Biodegradable Poly(Amino Ester) with Aromatic Backbone as Efficient Nonviral Gene Delivery Vectors

Article abstract of DOI:10.3390/molecules22040566

The development of gene delivery vectors with high efficiency and biocompatibility is one of the critical points of gene therapy. Two biodegradable poly(amino ester)s were synthesized via ring-opening polymerization between low molecular weight (LMW) PEI and diepoxide. The molecular weights of poly(amino ester)s were measured by GPC. Agarose gel retardation assays showed that these materials have good DNA-binding ability and can retard the electrophoretic mobility of plasmid DNA (pDNA) at a weight ratio of 1. The formed polyplexes have proper sizes of around 200 nm and zeta-potential values of about 30-40 mV for cellular uptake. In vitro experiments revealed that polymer P2 gave higher transfection efficiency than PEI 25KDa and Lipofectamine 2000 with less toxicity, especially in 293 cells. Results demonstrate that such a type of degradable poly(amino ester) may serve as a promising non-viral gene vector.

Full text of DOI:10.3390/molecules22040566

molecules
Article
Biodegradable Poly(Amino Ester) with Aromatic
Backbone as Efficient Nonviral Gene Delivery Vectors
Qiang Liu, Rong-Chuan Su, Wen-Jing Yi * and Zhi-Gang Zhao *
College of Chemistry and Environmental Protection Engineering, Southwest Minzu University,
Chengdu 610041, China; lqchem@163.com (Q.L.); Surongcc@163.com (R.-C.S.)
* Correspondence: ywjchem@163.com (W.-J.Y.); zzg63129@163.com (Z.-G.Z.);
Tel.: +86-28-85522315 (W.-J.Y.); +86-28-85522315 (Z.-G.Z.)
Academic Editor: Derek J. McPhee
Received: 4 March 2017; Accepted: 28 March 2017; Published: 31 March 2017
Abstract: The development of gene delivery vectors with high efficiency and biocompatibility is one
of the critical points of gene therapy. Two biodegradable poly(amino ester)s were synthesized
via ring-opening polymerization between low molecular weight (LMW) PEI and diepoxide.
The molecular weights of poly(amino ester)s were measured by GPC. Agarose gel retardation
assays showed that these materials have good DNA-binding ability and can retard the electrophoretic
mobility of plasmid DNA (pDNA) at a weight ratio of 1. The formed polyplexes have proper sizes of
around 200 nm and zeta-potential values of about 30–40 mV for cellular uptake. In vitro experiments
revealed that polymer P2 gave higher transfection efficiency than PEI 25 KDa and Lipofectamine
2000 with less toxicity, especially in 293 cells. Results demonstrate that such a type of degradable
poly(amino ester) may serve as a promising non-viral gene vector.
Keywords: gene delivery; non-viral vectors; aromatic backbone; cationic polymer; biodegradable
1. Introduction
Gene therapy affords a promising way to treat congenital and acquired diseases [
1
,2]. Safe and
efficient delivery of genetic material into target cells remains a challenging aspect of gene therapy [
3].
Although viral vectors are considered to have high gene transfer efficiency, their clinical applications
have been greatly restricted by a number of key issues such as immunogenicity, insertional mutagenesis,
oncogenic effects, and toxicity [
increasing attention for their easy preparation and modification [
have been developed for fabricating non-viral carrier systems, e.g., polyethylenimine (PEI) [
poly(L-lysine) (PLL) [11 12], polyamidoamine dendrimers (PAMAM) [13 14], and chitosan [15 16] etc.
4
,5
]. As safer alternatives, non-viral gene vectors have received
]. Numerous cationic polymers
10],
6–8
9,
,
,
,
Among these polymeric polymers, PEI is the most widely studied gene carrier [17].
High molecular weight (HMW) PEI, especially with the Mw of 25 KDa, has relatively higher
transfection efficiency (TE) both in vitro and in vivo
the design of six novel polymeric vectors [19 20]. In addition, a linear form with an average molecular
weight of 22 kDa has also been described with high transfection activity [21 22]. However, the high
charge density and lack of biodegradability contribute significantly to its evident cytotoxicity in HMW
PEI-mediated gene delivery [23 25]. Compared to the HMW PEI, the low molecular weight (LMW) PEI
demonstrates much lower toxicity, yet almost no transfection activity [17 26 27]. Therefore, to develop
[18]. This made it the “golden standard” for
,
,
–
,
,
novel PEI-based vectors with both high TE and low cytotoxicity is of great importance. Linking LMW
PEI to some special groups, such as ester, phosphoester, ketal, disulfide, carbamate, amide, etc. to
form a biodegradable cationic polymer is an effective strategy [28–32]. These degradable PEIs have
shown significantly enhanced transfection activity compared to the parent LMW PEI and retain their
biocompatibility due to groups or bonds degradation in cellular environments.
Molecules 2017, 22, 566; doi:10.3390/molecules22040566
www.mdpi.com/journal/molecules

Molecules 2017, 22, 566
2 of 12
Our group recently revealed that cationic polymers with rigid aromatic structure in the backbone
showed significantly increased TE [33 34]. The aromatic moiety might play an important role in
enhancing the DNA condensation ability of cationic polymers by -stacking interaction with DNA,
,
π
which contribute to more stable polyplexes and favor cellular uptake. Additionally, we also found
that ring-opening polymerization from bis-epoxides is an effective synthetic approach toward gene
delivery materials with high biological activity [35–37]. The newly formed hydroxyls and oxygen
atoms in the backbones could effectively enhance TE and serum-tolerance similar to PEG structure.
Herein in this report, we synthesized two kinds of LMW PEI-based biodegradable poly(amino ester)
with aromatic backbones through the epoxide ring-opening polymerization. We characterized the
molecular weights and structures of the novel polymers. Their gene delivery properties were also
systematically investigated by acid-base titration, DNA-binding assay, cytotoxicity assay, and in vitro
cell transfection studies.
2. Results and Discussion
2.1. Synthesis and Characterization of Target Polymers
The aim of this study was to rationally design and develop a novel gene delivery vector based on
degradable poly(amino ester). As shown in Scheme 1, the diglycidyl ethers 2a, 2b as linking moiety
were separately prepared by oxidation and ester condensation reaction. Subsequently, ring-opening
polymerization was simply processed by mixing 2a or 2b and PEI 600 Da with an appropriate amount
ratio (mole ratio = 1:1 of monomer) under reflux temperature. The crude product was recrystallized
3 times with anhydrous DCM and cyclohexane to ensure their polydispersity. The structural difference
between P1 and P2 is the orientation of the ester group, helping us to elucidate the structure-activity
relationships (SAR). Additionally, it is expected that the presence of different types of amino groups in
the LWM PEI cationic moiety could efficiently improve gene delivery efficiency. The primary amines
the polymer bears are capable of condensing DNA into complexes through electrostatic interaction,
while the secondary and tertiary amines provide the polycations with high buffer capacity, which can
facilitate the endosomal escape [6,7,17]
Gel permeation chromatography (GPC) was used to measure the molecular weights of target
compounds. Results show that the cationic polymer P2 (Mn = 24,550) has a larger molecular weight
than P1 (Mn = 17,561). As is well-known, the degradation of polymers makes them easy to be
eliminated through the excretion pathway in vivo [38]. Further, the degradability of the polymeric
material was also studied by GPC. In 48 observation hours, the Mn of P2 was finally decreased to
20,218 Da (Figure S1 in Supplementary Materials). Results affirmed the degradation of the target
polymers in physiological solution.
Scheme 1. Synthetic route of the target polymers.

Molecules 2017, 22, 566
3 of 12
2.2. Buffer Capacity
It was assumed that the buffering capacity of cationic polymers facilitates the escape of
polymer/DNA complexes from the endosome, and to promote TE, which was known as the
“proton-sponge effect” [39]. The buffering capacities of polymers, expressed as the percentage of
amino nitrogen that could be protonated in the endosome pH range of 7.4–5.1, were examined by
acid-base titration. The results are shown in Table 1 and Figure S2 in the supplementary information.
The target polymers, especially P2, definitely showed a higher buffering capacity (28.37%) than “golden
standard” PEI 25 KDa (18.09%) and PEI 600 Da (11.20%), indicating their excellent buffering capacities.
The lower buffering capability of P1 (25.85%) may be ascribed to its lower molecular weight.
Table 1. The buffer capacity of newly synthesized polymers and polyethylenimine (PEI).
Polymer
PEI 600
PEI 25K
P1
P2
Buffering capacity (%)
11.20
18.09
25.85
28.37
2.3. The Formation and Properties of Polymer/DNA Polyplexes
Good DNA condensation capability of polycations is a prerequisite for cationic gene carriers.
Electrostatic interaction is the predominant mode of polycations–nucleic acid interactions. Besides,
site- and structure-specific binding has also been recognized [40–42]. 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
performed to investigate the binding capability of P1 and P2 toward plasmid DNA at various weight
ratios (polymer/DNA, w/w). As shown in Figure 1, full retardation of plasmid DNA was observed
from the w/w ratio of 1. The result demonstrated that both polymers can effectively bind DNA at a
relatively low dosage with excellent DNA binding capacity.
Figure 1. Agarose gel electrophoresis of cationic polymers complexed with DNA to form polyplexes at
different weight ratios: 0.5 (N/P = 2.5), 1 (N/P = 4.9), 2 (N/P = 9.8), 4 (N/P = 19.6), and 6 (N/P = 29.4).
The proper particle size and positive surface charge of polyplexes are also important for cellular
uptake and efficient gene delivery. As reported in the literature, the particles with mean diameters
between 54 and 625 nm are most liable to endocytosis by cells [44]. These properties of complexes
formed at various weight ratios ranging from 0.25 to 8, and were measured by dynamic light scattering
(DLS). Results in Figure 2A reveal that, with the increase of w/w ratio (>2), the sizes of nanoparticles
plateaued with mean diameters of 150–230 nm. On the other hand, a cationic surface charge is
beneficial because it aids in the electrostatic interaction between polyplexes and negatively charged
cellular membranes, leading to efficient intracellular uptake. As shown in Figure 2B, zeta-potential of
the polyplexes increased along with the increase of w/w ratio, and the surface charge was observed
about +31–41 mV above w/w ratio of 2. The lower zeta-potential of P1 might be attributed to the closer
oxygen-rich structure with amino groups, resulting in the screening of positive charges.

Molecules 2017, 22, 566
4 of 12
Figure 2. Mean particle size (A) and zeta potential (B) of polyplexes obtained at various weight ratios
by dynamic light scattering (DLS). Data represent mean ± SD (n = 3).
2.4. In Vitro Transfection
Luciferase reporter gene was used to quantitatively assess the in vitro TEs of the new polyplexes
in several cell lines, including human embryonic kidney cell lines (HEK 293), human cervix carcinoma
cell lines (HeLa), and human hepatoma cell lines (HepG2). Figure 3 exhibits the TEs of P1 and P2 at
various w/w ratios as the relative fluorescence intensity per mg protein (RLU/mg protein), and PEI 25
KDa and Lipofectamine 2000 were used for comparison. The new cationic polymer P2 showed much
higher TEs in all tested cell lines, and the TEs were greatly dependent on the cell type. As shown in
Figure 3A, the complexes made by P2 shows 1.63-fold higher TE than PEI 25 KDa, and 1.85-fold higher
TE than Lipofectamine 2000, toward HEK 293 cells at the optimized w/w ratio of 4. Meanwhile, P2
also gave comparable TEs to PEI 25 KDa and Lipofectamine 2000 in tumor cells (Figure 3B,C). Under
the same condition, P1 showed much lower TEs than P2. For the ester-contained polymers P1 and P2
such subtle differences in their structure led to completely different TEs. We speculate that P2, with
higher surface charge and better buffering capacity, may promote more efficient cellular uptake and
endosome escape of the complexes. The detailed transfection mechanisms leading to such differences
are under further investigation.
,
Figure 3. Luciferase gene expression transfected by polyplexes at different weight ratios: 2 (N/P = 9.8),
4 (N/P = 19.6), and 6 (N/P = 29.4) in comparison with Lipofectamine 2000 and 25 KDa PEI (w/w = 1.4
N/P = 10) in human embryonic kidney cell lines (HEK 293) ( ); human cervix carcinoma cell lines
(HeLa) ( ), and human hepatoma cell lines (HepG2) ( ) cells in the absence of serum. Data represent
mean ± SD (n = 3).
,
A
B
C

Molecules 2017, 22, 566
5 of 12
To directly visualize the infected cells expressing the pEGFP-Nl reporter gene, polymers P1 and
P2 mediated enhanced green fluorescent protein (eGFP) expression in HepG2 and HeLa cells was
observed by an inverted fluorescent microscope. The weight 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 P1 gave much better GFP expression than the others involving controls, and much
higher green fluorescence density could be observed.
Figure 4. Fluorescence microscope image of pEGFP-transfected HepG2 and HeLa cells in the absence
of serum at optimal weight ratio.
Furthermore, to estimate the in vivo transfection potential, we studied the serum-present gene
delivery ability of cationic polypexes as a fundamental predictive model. The serum-triggered
transfection inhibition was believed to have a strong association with the non-specific interaction
between complexes and negatively charged proteins [45,46]. For the higher TEs of P2 in preceding
serum-free studies, this polymer was subsequently chosen to study its transfection behavior in all the
cell lines in the presence of 10% serum. As shown in Figure 5, the TEs of P2/DNA polyplexes were
lower than that obtained in the absence of serum and the controls PEI 25 KDa and Lipofectamine 2000.
Further studies to modify of these types of polymers to improve the TE are now in progress.
Figure 5. Transfection by P2 at various weight ratios: 2 (N/P = 9.8), 4 (N/P = 19.6), and 6 (N/P = 29.4)
in HEK 293 (
A); HeLa (B) and HepG2 (C) cells in the presence of 10% serum. Lipofectamine 2000 and
PEI 25 KDa (w/w = 1.4, N/P = 10) were used as controls. Data represent mean ± SD (n = 3).

Molecules 2017, 22, 566
6 of 12
3. Cytotoxicity
The negatively-charged cellular and blood components would interact with cationic polyplexes,
leading to inherent toxicity [47]. Therefore, cytotoxicity is a crucial factor in assessing biosafety for
polymeric gene carriers. A successful delivery system should have high TE and low toxicity. The cell
viability of the target polymer/DNA complexes was evaluated in HepG2 cells by CCK-8 assays,
using PEI 25 KDa and Lipofectamine 2000 as controls. It should be noted that the weight ratio for
CCK-8 assay was the same with that used in the transfection experiment. As shown in Figure 6, the
cytotoxicity of polyplexes gradually increased with increasing of the weight ratios, due to the presence
of higher cationic charge density. At a low weight ratio (=2), almost no cytotoxicity was observed, and
the cell viabilities of studied polyplexes (especially P1) were also higher than that involving PEI 25 KDa
and Lipofectamine 2000, even at higher weight ratios. Comparing with the structure of PEI 25 KDa,
we believe that the lower cytotoxicity may be attributed to the newly formed hydroxyl groups and
biodegradable ester linkages, which can benefit the biocompatibility of the polymeric materials used
for gene transfection [24,37]. It is also proved that the ring-opening polymerization from bis-epoxides
is an effective synthetic approach toward materials with low cytotoxicity.
Figure 6. Cytotoxicity of polyplexes at different weight ratios toward HepG2 cells. Lipofectamine 2000
and PEI 25 KDa were used as controls (w/w = 1.4). Data represent mean ± SD (n = 3).
4. Experimental Section
4.1. Materials and Methods
All chemicals and reagents were obtained commercially and were used as received. Anhydrous
ethanol and dichloromethane (DCM) were dried and purified under nitrogen by using standard
methods and were distilled immediately before use. (2-nitro-1,3-phenylene)bis(methylene) diacrylate
(1a) was prepared according to the literature [48]. LMW PEI (branched, average molecular weight
600 Da, 99%), branched PEI 25 KDa and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The plasmids used in the study
were pGL-3 (Promega, Madison, WI, USA) coding for luciferase DNA and pEGFP-N1 (Clontech,
Palo Alto, CA, USA) coding for EGFP DNA. The Dulbecco’s Modified Eagle’s Medium (DMEM) and
fetal bovine serum were purchased from Invitrogen Corp. MicroBCA protein assay kit was obtained
from Pierce (Rockford, IL, USA). Luciferase assay kit was purchased from Promega (Madison, WI,
USA). Endotoxin free plasmid purification kit was purchased from TIANGEN (Beijing, China). HEK
293 cells, HeLa cells, and HepG2 cells were purchased from Shanghai Institute of Biochemistry and
Cell Biology, Chinese Academy of Sciences. All other reagents used in the synthesis, if not specified,
were obtained from Sigma-Aldrich Co. and used without further purification.
The 1H-NMR and 13C-NMR spectra were obtained on a Varian INOVA-400 spectrometer. CDCl3
or D2O was used as the solvent, and TMS was used as the internal reference. The MS (ESI) spectra data
were recorded with a Finnigan LCQDECA and with a Bruker Daltonics BioTOF mass spectrometer
(Bruker Daltonics Inc., Billerica, MA, USA). The molecular weight of polymers was determined by
gel permeation chromatography (GPC) 515 pump, Waters 2410 Refractive Index Detector (Waters

Molecules 2017, 22, 566
7 of 12
◦
Corp, Milford, MA, USA), 25 C, incorporating Shodex columns OHPAK KB-803, (Showa Denko Corp.,
Tokyo, Japan). A filtered mixture of 0.5 mol
−1 HAc/NaAc buffer was used as the mobile phase, with
a flow rate of 0.5 mL
min−1. Molecular weights were calculated against polyethylene glycol standards,
·
L
·
with average molecular weights ranging from 900 to 80,000.
4.2. General Method for Preparation of Biodegradable Poly(Amino Ester) with Aromatic Backbone
4.2.1. Synthesis and Characterization Linker of 2a
(2-nitro-1,3-phenylene)bis(methylene) diacrylate (1a) (492 mg, 2 mmol) was oxidized by
meta-chloroperoxybenzoic acid (m-CPBA, 1.73 g, 8 mmol, 80% purity) in 30 mL DCM. The mixture
◦
was refluxed at 55 C for 8 h. After the reaction, the solution was washed with sat. NaHCO3
(2
×
50 mL), and brine (2
×
50 mL). The organic layer was dried over MgSO4, and concentrated
under reduced pressure. The residue was purified by silica gel column chromatography (silica,
ethyl acetate/petroleum ether: v/v = 1/4) to give product 2a (pale-yellow oil, 440 mg, yield 79%).
1H-NMR (CDCl3, 400 MHz)
δ
(ppm) 7.40–7.34 (m, 4H, PhH), 5.23 (q, J = 12.3 Hz, 4H, -CH2PhCH2-),
3.49 (dd, J = 4.1, 2.4 Hz, 2H, -OCOCH), 3.01–2.95 (m, 4H, ring CH2). 13C-NMR (100 MHz, CDCl3):
δ
169.04, 135.53, 129.05, 128.62, 128.38, 128.37, 66.86, 47.30, 46.41. HRMS (ESI): m/z calcd for
C14H14O6 (M+) 278.0790; found = 301.0680 ([M + Na]+, 100). The NMR spectra of 2a are shown
in the supplementary information.
4.2.2. Synthesis and Characterization Linker of 2b
2, 3-epoxy-1-propanol (1.56g, 21 mmol) was added to the 50 mL anhydrous DCM solution of
1, 4-dicarboxybenzene (2a) (1.66g, 10 mmol), dicyclohexylcarbodiimide◦(DCC, 4.1 g, 20 mmol) and
N,N-dimethylaminopyridine (DMAP, 27 mg, 2 mmol) in ice-bath at 0 C for 1 h and then 24 h at
room temperature and filtered. The filtrate was evaporated under reduced pressure, and a little ethyl
acetate was added. The mixture was maintained at 0 ◦C for half an hour and filtered. The filtrate was
evaporated under reduced pressure to give the crude products which were purified by silica gel column
chromatography (silica, ethyl acetate/petroleum ether: v/v = 1/4) to give product 2b (white solid, 1.2 g,
1
yield 45%). H-NMR (CDCl3, 400 MHz)
δ
(ppm) 8.74–7.55 (m, 4H, PhH), 4.70–4.19 (m, 4H, -COOCH2),
165.34, 134.22, 130.96,
3.37 (s, 2H, ring CH), 2.93–2.75 (m, 4H, ring CH2). 13C-NMR (100 MHz, CDCl3):
δ
130.17, 128.74, 65.91, 49.32, 44.74. HRMS (ESI): m/z calcd for C14H14O6 (M+) 278.0790; found = 301.0679
([M + Na]+, 100). The NMR spectra of 2b are shown in the supplementary information.
4.2.3. Synthesis and Characterization of Target Polymers P1, P2
PEI 600 (0.5 mmol) was dissolved in 1 mL anhydrous EtOH, then diglycidyl ether linkers (2a or 2b
0.5◦mmol) were added to the solution. Under the protection of N2, the reaction mixture was stirred at
80 C for 72 h. After the reaction, the mixture was diluted with 2 mL of anhydrous EtOH, and the crude
product was precipitated by the addition of anhydrous DCM/cyclohexane (v/v = 1/1). The precipitate
was collected and dried in vacuum to get the product as a pale-yellow oil. The molecular weights of
compounds P1, P2 were measured by GPC.
,
1
P1: Yield 30%. H-NMR (400 MHz, D2O):
δ (ppm) 8.16–7.35 (m, 4H, PhH), 4.67–2.15 (m, -CH2PhCH2-,
-OCOCH2CH-, -NHCH2CH2-). GPC: Mn = 17,561, PDI = 1.18.
P2: Yield 35%. 1H-NMR (400 MHz, D2O):
-COOCH2CH(OH)CH2-, -NHCH2CH2-). GPC: Mn = 24,550, PDI = 1.87. The H-NMR spectra of
P1 and P2 were shown in the supplementary information.
δ
(ppm) 7.87–7.57 (m, 4H, PhH), 4.42–1.67 (m,
1
4.3. Amplification and Purification of Plasmid DNA
pGL-3 and pEGFP-N1 plasmids were used. The former one as the luciferase reporter gene was
transformed in E. coli JM109 and the latter one as the enhanced green fluorescent protein gene was

Molecules 2017, 22, 566
8 of 12
◦
transformed in E. coli DH5α. Both plasmids were amplified in Luria-Bertani broth at 37 C and 220 rpm
overnight. The plasmids were purified by an EndoFree TiangenTM Plasmid Kit. Then, the purified
plasmids were dissolved in TE (Tris + EDTA) buffer solution and stored at
plasmids was confirmed by agarose gel electrophoresis. The purity and concentration of plasmids
were determined by ultraviolet (UV) absorbance at 260 and 280 nm.
−
20 ◦C. The integrity of
4.4. Acid-Base Titration
Briefly, 10 mg of polymers were dissolved in 5 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, Shanghai instrument electric science Inc., Shanghai, China) after
each addition. For comparison, NaCl (150 mM), PEI 600 and PEI 25 KDa were used under the same
experimental conditions. The buffering capacity, defined as the percentage of amine groups becoming
deprotonated from pH 5.1 to 7.4, was calculated from equation:
Buffer capacity (%) = 100[(∆V1 NaOH - ∆V2 NaOH) × 0.1 M]/N mol
(1)
wherein
∆V1 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, V2 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.
4.5. Polymer Degradation Study
For the polymer degradation study, P2 (Mn = 24,550) was dissolved in a single-strength (1
×
)
◦
phosphate-buffered saline (PBS) solution and constantly shaken in a 37 C incubator at 100 rpm. The P2
solution was withdrawn at different time points and then lyophilized. The relative molecular mass of
degraded products was determined by GPC using a Waters 515 pump and a Waters 2410 refractive
index detector (25 ◦C).
4.6. Agarose Gel Retardation
Polyplexes at different w/w ratios (weight ratio of polymer relative to pDNA) were prepared by
adding an appropriate volume of the polymer solution to 5
complex solution was then diluted to the total volume of 10
the polyplexes were electrophoresed on a 0.7% (w/v) agarose gel containing GelRedTM in Tris-acetate
(TAE) running buffer at 120 V for 40 min. DNA was then visualized under an ultraviolet lamp by
using a Vilber Lourmat imaging system (Paris, France).
µ
L of Puc-19 (0.025 mg/mL). The obtained
µ
L. After incubation at 37 ◦C for 0.5 h,
4.7. Particle Size and Zeta Potential Measurements
◦
Complex size and zeta potential were evaluated by dynamic light scattering (DLS) at 25 C
by Nano-ZS ZEN3600 (Malvern Instruments, Malvern, UK). The polymer/DNA complexes with
various w/w ratios were prepared by adding 1 µg of DNA to the appropriate volume of the polymer
solution. The complex solution was then vortexed for 30 s before being incubated at 37 ◦C for 0.5 h
and then diluted up to 1 mL by purity water solution prior to being measured. Data were shown as
mean ± standard deviation (SD) based on three independent measurements.
4.8. Cell Culture
HEK 293, HepG2, and HeLa cell lines were maintained in DMEM supplemented with 10% (v/v)
heat-inactivated fetal bovine serum, 100 µg/mL streptomycin and 100 IU/mL penicillin at 37 ◦C in a
humidity atmosphere of 5% CO2 incubator.

Molecules 2017, 22, 566
9 of 12
4.9. In Vitro Transfection Experiments
Gene transfection of a series of complexes was investigated in HEK 293, HepG2, and HeLa
cells. Cells were seeded in 24-well plates (8
×
104 cells/well for HEK 293, 7
×
104 cells/well for
HepG2 and HeLa) and grown to reach 70–80% cell confluence 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/DNA (1 µg) complexes at various weight ratios. After 4 h under standard
incubator conditions, the medium was replaced with fresh medium containing serum and incubated
for another 20 h.
For fluorescent microscopy assays, cells were transfected by complexes containing pEGFP-N1.
After 24 h incubation, GFP-expressed cells were observed with an inverted fluorescence microscope
(Nikon Eclipse TE 2000E) equipped with a cold Nikon camera. Control transfection was performed in
each case using a commercially available transfection reagent PEI 25 KDa and Lipofectamine 2000,
based on the standard conditions specified by the manufacture.
For luciferase assays, cells were transfected by complexes containing pGL-3. For a typical assay
in a 24-well plate, 24 h post transfection as described above, cells were washed with cold PBS and
lysed with 100
µ
L 1
×
Lysis reporter buffer (Promega, Madison, WI, USA). The luciferase activity was
measured by microplate reader (Model 550, BioRad, Hercules, CA, USA). Protein content of the lysed
cell was determined by BCA protein assay. Gene transfection efficiency was expressed as the relative
fluorescence intensity per mg protein (RLU/mg protein). All of the experiments were carried out
in triplicate.
4.10. Cytotoxicity Assay
The cytotoxicity studies were examined by Cell Counting Kit-8 (CCK-8) assay. Briefly, HepG2
cells were seeded at 0.8
confluence. The medium was replaced with 50
various concentrations were added to achieve a final volume of 100
hours later, 10 L CCK-8 mixed in 90 L PBS was added to each well for an additional 1 h incubation.
×
104 cells/well in 96-well plates and cultured overnight for 70–80% cell
µL of fresh medium, to which 50
µL polyplexes at
µ
L (0.2 g DNA/well). Twenty-four
µ
µ
µ
The absorbance was measured in an ELIEA plate reader (model 550, BioRad, Hercules, CA, USA) at a
wavelength of 450 nm. The metabolic activity of the polyplex-treated cells was expressed as relative to
untreated cell controls, taken as 100% metabolic activity. Moreover, the cell viability of PEI 25 KDa and
Lipofectamine 2000 were also determined.
4.11. Statistical Analysis
Data are presented as mean
±
standard deviations ( S.D.) of at least three independent samples
±
and each measurement was performed in triplicate. Statistical analysis was determined by analysis of
variance tests (ANOVA), using the software of Microsoft Excel 2007. Data sets were compared using
two-tailed, unpaired t-tests, and a p value of <0.05 was considered to be statistically significant.
5. Conclusions
A new class of biodegradable poly(amino ester) compounds were synthesized by the ring-opening
polymerization between diepoxide and LWM PEI. These materials were applied in the gene delivery
as non-viral vectors to study their structure-activity relationships (SAR). The tiny difference of
structure largely influenced the buffering capacity, surface properties, and transfection efficiency of the
formed polyplexes. Polymer P2 exhibited higher buffering capacity, proper size, and zeta-potential,
which might lead to easier endocytosis and subsequently higher TE. Compared to PEI 25 KDa and
Lipofectamine 2000, P2 could give higher TE and lower cytotoxicity in several cell lines. For example,
up to ~2 times higher TE than PEI and Lipofectamine in HEK 293 cells was achieved by employing P2
as transfection reagent. The lower cytotoxicity of the polymers might come from their biodegradable
backbones and hydroxyl groups. Results suggest that such a polymer might be a promising non-viral

Molecules 2017, 22, 566
10 of 12
gene carrier in future in vivo applications. Further modification of the polymer structure and relative
mechanism studies are now in progress.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This work was financially supported by the Science and Technology Department of Sichuan
Province (No. 2015JY0115), the Fundamental Research Funds for the Central Universities, Southwest University
for Nationalities (No. 2016NZYZQ66). 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.
Author Contributions: Q.L. and W.-J.Y. contributed to conceive and design the study; W.-J.Y. and R.-C.S.
contributed to perform the experiments; W.-J.Y. performed the data analyses and wrote the manuscript; Q.L.
helped perform the analysis with constructive discussions; Q.L. and Z.-G.Z. reviewed and edited the manuscript;
All the authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
He, H.; Bai, Y.; Wang, J.; Deng, Q.; Zhu, L.; Meng, F.; Zhong, Z.; Yin, L. Reversibly cross-linked
polyplexes enable cancer-targeted gene delivery via self-promoted DNA release and self-diminished toxicity.
Biomacromolecules 2015, 16, 1390–1400. [CrossRef] [PubMed]
Medina, S.H.; Li, S.; Howard, O.M.Z.; Dunlap, M.; Trivett, A.; Schneider, J.P.; Oppenheim, J.J. Enhanced
immunostimulatory effects of DNA-encapsulated peptide hydrogels. Biomaterials 2015, 53, 545–553.
[CrossRef] [PubMed]
Yu, M.; Zhang, L.; Wang, J.; Tang, R.; Yan, G.; Cao, Z.; Wang, X. Acid-labile poly(ortho ester amino alcohols)
by ring-opening polymerization for controlled DNA release and improved serum tolerance. Polymer 2016
,
96, 146–155. [CrossRef]
Chen, Y.; Li, Y.; Gao, J.; Cao, Z.; Jiang, Q.; Liu, J.; Jiang, Z. Enzymatic PEGylated Poly(lactone-co-β-amino
ester) Nanoparticles as Biodegradable, Biocompatible and Stable Vectors for Gene Delivery. ACS Appl.
Mater. Interfaces 2016, 8, 490–501. [CrossRef] [PubMed]
5.
6.
7.
8.
9.
Suzuki, Y.; Okuda, T.; Okamoto, H. Development of New Formulation Dry Powder for Pulmonary Delivery
Using Amino Acids to Improve Stability. Biol. Pharm. Bull. 2016, 39, 394–400. [CrossRef] [PubMed]
Bansal, R.; Gupta, K.C.; Kumar, P. Biodegradable and versatile polyethylenimine derivatives efficiently
transfer DNA and siRNA into mammalian cells. Colloids Surf. B 2015, 135, 661–668. [CrossRef] [PubMed]
Yue, Y.; Wu, C. Progress and perspectives in developing polymeric vectors for in vitro gene delivery.
Biomater. Sci. 2013, 1, 152–170. [CrossRef]
Mintzer, M.A.; Simanek, E.E. Nonviral vectors for gene delivery. Chem. Rev. 2009, 109, 259–302. [CrossRef]
[PubMed]
Liu, J.; Feng, M.; Liang, D.; Yang, J.; Tang, X. Vitamin E-Labeled Polyethylenimine for in vitro and in vivo
Gene Delivery. Biomacromolecules 2016, 17, 3153–3161. [CrossRef] [PubMed]
10. Ahn, H.H.; Lee, J.H.; Kim, K.S.; Lee, J.Y.; Kim, M.S.; Khang, G.; Lee, I.W.; Lee, H.B.
Polyethyleneimine-mediated gene delivery into human adipose derived stem cells. Biomaterials 2008
29, 2415–2422. [CrossRef] [PubMed]
,
11. Hwang, H.S.; Hu, J.; Na, K.; Bae, Y.H. Role of polymeric endosomolytic agents in gene transfection:
A comparative study of poly(L-lysine) grafted with monomeric l-histidine analogue and poly(L-histidine).
Biomacromolecules 2014, 15, 3577–3586. [CrossRef] [PubMed]
12. Zhou, D.; Li, C.; Hu, Y.; Zhou, H.; Chen, J.; Zhang, Z.; Guo, T. The effects of a multifunctional oligomer
and its incorporation strategies on the gene delivery efficiency of poly(L-lysine). Chem. Commun. 2012, 48,
4594–4596. [CrossRef] [PubMed]
13. Wu, L.; Ficker, M.; Christensen, J.B.; Trohopoulos, P.N.; Moghimi, S.M. Dendrimers in Medicine: Therapeutic
Concepts and Pharmaceutical Challenges. Bioconj. Chem. 2015, 26, 1198–1211. [CrossRef] [PubMed]
14. Vaidyanathan, S.; Orr, B.G.; Holl, M.M.B. Role of cell membrane–vector interactions in successful gene
delivery, Acc. Chem. Res. 2016, 49, 1486–1493. [CrossRef] [PubMed]

Molecules 2017, 22, 566
11 of 12
15. Taranejoo, S.; Chandrasekaran, R.; Cheng, W.; Hourigan, K. Bioreducible PEI-functionalized glycol chitosan:
A novel gene vector with reduced cytotoxicity and improved transfection efficiency. Carbohydr. Polym. 2016
153, 160–168. [CrossRef] [PubMed]
,
16. Islam, N.; Ferro, V. Recent advances in chitosan-based nanoparticulate pulmonary drug delivery. Nanoscale
2016, 8, 14341–14358. [CrossRef] [PubMed]
17. 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]
18. Lungwitz, U.; Breunig, M.; Blunk, T.; Gopferich, A. Polyethylenimine-based non-viral gene delivery systems.
Eur. J. Pharm. Biopharm. 2005, 60, 247–266. [CrossRef] [PubMed]
19. Baker, A.; Saltik, M.; Lehrmann, H.; Killisch, I.; Mautner, V.; Lamm, G.; Christofori, G.; Cotton, M.
Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid
DNA to adenovirus for gene delivery. Gene Ther. 1997, 4, 773–782. [CrossRef] [PubMed]
20. Boussif, O.; Lezoualch, F.; Zanta, M.A.; Mergny, M.D.; Scherman, D.; Demeneix, B.; Behr, J.P. A versatile
vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl.
Acad. Sci. USA 1995, 92, 7297–7301. [CrossRef] [PubMed]
21. Wightman, L.; Kircheis, R.; Rössler, V.; Carotta, S.; Ruzicka, R.; Kursa, M.; Wagner, E. Different behavior of
branched and linear polyethylenimine for gene delivery in vitro and in vivo. J. Gene Med. 2001, 3, 362–372.
[CrossRef] [PubMed]
22. Venkiteswaran, S.; Thomas, T.; Thomas, T.J. Selectivity of polyethyleneimines on DNA nanoparticle
preparation and gene transport. ChemistrySelect 2016, 6, 1144–1150. [CrossRef]
23. 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]
24. Luo, X.H.; Huang, F.W.; Qin, S.Y.; Wang, H.F.; Feng, J.; Zhang, X.Z.; Zhuo, R.X. A strategy to improve
serum-tolerant transfection activity of polycation vectors by surface hydroxylation. Biomaterials 2011, 32,
9925–9939. [CrossRef] [PubMed]
25. Teo, P.Y.; Yang, C.; Hedrick, J.L.; Engler, A.C.; Coady, D.J.; Ghaem-Maghami, S.; George, A.J.; Yang, Y.Y.
Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection.
Biomaterials 2013, 34, 7971–7979. [CrossRef] [PubMed]
26. Fischer, D.; Bieber, T.; Li, Y.; Elsasser, H.P.; Kissel, T. A novel non-viral vector for DNA delivery based on low
molecular weight, branched polyethylenimine: Effect of molecular weight on transfection efficiency and
cytotoxicity. Pharm. Res. 1999, 16, 1273–1279. [CrossRef] [PubMed]
27. Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery.
J. Control. Release 2006, 114, 100–109. [CrossRef] [PubMed]
28. 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]
29. Zheng, M.; Yang, C.; Meng, F.; Peng, R.; Zhong, Z. pH-sensitive degradable hydrophobe modified 1.8
kDa branched polyethylenimine as “artificial viruses” for safe and efficient intracellular gene transfection.
Macromol. Res. 2012, 20, 327–334.
30. Zhao, D.; Gong, T.; Zhu, D.; Zhang, Z.; Sun, X. Comprehensive comparison of two new biodegradable gene
carriers. Int. J. Pharm. 2011, 413, 260–270. [CrossRef] [PubMed]
31. Xiong, M.P.; Forrest, M.L.; Ton, G.; Zhao, A.; Davies, N.M.; Kwon, G.S. Poly(aspartate-g-PEI800),
a polyethylenimine analogue of low toxicity and high transfection efficiency for gene delivery. Biomaterials
2007, 28, 4889–4900. [CrossRef] [PubMed]
32. 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]
33. 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]
34. 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]

Molecules 2017, 22, 566
12 of 12
35. 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]
36. Yi, W.-J.; Zhang, Q.-F.; Zhang, J.; Liu, Q.; Ren, L.; Chen, Q.-M.; Guo, L.; Yu, X.-Q. Cyclen-based lipidic
oligomers as potential gene delivery vehicles. Acta Biomater. 2014, 10, 1412–1422. [CrossRef] [PubMed]
37. Zhang, Q.-F.; Yi, W.-J.; Wang, B.; Zhang, J.; Ren, L.; Chen, Q.-M.; Guo, L.; Yu, X.-Q. Linear polycations by
ring-opening polymerization as non-viral gene delivery vectors. Biomaterials 2013, 34, 5391–5401. [CrossRef]
[PubMed]
38. Xun, M.-M.; Liu, Y.-H.; Guo, Q.; Zhang, J.; Zhang, Q.-F.; Wu, W.-X.; Yu, X.-Q. Low molecular weight
PEI-appended polyesters as non-viral gene delivery vectors. Eur. J. Med. Chem. 2014, 78, 118–125. [CrossRef]
[PubMed]
39. Morille, M.; Passirani, C.; Vonarbourg, A.; Clavreul, A.; Benoit, J.P. Progress in developing cationic vectors
for non-viral systemic gene therapy against cancer. Biomaterials 2008, 29, 3477–3496. [CrossRef] [PubMed]
40. Thomas, T.J.; Tajmir-Riahi, H.A.; Thomas, T. Polyamine–DNA interactions and development of gene delivery
vehicles. Amino Acids 2016, 48, 2423–2431. [CrossRef] [PubMed]
41. Vijayanathan, V.; Thomas, T.J.; Thomas, T. DNA Nanoparticles and Development of DNA Delivery Vehicles
for Gene Therapy. Biochemistry 2002, 41, 14085–14094. [CrossRef] [PubMed]
42. Sun, C.; Tang, T.; Uludag, H. Molecular Dynamics Simulations of PEI Mediated DNA Aggregation.
Biomacromolecules 2011, 12, 3698–3707. [CrossRef] [PubMed]
43. Farber, I.Y.; 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. Son, S.; Kim, W.J. Biodegradable nanoparticles modified by branched polyethylenimine for plasmid DNA
delivery. Biomaterials 2010, 31, 133–143. [CrossRef] [PubMed]
46. Jones, C.H.; Chen, C.-K.; Jiang, M.; Fang, L.; Cheng, C.; Pfeife, B.A. Synthesis of Cationic Polylactides with
Tunable Charge Densities as Nanocarriers for Effective Gene Delivery. Mol. Pharm. 2013, 10, 1138–1145.
[CrossRef] [PubMed]
47. Fischer, D.; Li, Y.X.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. In vitro cytotoxicity testing of polycations:
Influence of polymer structure on cell viability and hemolysis. Biomaterials 2003, 24, 1121–1131. [CrossRef]
48. Deng, X.; Zheng, N.; Song, Z.; Yin, L.; Cheng, J. Trigger-responsive, fast-degradable poly(β-amino ester)s for
enhanced DNA unpackaging and reduced toxicity. Biomaterials 2014, 35, 5006–5015. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 2a and 2b are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).