Diol glycidyl ether-bridged cyclens: Preparation and their applications in gene delivery

Article abstract of DOI:10.1039/c0ob00879f

Polymeric 1,4,7,10-tetraazacyclododecanes (cyclens) using diol glycidyl ether with different chain length as bridges (5a-e) were designed and synthesized from various diols, 1,7-diprotected cyclen and epichlorohydrin. The molecular weights of the title polymers were measured by GPC with good polydispersity. Agarose gel retardation and fluorescent titration using ethidium bromide showed good DNA-binding ability of 5. They could retard plasmid DNA (pDNA) at an N/P ratio of 4-6 and form polyplexes with sizes around 100-250 nm from an N/P ratio of 10 to 60 and relatively low zeta-potential values (5-22 mV). The cytotoxicity of 5 assayed by MTT is much lower than that of 20 kDa PEI. In vitro transfection against A549 and 293 cells showed that the transfection efficiency (TE) of 5c/DNA polyplexes is close to that of 20 kDa PEI at an N/P ratio of 5. Structure-activity relationship (SAR) of 5 was discussed in their DNA-binding, cytotoxicity, and transfection studies. The TE of 5c/DNA polyplexes could be improved by the introduction of 50 μM of chloroquine, the endosomolytic agents, to pretreated cells. These studies may extend the application areas of macrocyclic polyamines, especially for cyclen.

Full text of DOI:10.1039/c0ob00879f

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Organic &
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Chemistry
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PAPER
Diol glycidyl ether-bridged cyclens: preparation and their applications in gene
delivery†
Wen-Jing Yi,‡ Zhi-Hua Feng,‡ Qin-Fang Zhang, Ji Zhang,* Ling-Dong Li, Wen Zhu* and Xiao-Qi Yu*
Received 14th October 2010, Accepted 6th January 2011
DOI: 10.1039/c0ob00879f
Polymeric 1,4,7,10-tetraazacyclododecanes (cyclens) using diol glycidyl ether with different chain
length as bridges (5a–e) were designed and synthesized from various diols, 1,7-diprotected cyclen and
epichlorohydrin. The molecular weights of the title polymers were measured by GPC with good
polydispersity. Agarose gel retardation and fluorescent titration using ethidium bromide showed good
DNA-binding ability of 5. They could retard plasmid DNA (pDNA) at an N/P ratio of 4–6 and form
polyplexes with sizes around 100–250 nm from an N/P ratio of 10 to 60 and relatively low
zeta-potential values (5–22 mV). The cytotoxicity of 5 assayed by MTT is much lower than that of 20
kDa PEI. In vitro transfection against A549 and 293 cells showed that the transfection efficiency (TE)
of 5c/DNA polyplexes is close to that of 20 kDa PEI at an N/P ratio of 5. Structure–activity
relationship (SAR) of 5 was discussed in their DNA-binding, cytotoxicity, and transfection studies. The
TE of 5c/DNA polyplexes could be improved by the introduction of 50 mM of chloroquine, the
endosomolytic agents, to pretreated cells. These studies may extend the application areas of macrocyclic
polyamines, especially for cyclen.
Introduction
the high gene transfection efficiency (TE) observed with PEI might
be attributed its comparatively low pK values. The intracellular
a
The efficient delivery of nucleic acids into cells is critical for
release of PEI-complexed nucleic acids from endosomes is hypoth-
esized to rely on the protonation of amines in the PEI molecule,
1
,2
successful gene delivery or gene transfection. Among the various
viral and non-viral vectors, cationic polymers have been receiving
more and more attention and have been considered to be the
most substantial competitors to sometimes unsafe viral vectors
for gene therapy. These materials can potentially avoid toxicity
and immunogenicity, efficiently condense DNA into compact
particles, facilitate cellular uptake of nanosized polyplexes, and
achieve prolonged gene expression. Since the first performance
of polycation-mediated gene delivery in 1987, many polycation
14
which has been explained by the “proton sponge effect”. High-
molecular weight PEI has higher TE than low-molecular weight
3
15,16
PEI, but its cytotoxicity is also higher.
For further enhancement
4
of TE and biocompatibility, some functional moieties such as
5
17
18
19
poly(ethylene glycol) (PEG), cyclodextrin and chitosan have
been introduced into PEI. Although several copolymers displayed
lower cytotoxicities along with comparable, or even higher TEs
compared with PEI, seeking other materials with novel structures
is of great importance.
6
7
materials have been explored for gene transfection applications,
with the most intensively studied examples polyethyleneimine
Our previous studies focused on the synthesis of 1,4,7,10-
8
9
10
(
PEI), chitosan, polyamidoamine dendrimers (PAMAM) etc.
tetraazacyclododecane (cyclen) derivatives and their interactions
Among non-viral vectors applied in gene therapy, polyethylen-
imine (PEI) has successfully been used both under in vitro and
in vivo conditions.
plexes with DNA and, recently, have also been reported for
the trafficking of siRNA to induce RNAi because of their high
20–22
with plasmid DNA.
The cyclen moiety also has repeated
ethylenediamine units which is similar to those in PEI. Further,
like branched PEI, the four N atoms on the cyclen cycle also
1
1,12
PEIs are able to form non-covalent com-
show different properties, especially pK . Thus, for the easily
a
modification of cyclen derivatives, we had hoped to design and
synthesize novel cyclen-based polymeric structures which could be
used as efficient non-viral gene vectors. Some bicyclam derivatives
1
3
cationic charge density at physiological pH. It is assumed that
23
have been used as non-viral vectors for specific gene delivery. We
Key Laboratory of Green Chemistry and Technology (Ministry of Educa-
tion), College of Chemistry, and State Key Laboratory of Biotherapy, West
China Hospital, Sichuan University, Chengdu 610064, People’s Republic of
China. E-mail: xqyu@tfol.com, zwjulia@163.com; Fax: +86 28 8541 5886;
Tel: +86 28 8546 0576; Tel: +86 28 8516 4063
24
25
also reported cyclen-based cationic lipids or reticular polymers,
which could transfer plasmid DNA into cells without the use of
extraneous agents. However, the TE was not satisfying. More
recently, we prepared a novel linear cyclen based polyamine
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c0ob00879f
These authors contributed equally to this work.
(
LCPA), and it showed comparable TE to that of 25 kDa PEI
26
with relatively low cytotoxicity. It is of great importance to
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Scheme 1 Synthetic route of linear cyclen-based cationic polymers. (i) Epichlorohydrin, Bu
iii) Anhydrous HCl, CH Cl . (iv) aqueous NaOH.
4
NBr, NaOH, H
2
O. (ii) 1,7-diBoc cyclen, EtOH, reflux, 40 h.
(
2
2
further investigate the structure–activity relationship (SAR) of this
type of cationic polymer. In this report, we prepared a series of
cyclen-based linear polymers with different diol glycidyl ethers as
a bridge. Their interactions with plasmid DNA and the properties
of formed polyplexes were examined. The in vitro TEs towards
two cell-lines were investigated to reveal the SAR of this type of
cationic polymers in gene delivery.
Table 1 Molecular weights of polymers 5 measured by GPC
Polymer
M
w
M
n
PDI
Calculated n value
5
a
4349
3519
8416
3491
3666
3084
2607
3932
2586
2657
1.41
1.35
2.14
1.35
1.38
9
7
10
7
6
5b
5
5
5
c
d
e
All of the target polymers could dissolve well in water under
studied concentrations.
Results and discussion
Synthesis and characterization of compounds 5
Buffer capability
The preparation route of polymers 5 is shown in Scheme 1.
Diols of various chain length reacted with epichlorohydrin in the
Efficient gene transfer mediated by PEI is in part due to its high
buffering capacity, which is responsible for the timely release of
polyplexes from the endosome and the lysosome by the “proton
presence of Bu
4
NBr–NaOH–H O to give the diglycidyl ethers 2.
2
The protected cyclen-based polymers 3, in which the new-formed
hydroxyls and ether bonds might increase their solubility and
biocompatibility, were prepared by the ring-opening polymeriza-
tion between equal molar amounts of 1,7-diprotected cyclen and
27
sponge effect”. Acid–base titration experiments were carried
out for PEI and 5 to evaluate the buffering capacity of the new
polymers. As shown in Fig. 1, the buffer capabilities of 5 were
slight lower than that of 20 kDa PEI. This lower capability could be
ascribed to the lower molecular weight and lower density of amino
groups relative to PEI. On the other hand, we speculated that the
2
in refluxing ethanol under nitrogen. The bulky and electron-
withdrawing Boc groups on cyclen might hamper the reaction,
and it needs 40 h to achieve a satisfying polymerization degree.
Before deprotection, crude products of 3 were recrystallized by
chloroform/cyclohexane to remove the relatively lower molecular
weight portion. Anhydrous HCl gas was then used to remove Boc
groups on the cyclen ring. During the deprotection process, the
four amino groups with different pK
would contribute to the buffer capability.
a
values on the macrocycle
28
Formation of 5/DNA complexes
clear CH
2
Cl solution of 3 turned turbid, and the hydrochloride
2
1
salt of 4 was precipitated. H NMR (ESI†) showed the absence of
a singlet at 1.42–1.44 ppm, indicating that the Boc groups were
completely cleaved from the polyamine backbone. Polymers 4 were
dialyzed against pure water for 2 d to ensure their polydispersity,
One prerequisite for a polymeric gene carrier is the ability to
condense DNA into compact structures. The formation of a
polyplex can reduce the electrostatic repulsion between DNA
and the cell surface and can protect DNA against enzymatic
29
and the molecular weights (M
w
) of 5 measured by GPC are listed
degradation by nucleases in cytoplasm or serum. 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 5, and total DNA
retardation was detected at and above N/P ratios of 4 (for 5b and
5d) and 6 (for 5a, 5c and 5e). The results suggested that 5 can
bind to DNA through electrostatic interactions between the DNA
backbone and the cationic nitrogen atoms in the cyclen moiety.
in Table 1. Higher M was obtained for 5c, however, the M s
w
n
of the polymers did not have large difference, suggesting that
similar polymerization degrees were achieved for the five polymers.
The relatively low degree of polymerization might be attributed
to the two bulky and electron-withdrawing Boc groups. The
ring-opening reactions using some 1,7-dialkyl substituted cyclens
always lead to high polymerization degrees (unpublished results).
2
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Fig. 1 Determination of the buffering capacity of PEI, water and 5 by
acid–base titration. After polymer solution (0.25 mmol of amino groups)
was prepared, the pH of the solution was raised to 2.0 with 1 M HCl, and
then the solution pH was measured after each addition of 50 mL of 0.1 M
NaOH. The pH of all solutions was measured at room temperature. The
relatively flat curve of PEI indicates its higher buffer capability than those
of 5.
Fig. 3 Change of relative fluorescence of EB bound to DNA by the
addition of 5 to different N/P ratios. All the samples were excited at
497 nm and the emission was measured at 600 nm. DNA concentration
-1
was 10 mg mL in NaCl solution.
30
target cells. The desirable in vivo vectors should efficiently
compact pDNA into small (below 200 nm) nanoparticles, which
is very useful for particle stabilization, efficient endocytosis and
Ethidium bromide (EB) is a cationic dye that is widely used to
probe DNA–cationic agent interactions. The ethidium ion shows
a dramatic increase in fluorescence efficiency when it intercalates
into DNA. The displacement of EB from DNA upon complex
formation with polycations has been used widely to test the ability
of polymers to bind with DNA. Fluorescence intensity may be
quenched with the addition of cationic agents which can bind
to DNA with a higher binding affinity to premixed DNA–EB
solutions. Fig. 3 shows that the addition of 5 to EB pretreated
with DNA caused appreciable decrease in the emission intensity,
indicating that EB that was bound to DNA was partially replaced
by 5. The bridge length might affect the extent of fluorescence
quenching, and the polymer with the longer bridge showed
the better fluorescent quenching ability. For instance, although
polymer 5d has the lowest density of amino groups, it could quench
the fluorescence of EB to the greatest extent. We supposed that the
distance between two cationic cyclen moieties in polymer 5d might
fit that between two anionic phosphate groups in DNA, leading
to more compact condensation. For polymer 5e, the multi-ether
bridge might indeed increase its biocompatibility, but at the same
time, the oxygen-rich chain would harshly decrease its cationic
charge density. As a result, 5e displayed the lowest fluorescence
quenching ability.
31
gene transfer. The particle sizes of 5/pDNA complexes were
measured at various N/P ratios, and the results are shown in
Fig. 4A. As the polyamines were added, the particle sizes of the
polymer/DNA complexes decreased sharply as a result of the
electrostatic interaction and then hardly changed with further
increase of N/P ratio. All five polymers could efficiently compact
pDNA into small nanoparticles with diameters of 100–200 nm,
which are prone to endocytosis. The polyplex formed from multi-
ether-bridged polymer 5e and pDNA gave the biggest size of
almost all studied 5/DNA complexes, especially under N/P ratios
from 10 to 40. This could be also explained by its lower cationic
charge density which led to weaker electrostatic interaction with
DNA and the hydration effect due to the presence of ethylene
glycol units on the bridge.
The zeta-potential could indicate the surface charges on the
polymer/DNA nanoparticles. The zeta-potential of carrier/DNA
complexes is closely connected with cellular uptake, the cytoplas-
32
matic transport, and the migration through the nucleopore. The
zeta-potential values of 5/DNA complexes were also measured at
N/P ratios ranging from 5 to 60 (Fig. 4B). At low N/P ratio of
5–10, the zeta-potentials of the complexes increased sharply with
the increase of N/P ratio. On the other hand, at higher N/P ratios
of 15–60, these values appeared to reach a maximum plateau of
The appropriate size of the polymer/DNA nanoparticle is
of critical important for polyamines as gene vectors into the
Fig. 2 Agarose gel electrophoresis retardation assays of 5/DNA complexes. Plasmid DNA was mixed with 5 at different N/P ratios: 2, 4, 6, 8, and 10,
respectively. Mark “0”: pDNA control.
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Fig. 4 Average particle size (A) and zeta-potential (B) of 5/pDNA at N/P ratios of 5, 10, 15, 30, 40, 50 and 60.
about 5–20 mV, which was in the range of most reported bio-active
polycation/DNA complexes. The bridge length seemed to have no
regular influence on zeta-potentials, and 5a–d/DNA complexes
gave the values of 5–10 mV at the N/P ratios of 10–40. These
values were higher than that of previously studied LCPA, which
has a secondary N atom in the bridge (1.6–3.0 mV). To our
surprise, the zeta-potential values increased with the decrease of
the amino density in the order of LCPA, 5a–d, and 5e. The polyplex
formed from multi-ether-bridged polymer 5e and pDNA gave the
lowest zeta-potential at low N/P ratio, but the value increased to
about 20 mV when the N/P increased to 15, and the value was
much higher than those of the polyplexes formed from 5a–d.
compared to 20 kDa PEI, which displayed serious cytotoxicity in
both of the two cell lines (less than 20% of cells survived when its
-
1
concentration was up to 30 mg mL ). In comparison, 5 displayed
-
1
relatively high cell viabilities at 30 mg mL concentration (>70%
cell viability for both cell lines). The reduced cytotoxicity of 5
may be ascribed to the more biocompatible hydroxyl- and ether-
containing bridge. The introduction of the bridge moiety could
also reduce the density of amino groups, and therefore decrease
charge density. There was no distinct relationship between the
bridge length (5a–d) and cytotoxicity, and 5b seemed to be the
most biocompatible one of the four polymers. For 5e, the PEG-like
multi-ether bridge might benefit its biocompatibility and lead to
lower cytotoxicity. Additionally, cell-dependent cytotoxicities were
found for 5, and A549 cells showed a relative weakness against the
cytotoxicity of the prepared materials.
26
Cytotoxicity
The cytotoxicity of cationic polymers is thought to be caused
by damage from the interaction with plasma membrane or other
cellular compartments, and researchers have found a rough corre-
lation between toxicity and TE. Fig. 5 shows the viability of A549
and 293 cells after incubation for 24 h with various concentrations
of 5 assessed by the MTT assay, and 20 kDa PEI was used as
the control. Polyamines 5 exhibited improved compatibility as
In vitro transfection
The gene transfection efficiencies of 5/DNA complexes were
assessed by in vitro delivery experiments of luciferase reporter gene
(plasmid pGL-3) into A549 and 293 cells. PEI (20 kDa) was used as
the control in this work, and the PEI/DNA complex was prepared
Fig. 5 Relative cell viabilities of 5 and 20 kDa PEI in (A) A549 cells and (B) 293 cell.
416 | Org. Biomol. Chem., 2011, 9, 2413–2421
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Fig. 6 (A) Luciferase expression in A549 cells transfected by 20 kDa PEI/DNA (N/P = 10) and 5 at N/P ratios 5, 15 and 30. (B) Lucifecrase expression
in 293 cells transfected by 20 kDa PEI/DNA (N/P = 10) and 5 at optimal N/P ratios obtained in (A). For comparison with Fig. 5, the concentrations of
-1
5
a–e in the transfection experiments at N/P of 5 were 4.20, 4.37, 4.55, 4.89, 5.26 mg mL , respectively.
35,36
at an N/P ratio of 10 (optimal ratio for PEI). Fig. 6A shows the
complexes.
In quite a few literatures using CQ to improve
TEs of 5/DNA complexes in A549 cells at various N/P ratios of 5,
the transfection, 100 mM was a generally used concentration,
indicating that a certain concentration (could not be too low)
of CQ was needed for the improvement. Indeed, as shown in
Fig. 7, lower concentrations of CQ (12.5 and 25 mM) significantly
decreased the TE. We speculated that under lower concentrations,
CQ could not exert its buffer capacity smoothly, while on the
contrary, its toxicity negatively affected the transfection. It was
found that 50 mM of CQ seemed to be the optimal concentration,
and almost double TE was obtained in the transfection using
this dosage of CQ. Further increase of the CQ concentration led
to a harsh decrease of TE and mass cell death due to its high
cytotoxicity. In summary, as the buffer reagent CQ could notably
promote the TE of these polymers, we believed that the TE of
the linear cyclen-based polymer could be enhanced by proper
modifications towards their buffer capability.
1
5, and 30. Results showed that, except for 5c, whose complex with
DNA gave the best TE at the N/P of 5, all other polymer/DNA
complexes gave the best TEs at N/P of 15. Further increase of the
N/P ratio might lead to higher cytotoxicities and decreased TEs
(
data not shown). The binding affinity of the polymer towards
DNA and the stability of the formed polyplexes are already
33
well-recognized as the key factors in determining the TE. Both
the weak condensation ability of the cationic polymers and the
excessively stable polyplex are harmful to the gene transfection.
Usually, complexes which decompose too early always lead to
little or no gene expression, while highly tight combination may
result in slow release of DNA and therefore low TE. As different
bridge lengths would affect the DNA binding ability of polymer
5, we hope to find a balance between DNA binding ability and
TE. As shown in Fig. 6A, the TE was significantly influenced by
the bridge length. The highest binding ability (5d, Fig. 3) did not
result in highest TE, however, the 5c/DNA complex showed the
best TE at the N/P of 5, which was comparable to that of PEI. As
the weakest material in DNA binding among the five polymers,
5e showed higher TE than those of 5a, 5b and 5d. This might
be due to the higher biocompatibility of its bridge structure and
lower cytotoxicity. Similar results were obtained in the experiments
involving 293 cells (Fig. 6B), in which the N/P ratios were used
according to the best value in Fig. 6A.
The unique structure of cyclen affords four nitrogen atoms
34
with different pK
higher pK would help DNA binding and formation of polyplexes,
while the other two with lower pK would remain unprotonated
a
values. We had hoped that the two with
a
a
in the polyplex, directing the enhanced buffering capacity in
the endosomal compartment. However, the basicity of the latter
Fig. 7 Luciferase expression in A549 cells transfected by 5c/DNA
complex at N/P ratios 5 in the presence of 0, 12.5, 25, 50mM of chloroquine.
two N atoms is too weak (pK < 2) to be protonated in the
a
endosomal environment (pH 5.0–6.2), thus will not benefit the
buffer capability and subsequent transfection. The well-known
lysosomotropic reagent chloroquine (CQ) was tested for the
purpose of promoting the TE of 5c. CQ is a weak base that
is known to buffer the endosome and is often used to enhance
the transfection efficiency of some cationic polycation/DNA
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. The N/P
ratios were used according to the optimal results of the luciferase
assay, and the pictures are shown in Fig. 8. These 4 cm wide images
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Fig. 8 Fluorescent microscope images of pEGFP-transfected cells: A–F: A549 cells; G–L: 293 cells. A and G: 5a at N/P of 15; B and H: 5b at N/P of
1
5; C and I: 5c at N/P of 5; D and J: 5d at N/P of 15; E and K: 5e at N/P of 15, F and L: 20 kDa PEI at N/P of 10.
were obtained after 68-fold reduction from original pictures, which
were recorded at the magnification of 100¥. Polymer 5c gave the
strongest fluorescence among the five polymers (Fig. 8C, 8I),
nevertheless the density of transfected cells was less than that
observed in the experiment using PEI/DNA complex (Fig. 8F,
gene delivery. These studies may extend the application areas of
macrocyclic polyamines, especially for cyclen, and related studies
that focus on the purposeful modification of such a kind of
polycations are now in progress.
8
L). The TEs according to the density of transfected cells gave
the order of 5c > 5e > 5d > 5a–b, which was in accordance
with the results in luciferase assays processed in the same two cell
lines.
Experimental section
General methods and materials
All chemicals and reagents were obtained commercially and
were used as received. Anhydrous ethanol (EtOH), anhydrous
Conclusions
dichloromethane, triethylamine (Et N) and epichlorohydrin were
3
A new type of cationic polymer, linear cyclen-based polyamines
bridged by diol glycidyl ethers with different chain lengths
were designed and synthesized from 1,7-diprotected 1,4,7,10-
tetraazacyclododecane (cyclen), diols and epichlorohydrin. These
new polymeric materials could efficiently bind to plasmid DNA at
low N/P ratios and condense DNA to nanoparticles with proper
sizes and zeta-potentials. The cytotoxicity of these polymers
assayed by MTT is much lower than that of 20 kDa PEI. In vitro
transfections of reporter genes of luciferase and enhanced green
fluorenscent protein against A549 and 293 cell lines suggested that
higher binding ability might not result in higher TE, however, the
dried and purified under nitrogen by using standard meth-
ods and were distilled immediately before use. 1,7-Bis-(tert-
butyloxycarbonyl)-1,4,7,10-tetraazacyclodocane was prepared ac-
cording to the literature. High molecular weight PEI (branched,
average molecular weight 20 kDa: 20 kDa PEI), chloroquine
37
diphosphate (C18
H
26ClN
3
·2H
3
PO
4
, M = 515.87) and MTT (3-
w
(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, pEGFP-N1 (Clontech, Palo
Alto, CA, USA) coding for EGFP DNA and calf thymus DNA
(CT DNA) (Sigma Aldrich). 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). The luciferase
assay kit was purchased from Promega (Madison, WI, USA).
An endotoxin-free plasmid purification kit was purchased from
TIANGEN (Beijing, China).
5c/DNA complex showed the best TE at the N/P of 5. 50 mM
of chloroquine was found to benefit the transfection, and almost
double TE was obtained compared to the experiment without the
use of CQ. According to these results combined with our previous
studies, we considered that after proper structure modifications,
this new type of marcrocyclic polyamine-based polymeric material
would become a promising non-viral polycationic reagent for
2
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1
The H NMR spectra were obtained on a Varian INOVA-400
spectrometer. CDCl was used as solvent and TMS as the internal
The reaction mixture was stirred overnight at room temperature,
and white solid precipitated. The solvent was then removed under
reduced pressure to produce the hydrochloride product 4. After
dialysis (MWCO 3.5 kDa) against pure water for 2 d, the water
solution of 4 was treated with 2 M NaOH aqueous solution to
adjust the pH to 12. The alkaline solution was extracted with hot
3
reference. Fluorescence spectra were measured by a Horiba Jobin
Yvon Fluoromax-4 spectrofluorometer. The molecular weight of
polyamine was determined by gel permeation chromatography
GPC) (Waters 515 pump, Waters 2410 Refractive Index Detector
25 C), incorporating Shodex columns OHPAK KB-803). A
filtered mixture of 0.2 mol L HAc/NaAc buffer which contained
0% CH CN (volume ratio) was used as mobile phase with
a flow rate of 0.5 mL min . Molecular weight analyses were
calculated against poly(ethylene glycol) standards of number
average molecular weights ranging from 200 to 80 000.
(
(
◦
CHCl
3
. The organic layer was dried over anhydrous Na
2
SO . The
4
-
1
final products 5 were obtained after removing the solvent under
1
2
3
reduced pressure. These polymers were characterized by IR, H
-
1
NMR and GPC (see the ESI for the spectra†).
Acid–base titration
Polymer 5 (0.25 mmol of amino groups) was dissolved in 5 mL
of 150 mM NaCl aqueous solution, and 1 M HCl was added to
adjust the 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
Preparation of diglycidyl ether intermediates 2
38
Compounds 2 was prepared according to the literature. In
a typical procedure, a mixture of epichlorohydrin (9.34 mL,
(
pHS-25) after each addition. For comparison, PEI (20 kDa) and
0
0
.12 mol), glycol (0.02 mol) sodium hydroxide pellets (4.8 g,
.12 mol), water (0.5 mL, 0.028 mol), and tetrabutylammonium
a blank were used under same experimental conditions.
chloride (0.3224 g, 0.001 mol) was vigorously stirred for 2 h at
◦
40
C. The solid produced in the reaction was filtered off and
Amplification and purification of plasmid DNA
washed with dichloromethane. The combined organic layer was
dried with anhydrous magnesium sulfate. The solvent and excess
epichlorohydrin were distilled off to give a yellow oil. The residue
was purified by silica gel column chromatography to give the
product 2. The analytical data of compounds 2a–2c were identical
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
◦
media at 37 C overnight. The plasmids were purified by an
38
with those in our previous reports. New compounds 2d and 2e
are described as follows:
TM
EndoFree Tiangen Plasmid Kit. Then the purified plasmids
◦
were dissolved in TE buffer solution and stored at -20 C. The
1
,6-Hexanediol diglycidyl ether 2d. Colorless liquid, yield:
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.
6
8.2%. Eluent for silica gel column chromatography: n-
-
1
hexane/ethyl acetate = 10 : 1 (v/v). IR (neat): n (cm ) 3055, 2995,
1
2933, 2756, 1635, 1459, 1391, 1340, 1253, 1107, 909, 851, 761. H
NMR (400 MHz, CDCl
3
): d (ppm) 3.72-3.68 (m, 2H, CHCH
CH O), 3.45–3.43 (m, 2H, CHCH
.72–3.43 (m, 2H, CH), 2.80–2.78 (m, 2H, ring CH
2
O),
O),
), 2.61–2.59
), 1.38–1.35
Agarose gel retardation assay
3
3
.53–3.47 (m, 4H, CH
2
2
2
2
5/DNA complexes at different N/P ratios (the amino groups of 5
(
(
(
m, 2H, ring CH
2
), 1.59–1.57 (d, 4H, OCH
2
CH
2
CH
2
to phosphate groups of DNA) ranging from 2 to 10 were prepared
by adding an appropriate volume of 5 (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
1
3
m, 4H, OCH
2
CH
2
CH
2
CH
2
). C NMR (400 MHz, CDCl ): d
3
ppm) 71.30, 71.22, 50.61, 43.89, 29.45, 25.74. MS (ESI): m/z =
53.12 ([M + Na] , 100).
-1
+
2
Triethylene glycol diglycidyl ether 2e. Colorless liquid, yield:
1.4%. Eluent for silica gel column chromatography: n-
solution to a total volume of 6 mL, and then the complexes were
4
◦
incubated at 37 C for 30 min. After that the complexes were
-
1
hexane/ethyl acetate = 4 : 1 (v/v). IR (neat): n (cm ) 3055, 2997,
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.
1
2
872, 1637, 1461, 1349, 1253, 1105, 912, 854, 759. H NMR (400
MHz, CDCl ): d (ppm) 3.78 (dd, J = 11.6, 2.8 Hz, 2H, CHCH O),
.71–3.65 (m, 12H, OCH CH O), 3.42 (dd, J = 11.6, 6 Hz, 2H,
O), 3.17–3.13 (m, 2H, CH), 2.78 (dd, J = 4.8, 4.8 Hz, 2H,
3
2
3
2
2
CHCH
2
1
3
ring CH
2
), 2.60 (dd, J = 2.4, 4.8 Hz, 2H, ring CH
2
). C NMR (400
Ethidium bromide displacement assay
MHz, CDCl
3
): d (ppm) 71.74, 70.43, 70.32, 70.30, 50.48, 43.78.
+
MS (ESI): m/z = 285.11 ([M + Na] , 100).
The ability of 5 to condense DNA was studied using ethidium bro-
mide (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 response. EB (5
Preparation of linear cyclen-based polymer 5
-
1
1
,7-Bis(tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclodocane (0.75
g, 2 mmol) was dissolved in 1 mL of C OH, then compound
(2 mmol) was added to the solution. Under the protection of
mL, 1 mg mL ) was put into quartz cuvette containing 2.5 mL of
2
H
5
150 mM NaCl solution. After shaking, the fluorescence intensity
-
1
2
of EB was measured. Then CT DNA (4.08 mL, 6.24 mg mL ) was
added to the solution and mixed symmetrically, and the measured
fluorescence intensity is the result of the interaction between DNA
◦
N
2
, the reaction mixture was stirred at 80 C for 40 h. The solvent
was removed under reduced pressure to give polymer 3, which
was then recrystallized by chloroform/cyclohexane. Polymer 3 was
-
1
and EB. Subsequently, the solutions of 5 (1 mg mL , 2.5 mL
dissolved in CH
2
Cl
2
, and HCl gas was imported to the solution.
for each addition) were added to the above solution for further
This journal is © The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2413–2421 | 2419

View Article Online
measurement. All the samples were excited at 497 nm and the
emission was measured at 600 nm.
light units (RLUs) were measured with a chemiluminometer
(FLOROSKANASCENT FL). The total protein was measured
according to a BCA protein assay kit (Pierce). Luciferase activity
was expressed as RLU/mg protein. Data are shown as mean ±
standard deviation (SD) based on 3 independent measurements.
The statistical significance between two sets of data was calculated
using the Student’s t-test. A P value < 0.05 was considered
statistically significant.
Particle size and zeta-potential measurements
Particle size and zeta-potential were measured by a Zeta Nano
◦
Series (Malvern Instruments Led) at 25 C. The 5/DNA com-
plexes at various N/P ratios ranging from 10 to 60 were prepared
by adding an appropriate volume of 5 solution (in 150 mM NaCl
In the transfection of plasmid pGL-3 involving chloroquine,
-
1
solution) to 100 mL of pEGFP-N1 DNA solution (50 mg mL in
0 mM Tris-HCl buffer solution) with the final volume of 200 mL.
4
A549 cells were seeded at 7.5 ¥ 10 cells/well in the 24-well plate
4
with 0.5 mL of medium containing 10% FBS and incubated at
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.
◦
3
7 C for 24 h. 5c/DNA (N/P ratio = 5) complexes with different
concentration of chloroquine were prepared by adding appropriate
amount chloroquine to the 5c/DNA complex solution.
Cell culture
Green fluorescent protein assay. Transfections of pEGFP-N1
plasmid mediated by 5 in 293 cells and A549 cells were also
evaluated. The best N/P ratio of each polymer in 293 cells and
A549 cells determined from the luciferase assay was used. 293
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%
antibiotics (penicillin-streptomycin, 10000 U mL ) at 37 C in
a humidified atmosphere containing 5% CO .
5
cells and A549 cells were inoculated at a density of 2.4 ¥ 10
5
and 3 ¥ 10 cells/well in 24-well plates, respectively, 24 h prior
-
1
◦
to transfection. 5/DNA complexes were prepared by adding an
2
appropriate volume of 5 solution (in 150 mM NaCl solution) to
-
1
5
0 mL pEGFP-N1 DNA solution (30 mg mL in 40 mM Tris-
Cell viability assay
HCl buffer solution) with the final volume of 100 mL. Then the
complexes were incubated at room temperature for 30 min. The
plates were washed by PBS twice, and 100 mL of 5/DNA complexes
were then added to a well with an additional 150 mL of medium
without FBS. The final concentration of DNA in the complexes
Toxicity of 5 toward 293 cells and A549 cells was determined by us-
ing MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium
bromide) reduction assay following literature procedures. The
293 cells (6000 cells/well) and A549 cells (14000 cells/well) were
-
1
was calculated to be 6 mg mL . After 4 h of incubation, the
/DNA complex containing medium was replaced with 0.5 mL
seeded into 96-well plates. After being incubated for 24 h, the
culture medium was replaced with the medium containing 5 with a
particular concentration, and the cells were incubated for another
5
of fresh medium containing 10% FBS, and the cells were further
◦
incubated for 24 h at 37 C. The cells were directly observed by
2
4 h. After that, the medium was replaced again with 200 mL of
an inverted microscope (Olympus IX 71). The microscopy images
were obtained at the magnification of 100¥ and recorded using
Viewfinder Lite (1.0) software.
-
1
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, BioRad) at a wavelength of
Acknowledgements
5
70 nm. The cell survival was expressed as follows: cell viability =
This work was financially supported by the National Science
Foundation of China (Nos. 20902062, 20725206, and 20732004),
the Program for Changjiang Scholars and Innovative Research
Team in University, the Key Project of Chinese Ministry
of Education in China and the Scientific Fund of Sichuan
Province for Outstanding Young Scientist. We also thank An-
alytical and Testing Center of Sichuan University for NMR
analysis.
(
ODtreated/ODcontrol) ¥ 100%.
In vitro transfection
Luciferase assay. The 20 kDa PEI was used as the positive
control due to its high transfection efficiency in vitro and in vivo.
The plasmid pGL-3 was used as a reporter gene. Transfections
of pGL-3 plasmid mediated by 5 in 293 cells and A549 cells were
studied as compared with 20 kDa PEI. 293 cells or A549 cells were
4
seeded at a density of 6 ¥ 10 cells/well in the 24-well plate with
0
◦
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by serum-free medium, and then the 5/DNA complexes were
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