A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery.

Abstract

The success of gene therapy asks for the development of multifunctional vectors that could overcome various gene delivery barriers, such as the cell membrane, endosomal membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy operation which can be used for the assembly of multifunctional gene carriers. This work describes a pH-sensitive multifunctional gene vector that offered long circulation property but avoided the inhibition of tumor cellular uptake of gene carriers associated with the use of polyethylene glycol.
Deoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic core which was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic liposome-coated DNA/protamine/DNA complexes (CLDPD) was constructed. For in vitro test, the cytotoxicity and transfection investigation was carried out on HepG2 cell line. For in vivo evaluation, CMCS-CLDPD was intratumorally injected into tumor-bearing mice and the tumor cells were isolated for fluorescence determination of transfection efficiency.
CMCS-CLDPD had ellipsoidal shapes and showed "core-shell" structure which showed stabilization property in serum and effective protection of DNA from nuclease degradation. In vitro and in vivo transfection results demonstrated that CMCS-CLDPD had pH-sensitivity and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect CMCS- CLDPD from serum interaction but also enhance gene transfection efficiency.
These results demonstrated that multifunctional CMCS-CLDPD had pH- sensitivity, which may provide a new approach for the antitumor gene delivery.

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© 2012 Li et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
which permits unrestricted noncommercial use, provided the original work is properly cited.
International Journal of Nanomedicine 2012:7 925–939
International Journal of Nanomedicine
A pH-sensitive multifunctional gene carrier
assembled via layer-by-layer technique
for efficient gene delivery
Peng Li
Donghua Liu
Lei Miao
Chunxi Liu
Xiaoli Sun
Yongjun Liu
Na Zhang
School of Pharmaceutical Science,
Shandong University, Jinan, Shandong,
People’s Republic of China
Correspondence: Na Zhang
School of Pharmaceutical Sciences,
Shandong University, 44 Wen Hua Xi
Road, Jinan 250012, Shandong,
People’s Republic of China
Tel +86 531 8838 2015
Fax +86 531 8838 2548
Email zhangnancy9@sdu.edu.cn
Background: The success of gene therapy asks for the development of multifunctional vectors
that could overcome various gene delivery barriers, such as the cell membrane, endosomal
membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy
operation which can be used for the assembly of multifunctional gene carriers. This work
describes a pH-sensitive multifunctional gene vector that offered long circulation property but
avoided the inhibition of tumor cellular uptake of gene carriers associated with the use of
polyethylene glycol.
Methods: Deoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic
core which was used as assembly template. Then, additional layers of anionic DNA, cationic
liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template
via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic
liposome-coated DNA/protamine/DNA complexes (CLDPD) was constructed. For in vitro test,
the cytotoxicity and transfection investigation was carried out on HepG2 cell line. For in vivo
evaluation, CMCS-CLDPD was intratumorally injected into tumor-bearing mice and the tumor
cells were isolated for fluorescence determination of transfection efficiency.
Results: CMCS-CLDPD had ellipsoidal shapes and showed “core-shell” structure which showed
stabilization property in serum and effective protection of DNA from nuclease degradation.
In vitro and in vivo transfection results demonstrated that CMCS-CLDPD had pH-sensitivity
and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect
CMCS-CLDPD from serum interaction but also enhance gene transfection efficiency.
Conclusion: These results demonstrated that multifunctional CMCS-CLDPD had
pH- sensitivity, which may provide a new approach for the antitumor gene delivery.
Keywords: layer-by-layer, multifunctional nanovector, pH-sensitivity, gene delivery
Introduction
Gene therapy has offered highly possible promises for treatment of cancers, as many
potential therapeutic genes involved in regulation of molecular processes may be
introduced by gene transfer, which can arrest angiogenesis, tumor growth, invasion,
metastasis, and/or stimulate the immune response against tumors.1 However, the initial
enthusiasm for cancer gene therapy has been dampened by poor efficiency of the
delivery systems.2 To date, viral vectors are still the most efficient systems for gene
transfer but the limitations associated with their use, in terms of risks of mutagenesis,
safety, immunogenicity, and high production costs, have encouraged researchers to
focus on alternative nonviral systems.3,4
In order to achieve efficient gene delivery into the nucleus of target cells, nonviral
systems must overcome several barriers, such as circulation barriers, target tissue
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International Journal of Nanomedicine 2012:7
distribution, cellular endocytosis, endosomal escape, and
nucleus entry. Therefore, various functional elements, such
as ligands for specific targeting, fusogenic lipids or
pH-sensitive polymers for endosomal escape, and nuclear
localization signals for enhanced nuclear entry, are needed
to be equipped into the nonviral gene delivery system to form
a multifunctional gene vector.5 However, it is difficult to
integrate all these functional devices into a single system by
simple mixing and to have each function exerted at the
appropriate time and correct place.6 To date, two approaches
have been widely used to construct multifunctional vectors:
chemical modification of block copolymers subsequently
assembled with deoxyribonucleic acid (DNA) to form
micelles and incorporation of functional devices into lipid-
based gene vectors. In this study, the intention was to propose
a new approach called layer-by-layer (LbL) self-assembly
to construct multifunctional vectors.
LbL technique, first introduced at the beginning of the
1990s by Gero Decher, was used to assemble multilayered
films with alternate deposition of polycations and polyanions.7
Based on electrostatic or other intermolecular forces, it
presents a new approach to the formation of supramolecular
architectures. As DNA is anionic charged and can be self-
assembled with cationic polymers, it can be incorporated
into multilayered films by LbL technique and these DNA-
loaded multilayered films have been used as nonviral gene
delivery system.8,9 LbL assembly offers numerous advantages.
Firstly, it is entirely aqueous and scarcely involves organic
solvents, which is beneficial to maintain the activity of
DNA.10 Secondly, it can control DNA loading by controlling
film thickness or the number of layers of DNA deposited
during fabrication.11 Thirdly, application of functional poly-
electrolytes can assign multilayered films with different
functions including cell targeting, controlled release of DNA
in physiological condition, enhanced membrane penetration,
and intracellular targeting.12,13 With the rapid development
of LbL technique, gene delivery systems constructed via LbL
technique have been extended from planar films to three-
dimensional “core-shell” nanoparticles. For example,
Trubetskoy et al demonstrated that the recharging of poly-
ethylenimine (PEI)/DNA polyplexes using poly(acrylic acid)
could be used to increase levels of cell transfection in vitro
in the presence of serum and increase levels of gene
expression in the lung in vivo.14 Saul et al constructed PEI-
DNA-poly(acrylic acid)-PEI quaternary complexes and the
increased association of PEI achieved by the multilayer
approach lead to substantial increases in expression of trans-
gene for reporter plasmids without the need for excess free
polymer typically required for nonviral gene delivery.15 With
programmed deposition of required functional polyanions
and polycations, LbL assembly has the potential to achieve
highly sophisticated programmable control of gene delivery.
Therefore, it can be concluded that a multifunctional nano-
vector could be designed with programmed assembly of
functional polyanions and polycations to help overcome
biological barriers for gene transfer. Compared with the
widely used chemical modification method to fabricate
multifunctional gene vectors, LbL is an easy method without
complicated chemical modification which avoids losing
original functionality of polymers or lipids.16 As multilayers
built by the LbL method afford a more stable coating than
those prepared by lipid incorporation because of the strong
electrostatic attractions between layer to layer and layer to
substrate, LbL also solves the problem of randomness and
poor reproducibility associated with lipidic incorporation of
functional ligands and LbL offers a degree of stable modifica-
tion to functional ligands.17
In this study, after choosing the required functional
components to overcome the barriers of gene delivery, such
as pH-sensitive polymer for long circulation time, fusogenic
lipids for endosomal escape, and a nuclear localization
signal for enhanced nuclear delivery, a new multifunctional
nanovector was constructed via LbL technique. In order to
have each function exerted at the appropriate time and cor-
rect place, programmed assembly of these functional
materials was indispensable. Firstly, protamine (Pro), which
showed improvement in both nuclear targeting and intra-
nuclear transcription,18 was used to condense DNA into a
compact core as the template for the following assembly.
Then, an additional layer of DNA was deposited onto the
template to form DNA/Pro/DNA complexes (DPD). This
additional DNA layer could increase DNA loading and also
reverse the cationic surface charge of Pro/DNA for further
assembly. After that, a cationic lipid layer of cationic lipo-
somes for endosomal escape, which were prepared with
cationic lipid 6-lauroxyhexyl lysinate (LHLN) previously
synthesized by the authors’ group and 1,2-dioleoyl-
sn-glycero-3-phosphoethanolamine (DOPE), was assem-
bled onto the surface of anionic DPD, and thus cationic
liposome-coated DPD (CLDPD) were obtained. As polysac-
charide coating is the most common biomimic strategy, a
derivate of chitosan – anionic o-carboxymethyl-chitosan
(CMCS) – was chosen as the outermost layer which would
be assembled onto CLDPD to form multifunctional CMCS-
CLDPD. The CMCS coating offered long circulation
property but avoided the inhibition of tumor cellular uptake
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of gene carriers associated with the use of hydrophilic
polymers (ie, polyethylene glycol). It was interesting that
the outermost CMCS layer had pH- sensitivity, which pos-
sessed cationic charges under pH 6.5 and was anionic
charged above pH 7.0. Therefore, in blood circulation,
CMCS was anionic charged and adsorbed on the surface
of CLDPD, which could protect the cationic CLDPD against
serum interaction and offered long circulation capability.
When arriving at the acidic environment of tumor tissue,
CMCS was cationic charged, repelled with CLDPD, and
fell off. The exposed cationic CLDPD combined with
negatively-charged cell membrane was internalized via
endocytosis. With the help of the fusogenic lipid DOPE,
CLDPD could escape from endosomes and release the DPD
in cytoserum. The loaded DNA was then translocated into
nucleus under the guidance of the nuclear targeting poly-
peptide Pro. Therefore, a high efficient multifunctional gene
vector which offered long circulation, efficient cellular
membrane transport, endosomal escape, high DNA loading,
and nuclear targeting was fabricated for tumor gene
delivery. This delivery process of CMCS-CLDPD is illus-
trated in Figure 1. The physicochemical property and
in vitro and in vivo transfection efficiency of CMCS-
CLDPD were tested in this study. In vitro and in vivo studies
showed that the outermost CMCS layer could fall off in the
acidic environment of tumor tissue and expose CLDPD,
which enhances gene transfection efficiency. Results indi-
cated that the multifunctional CMCS-CLDPD might pro-
vide a new approach for antitumor gene delivery.
Materials and methods
Materials
The pEGFP-N1 vector was provided by Zhejiang University
(Hangzhou, China). PicoGreen® reagent was purchased from
Invitrogen Life Technologies (Carlsbad, CA). Pro was
obtained from Sigma-Aldrich Corporation (St Louis, MO).
DOPE was obtained from Avanti Polar Lipids, Inc,
(Alabaster, AL). CMCS (Mr 50000, carboxymethyl degree
of substitution 60%) was purchased from Jinan Haidebei
Marine Bioengineering Co, Ltd, (Jinan, China). Agarose
was purchased from Biowest (Nuaille, France). Goldview
was obtained from Beijing Saibaisheng Biological Engineer-
ing Co, Ltd, (Beijing, China). MTT (3-[4,5-dimethyl-2-
thiazolyl]-2,5-diphenyl- 2H-tetrazolium bromide) was
purchased from Sigma-Aldrich. Lipofectamine™ 2000 was
obtained from Invitrogen. Human liver carcinoma (HepG2)
and mouse melanoma (B16) cells were obtained from the
American Type Culture Collection (Manassas, VA). DNase I
enzyme and buffer were obtained from Beijing Yinfeng
Century Scientific Develop Co, Ltd, (Beijing, China).
CMCS deshield
Endocytosis
Long
circulation
1
2
3
4
5
In circulation
pH 7.4
At tumor tissue
pH 6.5
CMCS-CLDPD
Additional DNA layer
Protamine
DOPE
Endosomal
escape
Translocation
into the nucleus
Nucleus
CMCS
Figure 1 Schematic representation of multifunctional o-carboxymethyl-chitosan cationic liposome-coated deoxyribonucleic acid/protamine/deoxyribonucleic acid complexes
delivery process.
Abbreviations: CMCS, o-carboxymethyl-chitosan; CLDPD, cationic liposome-coated deoxyribonucleic acid/protamine/deoxyribonucleic acid complexes; DNA, deoxyribonucleic
acid; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.
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Heparin was delivered by the Institute of Biochemical and
Biotechnological Drug of Shandong University (Jinan,
China). Deionized water was used throughout the experi-
ment. All other chemicals and reagents used were of analyti-
cal purity grade or higher, obtained commercially.
Animals
Kunming mice weighing 18–22 g were supplied by the
Medical Animal Test Center of the New Drugs Evaluation
Center, Shandong University (Jinan, China). All animal
experiments complied with the requirements of the National
Act on the Use of Experimental Animals (China).
Preparation of CMCS-CLDPD
Initially, 100 µL of 50 µg/mL aqueous solution of plasmid
DNA was added dropwise to 100 µL of 100 µg/mL Pro
solutions under gentle vortexing for 20 seconds. The sample
was incubated at room temperature for 30 minutes to facilitate
the formation of Pro/DNA complexes.19 Then, an isovolumic
solution of DNA was added dropwise to Pro/DNA and the
sample was incubated at room temperature for 30 minutes
to facilitate the formation of DPD. After that, DPD were
stored at 4°C until use. LHLN was synthesized using the
method described previously.20 LHLN was dissolved in
dehydrated alcohol and mixed with a helper lipid DOPE
(1:1 molar ratio). The mixture was evaporated to dryness in
a round-bottomed flask using a rotary evaporator at room
temperature. The resulting lipid film was dried by nitrogen
for an additional 10 minutes to evaporate any dehydrated
alcohol. After that, DPD was added to the round-bottomed
flask. The flask was then gently vibrated to facilitate hydra-
tion of the lipid film and sonicated using a Sonic Dismembra-
tor (Model 500; Thermo Fisher Scientific, Pittsburgh, PA)
for 5 minutes at room temperature to form homogenized
CLDPD. The obtained CLDPD suspension was put into an
Eppendorf tube (Eppendorf AG, Hamburg, Germany). Under
weak vortex conditions, CMCS was added dropwise and
incubated for 30 minutes at room temperature. Finally,
CMCS-CLDPD was obtained. The preparation process of
CMCS-CLDPD is illustrated in Figure 2.
DNA
DNA
Pro/DNA
Pro
DNA/Pro/DNA
(DPD)
CLDPD
pH 7.4
CMCS
CMCS-CLDPD
Cationic liposomes
(CL)
Figure 2 Schematic representation of the assembly process of multifunctional o-carboxymethyl-chitosan cationic liposome-coated deoxyribonucleic acid/protamine/
deoxyribonucleic acid complexes.
Abbreviations: CMCS, o-carboxymethyl-chitosan; CLDPD, cationic liposome-coated deoxyribonucleic acid/protamine/deoxyribonucleic acid complexes; DNA, deoxyribonucleic
acid; Pro, protamine.
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Characterization of DPD, CLDPD,
and CMCS-CLDPD
The surface morphology of DPD, CLDPD, and CMCS-
CLDPD was examined by transmission electronic microscopy
(JEM-1200EX; JEOL Ltd, Tokyo, Japan). Samples were
prepared respectively by placing a drop of three kinds of
nanoparticle suspensions onto a copper grid and air drying,
followed by negative staining with one drop of 3% aqueous
solution of sodium phosphotungstate (Sigma-Aldrich) for
contrast enhancement. The air-dried samples were then
directly examined under the transmission electron microscope.
Mean particle size and zeta potential of the three kinds of
nanoparticle suspensions were analyzed by photon correla-
tion spectroscopy with a Zetasizer® 3000 (Malvern
Instruments, Malvern, UK). All measurements were carried
out in triplicate. The average particle size was expressed as
volume mean diameter and the reported value was repre-
sented as mean ± standard deviation (n = 3).
pH sensitivity analysis of CMCS-CLDPD
In previous studies focused on drug delivery, the determi-
nation of drugs released from carriers at different pH
values was often used to evaluate the pH-sensitivity of
carriers.21 It was expected that the outermost layer of
CMCS would fall off from the surface of CMCS-CLDPD
in the acidic environment of tumors. Therefore, in order
to evaluate the pH sensitivity of CMCS-CLDPD, the in
vitro release of DNA from CMCS-CLDPD in 50 mM
phosphate buffer solution (PBS) with different pH values
(pH 5.5, 6.0, 6.5, 7.0, 7.4) was determined. Typically, an
aliquot of CMCS-CLDPD (equivalent to 2.5 µg DNA) was
suspended in 1 mL PBS at pH 5.5 in Eppendorf tubes in
a 37°C shaking water bath at 100 rpm. Separate tubes were
used for each time point. At predetermined time intervals,
the CMCS-CLDPD suspensions were centrifuged (15,000
rpm for 30 minutes) and the amount of DNA released in
the supernatant was analyzed using fluorospectrophotometry
with PicoGreen™ double-stranded DNA quantitative
reagent.22 Background readings were obtained using the
supernatants from PBS at pH 5.5. Fluorescence was mea-
sured by fluorescence spectrophotometer (model 850;
Hitachi High-Technologies, Tokyo, Japan) at excitation
and emission wavelengths of 480 nm and 520 nm, respec-
tively. The amount of DNA was calculated according to
the linear calibration curve of DNA (F = 0.0850C + 0.1132,
R = 0.9996). The in vitro release of DNA at other values
(pH 6.0, 6.5, 7.0, 7.4) was respectively performed with
the same procedure. Also, zeta potential of CMCS-CLDPD
after incubation in PBS with different pH values (pH 5.5,
6.0, 6.5, 7.0, 7.4) was analyzed by photon correlation
spectroscopy.
DNase I protection assay
To test whether CMCS-CLDPD could protect loaded DNA
from nuclease digestion, DNase I-mediated digestion was
evaluated using agarose gel electrophoresis. In brief, 20 µL
of CMCS-CLDPD containing 1 µg of DNA was incubated
with DNase I (0.2 U/µg DNA) in DNase I buffer. The suspen-
sions were incubated in a shaking water bath at 37°C and 100
rpm for 1, 2, and 4 hours, respectively. Naked DNA (1 µg)
treated with DNase I at 0.2 U/µg DNA in DNase I buffer were
incubated in a shaking water bath at 37°C and 100 rpm for
30 minutes. The enzymatic digestion reaction was terminated
with ethylenediaminetetraacetic acid solution (0.5 M, pH 8.0).
To separate DNA from CMCS-CLDPD, TE buffer (10 mM
tris(hydroxymethyl)aminomethane-hydrochloride, 1 mM
ethylenediaminetetraacetic acid, pH 7.4) containing 1%
heparin and 1% Triton X-100 (Sigma-Aldrich) was added to
the suspensions. The suspensions were then placed in a 37°C
shaking water bath at 100 rpm for 2 hours. After that, the
suspension was analyzed by gel electrophoresis as described
above. Untreated DNA was applied to the gel as a control.
Stability study in the presence of serum
To estimate the stability of CMCS-CLDPD, the turbidity
change of CMCS-CLDPD suspension in the serum was
evaluated in a previous report.23 A freshly prepared CMCS-
CLDPD solution (250 µL) was added to 1 mL of serum at
pH 7.4 and the mixed solution was incubated at 37°C with
mild stirring. The turbidity change of the mixed solution was
measured using an ultraviolet-visible spectrophotometer
(UV-2102PC; UNICO, Dayton, NJ) at 450 nm.
Cells culture
HepG2 and B16 cells were cultured in Roswell Park Memo-
rial Institute (RPMI) 1640 medium supplemented with 10%
fetal bovine serum, streptomycin at 100 µg/mL, and penicillin
at 100 U/mL. All cells were cultured in a 37°C incubator
with 5% carbon dioxide.
In vitro cell viability
Cell viability studies were investigated by MTT assay using
HepG2 cells as model cells.24 HepG2 cells were seeded in
Corning® Costar® 96-well plates (Sigma-Aldrich) at a density
of 8 × 103 viable cells per well in 0.2 mL RPMI 1640 supple-
mented with 10% fetal bovine serum and antibiotics. After
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