Linear cationic click polymer for gene delivery: Synthesis, biocompatibility, and in Vitro Transfection

Article abstract of DOI:10.1021/bm100906m

Sixteen novel cationic click polymers (CPs) were parallelly synthesized via the conjugation of four alkyne- functionalized monomers to four azide-functionalized monomers by "click chemistry". The biocompatibility of CPs was evaluated by in vitro cytotoxicity (MTT assay, Hoechst/PI apoptosis/necrosis assay, and cell cycle analysis) and blood compatibility tests (hemolysis and erythrocyte aggregation). The experimental results showed that the kind of amine groups, charge density, and number of methylene or ethylene glycol groups brought about the effect on toxicity of CPs. Among all polymers, two polymers (B₁ and B₂) showed good biocompatibility, inducing neither apoptosis nor necrosis at the test concentration and low hemolysis ratio and erythrocyte aggregation. In particular, B₁ and B₂ exhibited the comparable transfection efficiency compared with PEI (25 kDa) but much lower cytotoxicity. These results suggested that the novel cationic CPs could be promising carriers for gene delivery.

Full text of DOI:10.1021/bm100906m

3
102
Biomacromolecules 2010, 11, 3102–3111
Linear Cationic Click Polymer for Gene Delivery: Synthesis,
Biocompatibility, and In Vitro Transfection
Yu Gao, Lingli Chen, Zhiwen Zhang, Wangwen Gu, and Yaping Li*
Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
Received August 5, 2010; Revised Manuscript Received September 7, 2010
Sixteen novel cationic click polymers (CPs) were parallelly synthesized via the conjugation of four alkyne-
functionalized monomers to four azide-functionalized monomers by “click chemistry”. The biocompatibility of
CPs was evaluated by in vitro cytotoxicity (MTT assay, Hoechst/PI apoptosis/necrosis assay, and cell cycle analysis)
and blood compatibility tests (hemolysis and erythrocyte aggregation). The experimental results showed that the
kind of amine groups, charge density, and number of methylene or ethylene glycol groups brought about the
effect on toxicity of CPs. Among all polymers, two polymers (B
neither apoptosis nor necrosis at the test concentration and low hemolysis ratio and erythrocyte aggregation. In
particular, B and B exhibited the comparable transfection efficiency compared with PEI (25 kDa) but much
1 2
and B ) showed good biocompatibility, inducing
1
2
lower cytotoxicity. These results suggested that the novel cationic CPs could be promising carriers for gene
delivery.
14,15
1
. Introduction
still very few,
and the structure-biocompatibility relation-
ship of the linear CP has not been investigated until today.
Parallel synthesis has gained increasing popularity as a means
Cationic polymers have attracted the great interest and
attention as nonviral gene vectors because of their merits in
avoidance of potential immunogenicity, large gene loading
to synthesize the diverse polymer libraries with different
16,17
chemical or biological properties.
Screening of the library
1,2
capacity, and the ease of large-scale preparation. The cationic
polymers could electrostatically interact with negatively charged
DNA or RNA and condense them into polyplexes, which could
enter cells via endocytosis. However, the current several
important cationic polymers for gene delivery are not fully
effective in vivo because of their toxicity or the instability of
of polymers could rapidly identify the novel polymers for gene
delivery with the high transfection efficiency and low cytotoxi-
1
6-19
city.
We are interested in developing a library of novel
linear cationic CPs with good biocompatibility for gene delivery.
In this work, 16 cationic CPs were parallelly synthesized via
the conjugation of four alkyne-functionalized monomers to four
azide-functionalized monomers by “click chemistry”, and the
biocompatibility of CPs was evaluated by in vitro cytotoxicity
and blood compatibility. Finally, the in vitro transfection
efficiency of two CPs with good biocompatibility was evaluated
in MDA-MB-468 cells.
3
-5
polyplexes in serum.
Therefore, it is still challenging work
to design rationally the new cationic polymers with defined
molecular structure that are biocompatible and applicable for
more efficient gene delivery.
In recent years, the concept of click chemistry, introduced
2
8
by Sharpless et al., has been regarded as a powerful tool in
the design and synthesis of materials. The copper-catalyzed
azide-alkyne cycloaddition (CuAAC), which is a unique
ligation method combining selective reactivity and easy manu-
facturing, is generally regarded as the quintessential example
2
. Experimental Section
2
.1. Materials. Diethylenetriamine, triethylenetetramine, diethylene
glycol, triethylene glycol, diethanolamine, N-methyldiethanolamine, di-
tert-butyl dicarbonate, 4-dimethylamino pyridine (DMAP), copper(II)
sulfate, sodium azide, trifluoroacetic acid, and p-toluenesulfonyl chloride
TsCl) were obtained from Sinopharm Group Chemical Reagent
China). N-(tert-Butoxycarbonyl)-L-aspartic acid, N,N′-dicyclohexyl-
6
,7
of click chemistry, which has rapidly found utilization in
numerous fields including in a variety of polymer syntheses such
(
(
8
9,10
11
as the block copolymers, dendrimers,
graft copolymers,
1
2
and adhesive polymers. In the linear polymer synthesis, the
azide- or alkyne-functionalized monomers could be easily
tailored to form the different copolymers with different chemical
carbodiimide (DCC), 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide
hydrochloride (EDC ·HCl), and N-(tert-butoxycarbonyl)-L-glutamic acid
were purchased from GL Biochem (Shanghai, China). Trypsin-EDTA
and phosphate-buffered saline (PBS) were obtained from Gibco-BRL
(Burlington, ON, Canada). The RPMI 1640 medium, antibiotics, and
fetal bovine serum (FBS) were purchased from Invitrogen GmbH
(Karlsruhe, Germany). Branched polyethylenimine (PEI, 25 kDa),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
DNA-free RNase A, Hochest 33342, propidium iodide (PI), propiolic
acid, propargylamine, and ethyl trifluoroacetate were purchased from
Sigma (St. Louis, MO). All other chemicals and solvents, if not
mentioned, were of analytical grade and used as received without
additional purification.
1
3
and biological properties. The unique 1,2,3-triazole-amide
moiety, which was formed during polymerization, could be
H-bond acceptors and increase hydrophobic interactions of the
1
4
polymer with DNA. This characteristics makes the linear
cationic click polymer (CP) bind effectively with DNA to
improve stability of polyplexes. Although the linear cationic
CP demonstrated the great potential for gene delivery, the
syntheses and applications of cationic CP for gene delivery are
*
To whom correspondence should be addressed. Address: 501 Haike
Road, Shanghai 201203, China. Phone: +86-21-2023-1979. Fax: +86-21-
023-1979. E-mail: ypli@mail.shcnc.ac.cn.
Plasmid EGFP-N1 (4.7 kb) encoding enhanced green fluorescent
protein driven by immediate early promoter of CMV was purchased
2
10.1021/bm100906m
 2010 American Chemical Society
Published on Web 09/28/2010

Linear Cationic Click Polymer for Gene Delivery
Biomacromolecules, Vol. 11, No. 11, 2010 3103
layers were combined, dried over Na SO , and evaporated to yield the
Scheme 1. Synthetic Routes for Monomers
2
4
compound bis-(2-amino-ethyl)-carbamic acid tert-butyl ester as a waxy
solid.
Propiolic acid (0.38 g, 5.4 mmol) was added to a solution of DCC
(
2
1.25 g, 6 mmol) in dichloromethane (100 mL) at 0 °C under N and
stirred for 2 h. A solution of bis-(2-amino-ethyl)-carbamic acid tert-
butyl ester (0.50 g, 2.4 mmol) dissolved in dichloromethane was then
added dropwise to this mixture, and the reaction mixture was stirred
for 2 h at 0 °C. The mixture was then warmed to room temperature
and stirred for an additional 24 h. After the white solid was filtered,
the solvent was evaporated, and the resulting crude product was purified
via silica gel chromatography using a gradient elution of 2 to 4%
methanol in chloroform (Rf ) 0.3 for A in 4% MeOH/CHCl
3
) to yield
1
the final product as pale yellow solid. H NMR (CDCl
.49-3.41 (m, 8H), 2.82 (s, 2H), 1.49 (s, 9H).
.3.3. {2-[tert-Butoxycarbonyl-(2-propynoylamino-ethyl)-amino]-eth-
3
): 6.86 (bs, 2H),
3
2
yl}-(2-propynoylamino-ethyl)-carbamic Acid tert-Butyl Ester (B). Com-
pound B was prepared using a four-step synthesis procedure with
triethylenetetramine as the starting material in a similar manner as
diethylenetriamine. Compound B was obtained as pale yellow solid.
1
H NMR (CDCl
.49 (s, 18H).
.3.4. (1,2-Bis-prop-2-ynylcarbamoyl-ethyl)-carbamic Acid tert-Bu-
3
): 8.14 (bs, 2H), 3.22-3.48 (m, 8H), 2.75 (s, 2H),
1
2
tyl Ester (C). To a stirred solution of (1,2-dicarbamoyl-ethyl)-carbamic
acid tert-butyl ester (2.33 g, 10 mmol) in dichloromethane (100 mL),
EDC·HCl (4.34 g, 22 mmol) and DMAP (0.24 g, 2 mmol) were added
and stirred for 2 h. To this mixture, a solution of propargylamine (1.21
g, 22 mmol) dissolved in 25 mL of dichloromethane was added at 0-10
from Clontech (Palo Alto, CA). The plasmid DNA (pDNA) grown in
DH5R strain of E. coli was isolated with the EndFree Plasmid Mega
Kit (Qiagen GmbH, Hilden, Germany). The purity was confirmed by
spectrophotometry (A260/A280), and DNA concentration was measured
by UV absorption at 260 nm.
2
°C. Then, the reaction mixture was stirred under N for 24 h at room
temperature. After the solvent evaporated, the resulting crude product
was purified via silica gel chromatography using a gradient elution of
2.2. Cell Culture. The cell line MDA-MB-468 (human breast cancer
2
to 4% methanol in chloroform (Rf ) 0.3 for C in 4% MeOH/CHCl
3
).
cell) was obtained from the American Type Culture Collection (ATCC,
Manassas, VA). MDA-MB-468 cells were cultured in RPMI 1640
medium supplemented with 10% FBS and 1% antibiotics (100 U/mL
penicillin G and 0.1 mg/mL streptomycin). Cells were maintained at
The proper fractions were combined, the solvent was evaporated, and
1
the resulting white solid was isolated. H NMR (CDCl
.26 (bs, 1H), 6.11 (m, 1H), 4.46 (m, 1H), 3.93-3.10 (m, 4H), 2.88
dd, J ) 3.9 Hz, 4H), 2.55 (dd, J ) 6.4 Hz, 1H), 2.52-2.59 (m, 1H),
.22-2.23 (m, 1H), 1.45 (s, 9H).
.3.5. (1,3-Bis-prop-2-ynylcarbamoyl-propyl)-carbamic Acid tert-
3
): 7.15 (bs, 1H),
6
(
2
3
7 °C in a humidified and 5% CO
.3. Synthesis of Monomers. 2.3.1. General. The synthetic routes
for monomers were shown in Scheme 1. TLC was performed on silica
gel and detected by UV light, KMnO , and I wherever applicable.
2
incubator.
2
2
Butyl Ester (D). To a stirred solution of (1,3-dicarbamoyl-propyl)-
carbamic acid tert-butyl ester (2.47 g, 10 mmol) in dichloromethane
(100 mL), EDC·HCl (4.34 g, 22 mmol), and DMAP (0.24 g, 2 mmol)
were added and stirred for 2 h. To this mixture, a solution of
propargylamine (1.21 g, 22 mmol) dissolved in 25 mL of dichlo-
romethane was added at 0-10 °C. The reaction mixture was then stirred
4
2
1
The H spectra were recorded on a Bruker 400 MHz NMR spectrometer.
The H NMR spectrum was as follows: chemical shift (δ), multiplicity
1
(
s ) singlet, d ) doublet, t ) triplet, m ) multiplet, dd ) double
doublets, bs ) broad singlet, bm ) broad multiplet), J coupling constant
hertz), and the peak integration. Gel permeation chromatography (GPC)
(
2
under N for 24 h at room temperature. After the solvent evaporated,
the resulting crude product was purified via silica gel chromatography
was conducted on a Waters 2695 controller equipped with an UL-
TRAHYDROGEL column 250 PKGD and a refractive index detector
(
model 2414) for the estimation of the molecular weight of CPs. The
using a gradient elution of 2 to 4% methanol in chloroform (Rf ) 0.3
mobile phase is the water-containing sodium azide (0.05%, w/v) at a
flow rate of 0.5 mL/min with a column temperature of 30 °C. The
polymer was dissolved in water (0.05% sodium azide), and the sample
was analyzed and calibrated by DEXTRAN standards (Mp )
for D in 4% MeOH/CHCl
solvent was evaporated, and the resulting white solid was isolated. H
NMR (CDCl ): 7.21 (bs, 1H), 6.58 (bs, 1H), 5.73 (d, J ) 7.5 Hz),
4.17-4.19 (m, 1H), 4.02-4.06 (m, 4H), 2.32-2.40 (m, 2H), 2.21-2.25
(m, 2H), 1.96-2.11 (m, 2H), 1.46 (s, 9H).
2.3.6. 1-Azido-2-(2-azido-ethoxy)-ethane (1). To a stirred solution
of diethylene glycol (2.12 g, 20 mmol) in dichloromethane (250 mL)
at 0 °C, triethylamine (4.25 g, 42 mmol), DMAP (0.12 g, 0.1 mmol),
and TsCl (7.62 g, 42 mmol) were added. The mixture was stirred at
0-10 °C for 1 h, then at room temperature for 12 h. The reaction
3
). The proper fractions were combined, the
1
3
4
400-401 000 Da, American Polymer Standards Corporation).
.3.2. Bis-(2-propynoylamino-ethyl)-carbamic Acid tert-Butyl Ester
A). Bis-(2-propynoylamino-ethyl)-carbamic acid tert-butyl ester was
obtained using a four-step synthesis procedure, as previously reported
2
(
1
4
with minor modification. In brief, ethyl trifluoroacetate (2.95 g, 21
mmol) was added dropwise to diethylenetriamine (1.03 g, 10 mmol)
dissolved in dichloromethane (50 mL) at 0 °C. After being stirred for
solution was extracted with saturated NaHCO
and then with saturated NaCl solution (300 mL). The organic layers
were dried over Na SO , and the dichloromethane was evaporated to
yield the off-white solid. The crude product was purified by recrys-
tallization from CH Cl /petroleum ether to yield white solid 2,2′-
3
solution (2 × 300 mL)
1
h at room temperature, a solution of di-tert-butyl dicarbonate (3.28
g, 15 mmol) in dichloromethane and triethylamine (1.52 g, 15 mmol)
were added dropwise to this mixture. The solution was stirred overnight
2
4
and then washed with aqueous NaHCO
separated, dried over Na SO , and evaporated. The crude product was
purified by recrystallization from CH Cl /hexane. The yielding protected
oligoamine (2.00 g) was then refluxed in methanol/water (volume ratio,
0:1, containing 1.80 g K CO ) for 4 h. After the methanol was
evaporated, the residue was extracted with dichloromethane. The organic
3
. The organic layer was
2
2
2
4
oxybis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate).
2
2
To a solution of 2,2′-oxybis(ethane-2,1-diyl) bis(4-methylbenzene-
sulfonate) (4.1 g, 10 mmol) in acetone, sodium azide (1.9 g, 30 mmol)
was added, and the mixture was refluxed for 48 h. After the solvent
was evaporated, the resulting crude product was purified via silica gel
2
2
3

3
104 Biomacromolecules, Vol. 11, No. 11, 2010
Gao et al.
Scheme 2. Synthetic Routes for Polymers
chromatography using an elution of 50% ethyl acetate in petroleum
ether (Rf ) 0.8 for 1 in 50% ethyl acetate/petroleum ether). The proper
To a solution of bis-(2-chloro-ethyl)-methyl-amine (1.6 g, 10 mmol)
in acetone, sodium azide (3.3 g, 50 mmol) was added, and the mixture
was refluxed for 48 h. After the solvent evaporated, the resulting crude
product was purified via silica gel chromatography using an elution of
50% ethyl acetate in petroleum ether (Rf ) 0.5 for 4 in 50% ethyl
fractions were combined, and the solvent was evaporated to yield 1 as
1
yellow oil. H NMR (CDCl
3
): 2.87 (s, 4H), 2.70 (s, 4H).
2
.3.7. 1-Azido-2-[2-(2-azido-ethoxy)-ethoxy]-ethane (2). To a stirred
solution of triethylene glycol (3.00 g, 20 mmol) in dichloromethane
250 mL) at 0 °C, triethylamine (4.25 g, 42 mmol), DMAP (0.12 g,
.1 mmol), and TsCl (7.62 g, 42 mmol) were added. The mixture was
stirred at 0-10 °C for 1 h, then at room temperature for 12 h. The
reaction solution was extracted with saturated NaHCO
solution (2 ×
00 mL), then with saturated NaCl solution (300 mL). The organic
layers were dried over Na SO , and the dichloromethane was evaporated
to yield the off-white solid. The crude product was purified by
recrystallization from CH Cl /petroleum ether to yield white solid 2,2′-
ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzene-
sulfonate).
acetate/petroleum ether). The proper fractions were combined, and the
1
(
0
solvent was evaporated to yield 4 as yellow oil. H NMR (CDCl
3.33 (t, J ) 6.0 Hz, 4H), 2.63 (t, J ) 6.0 Hz, 4H), 2.32 (s, 3H).
3
):
2.4. Synthesis of CPs. The synthetic routes for CPs were shown in
3
Scheme 2. For synthesis of CP A , the solution of copper(II) sulfate
1
3
(0.032 g, 0.2 mmol) in water (0.5 mL) was added to the solution of
monomer A (0.31 g, 1 mmol) and monomer 1 (0.16 g, 1 mmol)
dissolved in tert-butanol (1 mL), and the mixture was introduced to a
small vial covered with a rubber cap. The vial was filled with nitrogen
in vacuum. The solution of sodium ascorbate (0.079 g, 0.4 mmol)
dissolved in 0.5 mL of water was added dropwise with a thin pinhead
piercing through the rubber cap. The mixture was heated to 50 °C in
an oil bath, stirred for 24 h. The solvents were evaporated, and the
resulting copolymer was then dissolved in 10 mL of a solution of
dichloromethane/trifluoroacetic acid (1:1 volume) with stirring for 3 h
and dried under vacuum. The final product after neutralization using 1
M NaOH solution was purified via dialysis against 0.01 M HCl for
2
4
2
2
(
To a solution of 2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)
bis(4-methyl -benzenesulfonate) (4.6 g, 10 mmol) in acetone, sodium
azide (1.9 g, 30 mmol) was added, and the mixture was refluxed for
4
8 h. After the solvent was evaporated, the resulting crude product
was purified via silica gel chromatography using an elution of 50%
ethyl acetate in petroleum ether (Rf ) 0.8 for 2 in 50% ethyl acetate/
petroleum ether). The proper fractions were combined, and the solvent
24 h, then exhaustively against ultra pure H
deprotected polymers were lyophilized to yield a brown solid. H NMR
(D O): 7.94 (s, 2H), 4.34-4.29 (m, 4H), 3.70 (bs, 4H), 3.39-3.45 (m,
2
O for 3 days. The final
1
1
was evaporated to yield 2 as yellow oil. H NMR (CDCl
3
): 3.65-3.69
(
m, 8H), 3.37 (t, J ) 5.2 Hz, 4H).
.3.8. Bis-(2-azido-ethyl)-amine (3). To a solution of diethanolamine
2
2
4H), 2.75-2.87 (m, 4H).
(
(
7.7 g, 100 mmol) in chloroform (100 mL) at 0 °C, thionyl chloride
The other CPs were prepared from alkyne-functionalized monomers
47.6 g, 400 mmol) dissolved in chloroform (20 mL) was added
and azide-functionalized monomers using a similar method as A
2 2
analyzed by H NMR (D O). The data are listed as below: A : H NMR
1
and
1
1
dropwise for 1 h. The solution was then stirred for 24 h at room
temperature. The solvent was evaporated, and the crude product was
purified by recrystallization from CHCl
(D
2
O): 8.05 (s, 2H), 4.50 (bs, 4H), 3.82 (bs, 4H), 3.61 (bs, 8H), 3.20
1
3
/petroleum ether to yield the
(bs, 4H). A
3
: H NMR (D
2
O): 8.59 (bs, 2H), 3.84-3.71 (m, 8H), 3.48
O): 8.00-8.02 (m, 2H), 4.35-4.62 (m, 4H),
1
bis-(2-chloro-ethyl)-amine hydrochloride as a white solid.
(m, 8H). A
4
: H NMR (D
2
A solution of bis-(2-chloro-ethyl)-amine hydrochloride (1.8 g, 10
mmol) and sodium azide (3.3 g, 50 mmol) in water (50 mL) was heated
at 80 °C for 24 h. After evaporating most of the water, the solution
was made basic with sodium hydroxide and then extracted with diethyl
ether. The organic phase was combined and dried over potassium
carbonate. After evaporating the solvent, the resulting crude product
was purified via silica gel chromatography using an elution of 50%
ethyl acetate in petroleum ether (Rf ) 0.5 for 3 in 50% ethyl acetate/
3.65-3.70 (m, 4H), 3.39-3.53 (m, 4H), 2.84-2.92 (m, 4H), 2.35 (s,
1
3H). B
3.61 (bs, 4H), 2.17-3.22 (m, 8H). B
4.54-4.60 (m, 4H), 3.88 (s, 4H), 3.53-3.63 (m, 8H), 3.14-3.20 (m,
1 2
: H NMR (D O): 8.07 (s, 2H), 4.53 (bs, 4H), 3.91 (bs, 4H),
1
2
2
: H NMR (D O): 8.23 (s, 2H),
1
8H). B
(m, 12H). B
3.70-3.73 (m, 4H), 3.26-3.32 (m, 8H), 2.96-2.98 (m, 4H), 2.37 (s,
3
: H NMR (D
2
O): 8.60 (bs, 2H), 3.83-3.86 (m, 8H), 3.41-3.59
1
4
: H NMR (D O): 8.10-8.18 (m, 2H), 4.43 (bs, 4H),
2
1
3H). C
(m, 5H), 3.70 (bs, 2H). C
4.47-4.48 (m, 4H), 4.40-4.42 (m, 2H), 4.35 (bs, 2H), 3.80-3.82 (m,
1 2
: H NMR (D O): 7.52 (s, 2H), 4.20-4.30 (m, 8H), 3.63-3.67
1
petroleum ether). The proper fractions were combined, and the solvent
2 2
: H NMR (D O): 7.83-7.84 (m, 2H),
1
was evaporated to yield 3 as yellow oil. H NMR (CDCl
3
): 3.40 (t, J
1
)
5.4 Hz, 4H), 2.79 (t, J ) 4.8 Hz, 4H).
.3.9. Bis-(2-azido-ethyl)-methyl-amine (4). To a solution of N-
methyldiethanolamine (11.9 g, 100 mmol) in chloroform (100 mL) at
°C, thionyl chloride (47.6 g, 400 mmol) dissolved in chloroform (20
mL) was added dropwise for 1 h. The solution was then stirred for
4 h at room temperature. The solvent was evaporated, and the crude
product was purified by recrystallization from CHCl /petroleum ether
5H), 3.51 (bs, 4H), 2.84-2.86 (m, 2H). C
3 2
: H NMR (D O): 7.94 (bs,
2
2H), 4.63-4.73 (m, 4H), 4.56-4.59 (m, 4H), 3.29-3.39 (bs, 5H), 2.96
1
(bs, 2H). C
2.82-3.15 (m, 5H), 2.25-2.46 (m, 5H). D
2H), 4.45-4.52 (m, 8H), 3.89 (bs, 4H), 3.79 (bs, 1H), 2.45 (bs,2H),
4 2
: H NMR (D O): 7.85-7.92 (m, 2H), 4.52-4.72 (m, 8H),
1
0
1 2
: H NMR (D O): 7.81 (s,
1
2
2.19 (bs, 2H). D
2 2
: H NMR (D O): 7.91-7.92 (m, 2H), 4.44-4.55 (m,
3
8H), 3.87 (bs, 4H), 3.52-3.66 (m, 5H), 2.32 (t, J ) 3.6 Hz, 2H), 1.95
1
to yield the bis-(2-chloro-ethyl)-methyl-amine hydrochloride as white
solid.
The obtained bis-(2-chloro-ethyl)-methyl-amine hydrochloride was
(bs, 2H). D
3.92 (bs, 1H), 3.08 (bs, 4H), 2.35-2.37 (m, 2H), 2.09 (bs, 2H). D
NMR (D O): 7.91 (s, 2H), 4.43-4.56 (m, 8H), 4.09 (bs, 1H), 3.16 (bs,
4H), 2.48 (bs, 5H), 2.20 (bs, 2H).
3 2
: H NMR (D O): 7.85-7.89 (m, 2H), 4.32-4.45 (m, 8H),
1
4
: H
2
dissolved in saturated solution of NaHCO
extracted with dichloromethane. The organic layers were dried over
Na SO , and the dichloromethane was evaporated to yield the bis-(2-
chloro-ethyl)-methyl-amine as a yellow oil.
3
, and the solution was then
2.5. MTT Assay. MDA-MB-468 cells were seeded at a density of
4
2
4
1 × 10 cells per well in 150 µL of growth medium in 96-well plates
and grown overnight. Immediately after growth medium was removed,

Linear Cationic Click Polymer for Gene Delivery
Biomacromolecules, Vol. 11, No. 11, 2010 3105
CPs were applied to the cells in fresh culture media with 10% FBS at
polymer concentrations from 5 to 200 µg/mL. Cells treated with fresh
culture media only were used as control. After 24 h of incubation, the
polymer-containing media were replaced with fresh culture media
containing MTT solution (0.2 mg/mL), and the cells were incubated
for an additional 4 h at 37 °C. Then, the medium was removed, and
DMSO was added to dissolve the crystal. The plates were mildly shaken
for 10 min to ensure the dissolution of formazan. The absorbency values
were measured by TECAN Infinite F200 multimode microplate reader
7.4). The transfection results were measured using a FACSCalibur
through FL1. As control, naked DNA was used on cell cultures and
examined as described above. As positive control, PEINs with mass
ratio 3 were prepared and transfected with media without FBS for 2 h
before the medium was replaced with fresh complete growth medium.
3
. Results and Discussion
3
.1. Monomer Choosing and Polymer Synthesis. Alkyne-
(
Salzburg, Austria) at wavelength 570 nm, blanked with DMSO
solution. Six replicates were counted for each sample.
.6. Apoptosis or Necrosis Observation. The apoptosis or necrosis
was detected by assessment of nuclear double-staining with Hoechst
functionalized monomers A-D and azide-functionalized mono-
mers 1-4 representing either structural or functional character
were selected for the synthesis of the CPs. Changing azide-
functionalized monomers while keeping the alkyne-functional-
ized monomer unaltered, the differences between polymers could
be measured, the different functions of the groups on the azide-
functionalized monomers could be deduced, and vice versa. All
polymers were designed to be water-soluble and had aliphatic
amines that were able to interact electrostatically with plasmid
DNA. Monomers containing aromatic amines were not chosen
for polymer synthesis because of the decreased alkalinity of
the amine and the hydrophobicity of the aromatic ring. Because
the types of amino groups and charge density have been
considered to be one of the most important factors influencing
2
6
3
1
3342 and PI. In brief, ∼1 × 10 MDA-MB-468 cells treated with
00 µg/mL CPs for 24 h were collected by centrifugation, washed twice
with PBS (pH 7.4), and resuspended in 250 mL of PBS (pH 7.4)
containing 10 µg/mL Hoechst 33342 and 20 µg/mL PI in dark at room
temperature for 15 min and then observed with a fluorescence
microscope (E800, Nikon) and documented by photography.
2
.7. Cell Cycle Analysis. Cell cycle analysis was assessed by flow
2
0
cytometry. In brief, cells seeded on the six-well plate were treated
with 100 µg/mL CPs for 24 h. Cells (1 × 10 ) were collected, washed
6
twice with PBS, and then fixed with 70% precooled ethanol and stored
at 4 °C for 24 h. Cells were centrifugated, washed with cold-PBS twice
and incubated with RNase A (10 mg/mL) for 20 min at 37 °C, and
stained with PI (2 mg/mL) for 30 min in the dark. The DNA content
was measured by FACSCalibur system (Becton Dickinson). The
percentage of DNA at G0/G1, S, and G2/M phases of the cell cycle
could be determined from the flow histograms, and apoptotic cells with
degraded DNA appeared in the area left to the G0/G1 peak, which
was the so-called “SubG1” phase on the DNA histogram.
14,21,22
the property of cationic gene vectors,
the monomers were
chosen with different types of amino groups and different
distance between charge groups. Comparing A with C or 3 with
4
, the different functions of the primary, secondary, or the
tertiary amine on the CP backbone could be found. Comparing
A with B, the influence of charge density on the property of
CP could be found. The monomers were also designed with a
different number of ethylene glycol or methylene groups because
the previous reports demonstrated that the degree of hydropho-
bicity and hydrophilicity of polymer played an important role
2
.8. Hemolysis Assay. Fresh blood from ICR rats was collected in
heparinized tubes. Erythrocytes were washed three times and suspended
in 0.9% NaCl solution (2% (v/v)). Washed erythrocytes were incubated
with CPs at polymer concentration of 100 µg/mL, 0.9% NaCl solution
2
3-26
(
negative control group with 0% hemolysis), or DD water (positive
in affecting the polymer function,
and the number of
control group with 100% hemolysis) for 3 h at 37 °C. After
centrifugation at 1000 rpm for 5 min, the absorbance of the supernatant
was determined at 540 nm. The percent of hemolysis was calculated
methylene on the polymer side chains showed the great effects
on the transfection efficiency and cytotoxicity of polymers.
2
7
The function of the ethylene glycol group could be found by
comparing 1 or 2 containing ethylene glycol with other azide-
functionalized monomers, and the effect of the number of
ethylene glycol on polymer properties could be found by
comparing 1 with 2. The role of the methylene on the backbone
of CP could be investigated by comparing C with D, between
which there was one methylene variation.
by (T
i
- T
0
)/(Tmax - T
0
) × 100%, where T
i
was the average absorbance
was the average absorbance of the negative
of the CP-treated group, T
0
control group, and Tmax was the average absorbance of the positive
control group.
2
.9. Erythrocyte Aggregation Assay. The washed erythrocyte
suspension was mixed with polymer solution to get a final polymer
concentration 100 µg/mL and incubated for 3 h at 37 °C. Aggregation
of erythrocytes was evaluated under an optical microscope.
Polymers were synthesized with alkyne-functionalized mono-
mers and azide-functionalized monomers in equal molar ratios
via “click polymerization” by a similar method exploited by
2
.10. In Vitro Transfection. For in vitro transfection, CP/DNA
complex nanoparticles (CPNs) were prepared. In brief, pDNA (100
µg/mL) dissolved in sterile water was added to the CP solution diluted
in sterile water with equal volume to obtain the desired polymer-amine
to DNA-phosphate ratio (N/P ratio). Subsequently, solution was
immediately vortexed for 30 s (XW-80A Votex mixer, Shanghai). The
resulting CPNs were allowed to sit at room temperature for 30 min.
As positive control, PEI 25K/DNA complex nanoparticles (PEINs) were
prepared with the same procedure as CPNs. The particle size and ꢀ
potential of CPNs were determined by the laser light scattering
measurement using a Nicomp 380/ZLS zeta potential analyzer (Particle
Sizing System).
2
8,29
Sharpless et al.
1:1, v/v) was chosen as a general solvent for polymer synthesis
Solution of tert-butyl alcohol and water
(
on the basis of solubility of all monomers. Copper(II) sulfate
pentahydrate and sodium ascorbate were chosen as catalysts,
and polymerization proceeded at 50 °C with similar monomer
concentration to yield polymers with the similar degree of
polymerization. To yield the final polymers, the Boc groups on
the alkyne-functionalized monomers were deprotected with
trifluoroacetic acid in dichloromethane. The final 16 products
(
4 alkynes × 4 azides ) 16 polymers) were all water-soluble,
MDA-MB-468 cells were seeded in 24-well plates at a density of 1
5
and the Boc protecting groups were completely cleaved from
the polymer backbone according to H NMR analysis. The
representative H NMR spectra of polymer B
×
10 cells per well in 500 µL of complete medium and incubated for
1
2
4 h prior to transfection. Then, the media were replaced with fresh
complete growth medium containing CPNs with DNA concentration
.5 µg/well at various N/P ratios. Media were replaced by fresh culture
1
1
and B
2
were
shown in Figure 1. The signals δ at ∼8 were assigned to the
2
medium after 24 h, and cells were incubated for an additional 24 h.
The expressed green protein could be observed under a fluorescence
microscope. Finally, cells were collected and resuspended in PBS (pH
triazole groups. The relative weight molecular weights of CPs
4
ranged from (1.2 to 1.8) × 10 measured by GPC. All polymers
were successfully synthesized with similar degrees of polym-

3
106 Biomacromolecules, Vol. 11, No. 11, 2010
Gao et al.
indicated that polymers synthesized using alkyne-functionalized
monomers with secondary amine were much more toxic than
polymers synthesized using alkyne-functionalized monomers
containing primary amine irrespective of the structure of the
azide-functionalized monomer. Polymers of A and B series
showed the same trend of toxicity of the azide-functionalized
monomers in the order of 4 > 3 > 1 > 2. It demonstrated that
the ethylene glycol unit in the polymer backbone could greatly
reduce polymer toxicity. A significant increase in polymer
toxicity could be observed once monomers containing ethylene
glycol unit were replaced with monomers containing amine
group. These results suggested that the amine density of polymer
played a critical role in the polymer toxicity. In C series, to our
2
surprise, C appeared to be far more toxic than other polymers.
Polymers synthesized using monomers D were a little less toxic
than polymers synthesized using monomers C, which indicated
that more methylene moiety in polymer backbone could reduce
the amine density of the polymers and thus reduce toxicity.
PEI is generally used as a positive control for nonviral gene
3
0
transfection in many previous papers; however, its severe
toxicity limited its in vivo use. By comparing the toxicity of
1
31
Figure 1. H NMR spectra of polymer (A) B
1
and (B) B
2
.
CPs with PEI, it could be found that the toxicity of CPs was
much lower than that of PEI. The 100 µg/mL B
PEI showed about the same amine concentration. The toxicity
of B was very low in MDA-MB-468 cells with cell viability
of 69.12% at 100 µg/mL. In contrast, for cells treated with PEI,
no cell was viable at polymer concentration of 20 µg/mL after
4
and 30 µg/mL
erization to elucidate the effect of different functional groups
on polymer backbone on biocompatibility of polymers (Table
2
).
4
3
.2. MTT Assay. Among all methods utilized for the
biocompatibility study, the in vitro experiment with the cultured
cell line is the most common. The cytotoxicity of CPs was
detected on MDA-MB-468 cell lines by MTT assay. Relative
2
4 h incubation. The reduced toxicity of CPs compared with
PEI could be attributed to the reduced charge density of
polymers by the 1,2,3-triazole-amide moiety in the polymer
2 3 4 3 4
to the polymers C , A , A , B , and B , the other 11 polymers
3
2
showed low cytotoxicity in MDA-MB-468 cells with cell
viability of ∼75% at 100 µg/mL polymer (Figure 2). The
toxicity of CPs increased with polymer concentration increasing.
In the cell viability curves, it was very obvious that C and D
series showed much lower cytotoxicity than A or B series, which
backbone to distribute the positive charge.
3.3. Apoptosis or Necrosis Observation. Apoptosis and
necrosis are two fundamental types of cell death. Apoptosis is
an active, genetically regulated disassembly of the cell, and
necrosis, unlike apoptosis, is considered to be a passive
Figure 2. Cytotoxicity of CPs in MDA-MB-468 cells. Cells were incubated with CPs at polymer concentrations ranging from 5 to 200 µg/mL for
4 h. Each data point represents the mean ( SD of six replicates of three times.
2

Linear Cationic Click Polymer for Gene Delivery
Biomacromolecules, Vol. 11, No. 11, 2010 3107
Figure 3. Fluorescence microscopic images of MDA-MB-468 cell nuclei following 24 h of incubation with CPs at polymer concentration of 100
µg/mL. Cell nucleus was stained with Hoechst 33342.
Figure 4. Fluorescence microscopic images of MDA-MB-468 cell nuclei following 24 h of incubation with CPs at polymer concentration of 100
µg/mL. Cell nucleus was stained with PI.
degenerative phenomenon induced by direct toxic and physical
PI is not permeant to live cells or apoptotic cells but stains
necrotic cells that have lost membrane integrity with red
fluorescence. In this work, apoptosis or necrosis was determined
by Hoechst 33342 or PI nuclear staining. By this method, it
3
3,34
injuries.
A cell undergoing apoptosis demonstrates nuclear
condensation and DNA fragmentation, which can be detected
by staining with Hoechst 33342 and fluorescence microscopy.

3
108 Biomacromolecules, Vol. 11, No. 11, 2010
Gao et al.
Figure 5. Effects of treatment with CPs at polymer concentration of 100 µg/mL for 24 h on the cell cycle of MDA-MB-468 cells.
a
Table 1. Hemolysis Assay of CPs
was possible to detect, in the same sample and at the same time,
intact cells, cells undergoing apoptosis, and dead cells resulting
from apoptotic or necrotic processes.
polymer
hemolysis rate (%)
polymer
hemolysis rate (%)
A
A
A
A
B
B
1
2
3
4
1
2
2.64 ( 0.58
0.73 ( 0.10
2.19 ( 0.30
3.96 ( 0.30
0.85 ( 0.15
0.86 ( 0.05
3.68 ( 0.63
3.87 ( 0.78
C
C
C
C
D
D
1
2
3
4
1
2
0.78 ( 0.11
1.92 ( 0.37
2.24 ( 0.09
2.64 ( 0.21
2.26 ( 0.09
2.10 ( 0.08
1.82 ( 0.19
1.62 ( 0.18
As shown in Figure 3, polymer A
severe apoptosis with condensed nuclear chromatin. From Figure
, the PI staining could be observed in cells treated with polymer
, which demonstrated that polymer A could cause cell death
through either apoptosis or necrosis. Slight DNA fragmentation
was observed in cells treated with A . The condensed nuclei
characteristic of apoptosis was also observed in cells treated
with polymer B or C . No induction of necrosis was observed
in cells treated with polymer C , which indicated that the death
of cells treated with polymer C was mainly through apoptosis.
From the PI stain study, it could be found that polymer A
, or B caused severe cell necrosis (Figure 4). The high cellular
toxicity of A , A , B , or B was consistent with the results of
MTT tests. Compared with polymers A , A , B , and B , the
high toxicity of A , A , B , or B could be attributed to the
high amine density of CPs, which could cause more severe cell
injury and resultant cell death. Except C , polymers of C or D
1
at 100 µg/mL could induce
4
A
1
1
B3
D3
B
4
D
4
3
a
Erythrocytes were incubated with CPs at polymer concentration 100
µg/mL for 3 h. 0.9% NaCl solution was used as the negative control with
% hemolysis, and DD H O was used as the positive control with 100%
4
2
0
2
2
hemolysis. All hemolysis data were presented as the percentage of the
complete hemolysis. Data were given as mean ( SD (n ) 3).
2
3
, A4,
B
3
4
also demonstrated cell-cycle-dependent toxicity. It was reported
that the toxicity of the synthetic peptide seemed to be strongly
related to the cell cycle, more precisely to the hyperpolarization
occurring at the G1/S transition.
Flow cytometric analysis (FCM) of cell cycle indicated that
polymers A , A , C exerted toxic effect on MDA-MB-468 cells
3
4
3
4
1
2
1
2
3
6
3
4
3
4
2
1
3
2
series did not induce apoptosis or necrosis in MDA-MB-468
cells. These results demonstrated that the toxicity of polymers
containing primary amine was much lower than that of polymers
containing secondary amine.
and induced cell apoptosis as highly definable sub-G1 peaks
occurred (Figure 5). An increase in G2/M peak could also be
observed in cells treated with polymer C
2
, which indicated that
polymer C could inhibit cell growth by inducing cell cycle
2
3
.4. Cell Cycle Analysis. Cell cycle analysis is a sensitive
arrest and apoptosis. No obvious changes of cell cycle phase
were found in all other CPs, which indicated that their cellular
toxicity was not related to cell cycle arrest.
3.5. Hemolysis Assay. When cationic polyplexes are ad-
ministrated intravenously, the interactions between positively
test for the toxicity of test materials to the proliferating cells.
Many conventional anticancer drugs such as taxol and anthra-
cyclines act by blocking one or more stages of the cell cycle to
3
5
inhibit cell proliferation. Many synthetic polymeric materials

Linear Cationic Click Polymer for Gene Delivery
Biomacromolecules, Vol. 11, No. 11, 2010 3109
Table 2. Characterization of CPs
polymer
M
w
(Da)
M
w
/M
n
yield (%)
polymer
M
w
(Da)
M
w
/M
n
yield (%)
A
A
A
A
B
B
B
B
1
2
3
4
1
2
3
4
15 480
17 221
15 557
14 014
16 940
15 166
14 923
15 139
1.13
1.15
1.21
1.14
1.14
1.16
1.22
1.12
31
28
25
36
34
38
31
39
C
C
C
C
D
D
D
D
1
2
3
4
1
2
3
4
15 640
15 990
15 778
12 857
14 050
12 879
16 207
14 966
1.16
1.21
1.15
1.12
1.20
1.11
1.18
1.13
43
56
39
47
54
49
41
64
charged polymers and negatively charged erythrocytes are
inevitable. Therefore, it is very necessary to test the blood
compatibility of new gene delivery vectors. Hemolysis was one
of the most important phenomena in the determination of the
biocompatibility of the synthetic materials. Toxicity of materi-
als to human red blood cells (RBCs) could disturb the
erythrocyte membrane and induce hemoglobin release. As
shown in Table 1, the hemolysis rates of blood samples treated
with 100 µg/mL CPs were all <5%, which was not considered
to be hemolytic. These results proved that CPs were not
erythrocyte membrane lytic. Among the 16 CPs, the percentage
Therefore, RBC aggregation has been considered to be one of
the major factors affecting transfection efficiency of gene
delivery carriers.
To investigate the interaction of CPs with erythrocytes, the
morphology of erythrocytes after treatment with CPs was
examined under light microscopy. It could be seen that all CPs
could induce erythrocyte aggregation due to their positive
37
charged property (Figure 6). Polymer B
relatively low levels of aggregation. Polymer A
and D could cause severe erythrocyte aggregation, which
1
, B
2
, D
1
, and D
2
showed
1
, A
3
, B , D
3
3
,
4
indicated that the CPs with higher amine density caused more
severe RBC aggregation. In the B, C, or D series, it could be
found obviously that the introduction of ethylene glycol group
in the polymer backbone could reduce the interaction between
polymers and erythrocytes. The interaction reduced as the
number of the ethylene glycol group in the polymer backbone
of hemolysis detected with polymer A
4 4
and B demonstrated
>
3.8%, which indicated that CPs containing both second amine
and ternary amine could interact more strongly with negatively
charged erythrocyte membrane than CPs containing other
amines.
3
.6. Erythrocyte Aggregation Assay. Because cationic
increased (A
was observed after treatment with A
membrane disturbance was found from the microscopic images.
These results demonstrated that A and B showed relatively
4 4
high hemotoxicity.
2
< A
1
, B
2
< B
1
). No obvious RBC aggregation
polymer could electrostatically interact with anionic cell mem-
brane and elevate the electrostatic repulsion among RBCs,
erythrocyte aggregation usually occurs when the cationic gene
delivery vectors are administrated intravenously. RBC aggrega-
tion not only affects blood viscosity and microvascular flow
dynamics but also alters pharmacokinetics, which in turn might
4
or B , but erythrocyte
4
3.7. In Vitro Transfection. By summing up all data from
the biocompatibility study, polymers B and B showed good
biocompatibility, inducing neither apoptosis nor necrosis at the
1
2
3
8
affect the tissue distribution of injected delivery vectors.
Figure 6. Microscopic images of erythrocytes following incubation with CPs at polymer concentration of 100 µg/mL for 3 h.

3
110 Biomacromolecules, Vol. 11, No. 11, 2010
Gao et al.
Figure 7. In vitro transfection efficiency of CPNs against MDA-MB-468 cells. (A) Transfection efficiency of B
and B /DNA complex nanoparticles (B Ns) at different N/P ratios. (B) Transfection efficiency of B Ns and B
that of PEINs at mass ratio 3 and naked DNA with plasmid dosage of 2.5 µg/well.
1
/DNA complex nanoparticles (B
1
Ns)
2
2
1
2
Ns at N/P ratio of 32 compared with
test concentration. They also demonstrated low hemolysis ratio
cytotoxicity and blood compatibility tests. In A and B series,
polymers B and B showed good biocompatibility, inducing
and erythrocyte aggregation. Therefore, we chose B
1
and B
2
to
1
2
investigate preliminarily their transfection ability.
neither apoptosis nor necrosis at the test concentration. They
For effective gene delivery, cationic polymer should have
the ability to condense DNA into nanoparticles. Both B and
could form nanoparticles with DNA in water at N/P ratio
g1. The particle sizes of B /DNA complex nanoparticles (B Ns)
and B /DNA complex nanoparticles (B Ns) decreased with the
also demonstrated low hemolysis ratio and erythrocyte aggrega-
tion. In C and D series, except C , the cytotoxicity of all
2
polymers was low, inducing neither apoptosis nor necrosis at
the test concentration. All polymers in C and D series could
1
B
2
1
1
2
2
1 2 1
cause RBC aggregation, except D and D . Polymers B and
N/P ratio increasing and decreased to constant value in the range
2
B with good biocompatibility exhibited comparable transfection
of 160 to 220 nm when the N/P ratios increased from 8 to 64.
The particle sizes of B Ns and B Ns at N/P ratio of 32 were
1 2
efficiency in comparison with PEI (25 kDa) but much lower
cytotoxicity. These results suggested that the new cationic CPs
could be promising carriers for gene delivery. Future work will
involve in the evaluation of the structure-activity relationship
of CPs.
1
71.3 ( 17.3 and 208.3 ( 10.0 nm, respectively. The ꢀ
1 2
potentials of B Ns and B Ns were 20.6 ( 0.8 and 21.6 ( 1.0
mV, respectively.
The ability to withstand serum challenge to some extent is a
very important property for cationic polymer/DNA complex
nanoparticles because the instability of polyplexes in serum
Acknowledgment. The National Basic Research Program of
China (2010CB934000 and 2007CB935800), the National
Natural Science Foundation of China (30925041 and 30901866),
National Science & Technology Major Project “Key New Drug
Creation and Manufacturing Program” (No. 2009ZX09501-024
and 2009ZX09301-001), and Shanghai Nanomedicine Program
(0852 nm05700) are gratefully acknowledged for financial
support.
39
could greatly limit their transfection efficiency. It was reported
that the presence of serum had great effects on the transfection
3
9,40
efficiency of many kinds of polyplexes including PEINs.
But no obvious effect of serum on the transfection efficiency
of B Ns and B Ns was found in the transfection experiment,
1
2
which could attribute to the triazole-amide moiety in the polymer
backbone, which served as H-bond acceptors and could increase
hydrophobic interactions of the polymer with DNA and thus
increase the stability of CPNs in serum.
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