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Z. Wang et al. / International Journal of Pharmaceutics 505 (2016) 24–34
6-Diamidino-2-phenylindole dihydrochloride (DAPI) were
obtained from Sigma-Aldrich. RPMI-1640 medium, fetal bovine
serum (FBS) and trypsin were obtained from Gibco.
Lipofectamine2000 and TRIzol reagent were purchased from
Invitrogen. Annexin V-FITC apoptosis detection kit was purchased
from shanghai BestBio biology co. Reverse Transcription System
and Go Taq qPCR Mastermix were purchased from Promega
(Madison, WI, USA).
The siRNA targeting survivin mRNA was synthesized by
Ribobio Co. FAM-labeled siRNA and negative control siRNA were
purchased from Gene PharmCo. Ltd. Survivin and GAPDH primer
were synthesized by Invitrogen. The sequences are as follows: si-
survivin: sense: 50-UCCGGUUGCGCUUUCCUUUdTdT-30; antisense:
50-AAAGGAAAGCGCAACCGGAdTdT-30. Negative control siRNA:
sense: 50 -UUCUCCGAACGUGUCACGUTT-30; antisense: 50-ACGU-
GACACGUUCGGAGAATT-30. Survivin primer: forward: 50- CAACCG-
GACGAATGCTTTT-30; reverse: 50-AAGAACTGGCCCTTCTTGGA-30.
GAPDH primer: forward: 50-GAACGGGAAGCTCACTGG-30; reverse:
50-GCCTGCTTCACCACCTTCT-30.
chosen as model dispersion media. The PPGS/siRNA polyplex
dispersion were balanced in room temperature for 10 min, then
their particle size was measured by Malvern Zetasizer NS90
(Malvern Instruments, U.K.). To evaluate the changes in particle
size of the PPGS/siRNA polyplex when it was diluted, serial
dilutions of polyplex were carried out and polyplex dispersion
were balanced in room temperature for 10 min, then their particle
size was measured by Malvern Zetasizer NS90 (Malvern Instru-
ments, U.K.). PEI 25KDa/siRNA polyplex at the N/P ratio of 10/1 was
used as a control.
2.5. Cytotoxicity of PPGS and PPGS/siRNA polyplex
The cytotoxicity of PPGS and PPGS/siRNA was measured by
using MTT assay. A549/DDP cells were seeded at 5000 cells per
well in 96-well plates, with 100 mL RPMI 1640 containing 10% FBS.
After further cultured for 24 h, the culture media were replaced by
fresh medium containing 10% FBS with various amounts of PPGS or
PPGS/siRNA. Following incubation for 4 h, the medium was
replaced by fresh medium containing 10% FBS and further cultured
for 48 h. MTT was added to each well at a final concentration of
0.5 mg/mL, and following incubation for 4 h, the supernatant was
2.2. Synthesis of mPEG-b-PG-g-spermine
Polyglutamate derivative polymer brush, mPEG-b-PG-g-sper-
mine (PPGS), was synthesized as follows. Firstly, the synthesis of
poly(ethyleneglycol) monomethyl ether-b-poly(benzyl-gluta-
mate)(mPEG-b-PBLG) was according to a similar procedure (Deng
removed, and the resulting formazan was dissolved in 150 mL
DMSO. The absorbance at 490 nm was measured by microplate
reader (ELX800, Bio-Tek, USA.). The cell viability was expressed as
the percentage of the absorbance of sample to that of the untreated
cells.
et al., 2004; Tian et al., 2005; Wen et al., 2009). In brief,
glutamic (BLG) was synthesized through esterification reaction of
-glutamic acid and benzyl alcohol. Then 10 g BLG was dissolved in
g-benzyl-L-
L
2.6. Effects of serum concentration on cellular uptake
80 ml anhydrous tetrahydrofuran, and 6 g triphosgene was added
to the reaction mixture. After stirring at 60 ꢀC for 40 min the
reaction solution was precipitated using cold petroleum ether and
Flow cytometry (FCM) and confocal laser scanning microscopy
(CLSM) assays were used to evaluate the effects of serum
concentration on cellular uptake. A549/DDP cells were seeded
onto 12-well plates at a density of 1 ꢁ105cells per well, with 1 mL
RPMI 1640 medium containing 10% FBS. After the cells reaching
70–80% confluence, the culture medium were replaced with fresh
complete medium with varying concentration of serum (0–50%)
and PPGS/FAM-siRNA polyplex at varying N/P ratios were added to
the cells. PEI 25KDa/FAM-siRNA polyplex (N/P = 10/1) and
Lipofectamine2000/FAM-siRNA complex (w/w = 2/1) were used
as positive controls according to manufacturer’s instruction.
g
-benzyl-L-glutamic-N-carboxyanhydride
(BLG-NCA)
was
obtained. Secondly, mPEG-b-PBLG was prepared by ring opening
polymerization of BLG-NCA in anhydrous chloroform using
PEG-monomethyl ether 2k as
a
macromolecule initiator
with the molar ratio of Monomer to Initiator = 100:1. Thirdly, 1 g
mPEG-b-PBLG was dissolved in dimethylformamide (DMF)
(w/v 1/20). Spermine (10-fold mol of the ester groups of
mPEG-b-PBLG) and 2-hydroxypyridine (5-fold mol of the ester
groups of mPEG-b-PBLG) were added to the reaction mixture, and
then the solution was stirred at 40 ꢀC for 48 h. Finally, PPGS was
obstained by precipitation in cold diethyl ether, followed by
dialyzed for 48 h in 6–8 kD MWCO dialysis bag to remove
unreacted spermine. The final product PPGS was lyophilized and
characterized by 1H NMR.
Naked FAM-siRNA served as
a negative control. After 4 h
incubation, the media were removed and the cells were washed
with PBS for three times. Subsequently, transfected cells were
treated with trypsin/EDTA for 2 min, collected by centrifugation
and suspended in 500 mL PBS. The cell samples were measured by
FCM using a FACS-Calibur Instrument (Beckman, USA.) and data
was analyzed with Winmdi.
2.3. Preparation of PPGS/siRNA polyplex
CLSM was used to observe the intracellular distribution and the
changes of fluorescence intensity with the increase of serum
concentrations (0–50%) in media. Coverslips putting in the 6-well
plates were seeded with A549/DDP cells at the density of 1 ꢁ105
cells per well. After incubated for 24 h, the medium were replaced
by fresh culture media with varying concentration of serum
(0–50%). PPGS/FAM-siRNA polyplex (N/P = 40/1) and Lipofect-
amine2000/FAM-siRNA complex (w/w = 2/1) were added to the
cells, respectively. After 4 h incubation, the media were removed
and the cells were washed with PBS for three times. The
extracellular fluorescence was quenched with 0.4% trypan blue
for 3 min. Then the cells were fixed with 4% paraformaldehyde for
30 min and stained with 10 mg/mL of DAPI for 20 min at 37 ꢀC in
dark. After that, cells were observed using Zeiss LSM710CLSM (Carl
Zeiss, DE) at excitation wavelength of 405 nm and 488 nm to
visualize nuclei (blue fluorescence) and FAM- labeled siRNA,
respectively.
PPGS polymer brush/siRNA polyplex was prepared by adding
20 mM of siRNA in DEPC-treated water to equal volumes of PPGS by
gentle vortexing. The working concentration of siRNA was 100 nM,
and the amounts of PPGS based on the molar ratio of amino groups
of PPGS to phosphate groups of siRNA (N/P). The mixture was
incubated for 30 min at room temperature to form PPGS/siRNA
polyplex.
2.4. Particle size of PPGS/siRNA polyplex in ultra-high dilution, saline
and in the presence of serum
PPGS/siRNA polyplex was prepared as described above with the
N/P ratio of 40:1. To evaluate the particle size of the PPGS/siRNA
polyplex in the presence of salt and serum, media containing series
concentration of NaCl and 10% final concentration of serum were