A Serum-Tolerant Hydroxyl-Modified Polyethylenimine as Versatile Carriers of pDNA/siRNA

Article abstract of DOI:10.1002/mabi.201200346

The polyethylenimine (PEI) derivatives (PTn) are prepared by treating PEI25k with Tris(hydroxymethyl) acrylamidomethane via the Michael addition. These PTns can effectively condense nucleic acids into nanosized particles with positive surface charges. The PTns show lower cytotoxicity and better serum-resistant capacity than PEI25k. Specially, the transfection efficiency of PT26/DNA is 29-fold higher than that of PEI25k in HeLa cells in serum-containing medium. The PTn/siRNA complexes show superior knockdown effect in CT26 cells in serum-containing medium. In addition, flow cytometry analysis shows that the PTns can efficiently mediate the entry of nucleic acids into the cell. Thus, PTns are potentially applicable as non-viral carriers of nucleic acids and warrant further development for use in gene therapy. A series of serum-tolerant PTn conjugates, by introduction of hydroxyl groups onto PEI25k, is designed as dual carriers for nucleic acids delivery. The transfection efficiency of PTn can be up to 29.1-fold higher than that of PEI25k in the serum-containing medium. Meanwhile, PTn with higher endocytosis is more effective in luciferase knockdown compared to PEI25k in the serum-containing medium.

Full text of DOI:10.1002/mabi.201200346

Full Paper
A Serum-Tolerant Hydroxyl-Modified
Polyethylenimine as Versatile Carriers of
pDNA/siRNA
Xuan Dong, Lin Lin, Jie Chen, Zhaopei Guo, Huayu Tian,* Yuce Li, Yen Wei,*
Xuesi Chen
The polyethylenimine (PEI) derivatives (PTn) are prepared by treating PEI25k with Tris-
(hydroxymethyl) acrylamidomethane via the Michael addition. These PTns can effectively
condense nucleic acids into nanosized particles with positive surface charges. The PTns show
lower cytotoxicity and better serum-resistant capacity
than PEI25k. Specially, the transfection efficiency of
PT26/DNA is 29-fold higher than that of PEI25k in HeLa
cells in serum-containing medium. The PTn/siRNA com-
plexes show superior knockdown effect in CT26 cells in
serum-containing medium. In addition, flow cytometry
analysis shows that the PTns can efficiently mediate the
entry of nucleic acids into the cell. Thus, PTns are
potentially applicable as non-viral carriers of nucleic
acids and warrant further development for use in gene
therapy.
1. Introduction
much attention as a promising strategy for therapeutic
intervention of various human diseases.[4,5] Despite high
promise, safeandefficientdeliveryofnucleicacidsintocells
is still a bottleneck for the successful gene therapy or gene
knockdown.[6,7] Thus, there is an unmet need to develop
carriers, which can protect the therapeutic genes and
transport them into targeted sites where they can exert
theirfunctionswhileretainingtheirtransfectionefficiency.
Although viral vectors are shown to be highly efficient in
gene delivery, their clinical application is limited by safety
concerns such as virus replication, inflammatory response,
and immunogenicity risks.[8,9] Linear or branched poly-
ethylenimine (PEI) has generally been accepted as a non-
viral cationic gene carrier for DNA and siRNA delivery over
the past decade.[9–11] PEI25k, a high molecular weight PEI
(25 kDa) typically shows high transgene expression both
in vitro and in vivo; however, low biocompatibility and
limited stability in the presence of serum have hindered
their clinical applications.[12,13]
Gene therapy holds promise for treating inherited and
acquired diseases or genetic disorders.[1–3] In the past
decade, developing the therapeutic plasmid DNA (pDNA)
and RNA interference (RNAi) technology has attracted
X. Dong, L. Lin, J. Chen, Z. Guo, H. Y. Tian, Y. C. Li, X. S. Chen
Key Laboratory of Polymer Ecomaterials, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun
130022, P. R. China
E-mail: thy@ciac.jl.cn
X. Dong, Y. C. Li
Graduate School of Chinese Academy of Sciences, Beijing 100039,
P. R. China
Y. Wei
Department of Chemistry, Tsinghua University, Beijing 100084,
P. R. China
E-mail: weiyen@tsinghua.edu.cn
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DOI: 10.1002/mabi.201200346

A Serum-Tolerant Hydroxyl-Modified Polyethylenimine as Versatile Carriers . . .
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Recently, tremendous research efforts have been
invested in overcoming the undesirable properties of
PEI-based carriers.[14–19] Our previous work showed PEI
modified with hydrophobic amino acid segments led to
enhanced transfection efficiency and biocompatibility.[14]
Wagner’s team showed that the introduction of ethyl
acrylate or acetyl functionality, or the introduction of a
negatively charged acid to PEI improved siRNA-mediated
gene silencing efficacy.[15] Bertrand’s work showed that
hisdinylated linear PEI was a new efficient non-toxic
polymer for efficient gene delivery with improved transfec-
tion efficacy and biocompatibility.[16] The cytotoxicity was
reduced to some degree by all the above-metioned
modification. In addition, PEI modified with poly(ethylene
glycol) (PEG) has been shown to shield the positive charges
on complex effectively for improving the serum toler-
ance.[17,18,20,21] Similarly, these studies found that the
introduction of PEG significantly reduced the transfection
efficacy and siRNA silencing effects.
tration, PTn conjugates showed superior siRNA silencing
effects in comparison with PEI25k.
2. Materials and Methods
2.1. Materials
Branched PEI25k, acryloyl choride, Trinitrobenzenesulfonic
acid (TNBS), and Tris(hydroxymethyl) aminomethane were
purchased from Aldrich (St. Louis, MO, USA). N,N-dimethyl-
formamide (DMF) was treated with CaH2 and distilled
before use. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) was supplied by Amresco (Solon,
Ohio, USA). TheDulbecco’s modified Eaglemedium (DMEM)
and fetal bovine serum (FBS) were purchased from
Gibco (Grand Island, USA). The plasmid encoding firefly
luciferase (pGL3), cell lysis buffer and luciferin substrate
were purchased from Promega (Madison, WI, USA). Cell
culture flasks were obtained from Wuxi NEST Biotechno-
logy. Co., Ltd. (Shanghai, China). Control siRNA that
targets the sequence 50-GTCGAACGTCGTGAACCTAC-
CATG-30, GL3-siRNA that targets the sequence 50-CTTACG-
CTGAGTACTTCGA-30 of luciferase GL3 plasmid and were
purchased from GenePharma Co. Ltd. (Shanghai, China).
The control sequence does not match any human genome
sequence. Other materials and solvents were used as
received without further purification.
In the current study, PEI derivatives, denoted as PEI-g-
THAn (PTn, n equals to the average Tris(hydroxymethyl)
acrylamidomethane (THA) units per PEI molecule), were
prepared by modifying PEI25k with THA via the Michael
addition (Scheme 1). The introduction of hydroxyl groups
on PEI surface can markedly improve serum-tolerant
capacity[13] and reduce cytotoxicity of carriers for nucleic
acids delivery. The physiochemical properties of PTn/
nucleic acids complexes were studied by measurement
of the particle size and zeta potential, as well as DNA
condensing capacity. The in vitro transfection efficiencies
were also investigated, which showed that the increase of
transgene efficiency mediated by PTn conjugates was
directly correlated to the improved serum-tolerant capa-
city. Furthermore, when used for siRNA-mediated gene
silencing under the condition of increasing serum concen-
2.2. Synthesis of Conjugates
2.2.1. Synthesis of Tris(hydroxymethyl)
Acrylamidomethane (THA)
THA was synthesized according to Chabre’s method.[22]
Acryloyl choride (1.20 mL, 14.8 mmol) was added to a
Scheme 1. Synthetic procedures of the PEI-g-THAn (PTn) conjugates.
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the amounts of grafted THA on primary amines were
calculated by the difference in the amounts of primary
amines between PEI25k and the derivatives. For purpose of
this calculation, it is naturally assumed the polydispersity
ofPEI25kwas1;thebranchedPEIshouldcontainanaverage
of25%primary(18),50%secondary(28),and25%tertiary(38)
amines.[24] The amounts of primary amines in the PEI25k
derivatives are equal to that in the equivalent PEI25k
to obtain the following equation:
a ꢃ ½ð25000=43Þð1=4Þ ꢂ xꢄ b ꢃ ½ð25000=43Þð1=4Þꢄ
¼
ð25 000 þ mnÞ
25 000
where a is the mass of PEI25k derivatives, x is the amount
of reacted primary amines in the PEI25k derivative, m is
the molecular weight of THA, n is the number of THA in
the PEI25k derivatives calculated from 1H NMR, b is the
equivalent mass of PEI25k obtained by interpolation of the
standard curve, 25 000 is the molecular weight of PEI25k,
43 is the average molecular weight of PEI monomer, and
1/4 is the fraction of primary amines in the PEI.
Figure 1. The 1H NMR spectra of (A) THA in DMSO-d6, and (B) PEI-g-
THA46 (PT46, Table 1, Entry 2) in D2O.
solution of Tris(hydroxymethyl) aminomethane (1.00 g,
8.2 mmol) in methanolic potassium hydroxide 3N and
stirred vigorously at 0 8C (pH 11 ꢀ 12) for 1 h. Next, the
reaction mixture was stirred for 4 h at room temperature.
After reaction, the solution was filtered and the filtrate
evaporated in vacuum to dryness. THA was obtained as a
whitepowder afterprecipitation and recrystallization from
methanol (yield: 1.20 g, 83%). 1H NMR (400 MHz, DMSO-d6):
d 7.44 (s, 1H), d 6.38 (dd, 1H), d 6.07 (dd, 1H), d 5.56 (dd, 1H),
d 4.76 (t, 3H), d 3.58 (d, 6H) (Figure 1A).
2.5. Gel Retardation Assay
Ten microliters polymer/DNA (0.2 mg pGL3) complex
solution at varying polymer/pDNA (mass ratio, w/w) ratios
was prepared for gel retardation assay. After 20 min
incubation at room temperature, complex solutions were
electrophoresed on 1% agarose gel in TAE buffer solution
(100 V, 30 min). DNA bands were visualized under a UV
illuminator using a UVP EC3 bioimaging system (Upland
CA, USA). For siRNA, the assay was carried out under the
same condition except for the voltage of 60 V.
2.2.2. Modification of PEI25k by THA
The PEI25k was dissolved in anhydrous DMF (1 g ꢁ 10 mLꢂ1),
and then the desired amount of THA based on molar ratios
was added. The reaction mixture was stirred for 24 h at
50 8C, after which the solution was dialyzed against
distilled water. The final products were collected after
lyophilization. Three derivatives with average 26, 46, 57
THA units per PEImolecule were prepared by varying molar
feed ratios of THA to PEI (40:1, 60:1, and 80:1, respectively),
and the corresponding yield of each product was 85, 82, and
88% for PT26, PT46, and PT57, respectively.
2.6. Particle Size and Zeta Potential Measurements
The cationic polymers and nucleic acids (pDNA or siRNA)
complexes were prepared by diluting appropriate amounts
ofacationiccomponentandnucleic acidsinaPBS(pH ¼ 7.4)
buffer solution. A cationic component was added to the
nucleic acids at varying mass ratios, with a final complex
concentration of 0.1 mg ꢁ mLꢂ1. Then the solution was
incubated for 30 min at room temperature. The particle
size and zeta-potential of the complexes were measured
at 25 8C under a Zeta Potential/BI-90Plus Particle Size
Analyzer (Brookhaven, USA). The data are expressed as
mean value ꢅ SD of three experiments.
2.3. Characterization
1H nuclear magnetic resonance (1H NMR) spectra in D2O or
DMSO-d6 were recorded on a Bruker AV 400-MHz instru-
ment at room temperature. The degree of substitution was
defined by the 1H NMR.
2.7. Cell Culture
HEK293, HeLa, and CT26 (murine colorectal adenocarci-
noma cells stably transfected with luciferase gene) cells
were cultured in the DMEM medium supplemented
with high glucose and 10% v/v FBS and 1% penicillin-
streptomycin, at 37 8C under 5% CO2.
2.4. Determination of Primary Amine Content
The amounts of primary amines in each of PEI25k
derivatives were determined by the TNBS assay[23] and
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2.8. Cytotoxicity Evaluation
lysate buffer (Promega; 50 mL/well), 20 mL of cell lysate was
mixed with 100 mL of luciferin substrate (Promega). The
light emission was measured by a luminometer (GloMax
20/20, Promega) and the protein content was determined
by using BCA protein assay kit (Pierce). Luciferase activity
was expressed as RLU ꢁ mgꢂ1 protein. The relative luciferase
activity was compared with untreated control cells. Each
experiment was done in triplicate.
HEK293, HeLa, and CT26 cells were plated in 96-well plates
at 1.0 ꢃ 104 cells/well for 24 h as described above,
respectively. The PEI25k and PEI-g-THAn of varying con-
centrations were prepared in the PBS (pH ¼ 7.4) solution. A
polymer or the PEI solution of 20 ml was added to each well
and the cells were cultured for another 24 h. Twenty
microliters of stock solution MTT (5 mg ꢁ mLꢂ1 in PBS) was
then added to each well. After 4 h of incubation, unreacted
dye was carefully removed, and the formazan crystals were
dissolved by DMSO (200 mL/well). The absorbance at
492 nm was measured by an ELISA microplate reader
(Bio-Rad). The cell viability (%) was calculated as Cell
viability (%) ¼ (Asample/Acontrol) ꢃ 100, where Asample is the
absorbance of the cells treated by polymers and Acontrol is
the absorbance of the untreated cells. Each experiment was
done in triplicate. For the complexes formed by carriers and
nucleic acids (pDNA or siRNA) at their optimal transfection
mass ratios, the assay was carried out under the same
condition except for the cultured time of 44 h.
2.11. Protein Adsorption Assay
In brief, 0.5 mL of polymer solution (1 mg ꢁ mLꢂ1) was mixed
with 0.5 mL BSA solution (2 mg ꢁ mLꢂ1). The mixture
solutionwasshakenat37 8Cfor30 minandthesupernatant
was carefully collected after centrifugation. The concentra-
tion of BSA in the supernatant was calculated from the
UV absorbance at 280 nm, using a previously established
calibration curve. The protein adsorbed on the polyplexes
was calculated as: q ¼ (Ci ꢂ Cs)V ꢁ mꢂ1. where Ci is the initial
BSA concentration and Cs is the BSA concentration in the
supernatant after adsorption experiments; V is the total
volume of the mixture solution (1 mL); and m is the weight
of the polymer (0.5 mg) in the mixture solution.
2.9. In Vitro Transfection
HEK293 and HeLa cells were plated in 96-well plates at
1.0 ꢃ 104 cells/well prior to tansfection, and then the
medium was replaced with a 200 mL of the serum-free or a
10% serum-containing culture medium. Polymer/pGL3
(0.2 mg pDNA) complexes with varying mass ratios were
added to each well, and then the cells were incubated for 4 h
at 37 8C. Then the medium was replaced with a 10% serum-
containing medium and incubated for 44 h at 37 8C. The
medium was then discarded and the cells were washed
three times with PBS. After thorough lysis of the cells with
cell lysate buffer (Promega; 50 mL/well), the luciferase
gene expression was quantified using luciferase reporter
gene assay kit and photon counting with a luminometer
(GloMax 20/20; Promega), by adding 100 mL of luciferin to
20 mL of cell lysate in each well. The protein content was
determinedbyusingBCAproteinassaykit(Pierce, Rockford,
IL, USA). Each experiment was done in triplicate.
2.12. Cellular Uptake
For the in vitro cellular uptake, the polymer/Cy3-labeled
pGL3 pDNA (Cy3-DNA) and polymer/FAM-labeled siRNA
(FAM-siRNA) complexes were investigated by flow cyto-
metry. HeLa and CT26 cells were plated in six-well plates at
2.0 ꢃ 105 cells/well with 2 mL of DMEM containing 10% FBS
for24 h,respectively. Themediumwasreplacedwith2.0 mL
of the fresh 10% serum-containing culture medium
containing the complexes of pGL3 or siRNA. After incuba-
tion for 4 h at 37 8C, the harvested cells were washed with
PBS, detached with 0.25% trypsin and then suspended with
500 mL PBS (pH 7.4). Finally, the cellular uptake efficiency
was determined using a flow cytometer (FACS Caliber,
Becton-Dickinson, San Jose, CA, USA).
3. Results and Discussion
2.10. In Vitro Gene Silencing
3.1. Synthesis and Characterization of the PEI-g-THAn
To study luciferase knockdown efficiency of the carriers,
CT26 cells were seeded onto a 96-well plate at 1.0 ꢃ 104
cells/well prior to transfection. At the time of transfection,
themediumwasreplacedwith 200mLoftheserum-freeora
10% serum-containing culture medium containing poly-
mer/siRNA(0.2mgsiRNAand20mLsterilewater)complexes
at varying mass ratios. After incubation for 4 h at 37 8C, the
mediumwasreplacedwitha10%serum-containingculture
medium for 44 h incubation. The cells were washed three
times with PBS. After thorough lysis of the cells by a cell
Conjugates
Scheme 1 presents the synthetic procedure of the cationic
PEI-g-THAn (PTn) conjugates. Three derivatives (PT26, PT46,
and PT57) were obtained by the Michael addition, which
was straightforward without any strict conditions, cata-
lysts or activator reagents such as ethylcarbodiimidehy-
drochloride, carbodicyclohexylimide, and N-hydroxysucci-
nimide. As a result, the primary and secondary amine
functionalities of PEI25k decreased while threefolds of
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Table 1. Molecular characteristics of the PEI-g-THAn conjugates.
a)
b)
b)
Entry
PEI conjugates
NTHA
NTHA
The number
of reacted 1-
aminesc)
The number
of reacted 2-
amines
Mn
(design)
(1H NMR)
1
2
3
PEI-g-THA26 (PT26)
PEI-g-THA46 (PT46)
PEI-g-THA57 (PT57)
40
60
80
26
46
57
1.2
4.4
7.5
24.8
41.6
49.5
29 600
33 100
35 100
a)Designed number of THA moieties per PEI molecule. b)As determined by 1H NMR spectra. c)As determined by the TNBS assay.
hydroxyl groups were attached on PEI surface. The
structures of THA and PT46 were confirmed by the
1H NMR spectra (Figure 1). In Figure 1B, the broad peak
(2.48–3.42 ppm, ꢂCH2CH2NHꢂ) was characteristic of
PEI25k. Peaks a (3.72 ppm, ꢂCH2ꢂ) belonged to THA
moieties. The average THA units per PEI molecule (n) were
calculated from the integral values of signals at d 2.48–3.42
andd3.72. Table1summarizesPTnconjugateswithaverage
26, 46, 57 THA units per PEI molecule which were obtained
at THA/PEI mole feed ratios of 40/1, 60/1, and 80/1,
respectively. The detailed compositions and molecular
weights of the PTn conjugates are summarized in Table 1.
reason for the lower carrier content for mass ratio pDNA
than siRNA is likely due to that pDNA (ꢀ4.1 kbp) is
inherentlymuchlargerinsizethansiRNA(ꢀ21bp), andalso
thattheformerismoreflexibleandadaptableininteracting
with polycations.[6]
3.2.2. Particle Size Analysis
It is necessary for the polycation to compact nucleic acids
(pDNA/siRNA) into nanosized particles, because cells
typically take up particles ranging from micrometers to
nanometers.[25] The dynamic light scattering is used to
measure the diameters of complexes formed by the PTns
and pDNA or siRNA compared with those of PEI25k at
varying transfection mass ratios. As shown in Figure 3A,
PT26, PT46, PT57, and PEI25k could efficiently condense
3.2. Characterization of PTn/DNA Complexes
3.2.1. Gel Retardation Assay
The ability to condense pDNA and siRNA
is a fundamental and also critical require-
ment for polymeric gene carriers. The
pDNA and siRNA binding affinity of the
PTn conjugates were determined by gel
retardation assay at varying mass ratios,
using PEI25k as the positive control. The
Figure 2 presents the complete DNA
retardation induced by PT26, PT46, and
PT57 required the critical mass ratios 0.4,
0.5,and0.5,respectively, whereasthatfor
PEI25k was lower (0.2). With the increas-
ing degree of substitution, the binding
affinity of the PTn conjugates showed a
slight decline due to the partly shielded
surface positive charge after modifica-
tion. Generally, for any cationic poly-
mers, the complete siRNA retardation
requires a higher critical mass ratio
compared to that for DNA. In this study,
the carrier/siRNA mass ratios of PT26,
PT46 and PT57 for complete siRNA
retardation were as high as 0.8, 1.0, and
Figure 2. Gel retardation assay for DNA in the complexes formed by (A) PEI25k, (C) PT26,
(E) PT46 and (G) PT57 at varying mass ratios. Gel retardation assay of siRNA complexed
by (B) PEI25k, (D) PT26, (F) PT46, and (H) PT57 at various mass ratios.
1.0, respectively. EventhatforPEI25kwas
a relatively high mass ratio of 0.3. The
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Figure 3. Particle size (A) and zeta potential (B) of complexes formed by PEI-g-THAn conjugates and pDNA or siRNA in comparison with
PEI25k at various transfection mass ratios. The data are expressed as mean value ꢅ SD of three experiments.
pDNA or siRNA, at complex sizes ranging from 100 to
140 nm. Thesizesofcomplexesdeclinedwiththeincreasing
mass ratios. It is usually larger for polymer/siRNA
complexes due to the formation of loose and unstable
complex structures.[26] Our data show that the PTn
conjugates possessed the same abilities for compacting
nucleic acids to nanosized particles as those of PEI25k.
3.2.3. Zeta Potentials
Zeta potentials also play an important role in endocytosis
and material cytotoxicity. The positive surface charge of
nanoparticles formed by polymers and nucleic acids is
closely connected to the complex particle internalization
for the negative charged cell membrane.[27] Figure 3B
Figure 4. Viability of HEK293 (A), HeLa (B) and CT26 (C) cells exposed to various polymer concentrations for PT26, PT46, PT57 and PEI25k. The
apparent IC50 for the polymers are statistically different in all cell lines. Data represent mean ꢅ standard deviation (n ¼ 3), ꢆp < 0.01.
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presents the zeta potentials of the complexes formed by
polymers and pDNA or siRNA at varying transfection mass
ratios. With an increasing substitution degree, the zeta
potentials of the complexes declined, which is similar to
the former results. The charge of complexes gradually
decreased and eventually stabilized ranging from þ15 to
20 mV. The zeta potentials of nanoparticles formed by PTn
conjugates and pDNA or siRNA were lower than those
formed by PEI25k. The above results are in agreement
with the structures of PTn conjugates; the lower zeta
potentials of PTn conjugates are directly correlative to the
partly shielded surface positive charge after hydroxyl-
modification.[28]
decline in the order PT57 < PT46 < PT26. Generally, the
cytotoxicity of the PEI-derived gene vectors has a strong
correlation with surface charge density.[29] The above
results can be attributed to the decreased concentration of
primary and secondary amine groups (Table 1) thus the
decreased surface positive charge density after hydroxyl-
modification.
Figure 5 shows the cell viability of the complexes formed
by the cationic polymers and nucleic acids (pDNA and
siRNA) at their optimal transfection mass ratios. The
complexes formed by PTn conjugates and nucleic acids
exhibited high cell viability and reduced toxicity compared
with those formed by PEI25k.
3.3. In Vitro Toxicity
3.4. Transfection Activity
As shown in Figure 4, the in vitro cytotoxicities of PTn
conjugates and PEI25k were assessed by MTT assay at
varying concentrationsin the HEK293, HeLa, and CT26cells.
The IC50 values for PTn conjugates are much higher than
that of PEI25k indicating that all the PTn conjugates
exhibited lower cytotoxicity in comparison with PEI25k in
these cell lines. As the degree of substitution increased,
the cytotoxicity of the PTn conjugates showed a slight
The serum-free and serum-containing transfection effi-
ciency of the PTn conjugates and PEI25k was determined by
luciferase assay in HEK293 and HeLa cells, using the pDNA
(pGL3) as the marker gene. Figure 6 shows the gene
transfection efficiency mediated by the cationic PTn
conjugates and PEI25k for DNA delivery in the serum-free
medium. At the optimal carrier/DNA mass ratio, the
transfection efficiency of PT26, PT46, and PT57 in both cell
Figure 5. Viability of HEK293 (A), HeLa (B) and CT26 (C) cells exposed to optimal transfection mass ratios of carriers to nucleic acids(pDNA or
siRNA).
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Figure 6. In vitro gene transfection efficiency of the complexes of PTn/pGL3 in comparison with that of PEI25k in HeLa (A) and HEK293
(B) cells in the serum-free medium. Data represent mean ꢅ standard deviation (n ¼ 3).
lines was much higher than that of PEI25k. The transfection
efficiency increased in both the cell lines in the order of
PT26 > PT46 > PT57 > PEI25k as the degree of substitution
decreased. The optimal mass ratio of PT57 (20:1) in HEK293
cells was higher than that of PT26 (15:1) and PT46 (15:1) due
to the lack of primary and secondary amine groups and
decreased charge density via surface modification; this led
to increase DNA-binding ability efficiently. In HeLa cells,
transfectionefficienciesofPT26(15:1),PT46(20:1),andPT57
(20:1) at the optimal mass ratio were 2.4-, 2.1-, and 2.1-fold
higher than that of PEI25k (1:1), respectively (Figure 5B).
The ability of gene carriers to induce gene transfection
under serum-containing condition can serve as an essential
predictive parameter prior to in vivo efficiency evalua-
tion.[13,30] This parameter was assessed with polymer/DNA
polyplexes at the optimal transfection mass ratios (For
PEI25k, PT26, PT46, and PT57 polyplexes, the mass ratio was
1, 15, 15, and 20 in HEK293 cells and was 1, 15, 20, and 20 in
HeLa cells). As shown in Figure 7, the transfection efficiency
of PTn conjugates in the serum-containing medium was
much higher than that in the serum-free medium in both
cell lines, except for PEI25k, demonstrating that introduc-
tion of hydroxyl groups improved the serum tolerance of
the transfection system. PT26 effected higher expression
(sevenfolds) than PEI25k in the serum-containing condition
in HEK293 cells and 29 folds better in HeLa cells. These
results suggest that the PTn conjugates possess better
characteristics of serum tolerance as a result of surface
hydroxyl-modification.
3.5. In Vitro siRNA Gene Silencing Effect
For safe and effective siRNA-based therapy, a major hurdle
is the design and evaluation of better delivery vehicles.[7]
Many efficient carriers for pDNA are usually not effective
for siRNA delivery.[31] In this study, PTn conjugates and
Figure 7. Comparison between the serum-free and serum-conditioning medium in HeLa (A) and HEK293 (B) cells meditated by PTn/pGL3
complexes at the optimal mass ratios as detected in the serum-free transfection. Transfection mediated with PEI25k at mass ratio of 1:1 was
used as a control. For PT26 PT46and PT57 complexes, the mass ratio was 15, 15, and 20 in HEK293 cells while that was 15, 20, and 20 in HeLa
cells, respectively. Data represent mean ꢅ standard deviation (n ¼ 3).
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Figure 8. Luciferase gene silencing by the complexes of PTn/siRNA in comparison with that of siRNA and PEI25k in the serum-free (A) and the
serum-containing (B) medium at CT26 cells. Data represent mean ꢅ SD (n ¼ 3).
PEI25k were evaluated for their siRNA-mediated knock-
downefficienciesinCT26cellsintheserum-freeandserum-
containing media. As shown in Figure 8, gene silencing
activities were determined with cells treated with either
siLuc or a control Rev siRNA, and compared with the
luciferase activity of untreated control cells. If luciferase
expressiondecreased with bothtargeted andcontrolsiRNA,
‘‘knockdown’’ was mainly due to unspecific carrier toxicity.
The increasing serum concentration (0ꢀ10%) in medium
would reduce luciferase knockdown efficiency for PEI25k/
siRNA except for PTn/siRNA. At the same time, the
increasing degree of hydroxyl substitution could inhibit
luciferase knockdown efficiency PTn/siRNA at the optimal
mass ratio, due to the higher cationic density in the
complexes that is beneficial to intracellular release of
siRNA.[32] The optimal gene silencing efficiencies for
PEI25k/siRNA, PT26/siRNA, PT46/siRNA, and PT57/siRNA
are 82, 70, 54, and 51%, respectively in the serum-free
medium and are 17, 81, 60, and 50% in the serum-
containing medium. Clearly the efficiency of polycation-
mediated transfecion is affected by the presence of serum.
The hydroxyl introduction improved the serumtolerance of
PTn/siRNA comparedwith PEI25k/siRNA. The above results
indicate that the PTn conjugates could be used as versatile
carriers of nucleic acids (pDNA/siRNA) with the serum-
tolerant characteristic.
reasonable to assume that the introduction of hydroxyl
groups on PEI surface improved the serum tolerance by
protecting the polyplexes from the negative protein
interactions. Furthermore, thetolerance againstthe protein
adsorption may assist the nucleic acid polyplexes onto cell
membrane to uplift the endocytosis and gene expres-
sion.[35,36] To demonstrate this, BSA was used as a model
protein to simulate process of the electrostatic adsorption.
Figure 9 shows that PEI25k has the highest protein
adsorption. Since the main driving force for protein
adsorption is the electrostatic attraction between the
positively charged polymer and the negatively charged
BSA, protein adsorption onto polymer surface is correlative
to the charge density changes caused by the THA coating.
The tolerance efficiency of the varying THA substitution
degree seemed to be similar but far higher than that of
PEI25k. The results indicate that introduction of hydroxyl
groups contributes to the tolerance toward protein
adsorption beyond the effect of the decreased charge
density.
3.6. Protein BSA Adsorption
The efficiency of polycation-mediated transfection is
affected by the presence of serum.[33] As reported in earlier
studies, addition of polyanions such as hyaluronate could
provide a coating and serve to block the undesirable
interactions between polyplexes and serum proteins,
thereby improve the gene transfection efficiency of
PEI/DNA polyplexes in the serum.[12,34]
Figure 9. Comparison of protein adsorption between PTn and
PEI25k. 1 mL of polymer solution (1 mg ꢁ mLꢂ1) was added to 1 mL
BSA solution (2 mg ꢁ mLꢂ1) and incubated with shaking at 37 8C
for 30 min prior to measurement.
With the observation that PTns resulted in high
transfection activity in serum-containing medium, it is
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A Serum-Tolerant Hydroxyl-Modified Polyethylenimine as Versatile Carriers . . .
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The results of flow cytometric analysis further confirmed
that the PTn conjugates could efficiently mediate entry of
nucleic acids into cells. In conclusion, the serum-tolerant
PTn conjugates may serve as a promising candidate as
transfection reagents for future gene therapy.
Acknowledgements: We wish to thank the National Natural
Science Foundation of China (Grant Nos. 21074129, 50903009,
51021003, 51222307), the Ministry of Science and Technology of
China (International cooperation and communication program
2010DFB50890 and 2011RI0001), as well as Jilin province science
and technology development program (201000115, 20120306) for
financial support of this work.
Figure 10. Cellular uptake analysis by flow cytometry in HeLa (A)
and CT26 (B) cells, PBS, 1; PEI25k, 2; PT26, 3; PT46, 4; PT57, 5.
3.7. Intracellular Uptake of pDNA and siRNA
Complexes
Received: September 21, 2012; Revised: November 29, 2012;
Published online: January 8, 2013; DOI: 10.1002/mabi.201200346
Internalization efficiency of our carriers was monitored by
flow cytometric analysis in HeLa and CT26 cells at optimal
transfection mass ratios, using the Cy3-DNA and FAM-
siRNA. For DNA, fluorescent intensity of the PT57/DNA was
similar to that of PEI25k (Figure 10A). The internalization
efficiencies for PT26/DNA (92.6%) and PT46/DNA (91.9%)
showed significantly higher than that of PEI25k (77.2%) in
HeLacells,whereasthatforPBS(0.4%)waslower. Figure10B
shows the fluorescent peaks for the cells treated with PT26/
siRNA and PT46/siRNA are noticeably shifted to the right
due to lower levels of hydroxyl-modification. The inter-
nalization efficiencies for PT26/siRNA, PT46/siRNA, and
PT57/siRNA in CT26 cells were 98.7, 94.7, and 82.4%,
respectively. Incontrast, thecontrolexperimentsofPBSand
PEI25k showed lower internalization, at 0.3 and 68.9%,
respectively. Overall, our results suggest that the PTn
conjugates could efficiently mediate entry of nucleic acids
into cells in the serum-containing medium. All the results
indicate that PTn conjugates with serum-tolerant proper-
ties exhibited higher transportation capability than the
unmodified PEI25k.
Keywords: DNA delivery; hydroxyl-modified polyethylenimine;
non-viral carrier; serum-tolerant; siRNA delivery
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