Aromatic thioacetal-bridged ros-responsive nanoparticles as novel gene delivery vehicles

Article abstract of DOI:10.3390/molecules23082061

In this report, a series of polycations are designed and synthesized by conjugating reactive oxygen species (ROS)-responsive thioacetal-linkers to low molecular weight (LMW) polyethylenimine (PEI) via ring-opening polymerization. Their structure-activity

Full text of DOI:10.3390/molecules23082061

molecules
Article
Aromatic Thioacetal-Bridged ROS-Responsive
Nanoparticles as Novel Gene Delivery Vehicles
Guo-Qing Lin, Wen-Jing Yi *, Qiang Liu, Xue-Jun Yang and Zhi-Gang Zhao *
College of Chemistry and Environmental Protection Engineering, Southwest Minzu University, Chengdu 610041,
China; lgq1012966356@163.com (G.-Q.L.); lqchem@163.com (Q.L.); yangxuejun@swun.edu.cn (X.-J.Y.)
* Correspondence: ywjchem@163.com (W.-J.Y.); zzg63129@163.com (Z.-G.Z.);
Tel.: +86-8552-2315 (W.-J.Y. & Z.-G.Z.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 July 2018; Accepted: 11 August 2018; Published: 17 August 2018
Abstract: In this report, a series of polycations are designed and synthesized by conjugating reactive
oxygen species (ROS)-responsive thioacetal-linkers to low molecular weight (LMW) polyethylenimine
(PEI) via ring-opening polymerization. Their structure–activity relationships (SARs) as gene delivery
vectors are systematically studied. Although the MWs of the target polymers are only ~9 KDa,
they show good DNA binding ability. The formed polyplexes, which are stable toward serum but
decomposed under ROS-conditions, have appropriate sizes (180~300 nm) and positive zeta-potentials
(+35~50 mV). In vitro experiments reveal that these materials have low cytotoxicity, and higher
transfection efficiency (TE) than controls. Furthermore, the title polymers exhibit excellent serum
tolerance. With the present of 10% serum, the TE of the polymers even increases up to 10 times
higher than 25 KDa PEI and 9 times higher than Lipofectamine 2000. The SAR studies also reveal that
electron-withdrawing groups on the aromatic ring in 4a may benefit to balance between the DNA
condensation and release for efficient gene transfection.
Keywords: reactive oxygen species (ROS); stimuli responsive; gene delivery; cationic polymer;
non-viral vectors
1. Introduction
Gene therapy is considered to be one of the most promising approaches to treating various
human genetic and acquired diseases, such as cancers, aging, diabetes, and cardiovascular diseases [
1].
Safe and effective gene delivery vectors are a prerequisite for successful gene therapy [ ]. Due to
2,3
the high transfection efficiency (TE), viral carriers are a commonly used method of gene delivery.
However, their immunogenicity, toxicity, limited DNA packaging capacity, and difficulty in large-scale
production have impeded their development and clinic applications [4,5]. In attempt to overcome
these barriers, a broad range of versatile and efficient non-viral gene vectors have been developed.
Cationic polymers, as one class of non-viral gene vectors, have acquired increasing attention due to
their easy preparation and modification in recent years [6–8].
Gene transfection with cationic polymers needs to overcome multiple delivery barriers, including
the formation of polymers/DNA complexes (polyplexes) and their initial binding to the cell surface,
endocytosis, endosomal escape, nuclear entry, and final expression [
KDa PEI is the most widely studied polymeric carrier, both in vitro and in vivo
9
–
11]. “Golden standard” 25
12]. Unfortunately,
[
high molecular weight (HMW) PEI remains cationic under physiological conditions and is not
biodegradable, resulting in its well-known cytotoxicity, the difficult release of genes and low TE
under serum conditions [13
toxicity, yet almost no transfection activity [15]. Recently, plentiful approaches to obtaining high TE
using covalently conjugated and non-conveniently assembled LMW PEI have been investigated [16 17].
,14]. Compared to the HMW PEI, the LMW PEI illustrates much lower
,
Molecules 2018, 23, 2061; doi:10.3390/molecules23082061
www.mdpi.com/journal/molecules

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One of the feasible approaches is to cross-link LMW PEI with biodegradable linkers (ester, ketal and
disulfide etc.) [13 18 19] to form “smart” polymers that can eventually degrade into small molecules
,
,
and simultaneously release complexed nucleic acids in the internal or external stimuli (pH, redox,
temperature, light, and magnetic field etc.) [20–23].
Such endogenous stimuli that are drawing keen attention recently are reactive oxygen species
(ROS), which include hydrogen peroxides (H2O2), hydroxyl radicals (·OH), peroxynitrites (ONOO−)
and superoxides (O2−) [24
–27]. Commonly, ROS are generated from mitochondrial electron transport
as a byproduct, and they are used in cell signaling of normal cells or cause oxidative stress from
overproduction [28]. On the other hand, a number of evidences reveal that chronically increased levels
of ROS are closely associated with various pathological disorders including cancer, atherosclerosis,
diabetes, infections, inflammatory diseases, where ROS levels can be 10- to 100-times higher than those
of the normal levels [29–31]. Therefore, ROS can be considered as a target or an indicator for selectively
delivering cargo molecules to diseased sites by targeting oxidative microenvironment at different
levels. Some materials that can undergo phase transition or hydrolyze under oxidative conditions
therefore have been developed to construct ROS-sensitive carriers. Alternatively, some oxidation-labile
groups (thioether, thioketal [25
acid esters etc.) are incorporated into polymers as ROS-responsive groups [33
,
26
,
32], selenium-containing groups, oligoproline peptide and boronic
36]. It was hypothesized
–
that the ROS-responsive nanocarriers could facilitate the intracellular gene release and transfection
expression inspired by the high level of intracellular ROS.
Based on the above issues, the aim of this study was to develop novel biodegradable polymeric
nanocarriers based on LWM PEI and thioacetal-linker units, exploiting the high level of ROS in cancer
cells (as shown in Scheme 1). In addition, rigid aromatic structure containing polymers showed
increased TE was recently reported by our group [37–39]. The aromatic rings may help to improve the
DNA condensation ability of cationic polymers. Besides, we also found diepoxide ring-opening
polymerization is a practical synthetic method toward polymeric vectors with high biological
compatibility by the newly formed hydroxyls and oxygen atoms similar to PEG structure [38–42]
Hence, we prepared this series of polycations through the epoxide ring-opening polymerization
from bis-epoxides with various aromatic thioacetal groups. Then, their gene delivery properties
(such as DNA condensation, cytocompatibility, in vitro TE) and structure-property correlations were
systematically investigated.
.
Scheme 1. Illustration of intracellular delivery of plasmid DNA to the nucleus of HeLa cancer cell
using ROS-responsive polymer based LWM PEI and thioacetal-linker units.

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2. Results and Discussion
2.1. Polymer Synthesis and Characterization
The preparation route for the target polymers is shown in Scheme 2. Various aromatic aldehydes
reacted with 2-mercaptoethanol to give compound in the presence of catalyst NiCl2 6H2O.
Subsequently, diglycidyl ether was prepared by the reaction of with epichlorohydrin in the presence
of NaOH and water with Bu4NBr as a phase transfer catalyst. The ring-opening polymerization was
simply processed between equal molar amounts of diglycidyl ethers cross-linker and PEI 600 Da
1
2
·
3
2
3
in refluxed ethanol. The crude product was recrystallized for 3 times with anhydrous DCM and
methanol to ensure their polydispersity. The obtained polymers showed significant water solubility,
which was required for the subsequent studies. Moreover, four diglycidyl ether bridges with varied
electron-effects (electron-donating and electron-withdrawing substituents) were introduced for helping
us to elucidate the structure–activity relationships (SARs). These new compounds were characterized
by 1H-NMR (400 MHz) using D2O as a solvent. Although the molar ratio of the substrates (bridge
and PEI 600) for polymerization was 1:1, the integral ratio of the proton signals from the bridge (18H
each) and PEI (56H each) revealed that the units of the bridge were more than the latter, indicating the
formation of a reticular but not linear structure (Supplementary Materials). The molecular weights of
target polymers 4 were measured by GPC, and results in Table 1 showed that the Mws were found to
be in the range of 7–10 KDa and the PDIs (polymer dispersity index) were relatively high around 1.3~2.
Because it is well-known that the higher molecular weight of polymers always comes with higher
cytotoxicity [38], we hope our materials would have lower toxicity while maintaining high TE.
S
S
H
C O
O
S
S
O
H
O
H
O
O
O
O
l
C
H
S
H
O
i
ii
R
R
R
1
2
3
NH2
bPEI600
S
S
bPEI600
O
O
N
=
bPEI600
N
m
n
H
O
H
O
iii
H
x
R
4
=
=
=
=
:
a
:
:
c
:
;
;
H
;
F
R
b R
R
d R
H
OC
F
C
3
3
Scheme 2. Synthetic route of the target polymers. (i) NiCl2
NaOH, NaOH, H2O, 35 C. (iii) EtOH, reflux.
·
6H2O, rt. (ii) epichlorohydrin, Bu4NBr,
◦
Table 1. Molecular weights and polydispersities of the target polymers.
Polymers
Mw (Da)
PDI
4a
4b
4c
9871
10,916
7123
1.85
1.97
1.70
1.40
4d
8209

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2.2. Polymer Responds to Reactive Oxygen Species
With the polymer
4 in hand, we investigated its response to ROS under simulated conditions.
In a typical experiment, polymer
degradation. When the polymer was incubated at 37 C for different times, the disappearance
4
was dissolved in D2O containing 100 mM H2O2 to trigger its
◦
1
of thioketal linkage peak (δ ≈ 5.20 ppm) was monitored by H-NMR spectroscopy (Figure S2,
1
Supplementary Materials). The H-NMR spectrum confirmed that the thioketal linkages were
efficiently cleaved by ROS, generating substituted benzaldehyde (δ ≈ 8.25 ppm) as a byproduct
during the cleavage process.
2.3. Interaction with Plasmid DNA and Characterization of Polyplexes
Condensation of negatively charged DNA into nano-sized particles is a prerequisite of great
importance for well-designed polymeric vectors. The formation of a polyplex can reduce the
electrostatic repulsion between DNA and the cell surface, and can protect DNA against enzymatic
degradation by nucleases in cytoplasm or serum [43]. Agarose gel electrophoresis was used to evaluate
the DNA binding ability between the polymer and DNA at various weight ratios (polymer/DNA,
w/w), and results were shown in Figure 1. All 4 materials exhibited good DNA binding capacity,
and full DNA retardation could be observed at a relatively low dosage from the w/w ratio of ~0.5.
The structure of aromatic substituent seemed to have little effect on their DNA retardation ability.
However, polymers could not efficiently complex with DNA even at a high w/w ratio of 8 after exposed
to ROS (Figure S3, Supplementary Materials). This finding implied that polymer
following incubation with H2O2.
4 was decomposed
Figure 1. Agarose gel electrophoresis assays of polymer/DNA (pUC19) complexes at different w/w
ratios. In each image, the first lane is the DNA control.
Proper particle size and positive surface charge are essential for efficient gene delivery. It was
reported that complexes within the size range of 50 to several hundred nanometers would receive
easier endocytosis [44]. Dynamic light scattering (DLS) measurements showed that the particle sizes
of 4/DNA polyplexes changed dramatically at relatively low w/w ratios (0.5–1), and became constant
at 180~300 nm with the increase of w/w ratio (Figure 2A). The larger particles formed at the w/w ratio
of 0.5–1 might be attributed to their neutral zeta-potential, which led to the minimized repulsion and
resulting aggregation. Meanwhile, we also examined the possible changes of particle sizes of 4a/DNA
at a w/w ratio of 2 in the presence of 10% serum. Results suggested that the particle sizes of polyplexes
were still stable at ~250 nm and seldom altered, indicating that no apparent aggregation occurred
(Figure S1, Supplementary Materials). Zeta potential shown in Figure 2B indicated the surface charge
rose along with the increase of w/w ratios, and reached about +35~50 mV above a w/w ratio of 2.
Interestingly, the polyplexes with electron-donating substitute revealed lower zeta potential than the
electron-withdrawing ones, which might be due to the positive charge screening by the electron-rich
effect. Generally, the positively charged complexes can interact with the negatively charged cellular
membranes, leading to more efficient cellular uptake [40].

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Figure 2. Particle size (A) and zeta-potential (B) of polymer/DNA polyplexes obtained at various w/w
ratios by DLS. Data are represented by mean ± standard deviation (S.D.) (n = 3).
2.4. In Vitro Gene Transfection
The in vitro TE of the new polyplexes was evaluated in HeLa cells with high ROS levels by
using pGL3 plasmid as a luciferase reporter gene. Figure 3A shows the relative TEs at various
w/w ratios in a serum-free medium, and Lipofectamine 2000, 25 KDa PEI and PEI 600 were used
as controls. All the polymers at w/w ratios of 2 and 4 gave comparable TEs to commercial reagent
Lipofectamine 2000. To our delight, polymer 4a gave the highest TE, which was nearly 4.5 times
higher TE than Lipofectamine 2000 and 1.5 times higher TE than 25 KDa PEI at a w/w ratio of
2. The electron-withdrawing groups on the aromatic ring seemed to play an important role in the
transfection process. We speculate that the lower zeta potentials caused by the electron-rich effect in 4a
might benefit the dissociation of the polyplexes, leading to better release of DNA in the cytosol [40].
In general, the interaction between negatively charged serum proteins and polycationic gene
vector would inhibit efficient gene delivery [45]. Previous results in DLS experiment showed good
serum tolerance of our new material. The transfection ability of the polymers in the presence of
serum was also estimated by the same method. As shown in Figure 3B, it was found that the relative
TE was even higher for all synthesized polymers in the presence of 10% of serum, indicating that
these polyplexes were quite stable and were seldom affected by the serum. Especially 4a polyplexes,
demonstrated approximately 10 times higher TE than Lipofectamine 2000 and 9 times higher than
25 KDa PEI, respectively. Besides the aromatic group, the hydroxyls formed from ring-opening
polymerization would also contribute to the excellent serum tolerance [38,41].
Figure 3. Luciferase gene expression transfected by polyplexes at different weight ratios: 2, 4, and 6
in comparison with Lipofectamine 2000, PEI 600 (w/w = 6, N/P = 28.7) and 25 KDa PEI (w/w = 1.4,
N/P = 10) in human human cervix carcinoma cell lines (HeLa) in the absence of serum (A) and in the
presence of 10% serum (B). Data are represented by mean ± S.D. (n = 3).

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To directly visualize the infected cells expressing green fluorescent protein reporter gene
pEGFP-Nl, enhanced green fluorescent protein expression in HeLa cells was observed by an inverted
fluorescent microscope. The w/w ratios were used according to the optimal results from luciferase
assays, and the images are shown in Figure 4. Similar to the results in luciferase assay, polymer 4a
gave the strongest green fluorescence among the four polymers, and the density of expressed GFP was
also higher than that involving control PEI 25 KDa and Lipofectamine 2000. Serum seemed to have no
negative effect on the transfection mediated by 4; however, PEI 25 KDa-participated transfection was
inhibited by serum on account of high positive charges. All results further demonstrated the good
serum tolerance of our new vectors.
Figure 4. Fluorescence microscope image of pEGFP-transfected HeLa cells at optimal weight ratios in
the absence of serum (2 for 4a–d) or in the presence of 10% FBS (4 for 4a and 4d, 2 for 4b and 4c).
2.5. Cytotoxicity
Successful gene transfection requires efficient transfection and minimized cytotoxicity. However,
most of the polymeric are caught in the dilemma: either high TE associated with severe cytotoxicity,
or low TE with better compatibility [26]. These new polycations have molecular weight around
9 KDa, and previous experiments exhibited that although their molecular weights are relatively low,
they could completely condense DNA even at low w/w ratios (~0.5). We hope their small molecular
weights may result in low toxicity. The cytotoxicities of the polyplexes were studied and compared
with PEI 25 KDa and Lipofectamine 2000 in HeLa cells via CCK-8 assays. It should be noted that
the w/w ratio for CCK-8 assay was the same as that used in the transfection experiment. As shown
in Figure 5, the cell viabilities of the studied materials were higher than those of 25 KDa PEI and
Lipofectamine 2000 even at high w/w ratios in most cases, suggesting that these polymers had better
biocompatibility. Comparing to the structure of bPEI, we believe that the lower cytotoxicity may be
attributed to the newly formed hydroxyl groups and biodegradable thioacetal, which can benefit
the biocompatibility of the polymeric materials used for gene delivery. It was also proved that the
ring-opening polymerization from bis-epoxides is an effective synthetic approach toward materials
with low cytotoxicity.
Figure 5. Cytotoxicity of polyplexes at different w/w ratios toward HeLa cells. Lipofectamine 2000 and
PEI 25 KDa were used as controls (w/w = 1.4, N/P = 10). Data are represented by mean
±
S.D. (n = 3).

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3. Experimental Section
3.1. Materials and Methods
Anhydrous MeOH, EtOH and dichloromethane (DCM) were distilled immediately before use after
0
drying and purifying by using standard methods. 2,2 -((4-methoxyphenyl)methylene)bis(sulfanediyl))
diethanol (2a), 2,20-((phenylmethylene)bis(sulfanediyl))diethanol (2b) and 2,20-((4-fluorophenyl)
methylene)bis(sulfanediyl))diethanol (2c) were prepared according to the literature [46]. 0.6 KDa
and 25 KDa branched polyethylenimine (PEI) (99%, Sigma-Aldrich, St. Louis, MO, USA), Dulbecco’s
Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) (Invitrogen Corp., Carlsbad, CA,
USA), MicroBCA protein assay kit (Pierce, Appleton, WI, USA), Luciferase assay kit (Promega,
Madison, WI, USA), Endotoxin free plasmid purification kit (TIANGEN, Beijing, China) were used as
received. Luciferase (pGL-3) and green fluorescent protein (pEGFP-N1) plasmid DNA were purchased
from Promega and Clontech. The supercoiled plasmid DNA (pUC-19) used in agarose-gel assay was
purchased from Takara (Dalian, China). HeLa cells (human cervix carcinoma cell lines) were purchased
from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. All the
other chemicals reagents and solvents of analytical grade, if not specified, were used as received from
commercial sources without further purification.
1
The H-NMR spectra were recorded at 400 MHz and 13C spectra at 100 MHz on a Varian
INOVA-400 spectrometer (Varian, Palo Alto, CA, USA). The high-resolution mass spectrometry (HRMS)
experiments were performed using a Finnigan LCQDECA (Thermo Scientific, Waltham, MA, USA) and
a Bruker Daltonics BioTOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). The Mw and
polydispersity index (PDI) of the target polymers were determined by gel permeation chromatography
(GPC) system, which consisted of Shodex columns OHPAK KB-803, Waters 515 pump and Waters 2410
Refractive Index Detector. 0.5 mol
L−1 HAc/NaAc buffer was passed through a filter and used as
·
the mobile phase, at a flow rate of 0.5 mL
ranging from 900 to 80,000.
·
min−1. Mws were calculated against PEG standards of Mn
3.2. Synthesis of Title Polymers
3.2.1. Preparation of 2,20-((4-(Trifluoromethyl)phenyl)methylene)bis(sulfanediyl))diethanol
(Compound 2d)
4-(Trifluoromethyl)benzaldehyde (1d, 20 mmol, 3.48 g) was mixed with 2-mercaptoethanol
(40 mmol, 3.12 g). After addition of the catalyst NiCl2 6H2O (0.1 mmol, 23.8 mg), the solution
·
was stirred at room temperature for 1 h, and then 10 mL DCM was added to separate out the catalyst
by filtering off. The solvent was concentrated to afford a white solid, which was purified by column
chromatography on a silica gel (DCM:MeOH = 40:1, v/v) to give compound 2d as a white solid. Yield:
77.6%. 1H-NMR (400 MHz, CDCl3)
δ
(ppm) 7.69–7.48 (m, 4H, PhH), 5.18 (s, 1H, -SCHS-), 3.78 (t,
J = 5.8 Hz, 4H, -CH2OH), 2.96–2.66 (m, 4H, -CH2CH2OH). 13C-NMR (100 MHz, CDCl3)
δ
(ppm) 144.16,
128.10, 125.77, 125.73, 125.70, 125.66, 61.57, 52.57, 35.32. HRMS (ESI): m/z calcd. for C12H15F3O2S2
(M+) 312.0466; found = 335.0358 ([M + Na]+, 100).
3.2.2. Preparation of Diglycidyl Ether Linkers 3
Compounds
3 were obtained using the method previously reported [37,40]. Briefly, compound
2
(0.02 mol), epichlorohydrin (4.67 mL, 0.06 mol), NaOH (2.4 g, 0.06 mol), tetrabutylammonium
chloride (0.3224 g, 0.001 mol) and pure water (1 mL, 0.056 mol) were mixed at room temperature
and stirred at 35 ◦C overnight. The solid residue was separated out by filtering off and washed with
DCM. The combined solvent was concentrated, and the residue was purified by a silica gel column
chromatography to give the compound 3. Analytical data for new compounds 3a–d were:
1
3a: white solid, yield: 31.2%. Eluent: PE/EA = 3:1 (v/v). H-NMR (400 MHz, CDCl3):
δ (ppm)
7.39–6.84 (m, 4H, PhH), 5.12 (s, 1H, -SCHS-), 3.81–3.78 (m, 3H, -OCH3), 3.76–3.33 (m, 8H, -CH2OCH2-),

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3.13 (m, 2H, ring CH), 2.85–2.77 (m, 4H, -SCH2), 2.73–2.59 (m, 4H, ring CH2). 13C-NMR (100 MHz,
CDCl3):
δ (ppm) 159.18, 132.14, 128.92, 113.84, 71.50, 70.96, 55.25, 53.26, 50.72, 44.11, 31.73. HRMS (ESI):
m/z calcd. for C18H26O5S2 (M+) 386.1222; found = 409.1117 ([M + Na]+, 100).
1
3b: white solid, yield: 33.6%. Eluent: PE/EA = 3:1 (v/v). H-NMR (400 MHz, CDCl3):
δ (ppm)
7.45–7.25 (m, 4H, PhH), 5.12 (s, 1H, -SCHS-), 3.76–3.35 (m, 8H, -CH2OCH2-), 3.14–3.12 (m, 2H, ring
CH), 2.85–2.78 (m, 4H, -SCH2), 2.75–2.60 (m, 4H, ring CH2). 13C-NMR (100 MHz, CDCl3):
δ
(ppm)
140.20, 128.55, 127.97, 127.73, 71.52, 70.96, 70.93, 53.84, 50.74, 44.15, 31.74. HRMS (ESI): m/z calcd. for
C17H24O4S2 (M+) 356.1116; found = 379.1010 ([M + Na]+, 100).
1
3c: pale-yellow oil, yield: 28.6%. Eluent: PE/EA = 2:1 (v/v). H-NMR (400 MHz, CDCl3):
δ (ppm)
7.50–7.00 (m, 4H, PhH), 5.17 (s, 1H, -SCHS-), 3.79–3.33 (m, 8H, -CH2OCH2-), 3.19–3.10 (m, 2H, ring
CH), 2.90–2.76 (m, 4H, -SCH2), 2.74–2.61 (m, 4H, ring CH2). 13C-NMR (100 MHz, CDCl3):
δ
(ppm)
163.39, 160.94, 136.16, 136.13, 129.52, 129.44, 115.50, 115.29, 71.56, 71.02, 53.08, 50.74, 50.73,44.09, 31.82,
30.90. HRMS (ESI): m/z calcd. for C17H23FO4S2 (M+) 374.1022; found = 397.0919 ([M + Na]+, 100).
1
3d: pale-yellow oil, yield: 21.3%. Eluent: PE/EA = 2:1 (v/v). H-NMR (400 MHz, CDCl3):
δ (ppm)
7.62–7.57 (m, 4H, PhH), 5.24 (s, 1H, -SCHS-), 3.82–3.29 (m, 8H, -CH2OCH2-), 3.17–3.10 (m, 2H, ring
CH), 2.89–2.77 (m, 4H, -SCH2), 2.74–2.57 (m, 4H, ring CH2). 13C-NMR (100 MHz, CDCl3):
δ
(ppm)
144.52, 128.31, 128.20, 125.57, 125.53, 73.41, 72.45, 72.44, 72.20, 71.62, 71.08, 71.06, 69.51, 53.30, 50.77,
50.76, 45.78, 44.09, 31.88, 30.86. HRMS (ESI): m/z calcd. for C18H23F3O4S2 (M+) 424.0990; found =
447.0890 ([M + Na]+, 100).
3.2.3. Synthesis and Characterization of Target Polymers 4
In a typical polymerization procedure, PEI 600 Da (0.5 mmol) and diglycidyl ether linkers
3
(0.5 mmol) were dissolved with 1 mL of anhydrous EtOH in a container. Under the protection of
nitrogen (N2), the reaction mixture was stirred at 80 ◦C for 72 h. The resulting crude product was
recrystallized from anhydrous DCM/methanol (v/v = 1/1). The recrystallized oily precipitate was
isolated and dried until a constant weight was achieved. The molecular weights of compounds
4 were
measured by GPC.
1
4a: Yield: 35.6%. H-NMR (400 MHz, D2O):
δ (ppm) 7.55–6.80 (m, 4H, PhH), 5.18 (s, 1H, -SCHS-),
3.98–2.20 (m, 33H, -SCH2CH2O-, -OCH2CH(OH)CH2-, -NHCH2CH2-). GPC: Mw = 9871, PDI = 1.85.
1
4b: Yield: 37.1%. H-NMR (400 MHz, D2O):
δ (ppm) 7.63–7.00 (m, 5H, PhH), 5.17 (s, 1H, -SCHS-),
4.08–1.94 (m, 42H, -SCH2CH2O-, -OCH2CH(OH)CH2-, -NHCH2CH2-). GPC: Mw = 10,916, PDI = 1.97.
1
4b: Yield: 29.8%. H-NMR (400 MHz, D2O):
δ (ppm) 7.45–6.64 (m, 4H, PhH), 5.13 (s, 1H, -SCHS-),
4.11–2.01 (m, 35H, -SCH2CH2O-, -OCH2CH(OH)CH2-, -NHCH2CH2-). GPC: Mw = 7123, PDI = 1.70.
1
4b: Yield: 31.2%. H-NMR (400 MHz, D2O):
δ (ppm) 7.77–7.18 (m, 4H, PhH), 5.21 (s, 1H, -SCHS-),
4.02–2.05 (m, 38H, -SCH2CH2O-, -OCH2CH(OH)CH2-, -NHCH2CH2-). GPC: Mw = 8209 PDI = 1.40.
3.3. Reactive Oxygen Species-Triggered Degradation of Thioketal Linkages
The degradation of thioketal linkages in polymer
4
under ROS conditions was investigated by
◦
incubating
4
(5 mg) with 100 mM of H2O2 in 0.6 mL of D2O at 37 C for various periods of time.
The disappearance of the thioketal linkage peak (δ ~5.20 ppm) was analyzed by using 1H-NMR.
3.4. Agarose Gel Retardation
pUC-19 (5
the varied w/w ratios in H2O (a total volume of 10
the polyplex solution was loaded onto an agarose gel (0.7% w/v agarose gel containing GelRedTM
µ
L of 0.025 mg/mL in H2O) was mixed with the required amount of polymer to obtain
µ
L). After an incubation time of 30 min at 37 ◦C,
)
and electrophoresed at 120 V for 40 min. A Vilber Lourmat imaging system was utilized to image the
agarose gels [36].
To investigate the polymer stability under ROS conditions, the polymer solution (1 mg/mL) was
treated with 100 mM H2O2 for 48 h at 37 ◦C. Subsequently, 0.125
µg pUC-19 and the desired amount

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of polymers at vary w/w ratios were mixed in 10
µL of DI for 0.5 h. The samples were run at the same
electrophoresis condition as described above.
3.5. Particle Size and Zeta Potential Measurements
A Malvern Instruments Zetasizer Nano-ZS (ZEN3600, Malvern Instruments Ltd., Worcestershire,
UK) was used to measure the complexes size and zeta potential. Polymer/DNA complexes were
prepared at varying w/w ratios as described above. Samples were then diluted up to 1 mL using purity
water before being measured [36]. In the experiments with serum, FBS (10% final concentrations) was
added to the formulated polyplexes and mixed for 30 s. The particle sizes of the polyplexes were
determined using Dynamic light scattering (DLS). Data were shown as mean
(S.D.) based on three independent measurements.
±
standard deviation
3.6. Reactive Oxygen Species-Responsive of Polyplexes
The ROS-responsive tests were conduct◦ed by mixing the complexes (4a/DNA at w/w of 2) with
100 µM H2O2 (final concentrations) at 37 C for 1 days. After 12 and 24 h, the polyplexes were
measured through DLS to obtain particle sizes and size distributions.
3.7. In Vitro Transfection Experiments
Gene transfection of a series of complexes was investigated in HeLa cells using two different
plasmids encoding for green fluorescence protein (pEGFP-N1) and luciferase (pGL-3). Cells were
seeded at a density of 7
~70–80% confluency. The culture medium was replaced with FBS free or 10% FBS containing DMEM
medium before transfection. Polymer/DNA (1 g) complexes were added to each well and incubated
at 37 ◦C. Control reagents 25 KDa PEI (at a w/w ratio of 1.4, N/P = 10) [
14 17] and Lipofectamine
×
104 cells per well in 24-well plates and incubated for 24 h to achieve
µ
4, ,
2000 were formulated with DNA based upon their recommended protocols. After 4 h of incubation,
the transfection medium was replaced with fresh complete medium containing 10% FBS and further
incubated for 20 h. The images of GFP expressed cells were taken with an inverted fluorescence
microscope (Nikon Eclipse TE 2000E, Nikon, Tokyo, Japan).
For the luciferase expression, cells were washed twice with PBS (pH 7.4) and lysed using 100
µL
1
×
Lysis reporter buffer. The luciferase activity was quantified using the luciferase assay reagent
(Promega). The total protein content of the cell lysate was analyzed by BCA protein assay. The gene TE
was expressed as the relative fluorescence intensity per mg protein (RLU/mg protein). All transfection
experiments were performed as replicates of three [17].
3.8. Cytotoxicity Assay
The cytotoxicity studies were performed using Cell Counting Kit-8 (CCK-8) assay. HeLa cells were
seeded in 96-well plates at a density of 8000 cells per well and allowed to adhere for 24 h. Complexes
prepared at varied w/w ratios (2, 4, 6 corresponding to final concentrations were 4, 8, 12 µg/mL)
were added to the FBS free growth medium and left to incubated for 4 h. Following incubation,
the wells were replaced with fresh complete medium and incubated for a further 24 h. Subsequently,
10 µ
L of CCK-8 was then added to each well and the cells were incubated for 1 h at 37 ◦C. After that,
the aborbance was readed using enzyme-linked immunosorbent assay (ELISA) plate reader (Model
680, Bio-Rad, Hercules, CA, USA) at a wavelength of 490 nm. The relative cell viability (%) related
to untreated control cells was calculated by test/control
triplicates ± S.D.
×
100 [42]. Values were given as mean of
3.9. Statistical Analysis
Results are summarized as mean
by variance analysis (ANOVA): p values smaller than 0.05 were considered to be significant [39].
±
S.D., and statistical significance of differences was evaluated

Molecules 2018, 23, 2061
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4. Conclusions
In summary, we have successfully synthesized a series of ROS-responsive materials by the
ring-opening polymerization between PEI 600 and diepoxide containing stimuli-responsive thioacetal
groups. Four diglycidyl ethers with various electron-effects were employed in the side chain structure
to facilitate the study of the SAR. These materials could efficiently condense DNA into stable
nanoparticles with proper sizes and zeta-potentials. Besides, the thioketal linkages in polymers
could also be dissociated under ROS-rich conditions for efficient gene delivery. Compared with 25 KDa
PEI and Lipofectamine 2000, these polymers showed much higher TE in HeLa cells, especially polymer
4a with electron-withdrawing substituent on the aromatic ring. More importantly, unlike PEI, serum
had little negative effect on the transfection by these materials, and their TEs were even increased
with the presence of 10% serum. The better serum tolerance of the polymers might come from their
evenly distributed hydroxyl groups. Moreover, the cationic polymers exhibited good cell viabilities.
The collective data suggested such polymer could be a promising target nanocarrier exploiting the
high levels of intracellular ROS in cancer cells. Further modifications of the polymer structure and
in-depth mechanism studies are now in progress.
Supplementary Materials: The following are available online. Figure S1–S3 and Table S1. Additionally, 1H, 19
F
and 13C-NMR and GPC data for the compounds are provided.
Author Contributions: W.-J.Y. and Q.L. conceived and designed the experiments; G.-Q.L. performed the
experiments; W.-J.Y. wrote the paper; Z.-G.Z. and X.-J.Y. provide the resources.
Funding: This work was financially supported by the National Science Foundation of China (No. 2170021243),
the Science and Technology Department of Sichuan Province (No. 2015JY0115), and the Fundamental Research
Funds for the Central Universities, Southwest Minzu University (No. 2018HQZZ19).
Acknowledgments: Wen-Jing Yi thanks Xiao-Qi Yu and Ji Zhang from the Key Laboratory of Green Chemistry
and Technology (Ministry of Education), College of Chemistry, Sichuan University for their support.
Conflicts of Interest: The authors declare there is no conflicts of interest regarding the publication of this paper.
References
1.
Cho, H.; Cho, Y.-W.; Kang, S.-W.; Kwak, M.-K.; Huh, K.M.; Bae, Y.H.; Kang, H.C. Tempo-spatial activation of
sequential quadruple stimuli for high gene expression of polymeric gene nanocomplexes. Mol. Pharm. 2017
,
14, 842–855. [CrossRef] [PubMed]
2.
3.
4.
Ilarduya, C.T.D.; Sun, Y.; Düzgünes¸, N. Gene delivery by lipoplexes and polyplexes. Eur. J. Pharm. Sci. 2010
40, 159–170. [CrossRef] [PubMed]
Mintzer, M.A.; Simanek, E.E. Nonviral vectors for gene delivery. Chem. Rev. 2009, 109, 259–302. [CrossRef]
[PubMed]
,
Xu, C.-T.; Chen, G.; Nie, X.; Wang, L.-H.; Ding, S.-G.; You, Y.-Z. Low generation PAMAM-based nanomicelles
as ROS-responsive gene vectors with enhanced transfection efficacy and reduced cytotoxicity in vitro
.
New J. Chem. 2017, 41, 3273–3279. [CrossRef]
5.
6.
Bouard, D.; Alazard-Dany, N.; Cosset, F.L. Viral vectors: From virology to transgene expression. Br. J. Pharmacol.
2009, 157, 153–165. [CrossRef] [PubMed]
Guan, Y.; Lu, H.; Li, W.; Zheng, Y.; Jiang, Z.; Zou, J.; Gao, H. Near-infrared triggered upconversion polymeric
nanoparticles based on aggregation-induced emission and mitochondria targeting for photodynamic cancer
therapy. ACS Appl. Mater. Interfaces 2017, 9, 26731–26739. [CrossRef] [PubMed]
7.
8.
Chen, W.; Deng, W.; Goldys, E.M. Light-triggerable liposomes for enhanced endolysosomal escape and gene
silencing in PC12 cells. Mol. Ther. Nucleic Acids 2017, 7, 366–377. [CrossRef] [PubMed]
Lee, D.-J.; He, D.; Kessel, E.; Padari, K.; Kempter, S.; Lächelt, U.; Rädler, J.O.; Pooga, M.; Wagner, E. Tumoral
gene silencing by receptor-targeted combinatorial siRNA polyplexes. J. Control. Release 2016, 244 Pt B,
280–291. [CrossRef]
9.
Truong, N.P.; Gu, W.; Prasadam, I.; Jia, Z.; Crawford, R.; Xiao, Y.; Monteiro, M.J. An influenza virus-inspired
polymer system for the timed release of siRNA. Nat. Commun. 2013, 4, 1902. [CrossRef] [PubMed]

Molecules 2018, 23, 2061
11 of 12
10. Hartono, S.B.; Phuoc, N.T.; Yu, M.; Jia, Z.; Monteiro, M.J.; Qiao, S.; Yu, C. Functionalized large pore
mesoporous silica nanoparticles for gene delivery featuring controlled release and co-delivery. J. Mater.
Chem. B 2014, 2, 718–726. [CrossRef]
11. Rosariomeléndez, R.; Yu, W.; Uhrich, K.E. Biodegradable polyesters containing ibuprofen and naproxen as
pendant groups. Biomacromolecules 2013, 14, 3542–3548. [CrossRef] [PubMed]
12. Neu, M.; Fischer, D.; Kissel, T.J. Recent advances in rational gene transfer vector design based on
poly(ethylene imine) and its derivatives. J. Gene Med. 2005, 7, 992–1009. [CrossRef] [PubMed]
13. Xun, M.-M.; Zhang, J.-H.; Liu, Y.-H.; Zhang, J.; Xiao, Y.-P.; Guo, Q.; Li, S.; Yu, X.-Q. Polyethylenimine analogs
for improved gene delivery: Effect of the type of amino groups. RSC Adv. 2016, 6, 5391–5400. [CrossRef]
14. Li, H.; Jiang, H.; Zhao, M.; Fu, Y.; Sun, X. Intracellular redox potential-responsive micelles based
on polyethylenimine-cystamine-poly(ε-caprolactone) block copolymer for enhanced miR-34a delivery.
Polym. Chem. 2015, 6, 1952–1960. [CrossRef]
15. Sarkar, K.; Debnath, M.; Kundu, P.P. Preparation of low toxic fluorescent chitosan-graft-polyethyleneimine
copolymer for gene carrier. Carbohydr. Polym. 2013, 92, 2048–2057. [CrossRef] [PubMed]
16. Yoshitomi, T.; Hirayama, A.; Nagasaki, Y. The ROS scavenging and renal protective effects of pH-responsive
nitroxide radical-containing nanoparticles. Biomaterials 2011, 32, 8021–8028. [CrossRef] [PubMed]
17. Xun, M.-M.; Xiao, Y.-P.; Zhang, J.; Liu, Y.-H.; Peng, Q.; Guo, Q.; Wu, W.-X.; Xu, Y.; Yu, X.-Q. Low molecular weight
PEI-based polycationic gene vectors via Michael addition polymerization with improved serum-tolerance.
Polymer 2015, 65, 45–54. [CrossRef]
18. Mao, C.Q.; Du, J.Z.; Sun, T.M.; Yao, Y.D.; Zhang, P.Z.; Song, E.W.; Wang, J. A biodegradable amphiphilic and
cationic triblock copolymer for the delivery of siRNA targeting the acid ceramidase gene for cancer therapy.
Biomaterials 2011, 32, 3124–3133. [CrossRef] [PubMed]
19. Liu, S.; Huang, W.; Jin, M.-J.; Fan, B.; Xia, G.-M.; Gao, Z.-G. Inhibition of murine breast cancer growth and
metastasis by survivin-targeted siRNA using disulfide cross-linked linear PEI. Eur. J. Pharm. Sci. 2016, 82,
171–182. [CrossRef] [PubMed]
20. Yuan, Y.; Zhang, C.J.; Liu, B. A photoactivatable AIE polymer for light-controlled gene delivery: Concurrent
endo/lysosomal escape and DNA unpacking. Angew. Chem. Int. Ed. 2015, 54, 1–6. [CrossRef] [PubMed]
21. Kim, J.; Lee, Y.M.; Kim, H.; Park, D.; Kim, J.; Kim, W.J. Phenylboronic acid-sugar grafted polymer architecture
as a dual stimuli-responsive gene carrier for targeted anti-angiogenic tumor therapy. Biomaterials 2016, 75,
102–111. [CrossRef] [PubMed]
22. Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M. A review of stimuli-responsive nanocarriers for drug
and gene delivery. J. Control. Release 2008, 126, 187–204. [CrossRef] [PubMed]
23. Fleige, E.; Quadir, M.A.; Haag, R. Stimuli-responsive polymeric nanocarriers for the controlled transport of
active compounds: Concepts and applications. Adv. Drug Deliv. Rev. 2012, 64, 866–884. [CrossRef] [PubMed]
24. Li, Y.; Gao, J.; Zhang, C.; Cao, Z.; Cheng, D.; Liu, J.; Shuai, X. Stimuli-responsive polymeric nanocarriers for
efficient gene delivery. Top. Curr. Chem. 2017, 375, 27. [CrossRef] [PubMed]
25. Liu, X.; Wang, Y.; Chen, C.; Tintaru, A.; Cao, Y.; Liu, J.; Ziarelli, F.; Tang, J.; Guo, H.; Rosas, R.; et al.
A fluorinated bola-amphiphilic dendrimer for on-demand delivery of siRNA, via specific response to
reactive oxygen species. Adv. Funct. Mater. 2016, 26, 8594–8603. [CrossRef]
26. Shim, M.S.; Xia, Y. A reactive oxygen species (ROS)-responsive polymer for safe, efficient, and targeted gene
delivery in cancer cells. Angew. Chem. Int. Ed. 2013, 52, 6926–6929. [CrossRef] [PubMed]
27. He, X.; Chen, X.; Liu, L.; Zhang, Y.; Lu, Y.; Zhang, Y.; Chen, Q.; Ruan, C.; Guo, Q.; Li, C.; et al. Sequentially
triggered nanoparticles with tumor penetration and intelligent drug release for pancreatic cancer therapy.
Adv. Sci. 2018, 5, 1701070. [CrossRef] [PubMed]
28. Yoo, J.; Rejinold, N.S.; Lee, D.; Jon, S.; Kim, Y.C. Protease-activatable cell-penetrating peptide possessing
ROS-triggered phase transition for enhanced cancer therapy. J. Control. Release 2017, 264, 89–101. [CrossRef]
[PubMed]
29. Zhang, Q.; Zhang, F.; Chen, Y.; Dou, Y.; Tao, H.; Zhang, D.; Wang, R.; Li, X.; Zhang, J. Structure-property
correlations of reactive oxygen species-responsive and hydrogen peroxide-eliminating materials with
anti-oxidant and anti-inflammatory activities. Chem. Mater. 2017, 29, 8221–8238. [CrossRef]
30. Lee, S.H.; Boire, T.C.; Lee, J.B.; Gupta, M.K.; Zachman, A.L.; Rath, R.; Sung, H.J. ROS-cleavable proline
oligomer crosslinking of polycaprolactone for pro-angiogenic host response. J. Mater. Chem. B 2014, 2,
7109–7113. [CrossRef] [PubMed]

Molecules 2018, 23, 2061
12 of 12
31. Yu, L.-Y.; Su, G.-M.; Chen, C.-K.; Chiang, Y.-T.; Lo, C.-L. Specific cancer cytosolic drug delivery triggered by
oxygen species-responsive micelles. Biomacromolecules 2016, 17, 3040–3047. [CrossRef] [PubMed]
32. Wilson, D.S.; Dalmasso, G.; Wang, L.; Sitaraman, S.V.; Merlin, D.; Murthy, N. Orally delivered thioketal
nanoparticles loaded with TNF-α–siRNA target inflammation and inhibit gene expression in the intestines.
Nat. Mater. 2010, 9, 923–928. [CrossRef] [PubMed]
33. Song, C.-C.; Du, F.-S.; Li, Z.-C. Oxidation-responsive polymers for biomedical applications. J. Mater. Chem. B
2014, 2, 3413–3426. [CrossRef]
34. Cheng, R.; Meng, F.; Deng, C.; Zhong, Z. Bioresponsive polymeric nanotherapeutics for targeted cancer
chemotherapy. Nano Today 2015, 10, 656–670. [CrossRef]
35. Saravanakumar, G.; Kim, J.; Kim, W.J. Reactive-oxygen-species-responsive drug delivery systems: Promises
and challenges. Adv. Sci. 2017, 4, 1600124. [CrossRef] [PubMed]
36. Luo, C.Q.; Xing, L.; Cui, P.F.; Qiao, J.B.; He, Y.J.; Chen, B.A.; Jin, L.; Jiang, H.L. Curcumin-coordinated
nanoparticles with improved stability for reactive oxygen species-responsive drug delivery in lung cancer
therapy. Int. J. Nanomed. 2017, 12, 855–869. [CrossRef] [PubMed]
37. Yi, W.-J.; Yu, X.-C.; Wang, B.; Zhang, J.; Yu, Q.-Y.; Zhou, X.-D.; Yu, X.-Q. TACN-based oligomers with aromatic
backbones for efficient nucleic acid delivery. Chem. Commun. 2014, 50, 6454–6457. [CrossRef] [PubMed]
38. Luan, C.-R.; Liu, Y.-H.; Zhang, J.; Yu, Q.-Y.; Huang, Z.; Wang, B.; Yu, X.-Q. Low molecular weight oligomers
with aromatic backbone as efficient nonviral gene vectors. ACS Appl. Mater. Interfaces 2016, 8, 10743–10751.
[CrossRef] [PubMed]
39. Liu, Q.; Su, R.-C.; Yi, W.-J.; Zhao, Z.-G. Biodegradable poly(amino ester) with aromatic backbone as efficient
nonviral gene delivery vectors. Molecules 2017, 22, 566. [CrossRef] [PubMed]
40. Yi, W.-J.; Zhang, Q.-F.; Zhang, J.; Liu, Q.; Ren, L.; Chen, Q.-M.; Guo, L.; Yu, X.-Q. Cyclen-based lepidic
oligomers as potential gene delivery vehicles. Acta Biomater. 2014, 10, 1412–1422. [CrossRef] [PubMed]
41. Zhang, Q.-F.; Yu, Q.-Y.; Geng, Y.; Zhang, J.; Wu, W.-X.; Wang, G.; Gu, Z.; Yu, X.-Q. Ring-opening
polymerization for hyperbranched polycationic gene delivery vectors with excellent serum tolerance.
ACS Appl. Mater. Interfaces 2014, 6, 15733–15742. [CrossRef] [PubMed]
42. Guo, Q.; Liu, Y.-H.; Xun, M.-M.; Zhang, J.; Huang, Z.; Zhou, X.-D.; Yu, X.-Q. Diol glycidyl ether-bridged low
molecular weight PEI as potential gene delivery vehicles. J. Mater. Chem. B 2015, 3, 2660–2670. [CrossRef]
43. Yudovin-Farber, I.; Domb, A.J. Cationic Polysaccharides for Gene Delivery. Mater. Sci. Eng. C 2007, 27,
595–598. [CrossRef]
44. Liu, Y.M.; Reineke, T.M. Hydroxyl stereochemistry and amine number within Poly(glycoamidoamine)s
affect intracellular DNA delivery. J. Am. Chem. Soc. 2005, 127, 3004–3015. [CrossRef] [PubMed]
45. He, Y.; Cheng, G.; Xie, L.; Nie, Y.; He, B.; Gu, Z. Polyethyleneimine/DNA polyplexes with reduction-sensitive
hyaluronic acid derivatives shielding for targeted gene delivery. Biomaterials 2013, 34, 1235–1245. [CrossRef]
[PubMed]
46. Laskar, R.A.; Begum, N.A.; Mir, M.H.; Rohman, M.R.; Khan, A.T. Nickel(II) chloride hexahydrate catalyzed
reaction of aromatic aldehydes with 2-mercaptoethanol: Formation of supramolecular helical assemblage of
the product. Tetrahedron Lett. 2013, 54, 5839–5844. [CrossRef]
Sample Availability: Not available.
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