Linear cyclen-based polyamine as a novel and efficient reagent in gene delivery

Article abstract of DOI:10.1039/b914877a

Linear cyclen-based polyamine (LCPA, M_w = 7392, M _w/M_n = 1.19) as a novel non-viral gene vector was designed and synthesized from 1,7-diprotected 1,4,7,10-tetraazacyclododecane (cyclen), bis(β-hydroxylethyl)amine and epichlorohydrin. Agarose gel retardation and fluorescent titration using ethidium bromide showed the good DNA-binding ability of LCPA. It could retard pDNA at an N/P ratio of 4 and form polyplexes with sizes around 250-300 nm from an N/P ratio of 10 to 60 and relatively lower zeta-potential values (< +3 mV) even at the N/P ratio of 60. The cytotoxicity of LCPA assayed by MTT is much lower than that of 25 kDa PEI. In vitro transfection against A549 and 293 cells showed that the transfection efficiency of LCPA/DNA polyplexes is close to that of 25 kDa PEI at an N/P ratio of 10-15, indicating that the new material could be a promising non-viral polycationic reagent for gene delivery.

Full text of DOI:10.1039/b914877a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Linear cyclen-based polyamine as a novel and efficient reagent in gene
delivery†
a
b
a
a
b
a
b
Yong-Zhe Xiang,‡ Zhi-Hua Feng,‡ Ji Zhang,* Yi-Le Liao, Chuan-Jiang Yu, Wen-Jing Yi, Wen Zhu*
and Xiao-Qi Yu*
a
Received 22nd July 2009, Accepted 20th October 2009
First published as an Advance Article on the web 30th November 2009
DOI: 10.1039/b914877a
Linear cyclen-based polyamine (LCPA, M
w
= 7392, M
w
/M
n
= 1.19) as a novel non-viral gene vector
was designed and synthesized from 1,7-diprotected 1,4,7,10-tetraazacyclododecane (cyclen),
bis(b-hydroxylethyl)amine and epichlorohydrin. Agarose gel retardation and fluorescent titration using
ethidium bromide showed the good DNA-binding ability of LCPA. It could retard pDNA at an N/P
ratio of 4 and form polyplexes with sizes around 250–300 nm from an N/P ratio of 10 to 60 and
relatively lower zeta-potential values (< +3 mV) even at the N/P ratio of 60. The cytotoxicity of LCPA
assayed by MTT is much lower than that of 25 kDa PEI. In vitro transfection against A549 and
2
93 cells showed that the transfection efficiency of LCPA/DNA polyplexes is close to that of 25 kDa
PEI at an N/P ratio of 10–15, indicating that the new material could be a promising non-viral
polycationic reagent for gene delivery.
Introduction
molecular weight (LMW) PEI has much lower cytotoxicity than
that of high-molecular weight (HMW) PEI, but its transfection
efficiency is also lower. To obtain decreased cytotoxicity and
Safe and efficient gene delivery vectors, including viral and
non-viral vectors, are prerequisites for successful gene therapy.
As viral approaches often cause notable toxicity and immuno-
genicity, non-viral systems have received increased attention
because of their potential to overcome many inherent challenges
of viral vectors. Among non-viral vectors, cationic polymers
and cationic lipids are the two major systems. Compared with
cationic lipids which have been widely used in gene delivery,
cationic polymers possess many advantages such as a good
ability to condense DNA and ease of special modifications.
Many cationic polymers, such as polyethyleneimine (PEI),
polyamidoamine dendrimers, poly(L-lysine) (PLL), poly[a-(4-
aminobutyl)-L-glycolic acid], chitosan, poly(b-aminoesters),
14
1
2
improved transfection efficiency, much attention has been paid to
modified PEI. The commonly used methods are to block or graft
PEI with biocompatible moieties, such as poly(ethylene glycol)
15
16
17
(
PEG), chitosan and dextran. Although several copolymers
3
displayed lower cytotoxicities along with comparable, or even
higher transfection efficiencies compared with PEI, seeking other
materials with novel structures is of great importance.
4
The positive charge on amino groups of polyamines may
facilitate charge interactions with the phosphate backbone of
DNA. Increasing knowledge about the crucial role of 1,4,7,10-
tetraazacyclododecane (cyclen) in cell biology stimulated wide
basic and applied research interests. Macrocyclic complexes with
tetraazamacrocyclic ligands, such as cyclen, cyclam and bicyclam,
5
6
7
8
9
10
11
12
poly[N,N-(dimethylamino)ethyl methacrylate] and polycationic
13
cyclodextrin, have been used as gene delivery carriers with
individual properties.
18
exhibited antitumor or anti-HIV activity. These important and
versatile applications stimulate researchers to explore new cyclen-
based ligands and complexes with potential chemical, biological
and catalytic properties. Thus, many studies have been focused
on metal complexes of cyclen and their cleavage ability towards
Among various cationic polymers, PEI is the most studied ma-
terial for DNA delivery because of its strong buffering capability
in the pH region of 7.4–5.1 together with high binding capabil-
ity towards DNA and a relatively high transfection efficiency.
Therefore, PEI has been considered as the gold standard of gene
phosphoesters and DNA as artificial enzymes in chemical biology,
19-21
medicine and gene therapy.
Some bicyclam derivatives were
3
transfection. However, the high cytotoxicity partly caused by its
22
used as non-viral vectors for specific gene delivery. More recently,
we also reported a novel cationic macrocyclic polyamine lipid
containing an imidazolium salt group and a cyclen unit, which
could transfer plasmid DNA into cells without use of extraneous
agent. Moreover, we prepared a reticular cyclen-based polymer
from cyclen and epichlorohydrin (EPI), however, the transfection
efficiency was not satisfying.
high charge density seriously hampered its therapeutic use. Low-
a
Key Laboratory of Green Chemistry and Technology (Ministry of Edu-
cation), College of Chemistry, Sichuan University, Chengdu, 610064,
P. R. China. E-mail: xqyu@tfol.com; Fax: +86 28 8541 5886; Tel: +86
23
24
2
8 8546 0576; Tel: +86 28 8516 4063
b
State Key Laboratory of Biotherapy, West China Hospital, Sichuan
University, Chengdu, 610041, P. R. China. E-mail: zwjulia@163.com
Herein, we wish to report a novel linear cyclen-based polyamine,
which is prepared from cyclen, bis(b-hydroxylethyl)amine and
epichlorohydrin. The buffer capability of the title compound
was detected by acid–base titration. The interaction between the
polymer and plasmid DNA was studied by fluorescence titration
†
Electronic supplementary information (ESI) available: NMR spectra
for 1, 2 and LCPA and size-exclusion chromatography (SEC) and
multiple angle laser light scattering (MALLS) assay for LCPA. See DOI:
1
0.1039/b914877a
‡
These authors contributed equally to this work.
6
40 | Org. Biomol. Chem., 2010, 8, 640–647
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Scheme 1 Synthetic route to LCPA. a) (Boc)
tetraazacyclodocane, EtOH; d) HCl (g), CH Cl
2
O, Et
3
N, CHCl
3
; b) epichlorohydrin, Bu
4
NBr, NaOH, H
2
O; c) 1, 7-bis(tert-butyloxycarbonyl)-1,4,7,10-
2
2
; e) NaOH, CHCl .
3
and agarose gel electrophoresis. The properties such as particle
size and zeta-potential of the formed complexes were also studied.
In vitro transfection experiments demonstrate that this cyclen-
based polymer may act as efficient non-viral vector in gene delivery
with low cytotoxicity.
Results and discussion
Synthesis and characterization of LCPA
The preparation of LCPA is shown in Scheme 1. The nitrogen
atom of bis(b-hydroxylethyl)amine was first protected by a Boc
group to avoid a side reaction caused by the nucleophilic attack
of an N atom to epichlorohydrin. Then compound 1 reacted
Fig. 1 Acid–base titration profiles of PEI and LCPA. LCPA or PEI
(0.25 mmol of amino groups) was first treated with 1 N HCl to adjust pH
to 2.0, and then the solution pH was measure after each addition of 50 mL
of 0.1 N NaOH. The relatively flat curve of PEI indicates its higher buffer
capability than that of LCPA.
smoothly with epichlorohydrin in the presence of Bu NBr–
4
NaOH to give the bridge unit 2. The protected cyclen-based
polymer 3, in which many new-formed hydroxyls may increase
the water-solubility, was subsequently prepared by the reaction
between compound 2 and 1,7-bis(tert-butyloxycarbonyl)-1,4,7,10-
tetraazacyclodocane (diBoc-cyclen) in equal molar ratio. Polymer
Formation of LCPA/DNA complexes
DNA condensation capability is a prerequisite for polymeric gene
vectors. Gel retardation analysis was performed to confirm the
affinity between LCPA and plasmid DNA. As shown in Fig. 2, the
electrophoretic mobility of DNA was retarded by the introduction
of LCPA, and total DNA retardation was detected at and above
N/P ratios of 4. The results suggested that LCPA can bind to
DNA through electrostatic interactions between DNA backbone
3
could be recrystallized from EtOH–hexane. Finally, the water-
soluble LCPA could be obtained by deprotection of Boc groups
using HCl (g) in dichloromethane. NMR analysis of the final
product showed the absence of a singlet at 1.42–1.44 ppm,
indicating that the Boc groups were completely cleaved from
the polyamine backbone. The final LCPA was analyzed via size-
exclusion chromatography (SEC) in combination with multiple
angle laser light scattering to determine the molecular weight
(
M
w
= 7392, M
w
/M
n
= 1.19). In addition, we also tried to
use diethoxyphosphoryl (DEP) groups for the 1,7-diprotection
of cyclen. However, this method was abandoned because of the
difficulty of deprotection in the last step.
Buffer capability
Cationic polymers are assumed to have the ability to facilitate the
escape of polyamine/DNA complexes from the endosome by the
25
“
proton sponge effect” and to promote transfection efficiency. In
this study, the buffer capabilities of LCPA and 25 kDa PEI were
evaluated by acid–base titration. As shown in Fig. 1, the buffer
capability of LCPA is lower than that of PEI. It was reported
that the buffer capability of a polycation mainly depends on the
presence of primary, secondary and tertiary amines groups. Since
LCPA has a lower molecular weight and less density of amino
groups in the structure relative to 25 kDa PEI, a lower buffer
capacity of LCPA is reasonable.
Fig. 2 Electrophoretic mobility of plasmid DNA in the presence of LCPA
(ethidium bromide staining). Lanes 1–5 (from left): LCPA/pDNA complex
with N/P ratio of 2, 4, 6, 8, and 10, respectively; Lane 6: pDNA control.
This journal is © The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 640–647 | 641

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and the cationic nitrogen atoms both in the cyclen cycle and on
the bridge.
The ethidium bromide (EB) exclusion assay is another useful
method in the studies of binding ability of polyamines to DNA. EB
has weak fluorescence, but its emission intensity in the presence of
DNA could be greatly enhanced because of its strong intercalation
between the adjacent DNA base pairs. It was previously reported
that this enhanced fluorescence could be quenched, or at least
partly quenched by the addition of a second molecule with
higher DNA-binding ability, such as polycations. The percent of
decreased fluorescence valuecan be used as a parameter toevaluate
the DNA binding affinity of certain materials. Fig. 3 shows that
the addition of LCPA to EB pretreated with DNA caused an
appreciable decrease in the emission intensity, indicating that EB
which bound to DNA was partially replaced by LCPA. The relative
fluorescence decreased by approximately 50% at N/P ratio of
5
. The results, which are in agreement with the gel retardation
analysis results, may confirm the good binding ability of LCPA
towards DNA.
Fig. 4 Average particle size (A) and zeta-potential (B) of LCPA/pDNA
at N/P ratios of 5, 10, 15, 20, 30, 40, 50 and 60.
Zeta-potential is an indicator of surface charges on the poly-
mer/pDNA nanoparticles. A positively charged surface allows
electrostatic interaction with anionic cell surfaces and facilitates
14a,27
cellular uptake.
complexes were also measured at N/P ratios ranging from 5 to 60
Fig. 4B). The net surface charge of the LCPA/DNA complexes
The zeta-potential values of LCPA/DNA
Fig. 3 Change of relative fluorescence of EB bound to DNA by the
addition of LCPA to different N/P ratios. All the samples were excited at
(
4
97 nm and the emission was measured at 600 nm. DNA concentration
stabilized at the N/P range of 5–20, and increased at the N/P ratio
of 20 and above. This zeta-potential range of 1.6–3.0 mV is much
lower than that of most reported polycation/DNA complexes
-1
was 3.8 mg mL .
The appropriate polymer/DNA nanoparticle is of critical
important for polyamines used as gene vectors. And the particle
size would apparently affect the transfection efficiency of gene
vectors. The particle size depends on many parameters, such as
DNA concentration, sequence of addition of polyamines or DNA
during complex preparation, and ionic strength of the solvent.
The particle size of LCPA/pDNA complexes was measured
at various N/P ratios, and the results are shown in Fig. 4A.
LCPA can efficiently compact pDNA into small nanoparticles.
Generally, the hydrodynamic sizes of the complexes decrease
with increasing N/P ratios. After reaching the N/P ratio of 10,
LCPA can condense DNA into nanoparticles of 250–300 nm in
diameter. Gel retardation analysis and ethidium bromide exclusion
assay have shown that LCPA has a high bind affinity towards
plasmid DNA. Thus under relatively high N/P, more LCPA
molecules could bind to DNA without an increase of polyplex
size. Indeed, the particles with a size of 100–200 nm are prone to
endocytosis. However, a larger size of particles is favorable to cell
attachment and subsequent cell uptake by increased sedimentation
(
about 20 mV) and is comparable to the results of PEG-PEI
28
copolymers which act as effective gene carriers. This relatively
low zeta-potential might be attributed to the repeated hydroxyl
groups and ether bonds which can screen the positive charge in the
structure of LCPA. Moreover, the compartmentation of cationic
cyclen moieties by the bis(b-hydroxylethyl)amine-epichlorohydrin
bridges also decreased the density of amino groups. On the other
hand, the increase of zeta potential under higher N/P might be
owing to the larger number of LCPA molecules binding to DNA. It
is known that polyamines with high positive surface charge may
cause high cytotoxicity and decreased transfection efficiency. Thus,
we hope that the low positive charge of LCPA may decrease the
cytotoxicity and benefit gene transfection.
Cytotoxicity
The cytotoxicity of cationic polymers is thought to be caused
by damage from the interaction with plasma membrane or
other cellular compartments, and researches have found a rough
correlation between toxicity and transfection efficiency. The
cytotoxicity of LCPA was evaluated in A549 and 293 cells by
MTT assay, and the 25 kDa PEI was used as the control. As
26
29
of complexes. Meanwhile, larger nanoparticles might promote
the escape of gene from the polyamine/DNA complexes into the
27
nucleus.
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42 | Org. Biomol. Chem., 2010, 8, 640–647
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Fig. 6 Luciferase expression in A549 and 293 cells transfected by 25 kDa
PEI/DNA (N/P = 10) and LCPA/DNA complexes at different N/P
ratios. For comparison with Fig. 5, the concentrations of LCPA in the
-1
transfection experiments were 7.5, 15, 22.5, 30, 45, 60 mg mL for the N/P
ratio of 5, 10, 15, 20, 30, 40, respectively.
complexes is about 1.3 times better than that of LCPA/DNA
complexes. In addition, comparing the two types of cells, the
transfection efficiency in 293 cells is higher than those in A549 cells
within the range of N/P ratios tested. This difference may be
attributed to the lower cytotoxicity of LCPA in 293 cells than that
in A549 cells.
Results showed that LCPA can act as an effective non-viral gene
vector withrelativelylow cytotoxicityandcomparable transfection
efficiency to that of 25 kDa PEI. In to the chemical structure of
LCPA, the unique macrocyclic system affords four amino groups
Fig. 5 Relative cell viabilities of LCPA and 25 kDa PEI in (A) A549 cells
and (B) 293 cells.
with different pK values (>9 for two amino groups and <5 for
other two) for each cyclen moiety. These values give a relative
measure of the basicity of the amino groups. The ones with higher
a
shown in Fig. 5, 25 kDa PEI displayed serious cytotoxicity in two
cell lines and the relative cell viability of PEI were less than 20%
30
-
1
when its concentration was over 30 mg mL . In comparison with
5 kDa PEI, LCPA indicated relatively high cell viabilities at 30 mg
pK
a
would help DNA binding and the formation of polyplex, while
are expected to remain
2
the other amino groups with lower pK
a
-
1
mL concentration (65% and 80% relative cell viability for A549
and 293 cells, respectively). The much lower cytotoxicity of LCPA
may be ascribed to the low positive charge, which is caused by
the relatively lower density of amino groups, on the surface of the
complexes. Additionally, cell-dependent cytotoxicities were found
for LCPA, and A549 cells showed a relatively severe weakness
against the cytotoxicity of both PEI and LCPA (Fig. 5A).
unprotonated in the polyplex, presumably due to the lower proto-
nation power and the steric restriction, directing to the enhanced
31
buffering capacity in the endosomal compartment. To directly
visualize the infected cells expressing pEGFP-Nl, enhanced green
fluorescent protein expression in A549 and 293 cells was observed
by an inverted fluorescent microscope. According to the results
of the luciferase assay, LCPA/DNA and PEI/DNA (control)
complexes were used at the optimal N/P ratios of 15 and 10,
respectively. The results were in accordance with the luciferase
expression in the same two cell lines. As shown in Fig. 7, the images
also indicated that the density of transfected cells by LCPA/DNA
is close to that caused by 25 kDa PEI/DNA complexes in both
cell lines. For the N/P ratios of LCPA/DNA complexes in the
range of 10 to 40, good transfection with low cytotoxicity were
observed in both 293 and A549 cell lines. Further increase (>40)
or decrease (<10) of the N/P ratio led to much lower transfection
efficiency, only a few cells that expressed GFP were observed by
microscopy (data not shown). Similar results were obtained in
luciferase expression experiments. This low transfection efficiency
was attributed to the liability to degradation under low N/P and
the difficulty to release DNA under high N/P. The transfection
efficiency of LCPA/DNA complexes towards 293 cells is higher
than that towards A549 cells, which was consistent with luciferase
experiments.
In vitro transfection
The gene transfection efficiency of LCPA/DNA complexes was
assessed by in vitro delivery experiments of luciferase reporter
gene (plasmid pGL-3) into A549 and 293 cells. 25 kDa PEI was
used for comparison because of its high transfection efficiency
and easiness to get. PEI/DNA polyplexes were prepared at an
N/P ratio of 10, and LCPA/DNA complexes were prepared at
various N/P ratios ranging from 5 to 40. Fig. 6 showed the effect
of different N/P ratios on transfection efficiency. The transfection
efficiency of LCPA/DNA complexes increased with the increase of
N/P ratio, and the maximal transfection efficiency was observed
at N/P ratio of 15 in both of the two cell lines, while the optimal
N/P ratio for PEI is 10 (only the transfection data of PEI under
N/P of 10 are shown). A further increase of N/P ratio might lead
to higher cytotoxicity and decreased transfection efficiency. The
results showed that the transfection efficiency of the PEI/DNA
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Experimental procedures
Chemicals and instruments
All chemicals and reagents were obtained commercially and
were used as received. Anhydrous ethanol (CH CH OH), anhy-
drous dichloromethane, triethylamine (NEt ) and epichlorohy-
3
2
3
drin were dried and purified under nitrogen by using standard
methods and were distilled immediately before use. 1,7-Bis(tert-
butyloxycarbonyl)-1,4,7,10-tetraazacyclodocane was prepared ac-
32
cording to the literature. High molecular weight PEI (branched,
average molecular weight 25 kDa: 25 kDa PEI) and MTT
(3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). The
plasmids used in the study were pGL-3 (Promega, Madison, WI,
USA) coding for luciferase DNA and pEGFP-N1 (Clontech,
Palo Alto, CA, USA) coding for EGFP DNA. The Dulbecco’s
Modified Eagle’s Medium (DMEM), 1640 Medium and fetal
bovine serum were purchased from Invitrogen Corp. MicroBCA
protein assay kit was obtained from Pierce (Rockford, IL, USA).
Luciferase assay kit was purchased from Promega (Madison, WI,
USA). Endotoxin free plasmid purification kit was purchased from
TIANGEN (Beijing, China).
DECA
MS-ESI spectra data were recorded on a Finnigan LCQ
and a Bruker Daltonics BioTOF mass spectrometer respectively.
1
The H NMR spectra were obtained on a Varian INOVA-
4
00 spectrometer. CDCl was used as solvent and TMS as the
3
internal reference. IR spectra were measured with a Shimadzu
FTIR-4200 spectrometer. Fluorescence spectra were measured
by a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. The
molecular weight of polyamine was determined by size-exclusion
chromatography (SEC) in combination with multiple angle laser
light scattering (Wyatt Technology Corporation), incorporating
Shodex columns (OHPAK KB-803).
Fig. 7 Fluorescent microscope images of pEGFP-transfected cells: (A)
LCPA in A549 at N/P = 15, (B) 25 kDa PEI in A549 at N/P = 10, (C)
LCPA in 293 at N/P = 15, (D) 25 kDa PEI in 293 at N/P = 10. These
4
cm-wide images were obtained after 68-fold reduction from original
Preparation of title polyamine
pictures, which were recorded at the magnification of 100¥.
N-tert-Butyloxycarbonyl-bis(b-hydroxylethyl)amine (1). Bis-
(
b-hydroxylethyl)amine (1.05 g, 0.01 mol) was dissolved in CHCl
triethylamine was added to adjust the pH to 9. At room temper-
ature, a solution of (Boc) O (2.38 g, 0.011 mol) in CHCl was
3
,
Conclusions
A new type of polycation, linear cyclen-based polyamine (LCPA)
was designed and synthesized from 1,7-diprotected 1,4,7,10-
tetraazacyclododecane (cyclen), bis(b-hydroxylethyl)amine and
epichlorohydrin. This new polymeric material showed enough abil-
ity to condense DNA into nanoparticles with lower cytotoxicity
compared with 25 kDa PEI for its relatively low charge density.
In vitro transfections of reporter genes of luciferase and enhanced
green fluorescent protein against A549 and 293 cell lines suggested
that the transfection efficiency of LCPA/DNA complexes is close
to that of 25 kDa PEI/DNA complexes, indicating that the new
type of polyamine could be a promising non-viral polycationic
reagent for gene delivery. As a new type of polyamine which is
easily modified, some shorter or N-rich bridge groups can be
introduced to increase the charge density of LCPA. Considering
the possibility of increased cytotoxicity induced by the increase
of charge density, some bio-degradable groups such as ester and
disulfide bridges can be constructed. Relative studies that focus
on the elucidation of the structure–efficiency relationship of such
kinds of polycations are now in progress.
2
3
added dropwise to the above solution. After stirring overnight
at room temperature, the reaction mixture was condensed under
reduced pressure. The residue was purified by alumina gel column
chromatography (v/v 9 : 1, CH
as colorless oil. Yield: 82%. IR (CH
877, 1669, 1475, 1412, 1367, 1234, 1170, 1077, 880, 775. H NMR
400 MHz, CDCl ): d 4.25 (br, 1H, OH), 4.11 (br, 1H, OH), 3.78
t, J = 4.4 Hz, 4H, CH O), 3.42 (t, J = 4.8 Hz, 4H, NCH ), 1.46
s, 9H, Boc). C NMR (400 MHz, CDCl ): d 156.42, 80.31, 61.80,
2.24, 28.40. MS (ESI): m/z = 228.1 [M + Na] .
2
Cl
2
–MeOH) to give compound 1
-
1
2
Cl , cm ): 3389, 2971, 2931,
2
1
2
(
(
(
3
2
2
1
3
3
+
5
N-tert-Butyloxycarbonyl-bis(2-(oxiran-2-ylmethoxy)ethyl)amine
(2). A mixture of epichlorohydrin (2.1 mL, 26.3 mmol),
sodium hydroxide pellets (1 g, 26.3 mmol), water (0.12 mL,
6.6 mmol), tetrabutylammonium bromide (71 mg, 0.22 mmol)
and compound 1 (0.9 g, 4.4 mmol) was stirred for 4 h at
◦
40 C. The reaction mixture was filtered off and the solid was
washed with dichloromethane. The combined organic layer was
6
44 | Org. Biomol. Chem., 2010, 8, 640–647
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dried with anhydrous magnesium sulfate. The solvent and excess
epichlorohydrins were distilled off under reduced pressure and the
residue was purified by silica gel column chromatography (v/v
Amplification and purification of plasmid DNA
pGL-3 and pEGFP plasmids were used. The former one as the
luciferase reporter gene was transformed in E. coli JM109 and the
latter one as the green fluorescent protein gene was transformed
in E. coli DH5a. Both plasmids were amplified in terrific broth
2
6
1
3
3
3
2
5 : 1, CH
1%. IR (CH
366, 1250, 1151, 1111, 851, 737. H NMR (400 MHz, CDCl
.75 (dd, J = 11.2, 2.2 Hz, 2H, CH O), 3.64–3.58 (m, 4H, CH
.49–3.44 (m, 4H, CH O + NCH
.16–3.12 (m, 2H, CH), 2.79 (dd, J = 4.8, 4.8 Hz, 2H, ring CH
.61 (dd, J = 4.8, 2.8 Hz, 2H, ring CH ), 1.45 (s, 9H, Boc).
): d 155.44, 79.69, 71.85, 69.87, 50.80,
2
Cl –MeOH) to give compound 2 as colorless oil. Yield:
2
-
1
2
Cl , cm ): 2968, 2926, 2863, 1690, 1462, 1410,
2
1
3
): d
◦
media at 37 C overnight. The plasmids were purified by an
2
2
O),
TM
EndoFree Tiangen Plasmid Kit. Then the purified plasmids
2
2
), 3.41–3.36 (m, 2H, NCH
2
),
),
C
◦
were dissolved in TE buffer solution and stored at -20 C. The
2
1
3
integrity of plasmids was confirmed by agarose gel electrophoresis.
The purity and concentration of plasmids were determined by
ultraviolet (UV) absorbance at 260 and 280 nm.
2
NMR (400 MHz, CDCl
8.01, 44.23, 28.42. MS (ESI): m/z = 340.2 [M + Na] .
3
+
4
Agarose gel retardation assay
Polymer 3. 1,7-Bis(tert-butyloxycarbonyl)-1,4,7,10-tetraazacy-
clodocane (1.29 g, 3.47 mmol) was dissolved in C OH, then
2
H
5
LCPA/DNA complexes at different N/P ratios (the amino groups
of LCPA to phosphate groups of DNA) ranging from 2 to 10 were
prepared by adding an appropriate volume of LCPA (in 150 mM
NaCl solution) to 0.8 mL of pEGFP-N1 DNA (120 ng mL in
40 mM Tris-HCl buffer solution). The complexes were diluted
by 150 mM NaCl solution to a total volume of 6 mL, and then
compound 2 (1.1 g, 3.47 mmol) was added to the solution. Under
◦
the protection of N
2
, the reaction mixture was stirring at 80 C for
-
1
2
4 h. The solvent was removed under reduced pressure. The residue
was dissolved in CHCl , cyclohexane was added to precipitate
polymer 3 (1.15 g). IR (CH
691, 1461, 1412, 1366, 1248, 1159, 979, 774, 735. H NMR
400 MHz, CDCl ): d 3.85 (br, 2H, CH), 3.52–2.95 (m, 24H, other
Hs), 2.60–2.25 (m, 8H, cyclen CH adjacent to alkyl N), 1.44–1.42
m, 27H, Boc).
3
-
1
2
Cl , cm ): 3422, 2974, 2929, 2867,
2
1
◦
1
(
the complexes were incubated at 37 C for 30 min. After that
-
1
3
the complexes were electrophoresed on the 0.7% (W V ) agarose
gel containing EB and with Tris-acetate (TAE) running buffer at
110 V for 30 min. DNA was visualized with a UV lamp using a
BioRad Universal Hood II.
2
(
LCPA. Polymer 3 (1.1 g) was dissolved in CH
2
Cl , and HCl
2
Ethidium bromide displacement assay
gas was imported. The reaction mixture was stirring overnight
at room temperature. The solvent was removed under reduced
pressure, and the residue was dissolved in a small amount
of water. Then 5 N NaOH aqueous solution was added to
adjust pH to 12. The alkaline solution was extracted with hot
The ability of LCPA to condense DNA was studied using
ethidium bromide (EB) exclusion assays. Fluorescence spectra
were measured at room temperature in air by a Horiba Jobin Yvon
Fluoromax-4 spectrofluorometer and corrected for the system
CHCl
After removing the solvent, LCPA was obtained as a white
solid (510 mg). The molecular weight of LCPA (M
= 7392,
/M
= 1.19) was measured by size-exclusion chromatography
SEC) in combination with multiple angle laser light scattering.
3
. The organic layer was dried over anhydrous Na
2
SO
4
.
-1
response. EB (5 mL, 1 mg mL ) was put into quartz cuvette
containing 2.5 mL of 150 mM NaCl solution. After shaking, the
w
fluorescence intensity of EB was measured. Then DNA (9.5 mL,
M
(
w
n
-1
1
mg mL ) was added to the solution and mixed symmetrically,
and the measured fluorescence intensity is the result of the
interaction between DNA and EB. Subsequently, the solutions
-
1
IR (CH
2
Cl
2
, cm ): 3355, 2924, 2856, 1495, 1359, 1116, 948, 732.
): d 3.88 (m, 2H, CH), 3.58–3.44
NCH CH OCH ), 2.76–2.50 (m, 20H,
1
H NMR (400 MHz, CDCl
m, 12H, CH OCH CH
other Hs).
3
-1
of LCPA (0.55 mg mL , 4 mL for each addition) were added to
(
2
2
2
2
2
2
the above solution for further measurement. All the samples were
excited at 497 nm and the emission was measured at 600 nm.
Particle size and zeta-potential measurements
Acid–base titration
Particle size and zeta-potential were measured by a Zeta Nano
Series (Malvern Instruments Led) at 25 C. The LCPA/DNA
complexes at various N/P ratios ranging from 10 to 60 were
prepared by adding an appropriate volume of LCPA solution (in
Briefly, LCPA (0.25 mmol of amino groups) was dissolved in 5 mL
of 150 mM NaCl aqueous solution, and 1 N HCl was added to
adjust pH to 2.0. Aliquots (50 mL for each) of 0.1 M NaOH
were added, and the solution pH was measured with a pH meter
◦
1
50 mM NaCl solution) to 100 mL of pEGFP-N1 DNA solution
(
pHS-25) after each addition. For comparison, PEI (25 kDa) was
-
1
(
50 mg mL in 40 mM Tris-HCl buffer solution) with the final
used under same experimental conditions.
volume of 200 mL Then the complexes were incubated at room
temperature for 30 min. After that the complexes were diluted by
150 mM NaCl solution to 1 mL prior to measurement.
Cell culture
HEK (human embryonic kidney) 293 cells and human non-
small-cell lung carcinoma A549 cells were incubated respectively
in Dulbecco’s Modified Eagle’s Medium (DMEM) and 1640
Medium containing 10% fetal bovine serum (FBS) and 1%
Cell viability assay
Toxicity of LCPA toward 293 cells and A549 cells was deter-
mined by using MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide) reduction assay following literature pro-
cedures. The 293 cells (6000 cells/well) and A549 cells
-
1
◦
antibiotics (penicillin-streptomycin, 10000 U mL ) at 37 C in
a humidified atmosphere containing 5% CO
2
.
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(
14000 cells/well) were seeded into 96-well plates. The cells were
Acknowledgements
then incubated in a culture medium containing LCPA with a
particular concentration for 24 h. After that, the medium was
replaced with 200 mL of fresh medium, and 20 mL of sterile filtered
MTT (5 mg mL ) stock solution in PBS was added to each well.
After 4 h, unreacted dye was removed by aspiration. The formazan
crystals were dissolved in 150 mL DMSO per well and measured
spectrophotometrically in an ELISA plate reader (model 550, Bio-
Rad) at a wavelength of 570 nm. The cell survival was expressed
as follows: Cell viability = (ODtreated/ODcontrol) ¥ 100%.
This work was financially supported by the National Science
Foundation of China (Nos. 20725206 and 20732004), Specialized
Research Fund for the Doctoral Program of Higher Education
and Scientific Fund of Sichuan Province for Outstanding Young
Scientist. We also thank Sichuan University Analytical & Testing
Center for NMR spectra analysis.
-
1
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