Novel reduction-responsive cross-linked polyethylenimine derivatives by click chemistry for nonviral gene delivery

Article abstract of DOI:10.1021/bc100191r

Novel reducible disulfide-containing cross-linked polyethylenimines (PEI-SS-CLs) were synthesized via click chemistry and evaluated as nonviral gene delivery vectors. First, about four azide pendant groups were introduced into a low-molecular-weight (LMW) PEI (1.8 kDa) to get an azide-terminated PEI. Then, click reaction between a disulfide-containing dialkyne cross-linker and the azide functionalized LMW PEI resulted in a high-molecular-weight disulfide-containing cross-linked PEI composed of LMW constitute via a reducible cross-linker. The synthesized polymers were characterized by ¹H NMR, FTIR, and size-exclusion chromatography (SEC). It was shown that the obtained disulfide-containing cross-linked PEIs were able to condense plasmid DNA into positively charged nanoparticles. The degradation of the disulfide cross-linked polymers PEI-SS-CLs induced by DTT was confirmed by a gel retardation assay and SEC analysis. In vitro experiments revealed that the reducible PEI-SS-CLs were less cytotoxic and more effective in gene transfection (in both the presence and absence of serum) than the control nondegradable 25-kDa PEI. This study demonstrates that a reducibly degradable cationic polymer composed of LMW PEI cross-linked via a disulfide-containing linker possesses both higher gene transfection efficiency and lower cytotoxicity than PEI (25 kDa). These polymers are therefore attractive candidates for further in vivo evaluations.

Full text of DOI:10.1021/bc100191r

Bioconjugate Chem. 2010, 21, 1827–1835
1827
Novel Reduction-Responsive Cross-Linked Polyethylenimine Derivatives by
Click Chemistry for Nonviral Gene Delivery
Jia Liu,† Xulin Jiang,*,† Li Xu,† Xianmiao Wang,‡ Wim E. Hennink,§ and Renxi Zhuo†
Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, and Modern Virology Center,
College of Life Sciences, Wuhan University, Wuhan 430072, P. R. China, and Department of Pharmaceutics, Utrecht Institute
for Pharmaceutical Science (UIPS), Utrecht University, P.O. Box 80082, 3508 TB, Utrecht, The Netherlands. Received
April 21, 2010; Revised Manuscript Received July 5, 2010
Novel reducible disulfide-containing cross-linked polyethylenimines (PEI-SS-CLs) were synthesized via click
chemistry and evaluated as nonviral gene delivery vectors. First, about four azide pendant groups were introduced
into a low-molecular-weight (LMW) PEI (1.8 kDa) to get an azide-terminated PEI. Then, click reaction between
a disulfide-containing dialkyne cross-linker and the azide functionalized LMW PEI resulted in a high-molecular-
weight disulfide-containing cross-linked PEI composed of LMW constitute via a reducible cross-linker. The
1
synthesized polymers were characterized by H NMR, FTIR, and size-exclusion chromatography (SEC). It was
shown that the obtained disulfide-containing cross-linked PEIs were able to condense plasmid DNA into positively
charged nanoparticles. The degradation of the disulfide cross-linked polymers PEI-SS-CLs induced by DTT was
confirmed by a gel retardation assay and SEC analysis. In vitro experiments revealed that the reducible PEI-SS-
CLs were less cytotoxic and more effective in gene transfection (in both the presence and absence of serum) than
the control nondegradable 25-kDa PEI. This study demonstrates that a reducibly degradable cationic polymer
composed of LMW PEI cross-linked via a disulfide-containing linker possesses both higher gene transfection
efficiency and lower cytotoxicity than PEI (25 kDa). These polymers are therefore attractive candidates for further
in vivo evaluations.
cellular uptake, by which the polymers lose their DNA binding
capacity resulting in DNA release inside target cells. Generally,
ester linkages have a pH-dependent hydrolysis with half-life
time of hours to days. In contrast, disulfide linkages have been
shown to be stable in blood circulation (20), and their degrada-
tion is rapid (minutes to hours) in the presence of reductive
1,4-dithio-DL-threitol (DTT) or glutathione (GSH) mimicking
the reductive intracellular environment (20, 21). Therefore, the
introductionofdisulfidebridgesinthepolymermainchain(22-26)
or in the cross-links of polymers (27, 28) has been investigated
for the design of polymeric gene delivery vectors (8). Gopferich
et al. (29) recently published an excellent review on the redox-
sensitive, disulfide-based carriers for delivery of nucleic acids.
Lee et al. (27) first synthesized reducible PEI derivatives using
0.8 kDa PEI reacting with homobifunctional cross-linking
reagents, dithiobis(succinimidylpropionate) (DSP), or dimethyl-
3,3′-dithiobis-propionimidate-2HCl (DTBP). The resulting poly-
mers showed higher transfection efficiency compared to the
parent 0.8 kDa PEI and reached transfection levels close to that
of 25 kDa PEI. In a similar way, 1.8 kDa PEIs cross-linked
with DTBP were able to condense DNA, and the formed
polyplexes were sensitive to the GSH with significantly low
cytotoxicity (30). Also, 5-kDa PEIs cross-linked with DSP
formed stable complexes with DNA which could be destabilized
by a redox trigger (31). Kloeckner et al. (32) studied a library
of 37 soluble polycations synthesized by oligomerization of
LMW oligoamines with different cross-linkers. They concluded
that the polyplexes based on 0.8 kDa PEIs cross-linked with
DSP or DTBP showed negligible toxicity and efficient reporter
gene transfection at high N/P ratios. Goepferich et al. (33)
described that linear PEIs with molecular weight of 2.6, 3.1, or
4.6 kDa cross-linked with DSP or boc-cystine resulted in
branched structures. Using seven different cell lines, they
showed a high transfection efficiency (about 60%) and a high
INTRODUCTION
Gene therapy is a promising approach to treat human genetic
and acquired diseases. The key to the success of gene therapy
is the availability of safe and efficient vector systems (1, 2).
Cationic polymers, such as poly(L-lysine) (3), chitosan and its
derivatives (4, 5), poly(2-(dimethylamino)ethyl methacrylate)
(pDMAEMA) (6-8), and poly(ethylene imine) (PEI) (9), have
been widely explored as nonviral gene vectors. Among them,
PEI has been regarded as the “gold” standard showing relatively
high transfection efficiency, particularly in vitro, due to the
proton sponge effect (10, 11). Although transfection efficiency
of PEI-based complexes increases with PEI molecular weight
(12), cytotoxicity of PEI also simultaneously increases. High-
molecular-weight (HMW) PEIs, such as 25 kDa PEI, not only
are highly effective in gene transfection, but also exhibit
significant cytotoxicity. Low molecular weight (LMW) PEIs,
such as 1.8 kDa PEI, are less cytotoxic but less efficient (12, 13).
To deal with this dilemma between transfection efficiency
and cytotoxicity, biodegradable cationic polymers have been
designed with either hydrolytically degradable ester groups (8, 14)
or reducibly degradable disulfide linkages (15-19). It is
expected that these polymers complexed with plasmid DNA can
be degraded into less toxic low-molecular-weight structures after
* Corresponding author. Xulin Jiang Key Laboratory of Biomed-
ical Polymers of Ministry of Education and Department of Che-
mistry, Wuhan University, Wuhan 430072, P. R. China. Tel.:
+86-27-68755200; fax: +86-27-68754509. E-mail address: xljiang@
whu.edu.cn.
† Key Laboratory of Biomedical Polymers of Ministry of Education
and Department of Chemistry, Wuhan University.
‡ Modern Virology Center, College of Life Sciences, Wuhan
University.
§ Utrecht University.
10.1021/bc100191r  2010 American Chemical Society
Published on Web 09/09/2010

1828 Bioconjugate Chem., Vol. 21, No. 10, 2010
Liu et al.
Scheme 1. Synthetic Route Towards Reducible Disulfide-Contained PEI Derivatives (PEI-SS-CLs)
cell viability (>90%) of polyplexes based on the reversibly cross-
linked linear PEI polymers, which was better than seven
commercial transfection reagents including SuperFect, Lipo-
fectamine, and JetPEI. It was also reported (34-36) that the
disulfide cross-linked PEI derivatives using LMW PEI and DSP
were highly effective as gene delivery vectors and hardly
showed cytotoxicity. However, the cross-linking reaction be-
tween PEI and DSP (or DTBP) can easily lead to the formation
of microgels resulting in poorly controlled molecular weight
and a low yield (35). Peng et al. (15) used methylthiirane
reacting with low-molecular-weight PEI to get thiolated PEIs
(PEI-SHx), which were cross-linked into higher-molecular-
weight PEI by oxidation of the thiol groups avoiding signifi-
cantly changing the acid-base property of the PEI. However,
no reducible degradation was obviously observed. They claimed
that the majority of disulfide cross-links are intramolecular when
the thiolation degree is high.
Recently, “click” chemistry has rapidly become a very popular
method for polymer synthesis and modification due to the high
selectivity and high fidelity (8, 37, 38). However, no papers
using click chemistry for PEI cross-linking have been published
so far. In this study, a new strategy for synthesis of disulfide
cross-linked polyethylenimine (PEI-SS-CL) via click reaction
is investigated, as depicted in Scheme 1. First, a LMW PEI
(1.8 kDa), which has low transfection efficiency and low
cytotoxicity, was modified with azide pendant groups. Then,
this azide-functional PEI was reacted with disulfide-containing
dialkyne cross-linker through click chemistry to obtain a
reducible PEI derivative with high molecular weight. Click
chemistry allows easy coupling of poly(ethylene glycol) (PEG)
shielding, targeting ligands and fluorescent groups into the
polyplexes for further in vivo evaluation. This paper describes
the synthesis and reduction-triggered degradation of the disul-
fide-containing cross-linked PEI polymers and the gene trans-
fection activities of the corresponding polyplexes.
EXPERIMENTAL PROCEDURES
Materials. Branched polyethylenimine (PEI) with molecular
weight of 1.8 kDa was purchased from Alfa Aesar, and branched
PEI (25 kDa) was from Sigma-Aldrich. 1,1′-Carbonyldiimida-
zole (CDI) (Shanghai Medpep Co., Ltd., China), propargyl
alcohol (Wuhan FengFan Chemical Co., Ltd., China), cystamine
dihydrochloride (Jiangsu Jintan Medicine Chemical Factory,
Jiangsu, China), 1,4-dithio-DL-threitol (DTT, Shanghai Regal
Biotechnology Company, China), and 3-bromopropanol (Shang-
hai Zhuo Rui Chemical Co., China) were used as received. N,N′-
Dimethylformamide (DMF) was obtained from Shanghai Chemi-
cal Reagent Co., China, and used after distillation under reduced
pressure. Plasmid pcDNA3-Luc in TE buffer with concentration
of 5.0 mg/mL was from Plasmid Factory, Germany. Plasmid
pEGFP encoding a red-shifted variant of wild-type green

Reduction-Responsive Cross-Linked PEIs for Gene Delivery
Bioconjugate Chem., Vol. 21, No. 10, 2010 1829
1
1
Figure 1. (A) H NMR spectra of AP-CI and PEI-(N3)4 (with imidazole) in CDCl3. (B) H NMR spectra of PEI-SS-CL1 in D2O.
fluorescent protein (GFP) was purchased from Clontech,
Mountain View, CA, USA, and was transformed in E. coli
DH5R and propagated in Luria-Bertani (LB) medium at 37
°C overnight. Then, the plasmids were purified by means of
EndoFree plasmid purification as described previously (39). The
purified plasmids were diluted with TE buffer solution and
stored at -20 °C. The purity and concentration of plasmids were
determined by ultraviolet (UV) absorbance at 260 and 280 nm.
All other chemicals were analytical grade and used as received.
Synthesis of Disulfide-Containing Cross-Linked Polyethyl-
enimine (PEI-SS-CL). Synthesis of Bis[(Propargyl Carbamate)-
Ethyl] Disulfide (BPPA-Cyst, 2). Propargyl ester of carbonyl-
imidazole (PPA-CI, 1) was synthesized from 1,1′-carbonyldi-
imidazole (CDI) and propargyl alcohol (PPA) as described
previously (8). Cystamine dihydrochloride was neutralized by
4 M NaOH and extracted with dichloromethane to yield
cystamine. Then, BPPA-Cyst was synthesized from PPA-CI
with the cystamine, as shown in Scheme 1. Briefly, cystamine
(4.40 g, 28.52 mmol) and PPA-CI (6.93 g, 46.2 mmol) were
dissolved in 50 mL dichloromethane and subsequently stirred
at room temperature for 24 h. After evaporation of dichlo-
romethane, 100 mL of 1.0 M NaH2PO4 (pH 4.0) was added.
This aqueous solution was extracted with ethyl ether (40 mL, 3
times) to obtain, after evaporation of the solvent, BPPA-Cyst
with a yield of 80.2%. 1H NMR in CDCl3 (Supporting
Information Figure SI1): δ (ppm) 2.48 (s, 2H, CHtC), 2.82 (t,
4H, S-CH2-C), 3.51 (app. quartet, 4H, NH-CH2-C), 4.69(s, 4H,
O-CH2-CtC), 5.31(s, 2H, NH-CdO). IR analysis of the
resulting product showed an absorption peak at 2126 cm-1
corresponding to the alkyne group (see Supporting Information
Figure SI2, IR spectrum: 3293, 3061, 2943, 2126 (νC≡C), and
1709 cm-1).
Synthesis of 3-Azidopropyl Ester of Carbonylimidazole
(AP-CI, 3). 3-Azidopropanol was synthesized according to the
literature (40) with some modification. Briefly, 3-bromopropanol
(3.01 g, 21.65 mmol) and sodium azide (5.62 g, 86.46 mmol)
were dissolved in a mixture of 25 mL water and 10 mL acetone,
stirred under reflux for 24 h in an oil bath of about 80 °C. After
evaporation of acetone, the product was extracted with dichlo-
romethane (25 mL, 3 times). The organic layer was dried over
anhydrous magnesium sulfate, filtered, and evaporated to obtain
colorless oil with a yield of 87.6%. 1H NMR (DCCl3): δ (ppm)
1.80 (app. quint, 2H, C-CH2-C), 3.41 (t, 2H, CH2-N3), 3.70 (t,
2H, CH2-O).
AP-CI (3) was synthesized from the obtained 3-azidopropanol
with carbonyldiimidazole (CDI). Briefly, a dry, round-bottomed
flask was charged with CDI (4.80 g, 29.60 mmol) and 40 mL
dichloromethane yielding a suspension. Next, 3-azidopropanol
(1.50 g, 12.40 mmol) was added dropwise under vigorous
stirring while the reaction mixture turned into a clear solution.
After 4 h reaction at room temperature, the solution was washed
three times with water. The organic layer was dried over
anhydrous magnesium sulfate, filtered, and evaporated to obtain
colorless oil with a yield of 74.3%. 1H NMR in CDCl3 as shown
in Figure 1A: δ (ppm) 2.05 (app. quint, 2H, C-CH2-C), 3.47 (t,
2H, N3-CH2), 4.50 (t, 2H, CH2-O), 7.06 (s, 1H, CdCHsNd),
7.40 (s, 1H, NsCHdC), 8.12 (s, 1H, NsCHdN).

1830 Bioconjugate Chem., Vol. 21, No. 10, 2010
Liu et al.
Table 1. Characteristics of the Synthesized Disulfide Cross-Linked
PEI-SS-CLs
molar feed ratios
polymers
of alkyne/azide
yield(%)
Mw (kDa)
PDI
PEI-SS-CL1
PEI-SS-CL2
1:1
1:2
59
49
9.4
14.2
3.12
1.85
Synthesis of Azide-Terminated PEI [4, PEI-(N3)4]. AP-CI
(2.17 g, 11.13 mmol) dissolved in chloroform (20 mL) was
slowly dropped (around 2 h) into a solution of 1.8 kDa PEI
(5.00 g, 2.78 mmol, 29 mmol of primary amine groups, as-
suming that the primary amine content of the 1.8 kD PEI is
25% of the total amines (13) in chloroform (100 mL). Next,
the reaction mixture was refluxed using an oil bath (75 °C) for
10 h. Chloroform was removed by evaporation under reduced
pressure yielding a yellow liquid which was subsequently
dissolved in CDCl3 and characterized by 1H NMR. No unreacted
AP-CI was detected as shown in Figure 1A. The average number
of azide groups per PEI molecule determined by 1H NMR was
close to 4. IR analysis of the resulting product showed an
absorption peak at 2099 cm-1 corresponding to the asymmetric
stretching vibration of the azide group (see Supporting Informa-
tion Figure SI2, IR spectrum (neat): 2944, 2837, 2933, 2099
(νN3), and 1700 cm-1).
Synthesis of Disulfide Cross-Linked PEI-SS-CL (5) Via
Click Reaction. Azide-functional PEI (PEI-(N3)4, 0.75 g, 1 equiv
azide groups) and BPPA-cyst (0.16 or 0.08 g, 1 or 0.5 equiv
alkyne groups) were dissolved in DMF, and CuBr (1 equiv) as
catalyst was added under a nitrogen atmosphere. The reaction
mixture was stirred at 50 °C for 2 days. The resulting solution
was purified by dialysis against water (dialysis tube, MWCO
3.5 kDa), and the formed disulfide cross-linked PEI derivatives
PEI-SS-CL1 and PEI-SS-CL2 were collected after freeze-drying.
The structure of the PEI-SS-CLs was analyzed by 1H NMR and
FTIR. The molecular weights were obtained by size-exclusion
chromatography-multiangle light scattering (SEC-MALLS) as
described below.
Figure 2. Size by DLS (A) and zeta potential (B) of the PEI/DNA
polyplexes at various N/P ratios. Error bars are standard deviations
(SD) of 3 measurements.
1
Characterizations. H nuclear magnetic resonance (NMR)
spectra were measured with a Varian Unity 300 MHz spec-
trometer using CDCl3 or D2O as solvent. Fourier transformed
infrared (FTIR) spectra were recorded on a Lambda Bio40
UV-vis spectrometer (Perkin-Elmer). The molecular weights
and the molecular weight distributions of the polymers were
evaluated by a SEC-MALLS system consisting of a Waters
2690D separations module, a Waters 2414 refractive index
detector (RI), and a Wyatt DAWN EOS MALLS detector. Two
chromatographic columns (Shodex OHpak SB-803 and SB-
802.5, Showa Denko, Japan) with a precolumn (Shodex SB-G)
were used in series. A sodium acetate (NaAc) solution was
prepared by dissolving a calculated amount of sodium acetate
in reverse osmosis water (0.3 M), and the pH was adjusted to
4.4 with acetic acid (41). A mixture of 70% 0.3 M NaAc
solution, pH 4.4, and 30% acetonitrile was used as the eluent
at a flow rate of 0.6 mL/min. The eluent was filtered through a
0.22 µm HPLC filter and degassed prior to use by ultrasound.
The data were processed with Astra software (Wyatt Technol-
ogy).
100 ug/mL streptomycin at 37 °C under a humidified atmosphere
of 95% air and 5% CO2.
Cytotoxicity Assay. The cytotoxicity of the different poly-
mers was studied using the MTT assay (39) on 293T cells. The
cells were seeded into 96-well plates at a density of 3000 cells/
well, and 100 µL DMEM containing 10% FBS was added. The
cells were allowed to grow at 37 °C under a humidified
atmosphere of 95% air and 5% CO2 for 48 h. Thereafter, the
medium was replaced with 100 µL of fresh medium and 100
µL solutions of PEI-SS-CLs, 25 kDa PEI and 1.8 kDa PEI, were
added (final polymer concentrations ranging from 0.01 to 0.20
mg/mL). The cells were incubated for 48 h, and the medium
containing polymer solution was replaced with 200 µL fresh
medium. Next, the MTT reagent (20 µL in PBS, 5 mg/mL) was
added to each well for further 4 h incubation at 37 °C. Then,
the medium was removed and 100 µL of DMSO was added to
dissolve the formed formazan crystals. The absorbance at 570
nm was recorded using a Microplate Reader (BIO-RAD 550).
The relative cell viability (mean ( SD, n ) 4) was calculated
as cell viability ) (ODsample - ODblank)/(ODcontrol - ODblank) ×
100%, where ODsample is the absorbance of solution with cells
treated by polymers, ODcontrol is the absorbance for untreated
cells (without polymer), and ODblank is the absorbance without
cells.
The reductive degradation of the disulfide cross-linked
polymer PEI-SS-CL triggered by DTT was evaluated by SEC-
MALLS. The polymer PEI-SS-CL (5.0 mg) was dissolved in
the mobile phase (0.9 mL) followed by the addition of 0.1 mL
DTT in water (1.0 mol/L). After incubation at 37 °C for 2 h,
the molecular weight of the degraded polymer was determined
by SEC-MALLS.
Cell Culture. Human embryonic kidney transformed (293T)
cells and human cervix carcinoma (HeLa) cells were incubated
in Dulbecco’s Modified Eagle’s Medium (DMEM) containing
10% fetal bovine serum (FBS), 100 units/mL penicillin, and
Formation of PEI-SS-CL/DNA Polyplexes and Agarose
Gel Retardation Assay. PEI-SS-CL/DNA complexes (N/P
ratios ranging from 3 to 9) were prepared by addition of 5 µL
polymer solution with designed concentrations in 150 mM NaCl

Reduction-Responsive Cross-Linked PEIs for Gene Delivery
Bioconjugate Chem., Vol. 21, No. 10, 2010 1831
Figure 3. Agarose gel electrophoresis retardation assay of PEI-SS-CLs/DNA complexes at different N/P ratios (A) without DTT and (B) with 10
mM DTT.
(pH 7.4) to 1 µL of pcDNA3-Luc plasmid DNA (100 ng/µL in
TE buffer). The resulting polyplex dispersions were incubated
at 37 °C for 30 min and were then analyzed by electrophoresis
in a 0.7% (w/v) agarose gel containing GelRed and in Tris-
acetate (TAE) running buffer at 80 V for 60 min. DNA bands
were visualized with a UV (254 nm) illuminator and photo-
graphed with a Vilber Lourmat imaging system.
The DTT triggered reductive degradation of PEI-SS-CL based
polyplexes was also evaluated by agarose gel electrophoresis.
To 1 mL of dispersions of PEI-SS-CL/DNA polyplexes prepared
at N/P ratios ranging from 3 to 9, 100 µL of 100 mM DTT in
water was added. The polyplexes were incubated at 37 °C for
30 min and were then analyzed by electrophoresis in a 0.7%
(w/v) agarose gel containing GelRed and in TAE running buffer
at 80 V for 60 min.
was used instead of the serum-free DMEM medium. All the
transfection assays were carried out in triplicate.
Green Fluorescent Protein Assay. Transfection activity of
complexes of PEI-SS-CLs with pEGFP plasmid was evaluated
in 293T cells. The 293T cells were seeded at a density of 70 000
cells/well in a 24-well plate. Subsequently, 1 mL of DMEM
with 10% FBS was added, and the cells were incubated at 37
°C in a humidified atmosphere of 95% air and 5% CO2 for 24 h.
Next, the cells were incubated for 4 h at 37 °C, followed by
the addition of polyplexes of various N/P ratios prepared as
described above. After transfection for 48 h, the cells were
directly observed by an inverted microscope (Olympus IX 70).
The microscopy images were obtained at the magnification of
100× and recorded using CoolSNAP-Pro (v 4.5.1.1) software.
RESULTS AND DISCUSSION
Particle Size and ꢀ-Potential Measurements. Polyplexes
were prepared by the addition 90 µL of a PEI solution with
designed concentrations (in 150 mM NaCl, pH 7.4) to 10 µL
of pcDNA3-Luc plasmid DNA solution (0.1 mg/mL in 40 mM
Tris-HCl buffer). The amount of polymer used was calculated
on the basis of chosen N/P ratios, nitrogen atoms of the polymer
over phosphates of DNA. After the complexes were incubated
at 37 °C for 30 min, the polyplex dispersion was diluted with
water to 1 mL prior to measurement. Particle size and ꢀ-potential
were measured by Nano-ZS ZEN3600 (Malvern Instruments)
at 25 °C.
In Vitro Transfection Activity of Polyplexes. Luciferase
Assay. Transfection activity of polyplexes based on pcDNA3-
Luc plasmid, complexed with PEI-SS-CLs, 25-kDa PEI or 1.8-
kDa PEI, was evaluated in 293T and HeLa cells. The cells were
seeded at a density of 70 000 cells/well in a 24-well plate.
Subsequently, 1 mL of DMEM containing of 10% FBS was
added, and the cells were incubated at 37 °C in a humidified
atmosphere of 95% air and 5% CO2 for 24 h. Next, the medium
in each well was replaced by 0.9 mL of serum-free DMEM.
The polymer/DNA polyplexes (100 µL, N/P ratios ranging from
10 to 50) prepared as described above were then added and
incubated with cells for 4 h at 37 °C. The medium was replaced
by fresh DMEM containing 10% FBS, and the cells were
incubated for 48 h. Thereafter, the medium was removed, and
the luciferase assay was performed according to manufacturer’s
protocols. Relative light units (RLUs) were measured with
chemiluminometer (Lumat LB9507, EG&G Berthold, Ger-
many). The total protein was measured according to a BCA
protein assay kit (Pierce). Luciferase activity was expressed as
RLU/mg protein.
Synthesis and Characterization of Disulfide-Containing
PEI-SS-CL. The two-step synthetic route of disulfide cross-
linked polyethylenimine (PEI-SS-CL) via click reaction is
illustrated in Scheme 1. First, azide pendant groups were
introduced into a low-molecular-weight (LMW) PEI (1.8 kDa)
to obtain PEI-(N3)x, in which x stands for the average number
of azide groups per PEI molecule. Second, the azide-functional
PEI was reacted in DMF with the disulfide-containing dialkyne
cross-linker BPPA-Cyst, through click chemistry with CuBr as
catalyst, to obtain a reducible PEI derivative with high molecular
weight.
Azide-functionalized LMW PEI was synthesized by coupling
of activated azidopropanol, 3-azidopropyl ester of carbonylimi-
dazole (AP-CI, 3, Scheme 1), to PEI. H NMR analysis of the
1
obtained product (Figure 1A) shows the absence of the starting
compound AP-CI (comparing the proton NMR spectra of AP-
CI and PEI-(N3)4 in Figure 1A, no peaks at 8.12 (d) and 7.40
ppm (f) for PEI-(N3)4, the peaks at 4.50 (c), 3.47 (a), and 2.05
ppm (b) in AP-CI shifting to 4.09 (c′), 3.34 (a′), and 1.85 ppm
(b′) for PEI-(N3)4, respectively). Also, imidazole (with one
proton peak at 7.66 and two protons at 7.04 ppm) was detected
in the resulting product. The IR spectrum of the resulting product
shows an absorption peak at 2099 cm-1 corresponding to the
asymmetric stretching vibration of the azide group (see Sup-
porting Information Figure SI2). From NMR and IR analysis,
it is therefore concluded that azide-functionalized PEI has been
obtained. The average number of azide groups per PEI molecule
(x in PEI-(N3)x) was calculated on the basis of the ratio of the
integral from 2.30 to 2.90 ppm of the PEI protons to the protons
of azidopropanol at 3.34 ppm (2H, next to azide group),
assuming that the primary amine content of the PEI (1.8 kD) is
25% of the total amines (13, 27). 1H NMR analysis shows that
x in PEI-(N3)x is 4.1 when the AP-CI/PEI molar feed ratio was
Transfection experiments in 293T cells in the presence of
serum were carried out. The protocol was the same as described
above, except that the cell medium of DMEM with 10% FBS

1832 Bioconjugate Chem., Vol. 21, No. 10, 2010
Liu et al.
Figure 4. Cytotoxicity of cationic polymers PEI-SS-CLs and controls
1.8 kDa PEI and 25 kDa PEI to 293T cells after 48 h incubation (mean
( SD, n ) 4).
Table 2. IC50 Values of PEI-SS-CLs, 1.8 kDa PEI and 25 kDa PEI
polymers
PEI-SS-CL1 PEI-SS-CL2 1.8-kDa PEI 25-kDa PEI
52 47 162
IC50 (µg/mL)
6
4, which indicates that AP-CI had reacted quantitatively with
PEI. Because of the quantitative conversion, the reaction mixture
only contains PEI-(N3)x, imidazole, and solvent; no further
purification was required for the next click reaction after
evaporation removal of the solvent, chloroform.
The azide-functionalized LMW PEI-(N3)4 was cross-linked
with disulfide-containing dialkyne BPPA-Cyst by click reaction
in DMF at 50 °C for 48 h, using CuBr as catalyst, to obtain
reducible HMW PEI derivative, PEI-SS-CL. Two disulfide
cross-linked PEIs, named PEI-SS-CL1 and PEI-SS-CL2, were
obtained with molar feed ratios of alkyne/azide of 1:1 and 1:2,
respectively. Both polymers are water-soluble, which indicates
that the cross-linking reactions between dialkynes and azide
groups only take place within a definite number of LMW PEI
molecules. However, with increasing cross-linking ratio (molar
feed ratio alkyne/azide 1:0.75), microgels were formed. The
soluble polymers, PEI-SS-CL1 and PEI-SS-CL2, were further
investigated as gene vectors.
The structures of these soluble polymers were investigated
by 1H NMR and FTIR analysis. The 1H NMR spectrum of PEI-
SS-CL1 is shown in Figure 1B, from which it can be seen that
a new peak appeared at 7.8 ppm (H of the triazole ring)
indicating the formation of triazole groups by click reaction.
The absorption peaks at 2099 cm-1 and 2126 cm-1 in the IR
spectrum for polymer PEI-SS-CL1 are very weak (Supporting
Information Figure SI2), which indicates that the azide groups
reacted almost quantitatively with the alkyne groups.
Figure 5. Luciferase expression of the PEI-SS-CL/DNA polyplexes at
different N/P ratios with control 1.8 kDa PEI and 25 kDa PEI (at N/P
ratio of 10) in serum-free DMEM in 293T (A) and HeLa cells (B).
unable to condense DNA. Importantly, at N/P ratios above 10,
the two disulfide-containing PEI-SS-CLs prepared from the 1.8
kDa PEI were able to condense DNA into small particles with
size of around 200 nm and a positive zeta potential of
approximately +20 mV, similar to the 25 kDa PEI (Figure 2).
The gel retardation assay further confirmed that the two
disulfide-containing PEI-SS-CLs can bind plasmid DNA ef-
ficiently and retard DNA migration at N/P ratios above 7 (Figure
3). In agreement with DLS analysis, 1.8 kDa PEI was unable
to bind DNA even at an N/P ratio up to 50 (data not shown).
Reductive Degradation of the PEI-SS-CL. The DTT-
induced degradation of the disulfide cross-linked PEI-SS-CLs
was investigated using the agarose gel retardation assay and
SEC analysis. The SEC traces of PEI-SS-CL1 and PEI-SS-CL2
in the absence and presence of DTT (Supporting Information
Figure SI3) clearly show that the disulfide cross-linked polymers
PEI-SS-CLs were responsive to the reductive agent DTT. After
incubation with DTT for 2 h at room temperature, the weight
average molecular weight of PEI-SS-CL1 decreased from 9.4
kDa (molecular weight distribution was 3.1) to 2.1 kDa
(molecular weight distribution was 3.3), which is very close to
Mw of PEI (1.8 kDa). Agarose gel electrophoresis (Figure 3B)
shows the presence of free DNA in PEI-SS-CL/DNA polyplexes
incubated with 10 mM DTT (30 min), which indicates that in
a reductive environment PEI-SS-CL1 polyplexes released DNA.
In Vitro Cytotoxicity. The MTT assay for 293T cells was
carried out to determine the cytotoxicity of disulfide-containing
PEI-SS-CLs; 1.8 kDa PEI and 25 kDa PEI were used as controls.
Figure 4 shows that the PEI-SS-CLs had a lower cytotoxicity
than 25k Da PEI. The half inhibitory concentrations (IC50) of
The molecular weights and molecular weight distributions
of the PEI-SS-CLs were determined by SEC-MALLS (Table
1). The weight average molecular weights of PEI-SS-CL1 and
PEI-SS-CL2 were about 5 and 7 times that of the starting
1
polymer PEI 1.8 kDa. With all H NMR, FTIR, and SEC
analysis results taken together, it can be concluded that the click
reactions between BPPA-Cyst and PEI-(N3)4 were successful.
Characterization of PEI-SS-CL-Based Polyplexes. To be
effective as gene delivery vectors, cationic polymers must
possess the capacity to bind and condense plasmid DNA into
nanoparticles which can be taken up by cells. The DNA binding
and condensation properties of the starting LMW PEI (1.8 kD),
the synthesized disulfide-containing PEI-SS-CLs and the branched
PEI (25 kD), were investigated. It was shown by DLS mea-
surements that 1.8 kDa PEI, even at an N/P ratio up to 50, was

Reduction-Responsive Cross-Linked PEIs for Gene Delivery
Bioconjugate Chem., Vol. 21, No. 10, 2010 1833
Figure 6. Fluorescent and bright-field images of enhanced green fluorescent protein expression in 293T cells for PEI-SS-CL based polyplexes (N/P
ratio 20) with control 25 kDa PEI (N/P ratio 10). The images were obtained at magnification of 100×.
the polymers are summarized in Table 2 and are 52 (PEI-SS-
CL1) and 47 µg/mL (PEI-SS-CL2), about 8-10× higher than
that of 25 kDa PEI (6 µg/mL). The low cytocompatibility of
HMW PEI is probably due to its high binding and aggregation
on the cell surface and internalization, which results in
significant necrosis (13). The low cytotoxicity of disulfide cross-
linked PEI-SS-CLs indicates that, after cellular binding and
internalization, they are intercellularly degraded, triggered by
gluthatione, into relatively low-toxicity products.
In Vitro Transfection. The transfection activity of PEI-SS-
CLs based polyplexes was evaluated in 293T and HeLa cells
using the plasmid pcDNA3-Luc (expressing luciferase) and
pEGFP (expressing green fluorescence) as reporter genes.
Branched 25 kDa PEI and 1.8 kDa PEI, which were used for
preparation of PEI-SS-CLs, were used as controls.
Figure 5 shows that PEI-SS-CL polyplexes had significantly
higher transfection activity in both 293T and HeLa cells than
those based on 1.8 kDa PEI. Also, incubation of cells with the
Figure 7. Luciferase expression of PEI-SS-CL/DNA polyplexes at
different N/P ratios with control 25 kDa PEI (at N/P ratio of 10) in the
PEI-SS-CL2 polyplexes resulted in about 5-10× higher lu-
presence of 10% FBS in 293T cells.
ciferase expression in HeLa and in 293T cells, respectively, than
polyplexes based on 25 kDa PEI, although the molecular weight
of PEI-SS-CL2 is lower than that of branched PEI (25 kDa).
The reason may be that PEI-SS-CLs polyplexes are degraded
that the network poly(amino ester) (42, 43) and disulfide cross-
linked PEI (15) based polyplexes showed very high transfection
efficiency even in the presence of serum. One possible explana-
through cleavage of disulfide bonds inside the cells into nontoxic
tion is that the cross-linked polymers have outer shells with a
small PEI; DNA would be released selectively inside the cells
large proportion of the groups exposed at the surface, which
may facilitate multiple simultaneous interactions with the nucleic
aid phosphates and fairly maintain the stability of polymer/DNA
polyplexes in the presence of serum. The other possible reason
might be the introduction of reducibly cleavable disulfide bonds.
Thus, importantly, the polyplexes remain active in the presence
of serum proteins, which encourages further in vivo evaluation.
to mediate efficient gene transfection (15, 25, 27). The trans-
fection activity of the PEI-SS-CL2 polyplexes was higher than
those of PEI-SS-CL1 at all N/P ratios studied, which can
probably be ascribed to higher molecular weight of PEI-SS-
CL2 .
The outstanding transfection activity of PEI-SS-CL polyplexes
was visualized with a fluorescent microscope (Figure 6). The
cells incubated with PEI-SS-CLs and 25-kDa PEI polyplexes
showed many intracellular bright green fluorescent spots. As
shown in Figure 6, substantial more fluorescent spots were
observed using PEI-SS-CL1 and PEI-SS-CL2 polyplexes pre-
pared at an N/P ratio of 20 than those based on 25-kDa PEI,
which is in line with transfection data using the luciferase assay.
The transfection ability of the polyplexes of the disulfide
cross-linked PEI-SS-CLs with plasmid pcDNA3-Luc in the
presence of serum (10% FBS) was also investigated using 293T
cells. As shown in Figure 7, the transfection efficiencies in 293T
cells of the two PEI-SS-CL/DNA polyplex formulations were
higher than that of the 25 kDa PEI based polyplexes in DMEM
medium with 10% FBS. Especially, the PEI-SS-CL2 based
polyplexes at an N/P ratio of 40 showed about an 8-fold increase
in luciferase reporter gene expression compared to the control
25 kDa PEI polyplexes. This is in agreement with the reports
CONCLUSIONS
The disulfide-containing cross-linked polyethylenimine de-
rivatives (PEI-SS-CL1 and PEI-SS-CL2) were synthesized via
click reaction between disulfide-containing dialkyne cross-linker
BPPA-Cyst and azide-functionalized LMW PEI. The synthe-
sized polymers were characterized by 1H NMR, FTIR, and SEC.
The DNA binding and condensation properties of PEI-SS-CL
were investigated for the complexes at various N/P ratios by
DLS, zeta potential, and agarose gel retardation assay. We
showed that the disulfide-containing PEI-SS-CLs were able to
bind plasmid DNA efficiently to yield small polyplexes. The
degradation of the PEI-SS-CLs in the presence of DTT was
confirmed by gel retardation assay and SEC analysis. In vitro
experiments demonstrated that the reducible PEI-SS-CLs not

1834 Bioconjugate Chem., Vol. 21, No. 10, 2010
Liu et al.
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only had much lower cytotoxicity, but also posed superior
transfection activity (in both the presence and absence of serum)
as compared to the control nondegradable 25 kDa PEI. Our
results imply that the obtained disulfide cross-linked PEI-SS-
CLs might be suitable for further in vivo gene transfection.
Future work will focus on coupling of poly(ethylene glycol)
(PEG) shielding, targeting ligands and fluorescent groups in the
polyplexes for further in vivo evaluation.
ACKNOWLEDGMENT
(17) Jere, D., Arote, R., Jiang, H. L., Kim, Y. K., Cho, M. H., and
Cho, C. S. (2009) Bioreducible polymers for efficient gene and
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This research was financially supported by the National
Natural Science Foundation of China (20774068), the Ph.D.
Programs Foundation of Ministry of Education of China
(20070486035), and the National Key Basic Research Program
of China (2005CB623903, 2009CB930300).
Supporting Information Available: Supplementary figures.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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