Reversibly Cross-Linked Polyplexes Enable Cancer-Targeted Gene Delivery via Self-Promoted DNA Release and Self-Diminished Toxicity

Article abstract of DOI:10.1021/acs.biomac.5b00180

Polycations often suffer from the irreconcilable inconsistency between transfection efficiency and toxicity. Polymers with high molecular weight (MW) and cationic charge feature potent gene delivery capabilities, while in the meantime suffer from strong chemotoxicity, restricted intracellular DNA release, and low stability in vivo. To address these critical challenges, we herein developed pH-responsive, reversibly cross-linked, polyetheleneimine (PEI)-based polyplexes coated with hyaluronic acid (HA) for the effective and targeted gene delivery to cancer cells. Low-MW PEI was cross-linked with the ketal-containing linker, and the obtained high-MW analogue afforded potent gene delivery capabilities during transfection, while rapidly degraded into low-MW segments upon acid treatment in the endosomes, which promoted intracellular DNA release and reduced material toxicity. HA coating of the polyplexes shielded the surface positive charges to enhance their stability under physiological condition and simultaneously reduced the toxicity. Additionally, HA coating allowed active targeting to cancer cells to potentiate the transfection efficiencies in cancer cells in vitro and in vivo. This study therefore provides an effective approach to overcome the efficiency-toxicity inconsistence of nonviral vectors, which contributes insights into the design strategy of effective and safe vectors for cancer gene therapy. (Chemical Presented).

Full text of DOI:10.1021/acs.biomac.5b00180

Article
pubs.acs.org/Biomac
Reversibly Cross-Linked Polyplexes Enable Cancer-Targeted Gene
Delivery via Self-Promoted DNA Release and Self-Diminished
Toxicity
Hua He,† Yugang Bai,‡ Jinhui Wang,§ Qiurong Deng,§ Lipeng Zhu,§ Fenghua Meng,† Zhiyuan Zhong,*,†
and Lichen Yin*,§
†Biomedical Polymers Laboratory and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of
§
Chemistry, Chemical Engineering and Materials Science, and Jiangsu Key Laboratory for Carbon-Based Functional Materials and
Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou 215123, People’s Republic of
China
‡Department of Chemistry, University of Illinois at Urbana−Champaign (UIUC), 600 South Mathews Avenue, Urbana, Illinois
61801, United States
S
* Supporting Information
ABSTRACT: Polycations often suffer from the irreconcilable inconsistency
between transfection efficiency and toxicity. Polymers with high molecular
weight (MW) and cationic charge feature potent gene delivery capabilities,
while in the meantime suffer from strong chemotoxicity, restricted
intracellular DNA release, and low stability in vivo. To address these critical
challenges, we herein developed pH-responsive, reversibly cross-linked,
polyetheleneimine (PEI)-based polyplexes coated with hyaluronic acid (HA)
for the effective and targeted gene delivery to cancer cells. Low-MW PEI was
cross-linked with the ketal-containing linker, and the obtained high-MW
analogue afforded potent gene delivery capabilities during transfection, while
rapidly degraded into low-MW segments upon acid treatment in the
endosomes, which promoted intracellular DNA release and reduced material
toxicity. HA coating of the polyplexes shielded the surface positive charges to
enhance their stability under physiological condition and simultaneously
reduced the toxicity. Additionally, HA coating allowed active targeting to cancer cells to potentiate the transfection efficiencies in
cancer cells in vitro and in vivo. This study therefore provides an effective approach to overcome the efficiency-toxicity
inconsistence of nonviral vectors, which contributes insights into the design strategy of effective and safe vectors for cancer gene
therapy.
The first major inconsistence is related to the molecular weight
(MW) of polycations. Particularly, polymers with high MW
afford stronger DNA condensation capacities than their low-MW
analogues and thus mediate stronger intracellular gene delivery
capabilities and transfection efficiencies.6,7 However, the
excessively strong binding affinity toward nucleic acids will
impede the intracellular release of gene cargos, which will
otherwise fight against effective gene transfection.8,9 Addition-
ally, high-MW polymers will induce stronger post-transfection
cytotoxicity due to the higher amount of contact points with cell
membranes, especially at high polymer concentrations or after
long-term incubation.10 The appreciable chemotoxicity of
polymers not only raises safety issues against clinical applications,
but inversely hampers the transfection efficiency as well due to
remarkable loss of cell viability and function.11 To address such
INTRODUCTION
■
Gene therapy affords a promising and efficient way to treat
congenital and acquired diseases by delivering generic materials
into target cells to promote or rectify the expression of specific
genes.1,2 While viral vectors afford high transfection efficiencies
due to their innate ability to enter and utilize transcription
machinery of host cells, they suffer from inherent immunoge-
nicity, carcinogenicity, and insertional mutagenesis.3 Therefore,
the clinical application of viral vectors is greatly hurdled. Nonviral
vectors, exemplified by cationic polymers and lipids, are
characterized by minimal immunogenicity and carcinogenicity
and, thus, serve as ideal alternatives to viral vectors toward clinical
gene therapy.4,5 As an important category of nonviral vectors,
cationic polymers (also termed as polycations) are capable of
condensing the anionic nucleic acids into nanocomplexes and
thus facilitating their intracellular delivery. Despite such desired
properties, the inconsistence between the transfection efficiency
and chemotoxicity of polycations always remains as a critical
challenge against their wide utilities.
Received: February 8, 2015
Revised: March 7, 2015
Published: March 10, 2015
© 2015 American Chemical Society
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Scheme 1. Formation and Intracellular Kinetics of K-PEI/HA/DNA Polyplexes, Including DNA Condensation by K-PEI,
Followed by HA Coating via Electrostatic Interaction, Cell Uptake of Polyplexes via HA Receptor- and Clathrin-Mediated
Endocytosis, Acid-Triggered De-Cross-Linking of K-PEI, and Dissociation of Polyplexes, Endosomal Rupture, and Cytoplasmic
Release of DNA via PEI-Assisted “Proton Sponge” Effect, and Nucleus Transport of DNA towards Gene Transcription
critical challenge, it is thus highly demanded to develop a vector
that possesses sufficient MW and charge density during the
transfection process to overcome the cellular barriers against
gene delivery while can be triggered to instantaneously degrade
into small segments at the post-transfection state, such that “on-
demand” gene release can be allowed and material toxicity can be
diminished.12
With the attempt to address the aforementioned critical
challenges associated with polycation-mediated gene delivery, we
herein developed pH-responsive, reversibly cross-linked, poly-
etheleneimine (PEI)-based polyplexes coated with hyaluronic
acid (HA) for the effective and targeted gene delivery to cancer
cells in vitro and in vivo. PEI is a widely utilized transfection
reagent because of its potent pH buffering capability to mediate
effective endosomal escape via the “proton sponge” effect,24−27
which, however, also suffers from the MW-dependent efficiency-
toxicity inconsistency. Particularly, commercially available
branched PEI 25 kDa is a golden standard reagent with high
transfection efficiency, while at the meantime, induces notable
cytotoxicity due to cell membrane disruption followed by
induction of apoptosis via destabilization of mitochondrial
membranes. On the contrary, low-MW PEI (MW < 2 kDa)
demonstrates negligible cytotoxicity yet much lower transfection
ability. To solve this problem, low MW branched PEI (L-PEI,
600 Da) was cross-linked via a pH-labile ketal linker to obtain
PEI with high MW and cationic charge density (K-PEI). We
hypothesized that such reversibly cross-linked PEI would afford
greatly enhanced DNA condensation capability to promote the
intracellular internalization of DNA cargoes;28−30 upon treat-
ment with internal acid triggers in the endolysosome (pH ∼ 5.0),
it could be instantaneously degraded into low-MW pieces upon
cleavage of the ketal bonds such that the intracellular DNA
release could be enhanced to potentiate the gene transfection
efficiency and the post-transfection material toxicity can also be
largely diminished (Scheme 1).31 While other cross-linkers such
as disulfides30 have been used to cross-link L-PEI that undergoes
redox-triggered disulfide cleavage by cytoplasmic glutathione
(GSH), the pH-responsive ketal-based cross-linking could allow
rapid degradation of PEI in the endosomes directly upon cellular
Another critical inconsistency is associated with the cationic
charge density of polyplexes. While the positive surface charges
on polyplexes feature effective binding to the negatively charged
cell membrane and accordingly facilitate the cellular internal-
ization, the excessive cationic charge density may at the
meantime induce notable damage to the cell membranes
especially at high concentrations or after long-term incuba-
tion.13,14 Additionally, upon systemic administration, the
circulating polyplexes are prone to adsorb negatively charged
serum proteins via electrostatic interaction,15,16 resulting in
nanoparticle aggregation and recognition by the reticuloendo-
thelial system (RES) that ultimately leads to remarkable
entrapment in RES organs (such as liver and spleen) yet very
little accumulation in tumors.15−17 As such, the in vivo
application of polycations is greatly hurdled. PEGylation of
polycations serves as the most widely used strategy to improve
their safety as well as serum stability because the neutral and
hydrophilic PEG molecules create a hydrophilic corona on
polyplex surfaces to shield the positive charges and sterically
hinder the binding of blood components.18−20 However,
PEGylated polycations often suffer from significantly reduced
gene transfection capabilities.21−23 Such inconsistence thus
highlights alternative strategies that can address the charge-
associated safety and stability issues of polycations without
compromising the transfection efficiencies.
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Scheme 2. Synthesis of AEA (A) as the pH-Responsive Cross-Linker and its Nonresponsive Analogue AHA (B)
Scheme 3. Synthesis of pH-Responsive PEI (K-PEI) and Its Nonresponsive Analogue NK-PEI
internalization such that material toxicity could be diminished at
an earlier stage. Hyaluronic acid (HA) as a natural anionic
polysaccharide was further selected to coat the K-PEI/DNA
polyplexes because it can not only reduce the surface cationic
charge of polyplexes but also can recognize the abundant HA
receptors (such as CD44) on cancer cells.32 We thus
hypothesized that the stability as well as cell tolerability of
polyplexes could be greatly enhanced. Because HA was capable
of targeting cancer cells to potentiate the HA-receptor-mediated
endocytosis,33,34 we also hypothesized that HA coating would
strengthen the cancer-targeted gene delivery efficiency of K-PEI
instead of diminishing the transfection efficiency as a result of
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polymer was dialyzed against distilled water (pH = 8, MWCO = 1 kDa)
for 3 days and lyophilized. The MW of the polymer was determined by
MALDI-TOF (Bruker, ultraflextreme MALDI-TOF/TOF). To enable
direct comparison on the pH-responsiveness, cross-linked, non-
responsive PEI (NK-PEI) was synthesized from L-PEI and the
nonresponsive cross-linker AHA using the same method as described
above (Scheme 3). The following nomenclature “K-PEI a” was adopted
for the cross-linked K-PEI, where a represents the molar feed ratio of
primary amine (in L-PEI) to acrylate (in AEA) during the Michael
addition reaction. The nomenclature of “K-PEI” was used throughout
the study to represent K-PEI 2 unless otherwise specified.
Polymer Degradation. K-PEI was dissolved in D2O (pH 5.0) and
incubated at 37 °C for different time before being subjected to 1H NMR
analysis to evaluate polymer degradation. The alteration of polymer MW
upon acid (pH 5.0) treatment was also determined by MALDI-TOF.
Preparation and Characterization of Polyplexes. K-PEI and
DNA were separately dissolved in DI water at 1 mg/mL. K-PEI was
added to DNA at various weight ratios, and the mixture was vortexed for
10 s and incubated at 37 °C for 30 min to allow DNA condensation and
formation of the K-PEI/DNA binary polyplexes. HA (1 mg/mL) was
then added to the freshly prepared K-PEI/DNA polyplexes at various
HA/DNA weight ratios which were incubated at RT for 1 h to allow HA
coating and formation of the K-PEI/HA/DNA ternary polyplexes.
Freshly prepared polyplexes were also visualized by transmission
electron microscopy (TEM) after staining with 1% phosphotungstic
acid.
cationic charge shielding (Scheme 1). To prove these design
strategies, we mechanistically evaluated the pH-responsive gene
delivery properties by probing the acid-triggered polymer
degradation, DNA release, transfection efficiency, and cytotox-
icity. The effect of HA-mediated cancer targeting on gene
transfection as well as cytotoxicity was also investigated.
EXPERIMENTAL SECTION
■
Materials. Branched PEI (bPEI) with MW of 25 kDa (H-PEI) and
600 Da (L-PEI), N-(2-hydroxyethyl)-phthalimide, 2-methoxy propene,
p-toluenesulfonic acid, and wortmannin were purchased from J&K
(Beijing, China). Hyaluronic acid (HA) with MW of 35 kDa was
purchased from Shangdong Freda Biopharm Co. Ltd. Acryloyl chloride
was purchased from Energy Chemical (Shanghai, China). 1,7-
Diaminoheptane, chlorpromazine, and genistein were purchased from
TCI (Shanghai, China). Methyl-β-cyclodextrin was purchased from
Aladdin (Shanghai, China). YOYO-1 was purchased from Invitrogen
(Carlsbad, CA, U.S.A.).
HeLa (human cervix adenocarcinoma cells), 3T3-L1 (mouse
embryonic fibroblasts), B16F10 (mouse melanoma cells), and RAW
264.7 (mouse monocyte macrophages) were purchased from the
American Type Culture Collection (Rockville, MD, U.S.A.) and were
cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand
Island, NY, U.S.A.) containing 10% fetal bovine serum.
Male C57/BL6 mice (6−8 wk) were purchased from Shanghai
Slaccas Experimental Animal Co., Ltd. All animal study protocols were
reviewed and approved by the Institutional Animal Care and Use
Committee, Soochow University.
Synthesis of 2-[1-(2-Amino-ethoxy)-1-methyl-ethoxy]-ethyl-
amine (AEE). AEE was synthesized as described previously (Scheme
2A).35 Briefly, N-(2-hydroxyethyl)-phthalimide (25 g, 1 equiv) was
added to benzene (400 mL), and the solution was cooled to 0 °C in an
ice bath. 2-Methoxy propene (12.5 mL, 1 equiv) was slowly added into
the solution, and p-toluenesulfonic acid (234.1 mg, 0.01 equiv) was
further added. After being stirred for 1 h at 0 °C, the reaction mixture
was heated to evaporate the solvent. When cooled to RT, triethylamine
(TEA, 30 mL) and acetic anhydride (7.5 mL) were added, and the
mixture was stirred overnight at RT. The crude product was precipitated
from the solution using hexanes which was further purified by
recrystallization with ethyl acetate for twice to obtain the final product
compound 1 as yellow solid (7.8 g, yield 28.9%).
Compound 1 was deprotected in 6 M NaOH (50 mL) at 100 °C
overnight. The solution was extracted with CHCl3/iPrOH (1/1, v/v,
100 mL × 3) and the organic phase was combined and dried over
anhydrous Na2SO4. The solution was filtered and concentrated by rotary
evaporation. The excess of iPrOH was removed by repeated washing
with hexanes and the final product of AEE was obtained as yellow oil (2
g, yield 66.9%).
To evaluate the DNA condensation capability, freshly prepared
polyplexes were subjected to electrophoresis on a 1% agarose gel at 100
V for 40 min. To quantitatively determine the DNA condensation level,
the ethidium bromide (EB) exclusion assay was adopted.36 Briefly, EB
solution was added into the polyplexes at the DNA/EB weight ratio of
10:1. After incubation at 37 °C for 1 h, the fluorescence intensity was
measured by spectrofluorimetry (λex = 510 nm, λem = 590 nm). A pure
EB solution and the DNA/EB solution without any polymer were used
as negative and positive controls, respectively. The DNA condensation
efficiency (%) was defined as following:
⎛
⎞
⎟
⎠
F − FEB
DNA condensation efficiency(%) = 1 −
× 100
⎜
F − FEB
⎝
0
where FEB, F, and F0 denote the fluorescence intensity of pure EB
solution, DNA/EB solution with polymer, and DNA/EB solution
without polymer, respectively.
In order to investigate the alteration of the DNA condensation
capacity of K-PEI upon acid treatment, pH of the polyplexes was
adjusted to 5.0 and the polyplexes were incubated at 37 °C for 4 h before
the pH was adjusted back to 7.2. The DNA condensation level of acid-
treated polyplexes was evaluated by both the gel retardation assay and
EB exclusion assay as described above.
To evaluate the stability of polyplexes in salt and upon dilution,
freshly prepared polyplexes were diluted with normal saline (pH 7.2) for
10-fold and incubated with different time before the particle size and
zeta potential were monitored by dynamic laser scanning (DLS) on a
Malvern Zetasizer. The stability of polyplexes in serum was also studied
by addition of serum into the polyplex solution at the final concentration
of 10% and incubation at RT for different time before measurement of
particle size by DLS. Additionally, particle size and zeta potential of
freshly prepared polyplexes and acid-treated polyplexes (pH 5.0 for 4 h)
were also monitored to evaluate the pH-responsiveness of polyplexes.
DNA Release. The heparin replacement assay was adopted to
evaluate DNA release from polyplexes.37 Briefly, heparin was added to
the polyplex solution at various final concentrations and the polyplexes
were incubated at 37 °C for 1 h before quantification of the DNA
condensation level using the EB exclusion assay as described above.
Acid-treated polyplexes (pH 5.0 for 4 h) were also explored to study the
pH-responsive DNA release profiles.
Synthesis of N-{2-[1-(2-Acryloylamino-ethoxy)-1-methyl-
ethoxy]-ethyl}-acrylamide (AEA). AEE (0.5 g, 1 equiv) was dissolved
in CH2Cl2 (20 mL) containing TEA (2.6 mL, 6 equiv) at 0 °C. Acryloyl
chloride (0.74 mL, 3 equiv) was slowly added into the solution and the
reaction mixture was stirred overnight at room temperature. The solvent
was removed by rotary evaporation, and the residue was dissolved in
ethyl acetate (100 mL) and washed with 10% (w/v) K2CO3 solution (70
mL × 3). The organic phase was combined and dried over anhydrous
Na2SO4. Finally, the solution was filtered and concentrated by rotary
evaporation to obtain the product AEA as yellow oil (0.25 g, yield 30%).
Synthesis of N-(7-Acryloylamino-heptyl)-acrylamide (AHA).
AHA as a nonresponsive analogue of AEA was synthesized from 1,7-
diaminoheptane instead of AEE by following the same method as
described before (Scheme 2B).
Synthesis of Cross-Linked PEI. K-PEI was synthesized via Michael
addition reaction of AEA and L-PEI (Scheme 3).30 Briefly, L-PEI (30
mg) was dissolved in methyl alcohol (0.5 mL) and heated to 45 °C in a
three-necked flask under a nitrogen atmosphere. AEA (20 mg, molar
ratio of acrylate in AEA to primary amine in PEI = 1:2) was dissolved in
methyl alcohol (0.5 mL) and added into the L-PEI solution which was
stirred in the dark for 48 h under a nitrogen atmosphere. The obtained
In Vitro Transfection. HeLa cells were seeded on 96-well plates at 6
× 103 cells/well and cultured in DMEM containing 10% FBS for 24 h.
The medium was changed to opti-MEM (100 μL/well) into which
polyplexes were added at 0.5 μg DNA/well. After incubation at 37 °C for
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Figure 1. (A) 1H NMR spectra of K-PEI in D2O following acid treatment (pH 5.0) for different times. (B) MALDI-TOF spectra of K-PEI before and
after acid treatment (pH 5.0, 4 h).
4 h, the medium was replaced by DMEM containing 10% FBS and cells
were further incubated for another 20 h. Luciferase expression was
quantified in terms of luminescence intensity using a Bright-Glo
Luciferase assay kit (Promega), and the cellular protein level was
determined using a BCA kit (Pierce). The transfection efficiency was
expressed as relative luminescence unit (RLU) associated with 1 mg of
cellular protein (RLU/mg protein). In order to study the transfection
efficiency of polyplexes in the presence of serum, cells were incubated
with polyplexes in DMEM containing 10% FBS for 4 h before
refreshment of fresh media and further incubation for 20 h as described
above. To evaluate the generality of K-PEI, transfection studies were also
performed in different mammalian cell types, as described above,
including B16F10, RAW 264.7, and 3T3-L1 cells.
To further probe the HA-assisted targeting effect, cells were
pretreated with free HA (final concentration of 10 mg/mL) for 4 h,
washed with PBS for three times, and transfected with K-PEI/HA/DNA
polyplexes as described above. CD44+ cells expressing HA receptors
(HeLa and B16F10) and CD44− cells without HA receptors (3T3-L1)
were selected for the transfection study.
Intracellular Kinetics. DNA was labeled with YOYO-1 at one dye
molecule per 50 bp DNA,38 and the YOYO-1-DNA was allowed to form
polyplexes with polymers as described above. Cells were seeded on 96-
well plates at 1 × 104 cells/well and cultured for 24 h. The medium was
changed to opti-MEM and K-PEI/HA/DNA polyplexes were added at
0.1 μg YOYO-1-DNA/well. After incubation at 37 °C for 4 h, cells were
washed three times with PBS containing heparin (20 U/mL) before
being lysed with the RIPA lysis buffer (100 μL/well). YOYO-1-DNA
content in the lysate was quantified by spectrofluorimetry (λex = 485 nm,
λem = 530 nm) and the protein content was measured using the BCA kit.
Uptake level was expressed as ng YOYO-1-DNA associated with 1 mg
cellular protein. To study the HA-mediated targeting effect of
polyplexes, cells were pretreated with free HA (10 mg/mL) for 4 h
before addition of the K-PEI/HA/DNA polyplexes. The uptake level of
YOYO-1-DNA was quantified 4 h later as described above.
determined by the MTT assay, and results were presented as percentage
viability of control cells. To further study the cytotoxicity of polyplexes
by simulating the transfection process, cells were incubated with K-PEI/
DNA polyplexes prepared at various K-PEI/DNA weight ratios at 0.1 μg
DNA/well and 37 °C for 4 h. The polyplexes were removed, the
medium was replaced by fresh DMEM containing 10% FBS, and cells
were further incubated for another 20 h. Cell viability was evaluated by
the MTT assay as described above. In a parallel study, cells were
incubated with K-PEI/HA/DNA polyplexs (K-PEI/DNA weight ratio
of 5) at various DNA amount (0.2, 0.6, 1 μg/well) for 4 h, further
cultured for 20 h, and subjected to viability assessment using the MTT
assay.
In Vivo Transfection. Melanoma-bearing mice were obtained by
subcutaneous injection of B16F10 cells. Briefly, male C57/BL6 mice
(18−20 g) were unhaired, anesthetized by isoflurane, and subcuta-
neously injected with 1 × 107 cells in the right flank. When the tumor
volume reached 100 mm3 (within ∼7 days), mice were divided into six
groups (four mice per group), anesthetized, and intratumorally injected
with K-PEI/HA/DNA polyplexes, K-PEI/DNA polyplexes, NK-PEI/
DNA polyplexes, L-PEI/DNA polyplexes, H-PEI/DNA polyplexes, and
HEPES buffer containing 5% glucose (HBG) at 30 μg DNA/mouse
(∼50 μL/injection). Polyplexes were prepared in HBG as described
above with minor modifications. Briefly, K-PEI and DNA were
separately dissolved in HBG at 20 and 1 mg/mL. K-PEI was added to
DNA at weight ratio of 10, and the mixture was vortexed for 10 s and
incubated at 37 °C for 30 min to allow polyplex formation. HA (20 mg/
mL) was then added to the freshly prepared K-PEI/DNA polyplexes at
the HA/DNA weight ratio of 5 which were incubated at RT for 1 h to
allow HA coating and formation of the K-PEI/HA/DNA ternary
polyplexes.
Forty-eight hours post-injection, mice were sacrificed, and tumors
were harvested, washed three times with PBS, and homogenized with
passive lysis buffer containing protease inhibitor cocktail. The mixture
was frozen in liquid nitrogen and thawed in 37 °C water bath for three
cycles. The mixture was then centrifuged at 4 °C and 12000 rpm for 10
min. The supernatant was subjected to quantification of the luciferase
expression level using the Bright-Glo assay kit and protein level using the
BCA kit. Transfection efficiencies were represented as RLU/mg protein.
Statistical Analysis. Statistical analysis was performed using
Student’s t test. The differences between test and control groups were
judged to be significant at *p < 0.05 and very significant at **p < 0.01
and ***p < 0.001.
To study the internalization mechanism of the polyplexes, the cellular
uptake study was performed at 4 °C or in the presence of various
endocytic inhibitors. Briefly, cells were preincubated with chlorproma-
zine (10 μg/mL), genistein (100 μg/mL), methyl-β-cyclodextrin
(mβCD, 5 mM), and wortmannin (10 μg/mL) for 30 min prior to
polyplex addition and throughout the 4-h uptake experiment at 37 °C.
Results were expressed as percentage uptake of control cells that were
treated with polyplexes at 37 °C for 4 h in the absence of inhibitors.
The internalization and intracellular distribution of polyplexes were
also visualized by confocal laser scanning microscopy (CLSM). Briefly,
HeLa cells were seeded on coverslips in 24-well plate at 3 × 104 cells/
well and were cultured for 24 h before treatment with polyplexes in opti-
MEM at 1 μg YOYO-1-DNA/well for 4 h. Cells were washed three times
with heparin-containing PBS, stained with Lysotracker-Red (200 nM),
fixed with paraformaldehyde (4%), and stained with DAPI (5 μg/mL)
before CLSM observation.
RESULTS AND DISCUSSION
■
Characterization of Cross-Linked K-PEI. K-PEI was
synthesized via the Michael addition between AEA and L-PEI.
Ketal-containing cross-linker was selected here to cross-link L-
PEI because it had been extensively reported that ketal bond was
stable at neutral physiological pH while can be rapidly cleaved at
the acidic endosomal pH (∼5.0). As such, ketone group will be
generated and the cross-linked PEI can instantaneously degrade
into low-MW segments to promote DNA release and self-
diminish the cytotoxicity. As shown in Supporting Information,
Cytotoxicity. Cells were seeded on 96-well plates at 6 × 103 cells/
well and cultured in DMEM containing 10% FBS for 24 h. The polymers
were added at various concentrations and incubated for 24 h. Cells
without polymer treatment served as the control. Cell viability was
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Figure 2. DNA condensation by K-PEI at various polymer/DNA weight ratios and acid-triggered (pH 5.0, 4 h) DNA release as evaluated by the gel
retardation assay. N represents naked DNA.
Figure 3. (A) DNA condensation efficiency of K-PEI, NK-PEI, and L-PEI at various polymer/DNA weight ratios as evaluated by the EB exclusion assay
(n = 3). (B) Alteration of the DNA condensation efficiencies of K-PEI, NK-PEI, and L-PEI following acid treatment (pH 5.0, 4 h). Polymer/DNA
weight ratio was maintained constant at 5 (n = 3).
Figure 4. (A) Size and zeta potential of K-PEI/DNA polyplexes at various polymer/DNA weight ratios. (B) Size and zeta potential of K-PEI/HA/DNA
polyplexes at various HA/DNA weight ratios. K-PEI/DNA weight ratio was maintained at 5. (C) Alteration of the particle size of K-PEI/DNA
polyplexes (weight ratio of 5) and K-PEI/HA/DNA polyplexes (weight ratio of 5/2.5/1) in normal saline. (D) pH-trigged size augment of K-PEI/DNA
polyplexes (weight ratio of 5) following acid treatment (pH 5.0, 4 h).
Figure S5, the proton peaks of -NCH2CH2- in L-PEI (δ 2.5−3.0
ppm) and the proton peaks of the ketal bond (a−c) appeared
while the peaks from acrylate (δ 5.6−6.2 ppm in Supporting
Information, Figure S3) disappeared, indicating successful cross-
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Figure 5. DNA release from K-PEI/DNA polyplexes (A) and NK-PEI/DNA polyplexes (B) in the presence of heparin at various concentration before
and after acid (pH 5.0) treatment (n = 3).
linking of L-PEI by AEA. MALTI-TOF results revealed that MW
of L-PEI increased from 600 to 4856 Da after the Michael
addition reaction, indicating formation of high-MW polymer
after intermolecular cross-linking.
profile, longer time of acid treatment promoted DNA release
(Supporting Information, Figure S9). Particle size of the K-PEI/
DNA polyplexes greatly enhanced from 160 to 790 nm upon acid
treatment (Figure 4D), suggesting dissociation of the nanoscale
polyplexes as a result of diminished binding affinity toward DNA
that served as the driving force for polyplex formation.
In support of the above assessment on the pH-responsiveness
of K-PEI, the DNA condensation efficiency of NK-PEI, a
nonresponsive analogue of K-PEI which formed polyplexes with
similar diameter and positive surface charge to K-PEI
(Supporting Information, Figure S10), did not change upon
acid treatment (Figure 3B), which further substantiated that acid-
induced ketal cleavage contributed to the degradation of K-PEI
and accordingly promoted DNA dissociation.
When K-PEI/DNA polyplexes were coated with HA to form
ternary complexes, the DNA condensation level did not
appreciably change except the slight decrease at the HA/DNA
weight ratio of 10 (Supporting Information, Figure S11). This
could be attributed to the excessive amount of negatively charged
HA that competitively reduced the electrostatic interactions
between K-PEI and DNA and accordingly partly replaced DNA
out of the polyplexes. At the HA/DNA weight ratio lower than 4,
size of the K-PEI/HA/DNA ternary polyplexes remained around
200 nm and showed no significant difference to the K-PEI/DNA
binary polyplexes (Figure 4B). However, further increase in the
HA coating amount led to dramatic elevation of particle size up
to ∼1000 nm, which was mainly attributed to the neutralization
of the positive surface charge of polyplexes by the negatively
charged HA as evidenced by the continuously reduced zeta
potential that led to particle aggregation (Figure 4B). Further
increase in the HA amount led to complete coating of polyplex
surface with the anionic HA molecules, leading to negative
surface charge and accordingly improved particle dispersion due
to the electrostatic repulsion among each other.
Stabillity of polyplexes in salt solution or 10% serum was
further evaluated in terms of particle size alteration. As shown in
Figure 4C, particle size of K-PEI/DNA binary polyplexes
dramatically increased upon dilution with normal saline while
size of the K-PEI/HA/DNA ternary polyplexes were only slightly
increased to ∼280 nm within 2 h. Similar results were observed
after incubation with 10% serum (Supporting Information,
Figure S12), indicating that HA coating significantly improved
the stability of polyplexes against salt and dilution. As a negatively
charged polysaccharide, HA absorbed water and formed a
hydrophilic corona on polyplexes surface to reduce the “charge
shielding” effect induced by the salt in the solution. As such, the
polyplex stability was greatly improved, and the obtained K-PEI/
Polymer Degradation. Acid-triggered polymer degradation
1
was explored using H NMR and MALDI-TOF. Because the
endolysosomal pH was similar to 5.0, K-PEI was dissolved in pH
1
5.0 D2O and its H NMR spectrum was recorded at different
timed intervals. As shown in Figure 1A, the proton peak of the
ketal bond at 1.37 ppm gradually disappeared when K-PEI was
incubated at pH 5.0 up to 4 h, while the proton peak of the
ketone group at 2.2 pm appeared and the peak intensity increased
with the incubation time, which demonstrated acid-triggered
cleavage of ketal groups and generation of ketone groups.
MALDI-TOF further revealed that MW of K-PEI decreased from
4856 to 632 Da that was similar to the MW of non-cross-linked
L-PEI upon acid (pH 5.0) treatment for 4 h (Figure 1B). These
results collectively indicated that acidic pH triggered cleavage of
the pH-responsive ketal bonds in AEA that was used to cross-link
L-PEI, and thus, the cross-linked K-PEI with higher MW was
degraded into low-MW PEI segments.
DNA Condensation and pH-Responsive DNA Release.
The capacity of K-PEI to condense DNA was first characterized
by the gel retardation assay. Compared to L-PEI, K-PEI was able
to condense DNA at polymer/DNA weight ratio ≥1, as
evidenced by the restricted DNA migration in the loading well
(Figure 2 and Supporting Information, Figure S7). In
consistence with this qualitative observation, the quantitative
EB exclusion assay further revealed that ∼90% of the DNA was
condensed by K-PEI at polymer/DNA weight ratio higher than
1, while L-PEI afforded low DNA condensation efficiency of
below 40% even at high polymer/DNA weight ratio up to 5
(Figure 3A). Such disparity substantiated that the inefficient
DNA condensation capacity of L-PEI could be greatly improved
via chemical cross-linking that yielded high MW analogues. DLS
measurement showed that at K-PEI/DNA weight ratio higher
than 1.5, nanoscale polyplexes with diameter of ∼160 nm and
positive surface charge of ∼20 mV could be obtained (Figure
4A). TEM image showed that K-PEI/DNA polyplexes had a
spherical morphology with an average particle size of ∼140 nm
(Supporting Information, Figure S8), which was consistent with
the DLS measurement.
Upon treatment with acid (pH 5.0, 4 h), DNA completely
migrated in the agarose gel at polymer/DNA weight ratio of 1
(Figure 2) and the DNA condensation efficiency notably
dropped from 90.1 to 38.2% (Figure 3B), indicating that the
capability of K-PEI to condense DNA was weakened in a pH-
responsive manner. In accordance with the polymer degradation
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Figure 6. (A) Comparison of the transfection efficiencies K-PEI, NK-PEI, L-PEI, and H-PEI in HeLa cells in the absence of serum (n = 3). (B)
Transfection efficiencies of K-PEI in B16F10, RAW 264.7, and 3T3-L1 cells in the absence of serum (n = 3). (C) Transfection efficiencies of K-PEI/HA/
DNA polyplexes in HeLa cells in the absence or presence of serum (n = 3). K-PEI/DNA weight ratio was maintained constant at 5. (D) Transfection
efficiencies of K-PEI/DNA and K-PEI/HA/DNA polyplexes (weight ratio of 5/2.5/1) in HeLa, B16F10, and 3T3-L1 cells in the absence of serum with
or without pretreatment of free HA (n = 3). H-PEI was used as a control at the optimal polymer/DNA weight ratio of 1 in the above assessments.
HA/DNA ternary complexes may be more suitable for in vivo
use.
RAW 264.7 (macrophage), and 3T3-L1 (primary fibroblast),
wherein K-PEI again showed a 1−2 order of magnitude higher
transfection efficiencies than H-PEI (Figure 6B). These results
thus substantiated that cross-linking of L-PEI to form high-MW
K-PEI and acid-triggered de-cross-linking of K-PEI at
endolysosomal pH greatly potentiated the DNA transfection
capabilities by facilitating DNA condensation in the extracellular
compartment while promoting intracellular DNA release.
Cancer cells express HA receptors (CD44+) on cell
membranes, and thus, surface coating of HA should lead to
active targeting to cancer cells. As such, the transfection
efficiencies of HA-coated polyplexes were further evaluated in
CD44+ cancer cells. As shown in Figure 6C, K-PEI/HA/DNA
polyplexes (HA/DNA weight ratio of 2.5) showed a 2.5-fold
higher transfection efficiency than K-PEI/DNA polyplexes in
HeLa cells. Similar enhancement was also noted in B16F10 cells
(CD44+) but not in 3T3-L1 cells (CD44−), which thus
substantiated the HA-mediated targeting effect to potentiate
gene transfection in cancer cells. When HeLa and B16F10 cells
were pretreated with HA to competitively block the HA
receptors on cell membranes, the transfection efficiency of K-
PEI/HA/DNA ternary polyplexes dramatically decreased
(Figure 6D), which further confirmed that the augmented
transfection efficiency of ternary polyplexes was related to the
HA-mediated targeting effect. 3T3-L1 does not express HA
receptors, and thus pretreatment with HA did not lead to
appreciable effect on the transfection efficiency.
To reflect the intracellular DNA release in the presence of
anionic polysaccharides/proteins, the acid-triggered DNA
unpackaging was also monitored using the heparin replacement
assay. As shown in Figure 5A, DNA was released from K-PEI/
DNA polyplexes in the presence of 0.15 mg/mL heparin.
Comparatively, a 3-fold lower concentration (0.05 mg/mL) of
heparin was required to release DNA from acid-triggered K-PEI
polyplexes, suggesting promoted DNA dissociation as a result of
acid-triggered degradation of K-PEI. The heparin-induced DNA
release profiles of the nonresponsive NK-PEI did not appreciably
alter upon acid treatment (Figure 5B). These results collectively
substantiated our design strategy to promote “on-demand” DNA
release by cascading instantaneous polymer degradation in
response to the endolysosomal acidic pH.
In Vitro Transfection. The transfection efficiencies of
polyplexes were first evaluated in HeLa cells under serum-free
condition. A spectrum of K-PEI synthesized at different primary
amine (L-PEI)/acrylate (cross-linker) molar ratios was screened,
and K-PEI 2 at the polymer/DNA weight ratio of 5 turned out to
display the highest transfection efficiency (Supporting Informa-
tion, Figure S13). Therefore, it was selected as the top-
performing material to further evaluate the pH-responsive gene
transfection capabilities. As illustrated in Figure 6A, K-PEI
polyplexes showed remarkably higher transfection efficiency than
NK-PEI and L-PEI by 2−3 orders of magnitude. In direct
comparison with H-PEI as the benchmark positive control, a 20-
fold higher transfection efficiency was also noted at the optimal
K-PEI/DNA and H-PEI/DNA weight ratios of 5 and 1,
respectively.
Remarkably compromised transfection efficiency by serum is a
major challenge for polycation-based nonviral gene vectors
especially when used in vivo. We thus further evaluated the gene
transfection efficiencies of polyplexes in HeLa cells in the
presence of 10% serum. As depicted in Supporting Information,
Figure S14, the transfection efficiency of K-PEI was slightly
reduced by several fold compared to that under serum-free
The superiority and generality of K-PEI in mediating effective
gene transfection was also demonstrated in another three
mammalian cell types, including B16F10 (melanoma cells),
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Figure 7. Intracellular kinetics of polyplexes in HeLa cells. (A) Uptake level of K-PEI/HA/DNA polyplexes containing YOYO-1-DNA in HeLa cells
with or without pretreatment of HA following incubation at 37 °C for 4 h. K-PEI/DNA weight ratio was maintained constant at 5 (n = 3). (B) Uptake
level of K-PEI/DNA polyplexes (weight ratio of 5/1) and K-PEI/HA/DNA polyplexes (weight ratio of 5/2.5/1) at 4 °C or in the presence of various
endocytic inhibitors (n = 3). (C) CLSM images of HeLa cells following incubation with K-PEI/HA/YOYO-1-DNA polyplexes at 37 °C for 1, 2, and 4 h.
Cell nuclei were stained with DAPI and endosomes/lysosomes were stained with Lysotracker-Red. Bar represents 15 μm.
condition, and a 10-fold higher transfection efficiency was still
noted when compared to H-PEI, which collectively demon-
strated the superiority of cross-linked K-PEI under the serum-
containing condition. Comparatively, the K-PEI/HA/DNA
ternary polyplexes (HA/DNA weight ratio of 5) achieved
comparable transfection efficiency to that under serum-free
condition (Supporting Information, Figure S15). Such results
indicated that HA coating greatly enhanced the capability of
polyplexes to resist serum, mainly because HA reduced the
affinity between polyplexes and serum protein and thus greatly
enhanced the polyplexes stability. The desired serum-resistance
of HA-coated ternary polyplexes indicated their promising
potential for in vivo gene delivery.
Intracellular Kinetics. The gene transfection efficiencies of
nonviral vectors are closely related to their intracellular kinetics
and internalization pathways. Therefore, the cell uptake level and
internalization mechanisms of K-PEI polyplexes were explored.
As shown in Supporting Information, Figure S16, K-PEI notably
promoted the internalization of DNA at polymer/DNA weight
ratios of 2.5−10 and the cell uptake level increased with the
incubation time. In direct comparison with H-PEI and L-PEI, a
3−5-fold increment in terms of the cell uptake level was also
noted. In consistence with the transfection efficiency, HA-coated
ternary polyplexes exhibited a 2-fold higher cell uptake level than
the binary polyplexes via HA-receptor-mediated cancer cell
targeting and internalization. Pretreatment of HeLa cells with
free HA significantly reduced the cell uptake level of ternary
polyplexes, which again substantiated the HA-mediated targeting
effect (Figure 7A).
The internalization pathway of the polyplexes was then probed
by lowering the temperature or using different endocytic
inhibitors. Energy-dependent endocytosis is blocked at low
temperature (4 °C); chlorpromazine inhibits the clathrin-
mediated endocytosis (CME) by inducing the dissociation of
the clathrin lattice; genistein and mβCD inhibit caveolae by
inhibiting tyrosine kinase and depleting cholesterol, respectively;
wortmannin prevents micropinocytosis by inhibiting phospha-
tidyl inositol-3-phosphate.38,39 As shown in Figure 7B, the cell
uptake level was significantly decreased by 60% at 4 °C,
suggesting that the majority of the polyplexes entered the cell
through energy-dependent endocytosis while the remaining may
be physical adhesion or diffusion. The cell uptake level also was
significantly inhibited by chlorpromazine but not genistein,
mβCD or wortmannin, indicating that the polyplexes were
internalized mainly via CME rather than caveolae or macro-
pinocytosis. We further studied the internalization pathway of
the polyplexes in B16F10 and RAW 264.7 cells (Supporting
Information, Figure S17). The cell uptake level was significantly
inhibited by chlorpromazine in B16F10 cells and by wortmannin
in RAW 264.7 cells, which indicated that the polyplexes were
internalized mainly via CME in B16F10 cells and micro-
pinocytosis in RAW 264.7 cells.
Endosomal entrapment and lysosomal degradation of the gene
cargoes serve as a critical barrier against effective nonviral gene
delivery. Therefore, the capability of K-PEI polyplexes to avoid/
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escape endosomes/lysosomes was further studied. CLSM images
revealed that polyplexes were effectively taken up by HeLa cells
as evidenced by extensive cytoplasmic distribution of YOYO-1-
DNA post 4-h incubation (Figure 7C), and green fluorescence
(YOYO-1-DNA) greatly separated from red fluorescence of
Lysotracker-red-stained endosomes/lysosomes (Figure 7C),
which demonstrated that K-PEI was able to effectively avoid
endosomal/lysosomal entrapment due to the “proton sponge”
effect mediated by the amine groups on K-PEI (Scheme 1).
YOYO-1-DNA was also noted to be distributed in DAPI-stained
nuclei, which indicated that it can be effectively transported into
the nuclei where gene transcription was initiated.
Cytotoxicity. Transfection efficiency and cytotoxicity of
polycations are often inversely correlated. For instance,
polycations with higher MW often afford stronger DNA
condensation capacities and higher transfection efficiencies
than their low-MW analogues. However, at the same time,
polycations with higher MW also induce higher cytotoxicities
than their low-MW analogues that are much easier to be expelled
from the biological membranes because of fewer contact points
with the cell components for each individual polymeric chain.
The excessive cytotoxicities especially at high doses will lead to
irreversible damage to target cells and ultimately impair the
transfection efficiencies. To this end, it is ideal that the polycation
vector possess high MW before transfection to enable effective
DNA condensation and intracellular delivery, while degrade into
small segments post-transfection to diminish the material
toxicity. As shown in Figure 8A, H-PEI that is widely used as a
golden standard nonviral transfection reagent induced notable
cytotoxicites when the concentration reached up to 0.05 mg/mL.
Acid-treated NK-PEI showed appreciable cytotoxicity, which
could be attributed to its relatively high MW after cross-linking
and inability to degrade into low-MW segments upon acid
treatment. In comparison, acid-treated K-PEI showed signifi-
cantly lower cytotoxicity comparable to that of the L-PEI after
long-term (24 h) incubation, indicating that acid-triggered
degradation of K-PEI effectively contributed to the self-
diminishment of material toxicity. It was also noted that the
cytotoxicity of K-PEI was dramatically reduced upon acid
pretreatment while that of NK-PEI did not appreciably change
(data not shown), which served as an evidence for the acid-
triggered self-diminishment of the cytotoxicity of K-PEI.
To reflect the transfection process and explore the effect of
endolysosomal acidic pH on the cytotoxicity of K-PEI,
polyplexes were formed and incubated with cells for 4 h before
further incubation for 20 h and assessment of cell viability. As
shown in Figure 8B, K-PEI polyplexes showed notably lower
cytotoxicities than NK-PEI polyplexes at all test polymer/DNA
weight ratios, and the desired cell tolerance of K-PEI polyplexes
(viability above 90%) was comparable to that of the L-PEI
polyplexes. These findings thus demonstrated our proposed
design strategy to eliminate the long-term cytotoxicity of PEI by
self-reduction of the MW via endolysosomal pH-triggered de-
cross-linking. HA coating on polyplexes further reduced the
material toxicity, and higher amount of HA corresponded to
elevated cell viability, which could be attributed to the reduction
of surface positive charge density of polyplexes and formation of
a hydrophilic outer shell (Figure 8C).
Figure 8. Cytotoxicity of acid-pretreated free polymer following 24 h
incubation (A), polymer/DNA polyplexes following 4 h incubation (B),
and HA-coated K-PEI/DNA polyplexes, following 4 h incubation at
various HA/DNA weight ratios (C) in HeLa cells as determined by the
MTT assay (n = 3).
HA/DNA polyplexes showed a 2-fold higher transfection
efficiency than K-PEI/DNA polyplexes which was 1−2 orders
of magnitude higher than those of H-PEI and L-PEI polyplexes,
respectively (Figure 9). These results suggested the potential
utilities of K-PEI/HA/DNA ternary polyplexes for the topical
In Vivo Transfection. We further evaluated the in vivo
transfection efficiencies of polyplexes in melanoma-bearing C57/
BL6 mice following intratumoral injection to explore the
potential of K-PEI/HA/DNA ternary polyplexes for in vivo
gene delivery. In consistence with the in vitro results, K-PEI/
Figure 9. In vivo transfection efficiencies of polyplexes following
intratumoral injection in B16F10 xenograft tumor-bearing mice at 30 μg
DNA/mouse (n = 4).
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CONCLUSIONS
■
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ASSOCIATED CONTENT
* Supporting Information
■
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2005, 7, 657−663.
S
1H NMR spectra of cross-linkers and cross-linked polymers, DLS
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: lcyin@suda.edu.cn. Phone: 86-512-65882039.
*E-mail: zyzhong@suda.edu.cn. Phone: 86-512-65880098.
■
Notes
(30) Sun, Y.-X.; Zeng, X.; Meng, Q.-F.; Zhang, X.-Z.; Cheng, S.-X.;
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(32) Ito, T.; Iida-Tanaka, N.; Koyama, Y. J. Drug Target. 2008, 16,
276−281.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the financial support from the
National Natural Science Foundation of China (51403145),
the Science and Technology Department of Jiangsu Province
(BK20140333), and the Collaborative Innovation Center of
Suzhou, Nano Science and Technology.
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