Cationic methacrylate copolymers containing primary and tertiary amino side groups: Controlled synthesis via RAFT polymerization, DNA condensation, and in vitro gene transfection

Article abstract of DOI:10.1002/pola.24064

A versatile family of cationic methacrylate copolymers containing varying amounts of primary and tertiary amino side groups were synthesized and investigated for n vitro gene transfection. Two different types of methacrylate copolymers, poly(2-(dimethylamino)ethyl methacrylate)/aminoethyl methacrylate [P(DMAEMA/AEMA)j and poly(2-(dimethylamino)ethyl methacrylate)/aminohexyl methacrylate [P(DMAEMA/AHMA)j, were obtained by reversible addition-fragmentation chain transfer (RAFT) copolymerization of dimethylaminoethyl methacrylate (DMAEMA) with ZV-(tert-butoxycarbonyl)aminoethyl methacrylate (Boc-AEMA) or ZV-(ferf-butoxycarbonyl)aminohexyl methacrylate (Boc-AHMA) followed by acid deprotection. Gel permeation chromatography (GPC) measurements revealed that Boc-protected methacrylate copolymers had M _n in the range of 16.1-23.0 kDa and low polydispersities of 1.12-1.26. The copolymer compositions were well controlled by monomer feed ratios. Dynamic light scattering and agarose gel electrophoresis mea-surements demonstrated that these PDMAEMA copolymers had better DNA condensation than PDMAEMA homopolymer. The polyplexes of these copolymers revealed low cytotoxicity at an NIP ratio of 3/1. The in vifro transfection in COS-7 cells in serum free medium demonstrated significantly enhanced (up to 24fold) transfection efficiencies of PDMAEMA copolymer polyplexes as compared with PDMAEMA control. In the presence of 10% serum, P(DMAEMA/AEMA) and P(DMAEMA/AHMA) displayed a high transfection activity comparable with or better than 25 kDa PEI. These results suggestthat cationic methacrylate copolymers are highly promising for development of safe and efficient nonviral gene transfer agents.

Full text of DOI:10.1002/pola.24064

Cationic Methacrylate Copolymers Containing Primary and Tertiary
Amino Side Groups: Controlled Synthesis via RAFT Polymerization,
DNA Condensation, and In Vitro Gene Transfection
1
2
1
1
1
1
CAIHONG ZHU, SOOYEON JUNG, GUOYING SI, RU CHENG, FENGHUA MENG, XIULIN ZHU,
2
1
TAE GWAN PARK, ZHIYUAN ZHONG
1
Biomedical Polymers Laboratory, Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemistry, Chemical
Engineering and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China
2
Department of Biological Sciences, Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and
Technology, Daejeon 305-701, South Korea
Received 17 March 2010; accepted 7 April 2010
DOI: 10.1002/pola.24064
Published online in Wiley InterScience (www.interscience.wiley.com).
ABSTRACT: A versatile family of cationic methacrylate copoly-
mers containing varying amounts of primary and tertiary amino
side groups were synthesized and investigated for in vitro gene
transfection. Two different types of methacrylate copolymers,
poly(2-(dimethylamino)ethyl methacrylate)/aminoethyl meth-
acrylate [P(DMAEMA/AEMA)] and poly(2-(dimethylamino)ethyl
methacrylate)/aminohexyl methacrylate [P(DMAEMA/AHMA)],
were obtained by reversible addition-fragmentation chain trans-
fer (RAFT) copolymerization of dimethylaminoethyl meth-
acrylate (DMAEMA) with N-(tert-butoxycarbonyl)aminoethyl
methacrylate (Boc-AEMA) or N-(tert-butoxycarbonyl)aminohexyl
methacrylate (Boc-AHMA) followed by acid deprotection. Gel
permeation chromatography (GPC) measurements revealed that
surements demonstrated that these PDMAEMA copolymers had
better DNA condensation than PDMAEMA homopolymer. The
polyplexes of these copolymers revealed low cytotoxicity at an
N/P ratio of 3/1. The in vitro transfection in COS-7 cells in serum
free medium demonstrated significantly enhanced (up to 24-
fold) transfection efficiencies of PDMAEMA copolymer poly-
plexes as compared with PDMAEMA control. In the presence of
10% serum, P(DMAEMA/AEMA) and P(DMAEMA/AHMA) dis-
played a high transfection activity comparable with or better
than 25 kDa PEI. These results suggest that cationic methacrylate
copolymers are highly promising for development of safe and
efficient nonviral gene transfer agents. VC 2010 Wiley Periodicals,
Inc. J Polym Sci Part A: Polym Chem 48: 2869–2877, 2010
n
Boc-protected methacrylate copolymers had M in the range of
1
6.1–23.0 kDa and low polydispersities of 1.12–1.26. The copoly-
KEYWORDS: biological applications of polymers; copolymers;
DNA polyplexes; gene delivery; living radical polymerization;
nanoparticles; PDMAEMA; RAFT polymerization
mer compositions were well controlled by monomer feed ratios.
Dynamic light scattering and agarose gel electrophoresis mea-
INTRODUCTION In the past decade, polymer-based gene
delivery systems have attracted a tremendous amount of
attention for gene transfer due to many advantages they
offer over their viral counterparts including improved safety,
low immune responses, enabling repeated uses, and ease of
amine) render effective complexation and protection of DNA,
whereas amine groups with a low pK
tiary amine) are hypothesized to facilitate endosomal escape
(e.g., secondary or ter-
a
1
1–13
through proton sponge mechanism.
Langer and Cho
reported that transfection activity of PLL and chitosan can
be largely augmented by introduction of imidazole functions
1
–5
production.
However, polymeric vectors are associated
1
4,15
(
pK ꢀ 6.0).
It has been shown by different groups on
with several extracellular and intracellular barriers such as
inadequate protection of DNA from enzymatic degradation,
poor cellular uptake, insufficient endosomal escape, and/or
inefficient intracellular release of DNA, which result in usu-
a
various types of cationic polymers that combination of
assorted amine groups with a wide buffer range gives rise to
best transfection activity.
1
6–22
The endosomal escape of poly-
6
,7
plexes is, however, not always enhanced by polymers buffer-
ally low to moderate transfection efficiencies.
Unlike
2
3
ing at low pH.
poly(L-lysine) (PLL) and chitosan, which contain only pri-
mary amines, polyethylenimine (PEI) and poly(amido amine)
It is interesting to note that high molecular weight (Mw > 300
(
PAMAM) dendrimer (two best polymeric transfection
kDa) poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)
agents) contain multivalent amine groups with distinct pK
a
which contains solely tertiary amines mediates efficient trans-
8
–10
24–26
values.
The amine groups with a high pK (e.g., primary
fection in various types of cells.
PDMAEMA is shown to
a
Correspondence to: F. Meng (E-mail: fhmeng@suda.edu.cn) or Z. Zhong (E-mail: zyzhong@suda.edu.cn)
Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 2869–2877 (2010) V
2010 Wiley Periodicals, Inc.
C
CATIONIC METHACRYLATE COPOLYMERS, ZHU ET AL.
2869

JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
SCHEME 1 Synthetic pathway to
cationic methacrylate copolymers
containing primary and tertiary
amino side groups (x
¼
1:
AEMA; 3: AHMA). Condi-
x
¼
tions: (i) RAFT polymerization,
ꢁ
dioxane, 70 C, 40 h; (ii) de-pro-
tection, r.t., TFA, HCl. [Color fig-
ure can be viewed in the online
issue, which is available at
www.interscience.wiley.com.]
2
7
display high buffer capacity at endosomal pH. However,
PDMAEMA is not readily biodegradable, which may render
toxicity problems when high molecular weight polymer is
di-tert-butyl dicarbonate (99%, J & K Chemical), triethyl-
amine (TEA, 99%, Alfa Aesar), methacryloyl chloride (Wuxi
Chemical), and trifluoroacetic acid (TFA, 99%, Aldrich) were
used as received.
2
8,29
used. In the past years, bioreducible PDMAEMA polymers,
3
0–33
biodegradable
PDMAEMA
copolymers,
PHEMA-
3
4
Synthesis of N-(tert-butoxycarbonyl)aminoethyl
methacrylate (Boc-AEMA)
PDMAEMA-PEG-PDMAEMA-PHEMA pentablock copolymers,
and copolymers of DMAEMA and N-vinyl-pyrrolidone (NVP)
3
5
Under a nitrogen atmosphere and vigorously stirring, to a
solution of 2-aminoethanol (8.6 g, 143.1 mmol) in 100 mL of
have been investigated for DNA condensation and/or in vitro
transfection. Unlike other cationic polymers including PEI and
PAMAM dendrimer, PDMAEMA copolymers can be conven-
iently prepared with controlled macromolecular structures
and compositions by living radical polymerizations including
reversible addition-fragmentation chain transfer (RAFT)
ꢁ
anhydrous DCM at 0 C was added dropwise a solution
of di-tert-butyl dicarbonate (15.3 g, 70.1 mmol) in 70 mL of
DCM. The reaction mixture was warmed to r.t. and reacted
for additional 20 h. The reaction mixture was filtered, the fil-
trate was concentrated, and the residue was dissolved in
3
6,37
polymerization.
1
00 mL saturated NaCl solution. The resultant aqueous solu-
tion was acidified to pH 4–5 and extracted with ethyl acetate
3 ꢂ 40 mL). The collected organic fractions were dried over
Na SO overnight and ethyl acetate was evaporated to yield
In this article, we report a versatile family of DMAEMA-based
copolymers that contain varying amounts of primary and ter-
tiary amino side groups for enhanced gene transfection
Scheme 1). These copolymers were designed with relatively
low molecular weights (M < 25 kDa) and DMAEMA as the
(
2
4
(
N-(tert-butoxycarbonyl)-2-aminoethanol as a colorless oil.
Yield: 71.8%.
n
major component, thereby retaining a good buffer capacity
at endosomal pH. The incorporation of primary amino func-
tions was intended to increase their charge density at physi-
ological pH, which on one hand may improve DNA condensa-
tion ability and on the other hand may enhance water
solubility and colloidal stability of DNA polyplexes. In this
study, two series of well-defined PDMAEMA copolymers,
based on aminoethyl methacrylate (AEMA) or aminohexyl
methacrylate (AHMA), were prepared by controlled radical
polymerization approach. The influences of copolymer struc-
ture and composition on DNA condensation, transfection ac-
tivity and cytotoxicity were investigated.
1
H NMR (300 MHz, CDCl ): d 1.44 (s, 9H, AC(CH ) ), 2.47 (s,
3
3 3
1
H, AOH), 3.28 (m, 2H, ACONHCH A), 3.70 (m, 2H,
2
2
ACH OH), 4.96 (br.s, 1H, ANHBoc).
To a solution of N-(tert-butoxycarbonyl)-2-aminoethanol (8.1
g, 50.3 mmol) and TEA (15 mL, 103.8 mmol) in 75 mL of
anhydrous DCM at 0 C was added dropwise a solution of
methacryloyl chloride (9.8 mL, 100.9 mmol) in 25 mL of
DCM. The reaction mixture was warmed to r.t. and reacted
for additional 24 h. After removing white precipitate, the fil-
trate was washed with water (3 ꢂ 100 mL) and dried over-
night over Na SO . The resultant solution was concentrated
ꢁ
2
4
and purified through a silica gel column with petroleum
ether/ethyl acetate (7/1, v/v) to yield Boc-AEMA as a white
solid. Yield: 62.9%.
EXPERIMENTAL
Materials
2
-(N,N-dimethylamino)ethyl methacrylate (DMAEMA, 97%,
1
H NMR (300 MHz, CDCl ): d 1.44 (s, 9H, AC(CH ) ), 1.95 (s, 3H,
3
3 3
Alfa Aesar) was purified by passing through a basic alumina
column before use. 4-Cyanopentanoic acid dithionaphthale-
noate (CPADN) was synthesized according to a reported pro-
cedure for 4-(4-cyanopentanoic acid) dithiobenzoate. Azo-
bisisobutyronitrile (AIBN, 99%, Chemical) was
ACH ), 3.44 (m, 2H, ACH NHBoc), 4.20 (t, 2H, ACOOCH A),
4
3
2
2
.77 (br.s, 1H, ANHBoc), 5.59/6.13 (s, 2H, CH ¼¼ CH).
2
3
8
Synthesis of N-(tert-Butoxycarbonyl)aminohexyl
methacrylate (Boc-AHMA)
J
&
K
recrystallized twice from hexane and methanol, respectively.
Dichloromethane (DCM) was dried by refluxing over CaH2
and distilled before use. 2-Aminoethanol (Sinopharm Chemi-
cal Reagent Co. Ltd.), 6-amino-1-hexanol (97%, Aldrich),
Under a nitrogen atmosphere and vigorously stirring, to a
solution of 6-amino-1-hexanol (3.3 g, 28.4 mmol) in 30 mL
of anhydrous DCM at 0 C was added dropwise a solution of
ꢁ
di-tert-butyl dicarbonate (7.1 g, 32.4 mmol) in 20 mL of
2870
INTERSCIENCE.WILEY.COM/JOURNAL/JPOLA

ARTICLE
DCM. The reaction mixture was warmed to r.t. and reacted
for additional 20 h. The reaction mixture was then diluted
with 60 mL diethyl ether and successively washed with so-
dium phosphates buffer (3 ꢂ 30 mL, pH 5.4), saturated so-
dium dicarbonate solution (1 ꢂ 50 mL), and saturated NaCl
solution (1 ꢂ 50 mL). The organic phase was dried over-
night over Na SO and concentrated by rotary evaporation to
polymer was isolated by precipitation in acetone, filtration,
and drying in vacuo. Yield: 75–90%.
1
2
H NMR (300MHz, D O) for P(DMAEMA/AEMA): d 4.36,
3
0
.35, 2.02, 1.11/0.94 (PAEMA); 4.36, 3.55, 2.96, 2.02, 1.11/
1
.94 (PDMAEMA). H NMR (300MHz, D O) for P(DMAEMA/
2
AHMA): d 3.98, 2.99, 1.99, 1.67, 1.41, 1.11/0.94 (PAHMA);
.36, 3.56, 2.99, 1.99, 1.11/0.94 (PDMAEMA).
2
4
4
yield N-(tert-butoxycarbonyl)-6-amino-1-hexanol as a white
solid. Yield: 71.5%.
Polymer Characterization
H NMR spectra of copolymers were recorded on an INOVA
1
1
H NMR (300 MHz, CDCl
H, AOH), 1.56 (m, 2H, CH CH OH), 1.47 (m, 2H, ACH CH
2
3 3 3
): d 1.43 (s, 9H, AC(CH ) ), 1.75 (s,
3
00 MHz nuclear magnetic resonance instrument using
CDCl or D O as a solvent. The chemical shifts were cali-
brated against solvent signal of CDCl or D O. The molecular
1
2
2
2
3
2
NHBoc), 1.35 (m, 4H, ANHCH CH CH CH CH CH OH), 3.63
2
2
2
2
2
2
3
2
(
t, 2H, HOCH A), 3.10 (m, 2H, ACH NHBoc), 4.54 (br, 1H,
2 2
weight and polydispersity (PDI) of copolymers were deter-
mined with a Waters 1515 gel permeation chromatography
ANHBoc).
To a solution of N-(tert-butoxycarbonyl)-6-amino-1-hexanol
3
4
(
GPC) instrument equipped with 500, 10 and 10 Å col-
(
4.3 g, 19.6 mmol) and TEA (6 mL, 41.5 mmol) in 50 mL of
umns (MZ-Gel SD plus) and a differential refractive index de-
tector (RI2414). The measurements were performed using
DMF containing 0.05 M LiBr as eluent at a flow rate of 0.8
ꢁ
anhydrous DCM at 0 C was added dropwise a solution of
methacryloyl chloride (3.8 mL, 39.1 mmol) in 10 mL of DCM.
The reaction mixture was warmed to r.t. and reacted for
additional 24 h. After removing white precipitate, the filtrate
was washed with water (3 ꢂ 100 mL) and dried overnight
over Na SO . The resultant solution was concentrated and
ꢁ
mL/min at 30 C and a series of polystyrene as standards.
Buffer Capacity Measurements
The buffer capacity of PDMAEMA copolymers was deter-
2
4
3
9
purified through a silica gel column with petroleum ether/
ethyl acetate (5/1, v/v) to yield Boc-AHMA as a pale yellow
oil. Yield: 56.9%.
mined by acid–base titration. Briefly, polymer was dis-
solved in 150 mM NaCl aqueous solution to yield 20 mM
nitrogen concentration. The pH of the solution was firstly
brought to 2.0, and then titrated with 0.1 M NaOH. The
buffer capacity defined as the percentage of amine groups
becoming protonated from pH 5.1 to 7.4 was calculated from
equation:
1
H NMR (300 MHz, CDCl ): d 1.44 (s, 9H, AC(CH ) ), 1.67 (m,
3
3 3
2
4
4
1
H, CH CH OCOA), 1.50 (m, 2H, ACH CH NHBoc), 1.37 (m,
2 2 2 2
H, ANHCH CH CH CH CH CH OCOA), 1.94 (s, 3H, ACH ),
2
2
2
2
2
2
3
.13 (t, 2H, ACH OCOA), 3.10 (m, 2H, ACH NHBoc), 4.52 (br.,
2
2
H, ANHBoc), 5.55/6.09 (s, 2H, CH ¼¼ CH).
2
Buffer capacity ð%Þ ¼ 100ðDVNaOH ꢂ 0:1MÞ=N mol:
Synthesis of DMAEMA-Based Copolymers
RAFT Polymerization
The copolymerization of DMAEMA with Boc-AEMA or Boc-
AHMA was carried out using CPADN as a RAFT agent and
AIBN as a radical source. In a typical example, under a nitro-
gen atmosphere, DMAEMA (0.92 g, 5.83 mmol), Boc-AEMA
wherein DVNaOH is the volume of NaOH solution required to
bring the pH value of the copolymer solution from 5.1 to 7.4,
and N mol is the total moles of protonable amine groups
(
0.1 mmol).
Preparation and Characterization of DNA Polyplexes
The polyplexes were prepared by adding a HEPES buffer so-
lution (400 lL, 20 mM, pH 7.4) of polymer with the desired
concentration to a HEPES buffer solution (400 lL, 20 mM,
pH 7.4) of plasmid DNA (20 lg/mL), which resulted in poly-
plexes with N/P ratios ranging from 3/1 to 9/1. The disper-
sions were vortexed for 5 s and incubated at r.t. for 15 min
before n-potential and size measurements were carried out.
The n-potential and hydrodynamic diameters of polyplexes
(
(
0.33 g, 1.46 mmol), CPADN (12.0 mg, 0.0365 mmol), AIBN
0.6 mg, 0.0036 mmol), and 2.5 mL dioxane were charged
into a Schlenk flask. The polymerization was performed at
ꢁ
7
0 C for 40 h. The resulting copolymer was isolated by pre-
cipitation in cold hexane, filtration and drying in vacuo.
Yield: 72–84%.
1
H NMR (300 MHz, CDCl ) of P(DMAEMA/Boc-AEMA): d
3
4.06, 3.36, 1.81–1.95, 1.45, 1.04/0.88 (poly(Boc-AEMA));
ꢁ
prepared at an N/P ratio of 3/1 were determined at 25 C
1
4.06, 2.58, 2.28, 1.81–1.95, 1.04/0.88 (PDMAEMA). H NMR
using a Zetasizer Nano ZS Instruments (Malvern) equipped
with a standard capillary electrophoresis cell and dynamic
light scattering (DLS, 10 mW He-Ne laser, 633 nm wave-
length), respectively. The measurements were performed in
triplicate.
(
300 MHz, CDCl ) of P(DMAEMA/Boc-AHMA): d 3.91, 3.09,
3
1
2
.35–1.62, 1.45, 1.04/0.88 (poly(Boc-AHMA)); 4.07, 2.58,
.29, 1.80–1.93, 1.04/0.88 (PDMAEMA).
Acid Deprotection
Under vigorously stirring, to
a
solution of 0.9
g
of
Gel Retardation Assay
P(DMAEMA/Boc-AEMA) or P(DMAEMA/Boc-AHMA) copoly-
mer in 4 mL of DCM was added dropwise 4 mL of TFA. After
The DNA binding ability of PDMAEMA copolymers was stud-
ied by agarose gel electrophoresis. The polymer/DNA com-
plexes prepared at varying N/P ratios from 0.75/1 to 3/1
were electrophoresed through a 1% agarose gel containing
2
h reaction at room temperature, 2 mL of concentrated HCl
(
12 M) was added and stirred for 30 min. The resulting co-
CATIONIC METHACRYLATE COPOLYMERS, ZHU ET AL.
2871

JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
a
TABLE 1 Synthesis of P(DMAEMA/Boc-AEMA) and P(DMAEMA/Boc-AHMA) Copolymers
d
GPC
b
c
Entry
Polymer
f
DMAEMA (%)
Yield (%)
F
DMAEMA (%)
M
n
(kDa)
PDI
1
2
3
4
5
6
7
a
P(DMAEMA/Boc-AEMA) 91/9
P(DMAEMA/Boc-AEMA) 79/21
P(DMAEMA/Boc-AEMA) 70/30
P(DMAEMA/Boc-AHMA) 91/9
P(DMAEMA/Boc-AHMA) 79/21
P(DMAEMA/Boc-AHMA) 72/28
PDMAEMA
90
80
70
90
80
70
84.2
81.1
71.9
81.7
75.3
72.7
92.6
91
79
70
91
79
72
16.1
18.2
21.8
16.1
16.7
23.0
13.4
1.19
1.16
1.12
1.20
1.26
1.21
1.26
100
100
RAFT polymerization conditions: [monomer]total/[CPADN]/[AIBN] ¼ 200/1/0.1, dioxane, 70 ꢁC, 40 h.
b
c
Molar fraction of DMAEMA in the feed.
1
Molar fraction of DMAEMA units in the resulting copolymers determined by H NMR.
d
ꢁ
Determined by GPC measurements (eluent: DMF containing 0.05 M LiBr, standards: polystyrene, flow rate: 0.8 mL/min, 30 C).
ethidium bromide at 100 V in TAE solution (40 mM Tris-HCl,
obtained from ethanolamine and hexanolamine through
selective protection of amino group with Boc and subsequent
methacrylation with methacryloyl chloride. The copolymer-
1
v/v % acetic acid, and 1 mM EDTA).
In Vitro Transfection and Cell Viability Assays
ꢁ
ization was performed in dioxane at 70 C for 2 days using
Transfection experiments were performed in COS-7 cells using
the plasmid pCMV-Luc as a reporter gene. Transfections were
conducted using polyplexes formed at N/P ratios of 3/1, 6/1,
CPADN. CPADN is a versatile RAFT agent, through which we
have obtained degradable PDMAEMA-PCL-PDMAEMA tri-
31
block copolymers. The total monomer-to-CPADN mole feed
and 9/1. The cells were plated in a 12-well plate (cell density
ratio was set at 200/1 and mole fraction of DMAEMA in the
feed was varied from 70%, 80% to 90%.
5
1
.5 ꢂ 10 cells/well) and maintained in DMEM supplemented
ꢁ
with 10% FBS at 37 C in a humidified atmosphere containing
1
The copolymerization results are summarized in Table 1. H
5
% CO2 until 70% confluency. In a standard transfection
experiment, the cells were rinsed with PBS and incubated with
00 lL of polyplex dispersion (i.e., 1 lg of plasmid DNA per
NMR spectrum of P(DMAEMA/Boc-AEMA) showed clearly
resonances characteristic of DMAEMA and Boc-AEMA units
1
[
Fig. 1(A)]. The copolymer compositions could be calculated by
well) and 400 lL of culture medium with or without 10% se-
ꢁ
comparing the intensities of signals at d 2.56 and 3.38, which
were assignable to methylene protons neighboring to the
amino group of PDMEMA and methylene protons next to the
amide group of P(Boc-AEMA), respectively. In a similar way,
compositions of P(DMAEMA/Boc-AHMA) copolymers could
also be determined [Fig. 1(B)]. Interestingly, our results
pointed to excellent control over copolymer compositions
rum for 4 h at 37 C. Next, the polyplexes were removed, 1 mL
of fresh culture medium was added, and the cells were cul-
tured for 43 h. Luciferase quantification was done using a com-
mercial luciferase assay kit (Promega, Madison, WI) and a
Lumat LB 9501 illuminometer (Berthold, Milbach, Germany).
Transfection efficiency was expressed as relative light unit
(
RLU) per mg of protein. A 25 kDa bPEI/DNA formulation pre-
(
Table 1). The copolymer compositions had little change with
pared at an optimal N/P ratio of 10/1 was used as a reference.
increasing conversions, indicating similar reactivities of
DMAEMA with Boc-AEMA or Boc-AHMA. Furthermore, GPC
curves showed that all copolymers had a unimodal distribution
The cytotoxicity of polyplexes was evaluated with the same
cell culture procedure as the transfection with 10% serum,
followed by CCK assay. The cells were incubated with CCK-8
reagent solution for 3.5 h before measurement of absorption
at 450 nm using a microplate reader (BIORAD, Model 550).
The CCK value for the untreated cells (i.e., cells not exposed
to transfection systems) was taken as 100% cell viability.
with M in the range of 16.1–23.0 kDa and low polydispersities
n
of 1.12–1.26 (Table 1). These copolymers were subsequently
treated with CF COOH/CH Cl (1:1 v/v), to yield a freely water
3
2
2
1
soluble product. Notably, H NMR spectra (Fig. 2) showed com-
plete disappearance of signal at d 1.44 that is attributable to
the methyl protons of Boc group, indicating quantitative depro-
tection. The mole fraction of PDMAEMA in the resulting copoly-
mers, however, was not changed upon acid deprotection (Fig.
2), supporting successful preparation of P(DMAEMA/AEMA)
and P(DMAEMA/AHMA) copolymers. For comparison, we have
also obtained PDMAEMA homopolymer (Mn ¼ 13.4, PDI ¼
1.26) using the same procedure (Table 1, entry 7).
RESULTS AND DISCUSSION
Synthesis of DMAEMA-Based Copolymers
Cationic methacrylate copolymers containing varying
amounts of primary and tertiary amino side groups were
synthesized by RAFT copolymerization of DMAEMA with
Boc-AEMA or Boc-AHMA followed by acid deprotection
(
Scheme 1). RAFT polymerization is a versatile yet controlled
free radical polymerization technique that has been exploited
for synthesis of well-defined functional polymers and copoly-
Buffer Capacity of Methacrylate Copolymers
It has been reported that the high transfection activity of PEI
is intimately related to its buffer capacity at endosomal pH,
4
0,41
mers.
Boc-AEMA and Boc-AHMA monomers were readily
2872
INTERSCIENCE.WILEY.COM/JOURNAL/JPOLA

ARTICLE
and P(DMAEMA/AHMA) copolymers were prepared at an N/P
ratio of 3/1 in HEPES buffer (20 mM, pH 7.4). DLS mea-
surements showed that all copolymers could effectively con-
dense plasmid DNA to give small sized polyplexes with mean
diameters of 60–110 nm for P(DMAEMA/AEMA) and 60–
75 nm for P(DMAEMA/AHMA) [Fig. 4(A)]. The sizes of
P(DMAEMA/AEMA) polyplexes decreased from 110 nm to 60
nm with increasing contents of AEMA from 9 to 30%, which is
in accordance with our hypothesis that incorporation of pri-
mary amino groups will result in enhanced DNA condensation.
Notably, P(DMAEMA/AHMA) copolymers with only 9% AHMA
were shown to condense DNA into ꢀ60 nm particles. It has
been reported that cationic polymers with certain hydropho-
42
bicity often lead to small-sized DNA particles. In comparison,
polyplexes of PDMAMEA homopolymer at an N/P ratio of 3/1
had somewhat larger size of ꢀ120 nm. f-potential measure-
ments displayed that polyplexes of P(DMAEMA/AEMA) and
P(DMAEMA/AHMA) copolymers had positive surface charges
of þ15 mV and þ26 mV, respectively, wherein content of
AEMA or AHMA did not seem to play a significant role in f-
potentials of polyplexes [Fig. 4(B)]. Agarose gel electrophoresis
1
3
FIGURE 1 H NMR spectra (300MHz, CDCl ) of P(DMAEMA/Boc-
AEMA) 70/30 copolymer (A) and P(DMAEMA/Boc-AHMA) 72/28
copolymer (B). [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
which facilitates endosomal escape of DNA polyplexes by
1
1–13
‘‘proton sponge effect.’’
The buffer capacity of these
methacrylate copolymers, defined as the percentage of amino
functions becoming protonated in the pH range from pH 7.4
to 5.1, was determined by acid-base titration. The results
showed that the buffer capacity of methacrylate copolymers
in general decreased with increasing content of AEMA or
AHMA (Fig. 3). This is in line with our expectation since
AEMA and AHMA units present only primary amino groups
that usually have a high pK . It should be noted, neverthe-
a
less, that the buffer capacity of these methacrylate copoly-
mers remains rather high, ranging from 26.6% to 53.1%, as
DMAEMA units are still the major component (70–91 mol
%).
1
FIGURE
2
H
NMR spectra (300MHz,
D
2
O) of P(DMAEMA/
Biophysical Characterization of DNA Polyplexes
To investigate influences of AEMA or AHMA units on DNA
complexation behaviors, polyplexes of P(DMAEMA/AEMA)
AEMA) 70/30 copolymer (A) and P(DMAEMA/AHMA) 72/28 co-
polymer (B). [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
CATIONIC METHACRYLATE COPOLYMERS, ZHU ET AL.
2873

JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
FIGURE 3 Acid–base titration curves for PDMAEMA copolymers
(
at a concentration of 20 mM nitrogen) titrated by 0.1 M NaOH.
showed that P(DMAEMA/AEMA) and P(DMAEMA/AHMA)
copolymers were capable of condensing DNA at and above an
N/P ratio of 1.5/1 or 0.75/1 depending on contents of AEMA
and AHMA, whereas for PDMAEMA complete DNA retardation
was observed only at a higher N/P ratio of 3/1 (Fig. 5). These
results fully confirmed that incorporation of primary amines
into PDMAEMA is an effective approach to enhance its DNA
complexation capacity.
FIGURE 4 Average particle size (A) and zeta potential (B) of
DNA polyplexes based on P(DMAEMA/AEMA) and P(DMAEMA/
AHMA) copolymers at an N/P ratio of 3/1.
In Vitro Cytotoxicity and Transfection
The cytotoxicity of polyplexes based on P(DMAEMA/AEMA)
and P(DMAEMA/AHMA) copolymers was evaluated by CCK
assay using COS-7 cells at different N/P ratios ranging from
3/1 to 9/1. Interestingly, P(DMAEMA/AEMA) copolymers
displayed in general comparable or improved cell viability as
compared with PDMAEMA under otherwise the same condi-
tions, in which P(DMAEMA/AEMA) copolymer with 9%
AEMA revealed minimal cytotoxicity up to an N/P ratio of 9/
1
(Fig. 6). P(DMAEMA/AHMA) copolymers showed practi-
cally similar level of cytotoxicity with respect to PDMAEMA
at N/P ratios of 3/1 and 6/1, and the copolymer composi-
tion appeared to have little influence on toxicity. It should be
noted that polyplexes of all copolymers revealed a low cyto-
toxicity at an N/P ratio of 3/1, in which approximately 80%
or above cell viability was observed (Fig. 6).
The in vitro transfection activity of polyplexes based on
PDMAEMA copolymers in COS-7 cells was studied using the
plasmid pCMV-Luc as a reporter gene. Remarkably, poly-
plexes of all copolymers demonstrated much better
FIGURE 5 Agarose gel electrophoresis of polymer/DNA com-
plexes based on P(DMAEMA/AEMA) and P(DMAEMA/AHMA)
copolymers at varying N/P ratios. Lane 1 is free DNA; Lanes
2–4 correspond to N/P ratios of 0.75, 1.5, and 3, respectively.
2874
INTERSCIENCE.WILEY.COM/JOURNAL/JPOLA

ARTICLE
via the hydrophobic interactions. DLS measurements
showed that polyplexes of P(DMAEMA/AHMA) 91/9 and
P(DMAEMA/AEMA) 91/9 formed at an N/P ratio of 3/1 fol-
lowing 4 h incubation with transfection medium had average
diameters of 280 nm and over 400 nm, respectively. Kim,
Langer, and Feijen reported separately that linear and
branched poly(b-amino ester)s composed of relatively hydro-
2
0,42,43
phobic units exhibit best transfection activity.
It should
be noted that P(DMAEMA/AHMA) has achieved high trans-
fection at a low N/P ratio of 3/1. In comparison, high mole-
cular weight PDMAEMA (Mw > 300 kDa) and 25 kDa PEI
afforded optimal transfections at an N/P ratio of 6/1 and
4
4
10/1, respectively.
Interestingly, when transfection was carried out in the pres-
ence of 10% serum, polyplexes of P(DMAEMA/AEMA) 91/9
formed at N/P ratios of 6/1 and 9/1, which had minimal cyto-
toxicity (Fig. 6), showed transfection activity comparable with
FIGURE 6 Cell viabilities of DNA polyplexes based on
P(DMAEMA/AEMA) and P(DMAEMA/AHMA) copolymers in
COS-7 cells at varying N/P ratios from 3/1 to 9/1 in 10% serum
condition. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
25 kDa PEI control. Moreover, two copolymers, P(DMAEMA/
AHMA) 72/28 and P(DMAEMA/AEMA) 70/30, mediated signif-
icantly more efficient transfection at an N/P ratio of 3/1 and
9/1, respectively, than 25 kDa PEI (Fig. 8). Ulbrich and Sey-
mour have reported previously that copolymer of DMAEMA
and AEMA has a higher transfection activity than PDMAEMA,
likely due to their combination of primary and tertiary
transfection efficiencies than those of PDMAEMA control at
the same N/P ratios in serum free medium (Fig. 7). The
transfection activity seemed to follow the order P(DMAEMA/
AHMA) > P(DMAEMA/AEMA) > PDMAMEA, in which poly-
plexes of P(DMAEMA/AHMA) displayed up to 24-fold higher
transfection efficiency as compared with those of PDMAEMA.
The elevated transfection of P(DMAEMA/AHMA) polyplexes
is most likely due to their small particle sizes, high surface
charges, and enhanced association with cellular membranes
45
amines. The polymers were obtained by conventional free
radical polymerization and had high molecular weights and
broad molecular weight distributions. The aim of this study
was to develop structurally well-defined low molecular weight
cationic DMAEMA copolymers for enhanced transfection. It is
remarkable that low molecular weight DMAEMA copolymers
prepared by controlled RAFT polymerization have transfection
activities higher than or comparable to 25 kDa PEI, which is
FIGURE 7 Transfection efficiencies of DNA polyplexes based
on P(DMAEMA/AEMA) and P(DMAEMA/AHMA) copolymers in
COS-7 cells at N/P ratios of 3/1, 6/1, and 9/1 in serum-free con-
dition. 25k bPEI at an N/P ratio of 10/1 was used as a control.
FIGURE 8 Transfection efficiencies of DNA polyplexes based
on P(DMAEMA/AEMA) and P(DMAEMA/AHMA) copolymers in
COS-7 cells at N/P ratios of 3/1, 6/1, and 9/1 in 10% serum con-
dition. 25k bPEI at an N/P ratio of 10/1 was used as a control.
(Student’s t-test, *p < 0.05, **p < 0.01). [Color figure can be
viewed in the online issue, which is available at
www.interscience.wiley.com.]
(
Student’s t-test, *p < 0.05, **p < 0.01). [Color figure can be
viewed in the online issue, which is available at www.
interscience.wiley.com.]
CATIONIC METHACRYLATE COPOLYMERS, ZHU ET AL.
2875

JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
known as one of the best nonviral gene transfer agents. These
DMAEMA copolymers may be developed for safe and highly ef-
ficient gene transfection.
11 Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.;
Scherman, D.; Demeneix, B.; Behr, J. P. Proc Nat Acad Sci USA
1
995, 92, 7297–7301.
1
2 Cho, Y. W.; Kim, J. D.; Park, K. J. Pharm Pharmacol 2003,
CONCLUSIONS
5
5, 721–734.
We have demonstrated that PDMAEMA copolymers containing
varying amounts of primary amino side groups are a versatile
family of cationic polymers that mediate significantly enhanced
gene transfection as compared with PDMAEMA homopolymer
under otherwise the same conditions. These methacrylate
copolymers have several unique features: (i) they can be read-
ily prepared with controlled compositions and narrow molecu-
lar weight distributions using RAFT polymerization method fol-
lowed by acid deprotection; (ii) they have improved water
solubility and can effectively condense DNA into small sized
particles due to presence of primary amine groups; and (iii)
they have relatively low molecular weights (<25 kDa) and
afford optimal transfection efficiencies at a low N/P ratio of
13 Akinc, A.; Thomas, M.; Klibanov, A. M.; Langer, R. J. Gene
Med 2005, 7, 657–663.
1
4 Putnam, D.; Gentry, C. A.; Pack, D. W.; Langer, R. Proc Natl
Acad Sci USA 2001, 98, 1200–1205.
1
5 Kim, T. H.; Ihm, J. E.; Choi, Y. J.; Nah, J. W.; Cho, C. S. J
Control Release 2003, 93, 389–402.
16 Lin, C.; Zhong, Z. Y.; Lok, M. C.; Jiang, X. J.; Hennink, W. E.;
Feijen, J.; Engbersen, J. F. J. J Control Release 2007, 123,
67–75.
1
7 Green, J. J.; Zugates, G. T.; Tedford, N. C.; Huang, Y. H.;
Griffith, L. G.; Lauffenburger, D. A.; Sawicki, J. A.; Langer, R.;
Anderson, D. G. Adv Mater 2007, 19, 2836–2842.
3
/1. We are convinced that cationic methacrylate copolymers
1
8 Fukushima, S.; Miyata, K.; Nishiyama, N.; Kanayama, N.;
with versatile design of structures and controlled synthesis
have a great potential in development of safe and efficient non-
viral gene transfer agents.
Yamasaki, Y.; Kataoka, K. J Am Chem Soc 2005, 127, 2810–2811.
1
9 Bikram, M.; Ahn, C. H.; Chae, S. Y.; Lee, M. Y.; Yockman, J.
W.; Kim, S. W. Macromolecules 2004, 37, 1903–1916.
This work was supported by National Natural Science Foun-
dation of China (NSFC 20874070, 50803043, 50703028,
2
0 Ou, M.; Wang, X. L.; Xu, R. Z.; Chang, C. W.; Bull, D. A.;
Kim, S. W. Bioconjugate Chem 2008, 19, 626–633.
5
0973078, and 20974073), the Natural Science Foundation
of the Jiangsu Higher Education Institutions of China
08KJB150016), Science and Technology Development Plan-
21 Lin, C.; Blaauboer, C. J.; Timoneda, M. M.; Lok, M. C.; van
Steenbergen, M.; Hennink, W. E.; Zhong, Z. Y.; Feijen, J.; Eng-
bersen, J. F. J. J Control Release 2008, 126, 166–174.
(
ning of Jiangsu Province (No. BK2007702), Program of Innova-
tive Research Team of Soochow University, and the A3 (China-
Japan-Korea) Program on Gene Delivery from the National
Research Foundation of Korea (NRF), Republic of Korea.
2
2 Zhang, K.; Fang, H.; Wang, Z.; Li, Z.; Taylor, J.-S. A.; Woo-
ley, K. L. Biomaterials 2010, 31, 1805–1813.
2
3 Funhoff, A. M.; van Nostrum, C. F.; Koning, G. A.; Schuur-
mans-Nieuwenbroek, N. M. E.; Crommelin, D. J. A.; Hennink,
W. E. Biomacromolecules 2004, 5, 32–39.
REFERENCES AND NOTES
2
4 Verbaan, F. J.; Klouwenberg, P. K.; van Steenis, J. H.; Snel,
1
Li, S.; Huang, L. Gene Ther 2000, 7, 31–34.
C. J.; Boerman, O.; Hennink, W. E.; Storm, G. Int J Pharm 2005,
3
04, 185–192.
2
De Laporte, L.; Rea, J. C.; Shea, L. D. Biomaterials 2006, 27,
2
5 Cherng, J. Y.; van de Wetering, P.; Talsma, H.; Crommelin,
947–954.
D. J. A.; Hennink, W. E. Pharm Res 1996, 13, 1038–1042.
6 van de Wetering, P.; Cherng, J. Y.; Talsma, H.; Hennink, W.
E. J Control Release 1997, 49, 59–69.
7 van de Wetering, P.; Moret, E. E.; Schuurmans-Nieuwen-
3
Jeong, J. H.; Kim, S. W.; Park, T. G. Prog Polym Sci 2007, 32,
2
1239–1274.
4
Mastrobattista, E.; van der Aa, M.; Hennink, W. E.; Cromme-
2
lin, D. J. A. Nat Rev Drug Discov 2006, 5, 115–121.
broek, N. M. E.; van Steenbergen, M. J.; Hennink, W. E. Biocon-
jugate Chem 1999, 10, 589–597.
5
Mintzer, M. A.; Simanek, E. E. Chem Rev 2009, 109, 259–302.
2
8 You, Y. Z.; Manickam, D. S.; Zhou, Q. H.; Oupicky, D. J Con-
6
Ruponen, M.; Honkakoski, P.; Ronkko, S.; Pelkonen, J.;
trol Release 2007, 122, 217–225.
Tammi, M.; Urtti, A. J Control Release 2003, 93, 213–217.
2
9 Dai, F.; Sun, P.; Liu, Y.; Liu, W. Biomaterials 31, 559–569.
7
Wong, S. Y.; Pelet, J. M.; Putnam, D. Prog Polym Sci 2007,
3
0 Jiang, X.; Lok, M. C.; Hennink, W. E. Bioconjugate Chem
32, 799–837.
2
007, 18, 2077–2084.
8
Neu, M.; Fischer, D.; Kissel, T. J. Gene Med 2005, 7,
3
1 Zhu, C.; Jung, S.; Luo, S.; Meng, F.; Zhu, X.; Park, T. G.;
992–1009.
Zhong, Z. Biomaterials 2010, 31, 2408–2416.
9
Sonawane, N. D.; Szoka, F. C.; Verkman, A. S. J Biol Chem
3
2 Funhoff, A. M.; van Nostrum, C. F.; Janssen, A.; Fens, M.; Crom-
2003, 278, 44826–44831.
melin, D. J. A.; Hennink, W. E. Pharm Res 2004, 21, 170–176.
10 Dufe, C.; Uchegbu, I. F.; Schazlein, A. G. Adv Drug Deliv Rev
33 Zhang, W. L.; He, J. L.; Liu, Z.; Ni, P. H.; Zhu, X. L. J Polym
2005, 57, 2177–2202.
Sci Part A: Polym Chem 48, 1079–1091.
2876
INTERSCIENCE.WILEY.COM/JOURNAL/JPOLA

ARTICLE
34 Xu, F. J.; Li, H. Z.; Li, J.; Zhang, Z. X.; Kang, E. T.; Neoh, K.
40 Lowe, A. B.; McCormick, C. L. Prog Polym Sci 2007, 32, 283–351.
G. Biomaterials 2008, 29, 3023–3033.
4
1 Stenzel, M. H. Chem Commun 2008, 3486–3503.
35 van de Wetering, P.; Cherng, J. Y.; Talsma, H.; Crommelin,
4
2 Akinc, A.; Lynn, D. M.; Anderson, D. G.; Langer, R. J Am
D. J. A.; Hennink, W. E. J Control Release 1998, 53, 145–153.
Chem Soc 2003, 125, 5316–5323.
3
3
6 Dubruel, P.; Schacht, E. Macromol Biosci 2006, 6, 789–810.
7 Zhang, Y. F.; Gu, W. Y.; Xu, H. X.; Liu, S. Y. J Polym Sci
4
3 Zhong, Z. Y.; Song, Y.; Engbersen, J. F. J.; Lok, M. C.;
Hennink, W. E.; Feijen, J. J Control Release 2005, 109, 317–
Part A: Polym Chem 2008, 46, 2379–2389.
8 Thang, S. H.; Chong, Y. K.; Mayadunne, R. T. A.; Moad, G.;
Rizzardo, E. Tetrahedron Lett 1999, 40, 2435–2438.
9 Zhong, Z. Y.; Feijen, J.; Lok, M. C.; Hennink, W. E.; Christen-
3
29.
3
4
4 Lin, C.; Zhong, Z. Y.; Lok, M. C.; Jiang, X. L.; Hennink, W. E.; Fei-
jen, J.; Engbersen, J. F. J. Bioconjugate Chem 2007, 18, 138–145.
3
sen, L. V.; Yockman, J. W.; Kim, Y. H.; Kim, S. W. Biomacromo-
lecules 2005, 6, 3440–3448.
45 Reschel, T.; Konak, C.; Oupicky, D.; Seymour, L. W.; Ulbrich,
K. J Control Release 2002, 81, 201–217.
CATIONIC METHACRYLATE COPOLYMERS, ZHU ET AL.
2877