Biodegradable cross-linked poly(amino alcohol esters) based on LMW PEI for gene delivery

Article abstract of DOI:10.1039/c0mb00339e

Polyethylenimine (PEI, especially with M_w of 25000) has been known as an efficient gene carrier and a gold standard of gene transfection due to its high transfection efficiency (TE). However, high concomitant cytotoxicity limited the application of PEI. In this report, several cationic polymers derived from low molecular weight (LMW) PEI (M_w 600) linked with diglycidyl adipate (DA-PEI) or its analogs (diglycidyl succinate, DS-PEI and diglycidyl oxalate, DO-PEI; D-PEIs for all 3 polymers) were prepared and characterized. GPC gave M_ws of DA-PEI, DS-PEI and DO-PEI as 6861, 16015 and 35281, respectively. Moreover, degradation of the ester-containing DS-PEI was also confirmed by GPC. In addition, hydroxyls in these polymers could improve their water solubility. These polymers exhibited good ability to condense plasmid DNA into nanoparticles with the size of 120-250 nm. ζ-potentials of the polyplexes were found to be around +10-20 mV under weight ratios (polymer/DNA) from 0.5 to 32. Agarose gel retardation showed that DNA could be released from the polyplexes after being pre-incubated for 30 h. In vitro experiments were carried out and it was found that DS-PEI showed about 5 times of TE compared to that of the PEI/DNA polyplex under a weight ratio of 1 in A549 cells. Meanwhile, the cytotoxicity of D-PEIs assayed by MTT is lower than that of 25 kDa PEI in HEK293 cells. These results suggested that this series of PEI derivatives would be promising non-viral biodegradable vectors for gene delivery. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0mb00339e

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Cite this: Mol. BioSyst., 2011, 7, 1254–1262
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PAPER
Biodegradable cross-linked poly(amino alcohol esters) based on
LMW PEI for gene deliveryw
Shuo Li,z Yu Wang,z Ji Zhang,* Wei-Han Yang, Zhen-Hua Dai, Wen Zhu* and
Xiao-Qi Yu*
Received 18th December 2010, Accepted 12th January 2011
DOI: 10.1039/c0mb00339e
w
Polyethylenimine (PEI, especially with M of 25 000) has been known as an efficient gene carrier
and a gold standard of gene transfection due to its high transfection efficiency (TE). However,
high concomitant cytotoxicity limited the application of PEI. In this report, several cationic
polymers derived from low molecular weight (LMW) PEI (Mw 600) linked with diglycidyl adipate
(
DA–PEI) or its analogs (diglycidyl succinate, DS–PEI and diglycidyl oxalate, DO–PEI; D–PEIs
for all 3 polymers) were prepared and characterized. GPC gave M s of DA–PEI, DS–PEI and
w
DO–PEI as 6861, 16 015 and 35 281, respectively. Moreover, degradation of the ester-containing
DS–PEI was also confirmed by GPC. In addition, hydroxyls in these polymers could improve
their water solubility. These polymers exhibited good ability to condense plasmid DNA into
nanoparticles with the size of 120–250 nm. z-potentials of the polyplexes were found to be around
+
10–20 mV under weight ratios (polymer/DNA) from 0.5 to 32. Agarose gel retardation showed
that DNA could be released from the polyplexes after being pre-incubated for 30 h. In vitro
experiments were carried out and it was found that DS–PEI showed about 5 times of TE
compared to that of the PEI/DNA polyplex under a weight ratio of 1 in A549 cells. Meanwhile,
the cytotoxicity of D–PEIs assayed by MTT is lower than that of 25 kDa PEI in HEK293 cells.
These results suggested that this series of PEI derivatives would be promising non-viral
biodegradable vectors for gene delivery.
high TE. Therefore, 25 kDa PEI has been considered as the
Introduction
gold standard of gene transfection. However, the high cyto-
toxicity partly caused by its high molecular weight and
charge density seriously hampered its therapeutic use. On the
The success of gene therapy largely depends on the availability
of delivery vehicles. Although viral vectors display rather good
transfection properties both in vitro and in vivo, there are a
large number of problems associated with the use of these
other hand, low molecular weight (LMW) PEI (M
w
o 2000),
by contrast, has been demonstrated to have a relatively low
cytotoxicity. But it also cannot be used as gene vectors
due to rather unsatisfying TE. To overcome this inconsistency,
the use of biodegradable polymers, which can break down
into low molecular weight (LMW) segments, is a logical
choice. Several functional groups, such as polycaprolactone
1
2
vectors. Frequently studied cationic gene delivery polymers
include polyethylenimine (PEI), poly(2-dimethylaminoethyl
methacrylate) (pDMAEMA), poly-L-lysine (pLL), etc. Our
3
4
group also prepared some linear and reticular cyclen-based
polymers which have in vitro transfection efficiencies (TE)
close to that of PEI (25 kDa). Among these polymers, PEI is
the most studied material for DNA delivery because of its
strong buffering capability in the pH region of 7.4–5.1 together
with high binding capability towards DNA and a relatively
5
6
7
diacrylate linkage, cyclodextrin (CD), dextran, and dithiobis
8
(
succinimidylpropionate),
were introduced to modify
LMW PEI. And some non-PEI-based biodegradable gene
9
carriers such as linear poly(b-amino ester) (PBAE), poly
1
0
(
4-hydroxy-L-proline ester),
1
hyperbranched poly(amino
12
1
ester), poly[a-(4-aminobutyl)-L-glycolic acid] (PAGA) and
poly(2-aminoethyl propylene phosphate) (PPE-EA) have
Key Laboratory of Green Chemistry and Technology (Ministry of
Education), College of Chemistry, and State Key Laboratory of
Biotherapy, West China Hospital, Sichuan University, Chengdu
610064, People’s Republic of China. E-mail: xqyu@scu.edu.cn,
zwjulia@163.com; Fax: +862885415886;
Tel: +86 28 85460576, +86 28 85164063
w These authors contributed equally to this work.
z Electronic supplementary information (ESI) available: Character-
ization of 2 and 3. See DOI: 10.1039/c0mb00339e
13
also been prepared and applied to gene delivery. Although
the TEs of these materials were comparable to that of 25 kDa
PEI and the cytotoxicity was reduced to some degree, it
remains a challenge to obtain efficient gene vectors combining
the advantages of high TE and low cytotoxicity by careful
molecular design and simple preparation.
1
254 Mol. BioSyst., 2011, 7, 1254–1262
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Recently, the poly(amino alcohol esters) were found to be
ideal gene delivery vectors. With the biodegradable polyester
backbone and the hydrophilic hydroxyl groups, this type of
cationic polymers showed relatively lower cytotoxicity and
0.1% (v/v) sulfuric acid for 24 h. Excess sulfuric acid was
neutralized with sodium carbonate. Unreacted allyl alcohol
was evaporated off at 70 1C for 1 h under vacuum. The
product, diallyl adipate, was extracted with ethyl acetate and
dried with anhydrous Na SO . After filtration, the crude
1
4
good water solubility. He and co-workers reported novel
2
4
PEI-grafted polycarbonates, and high TE was obtained by
product was collected by evaporation under reduced pressure
and was used in the next step without further purification.
Diallyl adipate (11.34 g, 0.05 mol) was oxidized by meta-
chloroperoxybenzoic acid (m-CPBA, 19.02 g, 0.11 mol, 99%
purity) in 100 mL of methylene chloride. The mixture was
refluxed at 55 1C for 8 h. After the reaction, the byproduct
meta-chlorobenzoic acid was crystallized at ꢀ20 1C overnight
and filtered from the solution. The remaining byproducts were
removed by filtration followed by column chromatography
with ethyl acetate and petroleum ether (v/v 1 : 2) as eluent.
1
5
using these polymers as gene delivery vectors. Dong and Wei
also synthesized cross-linked poly(amino alcohol esters) of
glycidyl acrylates and PEI 600, however, the TE of these
1
6
polymers in in vitro transfection was low. In this report, we
would like to introduce a series of cross-linked PEI 600 by
using diglycidyl esters with different chain lengths as linkage.
Their interaction with plasmid DNA was investigated, and the
in vitro TEs in different cell-lines were studied. The relative
quantitative structure–activity relationship (QSAR) was
discussed.
1
Diglycidyl adipate (DA, 2a). Yield 46.8%. H NMR
(
400 MHz, CDCl ): d = 1.684–1.691 (d, J = 2.8 Hz, 4H),
3
2
2
3
.386–2.393 (d, J = 2.8 Hz, 4H), 2.635–2.659 (m, 2H),
Experimental section
.841–2.868 (m, 2H), 3.203–3.213 (t, J = 1.6 Hz, 2H),
1
3
.883–3.941 (m, 2H), 4.399–4.442 (m, 2H). C NMR (400
): d = 23.90, 33.25, 44.28, 49.00, 64.60, 172.59.
Materials and methods
MHz, CDCl
MS (ESI): m/z = 281.18 [M + Na] . IR (KBr, cm ):
3
All chemicals and reagents were obtained commercially
and were used as received. Anhydrous ethanol and dichlor-
omethane were dried and purified under nitrogen by using
standard methods and were distilled immediately before use.
LMW PEI (branched, average molecular weight 600 Da, 99%)
was purchased from Aladdin (Shanghai, China). 25 kDa PEI
+
ꢀ1
3
1
062.09, 3004.15, 2948.90, 2874.25, 1731.00, 1419.08,
135.94, 1081.34, 909.12, 798.01, 762.12.
1
Diglycidyl succinate (DS, 2b). Yield 26.0%. H NMR
(
400 MHz, CDCl ): d = 2.649–2.668 (m, 2H), 2.705 (s, 4H),
3
2
.852 (t, J = 4.4, 2H), 3.198–3.238 (m, 2H), 3.957 (dd, J =
13
2.4, 2.4 Hz, 2H), 4.445 (dd, J = 12.4, 2.8 Hz, 2H). C NMR
(
(
branched, average molecular weight 25 kDa) and MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
1
(
400 MHz, CDCl
3
): d = 28.62, 44.39, 49.05, 65.03, 171.72. MS
+
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
ꢀ1
(
ESI): m/z = 253.17 [M + Na] . IR (KBr, cm ): 3013.30,
2
9
934.80, 1733.95, 1425.43, 1352.74, 1310.09, 1148.86, 996.73,
08.58, 861.75.
1
Diglycidyl oxalate (DO, 2c). Yield 45.0%. H NMR
(
Clontech, Palo Alto, CA, USA, coding for EGFP DNA).
The Dulbecco’s Modified Eagle’s Medium (DMEM), 1640
Medium and fetal bovine serum were purchased from
Invitrogen Corp. The MicroBCA protein assay kit was
obtained from Pierce (Rockford, IL, USA). The luciferase
assay kit was purchased from Promega (Madison, WI, USA).
The endotoxin free plasmid purification kit was purchased
(
3
400 MHz, CDCl ): d = 2.724 (dd, J = 4.8, 2.4 Hz, 2H),
.900 (t, J = 4.8 Hz, 2H), 3.294–3.334 (m, 2H), 4.175 (dd, J =
13
2.4, 6.4 Hz, 2H), 4.595 (dd, J = 12.4, 3.2 Hz, 2H). C NMR
2
1
(
400 MHz, CDCl ): d = 44.63, 48.54, 67.35, 156.89. MS (ESI):
+
3
ꢀ1
m/z = 225.02 [M + Na] . IR (KBr, cm ): 3006.82, 2961.11,
743.86, 1447.60, 1189.20, 901.42, 749.16, 545.72.
1
from TIANGEN (Beijing, China).
DECA
MS-ESI spectral data were recorded on a Finnigan LCQ
.
Preparation of D–PEIs (title polymers)
IR spectra were measured with a Shimadzu FTIR-4200
1
spectrometer. H-NMR spectra were obtained on a Bruker
In a typical procedure, PEI 600 (1.50 mmol), diglycidyl
adipate or its analogs (1.33 mmol) and 3 mL of anhydrous
ethanol were mixed in a flask with magnetic stirring and
refluxed for 24 h (besides, 6 h, and 12 h for DS–PEI-6 and
DS–PEI-12, respectively) in an oil bath. After the reaction,
the mixture was diluted with 3 mL of anhydrous methanol,
and the crude product was precipitated by the addition of
anhydrous dichloromethane/cyclohexane (v/v 2 : 1). The
precipitation was collected and dried in vacuum to get the
product as colorless oil. The molecular weights of D–PEIs
AV400 spectrometer. CDCl or D O was used as the solvent
3
2
and TMS as the internal reference. The molecular weight of
polyamine was determined by gel permeation chromatography
(
GPC) (Waters 515 pump, Waters 2410 Refractive Index
Detector (25 1C, incorporating Shodex columns OHPAK
ꢀ
1
KB-803). A filtered mixture of 0.2 mol L HAc/NaAc buffer
which contained 20% CH CN (volume ratio) was used as
3
ꢀ
1
the mobile phase with a flow rate of 0.5 mL min . Molecular
weights were calculated against poly(ethylene glycol)
standards of number average molecular weights ranging from
were measured by GPC.
1
DA–PEI (3a). Yield 46.5%. H-NMR (400 MHz, D
2
00 to 80 000.
2
O): d
=
1.477 (br, 4H), 2.135–2.358 (br, 4H), 2.358–2.609 (m, 58H),
Preparation of diglycidyl esters 2
3
.174–3.212 (m, 4H), 3.346–3.465 (m, 4H), 3.678–3.801 (m,
ꢀ
1
These compounds were prepared following ref. 17. In a typical
procedure, allyl alcohol (0.22 mol) and adipic acid (0.100 mol)
were mixed and refluxed at 105 1C in the presence of
2H). Mw = 6861 (PDI = 1.82). IR (KBr, cm ): 3346.15,
2934.37, 2843.64, 1618.86, 1470.31, 1311.66, 1110.20, 1045.87,
816.39.
This journal is c The Royal Society of Chemistry 2011
Mol. BioSyst., 2011, 7, 1254–1262 1255

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1
DS–PEI (3b). Yield 24.9%. H-NMR (400 MHz, D
.410–2.644 (m, 58H), 3.190–3.232 (m, 4H), 3.371–3.475
m, 4H) 3.704–3.854 (m, 2H). M = 16 015 (PDI = 3.35).
2
O): d =
volume of D–PEIs (in 150 mM NaCl solution) to 0.8 mL of
ꢀ
1
2
pEGFP–N1 DNA (120 ng mL in 40 mM Tris–HCl buffer
solution). Posterior procedures were the same as above except
that the incubation times were 0.5 and 30 h.
(
w
ꢀ
1
IR (KBr, cm ): 3284.25, 2931.77, 2837.02, 1628.08, 1562.40,
1
472.60, 1311.25, 1110.07, 1046.09, 815.96.
1
DO–PEI (3c). Yield 13.3%. H-NMR (400 MHz, D O):
Polymer degradation study
2
d = 2.556–2.672 (m, 54H), 3.243–3.314 (m, 4H), 3.393–3.517
w
For the polymer degradation study, DS–PEI (M = 7860,
(
m, 4H), 3.716 (br, 2H). M
w
= 35 281 (PDI = 3.78). IR
KBr, cm ): 3292.71, 2933.38, 2825.86, 1665.42, 1510.41,
462.91, 1358.94, 1301.32, 1048.27, 810.89.
smaller than that used for transfection) was dissolved in a
single-strength (1ꢁ) phosphate-buffered saline (PBS) solution
and was constantly shaken in a 37 1C incubator at 100 rpm.
The DS–PEI 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 1C).
ꢀ1
(
1
Acid–base titration
In this assay, briefly, D–PEIs (0.25 mmol of amino groups)
was dissolved in 5 mL of 150 mM NaCl aqueous solution, and
1
N HCl was added to adjust the pH to 2.0. Aliquots (50 mL
Particle size and f potential measurements
for each) of 0.1 M NaOH were added, and the solution
pH was measured with a pH meter (pHS-25) after each
addition. For comparison, PEI (25 kDa) was used under same
experimental conditions.
Particle size and z potential measurements of polyplexes were
carried out using a Nano-ZS 3600 (Malvern Instruments,
USA) with a He–Ne Laser beam (633 nm, fixed scattering
angle of 901) at 25 1C. D–PEI/DNA polyplexes at weight
ratios ranging from 0.5 to 32 were prepared by the same
method with above-mentioned agarose gel electrophoresis.
After 30 min incubation in 100 mL ultrapure water, polyplex
solutions were diluted to a final volume of 1 mL before
measurements.
Cell culture
HEK (human embryonic kidney) 293 cells and human
nonsmall-cell lung carcinoma A549 cells were incubated,
respectively, in Dulbecco’s Modified Eagle’s Medium
(
DMEM) and 1640 Medium containing 10% fetal bovine
serum (FBS) and 1% antibiotics (penicillin–streptomycin,
ꢀ
1
Cell viability assay
1
0000 U mL ) at 37 1C in a humidified atmosphere containing
% CO
5
2
.
Toxicity of D–PEIs toward 293 cells and A549 cells
was determined by using MTT (3-(4,5-dimethylthiazol-2-yl)-
Amplification and purification of plasmid DNA
2
,5-diphenyltetrazolium bromide) reduction assay following
pGL-3 and pEGFP plasmids were used. The former one as the
luciferase reporter gene was transformed in E. coli JM109 and
the latter one as the green fluorescent protein gene was
transformed in E. coli DH5a. Both plasmids were amplified
in terrific broth media at 37 1C overnight. The plasmids were
purified by an EndoFree TiangenTM Plasmid Kit. Then the
purified plasmids were dissolved in TE buffer solution and
stored at ꢀ20 1C. The integrity of plasmids was confirmed by
agarose gel electrophoresis. The purity and concentration of
plasmids were determined by ultraviolet (UV) absorbance at
literature procedures. The 293 cells (6000 cells per well) and
A549 cells (14 000 cells per well) were seeded into 96-well
plates. The cells were then incubated in a culture medium
containing D–PEIs with a particular concentration for 24 h.
After that, the medium was replaced with 200 mL of fresh
medium, and 20 mL of sterile filtered MTT (5 mg mL ) stock
solution in PBS was added to each well. After 4 h, the
unreacted dye was removed by aspiration. The formazan
crystals were dissolved in 150 mL DMSO per well and measured
spectrophotometrically in an ELISA plate reader (model 550,
Bio-Rad) at a wavelength of 570 nm. The cell survival was
expressed as follows: cell viability = (ODtreated/ODcontrol)ꢁ 100%.
ꢀ
1
2
60 and 280 nm.
Agarose gel electrophoresis
In vitro transfection
D–PEI/DNA complexes at different weight ratios ranging
from 0.06 to 0.2 were prepared by adding an appropriate
volume of D–PEIs (in 150 mM NaCl solution) to 0.8 mL of
Luciferase assay. The 25 kDa PEI was used as the positive
control due to its high TE in vitro and in vivo. The plasmid
pGL-3 was used as a reporter gene. Transfections of pGL-3
plasmid mediated by D–PEIs in 293 cells and A549 cells were
studied as compared with 25 kDa PEI. 293 cells or A549 cells
ꢀ
1
pEGFP–N1 DNA (120 ng mL in 40 mM Tris–HCl buffer
solution). The complexes were diluted by 150 mM NaCl
solution to a total volume of 6 mL, and then the complexes
were incubated at 37 1C for 30 min. After that the complexes
were electrophoresed on the 0.7% (W/V) agarose gel containing
EB and with Tris–acetate (TAE) running buffer at 110 V for 30
min. DNA was visualized with a UV lamp using a Bio-Rad
Universal Hood II.
4
were seeded at a density of 6 ꢁ 10 cells per well in the 24-well
plate with 0.5 mL of medium containing 10% FBS and
incubated at 37 1C for 24 h. Then the complexes were prepared
at different weight ratios by adding 1.5 mg of plasmid DNA
to an appropriate volume of the D–PEI solution. Before
transfection, the cells were washed by a serum-free medium,
and then the D–PEI/DNA complexes were added with the
serum-free medium for 4 h at 37 1C. Then the serum-free
medium was replaced by the flash medium containing 10%
The property of DNA release associated with the degradation
of D–PEIs was further investigated by agarose gel electro-
phoresis. D–PEI/DNA complexes at different weight ratios
ranging from 0.1 to 0.5 were prepared by adding an appropriate
1
256 Mol. BioSyst., 2011, 7, 1254–1262
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FBS, and the cells were further incubated for 24 h. After that,
the medium was removed. The luciferase assay was performed
according to manufacture’s protocols (Promega). Relative
light units (RLUs) were measured with a chemiluminometer
succinic acid and epichlorohydrin. But this method failed as
the succinic acid anions would lead to the ring-opening of
epichlorohydrin. The polymerization was simply processed by
mixing 2a and PEI 600 in ethanol under reflux temperature.
The crude product was recrystallized for 3 times to ensure their
polydispersity. In the polymerization reaction, the amount of
PEI 600 should be slightly more than that of the diesters,
otherwise the reaction mixture would change to gel-like
materials. This could be attributed to the uncontrollable
crosslinking between the diglycidyl esters and LMW PEI when
(
FLOROSKAN ASCENT FL). The total protein was
measured according to a BCA protein assay kit (Pierce).
Luciferase activity was expressed as RLU per mg protein.
Data are shown as mean ꢂ standard deviation (SD) based
on 3 independent measurements. The statistical significance
between two sets of data was calculated using Student’s t-test.
An AP value o 0.05 was considered statistically significant.
w
excessive diesters were used. To optimize the M of D–PEIs 3,
which might strongly influence their transfection efficiency and
cytotoxicity, we prepared DS–PEI (3a) through different
reaction times of 6 h (DS–PEI-6), 12 h (DS–PEI-12), 24 h
Green fluorescent protein assay. Transfections of the
pEGFP-N1 plasmid mediated by D–PEIs in 293 cells and
A549 cells were also evaluated. The best weight ratios in 293
cells and A549 cells determined from the luciferase assay were
used. 293 cells and A549 cells were inoculated at a density of
(
DS–PEI), and 48 h (DS–PEI-48). GPC gave the corresponding
M s of 6065, 8689, 16 015, 61 600, for DS–PEI-6, DS–PEI-12,
w
DS-PEI and DS–PEI-48, respectively, indicating that the
molecular weights of these polymers increased associated with
reaction time. According to the data of luciferase assay
5
5
2
.4 ꢁ 10 and 3 ꢁ 10 cells per well in 24-well plates,
respectively, 24 h prior to transfection. D–PEI/DNA
complexes were prepared by adding an appropriate volume
(
Fig. 6), we chose 24 h as the reaction time in the preparation
of D–PEIs solution (in 150 mM NaCl solution) to 50 mL
of DA–PEI (3a) and DO–PEI (3c). The obtained polymers
ꢀ1
pEGFP-N1 DNA solution (30 mg mL in 40 mM Tris–HCl
buffer solution) with a final volume of 100 mL. Then the
complexes were incubated at room temperature for 30 min.
The plates were washed with PBS twice, and 100 mL D–PEI/
DNA complexes were then added to a well with an additional
showed significant water solubility, which was required for the
subsequent studies. GPC gave M
DO–PEI as 6861 (PDI = 1.82), 16 015 (PDI = 3.35) and
5 281 (PDI = 3.78), respectively. The increasing M s from
w
s of DA–PEI, DS–PEI and
3
w
DA–PEI to DO–PEI suggested that the polymerization would
be more difficult for the longer bridge (2a). Additionally, the
degradation of the ester-containing polymer was also studied
1
50 mL of medium without FBS. The final concentration of
ꢀ
1
DNA in the complexes was calculated to be 6 mg mL . After 4
h of incubation, the D–PEI/DNA complex containing medium
was replaced with 0.5 mL of fresh medium containing 10%
FBS, and the cells were further incubated for 24 h at 37 1C. The
cells were directly observed by an inverted microscope (Olympus
IX 71). The microscopy images were obtained at a magnification
of 100ꢁ and recorded using Viewfinder Lite (1.0) software.
by using DS–PEI (M
w
7860 Da) as an example via GPC. As
of DS–PEI decreased from 7860 to
shown in Fig. 1, the M
w
Results and discussion
Synthesis and characterization of title polymer D–PEIs
The preparation route of D–PEIs is shown in Scheme 1. In a
typical procedure, adipic acid reacted with allyl alcohol in the
presence of sulfuric acid to give the diester compound 1a.
Subsequent oxidation of the double bonds by meta-chloroper-
benzoic acid (m-CPBA) led to diglycidyl esters 2a. We also
tried the direct route to prepare 2a by using sodium salt of
Fig. 1 Change of molecular weight of DS–PEI (Mw = 7860 Da) with
degradation time in PBS (pH = 7.4).
Scheme 1 Preparation route of D–PEIs.
This journal is c The Royal Society of Chemistry 2011
Mol. BioSyst., 2011, 7, 1254–1262 1257

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relative to 25 kDa PEI, the lower buffer capacities of D–PEIs
are reasonable. The titration profiles between 3a–c were
almost identical, indicating that the length of bridged diacid
chains has little effect on the buffer capacity.
Formation of D–PEI/DNA complexes
DNA condensation capability is essential for polymeric gene
vectors. Gel retardation analysis was performed to confirm the
affinity between D–PEIs and plasmid DNA. As shown in
Fig. 3, the electrophoretic mobility of DNA was retarded by
the introduction of D–PEIs, and total DNA retardation was
detected at and above weight ratios of 0.1. The results
suggested that D–PEIs can bind to DNA through electrostatic
interactions between the DNA backbone and the cationic
nitrogen atoms in D–PEIs.
Fig. 2 Acid–base titration profiles of PEI 25 KDa and D–PEIs.
D–PEIs or PEI 25 KDa (0.25 mmol of amino groups) was first treated
with 1 N HCl to adjust pH to 2.0, and then the solution pH was
measured after each addition of 50 mL of 0.1 N NaOH. The relatively
flat curve of PEI indicates its higher buffer capability than those of
D–PEIs.
The potential advantage of biodegradable gene vectors
as compared to their non-degradable counterparts is their
reduced cytotoxicity and the avoidance of accumulation of
19
the polymer in the cells after repeated administration. The
degradability of the polymers can be helpful as a tool to release
the plasmid DNA into the cytosol. Hence, the release of
pDNA from the polyplexes was also studied by gel retardation
analysis. Comparative experiments about D–PEIs pre-incubated
in 150 mM NaCl solvent at 37 1C for different periods of time
were performed. Fig. 4A shows that the polymers and
pEGFP-N1 plasmid could form stable complexes even under
a weight ratio of 0.1. After incubation alone for 30 h, these
polymers could not efficiently retard DNA, indicating that no
distinct condensation was processed (Fig. 4B). This might be
attributed to the cleavage of ester bonds in the polymers,
which resulted in the degradation to small segments that could
not efficiently bind to DNA. For DA–PEI, the degradation
rate might be slower than those of other two materials, and as
a result, it could still condense and retard DNA under the
weight ratio of 0.5 (Fig. 4B, lane 6).
5
900 Da within 12 h. Further extension of time resulted in a
relatively slight decrease of M , and the Mw of 5312 Da was
w
detected after 36 h.
Buffer capability
Acid–base titration was performed to evaluate the proton
buffering effect of D–PEIs. PEI 25 kDa is well known for its
strong proton-sponge effect, leading to the disruption of
1
8
endosome in the transfection process. In this study, the
buffer capabilities of D–PEIs and 25 kDa PEI were evaluated
by acid–base titration. As shown in Fig. 2, the buffer
capability of D–PEIs is slightly lower than that of PEI. It
was reported that the buffer capabilities of these polycations
mainly depend on the presence of primary, secondary and
tertiary amines groups. Since D–PEIs have a lower molecular
weight and less density of amino groups in the structure
The appropriate size of polymer/DNA nanoparticles is of
critical importance for polyamines as gene vectors into the
target cells. The desirable in vivo vectors should efficiently
Fig. 3 Electrophoretic mobility of pEGFP-N1 in the presence of DA–PEI (lanes 2–7), DS–PEI (lanes 8–13) and DO–PEI (lanes 14–19) (ethidium
bromide staining). Lane 1: DNA control. The weight ratios of D–PEI/DNA are 0.06, 0.07, 0.08, 0.09, 0.1, 0.2 for lanes 2–7, 8–13 and 14–19.
Fig. 4 Electrophoretic mobility of pEGFP-N1 plasmid after pre-incubation for 0 h (A) and 30 h (B) of D–PEIs alone (ethidium bromide staining).
Lanes 2–6: DA-PEI/pDNA with a weight ratio of 0.1, 0.2, 0.3, 0.4, 0.5, respectively; lanes 7–11: DS-PEI/pDNA with a weight ratio of 0.1, 0.2, 0.3,
0.4, 0.5, respectively; lanes 12–16: DO-PEI/pDNA with a weight ratio of 0.1, 0.2, 0.3, 0.4, 0.5, respectively; lane 1: pDNA control.
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258 Mol. BioSyst., 2011, 7, 1254–1262
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Fig. 5 Average particle size (A) and z-potential (B) of D–PEI/DNA at weight ratios of 0.5, 1, 2, 4, 8, 16 and 32 (mean ꢂ SD, n = 3).
compact pDNA into small (below 200 nm) nanoparticles,
which was very useful for particle stabilization, efficient
DNA complexes did not display clear trends associated with
the weight ratio (0.5–32, Fig. 5B), and the range of +5–20 mV
is slightly lower than the most reported PEI/DNA complexes
(B+25–30 mV) and is higher than the results of PEG–PEI
copolymers which also act as effective gene carriers. This
relatively low z-potential might be attributed to the repeated
hydroxyl and ester groups which can screen the positive charge
of amino groups in the structure of D–PEIs.
2
0
endocytosis and gene transfer. The particle size of D–PEIs/
pDNA complexes was measured at various weight ratios, and
the results are shown in Fig. 5A. D–PEIs could efficiently
compact pDNA into small nanoparticles with the sizes of
around 120–250 nm in diameter at the weight ratio from 0.5 to
2
2
1
6. Distinctly smaller particles of D–PEIs were observed at the
weight ratios of 1 compared to those obtained at adjacent
weight ratios (0.5 and 2). Further increase of weight ratio
might lead to the accretion of nanoparticle sizes, especially for
DS–PEI-6 which has a relatively low molecular weight caused
by shorter preparation time, and it might be more liable to
aggregate under high concentration. Of the three polymers,
DO–PEI could form the polyplex with around 140 nm in
diameter, which was much smaller than those formed by DNA
and other D–PEIs. This could be explained by the larger
molecular weight and ion density of DO–PEI induced by its
shorter diester bridge. In a word, D–PEIs could compact
pDNA to form nanoparticles with proper sizes that are prone
Cytotoxicity
The cytotoxicity of cationic polymers is thought to be caused
by the damage due to the interaction with plasma membrane
or other cellular compartments, and researches have found a
2
3
rough correlation between toxicity and TE. The in vitro
cytotoxicity of D–PEIs was evaluated in A549 and 293 cells
by MTT assay, and the 25 kDa PEI was used as the control.
As shown in Fig. 6, 25 kDa PEI displayed serious cytotoxicity
in two cell-lines and the relative cell viability of PEI were less
ꢀ
1
than 20% when its concentration was over 20 mg mL . To
our surprise, D–PEIs showed high cytotoxicity in A549 cells,
especially under higher concentration (Fig. 6A). However,
under the concentrations in subsequent transfection studies
3
to endocytosis.
z-potential is an indicator of surface charges on the
polymer/pDNA nanoparticles. A positively charged surface
allows electrostatic interaction with anionic cell surfaces and
ꢀ1
(up to 6.0 mg mL ), the cytotoxicities were low. On the other
hand, D–PEIs indicated relatively high cell viabilities in 293
2
1
facilitates cellular uptake. The z-potential values of D–PEI/
Fig. 6 Relative cell viabilities of D–PEIs and 25 kDa PEI in A549 (A) and 293 (B) cells.
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cells, and about 40% cell viability was found even at
To our delight, D–PEIs displayed comparable TEs with PEI in
A549 cell-line. Generally, the increase of the molecular weight
led to higher TE. However, for the experiments with a weight
ratio of 2, TE was decreased for DS–PEI, which was obtained
after 24 h reaction. This might be attributed to the increased
cytotoxicity under higher concentration and higher molecular
weight. The best result was achieved when using DS–PEI
under a weight ratio of 1, in which more than 5 times of TE
was obtained compared to that of the PEI/DNA polyplex.
Meanwhile, although DS–PEI could bind and retard DNA
under a weight ratio of 0.5 in gel retardation analysis, low TEs
were observed at this weight ratio.
ꢀ1
1
00 mg mL (Fig. 6B). DO–PEI displayed higher cytotoxicity
than DA–PEI and DS–PEI, and this might be attributed to the
higher positive charge, which was caused by the shorter diester
bridge and therefore higher density of amino groups on the
surface of the complexes. The higher Mw of DO–PEI might
also contribute to its higher cytotoxicity. It is evident that cell-
dependent cytotoxicities were found for D–PEIs, and A549
cells showed a relatively severe weakness against the cytotoxicity
of both PEI and D–PEIs.
In vitro transfection
The TEs of three prepared D–PEIs in different cell-lines
were then studied, and the results are shown in Fig. 8. D–PEIs
can act as effective non-viral gene vectors with high TEs. By
using PEI as standard, better results were obtained in A549
cells than those achieved in 293 cells. As shown in Fig. 8A, all
three polymers showed 3–5 times of TE compared to that of
the PEI/DNA complex, and the best result belonged to
DS–PEI under a weight ratio of 1. For DA–PEI, the best
TE was obtained under a weight ratio of 0.5. In 293 cell-line,
the weight ratio of 1 was also the optimized usage, and under
this condition, DO–PEI displayed the best result, which was
about 60% TE compared to that of the PEI/DNA complex
The gene transfection efficiency of D–PEI/DNA complexes
was assessed by in vitro delivery experiments of luciferase
reporter gene (plasmid pGL-3) into A549 and 293 cells. PEI
2
5 kDa was used for comparison because of its high TE
and easy availability. PEI/DNA polyplexes were prepared at
an N/P ratio of 10 (weight ratio of 1.39, which is the accepted
optimal PEI usage for transfection), and D–PEI/DNA
complexes were prepared at various weight ratios including
ꢀ
1
0
.5, 1 and 2 (D–PEI concentrations of 1.5, 3.0, 6.0 mg mL ,
respectively). Firstly, the effect of molecular weight of DS–PEI
on the TE was investigated, and the results are shown in Fig. 7.
(
Fig. 8B). We speculated that the relatively small particles
formed at the weight ratio of 1 (o200 nm, Fig. 5A) might
facilitate the endocytosis and subsequent gene expression.
Additionally, compared to PEI, the D–PEIs showed better
cell selectivity towards the cancer cell A549.
To directly visualize the infected cells expressing pEGFP-Nl,
enhanced green fluorescent protein expression in A549 and
2
93 cells was observed by an inverted fluorescent microscope.
According to the results of the luciferase assay, D–PEI/DNA
and PEI 25 kDa per DNA (as control) complexes were used at
the optimal weight ratio of 1 (0.5 for DA–PEI in A549 cells)
and the N/P ratio of 10, respectively (Fig. 9). In A549 cell-line,
the images showed that the density of transfected cells by
DS–PEI/DNA and DO–PEI/DNA were similar to that caused
by 25 kDa PEI/DNA complexes (Fig. 9A–D), while DA–PEI
showed weaker GFP expression. On the other hand, the
images in Fig. 9E–H showed that the density of transfected
Fig. 7 Effect of molecular weight of DS–PEI and weight ratio on the TE
(
luciferase expression) in A549 cells (mean ꢂ SD, n = 3). For comparison
with Fig. 6, the concentrations of DS–PEIs in the transfection experiments
ꢀ1
were 1.5, 3, 6 mg mL for the weight ratio of 0.5, 1 and 2, respectively.
Fig. 8 Luciferase expression in A549 (A) and 293 (B) cells transfected by 25 kDa PEI/DNA (N/P = 10) and D–PEI/DNA complexes at different
weight ratios (mean ꢂ SD, n = 3).
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260 Mol. BioSyst., 2011, 7, 1254–1262
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Fig. 9 Fluorescent microscope images of pEGFP-transfected in A549 (A–D) and 293 (E–H) cells. N/P of PEI/DNA was 10 (weight ratio of 1.39),
D–PEI/DNA weight ratios were 1 : 1 except B (0.5): (A, E) PEI 25 kDa, (B, F) DA–PEI, (C, G) DS–PEI, (D, H) DO–PEI. These 4 cm-wide images
were obtained after 68-fold reduction from original pictures, which were recorded at the magnification of 100ꢁ.
2
93 cells by DS-PEI/DNA (Fig. 9G) was somewhat enhanced
Research Team in University, the Key Project of Chinese
Ministry of Education in China and the Scientific Fund of
Sichuan Province for Outstanding Young Scientist. We also
thank Analytical & Testing Center of Sichuan University for
NMR analysis.
compared to that caused by 25 kDa PEI/DNA complexes. In a
sense, the types of both the cells and guest genes would affect
the TEs of D–PEI. Further increase or decrease of the weight
ratio led to much lower TE, fewer cells that expressed GFP
were observed by microscopy (data not shown). Similar results
were obtained in luciferase expression experiments. This low
TE was attributed to the liability to degradation under a low
weight ratio and the difficulty to release DNA and higher
cytotoxicity under a high weight ratio. The transfection results
exhibited that the bridge length may have large influence on
the TE, and distinct difference in TE in both cell-lines could be
observed between the three D–PEI/DNA polyplexes.
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