Effective gene delivery using stimulus-responsive catiomer designed with redox-sensitive disulfide and acid-labile imine linkers

Article abstract of DOI:10.1021/bm2017355

A dual stimulus-responsive mPEG-SS-PLL15-glutaraldehyde star (mPEG-SS-PLL15-star) catiomer is developed and biologically evaluated. The catiomer system combines redox-sensitive removal of an external PEG shell with acid-induced escape from the endosomal c

Full text of DOI:10.1021/bm2017355

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Effective Gene Delivery Using Stimulus-Responsive Catiomer
Designed with Redox-Sensitive Disulfide and Acid-Labile Imine
Linkers
Xiaojun Cai,†,# Chunyan Dong,‡,# Haiqing Dong,† Gangmin Wang,† Giovanni M. Pauletti,∥ Xiaojing Pan,§
Huiyun Wen,† Isaac Mehl,† Yongyong Li,*,† and Donglu Shi*,†,⊥
‡
†The Institute for Advanced Materials and Nano Biomedicine, School of Medicine, Department of Oncology, Shanghai East
§
Hospital, and School of Life Science and Technology, Tongji University, Shanghai 200092, China
⊥
∥James L. Winkle College of Pharmacy and School of Electronic and Computing Systems, College of Engineering and Applied
Science, University of Cincinnati, Cincinnati, Ohio, 10 45221, United States
ABSTRACT: A dual stimulus-responsive mPEG-SS-PLL15-glutaralde-
hyde star (mPEG-SS-PLL15-star) catiomer is developed and biologically
evaluated. The catiomer system combines redox-sensitive removal of an
external PEG shell with acid-induced escape from the endosomal
compartment. The design rationale for PEG shell removal is to augment
intracellular uptake of mPEG-SS-PLL15-star/DNA complexes in the
presence of tumor-relevant glutathione (GSH) concentration, while the
acid-induced dissociation is to accelerate the release of genetic payload
following successful internalization into targeted cells. Size alterations of
complexes in the presence of 10 mM GSH suggest stimulus-induced
shedding of external PEG layers under redox conditions that intra-
cellularly present in the tumor microenvironment. Dynamic laser light
scattering experiments under endosomal pH conditions show rapid
destabilization of mPEG-SS-PLL15-star/DNA complexes that is followed
by facilitating efficient release of encapsulated DNA, as demonstrated by agarose gel electrophoresis. Biological efficacy
assessment using pEGFP-C1 plasmid DNA encoding green fluorescence protein and pGL-3 plasmid DNA encoding luciferase as
reporter genes indicate comparable transfection efficiency of 293T cells of the catiomer with a conventional polyethyleneimine
(bPEI-25k)-based gene delivery system. These experimental results show that mPEG-SS-PLL15-star represents a promising
design for future nonviral gene delivery applications with high DNA binding ability, low cytotoxicity, and high transfection
efficiency.
journey, the chemical stability of encapsulated genetic materials
must be guaranteed until it reaches the nucleus.8 Inefficient
escape from the endosomal compartment usually results in
chemical degradation of DNA following cellular internalization
of polyplexes.9 Similarly, premature and incomplete unpacking
of DNA from polyplexes significantly decreases transfection
efficiency.4,10 In addition, it has been recognized that cationic
polyplexes are rapidly cleared from the circulatory system
following interaction with negatively charged blood compo-
nents.11,12 Hence, innovative approaches that address these
critical issues of nonviral gene delivery vectors are required to
offer viable therapeutic options in the future.
1. INTRODUCTION
Gene therapy is considered to be one of the most promising
approaches to treat diseases of genetic defects. In gene therapy,
genetic materials, either RNA or DNA, are transferred into
specific human tissues or cells to replace defective genes,
substitute missing genes, silence unwanted gene expression, or
introduce new cellular biofunctions.1 Clinical success of these
interventions, however, relies on the development of efficient,
nontoxic gene delivery vectors that are capable of mediating
high and sustained levels of gene expression. Polyplexes formed
by electrostatic interaction between cationic polymers (i.e.,
catiomers) and plasmid DNA (pDNA) have gained much
attention as nonviral gene delivery vectors due to their
respectable DNA loading capacity, easy of fabrication, limited
immunogenicity, and versatility for chemical modifications.2−5
Therapeutic efficacy of polyplex-mediated gene delivery
strategies critically depends on effective condensation of genetic
materials, successful distribution to desired target cells, cellular
internalization, endosomal escape, efficient unpacking, and
nuclear transport of genetic payload.6,7 Throughout this
Surface functionalization of catiomers using hydrophilic
polymers such as poly(ethylene glycol) (PEG) has become a
viable strategy to reduce positive surface charge of polyplexes
and, consequently, the toxic side effects. In addition, surface
Received: December 6, 2011
Revised: March 2, 2012
Published: March 9, 2012
© 2012 American Chemical Society
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Scheme 1. Schematic Diagram Illustrating mPEG-SS-PLL15-star Catiomer for pDNA Encapsulation and the Intracellular
Stimulus-Responsive pDNA release
coverage with a PEG shell protects catiomer/DNA complexes
from uptake by the reticuloendothelial system (RES),
effectively prolonging in vivo circulation time.13−16 Never-
theless, PEG-shielding has been demonstrated to reduce DNA
binding,17 cellular uptake,18,19 and endosomal escape.20−22 To
solve this dilemma, previous research introduced catiomers
bearing chemically cleavable PEG shells.23,24 This design helps
maintain the stability of the catiomer/DNA complex during
biodistribution, at the desired target cell; however, removal or
“shedding” of this hydrophilic surface layer enhances cellular
interactions, thereby facilitating efficient internalization.15
Various experiments demonstrated that transfection efficiency
of lipid or polymer-based gene delivery systems fabricated with
a cleavable PEG layer is superior to that of conventional
polyplexes.25−27 In particular, PEG shedding in response to a
redox-sensitive stimulus has gained much interest for drug and
gene delivery in cancer therapy, as the tumor microenviron-
ment generally presents a higher concentration of the
physiological reducing agent glutathione (GSH).28,29 For
example, Takae and coworkers reported a three-fold increase
in gene transfection efficiency using PEG-detachable PEG-SS-
P[Asp(DET)] micelles as nonviral delivery vector.30 Similar
results were recently reported from our studies where
transfection efficiency in HeLa cells increased about three- to
six-fold when mPEG-SS-PLL45 polyplexes were used instead of
mPEG-PLL40 delivery vectors.31
Because the therapeutic payload of gene delivery vectors
must be delivered into the nucleus, successful endosomal
escape of the catiomer/DNA complex following cell internal-
ization presents a critical step in gene therapy. This behavior
was reported to be associated with the proton sponge effect of
catiomer.7 Protonation of catiomer induces rapid flux of ions
and water into the subcellular compartment, eventually
resulting in rupture of the endosomal membrane, followed by
release of the entrapped compartments into the cytosol.9 The
high density of ionizable functional groups in polyethylenei-
mine (PEI) has been demonstrated to accelerate effectively
endosomal escape.32,33 In contrast, transfection efficiency of
poly-L-lysine (PLL)-based gene delivery vectors is generally
unsatisfactory because of its lower amine group density,33 and
most of the amine group has already been protonated at pH
7.4. To increase biologically efficacy, PLL has been chemically
modified using various moieties that improve endosomal
escape.34 For example, incorporation of membrane-fusion
peptide PAsp(DET) induces remarkable destabilization of the
endosomal membrane structure during acidification.35,36
Similarly, histidine-modified PLL also efficiently augments
gene transfer.37,38 Alternate chemical designs based on
inclusion of acid-labile linkers such as acetal-ketal,39 hydra-
zone,15 orthoester,5,40 and imine moieties32 could also increase
the transfection efficiency by accelerating the degradation of
carriers in the acidic endosomal compartment and facilitate the
release of payload pDNA.
On the basis of these findings, we engineered and developed
a unique mPEG-SS-PLL15-star catiomer that can combine
redox-sensitive removal of an external PEG shell with acid-
induced escape from the endosomal compartment. In
particular, the rational design offers this catiomer with star
polymer’s distinct properties including a 3-D globular compact
architecture that has unique properties compared with its linear
analogs.41−43 It is hypothesized that cleavage of the PEG layers
in response to a reductive environment of intracellular or tumor
microenvironment can effectively increase cellular uptake, as
illustrated in Scheme 1. Subsequently, the acid-labile PLL
segments cross-linked by imine linkers are expected to degrade
in the acidic endosome, forming low-molecular-weight frag-
ments such as mPEG-SS-PLL15 that facilitates endosomal
escape of therapeutically active DNA payload. The phys-
icochemical properties of mPEG-SS-PLL15-star were charac-
terized to show the structural consequences of the polymer
system upon exposure to high GSH concentrations and
endosomal pH conditions. Gene transfection efficiency was
evaluated in vitro using the pEGFP and pGL-3 pDNA as the
reporter genes.
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(MW cutoff: 3500 Da) for 24 h against water. The purified dual
stimulus-responsive mPEG-SS-PLL15-star catiomer was collected after
lyophilization.
2. EXPERIMENTAL SECTION
2.1. Materials. Poly(ethylene glycol) monomethyl ether (mPEG,
MW = 2000 g/mol) was purchased from Yare Biotech, and ε-
benzyloxycarbonyl-L-lysine was purchased from GL Biochem. Succinic
anhydride, glutaraldehyde (50% in water), cysteamine hydrochloride
(98%), and triphosgene (99%) were purchased from Aladdin and used
as received. Triethylamine (Et3N, 99%, Sigma) was used as received.
Tetrahydrofuran (THF), dichloromethane (DCM), and N,N-dimethyl
formamide (DMF) were dried by refluxing over CaH2, distilled, or
vacuum-distilled before use. Hydrogen bromide (HBr) 33% (w/w)
solution in glacial acetic acid was purchased from ACROS Organics.
Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate
buffered saline (PBS), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT), trypsin-EDTA, fetal bovine serum (FBS),
and penicillin-streptomycin were purchased from Gibco Invitrogen.
Dialysis bags (Spectra/Por 7) were purchased from Spectrum
Laboratories. Branched poly(ethylenimine) (bPEI-25 k, MW = 25
000 g/mol) was obtained from Aldrich-Sigma Chemical. Block
copolymer mPEG-PLL40 without disulfide bonds was synthesized by
our laboratory. Label IT Tracker intracellular nucleic acid localization
kit Cy3 was purchased from Mirus Bio. BCA protein assay kit was
purchased from Pierce. Luciferase assay system and reporter lysis
buffer were purchased from Promega. The reporter plasmids, pEGFP-
C1 and pGL-3, were purchased from Invitrogen and stored at −20 °C
until transfection experiments.
2.2. Synthesis of mPEG-SS-PzLL15 Intermediate. The synthesis
of mPEG-SS-PzLL15 was accomplished according to the following
strategy: (i) preparation of zLL-NCA; (ii) preparation of mPEG-SS-
NH2; and (iii) ring-opening polymerization of zLL-NCA initiated by
mPEG-SS-NH2.
ε-Benzyloxycarbonyl-L-lysine (zLL, 5.6 g, 20 mmol) was suspended
in dry THF (50 mL) at 50 °C, and triphosgene (2.5 g, 8.4 mmol) was
slowly added under nitrogen. Following completion of the reaction,
crude zLL-NCA was precipitated with excess dry n-hexane and further
purified by crystallizing using THF/n-hexane (1:15, v/v).
The mPEG-SS-NH2 intermediate was prepared following a protocol
previously published by our laboratory.31 In brief, N-hydroxysuccini-
mide (NHS) (0.07 g, 0.6 mmol), N,N′-dicyclohexylcarbodiimide
(DCC) (0.12 g, 0.6 mmol), and mPEG-COOH (1.0 g, 0.5 mmol)
were dissolved in DCM (30 mL) at 0 °C for 5 h. Subsequently, a dry
solution of cysteamine (0.5 g, 3.5 mmol) in 5 mL of DCM was added
dropwise, and the reaction was maintained at room temperature (RT)
for additional 24 h under a dry nitrogen atmosphere. The product was
isolated by filtration to remove insoluble byproducts, precipitated
twice in cold diethylether and purified by 24 h of dialysis (MW cutoff:
1000 Da) against water. mPEG-SS-NH2 was collected after
lyophilization (NMR (ppm): 3.55 (−CH2CH2O−), 3.4 (−CH3O),
2.5 (−SS−CH2−CH2−NH−), 2.35 (−SS−CH2−CH2−NH−)).
mPEG-SS-NH2 and zLL-NCA were combined in dry DMF at a
molar ratio of 1:15. Ring-opening polymerization was allowed to
proceed for 3 days at RT. The desired mPEG-SS-PzLL15 was purified
by 24 h of dialysis (MW cutoff: 3500 Da) against water and collected
after lyophilization.
2.3. Synthesis of Redox-Sensitive mPEG-SS-PLL15 Catiomer.
mPEG-SS-PzLL15 was dissolved in 10 mL of trifluoroacetic acid (TFA)
and combined with two mole equivalents (with respect to the benzyl
carbamate group) of a 33% (w/w) HBr solution in acetic acid. The
solution was stirred at 0 °C for 1 h under nitrogen and precipitated in
excess diethyl ether. The crude product was dissolved in DMF and
dialyzed (MW cutoff: 3500 Da) for 6 h against distilled water
containing a few drops of ammonia solution (pH 9.0) to remove the
HBr. After refreshing with pure distilled water, the crude product was
dialyzed for an additional 48 h. Deprotected mPEG-SS-PLL15 was
collected after lyophilization.
2.5. Chemical Properties of mPEG-SS-PLL15-star Catiomer.
Proton nuclear magnetic resonance (1H NMR) spectra were recorded
on the Advance500 MHz spectrometer (Switzerland) using DMSO-d6
as solvent and TMS as standard. Molecular weight analysis was
performed using the Applied Biosystems 4700 Proteomics (TOF/
TOF) Analyzer (Framingham, MA). The UV Nd:YAG laser was
operated at a 200 Hz repetition rate wavelength of λ = 355 nm.
Accelerated voltage was operated at 20 kV under batch mode
acquisition control. The solution was 0.001:1:2 (v/v) TFA/acetonitrile
(ACN)/water. Mass spectral data were processed using Data Explorer
4.0 (Applied Biosystems).
2.6. Cell Viability Assay. Human embryonic kidney transformed
293 (293T) and human cervix carcinoma (HeLa) cells were obtained
from the Cell Center of the Tumor Hospital at Fudan University and
routinely maintained at 37 °C in a humidified 5% (v/v) CO2
atmosphere using DMEM supplemented with 10% FBS and 0.1%
(v/v) penicillin/streptomycin solution. Cell viability in the presence
and absence of various mPEG-SS-PLL15-star catiomers was deter-
mined using the MTT assay. mPEG-SS-PLL15 and bPEI-25k served as
control. In brief, 293T and HeLa cells were seeded into a 96-well plate
at a density of 5 × 103 cells/well. Following an overnight attachment
period, cells were exposed to various catiomer concentrations (13.2 to
225 mg/L) prepared in cell culture medium. After 24 h, the medium
was replaced with 200 μL of fresh DMEM, 20 μL of a MTT solution
(5 mg/mL) in PBS pH 7.4 was added, and the plate incubated at 37
°C for additional 4 h. Subsequently, the culture medium was removed
and 150 μL of DMSO was added to each well, and after an additional
10 min of incubation at 37 °C optical density (OD) was measured at λ
= 492 nm using the Multiscan MK3 plate reader (Thermo Fisher
Scientific, Waltham, MA). The relative cell viability in percent (%) was
calculated according to: (OD sample/OD control) × 100%, where OD
control was measured in the absence of the polymers and OD sample
in the presence of the polymers. Each concentration was studied using
six independent experiments.
2.7. Buffering Capacity of mPEG-SS-PLL15-star Catiomers.
Relative buffering capacities of mPEG-SS-PLL15-star catiomers and
bPEI-25k were compared using acid−base titration. Polymers were
dissolved at 200 mg/L in 50 mM NaCl solution. Initially, the pH value
of this solution was adjusted to pH 10 using 0.1 M NaOH.
Sequentially, 0.1 M HCl aliquots were added and pH value was
measured after each addition using a microprocessor pH meter.
2.8. Fabrication of mPEG-SS-PLL15-star/pDNA Complexes. A
pDNA stock solution (120 ng/μL) was prepared in 40 mM Tris-HCl
buffer (pH 7.4). Separately, mPEG-SS-PLL15-star was dissolved in 150
mM NaCl at 2 mg/mL, and the solution was cleared by filtration (0.22
μm). Polyplexes were formed by adding mPEG-SS-PLL15-star with a
desired concentration to pDNA stock solution, resulting in catiomer/
pDNA ratios ranging from 0:1 to 8:1 w/w for agarose gel retardation
assay and 1:1 to 10:1 w/w for particle size distribution and zeta
potential measurement. Complexes were vortexed gently, then allowed
to incubate for 30 min at 37 °C before use.
2.9. Agarose Gel Retardation Assay. To assess the ability of
catiomers to condensate DNA into electrostatically stabilized
polyplexes, we performed the agarose gel retardation assay. Routinely,
catiomer/pDNA complex suspensions containing 0.1 μg of pDNA
were loaded onto 1% (w/v) agarose gel containing ethidium bromide.
Electrophoretic separation was carried out for 40 min at 120 V in Tris-
acetate running buffer. DNA bands were visualized at λ = 254 nm
using an Imago GelDoc system.
2.10. Particle Size Distribution and Zeta Potential. To
determine relevant physicochemical properties of polyplexes, we
measured particle size distribution and zeta potential of fabricated
catiomer/pDNA complexes containing 1 μg pDNA by Nano-ZS 90
Nanosizer (Malvern Instruments, Worcestershire, U.K.) according to
the manufacturer’s instructions. If required, polyplex suspension was
diluted with 150 mM NaCl.
2.4. Synthesis of Dual Stimulus-Responsive mPEG-SS-PLL15-
star Catiomer. mPEG-SS-PLL15 was dissolved in double-distilled
water using a 50 mL two-necked flask, and an aqueous solution of
glutaraldehyde was added dropwise over 2 h under vigorous stirring at
RT. The reaction mixture was stirred for additional 4 h before dialysis
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Figure 1. Synthesis of novel mPEG-SS-PLL15 catiomers (A) and reaction scheme for copolymerization of mPEG-SS-PLL15 in the presence of
glutaraldehyde (B).
2.11. Transmission Electronic Microscopy (TEM). To visualize
mPEG-SS-PLL15-star/pDNA association complexes, polyplexes were
prepared at a weight ratio of 5:1 and observed in a Hitachi H-7100
transmission electron microscope using an acceleration voltage of 100
kV.
in 24-well plates at a density of 5 × 104 cells/well. pGL-3-containing
polyplexes fabricated at various w/w ratios ranging from 1:1 to 10:1
were suspended in serum-free DMEM and added to each well (1 μg
DNA/well). Following a 4 h incubation at 37 °C in a humidified
atmosphere with 5% (v/v) CO2, the medium was replaced with fresh
DMEM containing 10% FBS, and cells were incubated for additional
44 h to allow gene expression. For luciferase assay, the medium was
removed, and the cells were washed with 0.25 mL of PBS, pH 7.4, then
the cells were lysed using 200 μL of reporter lysis buffer (Promega,
USA). The luciferase activity was measured with chemiluminometer
(GloMax-Multi, Promega, USA) according to manufacture’s protocol.
Luciferase activity was normalized to the amount of total protein in the
sample, which was determined using a BCA protein assay kit (Pierce).
For flow cytometry study, pEGFP-containing polyplexes (w/w 5:1
and 10:1) were separately transferred to 293T cells in terms of the
aforementioned method. At 44 h post-transfection, pEGFP-expressing
cells were first visualized under a Nikon Ti−S inverted microscope
equipped with a fluorescence attachment. For flow cytometry
assessment, cells were washed with PBS, pH 7.4, trypsinized, and
collected in sterile tubes after a 5 min centrifugation at 1000 rpm. The
supernatant was discarded, and cells were washed twice with PBS, pH
7.4 containing 2% (v/v) FBS and 2 mM EDTA, respectively. Cells
were fixed in the dark at 4 °C for 5 min using a 2% (w/v)
paraformaldehyde solution prepared in PBS, pH 7.4. Quantitative
analysis of viable pEGFP-expressing cells was performed by flow
cytometry (FACScan, Becton & Dickinson). The instrument was
calibrated with nontransfected cells (negative control) to identify
viable cells, and the percent of pEGFP-positive cells was determined
from fluorescence scan performed with ∼1 × 104 cells using the FL1-
H channel.
2.12. Stability of mPEG-SS-PLL15-star/pDNA Complexes. The
rational design of mPEG-SS-PLL15-star predicts redox-induced
cleavage of external PEG shell and acid-induced hydrolysis of the
cross-linked polymer structure. Experimentally, the stability of mPEG-
SS-PLL15-star/pDNA complexes in response to 10 mM GSH was
determined following a protocol previously published.29 In brief,
mPEG-SS-PLL15-star/pDNA polyplexes were formed in 150 mM
NaCl at a weight ratio of 5:1, and an adequate amount of GSH was
added to establish a 10 mM GSH solution mimicking intracellular or
tumor microenvironment redox conditions. Time-dependent changes
in particle size distribution of this suspension were monitored by
dynamic laser light scattering (DLS) for up to 4.5 h. Similarly, physical
stability of fabricated polyplexes at a weight ratio of 5:1 was measured
at pH 5.0, mimicking the environment of acidified endosomes.
Following preparation of mPEG-SS-PLL15-star/pDNA complexes at
desired weight ratios, an adequate volume of HCl was added to adjust
the solution pH to 5.0. Subsequently, particle size distribution was
monitored for 2.5 h by DLS, as described above. For determination of
the implications of physical changes in particle size distribution on
genetic payload release from mPEG-SS-PLL15-star/pDNA complexes
in the presence of 10 mM GSH and pH 5.0, aliquots of the polyplex
suspension conditions were removed and subjected to gel electro-
phoresis as described above.
2.13. In Vitro Transfection Efficiency of mPEG-SS-PLL15-star
Polyplexes. Biological activity of fabricated gene delivery vectors was
assessed in vitro using pGL-3 and pEGFP pDNA as reporter gene,
mPEG-PLL40 and PEI at its optimal ratio (w/w = 1.3:1) as the
control.44,45 Transfection experiments were performed with 293T cells
2.14. Observation of the Intracellular DNA Transport. The
ability of fabricated gene delivery vectors to transport pEGFP to the
cytoplasm and nucleus was evaluated using confocal laser scanning
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