Synthesis and micellization of pH/temperature-responsive double-hydrophilic diblock copolymers polyphosphoester- block -poly[2-(dimethylamino)ethyl methacrylate] prepared via ROP and ATRP

Article abstract of DOI:10.1021/ma902658n

Novel pH- and temperature-responsive double-hydrophilic diblock copolymers, poly(ethylethylene phosphate)-block-poly[2-(dimethylamino)ethyl methacrylate] (PEEP-b-PDMAEMA), have been synthesized via the combination of ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP). The PEEP block with a bromine-terminated end (PEEP-Br) was first prepared by ROP of 2-ethoxy-2-oxo-1,3,2-dioxaphospholane (EEP) using 2-hydroxyethyl 2-bromoisobutyrate as a bifunctional initiator and stannous octoate as a catalyst. ATRP was then used to polymerize DMAEMA monomer in a methanol/water mixture with PEEP-Br as a macroinitiator, resulting in diblock copolymers of PEEP-b-PDMAEMA. Their chemical structures were respectively characterized by ¹H NMR, ¹³C NMR, ³¹P NMR, and FT-IR. Their molar mass distributions were determined by gel permeation chromatography (GPC). The critical aggregation concentration (cac) of PEEP-b-PDMAEMA in aqueous solution, which was measured by the fluorescence probe technique, depends on the block composition. The results measured by static laser light scattering (LLS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) reveal that these diblock copolymers are able to self-assemble into aggregates with different particle sizes and morphologies in aqueous solutions, depending on various pH media. On the other hand, the UV-vis measurement shows that these diblock copolymers exhibit a reproducible temperature-responsive behavior with a lower critical solution temperature (LCST) that is tunable by the block composition and pH. In addition, agarose gel retardation assays, TEM, and zeta potential measurements demonstrate that such double-hydrophilic diblock copolymers can effectively condense DNA, potentially useful for the gene delivery.

Full text of DOI:10.1021/ma902658n

Macromolecules 2010, 43, 4771–4781 4771
DOI: 10.1021/ma902658n
Synthesis and Micellization of pH/Temperature-Responsive
Double-Hydrophilic Diblock Copolymers Polyphosphoester-block-
poly[2-(dimethylamino)ethyl methacrylate] Prepared via ROP and ATRP
Xu Liu, Peihong Ni,* Jinlin He, and Mingzu Zhang
Key Laboratory of Organic Chemistry of Jiangsu Province, College of Chemistry, Chemical Engineering and
Materials Science, Soochow University, Suzhou 215123, China
Received December 1, 2009; Revised Manuscript Received March 12, 2010
ABSTRACT: Novel pH- and temperature-responsive double-hydrophilic diblock copolymers, poly(ethyl-
ethylene phosphate)-block-poly[2-(dimethylamino)ethyl methacrylate] (PEEP-b-PDMAEMA), have been
synthesized via the combination of ring-opening polymerization (ROP) and atom transfer radical polymeri-
zation (ATRP). The PEEP block with a bromine-terminated end (PEEP-Br) was first prepared by ROP of
2-ethoxy-2-oxo-1,3,2-dioxaphospholane (EEP) using 2-hydroxyethyl 2-bromoisobutyrate as a bifunctional
initiator and stannous octoate as a catalyst. ATRP was then used to polymerize DMAEMA monomer
in a methanol/water mixture with PEEP-Br as a macroinitiator, resulting in diblock copolymers of PEEP-
1
13
31
b-PDMAEMA. Their chemical structures were respectively characterized by H NMR, C NMR, P NMR,
and FT-IR. Their molar mass distributions were determined by gel permeation chromatography (GPC). The
critical aggregation concentration (cac) of PEEP-b-PDMAEMA in aqueous solution, which was measured
by the fluorescence probe technique, depends on the block composition. The results measured by static laser
light scattering (LLS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) reveal
that these diblock copolymers are able to self-assemble into aggregates with different particle sizes and
morphologies in aqueous solutions, depending on various pH media. On the other hand, the UV-vis mea-
surement shows that these diblock copolymers exhibit a reproducible temperature-responsive behavior with a
lower critical solution temperature (LCST) that is tunable by the block composition and pH. In addition,
agarose gel retardation assays, TEM, and zeta potential measurements demonstrate that such double-
hydrophilic diblock copolymers can effectively condense DNA, potentially useful for the gene delivery.
Introduction
In addition, for biomedical applications, biodegradability and
1
biocompatibility would be a desirable trait of DHBCs. However,
extensive design of biodegradable DHBCs has not been perfor-
med yet. The main limitation lies in the nondegradability of carbon-
Double-hydrophilic block copolymers (DHBCs) represent a
class of copolymers with two or more water-soluble blocks made
of different chemical species. They exhibit switchable amphiphilic
1
2
2
carbon backbone or ether-based backbone, although it may
be partially overcome by advanced polymer synthesis techni-
ques. For example, a degradable block copolymer consisting of
poly(ethylene glycol) (PEG) and PDMAEMA segments con-
nected through an acid-labile cyclic ortho ester linkage (PEG-
characteristics. That is, one hydrophilic segment undergoes physi-
cal or chemical transformations and becomes hydrophobic, whereas
another hydrophilic segment remains soluble in water. And the
transformation is often induced by subtle adjustment of tempe-
rature, pH, or ionic strength in solutions as well as complexation
with appropriate molecules. Thus, DHBCs provide a wide range
of applications including drug delivery and gene therapy, rever-
sible solution condition-induced micellization, crystal growth
modifiers, mineralization templates, nanoparticle fabrication,
and so on. It is believed that more applications in biomedical field
will be developed due to their stimuli responsiveness and easily
tunable functionality. The nature of the monomer repeat units
in each hydrophilic block can be either ionic or non-ionized.
Typical ionic segments include poly(acrylic acid) (PAA), poly-
styrenesulfonic acid) (PSSA), poly[2-(dimethylamino)ethyl
methacrylate] (PDMAEMA), and poly(4-vinylpyridine)
P4VP). However, most of DHBCs with nonionic block pre-
pared so far are based on hydrophilic poly(ethylene oxide) (PEO)
1
3
a-PDMAEMA) was synthesized by ATRP for gene delivery.
3
,4
Polyphosphoesters (PPE) have been pursued as biomaterials
due to their potential biodegradability, good biocompatibility,
5
1
4
6
7
8,9
and functionality of side chain as well as their structural simi-
larities to naturally occurring nucleic and teichoic acids. Inter-
estingly, they can degrade under the physiological conditions via
1
5,16
hydrolysis or enzymatic cleavage of the phosphoester bonds.
The degradation rates are controllable by adjusting the chemical
structure in the backbone and side chain. By choosing biocom-
patible building blocks of the polymer, degradation products of
(
1
7
PPE have minimal toxic effects and good biocompatibility. As a
result, polyphosphates have been proposed for use in many bio-
medical applications, ranging from drug and nonviral gene car-
1
(
1
8
19
20
10,11
riers to cell encapsulations and tissue engineering scaffolds.
and its derivants.
Therefore, it is necessary to diversify
Recently, polyphosphoesters (PPE) have been reported to be used
in amphiphilic polymers as a hydrophilic block to replace tradi-
tional PEO. For example, Leong et al. synthesized random
copolymers of poly(DL-lactide) (PLA) and poly(ethylethylene
phosphate) (PEEP) as drug carrier and found that the hydro-
philicity of the copolymers increased with the increasing EEP
systematically the nonionic hydrophilic block of DHBCs. The
aim is to extend the types of double-hydrophilic block copoly-
mers and to expect unique properties of their micelles in aqueous
media.
*To whom correspondence should be addressed. E-mail: phni@suda.
21
edu.cn.
content. Wang et al. reported the synthesis and degradation of
r 2010 American Chemical Society
Published on Web 04/26/2010
pubs.acs.org/Macromolecules

4
772 Macromolecules, Vol. 43, No. 10, 2010
Scheme 1. Synthesis Route of Double-Hydrophilic PEEP-b-PDMAEMA Diblock Copolymer
Liu et al.
PEEP-PLLA-PEEP triblock copolymers, in which PLLA repre-
then refluxed over sodium wire with benzophenone as an indi-
cator until the color turned to purple. Ethidium bromide (EtBr)
obtained from Fluka were used as received. Phosphate buffers
saline tablets (PBS) and tris-borate-EDTA buffer (TBE) were
obtained from Medicago. Plasmid pUC18 (pDNA) was purchased
from Takara. Other reagents were purchased from Sinopharm
Chemical Reagent Co. and used as received.
2
2
sents poly(
L-lactic acid). They have also widely researched the
synthesis and application of amphiphilic block copolymers of PEEP
2
3
and poly(ε-caprolactone) (PCL) as drug carrier. Meanwhile,
Iwasaki et al. recently reported that PEEP and its copolymers
with poly(isopropylethylene phosphate) (PiPEP) are thermosensi-
24
tive, exhibiting varied LCSTs, depending on the compositions.
Synthesis of 2-Hydroxyethyl 2-Bromoisobutyrate (HEBI). HEBI
Wang et al. also demonstrated that triblock copolymers of PEG,
PEEP, and PiPEP exhibited thermo-induced self-assembly beha-
vior, and their critical aggregation temperature can be conveni-
30
was synthesized by a method described previously. 2-Bromoiso-
butyryl bromide (4.998 g, 0.0218 mol) was added dropwise to a
cold solution of ethylene glycol (30.759 g, 0.4961 mol) and tri-
ethylamine (2.202 g, 0.0218 mol) at 0 ꢀC for 2 h. The reaction was
continued at 0 ꢀC for another 2 h and then heated to 40 ꢀC for 5 h.
The reaction mixture was cooled, added to 500 mL of water, and
extracted with chloroform three times, and then the chloroform
25
ently adjusted.
In this work, we have synthesized a series of diblock copolymers
consisting of PEEP and PDMAEMA with various molecular
weights and compositions through a combination of ring-opening
polymerization (ROP) and ATRP strategy, as shown in Scheme 1.
PDMAEMA has both pH- and temperature-sensitive behaviors
3
layer was washed successively with diluted HCl, saturated NaHCO ,
and water. The organic layer was dried over anhydrous magnesium
sulfate and evaporated to provide a product. The product was
vacuum-distilled (yield: 70%) and characterized with H NMR
in aqueous solutions, which acts as a weak polybase with pK of
a
26,27
1
about 8.0.
In acidic or neutral media, PDMAEMA can be
completely or partially protonated and has been reported to be one
of the efficient condensing agents for DNA delivery. Nevertheless,
(in CDCl ): (CH ) CBr- (δ 1.80; 6H), -CH -CH -OH (δ 3.70;
3
3 2
2
2
2H), and -CH
2
-CH -OH (δ 4.15; 2H).
2
28
low biodegradability limits its application in gene delivery. Thus,
focusing on the development of biodegradable DHBCs with
both pH and temperature sensitivity for potential biomedical
utility, we have synthesized PEEP-b-PDMAEMA diblock co-
polymers combining the advantages of polyphosphoester and
PDMAEMA together. The effects of pH and temperature on
the micellization under different conditions have been investi-
gated. To the best of our knowledge, these PEEP-b-PDMAEMA
diblock copolymers have not been reported previously. And
we further demonstrated their potential application for gene
delivery.
Synthesis of PEEP-Br Macroinitiator. Polyphosphoester end-
capped with bromo group (PEEP-Br) was prepared by ring-opening
polymerization (ROP) of 2-ethoxy-2-oxo-1,3,2-dioxaphospho-
lane (EEP) using HEBI as an initiator and Sn(Oct) as a catalyst.
2
The polymerization was conducted in a 50 mL round-bottom
flask. The flask was treated with trimethylchlorosilane solution
in methylene chloride for 12 h and flame-dried under vacuum,
and three exhausting-refilling argon cycles were performed
before use. A typical experimental procedure for polymerization
was as follows: EEP monomer (2.0 g, 13.2 mmol) was intro-
duced into the flask using a syringe, followed by adding 10 mL of
THF. The mixture was subsequently stirred in a bath at 35 ꢀC.
To this solution was added HEBI (0.139 g, 0.66 mmol in 1.3 mL
of THF) through a syringe, followed rapidly by addition of Sn-
Experimental Section
Materials. 2-Ethoxy-2-oxo-1,3,2-dioxaphospholane (EEP)
(Oct) (0.134 g, 0.33 mmol in 0.66 mL of THF). The solution was
2
deactivated with 1 mol L acetic acid after 1 h and precipitated
2
9
-1
was synthesized by a method described previously and distilled
under reduced pressure just before use. 2-(Dimethylamino)ethyl
methacrylate (DMAEMA, Aldrich) was dried over calcium hydride
into an excess of diethyl ether. The precipitate was dried under
vacuum until a constant weight at room temperature to obtain
the product.
Synthesis of PEEP-b-PDMAEMA Diblock Copolymer. PEEP-
b-PDMAEMA diblock copolymers were synthesized in a methanol/
water mixture at 25 ꢀC via ATRP using PEEP-Br as a macro-
initiator in a tightly sealed 50 mL round-bottom flask under an
(
nous octoate [Sn(Oct) ], 2,2 -bipyridine (bpy), 2-bromoisobuty-
CaH
2
) and distilled in vacuum immediately before use. Stan-
0
2
ryl bromide, and cuprous bromide (CuBr) were all purchased
from Aldrich and used without further purification. THF was
initially dried over potassium hydroxide for at least 2 days and

Article
Macromolecules, Vol. 43, No. 10, 2010 4773
3
1
argon atmosphere. As an example, the flask was charged with a
required amount of macroinitiator PEEP-Br (0.306 g, 0.09 mmol)
and DMAEMA monomer (0.424 g, 2.7 mmol). The solvent of
deoxygenated methanol/water (2/1 by volume ratio) mixture was
added to make the PEEP-Br solution of 0.1 mol L . Three
exhausting-refilling argon cycles were performed to remove oxy-
gen from the polymerization solution, and then the solution was
of LLS instrumentation and principles can be found elsewhere.
In static LLS, the scattering angle (θ) and polymer concentra-
tion (C, g/mL) dependence of the absolute time-averaged scat-
tered light intensity, known as the excess Rayleigh ratio (Rvv(q)),
of a sufficiently dilute polymer solution can lead to the weight-
-
1
averaged molar mass (M
and the z-average mean-square radius of gyration ÆR
w 2
), the second virial coefficient (A ),
2
g
æ as
purged with argon gas for 10 min. CuBr (0.013 g, 0.09 mmol) and
0
ꢀ
ꢁ
2,2 -bipyridine (bpy) (0.029 mg, 0.18 mmol) were added with a
KC
1
1
2
2
ꢁ
1 þ q ÆRg æ þ 2A2C
ð1Þ
slow argon purge. The flask was sealed under an argon atmosphere
and kept in a water bath at 25 ꢀC. The reaction was terminated by
adding THF with stirring, and the solution turned from brown to
blue. The reaction mixture was then allowed to pass through a
basic alumina column. After being concentrated in a rotary
evaporator, it was poured into cold hexane to precipitate the
polymer. Finally, the copolymer was dialyzed against deionized
water for 2 days to remove the unreacted PEEP-Br macroinitiator
with a cellulose tubular membrane (MWCO 5000), and then
lyophilized. The lost weight as unreacted PEEP-Br macroinitiator
was used to estimate the initiation efficiency of the PEEP-Br
macroinitiator. The yield of PEEP-b-PDMAEMA copolymer
was determined gravimetrically.
RvvðqÞ
Mw
3
2
2
4
where K [= 4πn (dn/dC) /(NAλ )] is a constant for a given poly-
0
) sin(θ/2) ] is the scat-
tering vector, with dn/dC, N , and λ being the specific refractive
0
mer solution/dispersion and q [= (4πn/λ
A
0
index increment, the Avogadro number, and the light wave-
length in vacuum, respectively. dn/dC was determined by using
the Jianke differential refractometer.
The particle sizes of the aggregates of PEEP-b-PDMAEMA
diblock copolymer in aqueous solution were measured by a Zetasizer
Nano-ZS dynamic light scattering instrument (Malvern) equip-
ped with a 633 nm He-Ne laser using backscattering detection.
All samples had a constant concentration of 2 g L . The
measurements were carried out at a scattering angle of 90ꢀ and
at ambient temperature and various pH.
3
2
-
1
1
13
31
Characterizations. H NMR, C NMR, and P NMR spec-
tra were recorded on an INOVA-300 NMR spectrometer at room
temperature with CDCl as a solvent and TMS as an internal refe-
3
Transmission electron microscopy (TEM) images were obta-
ined using a TEM instrument (TECNAI G 20, FEI Co.) in a
2
rence. FT-IR measurements were performed on a Nicolet AVATAR
3
60 Fourier transform infrared spectrometer using the KBr disk
200 kV. To prepare the TEM samples, 5 μL of the sample solu-
tion was dropped onto a carbon-coated copper grid (400 meshes),
and the water droplet was allowed to evaporate slowly in air.
The thermoresponsive properties of PEEP-b-PDMAEMA
diblock copolymers were investigated with a Shimadzu 3150
UV-vis-NIR spectrophotometer. PEEP-b-PDMAEMA sam-
ples were dissolved in deionized water (0.5 wt %), and a trans-
mittance curve through the solution at a wavelength of 500 nm
method. Relative molecular weights and molecular weight dis-
tributions of PEEP-Br polymers were measured by a gel permea-
tion chromatography (GPC) system equipped with a Waters 1515
Isocratic HPLC pump, a Waters 2414 refractive index detector
(
RI), a Waters 2487 dual-wavelength absorbance detector, and a
set of Waters Styragel columns (HR3, HR4, and HR5, 7.8 mmꢀ
00 mm). GPC measurements were carried out at 35 ꢀC using
dimethylformamide (DMF) as eluent with a flow rate of 1.0 mL
3
-
1
was recorded with temperature variation (0.1 ꢀC min ) between
30 and 80 ꢀC. The lower critical solution temperature (LCST) of
the polymer solution was defined as the temperature producing
a half decrease of the total decrease in optical transmittance.
The ability of compacting DNA for PEEP-b-PDMAEMA
diblock copolymer was investigated by several methods, including
gel retardation assay, zeta potential measurements, and TEM.
For gel retardation assay, polyplexes in different N/P ratios were
loaded onto 0.8% agarose gel containing ethidium bromide
-
1
min . The system was calibrated with polystyrene standards.
While the molecular weights and molecular weight distributions
of PEEP-b-PDMAEMA diblock copolymers were determined
by a Waters 1515 GPC instrument using a PLgel 5.0 μm bead-
2
size guard column (50ꢀ7.5 mm ), followed by two linear PLgel
˚
columns (500 A and Mixed-C) and a differential refractive index
detector. THF was used as the eluent at 30 ꢀC with a flow rate of
-
1
.0 mL min and a series of standard monodispersed polysty-
1
rene as the calibration.
-
1
(0.5 μg mL ) and then electrophoresed in Tris-borate-EDTA
buffer (TBE: 40 mM tris-borate, 1 mM EDTA, and pH 7.4) at
70 V for 1 h. The migrated pDNA was visualized on a UV illu-
minator (M-15E, UVP Inc., Upland, CA), and the determination
of aqueous microelectrophoresis of pDNA/PEEP-b-PDMAEMA
complexes was carried out in a JS94J microeletrophoresis instru-
ment (Shanghai Zhongchen Co., China). The device used a CCD
camera, frame grabber, and software to capture the image of the
moving particles. The zeta potential data were directly calcu-
lated by the instrument. The morphologies of pDNA and pDNA/
PEEP-b-PDMAEMA complexes were observed by TEM.
The critical aggregation concentration (cac) was investigated
by the fluorescence probe method. Fluorescence spectra were
recorded on a FLS920 fluorescence spectrofluorometer (Edinburgh
Co., UK), and pyrene was used as a hydrophobic fluorescent
probe. A predetermined amount of pyrene in acetone was added
into a series of ampules, and the acetone was allowed to evapo-
rate. 10 mL of aqueous solutions at different concentrations
of copolymers was then added to the ampules containing the
pyrene residue. It should be noted that all the aqueous solutions
contained excess pyrene residue at the same concentration of
-
7
6
ꢀ 10 M. All the solution pH was adjusted to 9.2 for the cac
measurement. The aqueous solutions of copolymers were allowed
to stir for 24 h at room temperature to reach the solubilization
equilibrium of pyrene. Excitation was carried out at 335 nm, and
emission spectra were recorded ranging from 350 to 500 nm.
Both excitation and emission bandwidths were set at 1 nm.
From the pyrene emission spectra, the intensity ratio (I /I ) of
Results and Discussion
Synthesis and Characterization of the Diblock Copolymers.
The synthetic route for the preparation of PEEP-b-PDMAEMA
diblock copolymer is outlined in Scheme 1. As reported by
Jakubowski et al., ring-opening polymerization (ROP) can
be hindered in the presence of DMAEMA because the com-
plexation of the amine site of PDMAEMA would deactivate
3
1
the third band (383 nm, I
3
) to the first band (372 nm, I
1
) was
analyzed as a function of polymer concentration. The cac value
was defined as the point of intersection of the two lines in the
plot of fluorescence versus concentration. The experiments were
conducted in triplicate, and the average values are reported.
A commercial LLS spectrometer (ALV/SP-125) equipped with
a multi-τ digital correlator (ALV-5000/E) and a 22 mW He-Ne
laser (JDS-Uniphase 1145P) was used. The incident beam was
vertically polarized with respect to the scattering plane. The details
30
tin(II) hexanoate, which was used as a catalyst for ROP.
Therefore, in the present work, DMAEMA monomer was
polymerized following PEEP block. PEEP-Br with linear
molecular structure was synthesized through ring-opening
polymerization of EEP in THF under co-initiation of difunc-
tional ATRP/ROP initiator 2-hydroxyethyl 2-bromoisobu-
tyrate (HEBI) and Sn(Oct) . The feed molar ratio of HEBI to
2

4
774 Macromolecules, Vol. 43, No. 10, 2010
Liu et al.
1
Figure 1. H NMR spectrum of the bromine-terminated polyphospho-
3
ester (PEEP32-Br) (solvent: CDCl ).
EEP was 1:30, while the reaction time was limited to 1 h since
the extension of reaction time will likely lead to chain ex-
change side reaction though EEP conversion can be increa-
3
3
sed. PEEP-b-PDMAEMA diblock copolymer with con-
trolled molecular weight and low polydispersity was then
prepared by ATRP technique. A CuBr/bpy catalyst system
was used for the polymerization of DMAEMA using PEEP-
Br as a macroinitiator in a methanol/water mixture accord-
3
4
ing to previous literature. The obtained diblock copolymer
PEEP-b-PDMAEMA was characterized by NMR as descri-
bed in the Experimental Section.
1
Figure 1 shows the H NMR spectrum of PEEP-Br. Reso-
nances at δ 1.37 ppm (peak d), δ 4.18 ppm (peak c), and
δ 4.26 ppm (peak b þ e) are assigned respectively to pendent
methyl [-P(=O)-OCH CH ], methylene [-P(=O)-OCH -
2
3
2
CH ] protons, and methylene protons [-P(=O)-OCH -
3
2
CH O-] from PEEP backbone. And the resonance at
2
δ 3.81 ppm (peak a) should be assigned to the methylene
protons conjoint to the end hydroxyl group of phosphoester
unit of block copolymer [-P(=O)-OCH CH OH]. From
2
2
the resonances of protons for initiator HEBI [δ 1.80 ppm;
(
CH ) -CBr-] appearing in Figure 1, one can observe that
3
2
HEBI had been involved in the ring-opening polymerization
of EEP. The typical H NMR spectrum of diblock copoly-
1
mer of PEEP-b-PDMAEMA is shown in Figure 2A. It is
found that all signals assigned to protons of PEEP block
(except for the terminal methyl directly linked to the bromine
group) in Figure 1 are also present in the spectrum of PEEP-
b-PDMAEMA diblock copolymer. Those newly appearing
signals in Figure 2A are assigned as δ 0.7-1.3 ppm for the
R-CH (peakf), δ1.9 ppm for the protons in -CCH - (peake),
3
2
δ 2.3 ppm for the protons in -N(CH ) (peak i), δ 2.6 ppm for
Figure 2. Typical characterization of NMR spectroscopy for PEEP32-
1 13
3
2
b-PDMAEMA56 diblock copolymers: (A) H NMR, (B) C NMR,
the -CH N= (peak h), and δ 4.1 ppm for the methylene neigh-
2
31
3
and (C) P NMR. CDCl was used as the solvent in each measurement.
boring to the ester group [-CH OC (= O)] (peak g) of the
2
13
PDMAEMA block. The C NMR spectrum also revea-
led that resonances of carbon atoms from the PEEP and the
PDMAEMA block were all presented as assigned in Figure 2B.
-1
is also verified at 988 cm in the FT-IR spectrum. Accord-
ing to the FT-IR spectrum shown in Figure 3b, the following
characteristic peaks of PDMAEMA can be found: 1735
3
1
The P NMR spectrum of PEEP-b-PDMAEMA diblock
polymer (Figure 2C) gave a strong resonance at δ -0.25 ppm
-
1
-1
cm , carbonyl (CdO) stretch vibration; 2920-2960 cm
,
-
1
(peak b), assigned to the phosphorus atoms in polyphos-
phoester block except the phosphorus atom at the end, which
-N(CH
3
)
2
stretch vibration; around 1476 cm , -N(CH )
3 2
deformational stretch vibration. Those absorptions, how-
ever, are absent in the FT-IR spectrum of PEEP homopolymer.
Thus, judging together with analysis by NMR spectrum, it
demonstrated the successful polymerization of diblock co-
generated the weak signal at δ 0.6 ppm (peak a).
FT-IR analysis results of PEEP homopolymer and PEEP-
b-PDMAEMA diblock polymer are shown in Figure 3. Absorp-
-
1
tion (bands) at 1276 and 1158 cm in Figure 3a can be
observed, which can be ascribed to the asymmetrical and sym-
metrical PdO stretching, respectively. The P-O-C stretching
polymer PEEP-b-PDMAEMA.
The number-average molecular weights (M
dispersity indexes (PDI) of PEEP-Br homopolymers and
_
) and poly-
n

Article
Macromolecules, Vol. 43, No. 10, 2010 4775
Table 1. Characterization Data of the Compositions,
Number-Average Molecular Weights, and Molecular Weight
Distributions of PEEP Macroinitiators and PEEP-b-PDMAEMA
Diblock Copolymers
_
M
_
M
_
M
c
d
e
sample
n,GPC
n,theor
n,NMR
PDI
a
PEEP21
7340
7840
3690
8260
10140
10390
4450
6560
5440
10000
11410
13920
15450
3400
9400
10860
10590
4150
7290
5130
11470
14270
14360
1.13
1.41
1.24
1.30
1.21
1.23
1.19
1.29
1.39
1.36
1.33
b
b
b
a
b
a
b
b
b
b
PEEP21-b-PDMAEMA31
PEEP21-b-PDMAEMA43
PEEP21-b-PDMAEMA45
PEEP26
PEEP26-b-PDMAEMA20
PEEP32
PEEP32-b-PDMAEMA31
PEEP32-b-PDMAEMA40
PEEP32-b-PDMAEMA56
9280
12280
9320
4400
12200
6920
8110
10450
11730
PEEP32-b-PDMAEMA67
a
18320
b
GPC in DMF/LiBr, using polystyrene as standards. GPC in THF,
using polystyrene as standards. Theoretical molecular weight. Calcu-
Figure 3. FT-IR spectra of (a) PEEP32-Br macroinitiator and (b) PEEP32
-
c
d
b-PDMAEMA56 diblock copolymer.
1
lated on the basis of H NMR measurements in CDCl
GPC in corresponding solvent.
e
3
.
Measured by
PEEP-b-PDMAEMA diblock copolymers were measured
by GPC. For both, we tried to perform GPC measurement
using DMF and THF as eluents. Unfortunately, signals of
PEEP homopolymers were only detected in DMF (refrac-
tive index: 1.4305) and PEEP-b-PDMAEMA in THF (refrac-
tive index: 1.4040). The reason lies in the difference of refractive
index of polymers and eluents. Although GPC analysis based
on linear polystyrene standards is not a reliable approach to
determining the accurate molecular weights of polyphos-
phoester, it is meaningful in estimating the PDI values. The
molecular weight distribution for PEEP-Br was narrow. The
actual molecular weights of PEEP-Br was calculated accord-
ing to the relative integration intensities of methylene pro-
from 21 to 32, while the DP of PDMAEMA was fixed at 31,
-1
the cac values increased from 0.74 to 0.91 g L . This trend is
in agreement with the reported result by Wang et al., in which
a block copolymer with a rather long PEEP segment tended
2
3b
to increase the cac value in aqueous media. When the DP
of PEEP was fixed at 32, but the DP of PDMAEMA was
varied from 31 to 67 units, the cac value decreased from 0.91
-
1
to 0.86 g L , indicating that the length of PDMAEMA
block plays an important role on the thermodynamic stabi-
lity of micelles. Compared with PEG, the PEEP block is
slightly hydrophobic. Therefore, we can believe that PEEP-
b-PDMAEMA micelles would be more thermodynamically
stable in aqueous media. In this sense, PEEP-b-PDMAEMA
micelles should be a potential candidate for biomedical
application.
1
tons of PEEP-Br (peak b of H NMR spectrum in Figure 1)
and the protons from HEBI group at one end of the polymer
chain (peak g in Figure 1). The molar masses of the copolymers
were calculated by comparing the intensity of the methy-
1
lene protons (peak h of H NMR spectrum, Figure 2A) of
pH-Responsive Properties of PEEP-b-PDMAEMA Diblock
Copolymers. It is well-known that PDMAEMA possesses
pH-responsive properties. Herein, we have investigated the
effect of pH variation on the aggregation behavior of PEEP-
b-PDMAEMA diblock copolymers by performing TEM and
laser light scattering (LLS).
PDMAEMA at 2.6 ppm with the methyl protons of PEEP at
δ 1.37 ppm (peak d, Figure 2A). Detailed information on the
PEEP-Br homopolymers and PEEP-b-PDMAEMA diblock
copolyme _r s has been summarized in Table 1. It can be seen
that the M
values of the diblock copolymers as calcu-
n,NMR
1
lated by the H NMR spectrum are slightly higher than the
theoretical ones. The actual initiation efficiency was about
Figures 5 to 7 show TEM images of the micelles obtained
by the self-assembly of PEEP32-b-PDMAEMA67 diblock
copolymer in aqueous solution at pH 3.0, 7.4, and 9.5, res-
9
0%. This is owing to the high molecular weight of PEEP
-
1
macroinitiator, which leads to high viscosity of the reaction
solution and enhanced embedment of the radicals at the
chain ends with long PEEP chains. Meanwhile, PDMAEMA-
containing polymers are difficult to be characterized by GPC
because of the adsorption of PDMAEMA block onto the
pectively. All solution concentrations were kept at 2.0 g L ,
which was above the cac of PEEP32-b-PDMAEMA67 solu-
tion. It is clear that the particle sizes of aggregates at pH 3.0
are larger than those at both pH 7.4 and 9.5, and with
different morphologies, indicating that the diblock copoly-
mer has pH-responsive properties. We have proposed possi-
ble aggregating structures to explain the above-mentioned
phenomenon as shown in Scheme 2.
3
5
column. This usually results in broader peaks (larger poly-
dispersities) with higher retention times (lower molecular
weights).
Critical Aggregation Concentration (cac). The cac value of
a diblock copolymer is an important parameter that repre-
sents the self-assembling behavior of block copolymers in
solution. In this study, the fluorescence probe method was
used to determine the cac value of PEEP-b-PDMAEMA
copolymers, and all the solution pH was adjusted to 9.2 to
reach a complete deprotonation. Pyrene is a common probe
used to monitor micropolarity because the ratio of the third
to the first vibronic peaks (I /I ) in pyrene fluorescence spec-
Under acidic conditions (pH 3.0), the PEEP-b-PDMAE-
MA diblock copolymers were expected to be individual
unimers because both PEEP block and DMAEMA segments
with sufficient protonation were hydrophilic. Tam et al.
reported the unimer state of PEO-b-PDEAEMA in acidic
3
7
solution. In our test, however, aggregates were surprisingly
observed as shown in Figure 5. It is because of the hydrolysis
of PEEP segments. Penczek et al. explored the stability of the
poly(alkylene phosphates) in aqueous solutions at various
pH values and the relative rates of hydrolysis for the main
chain and methyl substituent on phosphorus via direct titrimetric
3
1
trum is sensitive to the polarity, the I /I ratio being larger in
3
1
3
less polar media. And I /I ratio variations with polymer
6
3
1
38
concentrations can be monitored. The cac values were plot-
ted against the degrees of polymerization (DPs) of both
PDMAEMA and PEEP blocks in the diblock copolymers
and shown in Figure 4. When the DP of PEEP block increased
and NMR methods. In acidic conditions the methyl group
hydrolyzes faster, whereas at basic conditions both the methyl
group and the main chain depart with approximately similar
rates. This difference results from different mechanisms of

4
776 Macromolecules, Vol. 43, No. 10, 2010
Liu et al.
Figure 4. Intensity ratios (I383/I372) as a function of concentration of PEEP-b-PDMAEMA (PEEP-b-PDMA in short) with different degrees of
polymerization for (a) changed PEEP length and (b) changed PDMAEMA length.
Figure 7. TEM micrographs of (a) aggregates from PEEP32-b-PDMA-
EMA67 diblock copolymer in solution at pH 9.5 with a concentration of
Figure 5. TEM micrographs of aggregates from PEEP32-b-PDMAE-
-
1
2
.0 g L , bar = 0.5 μm, and (b) the high-magnification image of (a),
MA67 diblock copolymer in solution at pH 3.0 with a concentration of
-1
bar = 50 nm.
2
.0 g L , bar = 0.5 μm.
Scheme 2. Proposed Microstructure Transformation of the
PEEP-b-PDMAEMA Diblock Copolymer at Different pH Values
Figure 6. TEM micrographs of (a) aggregates from PEEP32-b-PDMA-
EMA67 diblock copolymer in solution at pH 7.4 with a concentration of
-
1
2
.0 g L , bar = 0.5 μm, and (b) the high-magnification image of (a),
bar = 0.2 μm.
hydrolysis: in acidic conditions the carbon atoms are attac-
ked (as indicated in Figure 8), whereas in basic conditions the
phosphorus atom is attacked. The period breaking 1% of bonds
in the macromolecule was about 150 h at pH 2.0 and 45 ꢀC.
Furthermore, Wang et al. investigated the degradation of
2
2
PEEP-b-PLA-b-PEEP block polymers at various pH. They
found that the M of PEEP-b-PLA-b-PEEP block polymers
that a slight portion of the PEEP segments have been hydro-
lyzed, especially the ethyl group, resulting in an ionic block
bearing negative charges. Wang et al. proved the hydrolytic
products of PEEP at acidic condition, as shown in Figure 9.
After hydrolysis, PEEP-b-PDMAEMA copolymer turned into
polyampholyte species. Therefore, it is reasonable to assume
that the aggregates arise mainly from electrostatic interac-
tions between the partially negatively charged PEEP segments
w
-
1
decreased from 13 740 to 9230 g mol after 21 day in acidic
conditions, demonstrating a slow degradation rate of PEEP
chain.
As PEEP-b-PDMAEMA aqueous solutions with pH 3 were
prepared from the original one (with pH 8.2) by adding hydro-
chloric acid solution and stirred overnight, it is not surprising

Article
Macromolecules, Vol. 43, No. 10, 2010 4777
Figure 8. Chemical structure and its hydrolysis mechanism in acidic
3
8
conditions.
22
Figure 9. Degradation products of PEEP.
Figure 10. BerryplotofPEEP32-b-PDMAEMA67 aggregates dispersed
and the positively charged PDMAEMA segments. A similar
result has been reported by Tsitsilianis et al., who found a
three-dimensional physical network formed in a limited pH
range through electrostatic interactions between the few
negatively charged PAA segments located in the end blocks
and the positively charged P2VP segments located in the
-
3
in water at pH 3.0. The polymer concentration is for (A) 1 ꢀ 10 , (B) 1.5 ꢀ
-3
-3
-1
1
0
, and (C) 2.5 ꢀ 10 g mL
number Nagg was about 1390, since the M of a single poly-
.
w
1
mer chain could be determined from H NMR (M ) and
n
GPC measurement of M /M . This suggests that the micro-
n
w
3
9
middle block of PAA134-P2VP628-PAA134
.
structure is probably a collection of about 1390 polymer
chains. Similarly, the Nagg was about 800 at pH 7.4. How-
ever, the aggregation number was quite small in basic
conditions (pH 10.0) with Nagg of about 3, indicating the
formation of micelles.
On the other hand, the fact that particle size of the aggre-
gates was much larger in acid solutions than those in neutral
and basic solutions can be mainly attributed to the full proto-
nation of the tertiary amine functional groups, which caused
PEEP-b-PDMAEMA micelles to swell as the copolymer chains
Figure 11 tracked the change of the average particle size
as a function of pH values at the polymer concentration of
40
repel each other through electrostatic interactions.
Under the condition of pH 7.4 in Scheme 2, the hydrolysis
of PEEP segments is negligible because of the slow degrada-
-
1
2.0 gL , which was above the cac of PEEP -b-PDMAEMA
32 67
solution by DLS measurement. From Figure 11a-c (pH 2.93-
5.00), the coexistence of large aggregates (about 500 nm) and
single polymer chains (about 7 nm) could be obtained from
the DLS results. This is because of the different degradation
degrees of the polymer chains. A great number of the PEEP-
b-PDMAEMA block copolymers with negatively charged
PEEP block aggregated together into large particles out of
the strong electrostatic interactions. And the Nagg was 1390,
which was much bigger than those formed in both neutral
and basic conditions. At the same time, a few undegraded
polymers existed in the form of unimers due to the repellence
behavior of highly protonated PDMAEMA block. As seen,
the number of unimers decreased with the increase of pH
values and yielded less degradation. Under neutral condi-
tions (Figure 11d-f, ranged from pH 6 to 8), the hydrolysis
of PEEP segments is negligible. Relatively uniformed aggre-
gates of about 300 nm were thus formed due to the interchain
entanglements of those randomly distributed short hydro-
phobic PDMAEMA segments, and the Nagg decreased to
about 800 at pH 7.4. In contrast, when pH was increased to
9.06 or higher, the PDMAEMA block got fully deproto-
nated and became hydrophobic; meanwhile, the hydrolysis
of PEEP block was accelerated. Thus, the association of
PEEP-b-PDMAEMA chains was hindered by the repellence
2
2
tion rate and short time of stirring (12 h). In this case, the
repulsion effect of positive charges of protonated PDMAE-
MA block decreases, and partially deprotonated amine groups
may produce pockets of hydrophobic segments that are ran-
domly distributed along the PDMAEMA chains. Relatively
small and loose aggregates are thus thought to be formed due
to interchain entanglements and association of these short
hydrophobic segments.
In basic conditions as shown in Scheme 2, the PDMAEMA
block is fully deprotonated and becomes a hydrophobic
chain, causing the PEEP-b-PDMAEMA diblock copolymer
to become amphiphilic. Therefore, micelles are formed with
the deprotonated PDMAEMA blocks as a hydrophobic core
and the PEEP blocks as the hydrophilic corona. As described
above, the degradation of PEEP could be accelerated in basic
conditions and degradation rate of main chain was faster
than that of ethyl groups, resulting in a series of negative
charged PEEP-b-PDMAEMA chains with different degrees
of degradation. Thus, large and loose aggregates formed
with those different micelles together.
SLS and DLS analysis can also present evidence for the
above explanations. The weight-average molar mass (M ) of
w
the aggregates at various pH were measured using the SLS
technique via eq 1. Berry plots were used to determine the
molecular parameters of large aggregates, which were con-
23b
of negative charges on hydrolyzed PEEP blocks.
As a
consequence, micelles of about 17 nm in size emerged at pH
9.06. Because the degradation rate of main chain was faster
than the ethyl groups, the degradation degree, the residue
molecular weight, and quantities of negative charge could be
varied. And different micelles aggregated together into
loosely packed micelles aggregates for this reason. As shown
in Figure 11g-i, the number of micelles (17 nm) increased
greatly from pH 9.06 to pH 10.98 with an increased degrada-
tion degree and the consequent enhanced repellence between
PEEP blocks with negative charges.
1
/2
structed by plotting [KC/R (q)] measured at different
vv
2
concentrations and scattering angles against (q þ kC), where
1/2
k is an arbitrary constant. Extrapolation of [KC/R (q)] to
vv
zero concentration or angle yields M , R , and A . Figure 10
2
w
g
shows a typical Berry plot for the large PEEP -b-PDMAE-
3
2
MA67 aggregates at pH 3.0 in deionized water at 22 ꢀC obta-
ined from SLS measurements at scattering angles ranging
from 30ꢀ to 90ꢀ at 10ꢀ intervals. From the Berry plot, M of
w
7
-1
the aggregates was about 3.39ꢀ10 gmol , and the aggregation

4
778 Macromolecules, Vol. 43, No. 10, 2010
Liu et al.
Figure 12. Change in the transmittance of PEEP32-b-PDMAEMA67
polymer solution by repeated thermal cycling.
Figure 11. pH-responsive behaviors of aggregates of PEEP-b-PDMA-
-1
EMA at polymer concentration of 2 g L in H
2
O via DLS measure-
ments at various pH values: (a) pH 2.93, (b) pH 4.04, (c) pH 5, (d) pH
6
.02, (e) pH 6.97, (f) pH 8.01, (g) pH 9.06, (h) pH 9.94, and (i) pH 10.98.
Thermal Properties of PEEP-b-PDMAEMA Diblock Co-
polymers. PEEP homopolymer and copolymer with PPEP
2
4
are thermosensitive with a LCST at around 38 ꢀC or lower.
Meanwhile, PDMAEMA exhibits temperature sensitivity
resulting from (dimethylamino)ethyl groups and generates
4
1
the LCST at around 50 ꢀC. In order to determine the
dependence of the thermal properties on temperature, the
transmittance of light through aqueous solutions of the poly-
mer was measured on a UV-vis spectrometer. The trans-
mittance of 0.5 wt % aqueous solution of the polymers
was monitored at 500 nm at a heating or cooling rate of
Figure 13. Effects of PDMAEMA lengths on temperature dependence
of transmittance changes for 0.5 wt % aqueous solution of PEEP-
b-PDMAEMA: (A) PEEP -b-PDMAEMA ; (B) PEEP -b-PDMAE-
32
31
32
MA40; (C) PEEP32-b-PDMAEMA67
.
-
1
0
.1 ꢀC min . We have found that all solutions exhibited a
the increasing molar fraction of PDMAEMA. Matyjaszewski
et al. reported that the LCST of MEO MA-stat-DMAEMA
copolymers increased with the increasing DMAEMA content.
LCST, and their thermoresponsive behaviors were consis-
tently reversible.
2
27
Figure 12 shows the repeated temperature dependence of
the transmittance of light through PEEP -b-PDMAEMA
67
In fact, the LCST of thermoresponsive polymers can be
controlled by compositions of hydrophobic and hydro-
3
2
43
aqueous solution. The curve coincided well with the varia-
tions in temperature regardless of the number of repetitions
although hysteresis of change in transmittance between the
variations was observed. The phase separation behavior of
the copolymer was easily reproducible. At temperatures
below the LCST of PEEP -b-PDMAEMA diblock copo-
philic units. Thus, LCST can also be further tuned by adjust-
ing the composition of the polyphosphoester block or adding
new hydrophobic blocks to find its application under physio-
logical temperatures.
The effect of pH on the thermal properties of PEEP -b-
32
PDMAEMA67 copolymer was investigated. It showed a sharp
decrease in transmittance around their LCST in neutral
and basic conditions. The changes, which are summarized
in Figure 14, were consistently reversible. The LCST of PEEP -
3
2
67
lymer, the hydrogen bonding between PEEP backbone
-OP(=O)O-] and water molecules is dominant. The PEEP
[
block becomes more hydrophobic with an increase of tem-
perature, which would cause the disruption of hydrogen
bonding and dehydration. Therefore, the balance of hydro-
32
b-PDMAEMA67 at pH 7.5 was 55 ꢀC. And at pH 8.4, the
polymer displayed a lower LCST than that at pH 7.5, sug-
gesting the deprotonation of DMAEMA under basic condi-
tions. Additionally, the LCST of solutions at pH 10 and pH 11
were very close to each other, implying the severe deprotona-
tion of DMAEMA. These results indicate that the PEEP-
b-PDMAEMA diblock copolymer has been successfully syn-
thesized and possesses pH/temperature-responsive behavior.
Characteristics of Polymer/DNA Complexes. Several re-
search groups have investigated PDMAEMA-PEG copoly-
mers with various architectures in order to address the poor
colloidal stabilities of DNA complexes produced by PDMA-
EMA homopolymers and improve their biological proper-
2
5
philicity and hydrophobicity would shift. Meanwhile, hydro-
gen bonding formed between (dimethylamino)ethyl groups
of PDMAEMA and water molecules can also be disrupted
above the LCST, leading to the polymers being hydrophobic
and precipitating from the solution.
Figure 13 shows the effect of the composition of the mono-
mer unit on the LCST of the diblock copolymers. The LCST
of PEEP -b-PDMAEMA was 66 ꢀC, and it was obvious
3
2
31
that LCST of block copolymers decreased when the mole-
cular weight of PDMAEMA increased, while the molecular
weight of PEEP block was roughly constant (decreasing
from 52 ꢀC of PEEP -b-PDMAEMA to 48 ꢀC of PEEP -
1
3,44
ties.
Considering PEEP has good biocompatible and
3
2
40
32
b-PDMAEMA ). In general, the LCST decreases with dec-
biodegradable properties, we used the PEEP-b-PDMAEMA
diblock copolymer to complex with pDNA because it can offer
a potential alternative chemical structure for conferring the
necessary steric stabilization and reducing the cytotoxicity.
6
7
4
2
reasing hydrophilicity of the polymer. Considering the fact
that PDMAEMA block is more hydrophobic compared with
PEEP block, it is reasonable that the LCST decreases with

Article
Macromolecules, Vol. 43, No. 10, 2010 4779
One of the prerequisites as a polymeric gene carrier is
DNA condensation. Agarose gel retardation electrophoresis
was used to study DNA binding affinity of the polymers.
We chose PEEP -b-PDMAEMA diblock copolymer as a
3
2
67
representative sample for use in the DNA complex. The forma-
tion of polyplexes at various N/P ratios was investigated
by gel retardation assay, as shown in Figure 15. pDNA was
totally retained by the presence of PEEP -b-PDMAEMA
3
2
67
at N/P ratio of 3/1 (lane 4). This result revealed that amine
groups of PEEP-b-PDMAEMA carrying positive charges,
which could interact with pDNA phosphate groups with
negative charges to form close-neutral complexes. Mean-
while, for PDMAEMA homopolymer of similar molecular
weight, complete retardation of free pDNA occurred when
the N/P ratio was about 1.0 (date not shown). This phenom-
enon indicates that the complexation of pDNA with the
block copolymers may be partially hindered by the PEEP
chains. Similar results have been reported that the PEG
segment in block copolymers has interference on their
binding affinity to DNA as compared to the homopoly-
Figure 14. Effect of pH values on LCST of PEEP32-b-PDMAEMA67
(0.5 wt % aqueous solution).
4
5
mer. An important advantage of PPE over conventionally
used PEG is that polyphosphoester is biodegradable, and the
degradation rate of polyphosphoester may be adjusted by
controlling the chemical structure of the backbone and side
chain.
Transport of the gene transfer vector into the target cell
is required through different barriers, across the centimeter
Figure 15. Agarose gel electrophoresis of PEEP32-b-PDMAEMA67
/
(in blood circulation) to nanometer size ranges (intracellu-
larly). For efficient endocytosis and gene transfer, the com-
plex must be small (below 150 nm) and compact. Examination
Plasmid pUC 18 DNA complexes at various N/P ratios, as indicated on
the top of the lanes. In the panel, the rightmost lane represents the
migration of the uncomplexed pUC 18 marker.
4
6
47
Figure 16. TEMmicrographs ofvarious particles formed in 10 mM PBS buffer at pH7.4: (a) naked DNA particles fromPlasmid pUC 18 DNA of1.0 g
-1
L
, bar = 50 nm; (b) PEEP32-b-PDMAEMA67/pDNA complexes at N/P ratio =1, bar = 50 nm; (c) PEEP32-b-PDMAEMA67/pDNA complexes at
N/P ratio = 2, bar = 100 nm; (d) PEEP32-b-PDMAEMA67/pDNA complexes at N/P ratio = 3, bar = 100 nm.

4
780 Macromolecules, Vol. 43, No. 10, 2010
Liu et al.
small (about 95 nm in size) and positively charged complexes
which are suitable for gene delivery. Further modification of the
block copolymers and related bioresearch are in progress in our
laboratory now. We expect that the polymer would provide
potential applications in gene therapy.
Acknowledgment. The authors are thankful for the financial
support from the National Natural Science Foundation of China
(
(
20974074), the Natural Science Foundation of Jiangsu Province
BK2008157), Qing Lan Project for Innovation Team of Jiangsu
Province, and the Program of Innovative Research Team of
Soochow University. The authors are indebted to Mr. Weizhong
Chen, from University of Science and Technology of China, for
his valuable help in LLS experiments and discussion. J.H. has
received the financial support from the Innovation Project of
Graduate Students of Jiangsu Province, China.
Figure 17. Zeta-potential of PEEP32-b-PDMAEMA67/pDNA complex
at various N/P ratios.
of the morphology of the PEEP -b-PDMAEMA /pDNA
3
References and Notes
2
67
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and the condensation process. Figure 16 shows the transmis-
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Conclusions
(
In summary, we have utilized the combination of ring-opening
polymerization (ROP) and ATRP to synthesize a series of well-
defined double-hydrophilic diblock copolymers containing
polyphosphoester block and PDMAEMA block. These PEEP-
b-PDMAEMA diblock copolymers show obvious pH- and tem-
perature-responsive behavior, so they can self-assemble into nano-
particles with different sizes and morphologies when the pH values
of aqueous solution were adjusted in the range of 3.0-10. The
LCSTs of the diblock copolymers depend on the degrees of
polymerization of each block. With the decrease of PDMAEMA
units, the increasing LCST can be observed. Besides, PEEP-
b-PDMAEMA diblock copolymers showed excellent DNA bind-
ing characteristics. The PEEP-b-PDMAEMA diblock copolymer
can effectively condense pDNA at N/P ratio = 3, resulting in
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