Linear TACN-based cationic polymers as non-viral gene vectors

Article abstract of DOI:10.1039/c4ra11094c

A series of linear cationic polymers were synthesized by the ring-opening polymerization between diglycidyl ethers and 1-Cbz-1,4,7-triazacyclononane (TACN). Besides the good pH buffering capacity in endosome pH range caused by TACN, these polymers have ev

Full text of DOI:10.1039/c4ra11094c

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Linear TACN-based cationic polymers as non-viral
gene vectors
Cite this: RSC Adv., 2014, 4, 59164
*
Qin-Fang Zhang, Bing Wang, Dong-Xiao Yin, Ji Zhang, Wan-Xia Wu, Qing-Ying Yu
*
and Xiao-Qi Yu
A series of linear cationic polymers were synthesized by the ring-opening polymerization between
diglycidyl ethers and 1-Cbz-1,4,7-triazacyclononane (TACN). Besides the good pH buffering capacity in
endosome pH range caused by TACN, these polymers have evenly distributed hydroxyl groups, which
may benefit not only the water solubility but also their biocompatibility and serum tolerance. The
polymers could condense DNA into nanoparticles with appropriate sizes and zeta-potentials.
Cytotoxicity assays reveal that most of the polyplexes formed from title polymers have lower cytotoxicity
than those derived from PEI. In vitro transfection assays show that some of these materials have higher
transfection efficiency than bPEI, especially in tumor cells with the presence of serum. Flow cytometry
and confocal microscopy were applied to further confirm their good serum tolerance. The structure–
activity relationship of such type of polymeric vectors was also discussed. Results suggest that the ring-
opening polymerization may be an effective synthetic approach toward gene delivery materials with high
biological activity.
Received 24th September 2014
Accepted 3rd November 2014
DOI: 10.1039/c4ra11094c
www.rsc.org/advances
effect”.15–17 However, PEI/DNA complexes (polyplexes) have two
main problems. Firstly, PEI with high molecular weight is
Introduction
highly effective in gene transfection but also shows high cyto-
toxicity, while PEI with low molecular weight displays lower
cytotoxicity but limited delivering efficiency.18 Secondly, PEI
lacks serum-tolerance ability, which is necessary for in vivo
application.19
Gene therapy has evolved as a promising therapeutic modality
for the treatment of a wide range of diseases of both innate and
acquired origin.1,2 The naked DNA is highly susceptible to
nuclease degradation and displays poor cellular uptake, as well
as low transfection efficiency (TE).3,4 Therefore, the basic chal-
lenge for gene therapy is to design safe and effective carriers
that assist efficient transfer of the therapeutic gene to targeted
cells without degradation of the delivered gene. The advent of
nanotechnology offers numerous non-viral vectors suitable for
transmitting DNA and other biopharmaceuticals into cells and
subcellular compartments.5–7 Polymer-based non-viral vectors
remain an attractive alternative. Although their delivery effi-
ciency is still lower than viral vector systems, they possess some
unique advantages for practical use such as easy manipulation,
controlled release, stimuli-reactivity, and even targeted
delivery.8–12 Polyethylenimine (PEI) is a promising candidate
among polycationic polymers used for transfection.13,14 It is
assumed that the high TE observed with PEI might be attributed
to effective DNA condensation, endocytotic uptake pathway,
and especial endosomal escape capacity. The intracellular
release of PEI-complexed nucleic acids from endosomes is
hypothesized to rely on the protonation of amines in the PEI
molecule, which has been explained by the “proton sponge
The design of new vectors with high TE and low cytotoxicity
both in the presence and absence of serum has become
signicant to improve clinically efficient gene therapy. Poly-
ethylene glycol (PEG) chains or other hydrophilic neutral poly-
mers were previously introduced to polymers to reduce the
surface charge, leading to reduced cytotoxicity and improved
serum-tolerance.20,21 However, conjugation of PEG to the
surface of cationic polymer particles might decrease their TE,
and the surface-conjugated PEG hindered the interaction
between the functional groups on polymer and receptors on cell
surface.22,23 Hydrophobic modications has also been demon-
strated to have the ability to reduce surface charge and to
improve serum-tolerance and TE.24–26 Nevertheless, hydro-
phobic groups may cause hemolytic damage and cell
toxicity.19,27 Therefore, adjusting the surface charge and the
balance between hydrophobic–hydrophilic components are
required for in-depth structure–activity relationship studies.28
As an important type of macrocyclic polyamine, 1,4,7-tri-
azacyclononane (TACN) also has repeated ethylenediamine
units like PEI. Moreover, the three N atoms on the TACN cycle
exhibit very different properties, especially pKa.29 The amine
with pKa of ꢀ11 has strong basicity, which makes it liable to
Key Laboratory of Green Chemistry & Technology (Ministry of Education), College of
Chemistry, Sichuan University, Chengdu 610064, P. R. China. E-mail: xqyu@scu.
edu.cn; jzhang@scu.edu.cn; Fax: +86 28 85415886; Tel: +86 28 85415886
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capture protons, leading to the cationic property and good mixture was stirred for 24 h at room temperature. Then 10 mL
binding ability toward DNA. Another amine with pKa of ꢀ6.5 of sodium hydroxide solution (3 M) was added and stirred for 30
may have the ‘proton sponge’ effect, which may benet the min, dispensed and washed with saturated sodium bicarbonate
endosomal release of DNA. On the other hand, evenly distrib- solution, the organic layer was dried with anhydrous magne-
uted hydroxyl groups from epoxide ring-opening polymeriza- sium sulfate. The solvent was distilled off under reduced pres-
tion endow the vectors good water solubility, biocompatibility sure and the residue was puried by silica gel column
and serum-tolerance.30 In the present study, a series of cationic chromatography (v/v 1 : 10, PE–EA) to give compound 1 as
polymers based on TACN through ring-opening polymerization colorless oil. Yield: 90%. 1H NMR (400 MHz, CDCl3, d): 1.45
were prepared. To investigate the structure–activity relationship (s, 18H; 2Boc), 3.45 (m, 12H; TACN), 5.16 (s, 2H; OCH2), 7.34
of such type of cationic polymers, we chose the diepoxide (m, 5H; Ph).
linking compounds with different hydrophobicity and amino
density, which may inuence the surface charge and DNA
binding ability of the gene carrier. These polymers showed
better pH buffering capacity, serum resistance and TE than 25
Preparation of 1-Cbz-1,4,7-triazacyclononane (compound 2)
kDa PEI, indicating their potential as promising non-viral gene MeOH–HCl solution (100 mL) was added to a stirred solution of
vectors.
compound 1 in anhydrous chloroform (20 mL), and then the
reaction mixture was stirred for 24 h at room temperature. The
solvent was distilled off under reduced pressure and the residue
was dissolved in water, and then treated with anion exchange
resin and lyophilized to give compound 2 as colorless solid.
Yield: 87%. H NMR (400 MHz, CDCl3, d): 2.70 (m, 4H, TACN),
3.01 (m, 4H, TACN), 3.40 (m, 4H, TACN), 5.16 (s, 2H, OCH2),
7.34 (m, 5H, Ph).
Experimental section
Materials
1
Unless otherwise stated, all chemicals and reagents were
obtained commercially and used without further purication.
Absolute chloroform (CHCl3) and dichloromethane (CH2Cl2)
were distilled aer being dried with calcium hydride (CaH2).
The
starting
material
1,4-di-tert-butoxycarbonyl-triaza-
cyclononane was prepared according to literature.29 Column
chromatography was performed using 200–300 mesh silica gel
or 200–300 mesh Al2O3. All aqueous solutions were prepared
Preparation of polymers (P1–P6)
Epoxy compound (R1–R6)30,31 (1 mmol) was dissolved in
C2H5OH, respectively. Then compound 2 (263 mg, 1 mmol) was
added to the solution. Under the protection of N2, the reaction
mixture was stirring at 80 ꢁC for 72 h. The solvent was removed
under reduced pressure. The protect groups of Cbz was
removed by H2 catalytic with Pd/C. For polymers P5 and P6,
MeOH–HCl solution was added to remove of protecting t-buty-
loxycarbonyl (Boc) group. The residue was dissolved in a small
amount of water. Then 5 N NaOH aqueous solution was added
to adjust pH to 12 and dialyzed (MWCO 3500 kDa) against
deionized water for 3 days. The product was obtained as yellow
solid aer lyophilization. Yield: 35–68%.
1
from deionized or distilled water. The H NMR and 13C NMR
spectra were measured on a Bruker AM400 NMR spectrometer.
Proton Chemical shis of NMR spectra were given in ppm
relative to internals reference TMS (1H, 0.00 ppm). MicroBCA
protein assay kit was obtained from Pierce (Rockford, IL, USA).
Luciferase assay kit was purchased from Promega (Madison,
WI, USA). Endotoxin-free plasmid purication kit was
purchased from TIANGEN (BeiJing, China). 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 modied Eagle's medium
(DMEM), and fetal bovine serum were purchased from Invi-
trogen Corp. Cy5™ was obtained from Molecular Probe (Mirus,
Madison, WI, USA). HEK (human embryonic kidney) 293 cell
lines, HeLa cell lines and U-2OS human osteosarcoma cancer
cells were purchased from Shanghai Institute of Biochemistry
and Cell Biology, Chinese Academy of Sciences. Cell counting
Kit-8 was purchased from dojindo laboratories. Branched pol-
yethyleneimine (PEI 25 kDa, Mw ¼ 25 000) was supplied by
Sigma-Aldrich Co. (St. Louis, MO).
P1. 1H NMR (400 MHz, D2O, d): 1.51 (s, 4H; 2CH2), 2.51–2.84
(m, 12H; 6CH2), 2.99 (s, 4H; 2CH2), 3.34–3.45 (m, 8H; 4CH2),
3.85 (m, 2H; 2CH). GPC: Mw ¼ 5348 Da, PDI ¼ 1.64.
1
P2. H NMR (400 MHz, D2O, d): 1.24 (s, 4H; 2CH2), 1.48 (s,
4H; 2CH2), 2.55–3.18 (m, 12H; 6CH2), 3.31–3.58 (m, 12H; 6CH2),
3.85 (m, 2H; 2CH). GPC: Mw ¼ 4279 Da, PDI ¼ 1.44.
1
P3. H NMR (400 MHz, D2O, d): 1.20 (s, 8H; 4CH2), 1.44 (s,
4H; 2CH2), 2.55–3.16 (m, 12H; 6CH2), 3.37–3.51 (m, 12H; 6CH2),
3.80–3.91 (m, 2H; 2CH). GPC: Mw ¼ 10 017 Da, PDI ¼ 1.57.
1
P4. H NMR (400 MHz, D2O, d): 2.53–2.86 (m, 12H; 6CH2),
3.93–3.00 (m, 4H; 2CH2), 3.35–3.59 (m, 16H; 8CH2), 3.87–3.89
(m, 2H; 2CH). GPC: Mw ¼ 10 150 Da, PDI ¼ 1.70.
Preparation of 1,4-2Boc-7-Cbz-1,4,7-triazacyclononane
(compound 1)
1
P5. H NMR (400 MHz, D2O, d): 2.53–2.86 (m, 12H; 6CH2),
Benzyl chloroformate (2.4 g, 14 mmol) in anhydrous chloroform 2.93 (m, 4H; 2CH2), 3.36–3.64 (m, 12H; 6CH2), 3.82–3.89 (m, 2H;
(500 mL) was added dropwise to a stirred solution of 1, 4-di-tert- 2CH). GPC: Mw ¼ 5832 Da, PDI ¼ 1.74.
1
butoxycarbonyl-triazacyclononane (3.3 g, 10 mmol) and
P6. H NMR (400 MHz, D2O, d): 2.53–3.00 (m, 16H; 8CH2),
4-dimethylaminopyridine (2.5 g, 20 mmol) in anhydrous chlo- 3.35–3.59 (m, 16H; 8CH2), 3.87–3.89 (m, 2H; 2CH). GPC: Mw
roform (50 mL) under the ice bath. The resulting reaction 10 043 Da, PDI ¼ 1.20.
¼
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Cell culture
Particle size and z-potential measurement in water
Human osteosarcoma (U-2OS) cells, HeLa cells and human Zeta potential (z-potential) and particle size were measured by
embryonic kidney transformed 293 (293) cells were incubated in Nano-ZS ZEN3600 apparatus (Malvern Instruments) at 25 ꢁC.
DMEM with 10% (v/v) fetal bovine serum (FBS) and 1% antibi- Polyplexes with various w/w ratios were prepared by adding 1 mg
otics (penicillin–streptomycin, 10 000 U mLꢂ1) at 37 ꢁC in a of pUC-19 to the appropriate volume of the polymer solution (in
humidied atmosphere containing 5% CO2. The medium was PBS). Then the solution of the polyplexes was incubated at 37 ꢁC
replenished every other day.
for 30 min and then diluted with deionized water to 1 mL prior
to measurement. Data were shown as mean ꢄ standard devia-
tion (SD) based on triplicate independent experiments.
Amplication and purication of plasmid DNA
pGL-3 and pEGFP-N1 plasmids were used. The former one was
seed as the luciferase reporter gene, which was transformed in
M109 Escherichia coli, and the latter one was used as the
enhanced green uorescent protein reporter gene, which was
transformed in E. coli DH5a. Both plasmids were amplied in
Cytotoxicity assay
Toxicity toward 293, U-2OS and HeLa cells was determined by
cell counting kit-8 (CCK-8).24 About 7000 cells per well were
seeded in 96-well plates and cultured overnight for 70–80% cell
conuence. The medium was replaced with 50 mL of fresh
medium without FBS, to which 50 mL of polyplexes at various
concentrations was added to achieve nal volume of 100 mL.
Aer 24 h of incubation, polyplexes solutions were removed,
10 mL of CCK-8 solution in 90 mL PBS was added to each well for
additional 1 h incubation. This assay was also carried out in the
presence of 10% serum in HeLa cells with 48 h incubation. The
absorbance was measured in an ELISA plate reader (model 550,
BioRad) at a wavelength of 450 nm. The metabolic activity of the
polyplexes treated cells was expressed as a relative to untreated
cell controls taken as 100% metabolic activity. Besides, the cell
viability of 25 kDa PEI was also performed with CCK-8 assay.
ꢁ
E. coli grown in LB medium at 37 C and 220 rpm overnight.
The plasmids were puried by an EndoFree TiangenTM
Plasmid Kit. Then, the puried plasmids were dissolved in TE
(Tris + EDTA) buffer solution and stored at ꢂ80 ꢁC. The integrity
of plasmids was conrmed by agarose gel electrophoresis.
The purity of plasmids was determined by the ratio of
ultraviolet (UV) absorbances at 260 nm to 280 nm, and the result
was 1.91.
Acid–base titration
Briey, 5 mg of polymers were dissolved in 10 mL of 150 mM
NaCl aqueous solution, and 1 N HCl was added to adjust pH to
2.0. Aliquots of 0.1 N NaOH were added, and the solution pH
was measured with a pH meter (pHS-25) aer each addition. For
comparison, NaCl (150 mM) and 25 kDa PEI were used under
the same experimental conditions. The buffering capacity,
dened as the percentage of amine groups becoming deproto-
nated from pH 5.1 to 7.4, was calculated from equation
Transfection procedure
Gene transfection of a series of complexes was investigated in
HEK 293, U-2OS, and HeLa cells. Cells were seeded in 24-well
plates (8.5 ꢃ 104 cells per well for HEK 293 and HeLa cells, 10 ꢃ
104 cells per well for U-2OS cells) and grown to reach 70–80%
cell conuence at 37 ꢁC for 24 h in 5% CO2. Before transfection,
the medium was replaced with a serum-free or a 10% serum-
containing culture medium containing polymer/pDNA (1 mg)
complexes at various weight ratios. Aer 4 h under standard
incubator conditions, the medium was replaced with fresh
medium containing serum and incubated for another 20 h.
For uorescent microscopy assays, cells were transfected by
complexes containing pEGFP-N1. Aer 24 h incubation, GFP-
expressed cells were observed with an inverted uorescence
microscope (Nikon Eclipse TE 2000E) equipped with a cold
Nikon camera. Control transfection was performed in each case
using a commercially available transfection reagent 25 kDa PEI
based on the standard conditions specied by the manufacture.
For luciferase assays, cells were transfected by complexes
buffer capacity (%) ¼ 100[(DV1 NaOH ꢂ DV2 NaOH)
ꢃ 0.1 M]/N mol
wherein DV1 NaOH is the volume of NaOH solution (0.1 M)
required to bring the pH value of the polymer solution from 5.1
to 7.4, DV2 NaOH is the volume of NaOH solution (0.1 M)
required to bring the pH value of the NaCl solution from 5.1 to
7.4, and N mol is the total moles of protonatable amine groups
in the polymer.
Agarose gel retardation assay
Polyplexes at different w/w ratios (weight ratio of polymer rela- containing pGL-3. For a typical assay in a 24-well plate, 24 h post
tive to pDNA) were prepared by adding appropriate volume of transfection as described above, cells were washed with cold
the polymer solution to 5 mL of Puc-19 (0.025 mg mLꢂ1). The PBS and lysed with 100 mL 1ꢃ lysis reporter buffer (Promega).
obtained complex solution was then diluted to the total volume The luciferase activity was measured by microplate reader
ꢁ
of 15 mL. Aer incubation at 37 C for 30 min, the polyplexes (Model 550, Bio-Rad, USA). Protein content of the lysed cell was
were electrophoresed on a 1% (w/v) agarose gel containing determined by BCA protein assay. Gene transfection efficiency
GelRed™ in Triseacetate running buffer at 120 V for 30 min. was expressed as the relative uorescence intensity per mg
Then DNA was visualized under an ultraviolet lamp using a protein (RLU per mg protein). All the experiments were done in
Vilber Lourmat imaging system.
triplicates.
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laser (633 nm). Data from 20 000 events was gated using
forward and side scatter parameters to exclude cell debris.
Protein adsorption assay
In brief, 1 mL of polymer solution (1 mg mLꢂ1) was added to 1
mL of bovine serum albumin (BSA) solution (2 mg mLꢂ1). Aer
ꢁ
Confocal laser scanning microscopy (CLSM) analysis
shaking at 37 C for 30 min and following centrifugation, the
HeLa cells were seeded at a density of 2 ꢃ 104 cells per well in 35
mm confocal dish (F ¼ 15 mm), 24 h prior to transfection. For
transfection in the absence of serum, the medium was exchange
with serum-free medium. As for transfection in the presence of
serum, the medium was exchanged with serum-containing
medium. Cells were incubated with Cy5 labelled DNA
complexes (2 mg DNA per well, optimal weight ratio of each
sample) in media for 4 h at 37 ꢁC. Subsequently, cells were
rinsed twice with PBS (pH 7.4) to remove complexes that were
not uptaken by cells, xed with 4% paraformaldehyde (dis-
solved with PBS buffer) for 10 min, nuclear staining was done
with DAPI. The CLSM observation was performed using Leica
TCS SP5 at excitation wavelengths of 405 nm for DAPI (blue),
633 nm for Cy5 (red), respectively.
upper layer was carefully collected. The concentration of BSA in
the supernatant was determined by BCA protein assay. The
protein adsorbed on the polyplexes was calculated using
following equation:
q ¼ (Ci ꢂ Cs) ꢃ V/m
where Ci and Cs are the initial BSA concentration and the BSA
concentration in the supernatant aer adsorption experiments,
respectively; V is the total volume of the solution (2 mL); and m
is the weight of the polymer (1 mg) added into the solution.
Cellular uptake of plasmid DNA
The cellular uptake of the polymer/uorescein labelled-DNA
complexes was analyzed by ow cytometry. The Label IT Cy5
Labelling Kit was used to label pDNA with Cy5 according to the
manufacturer's protocol. Briey, HeLa cells were seeded in 12-
well plates (2.0 ꢃ 105 cells per well) and allow attach and grow
Results and discussion
Synthesis of target polymers
for 24 h. For transfection in the absence of serum, the medium As shown in Scheme 1, target polymers P1–P6 were prepared
was exchange with serum-free medium. As for transfection in from TACN and a series of diepoxides with different chain
the presence of serum, the medium was exchanged with serum- lengths and hydrophilic–hydrophobic properties. To avoid the
containing medium. Cells were incubated with Cy5 labelled formation of reticular polymers, TACN must be transformed to
DNA complexes (2 mg DNA per well, optimal mass ratio of each mono-N-protected product. However, direct mono-N-protection
sample) in media for 4 h at 37 ꢁC. Subsequently, the cells were of TACN is not possible. Thus TACN was rst reacted with di-
washed with 1ꢃ PBS containing heparin (120 U mLꢂ1) and tert-butyl dicarbonate (Boc2O) to give di-protected product,
harvested with 0.25% trypsin/EDTA and resuspended in 1ꢃ which was then treated with benzyl chloroformate (CbzCl) to
PBS. Mean uorescence intensity was analyzed using FC 500 yield compound 1. Selective deprotection by acid could remove
ow cytometer (Beckman Coulter). Cy5-labelled plasmid DNA the Boc group, and the mono-N-protected unit
2 for
uptake was measured in the FL4 channel using the red diode polymerization was obtained. Subsequently, ring-opening
Scheme 1 Synthetic route of cationic polymers.
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Table 1 The buffer capacity of newly synthesized polymers and
25 kDa PEI
shown in Table 1, 19.4–24.9% of amine groups could be
protonated in the endosomal–lysosomal pH range. These values
are a little higher than that of 25 kDa PEI (18.9%), indicating
their good buffering capacity, which most likely comes from the
suitable pKa of TACN. Increase of the length of hydrophobic
linkage would lead to decreased density of amino groups,
resulting in weaker buffering capacity (P1–P3). On the other
hand, P5 and P6 with secondary amino groups in the linkage
showed higher buffering capacity than their analogs P1–P4, in
which no N atom exists on the spacer chain.
P1
P2
P3
P4
P5
P6
PEI
Buffering capacity (%) 22.1 21.5 19.4 19.5 24.3 24.9 18.9
polymerization was carried out between 2 and various diep-
oxides R1–R6. The nal polymers were obtained by deprotection
of Cbz group with Pd/H2. For polymers P5 and P6, the amino
groups on diepoxides R5 and R6 must be protected by Boc2O
before the polymerization. The structures of the nal products
were characterized by 1H NMR and gel permeation chroma-
tography (GPC). The Mws of P1–P6 were found to be in the range
of 4–10 kDa. R3, R4 and R6 with longer spacer seemed to have
slightly higher reactivity with compound 2, giving relative
polymers with larger molecular weights.
Formation of polymer/DNA complexes (polyplexes)
An important requirement for cationic transfection reagents is
the ability to bind and condense plasmid DNA. The condensed
form of the polymer/DNA complexes can protect the plasmid
DNA from degradation during gene delivery. Agarose gel retar-
dation assays were rst performed with naked pDNA as control.
As shown in Fig. 1, commonly, the presence of partially disso-
ciated pDNA in lane 4 (at a polymer/DNA weight ratio of 1)
suggests the formation of physically unstable polyplexes.
Further increase of weight ratio (w/w) led to complete retarda-
tion of the DNA migration on the gel, indicating full conden-
sation of DNA. P5 and P6 with secondary amine in the spacer
have better DNA condensation ability compared to P1–P4, and
the full retardation was observed at w/w of 1. On the contrary,
the oxygen-rich polymer P4 showed weaker binding ability, and
at w/w of 2, there was still free DNA observed. This might be
attributed to the negatively charged oxygen atoms, which can
screen the positive charge of the polymer.
Buffering capability
A good non-viral gene delivery vector should have the ability to
escape from the endolysosomal degradation.32 The buffering
capacity of a cationic polymer is an important feature which can
facilitate the endolysosomal escape of polyplexes via the so-
called “proton-sponge” mechanism.33 The buffering capacity
of each TACN based polymer was calculated as the percentage of
amine groups that become protonated between pH 7.4 to 5.1. As
Subsequently, physical properties of the polyplexes such as
particle size and zeta potential were investigated. Previous study
reported that cells usually uptake complexes within the size
range of 50 to several hundred nanometers.34 These parameters
of the polyplexes at different weight ratios were determined by
dynamic light scattering (DLS) assay. Results in Fig. 2A show
that the particle size of the polyplexes tended to decrease with
the rise of weight ratio, suggesting the formation of condensed
particles due to the electrostatic interaction between the
protonated amines and negatively charged DNA. Being consis-
tent with the gel electrophoresis results, P4 could not condense
DNA well at w/w of 2. Meanwhile, polymers with higher binding
Fig. 1 Agarose gel electrophoresis of cationic polymers complexed
with DNA to form polyplexes at different weight ratios. In each image,
the first lane is DNA control. For the w/w of 1, the corresponding N/P
ratios for P1–P6 were 2.8, 2.6, 2.4, 2.4, 3.6 and 4.0, respectively.
Fig. 2 Particle size (A) and zeta potential (B) of polyplexes obtained at various weight ratios by DLS. Data represent mean ꢄ SD (n ¼ 3).
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Fig. 3 Cytotoxicity of title polymers in comparison with 25 kDa PEI in HEK 293 (A), U-2OS (B), and HeLa cells (C) in the absence of serum for 24 h
incubation. Cytotoxicity in HeLa cells (D) in the presence of serum for 48 h incubation.
Fig. 4 Luciferase gene expression transfected by polyplexes at different weight ratios in comparison with 25 kDa PEI (N/P ¼ 10) in the absence of
serum in HEK 293 cells (A), U-2OS cells (B), and HeLa cells (C).
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Fig. 5 Fluorescence microscope image of pEGFP-transfected HEK 293 cells at optimal weight ratio.
ability (P5 and P6) may condense DNA into smaller particles. polyplexes with more clarity. At the w/w of 1, the polyplexes
The particle sizes began to stabilize aer complete condensa- formed from P1–P4 gave negative potentials, while those
tion (starting from w/w of 4). Moreover, the zeta potential trend derived from P5 and P6 were positively charged. Such difference
shown in Fig. 2B may reveal the difference between the further proves that the secondary amines in the spacer may
Fig. 6 Luciferase gene expression transfected by polyplexes at different weight ratios in comparison with 25 kDa PEI (N/P ¼ 10) in the presence
of 10% serum in HEK 293 cells (A), U-2OS cells (B), and HeLa cells (C).
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indicating that the serum and incubation time have little effect
on their cytotoxicity.
In vitro gene transfection
The TE of the polyplexes derived from P1–P6 was rstly evalu-
ated in HEK 293 cells by luciferase transfection assay. Fig. 4A
shows the TE of P1–P6 at various w/w ratios in comparison with
25 kDa PEI, which was used at its optimal w/w ratio of 1.4 (N/P
ratio of 10).37 It is known that hydrophobic chains may not only
promote cellular uptake38 and the stability toward competitive
interaction of polyanions,39–41 but also induce cell cytotoxicity.36
Thus an optimized chain length is signicant for the hydro-
phobic modied vectors. Among the polymers with hydro-
phobic spacers (P1–P3), P2 showed the highest TE, suggesting
that six carbon chains may be suitable length for such kind of
polymeric vectors, and both longer and shorter chain would
lead to reduced TE. Compared to P3, its analog with oxygen-
contained backbone (P4) gave much higher TE. Such superi-
ority might come from the reduced cytotoxicity (Fig. 3), which
most probably resulted from the introduced oxygen atoms.
Moreover, by comparing the TEs of P3–P6, it was found that
secondary amines on the spacer (P5 and P6) would favor the
gene transfection.30 We speculate that the amino groups in
Fig. 7 Protein adsorption of synthesized polymers and PEI.
facilitate DNA binding, resulting in lower weight ratio for DNA
condensation.
Cytotoxicity
The cationic polymers are reported to comprise some inherent spacer would increase the zeta potential of the polyplexes
cytotoxicity due to their interactions with negatively charged (Fig. 2B) and therefore improve cell uptake due to the increased
cellular components, proteins, and blood components.35 In electrostatic interaction between the polyplexes and the nega-
order to evaluate the cytotoxicity of title polymers, the viability tively charged cell membrane.42 Besides, these amino groups
of three different cell lines was tested in the presence of poly- also increase the buffering capacity of the polymer (Table 1),
mers with various concentrations, and 25 kDa PEI was used for and facilitate the endosome escape.43 Subsequently, The TEs
comparison. As shown in Fig. 3, for the polymers with hydro- was also evaluated in tumor cell lines including U-2OS (Fig. 4B)
phobic spacers (P1–P3), the cytotoxicity dramatically increased and HeLa (Fig. 4C) cells. Compared to those in 293 cells, the
with the extension of hydrophobic chain, and P3 gave even relative TEs (compared to PEI) increased in these tumor cells,
higher toxicity than PEI in both cell lines. It was reported that especially in Hela cells, and the optimized TE was about 7.5
hydrophobic chain may improve the interaction with cell times higher than PEI. Such cell priority may offer the possi-
membrane, leading to enhanced cytotoxicity.36 On the contrary, bility of targeted therapy on clinical treatment.
polymers with heteroatoms-contained spacers exhibited much
To visually compare the gene transfer ability of the studied
lower toxicity, and P6 gave highest cell viability. It's worth cationic polymers, transfection experiments were also per-
mentioning that most polymers showed lower cytotoxicity in formed on HEK 293T cells using pGFP reporter gene. The
normal cells (HEK 293) than in tumor cells (U-2OS or Hela). weight ratios were used according to the optimal results from
Further, the cytotoxicity in HeLa cells in the presence of 10% luciferase assays, and the images are shown in Fig. 5A clear
serum with longer incubation time (48 h) was also investigated dependence of TE on polymer structure was observed. The
(Fig. 3D). The trend is very similar to the results in Fig. 3C, density of transfected cells by P2, P5 and P6 was higher than
Fig. 8 Cellular uptake of polyplexes at optimal transfection w/w ratio in HeLa cells quantified by flow cytometry analysis. (A) Without serum; (B) in
presence of FBS.
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Fig. 9 CLSM images of HeLa cells transfected with Cy5-labelled DNA by polymers at optimal weight ratio in absence of FBS. 25 kDa PEI was used
as control (w/w ¼ 1.4, N/P ¼ 10). For each triad image, left: cell nuclei stained by DAPI (blue); middle: Cy5-labelled pDNA (red); right: merged.
that obtained in the experiments using other vectors, and P3 inhibited by serum. The serum-triggered transfection inhibi-
gave lower TE among the polymers. These results are consistent tion was believed to have strong association with the non-
with those from luciferase assay (Fig. 4A).
specic interaction between polyplexes and negatively charged
One of the serious shortcomings of the cationic vector- proteins.44,45 For their relatively higher TEs in preceding studies,
mediated gene delivery is that the transfection is signicantly P2, P5 and P6 were subsequently chosen to study their
Fig. 10 CLSM images of HeLa cells transfected with Cy5-labelled DNA by polymers at optimal weight ratio in presence of FBS. PEI 25 kDa was
used as control (w/w ¼ 1.4, N/P ¼ 10). For each triad image, left: cell nuclei stained by DAPI (blue); middle: Cy5-labelled pDNA (red); right:
merged.
59172 | RSC Adv., 2014, 4, 59164–59174
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transfection behavior in the same cell lines in the presence of involving P3, suggesting its good cellular uptake, which is
10% serum. As shown in Fig. 6, the trends of TE are similar to consistent with the ow cytometry results (Fig. 8A). However,
those in the absence of serum. However, the relative TEs quite a few DNA located in cytoplasm but not around or inside
(compared to PEI) increased with the presence of serum, espe- nuclei. Given that nucleus trafficking of DNA is a prerequisite
cially in U-2OS cells (Fig. 4B and 6B), indicating their good for efficient gene expression,46 these observations suggest a
serum tolerance in such cell line. This might be attributed to its possible reason for the lower TE of P3. When 10% FBS was
evenly distributed hydroxyl groups generated from ring-open introduced to this assay (Fig. 10), the signals of labelled-DNA
polymerization. It was reported that the high resistance of was obviously weakened for PEI, but maintained for the
polymers against the protein adsorption would help to stabilize TACN-based polymers, especially for P5 and P6, also indicating
the polyplexes in the presence of serum and facilitate their gene their good serum tolerance.
delivery.46 Therefore, to examine the protein adsorption of the
polymers, their stability in the presence of bovine serum
albumin (BSA) was studied. Results in Fig. 7 show that the BSA
Conclusions
adsorption of PEI is much higher than the studied polymers.
A series of linear cationic polymers based on TACN were
We speculate that the studied polymers have a unique structure
synthesized by the ring-opening polymerization and applied as
which can prevent the electrostatic protein adsorption, and this
non-viral gene vectors. Several diglycidyl ethers were employed
most likely comes from the “hydroxyl effect”19,30 of the oxygen-
in the synthesis to facilitate the study of their structure–activity
rich backbone. The formed hydroxyl groups during polymeri-
relationship. In the polymer structures, TACN may give these
zation play the role similar to PEG to potentially enhance their
polymers good pH buffering capacity, which may facilitate the
escape of polyplex from endosome. On the other side, these
biocompatibility.47
polymers have evenly distributed hydroxyl groups formed by the
polymerization. The oxygen-rich backbone might benet their
biocompatibility and serum tolerance. Besides their good
buffering capacity and DNA binding ability, several assays reveal
that these materials (P5 and P6) have higher transfection effi-
Cellular uptake and intracellular distribution
Cy5-labelled DNA was used to estimate the polyplexes uptake by
HeLa cells through uorescence-activated cell sorting (FACS)
technique. Aer 4 h cell incubation with the polyplexes, the
ciency than 25 kDa PEI, especially in tumor cells with the
percentage of cells positive for Cy5-labelled pDNA was calcu-
presence of serum. Flow cytometry and confocal microscopy
lated and shown in Fig. 8A Combining the luciferase assay
were applied to further conrm their good serum tolerance.
results (Fig. 4C), we can conclude that polyplexes with better
Moreover, most of the materials showed lower cytotoxicity than
cellular uptake always gave higher TE. P5 and P6 with secondary
amines in spacer showed higher cellular uptake than others.30
This might be attributed to their higher zeta potential, which
PEI. Results reveal that such kind of cationic polymers might be
promising non-viral vectors for gene delivery.
has been discussed previously. On the other hand, the hydro-
phobic bridge seemed to have limited positive effect on the Acknowledgements
cellular uptake, and P1–P3 derived polyplexes encountered less
This work was nancially supported by the National Program on
Key Basic Research Project of China (973 Program,
uptake cell percentage. However, a linear correlation was found
between cellular uptake and the hydrophobic chain length,
2012CB720603), and the National Science Foundation of China
suggesting longer carbon chain may facilitate the trans-
(no. 21232005 and 21472131). J. Z. thanks the Program for New
portation of the polyplexes through plasma membrane.48
Century Excellent Talents in University (NCET-11-0354).
Moreover, the cellular uptake assays were also carried out in the
presence of 10% serum in the same cell line. As shown in
Fig. 8B, the uptake of PEI-derived polyplex was obviously
inhibited, while the uptake of polyplexes formed from our
References
polymers was seldom affected. Such results further indicate that
these polymers are more suitable for the gene delivery in serum
circumstance than PEI.
1 B. Layek, M. K. Haldar, G. Sharma, L. Lipp, S. Mallik and
J. Singh, Mol. Pharmaceutics, 2014, 11, 982–994.
2 J. B. Opalinska and A. M. Gewirtz, Nat. Rev. Drug Discovery,
2002, 1, 503–514.
3 M. S. Al-Dosari and X. Gao, AAPS J., 2009, 11, 671–681.
4 T. Kim, H. Jiang, D. Jere, I. Park, M. Cho, J. Nah, Y. Choi,
T. Akaike and C. Cho, Prog. Polym. Sci., 2007, 32, 726–753.
5 S. Pan, D. Cao, W. Yi, H. Huang and M. Feng, Colloids Surf.,
B, 2013, 104, 294–302.
6 T. I. Kim, M. Lee and S. W. Kim, Biomaterials, 2010, 31, 1798–
1804.
7 H. P. Song, J. Y. Yang, S. L. Lo, Y. Wang, W. M. Fan,
X. S. Tang, J. M. Xue and S. Wang, Biomaterials, 2010, 31,
769–778.
Confocal microscopy was utilized to provide a visual
inspection for intracellular trafficking of pGL-3 plasmid deliv-
ered by the polymers at optimized transfection weight ratio in
HeLa cells in the absence/presence of 10% serum. DNA was
labelled by Cy-5 (red), while the cell nuclei were stained with
40,6-diamidino-2-phenylindole (DAPI, blue). As shown in Fig. 9,
considerable amount of labelled DNA were delivered to the
perinuclear region aer 4 h of incubation with polyplexes
derived from P2, P5 and P6, while only weak DNA (red) signals
were observed for P1 and P4-mediated transfection. Besides,
strong DNA (red) signal was also observed for the transfection
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 59164–59174 | 59173

View Article Online
RSC Advances
Paper
8 U. Lungwitz, M. Breunig, T. Blunk and A. Gopferich, Eur. J. 28 Q.-F. Zhang, W.-H. Yang, W.-J. Yi, J. Zhang, J. Ren, T.-Y. Luo,
Pharm. Biopharm., 2005, 60, 247–266.
9 M. Neu, D. Fischer and T. Kissel, J. Gene Med., 2005, 7, 992–
1009.
10 J. H. Williams, S. R. Sirsi, D. R. Latta and G. J. Lutz, Mol.
Ther., 2006, 14, 88–96.
11 C. H. Jones, C. K. Chen, A. Ravikrishnan, S. Rane and
B. A. Pfeifer, Mol. Pharmaceutics, 2013, 10, 4082–4098.
12 K. Remaut, N. N. Sanders, B. G. De Geest, K. Braeckmans,
W. Zhu and X.-Q. Yu, Bioorg. Med. Chem. Lett., 2011, 21,
7045–7049.
29 B. Wang, W.-J. Yi, J. Zhang, Q.-F. Zhang, M.-M. Xun and
X.-Q. Yu, Bioorg. Med. Chem. Lett., 2014, 24, 1771–1775.
30 Q.-F. Zhang, W.-J. Yi, B. Wang, J. Zhang, L. Ren, Q.-M. Chen,
L. Guo and X.-Q. Yu, Biomaterials, 2013, 34, 5391–5401.
31 W.-J. Yi, Z.-H. Feng, Q.-F. Zhang, J. Zhang, L.-D. Li, W. Zhu
and X.-Q. Yu, Org. Biomol. Chem., 2011, 9, 2413–2421.
J. Demeester and S. C. De Smedt, Mater. Sci. Eng., R, 2007, 32 S. Mansouri, P. Lavigne, K. Corsi, M. Benderdour,
58, 117–161.
E. Beaumont and J. C. Fernandes, Eur. J. Pharm. Biopharm.,
2004, 57, 1–8.
13 O. Boussif, F. Lezoualc'h, M. Zanta, M. D. Mergny,
D. Scherman, B. Demeneix and J.-P. Behr, Proc. Natl. Acad. 33 M. X. Tang and F. C. Szoka, Gene Ther., 1997, 4, 823–832.
Sci., 1995, 92, 7297–7301.
34 Y. M. Liu and T. M. Reineke, J. Am. Chem. Soc., 2005, 127,
3004–3015.
¨
¨
14 J. Schafer, S. Hobel, U. Bakowsky and A. Aigner, Biomaterials,
2010, 31, 6892–6900.
15 U. Lungwitz, M. Breunig, T. Blunk and A. Gopferich, Eur. J.
35 D. Fischer, Y. X. Li, B. Ahlemeyer, J. Krieglstein and T. Kissel,
Biomaterials, 2003, 24, 1121–1131.
¨
Pharm. Biopharm., 2005, 60, 247–266.
16 K. Itaka, A. Harada, Y. Yamasaki, K. Nakamura,
36 H. Chang, H. Wang, N. Shao, M. Wang, X. Wang and
Y. Cheng, Bioconjugate Chem., 2014, 25, 342–350.
H. Kawaguchi and K. Kataoka, J. Gene Med., 2004, 6, 76–84. 37 Y. Hu, M. Y. Chai, W. T. Yang and F. J. Xu, Bioconjugate
17 Y. Hu, B. Xu, Q. Ji, D. Shou, X. Sun, J. Xu, J. Gao and
W. Liang, Biomaterials, 2014, 35, 4236–4246.
18 R. Deng, Y. Yue, F. Jin, Y. Chen, H. F. Kung, M. C. Lin and
C. Wu, J. Controlled Release, 2009, 140, 40–46.
Chem., 2013, 24, 1049–1056.
38 Y. H. Wang, M. Zheng, F. H. Meng, J. Zhang, R. Peng and
Z. Y. Zhong, Biomacromolecules, 2011, 12, 1032–1040.
39 A. M. Doody, J. N. Korley, K. P. Dang, P. N. Zawaneh and
D. Putnam, J. Controlled Release, 2006, 116, 227–237.
19 X. H. Luo, F. W. Huang, S. Y. Qin, H. F. Wang, J. Feng,
X. Z. Zhang and R. X. Zhuo, Biomaterials, 2011, 32, 9925– 40 S. Asayama, T. Hakamatani and H. Kawakami, Bioconjugate
9939. Chem., 2010, 21, 646–652.
20 G. P. Tang, J. M. Zeng, S. J. Gao, X. Y. Ma, L. Shi, Y. Li, 41 M. Thomas and A. M. Klibanov, Proc. Natl. Acad. Sci., 2002,
H. P. Too and S. Wang, Biomaterials, 2003, 24, 2351–2362. 99, 14640–14645.
21 W. C. Tseng and C. M. Jong, Biomacromolecules, 2003, 4, 42 J. J. Green, R. Langer and D. G. Anderson, Acc. Chem. Res.,
1277–1284. 2008, 41, 749–759.
22 S. Mishraa, P. Websterb and M. E. Davis, Eur. J. Cell Biol., 43 N. D. Sonawane, F. C. Szoka and A. S. Verkman, J. Biol.
2004, 83, 97–111. Chem., 2003, 278, 44826–44831.
23 M. Hana, Y. Bae, N. Nishiyama, K. Miyata, M. Oba and 44 S. Son and W. J. Kim, Biomaterials, 2010, 31, 133–143.
K. Kataoka, J. Controlled Release, 2007, 121, 38–48. 45 F. Y. Dai and W. G. Liu, Biomaterials, 2011, 32, 628–638.
24 F. Q. Hu, M. D. Zhao, H. Yuan, J. You, Y. Z. Du and S. Zeng, 46 M. Arsianti, M. Lim and C. P. Marquis, Biomacromolecules,
Int. J. Pharm., 2006, 315, 158–166. 2010, 11, 2521–2531.
25 K. Y. Lee, I. C. Kwon, W. H. Jo and S. Y. Jeong, Polymer, 2005, 47 B. Layek, M. K. Haldar, G. Sharma, L. Lipp, S. Mallik and
46, 8107–8112. J. Singh, Mol. Pharmaceutics, 2014, 11, 982–994.
26 W.-J. Yi, Q.-F. Zhang, J. Zhang, Q. Liu, L. Ren, Q.-M. Chen, 48 J. L. Santos, H. Oliveira, D. Pandita, J. Rodrigues, A. P. Pego,
ˆ
´
L. Guo and X.-Q. Yu, Acta Biomater., 2014, 10, 1412–1422.
27 R. Zhang, N. Zheng, Z. Song, L. Yin and J. Cheng,
Biomaterials, 2014, 35, 3443–3454.
P. L. Granja and H. Tomas, J. Controlled Release, 2010, 144,
55–64.
59174 | RSC Adv., 2014, 4, 59164–59174
This journal is © The Royal Society of Chemistry 2014