmol) in 12 mL of methanol under a nitrogen atmosphere, DMAAPS
(
436 mg, 1.6 mmol for the graft ratio of 55%) was added. The reaction
mixture was stirred at room temperature for 3 days and then dialyzed
MW cutoff: 3500) for 48 h against distilled water. The PEI-DMAAPS
The key difference between PEG and a poly(zwitterion) is
the surface properties associated with each polymer. PEG
interacts with water through hydrogen bonding leading to a
highly viscous, amphiphilic coating, whereas poly(zwitterion)s
associate with water through strong ionic interactions, creating
(
conjugate was collected after freeze-drying; the final yields 56% (476
mg, 0.01 mmol).
Characterization. H NMR spectra of the samples was recorded
1
on a Bruker Avance 400 MHz NMR spectrometer using D O as the
2
2
5,49,50
a superhydrophilic surface.
In consideration of the superb
solvents at 25 °C. The absorbance measurements were carried out
using a Hitachi U-3010 UV−vis spectrophotometer. The particle size
was determined by DLS using a Malvern Nano-ZS90 Zetasizer. The
cell imaging was performed on an Olympus IX71 inverted fluorescence
microscope equipped with a DP72 color CCD (excitation: 460−490
nm). The gene transfection efficiency was evaluated by scoring the
percentage of cells expressing EGFP using a Beckman Coulter Epics
XL flow cytometer with EXPO32 ADC software (Beckman Coulter,
U.S.A.).
surface properties brought by the zwitterion polymers, we
envision that the zwitterion monomers, if properly coated onto
the surfaces of PEI, the commonly used gene delivery vector,
may also create a superb surface for the vector and afford the
latter enhanced gene delivery properties along with good
biocompatibility. The conjugation of zwitterion monomer with
PEI is technically much simpler and more efficient than the
reaction of zwitterion polymer with PEI; thereby, conjugating
zwitterion monomer with PEI might become a technically
simple and highly efficient method for constructing biocompat-
ible and high-performance gene delivery vector.
Cell Culture. Two cell lines (HeLa and HepG2) were cultured in
DMEM supplemented with 10% FBS (penicillin/streptomycin 100 U/
mL). All cells were incubated at 37 °C in a humidified 5% CO
2
atmosphere.
Buffering Capacity. The buffering capacity of PEI-DMAAPS
samples were determined by acid−base titration assay. Briefly, 0.2 mg/
mL of each sample solution was prepared in 30 mL of 150 mM NaCl
solution. The pH was adjusted to 10 by using a 0.1 M NaOH solution.
The polymer solution was then titrated with 0.1 M HCl at room
temperature. The pH values for all solutions were measured using a
microprocessor pH meter (Sartorius PB-10).
With this idea in mind, in this study, we demonstrate an
alternative and facile strategy to significantly improve the
overall gene delivery properties of PEI (25 kDa) by coating a
large amount of betaine monomers (not its polymer) to PEI
through a Michael addition reaction. This betaine-monomer-
modified PEI exhibits extraordinary gene delivery efficacy, low
cytotoxicity and protein absorption, excellent anticoagulant
property, and excellent serum tolerance; in addition, for this
betaine-monomer-modified PEI, the overall performance
concerning gene transfection is outstanding among various
PEI derivatives. More importantly, this simple and safe
approach is highly reproducible, which is essential for the
practical applications of manufactured biomaterials.
Hemolysis Test. The hemolysis test was adapted from Standard
Practice for Assessment of Hemolytic Properties of Materials (ASTM
designation: F 756-08). The total blood was freshly collected from
three rabbits and mixed. Determination of total blood hemoglobin was
accomplished by using cyanmethemoglobin reagent (Van Kampen and
Zijlstar Solution) and measuring the absorbance of free hemoglobin in
solution at 540 nm. The total blood hemoglobin concentration was
used to adjust the hemoglobin content of the blood sample to 10 ± 1
mg/mL by adding blood and an appropriate amount of 1× PBS
solution. Rabbit blood, 1 mL, was incubated with 50 μL polymer or
complex solution in PBS at 37 °C for 3 h under gentle shaking. After 3
h, samples were centrifuged at 750 g for 15 min. Supernatant was
collected, added 1:1 to equivalent volume of cyanmethemoglobin
reagent and incubated for 5 min before measurement. Absorbance
measurements at 540 nm were then recorded using a Hitachi 3010
spectrophotometer. Triton X-100 and PBS were used as positive and
negative controls, respectively. Total hemoglobin released from whole
blood diluted in the cyanmethemoglobin reagent was determined as
EXPERIMENTAL SECTION
■
9
Materials. N-[3-(Dimethylamino)propyl] acrylamide (DMAPAA,
8%) and 1,3-propanesultone were purchased from TCI Shanghai.
The Dulbecco’s modified Eagle’s medium (DMEM), trypsin-ethyl-
enediaminetetraacetic acid (Trypsin-EDTA), penicillin−streptomycin,
and fetal bovine serum (FBS) were obtained from Invitrogen. A 25
kDa branched polyethylenimine (PEI, 25 kDa, primary amine content
∼25% in mole), ethidium bromide (EB), and 3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from
Sigma. The plasmid DNA [EGFP-N1 (4.7Kb) encoding enhanced
green fluorescent protein] was purchased from Clontech (Palo Alto,
CA) and stored at −20 °C until the transfection experiments. All other
reagents were of analytical grade and used as received. Van Kampen
and Zijlstar solution was purchased from Shanghai Rongbai Biological
Technology Co. Ltd.
1
00% hemoglobin release. Hemolysis was expressed as the percentage
of hemoglobin released to total content. Tests were done in triplicate.
Protein BSA Adsorption Assay. A total of 1 mL of polymer
solution (1 mg/mL) and 1 mL of BSA solution (2 mg/mL) were
mixed and shaken at 37 °C for 0.5 h. The mixtures were centrifuged
and the supernatants were collected carefully for obtaining the
characteristic UV absorbance at 280 nm. The BSA concentrations in
supernatants were determined using a calibration curve obtained from
Synthesis of N,N-Dimethyl(acrylamidopropyl)ammonium
Propane Sulfonate (DMAAPS). DMAAPS was synthesized by the
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Scheme 1. Synthesis of PEI-DMAAPS Conjugate
BSA solutions of known concentrations. The protein adsorption values
A are defined as
volumes were made up to 20 μL with 6× loading buffer containing
xylene cyanol dye. The stability of the complexes was analyzed on
0
.8% agarose gel in Tris-acetate-EDTA (TAE) buffer solution.
C V − C V
i
i
s s
A =
Electrophoresis was carried out at 100 V for 60 min in a Biorad
electrophoresis system (Biorad Laboratories, Hercules, CA, U.S.A.).
The DNA bands on the gels were visualized and photographed upon
ethidium bromide staining using a Gel Doc XR+ Imaging System
m
where C and C are the initial BSA concentration (2 mg/mL) and the
i
s
BSA concentration in the supernatant determined by adsorption
experiments, respectively; V is the initial volume of the BSA solution
1 mL); Vs is the total volume of the BSA after adsorption
(Biorad Laboratories, Hercules, CA).
i
Particle Size and Zeta Potentials Measurements. The
(
prepared complex solutions were diluted in 10 mM NaCl or 150
mM NaCl aqueous solution. The mean hydrodynamic diameter and
polydispersity of the complexes were determined by photon
correlation spectroscopy via dynamic light scattering measurement
using a Malvern NanoZS90 Zetasizer (Malvern Instruments, UK).
Measurements were performed at a temperature of 25 °C and at a
detection angle of 90° and with a He−Ne 633 nm laser as the light
source. The instrument was routinely calibrated using standard
reference latex particles (Malvern Instruments, U.K.). The particle
size of each sample was expressed as the mean hydrodynamic particle
diameter ± standard deviation of three measurements. Surface charge
of polymer/DNA complexes was also measured at 25 °C with the
same instrument after calibration with a polystyrene dispersion
(Malvern Instruments, UK) of known zeta potential. Given values
were the average of at least five runs. A PSS0012 Marvern Instruments’
DTS Application Software running on Windows environment was
used for the data analysis.
measurement (2 mL); and m is the weight of the polymer (1 mg)
added into the solution.
Cytotoxicity Assay. The cytotoxicities of the polymer vectors
were examined by MTT assay and 25 kDa PEI (PEI 25K) was used as
the positive control. For the assays, HeLa and HepG2 were seeded
4
into a 96-well plate at a density of 1 × 10 cells per well, respectively.
After 24 h of incubation at 37 °C, cells were washed with prewarmed
PBS buffer. Then, the cells were treated with PEI-DMAAPS conjugates
or PEI 25K dissolved in media. After 48 h of incubation, the plates
were washed with PBS buffer and incubated for another 4 h with
RPMI 1640 medium containing 0.5 mg/mL MTT. After discarding the
culture medium, 150 μL of DMSO was added to dissolve the
precipitates and the absorbance was recorded with a Thermo MK3
ELISA reader at 570 nm and the statistical mean and standard
deviation were used to estimate the cell viability. Untreated cells served
as control with 100% viability, and wells having MTT reagent only
(
without cells) were used as blank to calibrate the spectrophotometer
to zero absorbance.
In Vitro Transfection. HeLa and HepG2 cells were seeded in 24-
4
Preparation of Polymer/pDNA Complexes. All polymer/
pDNA complexes were freshly prepared before use. Complexes at
different N/P ratios were prepared by slowly adding appropriate
volume of polymer solution (1.0 mg/mL in 150 mM NaCl solution)
into 2 μL of plasmid EGFP-N1 DNA (1.0 μg/μL in pure water) with
gentle vortexing and incubated for 30 min at room temperature to
ensure effective formation of polymer/pDNA complexes. The N/P
ratio can be calculated using the following formula:
well plates at 5 × 10 cells/well and were incubated for 12 h. pEGFP
was complexed with PEI-DMAAPS samples (of varied DMAAPS graft
ratios) at N/P 15, 30, and 60, or with 25 kDa PEI (the control) at N/P
10. The medium was removed from each well and the cells were
washed once with 1× PBS (200 μL). Thereafter, the medium in each
well was replaced with serum-free media or fetal bovine serum media.
The complexes of PEI-DMAAPS/pDNA at different N/P ratios (with
2
μg pDNA in each sample) were incubated for 6 h. The transfection
medium was aspirated, replaced with 600 μL growth medium (DMEM
with 10% FBS), and the cells were further incubated for 42 h. The
transfected cells were washed once with PBS and detached with 0.25%
trypsin. Transfection efficiency was evaluated by scoring the
percentage of cells expressing pEGFP with the flow cytometer.
Moreover, the transfected cells were imaged using the fluorescence
microscope.
N/P ratio = M/(Q × 3)
where M represents the nitrogen atoms of PEI in the carriers (nmol)
and Q equals the quantity of DNA (μg).
Agarose Gel Electrophoresis. The efficiency of DNA con-
densation was assessed by agarose gel electrophoresis. The solutions of
complexes with varied N/P ratios were prepared and their final
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Statistical Analysis. All data are summarized as mean ± standard
deviation (SD). Significance in difference was assessed using GraphPad
Prism 5.0 (t tests).
RESULTS AND DISCUSSION
Synthesis of PEI-DMAAPS Conjugates. First, a vinyl-
containing betaine compound N, N-dimethyl-
■
(
acrylamidopropyl)ammonium propane sulfonate (DMAAPS,
namely the betaine monomer) was synthesized and charac-
terized, as shown in Scheme 1 and Figure S1, and three PEI-
DMAAPS conjugates with varied graft ratios were then
obtained through Michael addition reaction between DMAAPS
and PEI 25K in methanol. The unconjugated DMAAPS were
readily removed by dialysis. The mole percentages of DMAAPS
relative to the amino groups of PEI in the feed were set at 30,
Figure 2. Protein adsorption for PEI-DMAAPS conjugates and PEI 25
K. ***P < 0.001 vs PEI 25K. Two-tailed Student’s t-test (n = 3, error
bar represents SD).
1
6
0, and 100%, respectively. H NMR spectra for the three
1
samples were given in Figure 1. Based on the H NMR spectra,
level, about half of that for PEI 25K. The results also indicate
that the reduction of protein adsorption on the PEI-DMAAPS
conjugates was associated with the betaine monomer
(
DMAAPS) moieties on the PEI. Similarly, as shown in Figure
S5, the complexes formed by PEI-DMAAPS derivatives and the
pDNA exhibit much lower protein absorption than that formed
by PEI 25K and pDNA. Previously, the betaine polymers have
been shown to exhibit ultrahigh resistance to nonspecific
protein adsorption; while this result demonstrates that the
small molecular betaine, after being coated on PEI surfaces, also
displays resistance to protein adsorption.
52
Hemolysis Test for PEI-DMAAPS. The red blood cell
RBC) hemolysis assay was performed to evaluate the blood
(
compatibility of PEI-DMAAPS conjugates. The compatibility of
any gene delivery vector with blood indicates its suitability for
introduction into the systemic circulation. The hemolysis tests
were performed following ASTM F756-08. The materials are
usually considered with little to no hemolytic reaction when the
1
Figure 1. H NMR spectra (in D O) of PEI-DMAAPS conjugates with
2
varied graft ratios.
the graft ratio (mole percentage of DMAAPS relative to the
total amino groups in PEI 25K before the Michael addition
reaction) for the resulting conjugates were estimated to be 23,
5, and 95%, respectively, by calculating the ratio between the
integral area of the peaks for DMAAPS moieties and that of PEI
Figures S2−S4). The three samples are hereinafter labeled as
PEI-DMAAPS23%, PEI-DMAAPS and PEI-DMAAPS . In
this study, it was found that the Michael addition readily took
place and the graft ratio could be easily controlled, this ensures
repetitiveness of the facile approach.
Protein BSA Adsorption of PEI-DMAAPS Conjugates.
Proteins adsorbed onto the surface of biomaterials can trigger
thrombosis and blood coagulation, leading to life-threatening
situations or causing functional failure when biomaterials are
used in artificial organs, blood vessels, and other medical
devices in contact with blood. In system circulation, serum
proteins could bind to polymer/pDNA complexes in a
nonspecific manner, nonspecific protein adsorption is the first
interaction event that occurs at the interface between polymers
and human blood. We determined the BSA adsorption by the
polymer (Figure 2) and the polymer/pDNA complex (Figure
S5).
53
hemolysis rate was lower than 5%. Figure 3 shows the
hemolysis test results of the polymers (pristine PEI 25K and
the betaine monomer-modified PEIs) and the polymer/pDNA
complexes at two different concentrations. As shown in Figure
3A, the PEI-DMAAPS polymers exhibit much lower hemolysis
degrees than PEI 25K. At the polymer concentration of 300
and 500 μg/mL, PEI 25K displays a hemolysis rate of 6 and
17%, respectively, in contrast, all the PEI-DMAAPS polymers
exhibit hemolysis rates less than 3% at both concentrations. On
the other hand, the hemolysis tests were also conducted for
different polymer/pDNA complexes, and the PEI-DMAAPS/
pDNA complexes also display much lower hemolysis rates than
that of PEI 25K/pDNA complex, as shown in Figure 3B.
The high hemolytic damage of PEI could be owing to the
high charge density, which cause excessively strong interaction
with membrane proteins and phospholipids and thus disturbs
5
(
55%
95%
17
membrane structure and function. After surface modification
of PEI with DMAAPS, excessive amino groups were substituted
by DMAAPS, and these zwitterionic molecules on the polymer
surfaces can help to avoid the disruption of blood cell
membranes.
As shown in Figure 2, the BSA adsorption of PEI 25K is
much higher than PEI-DMAAPS derivatives. The absorption
was induced mainly by electrostatic interactions between
negatively charged BSA and the positively charged polymer,
leading to protein BSA adsorption. The protein adsorption
values for the three PEI-DMAAPS conjugates were at similar
In Vitro Cytotoxicity of PEI-Betaine Conjugates. Before
carrying out the transfection experiments, it is important to
evaluate the cell viability properties of the modified PEI gene
vector. The cytotoxicity of PEI-DMAAPS conjugates and PEI
25K in the concentration range from 10 to 150 mg/L were
evaluated in HeLa and HepG2 cell lines by MTT assays, as
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Figure 4. Cell viability profile for PEI-DMAAPS conjugates, PEI 25K
and betaine monomer (DMAAPS) in HeLa (A) and HepG2 (B) cells.
Percent viability of cells is expressed relative to control cells. **P <
.01, ***P < 0.001 vs PEI 25K. Two-tailed Student’s t-test (n = 3,
error bar represents SD).
Figure 3. Hemolysis test results for the polymers (A) and polymer/
pDNA complexes at N/P = 60 (B) at the polymer concentration of
0
300 or 500 μg/mL. The tests were performed following ASTM F756-
0
8. **P < 0.01, ***P < 0.001 vs PEI 25K. Two-tailed Student’s t-test
(n = 3, error bar represents SD).
zwitterionic polymers and highly resistant to nonspecific
protein adsorption, and this may contribute to the enhanced
biocompatibility and higher cell viability of the vector materials.
By comparing Figure 4A and B, we can find that the
polymers exhibit different cytotoxicities toward HeLa or
HepG2, we suppose the different cellular characteristics may
cause the different cytotoxicities.
shown in Figure 4. As anticipated, the highest cytotoxicity was
seen for PEI 25K, significant toxicity was observed when its
concentration was higher than 25 mg/L. In this case, the cell
viability for PEI was found to be lower than 20% for HeLa cell
line and 40% for HepG2 cell line as the concentration of PEI is
above 20 mg/L. On the other hand, the PEI-DMAAPS
conjugates exhibit much lower and graft-degree-dependent
cytotoxicity, and the betaine monomer DMAAPS exhibits no
cytotoxicity, as shown in Figure 4. The cytotoxicity of the
conjugates decreases with increasing graft degree. This result
agrees well with the accepted opinion that the cell
biocompatibility has a strong association with the charge
density on polycation surface. It is believed that the positive
charges facilitate endocytosis through negatively charged cell
surface, but such interactions also cause significant cell death
due to the aggregation of these polymers on the cell surface
impairing the important membrane function like protein kinase
Buffering Capacity of PEI-DMAAPS Conjugates.
Buffering capacity of the polymer is one of the important
properties that enables it to buffer the endosomes and helps in
the rupture and subsequent release of the genetic material into
the cytosol. Acid−base titration experiments were carried out
for PEI 25K and PEI-DMAAPS conjugates to evaluate the
buffering capacity of the new polymer. As shown in Figure S7,
PEI-DMAAPS conjugates exhibit lower buffering capacities
than PEI 25K, and with the increasing number of betaine
moieties on the polymer, the buffering capacity decreases. For
the three PEI-betaine conjugates, with increasing degree of
graft, the number of amines decreases and fewer amines are
available for charge neutralization. Interestingly, although the
efficient buffer capacity was important for endosomal release of
pDNA into the cytoplasm, previously published results also
indicated proper reduction of PEI buffering capacity sometimes
5
4,55
activity.
Moreover, we also determined the cytotoxicities
for the complexes formed by pDNA and PEI-betaine
conjugates or PEI 25K, as shown in Figure S6, which indicates
that the complexes formed by using PEI-betaine conjugates
exhibit lower cytotoxicity.
The conjugation of the betaine molecules on the PEI chains
can substantially alleviate the cytotoxicity, and we suppose the
betaine moieties have 2-fold role in relation to the cytotoxicity.
First, the conjugation of betaine can reduced the cation density
on the polymer surface, thereby lessening the damage to cell
membrane. Second, the betaine monomers, when uniformly
distributed on the polymer surfaces, are equivalent to
56
may be good for in vitro gene delivery activity.
Complex Formation of PEI-DMAAPS with pEGFP. To
examine the stability of the PEI-DMAAPS/pEGFP complexes,
the formation of the complexes was examined by their
electrophoretic mobility on an agarose gel. As shown in Figure
5, the three conjugates can efficiently condense pDNA and fully
retard the movement of pDNA along the gels. Moreover, the
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Figure 5. Agarose gel electrophoresis retardation assay of PEI-DMAAPS conjugates/pEGFP and PEI 25K/pEGFP at varied N/P ratios.
three complexes exhibit similar electrophoretic migration
patterns as a function of the N/P ratio, which refers to the
number of nitrogen atoms in PEI of the carrier per DNA
phosphate. For the conjugates, the complete retardation of
DNA was achieved at N/P ratios of 1.5, while for PEI 25K, the
ratio is 2. This result suggests that these betaine-modified PEI
polymers have satisfactory capacity to form stable complexes
with pEGFP. As can be seen from Scheme 1, the coupling of
each betaine monomer onto the PEI chain turns one primary
amino group into a tertiary amino group and introduces a
cationic quaternary amine and a negative sulfonic group. The
48
cationic quaternary amine group can also complex with DNA,
hence, PEI-DMAAPS conjugates still retain a better DNA
condensation capability, though the incorporation of betaine
molecules forms a barrier exerted by sulfo-anions on the PEI’s
surface. Moreover, for gene delivery, tertiary amino groups are
highly desirable because they have pKa values in the
physiological range and can be engineered into delivery
57
vehicles to buffer endosomes. In this experiment, we added
xylene cyanol into the gel as a color marker to monitor the
process of agarose gel electrophoresis, and we found the
addition of the dye did not cause obvious interference for the
result, as shown in Figure S8.
Size and Zeta Potential Measurements. The size and
zeta potential of the polymer/pDNA complexes in 10 mM
NaCl solution was determined by DLS method using a Malvern
Nano-ZS90 Zetasizer. The averaged sizes and zeta potentials as
a function of N/P ratio are displayed in Figure 6, while the size
distribution curves and the size polydispersity indices (PDI) are
displayed in Figure S9. The mean diameters of the complexes
determined in 10 mM NaCl solution were found to be in a
range of 220−500 nm, and for the complexes formed by pDNA
and PEI-betaine conjugates at higher N/P ratios (15, 30, and
Figure 6. Particle size (A) and zeta potential (B) for PEI-DMAAPS
conjugates/pDNA and PEI 25K/pDNA at varied N/P ratios (n = 3,
error bar represents SD).
6
0), the diameters are in the range of 260 to 380 nm,
complexes exhibit much higher positive zeta potentials (30−40
mV) at N/P = 3, and with increasing N/P ratio, zeta potentials
slowly increase and reach plateaus about 60 mV at N/P ratios
between 15 and 60. The high zeta potential for PEI-DMAAPS/
pDNA complexes at N/P = 3 is not quite clear at present; we
suppose, as the strong electrolyte cation, the quaternary
ammonium in the betaine moieties may contribute more
greatly to the positive zeta potential than the amines (weak
electrolyte cation) in PEI chains. The high positive zeta
potentials for the PEI-DMAAPS/pDNA complexes are
beneficial to their attachment to anionic cell surface and
suggesting that these complexes may be used for gene delivery.
In addition, as can be seen from Figure S9, the calculated PDI
for the complexes determined in 10 mM NaCl solution are
generally rather high, ranging from 0.30 to 0.65. In general, the
complexes with lower N/P ratios (N/P = 3 and 7.5) exhibit
relatively low PDI.
For surface charge measurement in 10 mM NaCl water
solutions, PEI 25K/pDNA complexes exhibit a slightly positive
zeta potential (+4 mV) at N/P = 3 and much higher positive
ones at higher N/P ratios. This result is largely consistent with
58
27
the literature reports. In contrast, the PEI-DMAAPS/pDNA
endocytosis, thus, improving the gene transfection.
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For comparison, we also measured the particle sizes and zeta
potentials in 150 mM NaCl water solution for the complexes
formed by pDNA and two betaine-modified PEIs (PEI-
DMAAPS23% and PEI-DMAAPS55%), and the results, together
with those measured in 10 mM NaCl solution, are listed in
Table S1. The particle sizes in 150 mM NaCl are slightly larger
than those in 10 mM NaCl. Hypothetically, complexes in 10
mM NaCl solution are more compact and smaller, because
there are fewer free ions in solution that can interact in complex
formation. In contrast, the zeta potentials in 150 mM NaCl are
lower than those in 10 mM NaCl, because the higher ionic
strength in solution impedes the ionizing of the amines more
greatly. These trends are in accordance with the literature
cells expressing EGFP at different N/P ratios using PEI-betaine
conjugates and PEI 25K. Interestingly, the complexes based on
PEI-DMAAPS23% and PEI-DMAAPS55% give remarkable levels
of transfection; their transfection efficiencies are substantially
higher than that of PEI 25K. Specifically, for the N/P ratios we
tested, both PEI-DMAAPS23%/pEGFP and PEI-DMAAPS55%
/
pEGFP complexes exhibit their maximum efficiency values at a
N/P ratio of 60. Up to ∼56% transfection efficiency was
achieved by the two complexes, which was statistically
significantly higher than the 21% transfection efficiency
obtained from PEI 25K in the absence of serum, at its optimal
N/P ratio of 10. In Figure 7B, the comparison of transfection
efficiency for PEI-betaine conjugates and PEI 25K is also
presented. Similarly, the transfection efficiencies for the
conjugates are also much higher than that for PEI 25K. In
addition, we found the sample PEI-DMAAPS95% displayed very
poor gene transfection efficiencies of ∼1% (with some typical
flow cytometry profiles and fluorescence images shown in
Figure S10), and this may be due to its poor buffering capacity
caused by the overly high graft ratio of betaine monomers on its
surface.
One of the major problems of the cationic vector-mediated
gene delivery is that the transfection is prominently inhibited in
the presence of serum. The study of gene vectors concerning
serum-conditioned transfection can serve as a fundamental
predictive model for their in vivo efficiency evaluation. In this
context, we investigated the effects of serum on the transfection
efficiency of the complexes based on PEI-DMAAPS23% and
PEI-DMAAPS55%, as revealed in Figures 7 and S11. Compared
to PEI 25K, the PEI-betaine conjugates exhibit much higher
transfection efficiencies in the presence of high serum
concentrations. In particular, for the pristine PEI 25K, its
transfection efficiency decreased drastically in 10% serum
compared to that in the absence of serum, while for the two
PEI-DMAAPS vectors, no significant difference could be
observed between the efficiencies in the presence and absence
of 10% serum, as shown in Figure 7. As revealed in Figure S11,
the pristine PEI 25 K exhibits very low transfection efficiency
5
9
report.
In Vitro Transfection. The in vitro transfection of PEI-
DMAAPS conjugates and PEI 25K were assessed in HeLa and
HepG2 cells using pEGFP as a reporter gene. Polymer/pDNA
complexes prepared at N/P ratios of 15:1, 30:1, and 60:1 were
selected for transfection studies; PEI 25K/pEGFP at its optimal
6
0,61
N/P ratio of 10:1 was chosen as the positive control.
The
transfection experiments were performed using different media,
that is, in the presence or absence of 10.0% serum. The
transfection efficiency of PEI-DMAAPS conjugates were
compared with that of PEI 25K.
The transfection efficiency of pEGFP in HeLa and HepG2
was quantified using flow cytometry in terms of cell population
expressing EGFP. Figure 7A shows the percentage of the HeLa
(
3
less than 5%) in 30 and 50% serum. On the other hand, at
0% serum, there are only ∼32% and ∼10% decreases in
transfection efficiency for PEI-DMAAPS2 and PEI-
3%
DMAAPS55%, respectively. In the case of 50% serum, in spite
of a decline in transfection level, PEI-DMAAPS23% and PEI-
DMAAPS55% still show 6 and 10 times higher transfection
efficiency than pristine PEI 25 K. These results clearly indicate
that the incorporation of DMAAPS onto PEI can significantly
improve serum-tolerant transfection activity for the polycation
vectors, suggesting that PEI-betaine conjugates hold a
promising potential in vivo application.
To visualize the transfection effect, the EGFP expression in
HeLa and HepG2 cells was observed with an inverted
fluorescent microscope. Figure 8 gives some typical fluo-
rescence images of HeLa cells transfected by PEI-DMAAPS23%
/
pEGFP, PEI-DMAAPS55%/pEGFP, and PEI 25K/pEGFP
complexes. The expression could not be detected when the
transfection was mediated by naked pEGFP, which was used as
a negative control (data not shown). As shown in Figure 8,
fluorescence images could be observed when the transfections
were mediated by PEI 25K; however, the cells mediated by
PEI-DMAAPS23% and PEI-DMAAPS55% show more bright
green fluorescent spots than that by PEI 25K. It appears that
the amount of green cells mediated by PEI-DMAAPS23% and
PEI-DMAAPS55% were similar. Furthermore, it can be seen
Figure 7. Percent transfection efficiency of PEI-DMAAPS/pEGFP
complexes and PEI 25K/pEGFP complexes in HeLa (A) and HepG2
(
B) at three N/P ratios with and without 10% serum. *P < 0.05, **P <
0.01, ***P < 0.001 vs PEI 25K (n = 3, error bar represents SD).
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Biomacromolecules
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ASSOCIATED CONTENT
Supporting Information
H NMR spectrum and mass spectrum for the synthesized
■
*
S
1
DMAAPS, PEI-DMAAPS23%, PEI-DMAAPS55%, and PEI-
DMAAPS95%. Protein adsorption for the complexes. Cell
viability profile for the complexes. Buffering capacity of PEI
2
5K and three PEI-betaine conjugates determined by perform-
ing acid−base titration from pH 10 to pH 3. Comparison of
agarose gel electrophoresis retardation assay for PEI-
DMAAPS55%/pEGFP complex with or without addition of
xylene cyanol dye. Diameter distribution for PEI/pEGFP and
three PEI-betaine/pEGFP polyplexes as determined by DLS.
Flow cytometry profiles and fluorescence images of PEI-
DMAAPS95%/pEGFP complex in HeLa at three N/P ratios.
Percent transfection efficiency for PEI 25K (at N/P = 10) and
two PEI-DMAAPS/pEGFP complexes in HeLa at N/P = 30
with 30% and 50% serum. Typical flow cytometry profiles for
pEGFP transfection in HeLa and HepG2 cell lines mediated by
PEI 25K, PEI-DMAAPS23% and PEI-DMAAPS55%. And size,
size polydispersity (PDI), and zeta potential measurement of
typical PEI-DMAAPS/pDNA polyplexes. This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 8. Fluorescence microscope images for HeLa and HepG2 cells
treated with PEI 25K/pEGFP, PEI-DMAAPS23%/pEGFP and PEI-
DMAAPS55%/pEGFP. The corresponding flow cytometry profiles for
the images are listed in Figure S9.
AUTHOR INFORMATION
Corresponding Author
Tel.: +86-20-22236262. Fax: +86-20-22236363. E-mail:
mcfzeng@scut.edu.cn; shzhwu@scut.edu.cn.
■
*
from the figure that the fluorescence was unaffected by the
presence of 10% serum for PEI-DMAAPS23% and PEI-
DMAAPS55%, except for a couple of observations for HepG2
cell line. The results presented in Figure 8 were largely in
accordance with the flow cytometry results (Figure S12), and
this gives additional evidence that betaine-modified PEI was
more effective as a gene carrier than PEI 25K.
The enhanced gene transfection efficiency of the two PEI-
DMAAPS samples, we suppose, is due to the improvement of
the surface properties of the vector as well as the PEI-
DMAAPS/pDNA complex. Unlike existing polybetaines, the
PEI-betaine conjugate of this study was obtained by “coating” a
layer of betaine monomer onto PEI. This strategy ensures
higher packing density of sulfobetaine groups on the PEI
surface and, eventually, significantly reduces the protein
adsorption and hemolytic reaction, as well as cytotoxicity, and
this is beneficial to the gene transfection. Moreover, similar to
phosphorylcholine headgroups, which are found in the outside
layer of cell membranes, the zwitterionic sulfobetaine groups
are also supposed to enhance cell binding and possibly
contribute to the endocytosis of vector/DNA complexes.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(
We gratefully acknowledge financial support from NSFC
Project No. 21025415, 21174040) and the National Key
Basic Research Program of China.
REFERENCES
■
(
1) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.;
Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A.
1
(
995, 92, 7297−7301.
2) Arote, R. B.; Lee, E. S.; Jiang, H. L.; Kim, Y. K.; Choi, Y. J.; Cho,
M. H.; Cho, C. S. Bioconjugate Chem. 2009, 20, 2231−2241.
3) Petersen, H.; Kunath, K.; Martin, A. L.; Stolnik, S.; Roberts, C. J.;
(
Davies, M. C.; Kissel, T. Biomacromolecules 2002, 3, 926−936.
(4) Zhong, Z. Y.; Feijen, J.; Lok, M. C.; Hennink, W. E.; Christensen,
L. V.; Yockman, J. W.; Kim, Y. H.; Kim, S. W. Biomacromolecules 2005,
6, 3440−3448.
(
5) Zintchenko, A.; Philipp, A.; Dehshahri, A.; Wagner, E.
Bioconjugate Chem. 2008, 19, 1448−1455.
6) Liu, L.; Zheng, M. Y.; Renette, T.; Kissel, T. Bioconjugate Chem.
012, 23, 1211−1220.
7) Park, J. W.; Bae, K. H.; Kim, C.; Park, T. G. Biomacromolecules
CONCLUSIONS
■
(
2
(
In summary, we have developed a facile and effective approach
to significantly improve the gene delivery performance of PEI.
By incorporating the sulfobetaine monomers onto PEI chains
via a simple Michael addition chemistry, we obtained a new
PEI-based gene vector featuring low protein adsorption, low
cytotoxicity, and high gene transfection efficiency. In addition,
the vectors display a satisfactory serum-tolerant property. In
this study, incorporating betaine monomer (not polybetaine)
onto PEI may ensure higher packing density of sulfobetaine
groups on the PEI surface. This approach is of low-cost and
facile in terms of synthesis and is much preferred in biological
applications. We suppose this new surface modification strategy
may also be suitable for fabricating some other materials with
enhanced biocompatibility.
2011, 12, 457−465.
(8) Suh, J.; Paik, H. J.; Hwang, B. K. Bioorg. Chem. 1994, 22, 318−
327.
(
9) Behr, J. P. Chimia 1997, 51, 34−36.
(10) Funhoff, A. M.; van Nostrum, C. F.; Lok, M. C.; Fretz, M. M.;
Crommelin, D. J. A.; Hennink, W. E. Bioconjugate Chem. 2004, 15,
212−1220.
11) Li, S.; Tseng, W. C.; Stolz, D. B.; Wu, S. P.; Watkins, S. C.;
Huang, L. Gene Ther. 1999, 6, 585−594.
12) Nimesh, S.; Aggarwal, A.; Kumar, P.; Singh, Y.; Gupta, K. C.;
1
(
(
Chandra, R. Int. J. Pharm. 2007, 337, 265−274.
(13) Doody, A. M.; Korley, J. N.; Dang, K. P.; Zawaneh, P. N.;
Putnam, D. J. Controlled Release 2006, 116, 227−237.
7
35
dx.doi.org/10.1021/bm301826m | Biomacromolecules 2013, 14, 728−736

Biomacromolecules
Article
(
14) Gabrielson, N. P.; Pack, D. W. Biomacromolecules 2006, 7,
427−2435.
15) Li, D.; Ping, Y.; Xu, F. J.; Yu, H.; Pan, H. M.; Huang, H. L.;
Wang, Q. Q.; Tang, G. P.; Li, J. Biomacromolecules 2010, 11, 2221−
229.
16) Zheng, M.; Zhong, Z. H.; Zhou, L.; Meng, F. H.; Peng, R.;
Zhong, Z. Y. Biomacromolecules 2012, 13, 881−888.
17) Luo, X. H.; Huang, F. W.; Qin, S. Y.; Wang, H. F.; Feng, J.;
Zhang, X. Z.; Zhuo, R. X. Biomaterials 2011, 32, 9925−9939.
18) Srinivasachari, S.; Fichter, K. M.; Reineke, T. M. J. Am. Chem.
Soc. 2008, 130, 4618−4627.
19) Lu, B.; Xu, X. D.; Zhang, X. Z.; Cheng, S. X.; Zhuo, R. X.
Biomacromolecules 2008, 9, 2594−2600.
20) Goyal, R.; Tripathi, S. K.; Vazquez, E.; Kumar, P.; Gupta, K. C.
Biomacromolecules 2012, 13, 73−83.
21) Liu, J.; Jiang, X. L.; Xu, L.; Wang, X. M.; Hennink, W. E.; Zhuo,
R. X. Bioconjugate Chem. 2010, 21, 1827−1835.
22) Park, K.; Lee, M. Y.; Kim, K. S.; Hahn, S. K. Biomaterials 2010,
1, 5258−5265.
23) Jiang, G.; Park, K.; Kim, J.; Kim, K. S.; Hahn, S. K. Mol.
Pharmaceutics 2009, 6, 727−737.
24) Wang, C. H.; Hsiue, G. H. Bioconjugate Chem. 2005, 16, 391−
96.
(47) Huang, C. J.; Li, Y. T.; Jiang, S. Y. Anal. Chem. 2012, 84, 3440−
3445.
2
(
(48) Dai, F. Y.; Liu, W. G. Biomaterials 2011, 32, 628−638.
(49) Parrott, M. C.; DeSimone, J. M. Nat. Chem. 2012, 4, 13−14.
(50) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Angew.
Chem., Int. Ed. 2010, 49, 6288−6308.
2
(
(
51) Takahashi, A.; Hamai, K.; Okada, Y.; Sakohara, S. Polymer 2011,
2, 3791−3799.
52) Cho, W. K.; Kong, B.; Choi, I. S. Langmuir 2007, 23, 5678−
682.
53) Risbud, M.; Saheb, D. N.; Jog, J.; Bhonde, R. Biomaterials 2001,
2, 1591−1597.
54) Kircheis, R.; Wightman, L.; Wagner, E. Adv. Drug Delivery Rev.
001, 53, 341−358.
55) Fischer, D.; Bieber, T.; Li, Y. X.; Elsas
Pharm. Res. 1999, 16, 1273−1279.
56) Forrest, M. L.; Gabrielson, N.; Pack, D. W. Mol. Ther. 2004, 9,
S138.
5
(
5
(
2
(
2
(
(
(
(
(
̈
ser, H. P.; Kissel, T.
(
(
(
3
(
(57) Zugates, G. T.; Little, S. R.; Anderson, D. G.; Langer, R. Isr. J.
Chem. 2005, 45, 477−485.
(58) Deng, R.; Yue, Y.; Jin, F.; Chen, Y.; Kung, H. F.; Lin, M. C. M.;
Wu, C. J. Controlled Release 2009, 140, 40−46.
59) Vroman, B.; Ferreira, I.; Jerome, C.; Jerome, R.; Preat, V. Int. J.
Pharm. 2007, 344, 88−95.
60) Kleemann, E.; Neu, M.; Jekel, N.; Fink, L.; Schmehl, T.; Gessler,
T.; Seeger, W.; Kissel, T. J. Controlled Release 2005, 109, 299−316.
61) Huang, F. W.; Wang, H. Y.; Li, C.; Wang, H. F.; Sun, Y. X.;
(
3
(
(
(
25) Szleifer, I. Curr. Opin. Solid State Mater. Sci. 1997, 2, 337−344.
(
26) Merdan, T.; Kunath, K.; Petersen, H.; Bakowsky, U.; Voigt, K.
H.; Kopecek, J.; Kissel, T. Bioconjugate Chem. 2005, 16, 785−792.
(
S.; Roberts, C. J.; Fischer, D.; Davies, M. C.; Kissel, T. Bioconjugate
Chem. 2002, 13, 845−854.
(
(
27) Petersen, H.; Fechner, P. M.; Martin, A. L.; Kunath, K.; Stolnik,
Feng, J.; Zhang, X. Z.; Zhuo, R. X. Acta Biomater. 2010, 6, 4285−4295.
28) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Angew.
Chem., Int. Ed. 2010, 49, 6288−6308.
29) Nishizawa, K.; Konno, T.; Takai, M.; Ishihara, K. Biomacromo-
lecules 2008, 9, 403−407.
30) Yu, B.; Lowe, A. B.; Ishihara, K. Biomacromolecules 2009, 10,
50−958.
31) Xu, Y.; Takai, M.; Ishihara, K. Biomacromolecules 2009, 10, 267−
74.
32) Nam, K. W.; Watanabe, J.; Ishihara, K. Biomacromolecules 2002,
, 100−105.
33) Ahmed, M.; Jawanda, M.; Ishihara, K.; Narain, R. Biomaterials
012, 33, 7858−7870.
34) Ahmed, M.; Bhuchar, N.; Ishihara, K.; Narain, R. Bioconjugate
Chem. 2011, 22, 1228−1238.
35) Bhuchar, N.; Deng, Z. C.; Ishihara, K.; Narain, R. Polym. Chem.
011, 2, 632−639.
36) Bhuchar, N.; Sunasee, R.; Ishihara, K.; Thundat, T.; Narain, R.
Bioconjugate Chem. 2012, 23, 75−83.
37) Lowe, A. B.; Billingham, N. C.; Armes, S. P. Macromolecules
999, 32, 2141−2148.
38) Kathmann, E. E.; White, L. A.; McCormick, C. L. Polymer 1997,
8, 879−886.
39) Moro, T.; Takatori, Y.; Ishihara, K.; Konno, T.; Takigawa, Y.;
Matsushita, T.; Chung, U.; Nakamural, K.; Kawaguchi, H. Nat. Mater.
004, 3, 829−836.
40) Snyder, T. A.; Tsukui, H.; Kihara, S.; Akimoto, T.; Litwak, K.
(
(
9
(
2
(
3
(
2
(
(
2
(
(
1
(
3
(
2
(
N.; Kameneva, M. V.; Yamazaki, K.; Wagner, W. R. J. Biomed. Mater.
Res., Part A 2007, 81A, 85−92.
(
(
41) Matsuno, R.; Ishihara, K. Macromol. Symp. 2009, 279, 125−131.
́
42) Zhang, Z.; Vaisocherova, H.; Cheng, G.; Yang, W.; Xue, H.;
Jiang, S. Y. Biomacromolecules 2008, 9, 2686−2692.
43) Keefe, A. J.; Brault, N. D.; Jiang, S. Y. Biomacromolecules 2012,
3, 1683−1687.
44) Ladd, J.; Zhang, Z.; Chen, S. F.; Hower, J. C.; Jiang, S. Y.
Biomacromolecules 2008, 9, 1357−1361.
45) Chang, Y.; Chen, S. F.; Yu, Q. M.; Zhang, Z.; Bernards, M.;
Jiang, S. Y. Biomacromolecules 2007, 8, 122−127.
46) Zhang, L.; Cao, Z. Q.; Li, Y. T.; Ella-Menye, J. R.; Bai, T.; Jiang,
S. Y. ACS Nano 2012, 6, 6681−6686.
(
1
(
(
(
7
36
dx.doi.org/10.1021/bm301826m | Biomacromolecules 2013, 14, 728−736