Download A novel environment-sensitive biodegradable polydisulfide with

GENE DELIVERY
Journal of Controlled Release 120 (2007) 250 – 258
www.elsevier.com/locate/jconrel
A novel environment-sensitive biodegradable polydisulfide with
protonatable pendants for nucleic acid delivery
Xu-Li Wang a , Randy Jensen b , Zheng-Rong Lu a,⁎
a
Departments of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84108, United States
b
Department of Neurosurgery, University of Utah, Salt Lake City, Utah 84108, United States
Received 9 March 2007; accepted 11 May 2007
Available online 21 May 2007
Abstract
Clinical application of nucleic acid-based therapies is limited by the lack of safe and efficient delivery systems. The purpose of this study is to
design and evaluate novel biodegradable polymeric carriers sensitive to environmental changes for efficient delivery of nucleic acids, including
plasmid DNA and siRNA. A novel polydisulfide with protonatable pendants was synthesized by the oxidative polymerization of a dithiol
monomer, which was readily prepared by solid phase chemistry. The polydisulfide exhibited good buffering capacity and low cytotoxicity. It
formed stable complexes with both plasmid DNA and siRNA. The particle sizes of the complexes decreased with the increase of the N/P ratios in
the range of 100 to 750 nm. The complexes were stable in the presence of salt and heparin under normal physiological conditions, but dissociated
to release nucleic acids in a reductive environment similar to cytoplasm. The polydisulfide demonstrated N/P ratio dependent transfection
efficiency for plasmid DNA and gene silencing efficiency for siRNA. The presence of an endosomal disrupting agent, chloroquine, did not affect
the DNA transfection efficiency of the polydisulfide. The transfection or gene silencing efficiency of the polydisulfide/DNA or siRNA complexes
was comparable to or slightly lower than that of corresponding PEI complexes. Moreover, the polydisulfide showed better serum-friendly feature
than PEI when delivering either DNA or siRNA in the presence of 10% FBS. This novel polydisulfide is a promising lead for further design and
development of safe and efficient delivery systems for nucleic acids.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Nonviral gene carrier; Polydisulfide; Plasmid DNA; siRNA; Environment sensitive gene delivery
1. Introduction
Nucleic acid-based therapies have emerged as exciting new
approaches to treat human diseases by transferring genetic
materials such as DNA, oligonucleotides (antisense) or small
interfering RNA (siRNA) into human cells to express or regulate genes for therapeutic purposes [1−7]. Unfortunately, clinical application of nucleic acid-based therapies is hindered by
lack of safe and efficient delivery systems. Efficient delivery of
nucleic acids is considered as the main challenge for the success
of nucleic acid-based therapies [8]. Viral delivery systems based
on attenuated viruses, such as adenoviruses, adeno-associated
viruses, herpesviruses, lentiviruses and retroviruses, have
shown high efficiency in delivering and expressing genes in
⁎ Corresponding author. 421 Wakara Way, Suite 318, Salt Lake City,
UT 84108, United States. Tel.: +1 801 587 9450; fax: +1 801 585 3614.
E-mail address: [email protected] (Z.-R. Lu).
0168-3659/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2007.05.014
cells. However, clinical application of viral vectors is limited by
their potential toxicity and immunogenicity [9]. In comparison,
synthetic delivery systems, including cationic lipids [10−12]
and polymers [13−16], are non-immunogenic, easy to use and
can be readily produced in a large scale at low cost. However,
the main drawbacks for catioinic delivery systems are their
cytotoxicity [17] and the low delivery efficiency of nucleic
acids. Innovative design of novel gene carriers is needed to
overcome these drawbacks of cationic gene delivery systems.
An ideal delivery system for nucleic acids should be a nontoxic and non-immunogenic system that can protect nucleic
acids from enzymatic degradation during the delivery process,
prevent rapid elimination from the body, and release nucleic
acid intracellularly at the site of action. Recently, there have
been significant efforts to design and develop biodegradable
cationic polymeric carriers to improve the delivery efficiency of
non-viral delivery systems and to reduce their toxic side effects
[18−22]. One of the approaches is to incorporate disulfide
bonds in the cationic polymers to prepare reducible polycations
for nucleic acid delivery. The disulfide bonds are stable in the
oxidative extracellular environment and degradable in the reductive intracellular environment, facilitating disassociation of
polyplexes and release of nucleic acids in cytoplasm. Initially,
disulfide-containing polycations were prepared from low molecular weight cationic peptides, which showed low cytotoxicity
but poor gene transferring capability [23,24]. Reducible polycations with improved transfection efficiency were then prepared by linking histidine-rich or PEI segments with disulfide
bonds [25−27]. Recently, Lin et al. have synthesized a series of
poly(amido amines) with a variable amount of disulfide linkers
[28,29]. These new reducible carriers have demonstrated good
buffering capacities and some of the agents have demonstrated
higher in vitro transfection efficiency than PEI.
In this study, we have designed and synthesized a novel
polydisulfide with protonatable pendants for nucleic acid delivery. In this novel cationic polydisulfide carrier, the side chains
containing multiple primary and secondary amines are designed
to form stable complexes with plamid DNA and siRNA. The
cationic side chains are more flexible than the polymer backbones and may be advantageous to form stable complexes with
small nucleic acids such as oligonucleotides or siRNAs. The
histidine, primary and tertiary amino groups are also incorporated in the backbone of the polydisulfide to enhance the
buffering capacity of the carrier. The complexes of nucleic acids
with the polydisulfide will be stable in the plasma and extracellular fluid during the delivery process and can readily escape
from endosomal-lysosomal compartments due to pH changes.
The complexes can then be reduced in cytoplasm to release
nucleic acids, resulting in effective transfection. The synthesis,
physicochemical property, cytotoxicity, and in vitro delivery
efficiency for plasmid DNA and siRNA of the novel cationic
polydisulfide are reported here.
2. Methods and materials
2.1. Materials
2-Chlorotrityl chloride resin (0.3 mmol/g), N-fluorenylmethoxycarbonyl-N-im-trityl-L-histidine, N-fluorenylmethoxycarbonyl-S-trityl-L-cysteine, 2-(1H-benzotriazole-1-yl)-1,1,3,
3-tetramethyluronium tetrafluoroborate (TBTU), N-hydroxybenzotriazole (HOBt) and 2-acetyldimedone (Dde-OH) were
purchased from EMD Biosciences (San Diego, CA). Triethylenetetramine, N,N-diisopropylethylamine (DIPEA), methyl acrylate, 1,2-ethylenediamine, hydrazine, 4-dithiothreitol (DTT),
piperidine and trifluoroacetic acid (TFA) were purchased from
Lancaster (Windham, NH). Hyperbranched PEI (Mw = 25 kDa),
chloroquine diphosphate (CQ) and 2,5-diphenyl-3-(4,5-dimethyl2-thiazolyl)tetrazolium bromide (MTT) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Anhydrous dimethyl
sulfoxide (DMSO), N,N-dimethylformamide (DMF) and dichloromethane (DCM) were purchased from Acros (Pittsburgh, PA).
ISOLUTE column reservoirs (Charlottesville, VA) were used for
the solid phase synthesis. Triethylenetetramine was purified by
distillation under reduced pressure. All other materials and
251
solvents were used without additional purification. gWiz™
reporter plasmid encoding luciferase was purchased from
Aldevron (Fargo, ND). siRNA targeting Firefly Luciferase was
purchased from Dharmacon (Chicago, IL). The sequence of
antisense is 5′-UCGAAGUACUCAGCGUAAGdTdT-3′ and
that of sense is 3′-dTdTAGCUUCAUGAGUCGCAUUC-5′.
2.2. Synthesis of dithiol monomer
2.2.1. Resin-(2-chlorotrityl)-1,4,7,10-tetraazadecane (resin 2)
2-Chlorotrityl chloride resin (300 mg, 0.3 mmol/g) was
extensively washed with anhydrous DCM. A mixture of triethylenetetramine (1.0 mL, excess) and DIPEA (64 mg) in DCM
was added to the resin, and the suspension was shaken for 2 h. The
solvent was drained and the resin was washed with DCM and
MeOH. The resin was further shaken with 10 mL DCM/MeOH/
DIPEA (17/2/1, v/v/v) for 20 min. The resulted resin 2 was
extensively washed with DCM and dried under reduced pressure.
2.2.2. Selective protection of secondary amines (resin 3–5)
A solution of 2-acetyldimedone (2.0 g) in 10 mL DMF was
added to the resin 2. The suspension was shaken at room
temperature for 12 h. The resin was extensively washed with
DMF and DCM to give Dde protected resin 3. Resin 3 was then
suspended in the solution of Boc2O (10 g) in 15 mL DCM. The
mixture was shaken at room temperature for 4 h. The resulted
resin was extensively washed with DMF and DCM, and dried
under reduced pressure to give resin 4. Resin 4 was suspended
in the solution of 2% hydrazine in DMF for 15 min to remove
the Dde protection at the primary amine. This step was repeated
three times to ensure complete removal of Dde group. The
resulted resin was extensively washed with DMF and DCM,
and dried under reduced pressure to give resin 5.
2.2.3. Coupling reaction with methyl acrylate, 1,2-ethylenediamine, N-α-Fmoc-N-im-trityl-L-histidine and N-α-Fmoc-S-tritylL-cysteine (resin 6–9)
Resin 5 was mixed with methyl acrylate (50 mL, excess) in
10 mL DMF to introduce methyl carboxylate via Michael
addition. The reaction was carried out in a rotary evaporator at
50 °C with continuous rotating. The reaction was monitored by
Kaiser ninhydrin test until complete consumption of primary
amine. The resulted resin 6 was extensively washed with DMF,
MeOH, DCM and dried under reduced pressure. A solution of
1,2-ethylenediamine (50 mL, excess) in 10 mL DMF was mixed
with resin 6. The mixture was rotated in a rotary evaporator at
50 °C for 5 days. The resin was extensively washed with DMF,
MeOH, DCM and dried under reduced pressure to give resin 7.
Resin 7 was transferred into an ISOLUTE column and mixed
with a solution of activated N-α-Fmoc-N-im-trityl-L-histidine
(2.0 g, excess) with TBTU/HOBt/DIPEA (excess) in DMF. The
mixture was shaken for 2 h and the reaction was followed by the
Kaiser test. The resin was subjected to a washing cycle and
Fmoc protecting group was removed with 20% piperidine in
DMF (20 min × 3) to give resin 8. The resin was then extensively washed with DMF and DCM, and dried under reduced
pressure. A solution of activated N-α-Fmoc-S-trityl-L-cysteine
GENE DELIVERY
X.-L. Wang et al. / Journal of Controlled Release 120 (2007) 250–258
GENE DELIVERY
252
X.-L. Wang et al. / Journal of Controlled Release 120 (2007) 250–258
(2.0 g, excess) with TBTU/HOBt/DIPEA (excess) in DMF was
added to resin 8 and the coupling reaction was continued for 2 h,
followed by the Kaiser test. The resin was extensively washed
and Fmoc protecting groups were removed with 20% piperidine
in DMF. The resulted resin 9 was then extensively washed with
DMF, MeOH and DCM.
illuminator. The DNA release from DNA/polydisulfide polyplexes was evaluated by incubating polyplexes (N/P = 3) with
0.1 M DTT at 37 °C for 4 h in the absence/presence of 0.15 M
NaCl or 1.0 mg/mL heparin. The samples were then analyzed
by gel electrophoresis as described above.
2.6. DNA-polydisulfide complexes size measurement
2.2.4. Recovery of dithiol monomer (10)
Resin 9 was suspended in a solution of TFA/H2O/EDT/TIBS
(94/2.5/2.5/1) and shaken for 3 h at room temperature. The
solution was collected and concentrated under reduced pressure.
The residue was washed with cold diethyl ether (40 mL × 5) and
dried. The dithiol monomer was further purified by preparative
HPLC to yield 60 mg product. 1H NMR (400 MHz, D2O): δ
8.45 (m, 2H), 7.2 (m, 2H), 4.5 (m, 2H), 4.05 (m, 2H), 3.2–2.8
(m, 24H), 2.7–2.5 (m, 8H), 2.4–2.3 (m, 4H). MS (m/z): 855.50
(found); 855.45 (calculated for C34H62N16O6S2 [M + H]+).
2.3. Oxidative polymerization in DMSO
The dithiol monomer (36 mg) was dissolved in 100 μL of
DMSO and kept at 37 °C for 5 days. The polymer was obtained
by precipitation in acetone (10 mL) and purified by dialysis
with a membrane of MWCO 2000 against ultrapure water. The
polydisulfide was obtained as a colorless powder after lyophilization. 1H NMR (400 MHz, D2O, δ, ppm): 8.34 (m, 2H), 7.12
(m, 2H), 4.5 (m, 2H), 4.1 (m, 2H), 3.2–2.8 (m, 24H), 2.7–2.5
(m, 8H), 2.4–2.3 (m, 4H). The molecular weight of the polymer was determined by size exclusion chromatography on an
AKTA FPLC system (Amersham Biosciences, Piscataway, NJ)
equipped with a Superose 12 column, and UV and refractive
index detectors, eluted with Tris buffer (20 mM, pH 7.4).
Molecular weights were calibrated with standard poly[N-(2hydroxypropyl)methacrylamide].
DNA-polydisulfide complexes were prepared as described
above. Particle size analysis was performed using a Zetasizer
3000 PCS (Malvern Instruments, Malvern, UK) equipped with
a 5 mW helium neon laser with a wavelength output of 633 nm.
Measurements were made at 25 °C at an angle of 90°. Measurements for each sample were repeated three times and reported as
mean values.
2.7. Cell culture
COS-7 (monkey SV40 transformed kidney fibroblast cells)
and MDA-MB-231 (human breast adenocarcinoma epithelial
cells) cell lines were obtained from ATCC (American Type
Culture Collection, Rockville, MD, USA). COS-7 cells were
maintained in Dulbecco's Modified Eagle's Medium (DMEM)
and MDA-MB-231 cells were maintained in ATCC Leibovitz's
L-15 medium at 37 °C in a humidified 5% CO2 atmosphere.
Growth medium was supplemented with fetal bovine serum
(10%), streptomycin (100 μg/mL) and penicillin (100 units/mL).
The human astrocytoma cell line U-373 MG with constitutive
firefly luciferase expression (U373-Luc) was generated by infecting U373 MG cells with recombinant retroviruses containing a
luciferase gene. U373-Luc cells were maintained in minimal
essential medium (ATCC) containing 10% FBS, G418 (300 μg/
mL), streptomycin (100 μg/mL) and penicillin (100 U/mL).
2.8. Cytotoxicity assay
2.4. Acid-base titration assay
The buffering capacity of the polydisulfide was determined
by acid-base titration with PEI and NaCl as controls. The
samples were dissolved in de-ionized water and pH of the
solutions was adjusted to 10. The polymer solutions (6.0 mL,
5.0 mM based on protonatable amino groups) were titrated with
aliquots of 2 μL 0.1 M HCl. pH of the solutions was measured
after each addition. A solution of NaCl (5.0 mM) was titrated
similarly as a control.
2.5. Gel electrophoresis assay
Agarose gel (0.8%, w/v) containing 0.5 μg/mL ethidium
bromide was prepared in TAE (Tris–Acetate–EDTA) buffer.
Plasmid DNA complexes were prepared by mixing DNA
(10 μL, 0.1 μg/μL) solution with an equal volume of polymer
solution or monomer solution at predetermined N/P ratios,
followed by 30-minute incubation. The samples (10 μL) were
mixed with 2 μL of 6× loading dye and the mixtures were
loaded onto an agarose gel. The gel was run at 100 V for 60 min
and the location of DNA bands was visualized with a UV
MDA-MB-231 cells were seeded in a 96-well plate at a
density of 10,000 cells/well and incubated for 24 h. The cells
were incubated with 200 μL of L-15 medium containing polymers at different concentrations for 24 h for assessing the
cytotoxicity of the polydisulfide with PEI as a control. The
cytotoxicity of the polyplexes of the polydisulfide or PEI at
different N/P ratios was evaluated by incubating with the cells
for 4 h followed by 44-hour incubation of the cells with fresh
complete medium. The concentration of plasmid DNA is fixed
at 5 μg/mL. MTT (25 μL, 5 mg/mL in PBS) was then added and
cells were further incubated for 2 h. The medium was removed
and 200 μL DMSO was then added to each well. The absorption
was measured at 570 nm using a microplate reader (Model 550,
Bio-Rad Lab. Hercules, CA). The relative cell viability was
calculated according to equation ([Abs]sample − [Abs]blank) /
([Abs]control − [Abs]blank) × 100%.
2.9. In vitro transfection
In vitro transfection efficiency of the polydisulfide was evaluated in COS-7 and MDA-MB-231 cells. COS-7 cells were
seeded 24 h prior to transfection into a 24-well plate. The
medium was then replaced with 1.0 mL fresh medium with or
without 100 μM chloroquine. The complexes of plasmid DNA
with polydisulfide or PEI at different N/P ratios were incubated
with the cells for 4 h at 37 °C. The medium was then replaced
with 1.0 mL fresh complete medium and cells were incubated for
an additional 44 h. The transfection tests were performed in
253
triplicates. After the incubation, cells were washed with prewarmed PBS and treated with 200 μL cell lysis buffer and then
subjected to a freezing-thawing cycle. Cellular debris was removed by centrifugation at 14,000 g for 5 min. The luciferase
activity in cell lysate (20 μL) was measured using a luciferase
assay kit (100 μL luciferase assay buffer) on a luminometer for
10 s (Lumat 9605, EG&G Wallac). The relative light units
Scheme 1. Resin-supported synthesis of dithiol-containing monomer possessing primary, secondary and tertiary charge groups. Reaction condition: (a) Triethylenetetramine, (b) Dde-OH, (c) Boc2O/DIPEA (d) 2% hydrazine in DMF, (e) Methyl acrylate, (f) 1,2-ethylenediamine, (g) Fmoc-His(Trt)-OH, TBTU/HOBt/
DIPEA, then 20% piperidine in DMF, (h) Fmoc-Cys(Trt)-OH TBTU/HOBt/DIPEA, then 20% piperidine in DMF, (i) TFA/H2O/EDT/TIBS (94/2.5/2.5/1).
GENE DELIVERY
X.-L. Wang et al. / Journal of Controlled Release 120 (2007) 250–258
GENE DELIVERY
254
X.-L. Wang et al. / Journal of Controlled Release 120 (2007) 250–258
(RLU) were normalized against protein concentration in the cell
extracts, measured by a BCA protein assay kit (Pierce, Rockford,
IL). The gene transfection efficiency of the polydisulfide in
MDA-MB-231 cells was similarly evaluated in the medium with
or without 10% serum.
2.10. Gene silencing with siRNA
U-373-Luc cells were used to evaluate the polydisulfide
mediated gene silencing efficiency. Cells were seeded 24 h prior
to transfection into a 96-well plate at a density of 2000 cells/
well. At the time of siRNA transfection, the medium in each
well was replaced with fresh serum-free or serum-containing
medium. The complexes of carrier/anti-luciferase siRNA at
different N/P ratios and a fixed siRNA concentration of 100 nM
were incubated with the cells and luciferase activities were
measured according to the same protocols described above. The
gene silencing efficiency was normalized against the luciferase
expression of untreated U373 MG-Luc cells.
3. Results
3.1. Synthesis of polydisulfide
A cationic dithiol monomer was first synthesized by solid
phase chemistry, Scheme 1. Triethylenetetraamine (large excess)
was attached to a 2-chlorotrityl chloride resin via one of the
primary amine. The other primary amino group of triethylenetetraamine in the resin was then protected by 2-acetyldimedone
(Dde-OH), a highly specific protection group for primary amines.
The secondary amines were protected by tert-butoxycarbonyl
(Boc) groups. Dde protecting group was removed by hydrazine
and the primary amine was converted into tertiary amine by
Michael addition reaction with methyl acrylate, followed by
Fig. 1. Acid titration profiles of NaCl, PEI and the polydisulfide (PDS).
Solutions were adjusted to pH 10 at room temperature and then titrated with
0.1 M HCl.
amidation with a large excess of ethylenediamine. Histidine
residues were then introduced by reacting with Fmoc-His(Trt)OH according to the standard Fmoc solid-phase peptide synthetic
procedures using TBTU/HOBt/DIPEA as coupling agents and
piperidine as a deprotecting agent. Finally, Fmoc-Cys(Trt)-OH
were similarly incorporated. The monomer was obtained by
treating the resin with 20% piperidine in DMF and then a TFA/
EDT/H2O/TIBS mixture, and purified by preparative HPLC.
Structure of the monomer was confirmed by 1H NMR and mass
spectroscopy. The molecular weight of the monomer was 855
(m/z). Polydisulfide was synthesized by oxidative polymerization of the dithiol monomer at 37 °C in DMSO, which was used
as a solvent as well as an oxidizing agent, Scheme 2. The average
molecular weight of the PDS was 6.2 kDa after purification with
dialysis.
3.2. Chemical and biophysical properties of polymer/polyplexes
The buffering capacity of the polydisulfide was determined
by acid titration assay. The polydisulfide exhibited high buffering capacity, the percentage of protonatable amine groups
becoming protonated, similar to that of PEI (branched, 25 KDa)
in endosomal-lysosomal pH range (5.0–7.4), Fig. 1. The buffering capacity in the range of pH 7.4 to 5.0 of the polydisulfide
Scheme 2. Synthesis of polydisulfide by oxidative polymerization.
Fig. 2. The size of PDS/DNA complexes at indicated N/P ratio.
and PEI was 34% and 32%, respectively. High buffering capacity of the polydisulfide might be attributed to the combination
effect of the primary, secondary, tertiary and aromatic amino
groups in the polymer.
The polydisufide carrier formed stable nanoparticles with
both plasmid DNA and siRNA. The size of nanoparticles
varied with the N/P ratios of the complexes, Fig. 2. A relatively
large particle size (600–700 nm) was observed for the complexes with both DNA and siRNA at a N/P ratio of 5. Significant size reduction was observed for the complexes with DNA
at an N/P ratio of 40 and the size then decreased to approximately 150 nm at an N/P of 80 or higher. For the complexes
with siRNA, their sizes also decreased with increased N/P ratio
and reached stable sizes of approximately 300 nm at an N/P
ratio of 40 or higher.
The formation of polydisulfide/DNA polyplexes was also
studied by gel electrophoresis shift assay. As shown in Fig. 3(a),
the monomer could not retard DNA migration in gel electrophoresis up to an N/P ratio of 9. In comparison, DNA was
partially retained by the polydisulfide at an N/P ratio of 1 and
completely retained at an N/P ratio of 1.5 or higher. The polyplexes exhibited high stability at an N/P ratio of 3 in the
presence of salt (0.15 M NaCl) or negatively charged heparin
(1.0 mg/mL). As shown in Fig. 2(b), no detectable dissociation
of the polyplexes was observed at the physiological ionic
strength and in the presence of competitive anionic biomacromolecules. However, the release of DNA from the polyplexes
was observed in a reductive environment. The presence of
disulfide reducing agent DTT led to the release of free DNA,
Fig. 3(b). High ionic strength or the presence of heparin further
facilitated the release of DNA in the reductive environment. The
results suggest that the DNA polyplexes with the cationic
polydisulfide are stable extracellularly and are able to release
DNA intracellularly to enhance transfection efficiency.
255
Fig. 4. Cell viability of polydisulfide and PEI at different concentrations
(a) and of polyplexes at different N/P ratios (b) with a DNA concentration
fixed at 5 μg/mL.
3.3. Cytotoxicity
The polydisulfide had much lower cytotoxicity than PEI as
shown by MTT assay in the incubation with MDA-MB-231
cells, Fig. 4. Cells incubated with the polydisulfide remained
100% viable at a concentration of 125 μg/mL and more than
60% viable at a concentration of 500 μg/mL, while only 10% of
cells incubated with PEI remained viable at a concentration of
125 μg/mL, Fig. 4(a). The cytotoxicity of the polydisulfide/
DNA complexes was also evaluated at high N/P ratios and a
fixed DNA concentration of 5 μg/ml. As shown in Fig. 4(b), the
polydisulfide/DNA polyplexes at the N/P ratios of 50 and 100
were much less toxic than PEI at the N/P ratio of 10.
3.4. In vitro gene transfection
Fig. 3. (a) Gel electrophoresis shift assay at the indicated N/P ratios. Lane 1:
DNA only; Lane 2–4: monomer/DNA complexes; lane 5–9: polymer/DNA
complexes. (b) Reduction destabilizes polyplexes in the presence of NaCl or
heparin. Polyplexes with N/P ratio of 3 were used in the study. Lane 1–4 were
performed in the absence of DTT, lane 5–8 were performed in the presence of
0.1 M DTT. Polyplexes were incubated for 4 h at 37 °C. Lane 1: DNA only; lane
2: polyplexes only; lane 3: polyplexes with 0.15 M NaCl; lane 4: polyplexes
with 1.0 mg/mL heparin. Lane 5: DNA only; lane 6: polyplexes only; lane 7:
polyplexes with 0.15 M NaCl; lane 8: polyplexes with 1 mg/mL Heparin.
(ND = naked DNA, O.C. = open circular, S.C. = supercoiled form of plasmid
DNA).
In vitro transfection efficiency of the polydisulfide/DNA
complexes was evaluated in COS-7 cells with gWiz plasmid
DNA encoding luciferase as a reporter gene. As shown in
Fig. 5(a), luciferase expression mediated by the complexes
was N/P ratio dependent. The transfection efficiency mediated
by the polydisulfide increased with the N/P ratios and high gene
transfection efficiency was observed at an N/P ratio of 100. A
relatively high charge ratio was required to achieve the high
transfection for the cationic polydisulfide, which is similar to
GENE DELIVERY
X.-L. Wang et al. / Journal of Controlled Release 120 (2007) 250–258
GENE DELIVERY
256
X.-L. Wang et al. / Journal of Controlled Release 120 (2007) 250–258
other reported biodegradable gene carriers [20,30]. The transfection efficiency may correlate to the size of the polyplexes
because the size decreased with the increase of N/P ratio, from
a diameter of 580 nm at N/P = 20 to 135 nm at N/P = 80. At the
N/P ratio of 100, the polydisulfide resulted in gene expression
approximately 3-fold lower than PEI in COS-7 cells. It is
interesting to note that the presence of chloroquine diphosphate
(CQ, 100 μM), a reagent known to disrupt endosomal membrane, did not significantly affect gene expression mediated by
the polydisulfide, indicating that the cationic polydisulfide
possessed high buffering capacity that may assist the polyplexes
escaping from endosomal compartments.
The transfection efficiency of the polydisulfide in MDAMB-231 cells peaked at an N/P ratio of 80 and was comparable
to that of PEI, Fig. 5(b). The polydisulfide also showed good
transfection efficiency in the presence of serum, particularly at
lower N/P ratios. The presence of 10% serum resulted in slight
decrease of transfection efficiency for the polydisulfide by 1.2,
2.8, 1.6, 4.5 and 6.5-folder at the N/P ratios of 20, 40, 60, 80
and 100, respectively, while PEI-mediated transfection de-
Fig. 6. Endogenous luciferase gene silencing efficiency mediated by the polydisulfide or PEI/siRNA (100 nM of siRNA) complexes in U373-Luc cells in the
absence or presence of 10% FBS. (Mean ± standard deviation (n = 3) for all
experiments).
creased by 10.4-folder under the same condition as compared to
that in the absence of serum.
3.5. In vitro gene silencing
The siRNA delivery efficacy of the polydisulfide was investigated with an anti-luciferase siRNA in U373-Luc cell line
with stable expression of firefly luciferase. As shown in Fig. 6,
luciferase silencing efficiency mediated by the polydisulfide/
siRNA complexes was as efficient as the PEI/siRNA complexes
in a serum-free medium. The luciferase expression was inhibited up to ∼ 70% for both polydisulfide/siRNA (N/P = 30) and
PEI/siRNA (N/P = 10) complexes. However, the PEI/siRNA
complexes had poor gene silencing efficacy (b 10%) in the
presence of 10% FBS, which might be attributed to unfavorable
interaction between the polyplexes and serum proteins. Under
the same condition, the polydisulfide/siRNA complexes still
exhibited approximately 40% gene silencing efficiency.
4. Discussion
Fig. 5. (a) Transfection efficiency of the polydisulfide/DNA (2 μg) complexes in
COS 7 cells at different N/P ratios in the absence or presence of 100 μM CQ
with PEI and naked DNA as controls. (b) Transfection efficiency of the
polydisulfide/DNA (2 μg) complexes in MB-231 cells at different N/P ratios in
the absence or presence of 10% FBS with PEI and naked DNA as controls.
(Mean ± standard deviation (n = 3) for all experiments.)
In this study, we have designed a novel environmental
sensitive polydisulfide with protonatable pendants for nucleic
acid delivery. The polydisulfide serve as a reducible backbone to
facilitate the release of nucleic acids in cytoplasm. The polydisulfide carrier has some unique features as compared to other
reported reducible carriers. Flexible side chains with multiple
protonatable amino groups are designed to form stable nanoparticulate complexes with nucleic acids. Histidine residues,
primary and tertiary amines are incorporated in the backbone to
enhance the buffering capacity of the carrier. The polydisulfide
showed higher buffering capacity than PEI at pH 5.0–7.4. The
carrier can form stable complexes with both plasmid DNA and
siRNA with sizes ranging from 100 to 750 nm, depending on the
N/P ratios of the complexes. The complexes with high N/P
ratios, up to a N/P ratio of 80 for DNA and 40 for siRNA,
resulted in small and compact nanoparticles. The relatively low
molecular weight of the polydisulfide might be the reason that a
high N/P ratio was required to form smaller nanoparticles. In
vitro cytotoxicity study demonstrated that the polydisulfide had
much lower cytotoxicity than PEI and could be a safer carrier for
nucleic acid delivery.
Gel electrophoresis showed that the polydisulfide carrier
formed stable complexes with plasmid DNA at an N/P ratio as
low as 1.5, while the monomer was not effective to form
complexes. The polydisulfide/DNA complexes at an N/P ratio
of 3 were stable in the presence of salt and polyanions.
However, the complexes can be dissociated to release DNA in
the reductive cytosolic environment. The results validated the
hypothesis that the polydisulfide with protonatable pendants
has the sensibility to reductive environment. It is an advantageous and necessary feature of the polydisulfide for effective
in vivo delivery of nucleic acids. High stability of the polydisulfide-nucleic acid complexes in oxidative extracellular
environment will protect nucleic acid from enzymatic degradation and dissociation of the complexes in the reductive
cytoplasm will release nucleic acids and enhance their biological activities.
In vitro gene transfection efficiency of the polydisulfide was
demonstrated in two different cell lines. The transfection efficiency of the polydisulfide/DNA complexes increased with
high N/P ratios of the complexes in both COS-7 and MDA-MB231 cell lines. The transfection efficiency may be related to the
size of the complexes because the transfection efficiency increased with decreasing sizes of the nanoparticles at high N/P
ratios. The best transfection efficiency of the polydisulfide/
DNA complexes was comparable to or slightly lower than PEI.
The presence of an endosomal membrane disrupting agent,
chloroquine diphosphate, did not affect the transfection efficiency at the test N/P ratios in COS-7 cell line. However, some
of the reported disulfide linked reducible cationic carriers
demonstrated low transfection efficiency and chloroquine assistance was required to achieve a comparable transfection efficiency to that of PEI [31]. The result indicates that the novel
polydisulfide is able to disrupt the endosomal membrane facilitating endosomal escape of the complexes because of high
buffering capacity due to its protonatable amines with different
pKas. It appears that the presence of serum resulted in less
reduction of transfection efficiency of the polydisulfide/DNA
complexes than PEI in MDA-MB-231 cell line, which is critical
for in vivo gene transfection.
The polydisulfide with protonatable pendants might be more
suitable for siRNA delivery. The side chains with multiple
protonatable amino groups would be effective to form stable
complexes with short nucleic acids because of the flexibility of
the side chains. It is shown here that the polydisulfide resulted in
comparable gene silencing efficiency as PEI. However, the gene
silencing efficiency of PEI dramatically reduced in the presence
of serum. Although the gene silencing efficiency of the polydisulfide also decreased in the presence of serum, the polydisulfide still retained much more significant gene silencing
efficiency than PEI (p b 0.05).
We have shown in this study that a novel polydisulfide with
protonatbale pendants and multifunctional amino groups, including primary, secondary, tertiary and aromatic groups, has
257
good buffering capacity, low cytotoxicity, environmental sensitivity and good in vitro delivery efficiency for both plasmid
DNA and siRNA. It demonstrated comparable delivery efficiency to that of PEI, a commonly used cationic polymeric
carrier with high cellular delivery efficiency. Although these
results are encouraging, the N/P ratio for efficient delivery is
still high for the polydisulfide and further structural modification is needed to improve its delivery efficiency of nucleic acids.
Increased molecular weight and elongated side chains with
more protonatable amino groups of the polydisulfide may be
necessary to form small and compact nanoparticles with nucleic
acids at low N/P ratios, which is important for high cellular
transfection. Our ongoing research is focused on the structural
modification of this lead to design and develop safe and effective carriers for nucleic acids.
5. Conclusion
The novel environment sensitive polydisulfide with protonatable pendants demonstrated low cytotoxicity, good buffering
capacity and formed stable polyplexes with nucleic acids. The
polydisulfide/DNA complexes were stable in normal physiological environment and readily dissociated to release DNA in
the reductive environment, resulting in good gene transfection
efficiency. The polydisulfide also resulted in efficient siRNA
delivery and effective gene silencing, particularly in the serumcontaining medium. The polydisulfide can be a safe and effective carrier for in vitro delivery of nucleic acids, including both
DNA and siRNA. It is a promising lead for further design and
development of safer and more efficient carriers for nucleic acid
delivery.
References
[1] I. McNeish, S. Bell, N. Lemoine, Gene therapy progress and prospects:
cancer gene therapy using tumour suppressor genes, Gene Ther. 11 (2004)
497–503.
[2] J. Rosenecker, S. Huth, C. Rudolph, Gene therapy for cystic fibrosis lung
disease: current status and future perspectives, Curr. Opin. Mol. Ther.
8 (2006) 439–445.
[3] U. Griesenbach, D. Geddes, E. Alton, Gene therapy progress and prospects: cystic fibrosis, Gene Ther. 13 (2006) 1061–1067.
[4] A. Fire, S. Xu, M. Montgomery, S. Kostas, S. Driver, C. Mello, Potent and
specific genetic interference by double-stranded RNA in caenorhabditis
elegans, Nature 391 (1998) 806–811.
[5] J. Couzin, Small rnas make big splash, Science 298 (2002) 2296–2297.
[6] G. Hannon, Rna interference, Nature 418 (2002) 244–251.
[7] C. Mello, D. Conte, Revealing the world of RNA interference, Nature
431 (2004) 338–342.
[8] D. Luo, W. Saltzman, Synthetic DNA delivery systems, Nat. Biotechnol.
18 (2000) 33–37.
[9] G. Odom, P. Gregorevic, J. Chamberlain, Viral-mediated gene therapy for
the muscular dystrophies: successes, limitations and recent advances,
BBA-Mol. Basis Dis. 1772 (2007) 243–262.
[10] S. Zhang, Y. Xu, B. Wang, W. Qiao, D. Liu, Z. Li, Cationic compounds
used in lipoplexes and polyplexes for gene delivery, J. Control. Release
100 (2004) 165–180.
[11] S. Li, L. Huang, Surface-modified LPD nanoparticles for tumor targeting,
Ann. N.Y. Acad. Sci. 1082 (2006) 1–8.
[12] K. Ewert, A. Ahmad, H. Evans, C. Safinya, Cationic lipid-DNA complexes for non-viral gene therapy: relating supramolecular structures to
cellular pathways, Expert Opin. Biol. Ther. 5 (2005) 33–53.
GENE DELIVERY
X.-L. Wang et al. / Journal of Controlled Release 120 (2007) 250–258
GENE DELIVERY
258
X.-L. Wang et al. / Journal of Controlled Release 120 (2007) 250–258
[13] W. Kim, J. Yockman, J. Jeong, L. Christensen, M. Lee, Y. Kim, S. Kim,
Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-gPEG-RGD/pcmv-SFLT-1 complexes in tumor-bearing mice, J. Control.
Release 114 (2006) 381–388.
[14] K. Wong, G. Sun, X. Zhang, H. Dai, Y. Liu, C. He, K. Leong, PEI-gchitosan, a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo,
Bioconjug. Chem. 17 (2006) 152–158.
[15] C. Lee, J. MacKay, J. Frechet, F. Szoka, Designing dendrimers for biological applications, Nat. Biotechnol. 23 (2005) 1517–1526.
[16] H. Mao, K. Roy, V. Troung-Le, K. Janes, K. Lin, Y. Wang, J. August, K.
Leong, Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency, J. Control. Release 70 (2001) 399–421.
[17] H. Lv, S. Zhang, B. Wang, S. Cui, J. Yan, Toxicity of cationic lipids and
cationic polymers in gene delivery, J. Control. Release 114 (2006) 100–109.
[18] D. Wu, Y. Liu, X. Jiang, L. Chen, C. He, S. Goh, K. Leong, Evaluation of
hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery, Biomacromolecules 6 (2005) 3166–3173.
[19] C. Ahn, S. Chae, Y. Bae, S. Kim, Synthesis of biodegradable multi-block
copolymers of poly(l-lysine) and poly(ethylene glycol) as a non-viral gene
carrier, J. Control. Release 97 (2004) 567–574.
[20] J. Green, J. Shi, E. Chiu, E. Leshchiner, R. Langer, D. Anderson, Biodegradable polymeric vectors for gene delivery to human endothelial cells,
Bioconjug. Chem. 17 (2006) 1162–1169.
[21] J. Kloeckner, E. Wagner, M. Ogris, Degradable gene carriers based on
oligomerized polyamines, Eur. J. Pharm. Sci. 29 (2006) 414–425.
[22] Z. Zhong, Y. Song, J. Engbersen, M. Lok, W. Hennink, J. Feijen,
A versatile family of degradable non-viral gene carriers based on hyperbranched poly(ester amine)s, J. Control. Release 109 (2005) 317–329.
[23] D. McKenzie, K. Kwok, K. Rice, A potent new class of reductively activated peptide gene delivery agents, J. Biol. Chem. 275 (2000) 9970–9977.
[24] D. Oupicky, A. Parker, L. Seymour, Laterally stabilized complexes of
DNA with linear reducible polycations: Strategy for triggered intracellular
activation of DNA delivery vectors, J. Am. Chem. Soc. 124 (2002) 8–9.
[25] D. Manickam, D. Oupicky, Multiblock reducible copolypeptides containing histidine-rich and nuclear localization sequences for gene delivery,
Bioconjug. Chem 17 (2006) 1395–1403.
[26] M. Gosselin, W. Guo, R. Lee, Efficient gene transfer using reversibly crosslinked low molecular weight polyethylenimine, Bioconjug. Chem. 12 (2001)
989–994.
[27] L. Christensen, C. Chang, W. Kim, S. Kim, Z. Zhong, C. Lin, J. Engbersen,
J. Feijen, Reducible poly(amido ethylenimine)s designed for triggered
intracellular gene delivery, Bioconjug. Chem. 17 (2006) 1233–1240.
[28] Z. Zhong, J. Feijen, M. Lok, W. Hennink, L. Christensen, J. Yockman,
Y. Kim, S. Kim, Low molecular weight linear polyethylenimine-b-poly
(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties, Biomacromolecules
6 (2005) 3440–3448.
[29] C. Lin, Z. Zhong, M. Lok, X. Jiang, W. Hennink, J. Feijen, J. Engbersen,
Novel bioreducible poly(amido amine)s for highly efficient gene delivery,
Bioconjug. Chem. 18 (2007) 138–145.
[30] M. Read, S. Singh, Z. Ahmed, M. Stevenson, S. Briggs, D. Oupicky, L.
Barrett, R. Spice, M. Kendall, M. Berry, J. Preece, A. Logan, L. Seymour,
A versatile reducible polycation-based system for efficient delivery of a
broad range of nucleic acids, Nucleic Acids Res. 33 (9) (2005).
[31] D. Manickam, H. Bisht, L. Wan, G. Mao, D. Oupicky, Influence of tatpeptide polymerization on properties and transfection activity of TAT/
DNA polyplexes, J. Control. Release 102 (2005) 293–306.