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Lysosome-independent intracellular drug/gene codelivery by lipoprotein-derived nanovector for synergistic apoptosis-inducing cancer-targeted therapy Wei Wang, Kerong Chen, Yujie Su, Jielei Zhang, Min Li, and Jianping Zhou Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01549 • Publication Date (Web): 15 Jan 2018 Downloaded from http://pubs.acs.org on January 15, 2018

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Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Biomacromolecules

Lysosome-independent co-delivery

by

intracellular

lipoprotein-derived

drug/gene

nanovector

for

synergistic apoptosis-inducing cancer-targeted therapy Wei Wang ‡, *, Kerong Chen ‡, Yujie Su, Jielei Zhang, Min Li, Jianping Zhou

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China

‡

W. Wang and K. Chen contributed equally to this work.

KEYWORDS: lysosome-independent; drug/gene co-delivery; reconstituted high density lipoprotein; synergistic apoptosis-inducing; tumor targeting therapy.

ABSTRACT: In this paper, reconstituted high-density lipoprotein (rHDL), a lipoprotein-derived nanovector, were constructed for co-delivery of paclitaxel (PTX) and wild type p53 gene (p53). The particle size and the zeta potential of PTX-DODAB/p53-rHDL nanoparticles were 177.2 nm and -20.06 mV, respectively. Meanwhile, they exhibited great serum stability and satisfactory sustained

release

characteristics

in

vitro.

PTX-DODAB/pDNA-rHDL

nanoparticles

simultaneously improved the cellular uptake of PTX and pDNA via scavenger receptor B type I (SR-BI) mediated lysosome-independent internalization and promoted the transfection of pDNA in MCF-7 cells, which were revealed by flow cytometry and confocal laser scanning microscopy

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analyses. The high p53 protein expression in MCF-7 cells after rHDL-mediated transfection was detected by western blotting assay. Moreover, PTX-DODAB/p53-rHDL nanoparticles showed superior cytotoxicity and significantly induced apoptosis in SR-BI over-expressed MCF-7 cells. In in vivo studies, PTX-DODAB/p53-rHDL nanoparticles without obvious toxic effects to vessels, blood or major organs exhibited efficient tumor targeting and encouraging antitumor effects on tumor-bearing nude mice compared with controls. All the results above indicated that PTX-DODAB/p53-rHDL

nanoparticles

held

broad

prospects

in

combination

of

chemotherapeutics and gene therapeutic agents for cancer-targeted therapy.

1. INTRODUCTION Advances in genomics and cell biology have emphasized the heterogeneity and complicacy of cancer1. Cancer is generally accepted as the result of combination of related disease pathways that generally cannot be healed effectively with monotonous therapeutic mechanisms2. The side effects of single-drug therapy often generate risks of drug resistance and tumor relapse, leading to tumor progression3-4. For this reason, combined treatment with synergistic mechanism is required for cancer treatment in order to avoid drug resistance5, improve antitumor effect and reduce the systemic toxicity and dosage of chemotherapeutics. An applicable vector with drug co-delivery and specific tumor targeting is urgently demanded to achieve this objective.

High density lipoprotein (HDL) is one of the four major species of lipoproteins, which is the smallest but densest. It plays a vital role in reverse cholesterol transport by promoting the return of excess cholesterol from peripheral cells to the liver for elimination6-7. HDL as an ideal drug


2

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carrier is completely biodegraded. With unique structure of hydrophilic shell-hydrophobic core and the small size, it can escape from the reticuloendothelial system (RES) recognition, showing longer residence time in body circulation8. The cellular uptake of HDL was mediated by scavenger receptor class B type I (SR-BI) which is mainly expressed in the liver9 and most malignant cells10-11. The process of this mechanism may be visualized as the development of a hydrophobic channel, through which lipid composition of HDL diffuse to the cell plasma while aqueous phase are excluded12-13. Reconstituted high density lipoprotein (rHDL) is composed of phospholipids, cholesterol, cholesteryl esters and apolipoproteins, etc.14-15, which is similar to endogenous HDL in composition and function16. Apolipoprotein A-I (apoA-I) is the most important and abundant protein constituent of HDL, which is utilized to fabricate rHDL by one-pot self-assembly or sonication with phospholipids, and it is considered to be responsible for scaffolding the size and shape of natural HDL species17. Moreover, the rHDL with simple preparation process can overcome biosafety issues, which possesses the extensive foreground as anticarcinogen nanovectors18.

A prepossessing way of inhibiting tumor growth is to reintroduce regulatory mechanisms regulated by antioncogene into tumor cells, thus suppressing tumor proliferation and metastasis19. Wild type p53 is a well-known antioncogene named for the expression of a protein with the molecular weight of 53 kDa. The main function of p53 protein is to inhibit tumor cell growth in cell cycle regulation and induce tumor cell apoptosis20. Studies have shown that almost every type of tumor cells present p53 pathway defects and more than 50 % kinds of tumors have p53


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mutation21-22. p53 gene therapy is to import wild type p53 gene into the tumor cells using a vector system23 and rebuild p53 gene function, thereby inducing apoptosis to inhibit tumor cell growth24. In order to enhance the efficiency of tumor therapy, paclitaxel (PTX), which is commonly used in drug delivery of anticancer therapy25, was combined with p53 to produce a synergistic apoptotic effects to overcome breast cancer26.

In this research, dimethyldioctadecylammonium bromide (DODAB), a cationic lipid, was used to condense plasmid DNA (pDNA) to form an amphiphilic binary complexes. Afterwards, hydrophobic chemotherapeutics PTX and hydrophilic gene therapeutic agents wild type p53 pDNA condensed by DODAB were simultaneously loaded into the lipid core of rHDL nanoparticles to treat SR-BI over-expressed MCF-7 human breast cancer27 (shown in Figure 1). This biomimimetic co-delivery system possessed unique mechanism of cellular uptake epitomized as lysosome-independent passway, through which p53 and PTX were simultaneously delivered into tumor cells to achieve synergistic antitumor effect. We studied the physicochemical properties, cellular targeting and lysosome-independent mechanisim of cellular uptake, synergistic anti-tumor effect and safety of PTX-DODAB/p53-rHDL in vitro and in vivo. Our research devoted to establish a theoretical and experimental basis for the neotype potential targeted drug co-delivery systems for clinical use in tumor therapy.


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Figure 1. Schematic design of PTX-DODAB/p53-rHDL as lysosome-independent intracellular co-delivery platform of chemotherapeutics and therapeutic gene for tumor-targeted synergistic therapy.

(A)

Preparation

process

of

PTX-DODAB/p53-rHDL

nanoparticles.

Receptor-meditated nanoparticles cellular internalization and intracellular trafficking:

(B)

(I) rHDL

specifically binds to SR-BI of tumor cells. (II) p53 protein expression. (III) PTX exerts apoptosis therapeutic effects synergistically with p53 protein.


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2. MATERIALS and METHODS 2.1. Materials, cell culture, and animals.

DODAB was purchased from Aladdin Reagent Inc. (Shanghai, China). The reporter plasmid (pEGFP-C3, 4.7 kb) encoding enhanced green fluorescent protein (GFP) and plasmid pCMV-Neo-Bam-p53wt (wt-p53, 8.4 kb) encoding wild type p53 gene (p53) driven by CMV promoter purchased from Addgene were respectively propagated in DH-5a Escherichia coli and purified by Endo Free Plasmid Maxi Kit (Qiagen, Germany). Soybean phospholipids was purchased from Shanghai A.V.T. pharmaceutical Co.,Ltd; cholesterol, cholesteryl esters and paclitaxel (PTX) was purchased from Sigma-Aldrich (MO, USA). Apolipoprotein A-I (apoA-I), the major protein component in HDL nanoparticles, was isolated and highly purified from the albumin byproduct in our laboratory according to an established protocol28. Deoxyribonuclease I (DNase I), DAPI, YOYO-1, coumarin-6 and Cy5.5-NHS were purchased from by Invitrogen Co. (Carlsbad, CA, USA). Cy5-dsDNA was purchased from RIBOBIO Bio-Technology Company (Guangzhou, China). Primary antibodies and Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from KeyGEN BioTECH Corp.,Ltd (Jiangsu, China). Annexin V-FITC apoptosis detection kit was purchased from BioVision (CA, USA). The near-infrared (NIR) dye DiR was purchased from Beijing Fanbo Science and Technology Co., Ltd. (Beijing, China). All chemicals were analytical grade.


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Human breast cancer cell line MCF-729 with low expression of p53 was obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM (HyClone, USA) containing 10% (v/v) fetal bovine serum (FBS) (Gibco, USA), 100 U/mL of penicillin and 100 µg/mL of streptomycin in an incubator (Thermo Scientific, USA) at 37 ± 0.5 °C under 5% CO2 and 90% relative humidity, and were subcultivated every 3 days at 80~90% confluence using 0.25% (w/v) trypsin at a split ratio of 1:5.

The adult female BALB/c nude mice (6 weeks, 18~22 g) were purchased from Shanghai Laboratory Animal Center (SLAC, China). All the mice were allowed to obtain food and water ad libitum in the SPF II lab and were conducted consistently with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

2.2. Preparation of PTX-DODAB/pDNA-rHDL nanoparticles

2.2.1. Preparation of DODAB/pDNA complexes. Cationic lipid DODAB was applied to the compression of pDNA at various N/P ratios. Briefly, pDNA was dissolved in distilled water to obtain a desired concentration of 1 mg/mL, then the solution was mixed with equal volume of DODAB/ethanol solution at different N/P ratio under gentle vortex for 30 s followed by incubation for 30 min at room temperature. The DODAB/pDNA complexes were confirmed by electrophoresis on a 0.6% agarose gel at 90 mV for 45 min in 0.5×Tris-Borate-EDTA (TBE) buffer solution. pDNA was visualized with staining of Goldview (Amresco, USA) and the gel image was taken under Gel-Pro analyzer (Genegenius, Syngene, UK). Subsequently, particle size


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and zeta potential of DODAB/pDNA complexes were detected by Zetasizer Nano-ZS90 (Malvern, UK) at 25 °C with 90 ° scattering angle.

2.2.2. Preparation of PTX-DODAB/pDNA-rHDL nanoparticles. PTX-DODAB/pDNA-rHDL nanoparticles were prepared by the solvent evaporation method. Briefly, 10 mg of soybean phospholipids (PC), 2 mg of cholesterol (C) and 4 mg of cholesteryl esters (CE) (5:1:2, w/w) were dissolved in chloroform. Meanwhile, 1.6 mg of PTX (weight ratio of total lipids vs PTX = 10:1) was added into the lipidic phase. The mixture was evaporated to remove organic solvent followed by vacuum desiccation for 6 h to remove residual organic solvent. After that, the film was dispersed using the solution of DODAB/pDNA complexes (concentration of pDNA was 2 µg/mL) diluted by 1 mL of phosphate buffered saline (PBS, pH 7.4). The solution was then dispersed by a probe-type ultrasonicator (JY 92-2D, Ningbo Scientz Biotechnology Co., Ltd, Nanjing, China) in ice bath at 100 W for 10 min. The unincorporated PTX was removed by a 0.22 µm cellulose nitrate membrane to obtain the drug co-delivery system of nanostructured lipid carriers (NLCs) (noted as PTX-DODAB/pDNA-NLC nanoparticles). Subsequently, the suspension was incubated with 2 mg of apoA-I (w/w 1:5 vs PC in PBS) overnight at 4 °C followed by loading to a Sepharose G-100 column (1.0×18 cm) and eluted with PBS (pH 7.4) to remove the extra apoA-I. Monotherapy groups as PTX-rHDL and DODAB/pDNA-rHDL were prepared and characterized as the description in Supporting Information. The encapsulation efficiency (EE) of PTX or pDNA (labeled by YOYO-1 ， noted as pDNAYOYO-1) in PTX-DODAB/pDNA-rHDL nanoparticles was calculated by the following formulation (1):


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EE%= C/C0×100% (1)

where C0 and C were the concentration of added drugs and the concentration of drugs in the nanoparticles, respectively. The concentration of PTX in samples was analyzed by high performance liquid chromatography (HPLC) while pDNAYOYO-1 was detected by fluorescence spectrophotometry.

2.3. Particle size, zeta potential and serum stability assays.

2.3.1. Particle size and zeta potential assays. The particle size and zeta potential of PTX-DODAB/pDNA-rHDL nanoparticles were measured by Zetasizer Nano-ZS90 (Malvern, UK) at 25 °C with 90 ° scattering angle after diluting 50 times with distilled water. An H-7650 transmission electron microscope (TEM) was used to visualize the morphology and size distribution of the PTX-DODAB/pDNA-rHDL nanoparticles.

2.3.2. Serum stability assays. Serum stability assays were carried out to demonstrate the effect of rHDL on the serum stability of the nanoparticles. The stability of PTX-DODAB/pDNA-rHDL nanoparticles was assayed in the presence of FBS. Nanoparticles were mixed with the equal volume of FBS and incubated for prearranged time (1, 3, 6, 12, 24 h) at 37 °C. After incubation, the samples were diluted 50 times with distilled water and the particle size of samples were measured by Dynamic Light Scattering (DLS) Analyzer (NanoZS-90, Malvern instruments, UK).

2.4. In vitro release of PTX.


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The release of PTX in vitro was performed using a dialysis method. PBS (pH 7.4) containing

0.1%

(w/v)

Tween

80

was

used

as

the

release

medium

for

PTX.

PTX-DODAB/pDNA-rHDL (1 mL, containing 0.5 mg of PTX) was placed into dialysis tubes (MWCO=10 kDa) respectively and tightly sealed. The dialysis tubes were added into 100 mL release medium and incubated at 37 °C with gentle oscillation for 24 h. At predetermined time points, 0.1 mL of release medium was sampled and replaced with equal volume of fresh release medium. Free PTX was set as control. The samples were then analyzed by HPLC to determine the concentrations of PTX.

2.5. Cellular uptake and intracellular distribution of PTX-DODAB/pDNA-rHDL.

2.5.1. Cellular uptake.

In order to verify the SR-BI targeting property while sensitively visualize the intracellular behavior of the PTX-DODAB/pDNA-rHDL, the hydrophobic fluorescent probe coumarin 6 (C6) was loaded into rHDL to indicate the position of PTX, while pDNA was replaced by Cy5 labeled dsDNA (noted as pDNACy5). The cellular uptake of C6-DODAB/pDNACy5-rHDL was analyzed by flow cytometry and confocal laser scan microscopy (CLSM, Leica TCS SP5, Germany). The MCF-7 cells (1×105 cells/well) were seeded in 6-well plates (Costar, USA), then the culture medium was replaced by 0.5 mL of culture medium (FBS free) containing 4 µg/mL of pDNACy5 and 0.1 µg/mL of C6 in C6-DODAB/pDNACy5-rHDL and C6-DODAB/pDNACy5-NLCs at 37 °C, respectively. For competition assay, excessive of apoA-I (a terminal concentration of 1 mg/mL)


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was added 2 h prior to C6-DODAB/pDNACy5-rHDL. After incubation for 4 h, the medium was removed, and the treated cells were washed three times by PBS (pH 7.4) and then fixed with 4% paraformaldehyde for 15 min, followed by adding DAPI to stain the cell nucleus. CLSM was utilized to observe cells, with excitation at 359 nm, 465 nm and 649 nm, and emission at 461 nm, 502 nm and 670 for DAPI, C6 and Cy5, respectively. Meanwhile, the fluorescent signal of C6 and Cy5 in cells was quantified by flow cytometer.

2.5.2. Intracellular distribution.

For intracellular trafficking experiments of PTX-DODAB/pDNA-rHDL, MCF-7 cells (1 × 105 cells/dish) were seeded in 35 mm confocal dishes (Φ = 15 mm). pDNA was labeled by YOYO-1 (noted as pDNAYOYO-1) to indicate the position. Cells were treated with PTX-DODAB/pDNAYOYO-1-rHDL and PTX-DODAB/pDNAYOYO-1-NLCs containing 4 µg/mL of pDNAYOYO-1 dispersed in culture medium (FBS free). For competition assay, excessive of apoA-I

(a

terminal

concentration

of

1

mg/mL)

was

added

2

h

prior

to

PTX-DODAB/pDNAYOYO-1-rHDL. At presupposed time points, cells were stained by 50 nM Lyso-Tracker Red for 1 h and rinsed three times with isotonic PBS (pH 7.4). The intracellular trafficking of nanoparticles was visualized using CLSM with excitation at 491 nm and 577 nm, and emission at 509 nm and 590 for YOYO-1 and Lyso-Tracker Red, respectively.

For the purpose of locating apoA-I, Cy5.5 was used to labeled apoA-I to form PTX-DODAB/pDNAYOYO-1-rHDL/Cy5.5-apoA-I on the basis of the established protocol18. Cells


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were treated in the same methods above followed by washing three times by isotonic PBS (pH 7.4) and then fixed with 4% paraformaldehyde for 15 min and adding DAPI to stain the cell nucleus. The location of Cy 5.5-apoA-I and pDNAYOYO-1 was observed by CLSM, with excitation at 359 nm, 491 nm and 675 nm, and emission at 461 nm, 509 nm and 695 for DAPI, YOYO-1-pDNA and Cy 5.5-apoA-I, respectively.

2.6. Transfection study of PTX-DODAB/pDNA-rHDL.

The pEGFP-C3 was utilized as a reporter gene to verify the transfection efficiency of PTX-DODAB/pDNA-rHDL. The MCF-7 cells were seeded in 6-well plates for 24 h at a density of 1×105 cells/well. The cells were treated with 1 mL of culture medium (FBS free) containing 4 µg/mL of pEGFP-C3 in PTX-DODAB/pEGFP-C3-rHDL, PTX-DODAB/pEGFP-C3-NLCs, respectively. Naked pEGFP-C3 with concentration of 4 µg/mL was set as control. For competition assay, excessive of apoA-I (a terminal concentration of 1 mg/mL) was added 2 h prior to PTX-DODAB/pEGFP-C3-rHDL. After incubation for 4 h, the formulations were replaced by fresh complete culture medium and the cells were incubated for 48 h. The expression of green fluorescent protein (GFP) in MCF-7 cells was observed under CLSM with excitation at 488 nm and emission at 509 nm for GFP and the transfection efficiency of complexes was quantified by flow cytometry.

2.7. Analysis of p53 protein expression.

The expression of p53 protein in MCF-7 cells treated with various formulations was


12

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evaluated by western blotting assay. MCF-7 cells (1×105 cells/well) were seeded in 6-well plates for 24 h followed by transfection with PTX-DODAB/p53-rHDL and PTX-DODAB/p53-NLCs (containing DNA 4 µg/mL in complete culture medium) for 48 h. Naked p53 with concentration of 4 µg/mL and PBS (pH 7.4) were acted as control. For competition assay, excessive of apoA-I (a terminal concentration of 1 mg/mL) was added 2 h prior to PTX-DODAB/p53-rHDL. After 48 h incubation, proteins in treated cells were isolated by the lysis buffer (Beyotime Institute of Biotechnology, China) and the concentration was quantified by standard BCA protein assay kit (Beyotime Institute of Biotechnology, China). Subsequently, the total protein samples were separated on a 15% SDS-PAGE and transferred onto the PVDF membranes (Merck Millipore, USA) followed by incubating with mouse anti-p53 protein antibodies and mouse anti-β-actin primary antibodies overnight at 4 ºC. Afterwards, the membranes were incubated with HRP-conjugated anti-mouse secondary antibodies and the blots were observed with ECL system (Pierce, USA).

2.8. Cytotoxicity study of PTX-DODAB/pDNA-rHDL in vitro.

The in vitro cytotoxicity effect of blank vector and drug loaded nanoparticles was evaluated by MTT assay with MCF-7 cells. The cells (5×103 cells/well) were seeded in 96-well plates (Costar, USA) and cultured at 37 °C for 24 h. After discarding the medium, the cells were incubated with 200 µL of PTX-DODAB/p53-rHDL, PTX-DODAB/p53-NLCs, PTX-rHDL, DODAB/p53-rHDL, PTX-DODAB/pDNA-rHDL and free PTX dispersed in complete culture medium with several concentrations for 48 h at 37 °C, respectively. Subsequently, 20 µL of MTT


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solution (5 mg/mL) was added into each well, and incubated with cells at 37 °C for 4 h. Finally, the solutions in each well were removed, and MTT formazan crystals were dissolved by 150 µL of dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm by an ELISA reader (Thermo Scientific, USA). The cytotoxicity of rHDL and NLCs blank vector were also evaluated. Cell viability was calculated by the following formulation (2):

Cell viability (%) = Asample/Acontrol×100% (2)

where Asample and Acontrol represented the absorbance of the cells treated with the corresponding sample and the blank culture medium (FBS free) as a negative control, respectively. Meanwhile, the half maximal inhibitory concentration (IC50) of each group was measured by GraphPad Prism 6.

2.9. Analysis of cell apoptosis in vitro.

To evaluate the cell apoptosis induced by PTX-DODAB/p53-rHDL, MCF-7 cells were treated with PTX-DODAB/p53-rHDL, PTX-DODAB/p53-NLCs, DODAB/p53-rHDL and PTX-rHDL (PTX concentration of 10 µg/mL or p53 concentration of 200 ng/mL in complete culture medium) for 48 h under 37 °C. Taxol® and PBS (pH 7.4) were used as control. The cells were then harvested, washed three times with PBS (pH 7.4) and resuspended in 500 µL binding buffer. Annexin V-FITC (5 µL) and PI (5 µL) were added and incubated with the cells for 10 min without light. Finally, the stained cells were analyzed by a flow cytometer (FACS Calibur, Beckman Coulter, USA).


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2.10. In vivo imaging and tumor targeting.

The orthotopic tumor-bearing nude mice were established by inoculating the suspension of MCF-7 cells (3 × 106 cells in 0.2 mL of PBS) to the mammary fat pad of female BALB/c mice. In vivo test was conducted with tumor volume of approximately 60 mm3. For in vivo imaging of PTX-DODAB/pDNA-rHDL, NIR dye DiR was labeled in PTX-DODAB/pDNA-rHDL (PTXDiR-DODAB/pDNA-rHDL) and injected into the tail vein of tumor-bearing nude mice at a DiR dose of 1 µg per mouse to investigate the biodistribution and tumor-targeting efficacy. PTXDiR-DODAB/pDNA-NLCs group was set as control. The fluorescence imaging were taken at 6 h, 12 h, 24 h and 48 h post-injection utilizing an in vivo imaging system (FX PRO, Kodak, USA) equipped with DiR filter sets (excitation/emission, 720/790 nm). Images were analyzed by the Kodak Molecular Imaging Software 5.1. After 48 h living imaging, the mice were sacrificed while tumor tissues and major organs were excised for ex vivo imaging using the same imaging system.

2.11. Antitumor efficacy.

The tumor-bearing nude mice were divided into the following six groups treated at 0, 2, 4, 6, 8, 10 and 12 days: PTX-DODAB/p53-rHDL, PTX-DODAB/p53-NLCs, PTX-rHDL, DODAB/p53-rHDL, Taxol® and saline (containing 10 mg/kg of PTX or 200 µg/kg of p53). The body weight was measured every other day. At day 14, one mouse of each group was sacrificed and the tumors were excised, weighed and fixed in 10% formalin. The fixed tumors were


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embedded in paraffin blocks to prepare hematoxylin and eosin (H&E) stained tumor sections, and then were observed by optical microscope (Olympus, Japan). The survival time of each group was simultaneously analyzed. The tumor inhibition rate was calculated using the following formula (3):

Inhibition rate (%) = (VControl – VAdministration) / VControl × 100% (3)

where VAdministration and VControl represented the average volume of the tumor treated with the corresponding formulation and the normal saline as a negative control, respectively.

2.12. Safety test.

2.12.1. Rabbit ear vein irritation test. Venous irritation was assess by administration of PTX-DODAB/p53-rHDL (containing 10 mg/kg of PTX and 200 µg/kg of p53) on New Zealand rabbits (2.2-2.5 kg) via marginal vain of left ear for 3 days while marginal vain of right ear was injected by the equal volume of saline as negative control. The administrated rabbits were sacrificed 24 h after final dosing and the treated ears were cut and fixed in 10% formalin for histopathological examination.

2.12.2. Safety profiles of PTX-DODAB/p53-rHDL. PTX-DODAB/p53-rHDL was injected into tail vein of healthy BALB/c female mice at a PTX dose of 10 mg/kg and p53 dose of 200 µg/kg to analyze the toxicity of the nanoparticles every other day for a total of three times with saline as control. After the final injection for 24 h, the routine blood tests were proceeded by collection of blood at Zhongda Hospital. Additionally, the blood was centrifuged to obtain plasma that was


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used to quantify the serum levels of aspartate amino transferase (AST), alanine amino transferase (ALT) and blood urea nitrogen (BUN) to assess hepatic and renal damage. After two weeks injection, the treated mice were sacrificed and the main organs containing heart, liver, spleen, lung and kidney were separated from each mouse for histological evaluation by H&E staining.

2.13. Statistical analysis.

All data were expressed as mean ± standard deviation (SD) and calculated using Student’s t-test and statistical significance was set at *P < 0.05, and extreme significance was set at **P < 0.01.

3. RESULTS and DISCUSSION 3.1. Characterizations of PTX-DODAB/pDNA-rHDL nanoparticles.

For encapsulating the hydrophilic pDNA into the hydrophobic core of rHDL, a cationic lipid DODAB was utilized to condense anionic pDNA through electrostatic interaction. Gel electrophoresis retardation assay, particle size and zeta potential results were shown in Figure 2A and Figure 2B. The electrophoretic migration of pDNA was completely retarded when the N/P of DODAB/pDNA complexes was more than 1, suggesting DODAB perfectly condensed pDNA at a low N/P ratio. The particle size of DODAB/pDNA complexes decreased with the increase of N/P, and was minimum (108.5 ± 3.1 nm) at the N/P of 4. The particle size and polydispersity index (PDI, in Table S1) did not changed significantly when N/P was larger than 4. The zeta potential of complexes increased with the increase of N/P (< +30 mV). As shown in


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Figure S1, the complexes proved an excellent stability against heparin, DNase I and serum at the N/P of 4. For this reason, we ultimately chose the DODAB/pDNA complexes at the N/P of 4 to be loaded into rHDL in the following experiments.

Subsequently, DODAB/pDNA complexes (N/P = 4) were incorporated into the lipid core of PTX-rHDL nanoparticles to construct drug/gene co-delivery system. As shown in Figure 2C and Figure 2D, PTX-DODAB/pDNA-rHDL nanoparticles had the resulting particle diameter of 177.2 ± 3.9 nm with the PDI of 0.106 ± 0.070 and the zeta potential of -20.06 ± 3.27 mV. The suitable size scale and negative zeta potential was beneficial for the in vivo stability, distribution and tumor targeting of the nanoparticles. The size and morphology of the nanoparticles were visualized directly by TEM as shown in the inset of Figure 2C. The observed size of nanoparticles was approximately 150 nm that was similar to hydrodynamic diameter obtained from the DLS measurements, and the quasi-spherical structure of nanoparticles was observed and confirmed. The encapsulation efficacy of pDNA in PTX-DODAB/pDNA-rHDL nanoparticles confirmed by the fluorescence spectrophotometry was (81.35 ± 2.41) %, and the encapsulation efficiency and drug loading of PTX was (92.67 ± 3.14) % and (8.86 ± 1.13) % analyzed by HPLC, respectively, indicating the drug combo of hydrophobic PTX and hydrophilic pDNA was efficiently loaded into rHDL nanoparticles. On the contrast, PTX-DODAB/pDNA-NLC nanoparticles were characterized as 148.8 ± 4.8 nm of particle size with 0.114 ± 0.038 of PDI and -10.38 ± 3.49 mV of zeta potential. The encapsulation efficacy of pDNA and PTX in the NLC-based nanoparticles was (77.93 ± 4.25) % and (85.34 ± 3.87) %,


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Biomacromolecules

respectively. The particle size, zeta potential and encapsulation efficacy of pDNA or PTX were shown in Table S2.

3.2. Serum stability assays of PTX-DODAB/pDNA-rHDL nanoparticles.

There were varieties of ingredients in serum that have influences on the stability of nanoparticles in vivo. It was essential for nanocarriers to avoid the attack of plasma ingredients until reaching the final tumor target. The size changes of the nanoparticles were represented as stability indexes in the presence of FBS in vitro. As shown in Figure 2E, during 24 h incubation with FBS, the particle size had no significant changes compared with the initial one and the change of PDI was acceptable (less than 0.3). Meanwhile, PTX-DODAB/pDNA-rHDL nanoparticles provided the protection of pDNA against DNase I for at least 24 h as shown in Figure S3. The results showed that PTX-DODAB/pDNA-rHDL nanoparticles exhibited satisfactory stability of particle size in FBS, demonstrating that rHDL could be a stable systemic co-delivery tool for PTX and pDNA.

3.3. In vitro release of PTX and pDNA in PTX-DODAB/pDNA-rHDL nanoparticles.

The release profile of PTX was studied in isotonic PBS (pH 7.4) containing 0.1% (w/v) Tween 80 (Figure 2F). PTX in rHDL nanoparticles exhibited a little release with approximately 30% within 6 h incubation. Moreover, the cumulative release of PTX in rHDL was less than 50% after 24 h incubation in the release medium, which demonstrated that the drug/gene sustained release behavior was performed by rHDL-based nanoparticles.


19


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Figure 2. (A) Agarose gel electrophoresis retardation assay of DODAB/pDNA complexes with different N/P ratio. (B) Effect of different N/P ratio on the particle size and zeta potential of DODAB/pDNA

complexes

(n

=

3).

(C)

Size

distribution

histogram

of

PTX-DODAB/pDNA-rHDL. Inset: TEM image of PTX-DODAB/pDNA-rHDL. Scar bar: 200 nm. (D) Zeta potential of PTX-DODAB/pDNA-rHDL. (E) Influence of FBS on the particle size and polydispersity index of PTX-DODAB/pDNA-rHDL (n = 3). (F) Release profiles of free PTX and PTX-DODAB/pDNA-rHDL at different time points tested in PBS (pH 7.4) containing 0.1% (w/v) Tween 80 (n = 3).

3.4. Cellular uptake and intracellular distribution of PTX-DODAB/pDNA-rHDL nanoparticles.

In order to demonstrate the intracellular co-delivery of PTX and pDNA in


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PTX-DODAB/pDNA-rHDL nanoparticles, the hydrophobic fluorescence dyes C6 replacing PTX and Cy5 labeled ds-DNA (pDNACy5) were co-loaded into rHDL. The cellular uptake of C6-DODAB/pDNACy5-rHDL nanoparticles was investigated by flow cytometry, while C6-DODAB/pDNACy5-NLC group, apoA-I pre-saturated C6-DODAB/pDNACy5-rHDL group and isotonic PBS (pH 7.4) were used as untreated groups. As shown in Figure 3A and Figure 3B, the cellular uptake of rHDL group was significantly increased compared with NLCs group at 4 h after incubation, owing to high affinity of apoA-I on rHDL to SR-BI over-expression in MCF-7 cells. The cellular uptake of C6-DODAB/pDNACy5-rHDL nanoparticles was also observed by CLSM (Figure 3C). The fluorescence intensity of C6 and pDNACy5 loaded in rHDL was obviously stronger than that of C6-DODAB/pDNACy5-NLC nanoparticles, which was consistent with the results of flow cytometry. Additionally, intracellular fluorescences of C6 and pDNACy5 were significantly distinctly weakened in MCF-7 cells pre-treated with excess apoA-I to competitively saturate SR-BI, which was observed from the results of flow cytometry and CLSM, indicating that the cellular uptake of C6-DODAB/pDNACy5-rHDL was mediated mainly by SR-BI on the surface of MCF-7 cells.


21


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Figure 3. Cellular uptake of C6-DODAB/pDNACy5-rHDL (containing 0.1 µg/mL of C6 and 4 µg/mL of Cy5-dsDNA) at 37 ºC incubation for 4 h in MCF-7 cells. C6-DODAB/pDNACy5-NLCs and apoA-I pre-treated C6-DODAB/pDNACy5-rHDL were performed as controls. (A) Flow cytometry measurements of C6 fluorescence intensity in MCF-7 cells (n = 3). (B) Flow


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cytometry measurements of Cy5 fluorescence intensity in MCF-7 cells (n = 3). (C) Representative

confocal

imaging

of

C6-DODAB/pDNACy5-rHDL

in

MCF-7

cells.

C6-DODAB/pDNACy5-NLCs and apoA-I pre-treated C6-DODAB/pDNACy5-rHDL were performed as controls. Scale bar: 50 µm.

The subcellular distribution of PTX-DODAB/pDNA-rHDL nanoparticles in MCF-7 cells was investigated by CLSM. The cellular uptake of rHDL-based nanoparticles was mediated by SR-BI hydrophobic channel through which the components from lipophilic core of the rHDL are selectively taken up into cells, rather than by only endosome/lysosome pathway30-32. In order to confirmed whether rHDL-based nanoparticles owned the ability of avoiding endosome/lysosome process, the co-localization of PTX-DODAB/pDNAYOYO-1-rHDL nanoparticles in tumor cells and acidic organelles (including endosomes and early lysosomes) stained with LysoTracker™ Red

was

studied.

As

shown

in

Figure

4A,

after

cells

incubated

with

PTX-DODAB/pDNAYOYO-1-rHDL for 2 h, the fluorescence signal of pDNAYOYO-1 (green) was mainly located on cytoplasm, and almost no co-localization of pDNAYOYO-1 and LysoTracker Red (red). On the contrast, the signal of pDNAYOYO-1 was mainly co-localized with the signal of LysoTracker™

Red

in

PTX-DODAB/pDNAYOYO-1-NLCs

and

apoA-I

pre-treated

PTX-DODAB/pDNAYOYO-1-rHDL group, which was reflected in obvious yellow fluorescence overlayed by green and red, indicating that the nanoparticles from two groups could be both entrapped into the endosomes/lysosome to lead to the degradation of cargos in rHDL by enzymes


23


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and acidic environment. Taken together, the cargos of PTX-DODAB/pDNAYOYO-1-rHDL nanoparticles could be specifically internalized into MCF-7 cells mediated by SR-BI by bypassing endosome/lysosome pathway. In order to demonstrate this detailed mechanism of the cellular uptake of rHDL-based nanoparticles, the locations of apoA-I and pDNAYOYO-1 in rHDL were detected in MCF-7 cells by CLSM. As shown in Figure 4B, Cy5.5-apoA-I mainly adhered to the surface of MCF-7 cells and pDNAYOYO-1 was primarily located in cytoplasm after 4 h incubation of DODAB/pDNAYOYO-1-rHDL/Cy5.5-apoA-I. This result indicated that SR-BI mediated cellular uptake was not a process of endocytosing the intact rHDL-based nanoparticles. Instead, when nanoparticles and cells encountered, apoA-I in rHDL was binded to SR-BI on the cell membrane and the lipid compositions from lipophilic core of rHDL-based nanoparticles were selectively taken up to cytoplasm by SR-BI based hydrophobic channels.

3.5. In vitro transfection mediated by rHDL-based nanoparticles.

To investigate the transfection efficiency of PTX-DODAB/pDNA-rHDL nanoparticles, plasmid coding enhanced green fluorescent protein plasmid (pEGFP-C3) was used to indicate the in vitro transfection efficiency by flow cytometry and CLSM. The representative flow cytometric analysis and CLSM images of GFP-positive cells treated with different formulations were shown in Figure 4C and Figure 4E, respectively (mean fluorescence intensity of GFPin MCF-7 cells was shown in Figure S4.). PTX-DODAB/pEGFP-C3-rHDL exhibited remarkable transfection

efficiency

in

MCF-7

cells,

which

was

2.34-fold

higher

than

PTX-DODAB/pEGFP-C3-NLCs group and 2.55-fold higher than apoA-I pre-treated


24

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PTX-DODAB/pEGFP-C3-rHDL group possiblely due to SR-BI mediated efficient cellular uptake as shown in Figure 3, while naked pEGFP-C3 group showed nearly no detectable fluorescence.

3.6. p53 protein expression after rHDL-mediated gene transfection.

The expression level of p53 protein treated by PTX-DODAB/p53-rHDL nanoparticles in MCF-7 cells was determined by western blotting assay. As shown in Figure 4D, at 48 h after transfection with PTX-DODAB/p53-rHDL nanoparticles, high-level expression of p53 protein was

observed

compared

with

PTX-DODAB/p53-NLCs

and

apoA-I

pre-treated

PTX-DODAB/p53-rHDL groups, while naked p53 and PBS group showed little expression of p53. These data demonstrated that therapeutic genes p53 were efficiently delivered into SR-BI over-expressed MCF-7 cells by rHDL nanoparticles and expressed at a higher protein level than untreated group, thus laying a basis on the synergistic therapy of PTX and p53 in MCF-7 cells.


25


Figure

4.

(A)

Representative

PTX-DODAB/pDNAYOYO-1-rHDL

in

image MCF-7

Page 26 of 43

intracellular cells

at

2

trafficking h

observed

of by

the CLSM.

PTX-DODAB/pDNAYOYO-1-NLCs and apoA-I pre-treated PTX-DODAB/pDNAYOYO-1-rHDL were performed as controls. For each panel, 1: YOYO-1 labeled pDNA (green); 2: lysosomes stained by LysoTracker Red (red); 3: overlay of 1, 2 and light field. Scale bars: 25 µm. (B) Representative

image

of

MCF-7

cells

treated

by


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PTX-DODAB/pDNAYOYO-1-rHDL/Cy5.5-apoA-I showing Cy5.5-apoA-I adhered on the cell surface and YOYO-1-pDNA located in the cytoplasm. For each panel, 1: nucleus of MCF-7 stained by DAPI (blue); 2: YOYO-1 labeled pDNA (green); 3: Cy5.5 labeled apoA-I (red); 4: overlay of 1, 2, 3 and light field. Scale bar: 25 µm. (C) Transfection efficiency in MCF-7 cells defined as the percentage of GFP positive cells detected by flow cytometry (n = 3). Cells were treated

with

PTX-DODAB/pEGFP-C3-rHDL,

PTX-DODAB/pEGFP-C3-NLCs,

apoA-I

pre-treated PTX-DODAB/pEGFP-C3 and naked pEGFP-C3. (D) Expression of p53 protein in MCF-7 cells tested by western blotting assay. Lane 1: PTX-DODAB/p53-rHDL; Lane 2: PTX-DODAB/p53-NLCs; Lane 3: apoA-I pre-treated PTX-DODAB/p53-rHDL; Lane 4: naked p53; Lane 5: PBS control. (E) Representative image of in vitro GFP expression in MCF-7 transfected by PTX-DODAB/pEGFP-C3-rHDL (containing 4 µg/mL of pEGFP-C3) obtained by CLSM. PTX-DODAB/pEGFP-C3-NLCs, apoA-I pre-treated PTX-DODAB/pEGFP-C3 and naked pEGFP-C3 were performed as controls. Scale bars: 150 µm.

3.7. In vitro cytotoxicity study of PTX-DODAB/p53-rHDL nanoparticles.

The

cytotoxicity

of

blank

vectors

as

well

as

PTX-DODAB/p53-rHDL,

PTX-DODAB/p53-NLCs, PTX-rHDL, DODAB/p53-rHDL nanoparticles and free PTX against MCF-7 cells was evaluated using MTT assay. As shown in Figure 5A, blank rHDL nanocarriers showed little cytotoxicity toward MCF-7 cells (cell viability was higher than 90%) as well as


27


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blank NLCs, indicating that the nanocarriers had no influence on therapeutic effects of drug-loaded formulations. On the contrary, PTX loaded nanoparticles showed appreciable cytotoxicity to MCF-7 cells as well as PTX-DODAB/pDNA-rHDL gene negative control. Meanwhile, the cytotoxicity of PTX-DODAB/p53-rHDL nanoparticles was significantly higher than that of other groups (IC50: 6.72 µg/mL shown in Figure 5B). Interestingly, p53 loaded rHDL (DODAB/p53-rHDL) exhibited weak cytotoxicity at investigated concentrations. These results demonstrated that co-delivery of PTX and p53 by rHDL developed a synergistic effect to enhance in vitro antitumor efficacy.

3.8. In vitro cell apoptosis after treatment of PTX-DODAB/p53-rHDL nanoparticles.

Cell apoptosis induction of co-delivery and mono-delivery fomulations was evaluated against MCF-7 cells. Figure 5C showed the percentages of apoptosis cells treated with different formulations (statistical analysis of apoptotic and necrotic cells was shown in Figure S2). The percentage of apoptosis cells was 53.29 % for PTX-DODAB/p53-rHDL, 32.24 % for PTX-DODAB/p53-NLCs, 30.92 % for apoA-I pre-treated PTX-DODAB/p53-rHDL, 22.92 % for PTX-rHDL and 12.19 % for DODAB/p53-rHDL. Drug co-loaded rHDL induced stronger cell apoptosis effect than mono therapy groups. The cytotoxicity and apoptosis studies of different formations confirmed that rHDL could remarkably improve the intracellular drug/gene co-delivery, and achieve strong proliferation inhibition by synergistic apoptosis-inducing effects in tumor cells.


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Figure 5. (A) Cytotoxicity of MCF-7 cells exposed to NLCs and rHDL (empty vectors) solution with different concentration of PC for 48 h (n = 5). (B) Cytotoxicity of MCF-7 cells exposed to different formulations and free PTX (n = 5). (C) Cell apoptosis of MCF-7 cells at 48 h induced by different formulations tested by flow cytometric analysis (n = 5).

3.9. In vivo imaging and tumor targeting of PTX-DODAB/pDNA-rHDL nanoparticles.

In order to verify the feasibility of applying PTX-DODAB/p53-rHDL nanoparticles for breast cancer therapy in vivo, MCF-7 orthotopic tumor-bearing mice was used to estimate the nanoparticles

biodistribution.

The

in

vivo

biodistribution

of

DiR-labeled

PTX-DODAB/pDNA-rHDL nanoparticles at indicated time points was shown in Figure 6A. PTXDiR-DODAB/pDNA-rHDL nanoparticles exhibited the greatest tumor targeting (the strongest fluorescence intensity at tumors site) at 48 h post-injection. The fluorescence intensity


29


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biodistribution of the harvested organs (shown in Figure 6B) showed superior accumulation of nanoparticles at the tumor site (over-expressed SR-BI) in the mice treated with PTX-DODAB/pDNA-rHDL nanoparticles. Moreover, the fluorescence intensity of rHDL-based group in tumor was 2.03-fold higher than that of NLCs based group (shown in Figure 6C). These results of in vivo biodistribution revealed the strong tumor targetability of PTXDiR-DODAB/pDNA-rHDL nanoparticles, which could be developed as an effective drug/gene co-delivery system for tumor-targeted therapy.

3.10. In vivo antitumor efficacy of PTX-DODAB/pDNA-rHDL nanoparticles.

The antitumor efficacy of co-delivery formulations was evaluated in MCF-7 tumor orthotopic mice. As shown in Figure 6D, the tumor volume of mice treated with saline rapidly increased, while PTX-DODAB/p53-rHDL nanoparticles exhibited a significant stronger inhibition effect on tumor growth (tumor inhibition rate: 64.88%) than any other formulations including NLCs-based nanoparticles, monotherapy groups and free PTX. The image of tumor tissue section stained by H&E showed the most abundant karyolysis and sporadic necrotic after administration of PTX-DODAB/p53-rHDL nanoparticles, which displayed their efficient antitumor activity in vivo (Figure 6E). Furthermore, PTX-DODAB/p53-rHDL nanoparticles also possessed the most notable effect on extending survival period of tumor-bearing nude mice (Figure 6F). Overall, these results suggested that PTX-DODAB/p53-rHDL nanoparticles could achieve the optimal synergetic antitumor efficacy in vivo probably because of efficient drug/gene intracellular co-delivery and SR-BI mediated tumor-homing ability.


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Figure 6. (A) In vivo time-dependent tumor-targeting images after intravenous injection of DiR-labeled rHDL and NLCs in MCF-7 tumor-bearing mice. (B-C) Accumulation of DiR in the tumor and different organs detected by ex vivo imaging at 48 h post-injection of different formulations (n = 6). (D) The changes of tumor volume of tumor-bearing nude mice treated by different formulations (n = 6). (E) Images of H&E-stained tumor tissue section of nude mice treated with different formulations observed by Light Microscopy. Scar bars: 100 µm. (F)


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Survival rates of tumor-bearing nude mice treated with different formulations after administration (n = 6).

3.11. Safety evaluation of PTX-DODAB/pDNA-rHDL nanoparticles.

Injection irritation was examined by administrating PTX-DODAB/p53-rHDL nanoparticles to rabbit via ear vein that was subsequently harvested and stained by H&E, while saline was also injected as negative control. Histopathological section in Figure 7A showed no dilation and congestion of blood vessel, no swelling and necrosis of vascular endothelial cells, no edema and bleeding around the blood vessel, suggesting that PTX-DODAB/p53-rHDL nanoparticles showed no venous irritation compared with saline group. Body weight analysis of tumor-bearing nude mice treated with different formulations was also performed to verify toxicity. There was no significant difference in the body weight of mice between each group after administration (shown in Figure 7B), indicating systemic injection of PTX-DODAB/p53-rHDL did not influence the living quality of mice.

Serum biochemistry, the whole blood analysis, and histological test were performed to assess the biological safety of PTX-DODAB/p53-rHDL nanoparticles on healthy mice compared to saline group as negative control. Blood of each group was collected to test liver functional biomarkers ALT, AST, and BUN in order to examine the liver and kidney toxicity of PTX-DODAB/p53-rHDL nanoparticles. As shown in Figure 7D, PTX-DODAB/p53-rHDL


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nanoparticles showed similar results with saline group, indicating insignificant hepatotoxicity and nephrotoxicity in mice. Furthermore, as shown in Figure 7E, white blood cells (WBC), red blood cells (RBC), hemoglobin, platelets, neutrophils, lymphocyte and monocyte of PTX-DODAB/p53-rHDL group were in the normal physiologic range of mice in whole blood analysis and were also analogous to those of saline group. Subsequently, main organs of treated healthy nude mice were harvested, and tissue sections were stained by H&E as shown in Figure 7C. Compared to saline group, no significant damage occurred on these organs in PTX-DODAB/p53-rHDL

group.

The

results

conclusively

demonstrated

that

PTX-DODAB/p53-rHDL nanoparticles owned superior safety and broad prospect in clinical application.


33


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Figure 7. (A) Pathological sections of vessel tissues of rabbit ear-rim (H&E staining, original magnification: × 200). (B) Body weight change curves of different formulations on tumor-bearing nude mice (n = 6). (C) Representative histological images of the H&E-stained heart,

liver,

spleen,

lung,

and

kidney

harvested

from

the

mice

treated

by

PTX-DODAB/p53-rHDL and saline. (D) Serum levels of AST, ALT and BUN at 24 h after intravenous injections in mice (n = 6). (E) Hematological parameters at 24 h after three


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intravenous injections in mice (n = 6).

4. CONCLUSIONS In summary, we developed a lipoprotein-derived nanovector rHDL co-encapsulating hydrophilic therapeutic gene (p53) and hydrophobic chemotherapy drugs (PTX) for active tumor-targeted drug delivery and synergistic treatment. The optimized rHDL formulation achieved high tumor cellular uptake mediated by SR-BI and potent tumor cell inhibition by synergistic apoptosis-inducing effects. Furthermore, rHDL-based formulation could avoid lysosome/endosome pathway to protect its drug/gene cargos from degradation of enzymes and acidic environment. Additionally, the rHDL-mediated drug/gene co-delivery system exhibited excellent tumor targetability, remarkable tumor inhibition efficacy and laudable biosafety in vivo. These encouraging results suggested that the rHDL-based PTX/p53 co-delivery system would be a promising strategy for clinical applications in breast cancer-targeted therapy.

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Heparin, DNase I and serum protection assay of DODAB/pDNA complexes; Determination method of PTX; Preparation and characterization of PTX-rHDL nanoparticles and


35


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DODAB/pDNA-rHDL nanoparticles; DNase I protection assay of PTX-DODAB/pDNA-rHDL nanoparticles; Effect of different N/P ratio on the PDI of DODAB/pDNA complexes; Flow cytometry measurements of GFP fluorescence intensity in MCF-7 cells; Statistical analysis of apoptotic and necrotic cells.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] (W. Wang).

Author Contributions ‡

W. Wang and K. Chen contributed equally to this work.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was financially supported by the National Major Scientific and Technological Special Project for "Significant New Drugs Development" (No. 2016ZX09101031), the National Natural Science Foundation of China (No. 81502680 and 81102398), the Graduate Cultivation Innovative Project of Jiangsu Province (No. SJLX16_0239), the National Foundation for


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Fostering Talents of Basic Science (No. J1030830) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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