Codelivery of Doxorubicin and shAkt1 by Poly(ethylenimine)-Glycyrrhetinic Acid Nanoparticles to Induce Autophagy-Mediated Liver Cancer Combination Therapy

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00879

Combination therapy has been developed as a promising therapeutic approach for hepatocellular carcinoma therapy. Here we report a low toxicity and high performance nanoparticle system that was self-assembled from a poly(ethylenimine)-glycyrrhetinic acid (PEI-GA) amphiphilic copolymer as a versatile gene/drug dual delivery nanoplatform. PEI-GA was synthesized by chemical conjugation of hydrophobic GA moieties to the hydrophilic PEI backbone via an acylation reaction. The PEI-GA nanocarrier could encapsulate doxorubicin (DOX) efficiently with loading level about 12% and further condense DNA to form PEI-GA/DOX/DNA complexes to codeliver drug and gene. The diameter of the complexes is 102 ± 19 nm with zeta potential of 19.6 ± 0.2 mV. Furthermore, the complexes possess liver cancer targeting ability and could promote liver cancer HepG2 cell internalization. Apoptosis of cells could be induced by chemotherapy of DOX, and PI3K/Akt/mTOR signaling pathway acts a beneficial effect on the modulation of autophagy. Here, it is revealed that utilizing PEI-GA/DOX/shAkt1 complexes results in effective autophagy and apoptosis, which are useful to cause cell death. The induction of superfluous autophagy is reported to induce type-II cell death and also could increase the sensity of chemotherapy to tumor cells. In this case, combining autophagy and apoptosis is meaningful for oncotherapy. In this study, PEI-GA/DOX/shAkt1 has demonstrated favorable tumor target ability, little side effects, and ideal antitumor efficacy.

Full text of DOI:10.1021/acs.molpharmaceut.5b00879

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Article
Co-delivery of doxorubicin and shAkt1 by poly(ethylenimine)-
glycyrrhetinic acid nanoparticles to induce autophagy-
mediated liver cancer combination therapy
Feng-zhen Wang, Lei Xing, Zheng-Hai Tang, Jin-Jian Lu, Peng-
Fei Cui, Jian-Bing Qiao, Lei Jiang, Hu-Lin Jiang, and Li Zong
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00879 • Publication Date (Web): 19 Feb 2016
Downloaded from http://pubs.acs.org on February 21, 2016
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Co-delivery of doxorubicin and shAkt1 by poly(ethylenimine)-glycyrrhetinic acid
nanoparticles to induce autophagy-mediated liver cancer combination therapy
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FengꢀZhen Wanga,d, 1, Lei Xinga, 1, Zhengꢀhai Tangc, JinꢀJian Luc, PengꢀFei Cuia,
JianꢀBing Qiaoa, Lei Jianga, HuꢀLin Jianga,b,* and Li Zonga,*
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a State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China
Pharmaceutical University, Nanjing 210009, China
b
Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China
Pharmaceutical University, Nanjing 210009, China
c State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
Medical Sciences, University of Macau, Macao, China
d
Department of Pharmaceutics, The Affiliated Hospital of Xuzhou Medical College,
99 West Huaihai Road, Xuzhou, 221002, Jiangsu, China
* To whom correspondence should be addressed.
Professor HuꢀLin Jiang, State Key Laboratory of Natural Medicines, Department of
Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China
Tel: +86ꢀ25ꢀ83271027; Fax: +86ꢀ25ꢀ83271027; Eꢀmail: jianghulin3@163.com
Professor Li Zong, Department of Pharmaceutics, China Pharmaceutical University,
Nanjing 210009, China
Tel: +86ꢀ25ꢀ83271317; Fax: +86ꢀ25ꢀ83271335; Eꢀmail: zong216@yahoo.com.cn
1 These authors contributed equally to this work.
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ABSTRACT: Combination therapy has been developed as a promising therapeutic
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approach for hepatocellular carcinoma therapy. Here we report a low toxicity and high
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performance
nanoparticle
system
that was selfꢀassembled from
a
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poly(ethylenimine)ꢀglycyrrhetinic acid (PEIꢀGA) amphiphilic copolymer as a
versatile gene/drug dual delivery nanoplatform. PEIꢀGA was synthesized by chemical
conjugation of hydrophobic GA moieties to the hydrophilic PEI backbone via an
acylation reaction. The PEIꢀGA nanocarrier could encapsulate doxorubicin (DOX)
efficiently with loading level about 12 %, and further condense DNA to form
PEIꢀGA/DOX/DNA complexes to coꢀdeliver drug and gene. The Diameter of the
complexes is 102 ± 19 nm with zeta potential of 19.6 ± 0.2 mV. Furthermore, the
complexes possess liver cancer targeting ability and could promote liver cancer
HepG2 cell internalization. Apoptosis of cells could be induced by chemotherapy of
DOX and PI3K/Akt/mTOR signaling pathway acts a beneficial effect on the
modulation of autophagy. Here, it is revealed that utilizing PEIꢀGA/DOX/shAkt1
complexes results in effective autophagy and apoptosis, which is useful to cause cell
death. The induction of superfluous autophagy is reported to induce typeꢀⅡcell death
and also could increase the sensity of chemotherapy to tumor cells. In this case,
combining autophagy and apoptosis is meaningful for oncotherapy. In this study,
PEIꢀGA/DOX/shAkt1 has demonstrated favorable tumor target ability, little side
effects and ideal antitumor efficacy.
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KEYWORDS: combination therapy, selfꢀassembled, glycyrrhetinic acid, liver cancer
targeting, autophagy.
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INTRODUCTION
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Hepatocellular carcinoma (HCC) is the second most lethal disease and the
incidence of HCC is increasing worldwide.1 Doxorubicin (DOX) is a widely used
chemotherapeutic drug for treatment of HCC. However, previous studies indicated
that various mechanisms could be developed to decrease the sensitivity of cancer cells
to DOX,2, 3 which leads to the reduction of drug potency and remains a formidable
challenge in the use of DOX in clinic.4 Combination therapy and coꢀdelivery of
gene/drug system through synergistic and enhancement effect are now becoming a
prospective approach for cancer therapy.4ꢀ10
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To our knowledge, the emergence of HCC is associated with the activation of the
PI3K/Akt/mTOR signaling pathway.11, 12 Akt, as a key factor in the pathway,
facilitates the development of tumor cell proliferation, angiogenesis, metastasis and
invasion. In addition, it occupies an important position in the increase of tumor
resistance to chemotherapy and inhibition of tumor cell apoptosis.13, 14 The signaling
of PI3K/Akt/mTOR also mediates an effect on the modulation of autophagy and
inhibition of the pathway might induce superfluous autophagy, which could induce
typeꢀⅡcell death. With the improved understanding of cancer at the molecular level,
a shRNA silencing Akt1 (shAkt1), as a therapeutic gene, has been widely studied.15, 16
It not only could confer the sensitivity of chemotherapeutics to tumor cells in
apoptosis competent cancer cells, but also could regulate cancer cell death through
superfluous autophagy in apoptosisꢀdefective cells.13, 14, 17, 18 It is wellꢀknown that
apoptosis and autophagy, through which cells selfꢀregulated cell death, are both
intimately related to cancer cell growth and were reported to be effective targets for
the treatment of cancer.19 Induction of apoptosis and autophagy has been evaluated as
a promising approach for interfering with the growth of cancer cells. Therefore, the
combination therapy of DOX and shAkt1 was investigated in the study.
To achieve the successful coꢀdelivery of DOX and shAkt1, it requires the
development of efficient and safe delivery vehicles.20, 21 As a promising polycationic
vector, poly(ethylenimine) (PEI) is often applied as a “golden standard” of polycation
due to its properties of highly positive charge to condense and protect gene from
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nuclease degradation as well as “proton sponge effect” to facilitate endosomal release
of gene.22 Another important advantage of PEI is its high density of primary amines in
the structure, making it easy to chemical modification.23ꢀ25 However, its high
cytotoxicity remains a great challenge in clinical application. By comparison, PEIs
with low molecular are safer to use, yet their comparatively low transfection
efficiency has limited their applications. To circumvent the problems associated with
low molecular weight, one rational strategy is to graft hydrophobic units on the
backbone of PEI. PEIs with the modification of hydrophobic anchors could cause the
reduction of the cytotoxicity of PEI by decreasing its high charge density. Besides, it
also could spark the increasement of transfection efficiency by modulating the
complex interactions with cells (facilitating adsorption on cell surfaces and cell
uptake). For instance, Bhattacharya et al. reported that lipopolymer based on low
molecular weight PEI and cholesterol had a higher transfection efficiency and better
serum stability than commercially available PEI25k.26 Liu reported that PEI with the
modification of hydrophobic anchors, such as fatty acids, lithocholate, cholesterol or
Vitamin E, could increase the transfection efficiency of siRNA.27 Park reported that
transꢀretinoic acid was grafted to PEI to synthesize a new polymer as gene carrier and
it showed less cytotoxicity and better characteristics than PEI without modification.28
These aforementioned results suggest that the means of modification of PEI should be
a promising method either to decrease its cytotoxicity or to increase its transfection
efficiency .23ꢀ28 As a metabolite of the natural product glycyrrhizin, glycyrrhetinic acid
(GA) is a preferential candidate as hydrophobic unit (lgP = 4.69)29 and could be
linked to a low molecular PEI. PEI with the modification of GA might achieve the
possibility of liver targeted delivery because of the targeting ability of GA to liver
tumor.25 In addition, amphiphilic derivative with GA conjugation could form
nanoꢀsized selfꢀaggregates for the delivery of hydrophobic drug.30 The construction of
PEIꢀGA nanoparticle might be used as an excellent drug and gene carrier.
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Herein, we successfully fabricated and synthesized
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poly(ethylenimine)ꢀglycyrrhetinic acid (PEIꢀGA) copolymers. PEIꢀGA could
selfꢀassemble into nanostructure. GA hydrophobic domain could load hydrophobic
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anticancer drug through the hydrophobic interaction and the modification of GA also
increased transfection activity and ensureed its effective delivery because of the
targeting property of GA to HepG2 cells.23ꢀ25, 29, 30 PEI was used as an efficient gene
carrier to deliver shAkt1. This nanoparticle could coꢀdeliver DOX and shAkt1 into the
same cell and achieve the synergistic/combined antiꢀtumor effect. Their
physicochemical properties, in vitro cytotoxicity, intracellular distribution, cellular
apoptosis and autophagy, and in vivo antitumor efficacy in xenograft mice model were
investigated in detail.
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Materials and methods
Materials. Nꢀhydroxysuccinimide (NHS), N,N′ꢀdicyclohexylcarbodiimide (DCC)
and GA were purchased from SigmaꢀAldrich (St. Louis, MO, USA). Branched PEI
(25, 10, 3.5, 1.8 and 0.6 kDa) were bought from Wako (Osaka, Japan). Doxorubicin
hydrochloride (DOX·HCl, purity > 98 %) was bought from Alliance Bernstein
Technology
Co.,
Ltd.
(Beijing,
China).
3ꢀ(4,
5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolm
(MTS) was bought from promega (promega, USA). Dulbeco`s modified eagle
medium (DMEM) was purchased from Gibico BRL (Paris, France). Fetal bovine
serum (FBS) was obtained from HyClon (Logan, Utah, USA). All other chemicals
were used with reagent grade or higher. Plasmids used in this study were generated in
Escherichia coli, then withdrew by the alkali lysis technique, and finally purified by a
E.Z.N.A.®Fastfilter Endoꢀfree Plasmid Maxi kit (Omega, USA). Sequence of the
targeted Akt1 mRNA was GAAGGAAGTCATCGTGGCC and the corresponding
cassette of oligonucleotide specific to the mRNA was designed.
Cell culture. HepG2 and Hepaꢀ1.6 cells were obtained from the Chinese
Academy of Sciences cell bank. Cells were cultured in DMEM medium supplemented
with 10 % FBS at 37 oC in 5 % CO2 atmosphere.
Animals. C57BL/6J mice (male, sixꢀweekꢀold, 20 ± 2 g) were bought from the
Model Animal Research Center of Nanjing University (Nanjing, China) and kept in
the animal room with the temperature and relative humidity controlled, under a 12 h
light/dark cycle. The mice were used in accordance to the policy and regulations for
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the care and use of laboratory animals of China Pharmaceutical University.
Synthesis and characteristics of PEI-GA copolymer. The synthesis route of
PEIꢀGA copolymer is presented in Figure. 1a. The GA moiety was conjugated to PEI
with amide linkage through the reaction of primary amines of PEI with carboxyl
groups of GA in DMSO in the presence of NHS. The characteristic of the PEIꢀGA
copolymer was analyzed by 1H nuclear magnetic resonance (1H NMR) (Avance™ 600,
Bruker, Germany), UV scanning spectra (Thermo, Bruker, China) and
Fourierꢀtransform infrared spectrometer (FTꢀIR) (Tenson, Bruker, Germany).
The buffering capability of PEIꢀGA copolymer was mensurated by acidꢀbase
titration with the pH ranging from 10.0 to 4.0. Briefly, 2 mg of PEIꢀGA copolymer
were dispersed in 10 mL of 150 mM NaCl solution under shaking. The solution was
adjusted to an initial pH of 10.0 with 0.1 M NaOH. It was then dropwised with 0.1 M
HCl and the pH was monitored using a pH meter (OHAUS, USA). PEIꢀ25kDa was
used as a control.
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The molecular weight of PEIꢀGA was analyzed by a gel permeation
chromatography column coupled with multiꢀangle laser scattering (GPCꢀMALS) with
690 nm laser wavelength (Dawn Eos, Wyatt, USA). Chloroform was used as a mobile
phase and the flow rate was 1.0 ml/min. The column temperature was maintained at
40 °C. Polystyrene with average molecular weights ranging from 1.0 ×103 to 9.5×103
Da were used as standards for calibration.
Preparation and characterization of PEI-GA/DOX/shAkt1 nanoparticles. As
a firstꢀline therapy for HCC, hydrophobic DOX was used for a model drug. It might
be loaded into the lipophilic cores during the selfꢀassembly process of PEIꢀGA using a
dialysis method and shAkt1 could be bound to the cationic backbone of the resulting
DOXꢀloaded nanoparticles (Scheme 1a). Briefly, DOX·HCl (100 mg) was stirred with
TEA (3ꢀfold molar of DOX·HCl) in 15 ml DMSO to obtain DOX base. PEIꢀGA (100
mg) were dispersed in another 15 ml DMSO (30 ml for blank PEIꢀGA). The DOX
base solution was then added and mixed with the DMSO solution. Subsequently, 10
ml deionized water was added to the mixture by dripping slowly. The obtained
solution was vigorously stirred for 2h. Afterwards, the solution was transferred into a
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dialysis bag (MWCO = 3500) and dialyzed against deionized water. The outer phase
was tipped away and fresh deionized water was added at 4, 8, 12, 18, 24, 36, 48 and
60 h. After 60 h, the solution inside the dialysis bag was recovered, and filtered. For
prepare PEIꢀGA/shAkt1 or PEIꢀGA/DOX/shAkt1 nanoparticles, PEIꢀGA or
PEIꢀGA/DOX solutions were further incubated with shAkt1. Then PEIꢀGA,
PEIꢀGA/DOX, PEIꢀGA/shAkt1 and PEIꢀGA/DOX/shAkt1 nanoparticles were
lyophilized. The powders produced were stored at ꢀ20 oC until use.
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Scheme 1. (a) A schematic diagram of PEIꢀGA/DOX/DNA nanoparticles’ formation.
(b) Schematic illustration of PEIꢀGA nanoparticle formation with different GA grafted
ratios.
The critical aggregation concentration (CAC) serves as an important indicator of
nanoparticle stability. In this study, the CAC of PEIꢀGA was measured using the
fluorescence probe technique with pyrene as the probe. Emission spectra of pyrene
incubated with different concentrations of PEIꢀGA (ranging from 1 to 1000 ꢁg/mL)
were observed by fluorescence spectroscopy (SpectraMax M5, Molecular Devices)
with excitation wavelength at 334 nm. The intensity ratio between the third peak (382
nm) of emission wave length and the first peak (371 nm) (intensity ratio, I382/I371) was
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determined for each sample in triplicate.
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The average particle size, polydispersity index (PI) and zetaꢀpotential
measurements were made using a ZetaPlus particle size and zeta potential analyzer
(Brookhaven Instruments, USA). The morphology of blank PEIꢀGA and
PEIꢀGA/DOX/shAkt1 nanoparticles were examined by EFꢀTEM (LIBRA 120, Carl
Zeiss, Germany). The entrapment efficiency (EE %) of DOX was acquired by
measuring the DOX encapsulated in assembled nanoparticles against that added in
fabrication. The drug loading (DL %) was obtained as the ratio between the mass of
DOX encapsulated and that of the DOXꢀloaded nanoparticles. The concentration of
DOX was calculated according to a standard curve by UV/vis spectrophotometry
(UVꢀ2450, Shimaꢀdzu, Japan) at 480 nm.
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The gel retardation assay. PEIꢀGA/DNA and PEIꢀGA/DOX/DNA complexes
were prepared freshly before use at various N/P ratios from 0.1 to 20 with 12 ꢁL final
volume. Luciferaseꢀencoding plasmid DNA (pGL3) was used. The complexes
including loading dye mixture were loaded on a 1 % agarose gel with Goldview
staining and run with Trisꢀacetate (TAE) buffer at 100 V for 30 min. The location of
the DNA was captured through gel image system (Tanon 1600, China).
Protection and release properties of DNA in PEIꢀGA/DOX/DNA nanoparticles
were analyzed electrophoretically by the method of Jiang et al.32 Briefly, DNase I or
PBS in DNase I/Mg2+ digestion buffer with the volume of 1 ꢁL was added to 4 ꢁL of
complexes solution (N/P ratio: 5:1) or to 0.4 ꢁg of naked plasmid DNA. The mixed
o
solutions were incubated at 37 C with continuous stirring at 100 rpm for 30 min.
Then all mixtures were maintained with 4 ꢁL EDTA (250 mM) for 10 min at 65 oC for
DNase I inactivation. Afterwards, the samples were mixed with 1 % sodium dodecyl
sulfate (SDS) dispersed in 1 M NaOH at a final volume of 18 ꢁL. The samples finally
were incubated for 2 h at room temperature, and were run electrophoretically in 1 %
agarose gel with TAE running buffer for 40 min at 50 V.
Release of DOX and DNA in vitro. The in vitro DOX and DNA release from
PEIꢀGA/DOX/DNA nanoparticles was investigated. Briefly,
1
mg/mL
PEIꢀGA/DOX/DNA nanoparticles dispersed in PBS buffer (pH 7.4) were placed in a
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dialysis bag (MWCO = 1000). The bag was suspended in 50 mL PBS buffer at 37 oC
with shaking speed of 100 rpm. At indicated time, 1 mL of the release medium was
withdrawn and fresh PBS buffer was added. The DOX and DNA content in the
samples were quantified spectrophotometrically by determining the absorbance at 480
and 260 nm, respectively. The experiment was performed in triplicate.
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Cellular uptake. Intracellular location of DOX was monitored by fluorescent
inverted microscope (Nikon tiꢀs, Japan). HepG2 cells were plated in a 6ꢀwell plate at a
density of 30×104 cells with 2 mL growth medium for 18 h in a 5 % CO2 incubator at
37 oC to allow the cells to adhere. The medium were then respectively renewed by the
medium containing PEIꢀGA/DOX/DNA complexes, the combined of GA and
PEIꢀGA/DOX/DNA complexes, or only DOX, respectively at a DOX concentration of
1 ꢁg/well. After treatment for 6 h, the cells were immediately washed three times with
PBS. Then cell fluorescence was analyzed.
To measure the fluorescence intensity, the detached and adherent cells were washed
three times with PBS and further collected by trypsinization. The cells were then
resuspended in PBS. The mean fluorescence intensity at 488/590 nm of DOX was
analyzed using flow cytometer (BD Accuri C6, USA).
In vitro cytotoxicity. To assess the cell proliferation, MTS assay was performed.
HepG2 cells were plated at a density of 1×104 with 200 µL growth medium per cell in
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96ꢀwell plates at 37 C under 5 % CO2 for 18 h. Then the cells were incubated with
the culture media containing saline, PEIꢀGA, DOX, PEIꢀGA/DOX, and
PEIꢀGA/DOX/shAkt1 complexes at different concentrations (0, 5, 10, 20, and 50
µg/mL), respectively. Each experiment was performed in triplicate. After treatment
for 24 h, the MTS solution (20 µL) was added to each well and the cells were
incubated for another 4 h at 37 oC
The formazan crystals were dispersed in the meida
.
by agitating the 96ꢀwell plates on an orbital shaker. The optical density values were
measured in each well by a microplate reader (Thermo Scientific Multiskan GO, USA)
at the absorbance of 490 nm. The Bliss method was applied to measure the half
maximal inhibitory concentration (IC50).
Cellular apoptosis and autophagy. HepG2 cells plated in 6ꢀwell plates were
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incubated for 24 h at 37 C with saline, PEIꢀGA, DOX, PEIꢀGA/DOX or
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PEIꢀGA/DOX/shAkt1 complexes to evaluate the antitumor efficiency. Annexin V and
Propidium Iodide (PI) double staining were used to detect cells apoptosis by flow
cytometry (BD Accuri C6, USA) using the Annexin VꢀFITC apoptosis detection kit I.
For autophagy investigation, transfected cells were washed thrice with iceꢀcold PBS.
Cells were lysed on ice for 30 min and meanwhile vortexed every 5 min. The lysates
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were centrifuged for 5 min at 10000 rpm at 4 C, and then detected by Western blot
analysis. Protein concentration was measured using a BCA Protein Assay Kit
according to the manufacturer’s instructions. Total protein (30 ꢁg) was subjected to
electrophoresis in 12 % BisꢀTrisꢀpolyacrylamide gels. Following electrophoresis, the
proteins were transferred to Immobilon Transfer Membranes (Millipore, Bedford,
USA) at 300 mA for 45 min. The membranes were then incubated with 5 % BSA in
PBS for 1 h and subsequently incubated with 1 % BSA in PBS with primary
antibodies against Akt1, LC3BꢀI and LC3BꢀII (1:1000) (Santa Cruz Biotech., Santa
o
Cruz, USA) at 4 C overnight. The membranes were washed thrice in TBST and
further incubated in 1 % BSA with horseradish peroxidaseꢀconjugated donkey
antiꢀgoat serum (1:10000) (Santa Cruz Biotech., Santa Cruz, USA) for 45 min. The
immune complexes were visualized using the ECL system (Pierce, Rockford, USA).
The relative levels for Akt1, LC3BꢀI and LC3BꢀII protein were normalized to the
levels of βꢀactin.
Hemolysis test and systemic toxicity. The rabbit red blood cells (RBC) in
heparin sodiumꢀcontaining tubes were collected by centrifugation at 2500 rpm for 10
minutes and washed thrice with physiological saline. After rinsing, the RBC were
dispersed in physiological saline at the concentration of 2 % (w:v). Different amounts
of PEIꢀGA/DOX/shAkt1 nanoparticles were resuspended in physiological saline and
incubated with the equal volume of 2 % RBC solutions. Following incubation for 1 h
at 37 oC, the samples were centrifugated at 3000 rpm for 5 min. The supernatant (200
ꢁL) was added in 96ꢀwell plate and the absorbance of sample (As), negative (An) and
positive controls (Ap) were analyzed for released hemoglobin at 540 nm using
ThermoMultiskan GO (Thermo Scientific, USA). 1 % Triton Xꢀ100 and physiological
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saline solutions were applied as positive and negative control, respectively.
Percentage hemolysis of RBC was computed according to the following formula:
[(AsꢀAn) / (ApꢀAn)] × 100 %.
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In vivo antitumor efficacy in xenograft model mice. C57BL/6J mice
(sixꢀweekꢀold, male) were randomly divided into 5 groups with 6 mice in each group:
1) saline; 2) DOX (10 mg/kg); 3) PEIꢀGA (10 mg/kg); 4) PEIꢀGA/DOX (10 mg/kg); 5)
PEIꢀGA/DOX/shAkt1 (10 mg/kg). After 14 days of Hepaꢀ1.6 cells (1 × 107)
implantation, the mice were used and treatment groups were administrated via tail
vein twice a week. Tumor growth was recorded by measuring length (l) and width (w)
and the volume (mm3) was computated according to the following formula: V = lw2/2.
The survival rates and body weights were monitored throughout the study.
Statistical analysis. Experiments were performed at least three times in a parallel
manne and all data were expressed as means ± standard deviation (SD). Statistical
analysis was performed using SPSS 19.0. Comparisons were performed using the
ANOVAꢀtest to determine whether data groups differed significantly from each other.
Statistically significant difference was considered at Pꢀvalues< 0.05.
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RESULTS AND DISCUSSION
Formation and characterization of PEI-GA, PEI-GA/DOX and
PEI-GA/DOX/shAkt1 nanoparticles. The characteristics of PEIꢀGA were verified
by UV scanning spectra, 1H NMR and FTꢀIR (Figure. 1b, 1c and 1d, respectively). As
displayed in Figure. 1b, the appearance of the UV absorbance peak of GA in PEIꢀGA
was demonstrated to the successful graft of GA on PEI backbone. The proton peaks of
PEI (–NHCH2CH2–) showed at 2.0ꢀ0.6 ppm and the proton peaks of GA at 3.5ꢀ2.5
ppm indicated that GA was introduced to the PEI chain (Figure. 1c). Moreover, the
FTꢀIR peak of ꢀCOꢀNHꢀ at 1651 and 1541 cmꢀ1 (Figure. 1d) in PEIꢀGA, further
indicated the successful synthesis of PEIꢀGA. In order to synthesis PEIꢀGA
copolymers with different modification of GA, we make a series of reaction of PEI
and GA with different feed ratio. The quantity of GA in the copolymer of PEIꢀGA was
detected by UV and the degree of GA grafting was between 15ꢀ75 % (Table 1).
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Figure. 1. (a) Synthesis of PEIꢀGA. Characterizations of PEIꢀGA, GA, and PEI via (b)
the ultra violet; (c) 1H NMR in d 6ꢀDMSO; and (d) IR.
As shown in Table 1, the critical association concentrations (CAC) of PEIꢀGA
became lower with the increase of the graft ratio of GA and the decrease of the
molecular weight of PEI. Owing to the high amount of amino groups on PEIꢀGA
nanoparticle surface, all of the nanoparticles were positively charged, with charge
decreasing as graft ratio of GA increased (Table 1). As shown in Scheme I (b), more
aggregation was observed with the increasing grafting ratio of GA. The sizes of the
formed nanoparticles ranged between 60 nm and 250 nm with narrow size
distributions (PI < 0.25). Conventional acidꢀbase titration was applied to investigate
the buffering capacity of PEIꢀGA. PEIꢀGA with different graft ratios of GA exhibited
nearly the same buffering capacity over the pH range 7.4 to 5.1 (14.3ꢀ14.8 %). For the
successful formation of nanoparticles using PEIꢀGA copolymer, the most important
prerequisite was to control the graft ratio of GA. PEIꢀGA with either low graft ratio or
high graft ratio of GA were hard to selfꢀassemble into nanoparticles in water. Yet
PEIꢀGA with suitable graft ratio (Table 2) of GA can easily selfꢀassemble into
nanoꢀaggregates based on the interaction of functional groups among GA. The CAC
is wellꢀcontrolled by the molecular weight of PEI and the GA graft ratio.
PEI3.5kꢀGA1/2 with the lowest CAC (22.94 µgꢂmLꢀ1) is selected for further study as it
is easy to selfꢀassemble into nanoparticles in water and could keep stable for a long
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time (more than one year). The molecular weight of PEI3.5kꢀGA1/2 was 7.27 kDa with
1.41 polydispersity index determined by GPC measurement.
Table 1
9
Physicochemical properties of synthesized PEIꢀGA.
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graft
(%)
ratio CAC
(µg mLꢀ1)
particle size (PI) Zeta Potential
Type of PEIꢀGA
(nm)
(mV)
28.3
ꢀ
PEI(1.8k)ꢀGA(1/4) 21.6
PEI(1.8k)ꢀGA(1/2) 43.4
PEI(1.8k)ꢀGA(1/1) 51.9
PEI(3.5k)ꢀGA(1/4) 19.6
PEI(3.5k)ꢀGA(1/2) 38.9
PEI(3.5k)ꢀGA(1/1) 47.5
PEI(10k)ꢀGA(1/4) 18.8
PEI(10k)ꢀGA(1/2) 32.1
PEI(10k)ꢀGA(1/1) 40.2
PEI(25k)ꢀGA(1/4) 16.8
PEI(25k)ꢀGA(1/2) 29.4
PEI(25k)ꢀGA(1/1) 44.7
38.63
ꢀ
67.4 (0.21)
ꢀ
ꢀ
ꢀ
ꢀ
46.82
22.94
ꢀ
91.2 (0.13)
60.7 (0.19)
ꢀ
29.6
19.9
ꢀ
79.32
54.16
28.28
103.83
68.81
35.83
110.5 (0.16)
96.4 (0.22)
89.7 (0.17)
243.9 (0.19)
162.5 (0.21)
113.7 (0.23)
37.3
25.7
16.3
30.8
24.2
15.7
To illustrate the detailed surface chemical composition of PEIꢀGA nanoparticles,
Xꢀray photoelectron spectroscopy (XPS) was performed. The data indicated that GA
is shown on the surface of our prepared PEIꢀGA nanoparticles (Table 2). A similar
result was obtained by Zhang et al.24 GA on the surface of nanoparticles is beneficial
to achieve the targeting efficiency to hepatocyte as the GA receptors of the liver.33, 34
Transmission electron microscopy (TEM) image indicated that the DOXꢀloaded
PEIꢀGA/DNA complexes still exhibited spherical morphology with good
monodispersity (Figure. 2a), suggesting that DOXꢀloaded PEIꢀGA/DNA complexes
did not collapse when DNA is combined. The encapsulation efficiencies (EE) were
measured by fluorescence spectra. As a modular system, DOX loading can be easily
achieved and an obvious increase in DOX loading was observed with the increase
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graft amount of GA as shown in Figure. 2b. The maximum drug loading (DL) could
reach about 15 %, which may attribute to the hydrophobic interaction between GA
and DOX. To clarify the structural integrity of DOXꢀloaded PEIꢀGA/DNA complexes
when DNA is combined, the aggregation behavior of PEIꢀGA was investigated by
fluorescence spectrophotometer using pyrene as a probe. The intensities of the
emission wavelength at 372 and 383 nm were monitored. The ratios of I372/I383 of
various samples are shown in Figure. 2c. A lower ratio would be obtained if pyrene
was encapsulated in a more hydrophobic core. In this study, the ratio of I372/I383
decreased when pyrene was encapsulated in the PEIꢀGA nanoparticles. Besides, the
ratio further decreased when the DNA is combined to PEIꢀGA/pyrene nanoparticles,
suggesting that pyrene was still in the core of PEIꢀGA/pyrene /DNA complexes even
if DNA is combined. These data demonstrated that the PEIꢀGA nanoparticles was
efficient for both drug and gene delivery.
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The condensability of DNA is required for polycationic vectors to effectively
achieve gene expression in target cell.34ꢀ36 The DNA binding ability of PEIꢀGA or
PEIꢀGA/DOX was investigated by gel retardation assay. As indicated in Figure. 2d,
either PEIꢀGA or PEIꢀGA/DOX was capable to bind DNA efficaciously, and the
intensity of the uncomplexed DNA band disappeared at N/P 1 and 2, respectively.
Higher N/P ratio was needed for PEIꢀGA/DOX, which may be related with the lower
zeta potential of PEIꢀGA/DOX (data not shown).
Table 2
XPS analysis of PEIꢀGA nanoparticle
Sample XPS elemental ratios XPS N1S envelope XPS O1S envelope ratios
(%)
ratios (%)
NꢀC
(%)
NꢀOꢀC=O OꢀC=O C=O
OꢀC
C
O
N
Bingding Energy (eV)
399.41 403.00
531.97
23.31
533.90 535.08
PEIꢀGA 76.15
19.24 4.61 28.62
71.38
11.09
65.6
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Figure. 2. The characterizations of the PEIꢀGA/DOX/DNA nanoparticles. (a) TEM
image of blank and both DOX and shAkt1 loaded PEIꢀGA nanoparticles in saline
solution (scale bar = 500 nm). (b) Drug loading of PEIꢀGA with different molecular
weight of PEI and different graft ratio of GA. (c) Intensity ratios (I372/I383) of the
emission spectra of pyrene, pyreneꢀloaded PEIꢀGA nanoparticles and pyreneꢀloaded
PEIꢀGA/DNA complexes at the N/P ratios specified. (d) DNA binding ability of
PEIꢀGA and DOX loaded PEIꢀGA nanoparticles (DNA, N/P ratios of 0.5, 1, 2, 5, 10,
20, and 30, respectively). (e) Protection study of DNA by agarose gel electrophoresis.
(f) Release profile of PEIꢀGA/DOX and PEIꢀGA/DOX/shAkt1 in saline medium with
0.2 % Tweenꢀ80 at 37 ◦C (n=3).
One of the principal obstacles for impactful gene delivery is to prevent DNA from
being degraded by enzymes in vitro and in vivo.35, 36 An efficient delivery system
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should be developed to avoid the degradation of DNA. The protection ability of
PEIꢀGA/DOX/DNA (N/P ratio:10) against DNase I was evaluated by the retardation
assay. As presented in Figure. 2e, naked DNA was totally degraded within 30 min
incubation with DNase I, as evidenced by the disappearance of the bright band
corresponding to free DNA. Whereas DNA in the complex was found to remain intact
and the band was clear, which demonstrated that DOX loaded PEIꢀGA could protect
DNA from degradation of DNase I effectively.
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In vitro release of DOX and DNA from PEIꢀGA/DOX/DNA (N/P ratio:10)
nanoparticles was shown in Figure. 2f. An obvious initial burst release of DNA is
displayed in the first 12 h. It can be attributed to the release of some DNA bound
weakly on the surface of the particles. In contrast, there is no initial burst release of
DOX, suggesting that the chemical drug was well loaded in the cores of nanoparticls. .
As indicated in Figure. 2f, PEIꢀGA/DOX nanoparticls demonstrated about 40%
release of DOX over 8h, while PEIꢀGA/DOX released only 25 % at the same
condition. The release of DOX from PEIꢀGA/DOX/DNA nanoparticles was relatively
slower. This might be because of stronger hydrophobic interaction between GA with
the DOX molecule or the electrostatic interaction of DNA and PEI to prevent the
release of DOX when DNA is complexed with PEIꢀGA/DOX nanoparticles.
Cellular uptake and transfection studies. In order to achieve efficient
transfection, it is integrant to develop a delivery system that will allow both chemical
drug and gene across cell membrane.7 Herein, we examined the effect of PEIꢀGA in
mediating the uptake of DOX and DNA into HepG2 cells. As the intracellular
fluorescence intensities varies directly as the internalized DOX, the cellular uptake of
DOX was investigated using fluorescent inverted microscope. Clear red fluorescence
of DOX was observed after 6 h transfection. PEIꢀGA/DOX/DNA group gave more
detectable fluorescence in cells compared with the group treated with both
PEIꢀGA/DOX/DNA complexes and GA or only DOX (Figure. 3a). As indicated in
Figure. 3b, the value of the average fluorescence intensity of PEIꢀGA/DOX/DNA
group detected by flow cytometer was remarkably greater than that of DOX and
PEIꢀGA/DOX/DNA add GA groups, indicating that cellular uptake of
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PEIꢀGA/DOX/DNA into HepG2 cells was highly influenced by GA moiety. Moreover,
we performed the luciferase assay in line with the manufacturer’s protocol to check
transfection efficiency. The transfection efficiency was expressed in relative light
units (RLU). As indicated in Figure. 3c, the transfection efficiency of luciferase of the
PEIꢀGA/DOX /DNA complexes exhibited about five times as big as that of the
PEI25k/DNA polyplexes. The results revealed that GA could functions effectively to
mediated PEIꢀGA/DOX/DNA nanoparticles into cytoplasm, which might be due to
the targeting ability of GA to liver tumor.23ꢀ25, 29ꢀ31, 33 The high transfection efficiency
of PEIꢀGA is fundamental for in vivo studies.
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Figure. 3. (a) Flow cytometric analysis of fluorescence intensity of PEIꢀGA, DOX,
PEIꢀGA/DOX/DNA+GA and PEIꢀGA/DOX/DNA nanoparticles treated cells. 10000
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cells were counted and analyzed in each experiment. (b) Luciferase activity of DNA
(pGL3ꢀcontrol), PEI25k, PEIꢀGA/DNA, PEIꢀGA/DOX/DNA and the combination of
PEIꢀGA/DOX/DNA and GA. The experiments were confirmed three times. (c)
Microscopic observations of DOX internalized in the HepG2 cells with treatment of
DOX, PEIꢀGA/DOX/DNA+GA and PEIꢀGA/DOX/DNA (at a functional N/P ratio)
following a 6 h period of incubation (scale bar = 100 ꢁm). (d) In vitro cytotoxicities
of PEIꢀGA with different molecular weight of PEI and graft ratio of GA at various
concentrations at 24 h in the HepG2 cells. The experiments were confirmed three
times. (e) Western blot analyses of Akt1, LC3BꢀI and LC3BꢀII proteins in HepG2
cells after incubation of PEIꢀGA/DOX and PEIꢀGA/DOX/shAkt1 formulations. (f)
Apoptosis analysis of HepG2 cell line after treatment with PEIꢀGA, DOX,
PEIꢀGA/DOX and PEIꢀGA/DOX/shAkt1, which was determined by Annexin
V/propidium iodide staining.
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In vitro cytotoxicity. Cytotoxicity is an important consideration for gene/drug
carriers. Cytotoxicity of PEIꢀGA against HepG2 cells with different degrees of
substitution and various concentrations was evaluated by MTS assay. As shown in
Figure. 3d, PEIꢀGA conjugates showed higher biocompatibility with decreasing
molecular weight of PEI and increasing degree of GA substitution. As PEI3.5KꢀGA1/2
was nontoxic even at 50 ꢁg/mL during 24 h incubation time, so it may be a safe
delivery carrier. This was probably attributed to its lower charge (19.9 mV) under
physiological pH condition. The in vitro cytotoxicity of PEI3.5KꢀGA1/2 was
determined in HepG2 cells using Light microscopic observation. The results indicated
that the polymer displayed almost no cytotoxicity even at high concentrations (data
not shown). The IC50 of free DOX, PEIꢀGA/DOX and PEIꢀGA/DOX/shAkt1
nanoparticles (N/P ratio:10) was estimated to be approximately 4.56, 2.07 and 0.99
µg/mL, respectively. The IC50 of PEIꢀGA/DOX/shAkt1 against HepG2 cells was
3.6ꢀfold lower than that of free DOX. This enhanced inhibitory effect could be
attributed to the combined therapy effect of DOX and shAkt1. For one thing, PEIꢀGA
nanoparticles could deliver more amounts of DOX into HepG2 cells by comparison
with free DOX solution. The phenomena were well matched with cellular uptake
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study as shown in Figure. 3a and 3b. For another thing, there is a link between the
activation of Akt kinase and human tumors,37 and inhibition of the PI3ꢀkinaseꢀAkt
signaling pathway by Akt1 shRNA might suppress the survival signal delivery that
ultimately leads to the induction of apoptosis.38 Therefore, it was reasonable to
assume that PEIꢀGA/DOX/shAkt1 complexes could works as an efficient platform for
cancer combination therapy.
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Apoptosis and autophagy. After treatment in HepG2 cell line with
PEIꢀGA/DOX/shAkt1 nanoparticles, cell growth slowed down, and furthermore the
cells became more circular in shape and were progressively distorted. At 24 h after
transfection with PEIꢀGA/DOX/shAkt1 nanoparticles, a marked decrease in the
expression of the Akt1 protein was observed as shown in Figure. 3e. We also found
that the expression of the protein of the cells treated with PEIꢀGA/DOX alone had
almost no change compared with the control. Furthermore, it was amusing to observe
that the transfection of PEIꢀGA/DOX/shAkt1 complexes up regulated the expression
of autophagyꢀrelated LC3BꢀII protein. It was already reported that inhibition of Akt1
could induce autophagy39 and autophagy constitutes an alternative pathway to cell
death that is called type II cell death.40 After treatment with PEIꢀGA/DOX/shAkt1
nanoparticles, the reduction of Akt1 protein level resulted in the up regulation of
LC3BꢀII protein, which might significantly induce autophagy. High transfection of
PEIꢀGA/DOX/shAkt1 nanoparticles in HepG2 cells (Figure. 3b) might be related with
the low expression of Akt1 protein level (Figure. 3e). To further assess whether
PEIꢀGA/DOX/shAkt1 complexes could efficiently induce apoptosis, results were
observed using annexin V/PI double staining and the percentage of apoptosis was
quantified. As indicated in Figure. 3f, when the HepG2 cell line was treated with
DOX, PEIꢀGA/DOX, and PEIꢀGA/DOX/shAkt1, apoptosis was induced 15.2, 27.6
and 55.7 %, respectively, suggesting that apoptosis in HepG2 cells could be
significantly enhanced by PEIꢀGA/DOX/shAkt1. In addition, apoptosis induction
from PEIꢀGA was 0.7 %, similar to controls (0.2 %). This suggests that blank PEIꢀGA
nanoparticles had no direct effect on apoptosis. The data achieved by apoptosis assay
and cell growth inhibition experiment in the HepG2 cell line are in good accordance.
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The apoptosis might be related with the induction of autophagy after the inhibition of
the expression of Akt in the HepG2 cell line treated with PEIꢀGA/DOX/shAkt1
complexes.
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Hemolysis test and systemic toxicity. The in vivo application of cationic
polymers is often hindered by its nonspecific interactions with blood components. The
compatibility of cationic vectors is a very important indicator for its introduction into
the systemic circulation.41 Figure. 4a displayed the effect of PEIꢀGA/DOX/DNA
complexes on hemolytic activity at the functional N/P ratio. PEI25k/DOX/DNA
nanoparticles resulted in notable hemolysis, yet lower hemolysis was found in
PEIꢀGA/DOX/DNA complex. Change in body weight and histological difference
were investigated to evaluate systemic toxicity in mice. As indicated in Figure. 4b, the
body weights of mice with PEIꢀGA nanoparticles treatment had no serious loss
compared with saline treatment for a 2ꢀweek observation period, which suggested that
no significant toxicity was induced by PEIꢀGA. There was also no obvious
histological difference of major organs between the two groups (Figure. 4c). These
findings suggested that PEIꢀGA is potential as an intravenous carrier for drug
delivery.
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Figure. 4. (a) Hemolysis of PEI25k, PEIꢀGA and PEIꢀGA/DOX/shAkt1 at various
concentrations. (b) Change in body weight. (c) Histological analysis for heart, liver,
spleen, lung and kidney organs of mice at 16 days post intravenous injection of
PEIꢀGA once a day (scale bar = 20 ꢁm). (d) Tumor growth curves and (e) survival
rates of saline, DOX, blank PEIꢀGA, PEIꢀGA/DOX and PEIꢀGA/DOX/shAkt1
formulations injected Hepaꢀ1.6 tumor bearing mice.
In vivo antitumor assay in xenograft mice model. The effect of
PEIꢀGA/DOX/shAkt1 complexes on tumor growth was investigated by in vivo tumor
suppression experiment. Hepaꢀ1.6 cells were inoculated subcutaneously in the
C57BLꢀ6J mice. As shown in Figure. 4d, we see no significant difference of tumor
volume between the PEIꢀGA group and the control group. In contrast, the group
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treated with PEIꢀGA/DOX/shAkt1 nanoparticles indicated outstanding growth
inhibition of Hepaꢀ1.6 cell grafted tumorꢀbearing C57BLꢀ6J mice. Survival rate at the
periods of time was indicated in Figure. 4e. Coꢀdelivery of shAkt1 and DOX by
PEIꢀGA was more powerful. According to reports, the efficacy of DOX in HCC is
frequently not satisfactory, which may be associaited with the activation of Akt
expression. RNA interference mediated by shRNA can specifically silence the targetd
gene. In this case, combining DOX with shAkt could be a promising strategy for HCC
therapy. To improve delivery to tumors, PEIꢀGA nanoparticls were developed and
utilized. The application of PEIꢀGA/DOX/shAkt1 complexes has resulted in
distinguished antitumor effect on the Hepaꢀ1.6 tumor bearing mice.
PEIꢀGA/DOX/shAkt1 complexes might provide a promising strategy for hepatic
carcinoma therapy.
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CONCLUSION
A low toxicity and high performance nanoparticle system was successfully
constructed. PEIꢀGA with suitable graft ratio of GA can easily selfꢀassemble into
nanoꢀaggregates based on the interaction of functional groups among GA. The graft
of GA not only could markedly increase the transfection efficiency of PEI, but also
could decrease the cytotoxicity of PEI. The formed nanoparticles showed suitable
physicochemical properties to coꢀdeliver gene/drug and effectively protected genes
from degradation by enzyme. The IC50 of PEIꢀGA/DOX/shAkt1 against HepG2 cells
was 3.6ꢀfold lower than that of free DOX. In addition, better antitumor efficacy of
PEIꢀGA/DOX/shAkt1 was observed on xenograft liver tumor. Therefore,
PEIꢀGA/DOX/shAkt1 showed great potential in hepatic carcinoma therapy and the
strategy of delivering chemotherapy drug and therapeutic genes using PEIꢀGA to
simultaneously induce apoptosis and autophagy may represent a platform for future
preclinical and clinical development.
AUTHOR INFORMATION
Corresponding Author
Professor HuꢀLin Jiang, Eꢀmail: jianghulin3@163.com, Phone: +86ꢀ25ꢀ83271027,
Fax: +86ꢀ25ꢀ83271027;
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Professor Li Zong, Eꢀmail: zong216@yahoo.com.cn, Phone: +86ꢀ25ꢀ83271317; Fax:
+86ꢀ25ꢀ83271335.
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Notes
9
The authors declare no competing financial interest.
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Acknowledgements
This work was supported by the Funds for Creative Research Groups of China,
National Natural Science Foundation of China (No. 81421005) and the Priority
Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Part of this work was supported by the Jiangsu Province Public Technology Service
Center of Nanodrug Preparation and Evaluation. Part of this work was also supported
by the National Natural Science Foundation of China (Grant No. 81573369 and
21301191), the Natural Science Foundation of Jiangsu Province (Grant No.
BK20130661 and BK20140659). Part of this work was also supported by the
Research Fund of the University of Macau (MYRG2015ꢀ00101ꢀICMSꢀQRCM). H.L.
Jiang was supported by the SpeciallyꢀAppointed Professors in Jiangsu Province
Program.
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Poly(ethylenimine)-glycyrrhetinic acid nanoparticles loaded with doxorubicin and shAkt1
88x35mm (300 x 300 DPI)
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