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Synergistic Therapy of Doxorubicin and miR-129-5p with Self-crosslinked Bioreducible Polypeptide Nanoparticles Reverses Multidrug Resistance in Cancer Cells Huqiang Yi, Lanlan Liu, Nan Sheng, Ping Li, Hong Pan, Lintao Cai, and Yifan Ma Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00141 • Publication Date (Web): 31 Mar 2016 Downloaded from http://pubs.acs.org on April 2, 2016

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Synergistic Therapy of Doxorubicin and miR-129-5p with Self-crosslinked Bioreducible Polypeptide Nanoparticles Reverses Multidrug Resistance in Cancer Cells

Huqiang Yi†, Lanlan Liu†, Nan Sheng†, Ping Li, Hong Pan, Lintao Cai, Yifan Ma*

Guangdong Key Laboratory of Nanomedicine, Key Lab for Health Informatics of Chinese Academy of Sciences, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China

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ABSTRACT:

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Although microRNAs (miRs) are short endogenous non-coding RNAs

playing a central role in cancer initiation and progression, their therapeutic potential in overcoming multidrug resistance (MDR) remains unclear. In the present study, we developed self-crosslinked biodegradable poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-cysteine) (LCss) polypeptide nanoparticles to co-deliver DOX and miR-129-5p, which aimed to overcome MDR in cancer cells. The results showed that LCss nanoparticles effectively co-encapsulated DOX and miR with great stability, but quickly disassembled and released their payload in a bioreducible environment. The co-delivery of miR-129-5p and DOX with LCss (DLCss/miR) significantly increased miR-129-5p expression over 100 folds in MCF-7/ADR cells, which effectively overcame MDR by directly inhibiting P-glycoprotein (P-gp), thereby increasing intracellular DOX accumulation and cytotoxicity in MCF-7/ADR cells. Furthermore, miR-129-5p also partially diminished cyclin dependent kinase 6 (CDK6), and synergized with DOX to simultaneously decrease S phase and induce G2 phase cell cycle arrest, thereby further enhancing the chemosensitivity of MCF-7/ADR cells. Hence, redox-responsive LCss nanoparticles are potent nanocarrier for combinational drug-miR therapy, which could be a promising strategy to overcome MDR in cancer cells.


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■ INTRODUCTION Chemotherapy is a leading strategy of cancer treatment,1 unfortunately, its therapeutic efficacy is often dampened by multidrug resistance (MDR) of tumor cells, which accounts for approximately 90% of failures in chemotherapy.2,

3

The development of MDR can be

attributable to multiple mechanisms, such as the over expression of drug efflux transporters, the activation of detoxification and DNA repair system, and the blockage of apoptotic pathways, etc.3-5 P-glycoprotein (P-gp) is an ATP binding-cassette (ABC) transporter encoded by MDR1 gene, which is responsible for shuttling drugs out of cells, thereby decreasing intracellular drug concentration and causing drug resistance.4,

6

P-gp expression can be

significantly up regulated by chemotherapeutic drugs, which is usually correlated with aggressive phenotype and poor prognosis.4, 7 Hence, P-gp has been considered as an important therapeutic target for overcoming MDR in cancer treatment. Although the co-administration of P-gp inhibitor with chemotherapy has been proposed to prevent and overcome MDR in cancer patients, its therapeutic efficacy remains unsatisfied due to the non-specificity and low efficiency as well as undesirable drug interactions with other anticancer drugs.8, 9 MicroRNAs (miRs) are short endogenous non-coding RNAs and master regulators of many biological processes, which directly bind to target gene mRNA, leading to mRNA degradation and/or translational inhibition.10-12 More recent studies have demonstrated a crucial role of miRs in regulating tumor initiation, progression, metastasis, as well as drug resistance.13, 14 Unlike siRNA, miRs are capable of regulating multiple genes and/or pathways, thereby holding great potential in cancer treatment.15,

16

The miR-129 family is a tumor

suppressor consisting of three mature miR members (miR-129-1-3p, miR-129-2-3p and 3 ACS Paragon Plus Environment

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miR-129-5p). The expression of miR-129 family members was shown to be significantly down regulated in majority of cancers,17 whereas increasing miR-129 inhibited tumor growth through causing G1/S cell cycle arrest and apoptosis.18 Furthermore, miR-129-5p was significantly reduced in chemo-resistant gastric cancer cells due to the hyper-methylation of miR-129-5p island, whereas increasing miR-129-5p effectively restored the chemo-sensitivity of the gastric cancer cells.19 These data suggest that miR-129-5p might be a potential target to overcome MDR and eradicate cancer. Combinational drug-RNA interference (RNAi) therapy with the ability of targeting multiple pathways has been reported as an effective approach to overcome MDR.20,

21

However, its clinical application is limited due to poor RNA stability, ineffective tumor targeting, and short circulation time, etc.20 Nanoparticle-based delivery systems are able to enhance RNA stability, prolong drug circulation, and facilitate the uptake of drug/gene by tumors, thereby offering great opportunities for successful drug-RNAi therapy.20 Previous studies mostly focused on nanoparticle-based drug-siRNA combination, which overcame MDR

by

targeting

multidrug

resistant

proteins

(P-gp

and

MRP-1)

or

tumor

proliferation/survival genes (e.g., BCL-2, PLK-1, VEGFR).20, 22 A recent study reported that co-delivering temozolomide and anti-miR221 with mesoporous silica nanoparticles successfully induced 70.9% of apoptosis in the temozolomide-resistant glioma cell line.23 These data suggest the combinational therapy of chemotherapy and miR might be a promising strategy to reverse drug resistance of cancer cells. Since miR-129-5p expression was reported to be directly associated with the chemo-sensitivity of certain cancer cells, it would be


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interesting to determine whether chemotherapy combined with miR-129-5p would be also able to reverse MDR in cancer cells. An ideal nanoparticle for combinational drug-miR therapy should not only be able to simultaneously encapsulate drug and miR with improved stability, but also facilitate their cellular uptake and intracellular release in order to overcome MDR. In the present study, we synthesized

hybrid

poly

(ethylene

glycol)-b-poly(L-lysine)-b-poly(L-cysteine)

(PEG-b-PLL-b-PLC) copolymers, which could self-assemble into redox-responsive polypeptide nanoparticles (LCss) with cross-linked poly(L-cysteine) as the hydrophobic core and PEGylated poly(L-lysine) as the shell. The positive charged poly (L-lysine) blocks could effectively encapsulate miR and improve RNA stability due to their electrostatic adsorption. Furthermore, the thiol groups of poly(L-cysteine) in situ crosslinked to form redox-responsive disulfide bonds, which not only enhanced DOX loading and nanoparticle stability, but also accelerated intracellular release of DOX and miR by redox-triggered nanoparticle disassembly

24, 25

. Since miR-129-5p was significantly decreased in DOX-resistant

MCF-7/ADR cells, we co-encapsulated DOX and miR-129-5p with LCss nanoparticles to generate DLCss/miR nanoparticles, which simultaneously delivered DOX and miR-129-5p into MCF-7/ADR cells. Our results showed that DLCss/miR robustly elevated miR-129-5p in MCF-7/ADR cells, which consequently inhibited P-gp expression and restored the chemosensitivity to DOX. In the meantime, miR-129-5p moderately suppressed cyclin dependent kinase 6 (CDK6), a cell-cycle check point, thereby synergizing with DOX to induce G2 phase cell cycle arrest.

■ EXPERIMENTAL SECTION 5 ACS Paragon Plus Environment

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Materials. S-benzyloxycarbonyl-L-cysteine(Cys(Z)) and ε-benzyloxycarbonyl-L-lysine (Lys(Z)) were purchased from GL Biochem (China). Triphosgene was purchased from J&K Chemical

Co.,

Ltd.

(USA)

and

recrystallized

with

diethyl

ether

before

use.

N-Carboxyanhydride (NCA) of ε-benzyloxycarbonyl-L-lysine (Lys(Z)-NCA) and NCA of s-benzyloxycarbonyl-L-cysteine (Cys(Z)-NCA) were prepared according to the previous method.26 Doxorubicin hydrochloride (DOX.HCl) (deprotonated before used), dry N,N-Dimethylformamide (DMF) DL-Dithiothreitol (DTT) and hydrogen bromide 33 wt% solution in acetic acid were purchased from Sigma-Aldrich (USA). MicroRNA-129-5p (miR-129-5p) and FAM-labeled miR-129-5p (FAM-miR-129-5p) were obtained from Gene Pharma Co., Ltd. (China). Human breast cancer MCF-7 cells were obtained from the cell bank of Chinese Academy of Sciences (China) and MCF-7 cells resistant to DOX (MCF-7/ADR) were purchased from Mei Xuan Biotech Co., Ltd. (China), both cells were cultured in RPMI 1640 media containing 10% FBS at 37 °C in a humidified atmosphere with 5% CO2. All other solvents were of analytical grade and obtained from Sigma-Aldrich (USA). Synthesis and characterization of PEG-b-PLL-b-PLC. PEG-b-PLLZ with the molar ratio of PEG:LysZ-NCA = 1:30 was synthesized according to our previous report.27 PEG-b-PLLZ-b-PLCZ was further synthesized by ring-opening polymerization of Cys(Z)-NCA initiated by PEG-b-PLLZ. In brief, PEG-b-PLLZ (0.382 g) and CysZ-NCA (0.22 g) were dissolved in 10 mL DMF (PEG-b-PLLZ:CysZ-NCA = 1:20, molar ratio) and stirred under N2 atmosphere at 35 °C for 72 h. The resulted product was precipitated by diethyl ether and dried under vacuum to obtain PEG-b-PLLZ-b-PLCZ. PEG-b-PLLZ-b-PLCZ (0.5 g) was next dissolved in 20 mL trifluoroacetic acid and 33% of HBr in HOAc solution 6 ACS Paragon Plus Environment

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and stirred at 0 °C for 2 h under N2, followed by precipitation with excessive amount of diethyl ether. The resulted product was then sequential dialyzed using a dialysis membrane (MWCO = 3500 Da) against 0.1% ammonia solutions, 0.1 mM H2O2 solution and ddH2O. The purified PEG-b-PLL-b-PLC was vacuum dried, and the 1H NMR spectra of the copolymers were recorded using a Unity Inova 400 spectrometer at 400 MHz with deuterated trifluoroacetic acid (CF3COOD) as solvent. The Fourier transform infrared spectroscopy (FT-IR) spectrum was recorded by Bruker Vertex 70 spectrophotometer in the region of 500-4000 cm-1 using KBr pellet technique. The average molecular weight (Mn, Mw) and polydispersity (Mw/Mn) of PEG-b-PLLZ-b-PLCZ and PEG-b-PLL-b-PLC were determined by gel permeation chromatography (GPC, Malven Company) at 40 °C and 2 × PLgel 5 µm MIXED-C 300 × 7.5 mm column was used.

DMF was utilized as the eluent with the

flowing rate at 1.0 mL/min, and polyethylene glycol (PEG) standards (K = 14.1 × 10-5 dL/g and α = 0.7 at 40 °C in DMF) were applied to calibrate the GPC traces. Preparation of redox-responsive PEG-b-PLL-b-PLC (LCss) nanoparticles and DOX-miR co-delivery. Redox-responsive PEG-b-PLL-b-PLC (LCss) nanoparticles were prepared by directly dissolving PEG-b-PLL-b-PLC copolymer in H2O. The size and zeta potential of nanoparticles were measured using Nano-ZS ZEN3600 (Malvern, UK). The secondary structure of polypeptides in LCss nanoparticles was characterized by circular dichroism (CD) spectra using Chirascan (Applied Photophysics, UK) at 25 °C. The redox-responsiveness of LCss nanoparticles was evaluated by incubation with PBS buffer ± 10 mM DTT at 37 °C for 48 h. The size and zeta potential of LCss nanoparticles were then monitored at different time intervals as indicated. The morphology of LCss nanoparticles 7 ACS Paragon Plus Environment

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were determined by transmission electron microscopy (TEM) using a FEI-F20 microscope (FEI, USA) operating at an acceleration voltage of 200 kVand the morphology changes of LCss nanoparticles were further investmented by TEM with the presence of 10 mM DTT incubated at 37 °C for 0 h, 4 h and 16 h. To prepare DOX-loaded (DLCss) nanoparticles, DOX solution in DMSO (1.5 mg, 5 mg/mL) was dropwisely added into PEG-b-PLL-b-PLC solution (7.5 mg, 1.0 mg/mL, and with weight ration: LCss: DOX = 5: 1) and stirred at room temperature in dark for 1 h. The reaction solution was extensive dialyzed against stirred ddH2O using a dialysis membrane (MWCO = 1000 Da) for 12 h, and ddH2O was replaced every 2 h. Encapsulated DOX was determined by fluorescence spectroscopy, and the loading capacity (LC) and encapsulation efficiency (EE) of DOX were calculated by following formula: LC (wt%) = weight of loaded DOX/weight of DLCss × 100%; EE (wt%) = weight of loaded DOX/weight of DOX in feed × 100%. For miR loading, LCss or DLCss nanoparticles were gently mixed with miR-129-5p at varied N/P ratios at room temperature for 20 min to obtain miR-loaded LCss nanoparticles (LCss/miR) and DLCss/miR, respectively. The stability of DLCss and DLCss/miR nanoparticles was determined by suspending nanoparticles in different medium at 4 °C, and the particle size was measured at different time points as indicated. In vitro release of DOX and miR. To determine the in vitro release of DOX or miR, DLCss (4 mg of DOX, 1.0 mg/mL) or LCss-encapsulated FAM-miR-129-5p nanoparticles (20 µg of miR, 0.5 mg/mL) were suspended in PBS (pH 7.4) ± 10 mM DTT and dialyzed in 15 mL PBS buffer (pH 7.4) with gently shaking (200 rpm) at 37 °C, respectively. At desired time intervals, 4 mL of released medium was collected and replenished with equal volume of 8 ACS Paragon Plus Environment

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fresh medium. To avoid oxidation of DTT, the released media were perfused with N2. The amount cumulative release of DOX and miR-129-5p was determined by measuring the amount of released DOX (λex = 480 nm, λem = 590 nm) or FAM-miR-129-5p (λex = 495 nm, λem = 520 nm) in external dialyzed PBS using fluorescence spectroscopy, respectively. Agrose gel electrophoresis and RNase protection assay. 0.4 µg of miR was encapsulated by LCss or DLCss at various N/P ratios and loaded on 1% agarose gel in Tris-Acetate-EDTA (TAE) buffer containing GelRed (Biotium, USA) for electrophoresis at 130 V for 20 min. For RNase protection assay, miR or miR-loaded nanoparticles were pretreated with RNase A (0.2 mg/mL) at 37 °C for 0.5 to 8 h, followed by incubation with heparin (1.0 mg/mL) for another 10 min. The sample mixtures were then subjected to gel electrophoresis, and miR was visualized using a Dolphin-Doc Molecular Imaging System (Wealtec, USA). In vitro cytotoxicity assay. MCF-7 or MCF-7/ADR cells were plated in 96-well plates (5 × 103 cells/well) and treated with LCss, DLCss, LCss/miR, DLCss/miR or negative control miR at 37 °C for 96 h. The dosages of DOX and miR-129-5p were 10 µg/mL and 100 nM, respectively. The cell viability was determined by MTT assay as previously reported,28 and the percentage of the viable cells was calculated using the following formula: Cell viability (%) = (ODexp-ODblank)/ (ODcontrol-ODblank) × 100%. To evaluate cell apoptosis, the cells were treated for 72 h, and labeled Annexin V apoptosis kit according to the manufacturer’s protocol. The apoptotic cells were detected by measuring Annexin V+ cells using Accuri C6 flow cytometer (BD Biosciences, Germany).


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Cellular uptake and intracellular distribution of DOX and miR by MCF-7/ADR cells. To quantify the cellular uptake of DOX and miR, MCF-7/ADR cells were incubated with miR, DOX, DLCss, LCss/miR or DLCss/miR nanoparticles at 37 °C for 4-24 h. The cellular uptake of DOX and miR was determined by measuring the percentage of DOX+ or miR+ cells using flow cytometry. To determine the intracellular distribution of DOX and miR, MCF-7/ADR cells were plated in eight-well Lab-Tek chamber slides (Thermo Fisher Scientific, USA) and incubated with different nanoparticles at 37 °C for 4-24 h. At the end of the experiment, the cells were labeled with hochest 33358 to identify cell nuclei, and fluorescent images were recorded by confocal laser scanning microscopy (Leica, Germany). Cell cycle analysis. For cell cycle analysis, MCF-7/ADR cells were treated with free miR-129-5p, LCss, LCss/NC, LCss/miR, DOX, DLCss, DLCss/NC or DLCss/miR for 48 h and fixed with 70% ethanol at -20 °C for 12 h. After wash, cells were incubated with 7-AAD (BBI Life Sciences, China) in dark for 15 min, and the cell cycle was measured by flow cytometry and analyzed using Flowjo software (Flowjo，USA). Quantitative real-time PCR. Total RNA was extracted using Trizol reagent (Life Technologies, USA), followed by reverse transcription to synthesize cDNA of miR using TOYOBO Reverse Transcription Kit. Quantitative real-time PCR (QRT-PCR) was then performed by using Thunder Bird SYBR qPCR mix (Toyobo, Japan) with U6 as an endogenous control for normalization. Primer sequences were shown in Supplementary Table S1.


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Western blot assay. Cells were lysed with RIPA buffer at 4 °C for 30 min and total protein concentration in cell lysates was measured by a BCA protein assay kit (Tiangen, China). Equal amount of cell lysates were separated by 12% SDS-PAGE gel and then transferred onto a PVDF membrane (Millipore, Germany). For immunoblotting, the membrane was incubated with anti-P-gp antibody (Abcam, USA, 1:1000) at 4 °C for 12 h, followed by incubation with horseradish peroxidase-labeled goat anti-mouse secondary antibody (KPL, USA) for 1 h, and then visualized with enhanced chemiluminescence system (Pierce, USA). The protein expression was semi-quantified using ImageJ software (National Institutes of Health, USA) and normalized with β-actin expression. Statistical analysis. Data are shown as mean ± SD from at least three independent experiments. The differences among groups were analyzed using one-way ANOVA analysis followed by Tukey’s post test using GraphPad software (Graphpad Prism, USA).

■ RESULTS AND DISCUSSION Synthesis and characterization of nanoparticles in different formulations. PEG-b-PLL-b-PLC was synthesized by ring-opening polymerization of NCA using PEG-NH2 and PEG-b-PLLZ as initiators and followed by deprotection of Z groups with HBr/HAc solution (Figure 1A). The structures of PEG-b-PLLZ, PEG-b-PLLZ-b-PLCZ and PEG-b-PLL-b-PLC copolymers were characterized by 1H NMR in CF3COOD (Figure 1B, C, and Supplementary Figure S1). The peaks around 7.3 ppm (i, q) and 5-5.5 ppm (h, p) were attributed to -C6H5 and -CH2-C6H5 protons, respectively. The peaks around 4.4 ppm (c) and 4.8 ppm (j) were attributed to -CH protons in the main chain of PLL and PLC, respectively. Compared with the spectrum of PEG-b-PLLZ-b-PLCZ in Figure 1B, the peaks p, q, h and i, 11 ACS Paragon Plus Environment

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which represented Z groups, were absent in the spectrum of PEG-b-PLL-b-PLC shown in Figure 1C, indicating completed deprotection of PEG-b-PLLZ-b-PLCZ. The polymerization degrees of PLLZ and PLCZ were determined by calculating the ratios of peak areas of c to b and j to b. The result showed the polymerization degrees of PLLZ and PLCZ were 28 and 12, respectively. The prepared copolymers were further characterized by FT-IR and GPC. According to the FT-IR spectra, the peaks at 1650 and 1550 cm-1 represented the amide I bond and amide II bonds in the PEG-b-PLLZ-b-PLCZ and PEG-b-PLL-b-PLC polypeptides. The peak at 1710 cm-1 was attributed to the C=O stretching vibration of protecting group Z of PEG-b-PLLZ-b-PLCZ. However, this peak was absent in the spectra of PEG-b-PLL-b-PLC (Figure 1D), indicating complete deprotection of PEG-b-PLLZ-b-PLCZ. Moreover, GPC chromatograms

demonstrated

that

the

average

molecular

weights

(Mw)

of

PEG-b-PLLZ-b-PLCZ and PEG-b-PLL-b-PLC were 12487 and 7132 according to the standard curve of PEG, respectively (Figure 1E and Supplementary Table S1), further confirming successful synthesis of PEG-b-PLL-b-PLC. The secondary structure of polypeptide within PEG-b-PLL-b-PLC was also determined using the CD spectra. The result showed a negative peak at 198 nm and a positive peak at 220 nm, suggesting that the secondary structure of PEG-b-PLL-b-PLC in H2O was predominantly a random coil conformation, which has been reported as an unique secondary structure essential for maintaining the stability of nanoparticles in complicated physiological environments (Supplementary Figure S2).29


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Preparation and characterization of redox-responsive PEG-b-PLL-b-PLC (LCss) nanoparticles. PEG-b-PLL-b-PLC copolymers were dissolved in H2O and simultaneously generated self-assembled PEG-b-PLL-b-PLC (LCss) nanoparticles, which consisted of self-crosslinked PLC as the hydrophobic core and cationic PEG-PLL as the hydrophilic corona. The obtained LCss nanoparticles had hydrodynamic size of 90 ± 4.2 nm with the zeta potential at 35 mv (Figure 2A). The TEM imaging showed a homogeneous distribution of spherical nanoparticles with a mean diameter at 30-50 nm (Figure 2C). The hydrodynamic size of LCss nanoparticles was much smaller than the size in TEM, which should be due to their hydrophilicity and changed secondary structure in H2O. The particle size of LCss nanoparticles did not significantly change during a 8-week storage in H2O, indicating their great stability (Supplementary Figure S3A). The redox-responsiveness of LCss nanoparticles was determined in PBS ± 10 mM DTT. As shown in Figure 2B and 2D, the size of the nanoparticles remained unchanged after 48 h incubation in PBS. However, the presence of DTT significantly increased the size of LCss nanoparticles from 80 nm to 350 nm within 16 h (Figure 2D). The morphological changes of LCss nanoparticles were monitored using TEM. At 4 h, the presence of 10 mM DTT led to significant swelling of LCss nanoparticle with a mean diameter at 50-100 nm.

After 16 h

incubation with DTT, majority of nanoparticles lost their spherical structure (Figure 2C). These results indicated reducible condition-triggered nanoparticle disassembly, which should be due to the cleavage of disulfide bond crosslinks in the core, thereby destroying the amphiphilic core-shell structure of LCss nanoparticles. The redox-responsiveness of LCss


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nanoparticles would allow maintaining superior colloidal stability in extracellular conditions, whereas facilitating the release of their payload in the reducible cytoplasm. An ideal drug delivery system should have minimal toxic effect in order to avoid potential adverse effect. Herein, the cytotoxic effect of LCss nanoparticles was evaluated in human MCF-7 cells and MCF-7/ADR cells using MTT assay (Figure 2E). The result showed that the treatment of 10-80 µg/mL of LCss nanoparticles did not significantly reduce the viability of either MCF-7 cells or MCF-7/ADR cells, suggesting their great biocompatibility. Preparation

and

characterization

of

DOX

and

miR-loaded

polypeptide

nanoparticles. DOX-loaded LCss (DLCss) nanoparticles were prepared by dialytic method as reported previously (Figure 3A).30 Although PEG-b-PLL-b-PLC copolymers lacked hydrophobic moieties, the self-assembled LCss nanoparticles encapsulated DOX with LC and EE at 9.3% and 51.3% respectively. The effective drug loading capacity of LCss nanoparticles should be attributed to self-crosslinked thiol groups, which formed a hydrophobic core for DOX encapsulation. Moreover, LCss and DLCss nanoparticles showed similar hydrodynamic size about 90 nm and zeta potential at +35 mV, suggesting DOX encapsulation did not affect the conformation of polypeptide nanoparticles. DLCss nanoparticles were further mixed with miR at room temperature to obtained DLCss/miR nanoparticles (Figure 3A). Compared with DLCss, DLCss/miR showed slightly increased size around 107.8 nm, and decreased zeta potential at 23.8 mV(Figure 3B), which should be due to the encapsulation of negatively charged miR. The RNA condensation capability is crucial for nanoparticle-based RNA delivery and gene silencing efficacy. We therefore evaluated the RNA binding capability of LCss and 14 ACS Paragon Plus Environment

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DLCss by gel electrophoretic assay at various N/P ratios. The results showed that both LCss and DLCss completely prevented miR migration at the N/P ratio of 6 or above (Figure 3C), which should be due to the electrostatic interaction between negatively charged RNA and positively charged PLL fraction. We next evaluated the RNA protection capability of LCss and DLCss nanocomplexes. As expected, naked miR was quickly degraded upon incubating with RNase A for 0.5 h. In contrast, miR encapsulated by either LCss or DLCss did not degrade even after 16 h incubation with RNAase A (Figure 3D), indicating enhanced RNA stability of polypeptide nanoparticles. The stability of DLCss/miRNA nanoparticles was further investigated in different buffers. During a 10-day storage, the particle size of DLCss/miRNA nanoparticles did not significantly change in H2O, PBS buffer, or RPMI 1640 with 10% FBS, indicating their great stability (Supplementary Figure S3B). Overall, LCss nanoparticles could effectively co-encapsulate DOX and miR to generate DLCss/miRNA nanocomplexes, which showed a great stability and ability to prevent RNA degradation. The in vitro release profiles of DOX and miR from nanocomplexes were evaluated in PBS ± DTT. Although the cumulative release of DOX from DLCss nanoparticles in PBS was less than 25% after 60 h, the presence of DTT dramatically increased DOX release up to 70% within 24 h (Figure 3E). These data indicated redox-triggered DOX release, which should be due to the cleavage of the disulfide bond by DTT. The release of miR was also evaluated with the same condition. The results showed that the release of miR by LCss nanoparticles was less than 10% in PBS, whereas DTT significantly enhanced the release of miR to 30% during 60 h of incubation, indicating redox-triggered miR release (Figure 3F). Notably, redox-induced miR release was slower than DOX, which might be due to the electrostatic interaction 15 ACS Paragon Plus Environment

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between miR and cationic PLL fragments. Nonetheless, redox-responsiveness of LCss nanoparticles could effectively prevent DOX and miR leakage in the extracellular environment, but significantly accelerate their release in a reducible condition. The cellular uptake and intracellular localization of miR-129-5p by human breast cancer MCF-7/ADR cells. Nanoparticle-based drug delivery has been reported to be able to overcome MDR through multiple mechanisms, such as inhibiting P-gp expression, altering internalization pathway, and enhancing sub-cellular drug accumulation.31 Hence, it would be necessary to determine whether DLCss nanoparticles by themselves could reverse MDR. Human breast cancer MCF-7 cells and DOX-resistant MCF7/ADR cells were treated with different concentrations of DLCss nanoparticles for 48 h, and the cell viability was determined using MTT assay. The results showed that although DLCss nanoparticles dose-dependently decreased the viability of MCF-7 cells, they failed to affect the viability of MCF7/ADR cells (Supplementary Figure S4A). We also evaluated the effect of DLCss nanoparticles on cellular uptake of DOX by confocal imaging. As expected, the administration of free DOX and DLCss both led to strong red fluorescence in MCF-7 cells, which diffused throughout the nuclei at 24 h (Supplementary Figure S4B). In contrast, free DOX led to weak red fluorescence in MCF-7/ADR cells at 24 h, confirming their resistance to DOX. Although DLCss nanoparticles slightly elevated intracellular fluorescent signals of DOX in MCF-7/ADR cells, the majority of DOX failed to enter into the nuclei but was retained in the cytosol (Supplementary Figure S4B), which should be due to the efflux of DOX caused by over-expressed P-gp.32, 33 These data suggested that DLCss nanoparticles by themselves were not capable of reversing MDR of MCF-7/ADR cells. 16 ACS Paragon Plus Environment

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MicroRNA-129 family, as a promising tumor suppressor, are significantly decreased in many types of cancers, such as colorectal cancer,34 cervical cancer,35 hepatocellular carcinoma,36 bladder cancer

37

and so on. More recently, the chemo-resistance of gastric

cancer has been reported to be directly related with the hyper-methylation and down-regulation of miR-129-5p.19 However, the role of miR-129-5p in DOX resistance of breast cancer remains unknown. Herein, the expression level of miR-129-5p was evaluated in MCF-7 and DOX-resistant MCF-7/ADR cell lines. Although MCF-7 cells expressed significant amount of miR-129-5p, the expression of miR-129-5p in MCF-7/ADR cells was less than 20% of that in MCF-7 cells (Figure 4A). These data suggest that the down-regulation of miR-129-5p might directly contribute to MDR in MCF-7/ADR breast cancer cells. To further determine whether increasing miR-129-5p could overcome MDR in breast cancer, miR-129-5p was encapsulated with/without DOX by LCss to form LCss/miR or DLCss/miR nanocomplexes. As is known, miR must be able to capture and transporte into the cytoplasm, where it is incorporated into the RNA-induced silencing complex (RISC) to conducting gene silencing.13 Hence, we evaluated the effect of polypeptide nanoparticles on miR uptake. In the present study, MCF-7/ADR cells were incubated with free FAM-miR-129-5p, LCss/miR or DLCss/miR nanoparticles for 24 h, and the cellular uptake of miR by MCF-7/ADR cells was evaluated using flow cytometry. The result showed that the uptake of free miR was less than 5%, indicating ineffective uptake of naked miR (Figure 4B). In contrast, the administration of LCss/miR and DLCss/miR both dramatically increased miR


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uptake to 80%, suggesting polypeptide nanoparticles as a potent carrier facilitating miR uptake. The intracellular localization of miR was investigated using confocal microscopy. Similar to the result of flow cytometry, the fluorescent signal of naked FAM-miR-129-5p in MCF-7/ADR cells was almost undetectable, whereas LCss/miR and DLCss/miR both robustly increased fluorescent signal of FAM-miR-129-5p in MCF-7/ADR cells (Figure 4C). Moreover, the fluorescent signals of FAM-miR-129-5p diffused throughout the cytosol at 24 h (Figure 4C), suggesting effective miR release from the polypeptide nanoparticles which should be due to intracellular bioreductant-triggered disulfide bond cleavage and nanoparticle disassembly. Hence, redox-responsive LCss and DLCss nanoparticles could not only enhance miR uptake, but also effectively release miR into the cytosol, both of which are essential for successful miR-based gene therapy. We further analyzed the expression of miR-129-5p by QRT-PCR after 24 h incubation. The result showed that free miR did not affect the expression of miR-129-5p in MCF-7/ADR cells, which should be due to the poor uptake of naked miR. In contrast, LCss/miR robustly elevated the expression of miR-129-5p level in MCF-7/ADR cells about 25-100 folds, whereas the replacement of miR-129-5p with negative control miR completely abolished the enhancement of miR-129-5p levels (Figure 4D). Interestingly, although LCss and DLCss nanoparticles demonstrated similar potency of facilitating miR uptake, DLCss/miR further enhanced miR-129-5p level about 10 folds in MCF-7/ADR cells as compared with LCss/miR did. Overall, the co-delivery of DOX and miR-129-5p with LCss nanoparticles not only


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enhanced the uptake of miR-129-5p, but also synergistically elevated miR-129-5p expression in MCF-7/ADR cells. Delivery of miR-129-5p by LCss and DLCss nanoparticles decreased MDR1 gene and P-glycoprotein in MCF-7/ADR breast cancer cells. P-glycoprotein (P-gp) encoded by MDR1 gene is a well-known ATP-binding cassette (ABC) transporters on the cell membrane responsible for pumping various molecules out of cells, playing a critical role in MDR of cancer patients.38, 39 Bioinformatic analysis and recent study have revealed that MDR1 gene is a target gene of hsa-miR-129-5p (Figure 5A). Since LCss and DLCss encapsulated miR-129-5p effectively increased the expression of miR-129-5p in MCF-7/MDR, it will be interesting to evaluate MDR1 gene and P-gp expression level. MCF-7/ADR cells were treated with free miR-129-5p, LCss/miR or DLCss/miR. The mRNA of MDR1 gene and protein level of P-gp were evaluated at 48 h after the treatment. As is expected, naked miR-129-5p failed to decrease either MDR1 mRNA or P-gp protein expression in MCF-7/ADR cells, indicating its poor gene silencing efficacy. However, miR-129-5p encapsulated by LCss or DLCss nanoparticles both significantly decreased MDR1 mRNA expression over 60%, indicating enhanced gene silencing efficacy by nanoparticle encapsulation. More importantly, both LCss/miR and DLCss/miR significantly reduced the protein level of P-gp in MCF-7/ADR cells, whereas the replacement of miR-129-5p by negative control miR completely restored the expression of MDR1 gene and P-gp proteins (Figure 5B, C). Hence, the delivery of miR-129-5p by redox-responsive polypeptide nanoparticles despite DOX encapsulation significantly deleted MDR1 gene and decreased P-gp expression in MCF-7/ADR cells. 19 ACS Paragon Plus Environment

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Co-delivery of miR-129-5p and DOX by LCss nanoparticles enhanced cellular uptake of DOX by MCF-7/ADR breast cancer cells. Since over expression of miR-129-5p significantly reduced P-pg expression on MCF-7/ADR cells, it would be interesting to determine whether co-delivery of DOX with miR-129-5p could block DOX efflux and enhance its intracellular accumulation. Herein, MCF-7/ADR cells were treated with free DOX or DLCss/miR for 4-24 h. The result of confocal imaging showed that the administration of DLCss/miR dramatically increased the intracellular DOX at 12-24 h (Figure 6A), which should be due to the deletion of MDR1 gene caused by miR-129-5p over expression. Moreover, DOX diffused throughout the cytoplasm at 12 h, and then transferred into the nuclei at 24 h (Figure 6A). As is known, DOX inhibits tumor growth through directly binding with DNA and inhibiting macromolecular biosynthesis. Therefore, enhanced DOX uptake and nuclear transportation by DLCss/miR were certainly important for overcoming the chemosensitivity of MCF-7/ADR cells. The DOX uptake was further quantified by flow cytometry. The results demonstrated that the uptake of DOX by MCF-7/ADR cells was less than 10% at 12 h, and gradually increased to 30% at 24 h. However, the co-delivery of miR-129-5p by DLCss nanoparticles not only increased DOX uptake to over 70%, but also significantly increased mean fluorescent intensity of DOX in the cells (Figure 6B, C), indicating significantly increased drug accumulation in MCF-7/ADR cells. Overall, the co-delivery of miR-129-5p by DLCss dramatically enhanced the cellular uptake and nuclear transportation of DOX in MCF-7/ADR cells, which would consequently enhance the chemosensitivity of cancer cells.


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Co-delivery of miR-129-5p and DOX by LCss nanoparticles induced DOX-induced apoptosis of MCF-7/ADR breast cancer cells. The chemotherapeutic efficacy of DOX with or without miR-129-5p co-delivery was evaluated by MCF-7/ADR cells. MTT assay showed free DOX did not affect the viability of MCF-7/ADR cells after 96 h incubation (Figure 7A), confirming the drug resistance to DOX. The treatment of DLCss nanoparticles failed to reduce cell viability as well (Figure 7A), further confirming our previous observation that DOX delivery with LCss nanoparticles couldn’t overcome the drug resistance of MCF-7/ADR cells. However, DLCss/miR nanoparticles significantly reduced the viability of MCF-7/ADR cells over 50%, whereas the replacement of miR-129-5p with negative control miR significantly recovered the cell viability (Figure 7A). These data indicated that the co-delivery of miR-129-5p by DLCss effectively reversed drug resistance of MCF-7/ADR cells and enhanced their chemosensitivity to DOX. Cell apoptosis is a key mechanism contributing to the anti-tumor effect of DOX. Herein, we showed that neither free DOX nor DLCss nanoparticles significantly induced cell apoptosis. However, DLCss/miR effectively caused over 30% of cell apoptosis (Figure 7B, C). These data confirmed that co-delivery of miR-129-5p and DOX by redox-responsive polypeptide nanoparticles could effectively reverse multi-drug resistance of MCF-7/ADR cells and recover the chemosensitivity of DOX. Co-delivery of miR-129-5p and DOX by redo-responsive polypeptide nanoparticles synergistically induced cell cycle arrest in MCF-7/ADR cells. MicroRNA-based gene therapy holds great potential for cancer treatment due to the ability of targeting multiple genes and pathways.15 Cyclin dependent kinase 6 (CDK6) is a cell cycle-associated protein essential 21 ACS Paragon Plus Environment

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for driving cell cycle progression from G1 to S phase.40 Previous studies showed that decreasing CDK6 led to G1/S phase cell cycle arrest, which implied CDK6 as a potential therapeutic target for cancer treatment.41 In the present study, since the biostatistic analysis showed a common standard target sequence of hsa-miR-129-5p in CDK6 3’-UTR (Figure 8A), it would be interesting to determine whether increasing miR-129-5p could suppress CDK6 gene expression. The results showed that the administration of LCss/miR significantly reduced CDK6 mRNA about 25% in MCF-7/ADR cells, whereas the replacement of miR-129-5p with negative control miR didn’t affect CDK6 mRNA at all. Hence, miR-129-5p transfection by LCss nanoparticles partially attenuated CDK6 gene expression (Figure 8B). The effect of miR-129-5p on the cell cycle of MCF-7/ADR cells was then investigated using flow cytometry. At 48 h, the administration of LCss/miR nanocomplexes decreased S phase by 20% but didn’t affect G2 phase at all (Figure 8C, D). These data suggested that overexpressing miR-129-5p moderately induced G1/S phase cell cycle arrest through targeting CDK6 gene. Since DOX has been reported to inhibit tumor growth by inducing G2/M phase cell cycle arrest, 42 we further evaluated the effect of DOX combined with miR-129-5p on cell cycle. The results showed that although DOX alone didn’t significantly increase G2 phase in MCF-7/ADR cells, DLCss/miR robustly induced G2 cell cycle arrest with increased G2 phase over 50% (Figure 8C, D). These results should be attributed to diminished P-gp by miR-129-5p, which consequently elevated intracellular DOX. Moreover, DLCss/miR also robust decreased S phase about 60%, further indicating a synergistic effect of miR-129-5p and DOX on inducing cell cycle arrest. Overall, co-delivery of miR-129-5p and DOX with


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redox-responsive LCss nanoparticles robustly induced cell cycle arrest, which consequently contributed to tumor suppression.

■ CONCLUSIONS Although miRs play a central role in MDR of human cancers, it remains unclear whether targeting miR could overcome MDR. In the present study, we reported self-crosslinked redox-responsive LCss nanoparticles with the capability of co-delivering DOX and miR-129-5p to overcome MDR in MCF-7/ADR cancer cells. The results showed that the co-delivery of miR-129-5p and DOX with LCss nanoparticles effectively elevated miR-129-5p in MCF-7/ADR cells, which reversed drug resistance by targeting P-gp, thereby robustly enhancing the cytotoxicity of DOX. Moreover, DOX combined with miR-129-5p synergistically induced both S phase reduction and G2 phase cell cycle arrest through targeting CDK6, which further enhanced chemosensitivity of MCF-7/ADR cells to DOX. Hence, redox-responsive LCss nanoparticles are potent nanocarrier for combinational drug-miR therapy, which could be a promising strategy to overcome MDR in cancer cells.


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Figure 1. Synthesis and characterization of copolymers. (A) Scheme of PEG-b-PLL-b-PLC Synthesis. (B-C) 1H NMR spectrum of PEG-b-PLLZ-b-PLCZ (B) and PEG-b-PLL-b-PLC (C). (D) FT-IR spectrum of PEG-b-PLLZ-b-PLCZ (black) and PEG-b-PLL-b-PLC (red). (E) GPC of PEG-b-PLLZ-b-PLCZ and PEG-b-PLL-b-PLC.


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Figure 2. Characterizations of PEG-b-PLL-b-PLC (LCss) nanoparticles. (A) Size distribution of LCss nanoparticles. (B) Size distribution of LCss nanoparticles in PBS ± 10 mM DTT at 37 °C. (C) Transmission electron microscopy (TEM) images ± 10 mM DTT at 37 °C for 0 h, 4 h and16 h in H2O. (D) Hydrodynamic sizes of LCss nanoparticles in PBS ± 10 mM DTT at 37 °C. (E) Cytotoxicity of LCss nanoparticles in MCF-7 and MCF-7/ADR cells using MTT assay. Data are shown as mean ± SD (n = 3-4).


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Figure 3. Preparation and characterization of DOX/miR-loaded LCss (DLCss) nanoparticles. (A) Schematic illustration of loading and releasing of DOX and miR by LCss nanoparticles. (B) The size and zeta potential of LCss, DLCss and DLCss/miR nanoparticles. (C) Binding ability of LCss and DLCss to miR at different ratios of nitrogen in carrier to phosphate in miR (N/P ratio) demonstrated by the agarose gel retardation assay. (D) RNAase protection assay of nanocomplexes. a: miR; b: LCss/miR; c: DLCss/miR. (E) In vitro release of DOX from DLCss in PBS (pH = 7.4) ± 10 mM DTT at 37 °C. (F) In vitro release of miR from LCss/miR in PBS (pH = 7.4) ± 10 mM DTT at 37 °C. Data are shown as mean ± SD (n = 3). 26 ACS Paragon Plus Environment

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Figure 4. The cellular uptake and intracellular localization of miR-129-5p in MCF-7/ADR cells. MCF-7/ADR cells were incubated with FAM-miR, LCss/miR or DLCss/miR at 37 °C for 24 h. (A) The expression of miR-129-5p in MCF-7 and MCF-7/ADR cells by QRT-PCR. (B) The uptake of miR was quantified using flow cytometry. (C) The intracellular distribution of FAM-miR-129-5p (Green) was recorded using confocal microscopy. Cells were labeled with hoechst to identify nuclei (Blue). (D) The expression of miR was quantified by QRT-PCR after 24 h incubation. Bars shown are mean ± SD (n = 3), and the differences between medium (Med) and other groups were determined using one-way ANOVA followed by Tukey’s post test. *: p < 0.05.


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Figure 5. Gene silencing efficacy of LCss/miR and DLCss/miR in MCF-7/ADR cells. (A) Shown is the alignment of hsa-miR-129-5p and its target sites in 3’-UTR of MDR1. (B-C) MCF-7/ADR cells were treated with DOX (10 µg/mL), miR-129 (miR, 100 nM) with or without nanoparticle encapsulation. The mRNA levels of MDR1 gene in MCF-7/ADR cells were determined by QRT-PCR (B), and protein levels of P-gp were determined by western blotting with β-actin as a loading control (C). Bars shown are mean ± SD, and differences between medium (Med) and other treatments were analyzed using one-way ANOVA. **: p < 0.01.


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Figure 6. The cellular uptake and intracellular localization of DOX in MCF-7/ADR cells. MCF-7/ADR cells were incubated with free DOX (10 µg/mL) or DLCss/miR at 37 °C. (A) The intracellular localization of DOX (red) in MCF-7/ADR cells was recorded at 4 h, 12 h and 24 h using confocal microscopy. Cells were labled with hoechst to identify nuclei (Blue). (B) The cellular uptake of DOX was determined by measuring DOX positive cells using flow cytometry. (C) Total fluorescent intensity (FI) = %of positive cells × mean fluorescent intensity. Bars shown are mean ± SD (n = 4), and the differences between two groups were analyzed using paired t test. *: p < 0.05. 29 ACS Paragon Plus Environment

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Figure 7. The cytotoxic effect of different nanoparticles on MCF-7/ADR cells. MCF-7/ADR cells were treated with free miR-129 (miR, 100 nM), DOX (10 µg/mL) or DLCss/miR nanoparticle. (A) The cell viability of MCF-7/ADR cells was determined by MTT assay at 96 h. (B) The apoptosis of MCF-7/ADR cells was determined using annevin V staining at 72 h. (C) The percentage of annexin V positive cells on MCF-7/ADR cells were measured by flow cytometry. Bars shown are mean ± SD, and differences between medium (Med) and other treatments were analyzed by one-way ANOVA. *: p < 0.05, **: p < 0.01.


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Figure 8. MiR-129-5p and DOX synergistically induce cell cycle arrest in MCF-7/ADR cells. (A) The alignment of hsa-miR-129-5p and its target sites in 3’-UTR of CDK6. (B-D) MCF-7/ADR cells were treated with free miR-129 (miR, 100 nM), DOX (10 µg/mL) or DLCss/miR nanoparticle. The mRNA levels of CDK6 gene in MCF-7/ADR cells were determined at 48 h by QRT-PCR (B), the cell cycle of MCF-7/ADR cells was analyzed at 48 h with flow cytometry (C-D). DNA histogram plots shown are the cell-cycle analysis of


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MCF-7/ADR cells. Bars shown are mean ± SD, and differences between medium (Med) and other treatments were analyzed by one-way ANOVA. *: p < 0.05, **: p < 0.01.

■ ASSOCIATED CONTENT Supporting Information GPC of polymers (Table S1), list of primers (Table S2), 1H NMR spectrum of PEG-b-PLLZ (Figure S1), CD spectrum of PEG-b-PLL-b-PLC(Figure S2), the stability of nanoparticles (Figure S3) and the cell viability and intracellular localization of DOX in MCF-7 and MCF-7/ADR cells (Figure S4).

■ AUTHOR INFORMATION Corresponding Author * Yifan Ma Fax/Tel: +86-755-86392229. E-mail: [email protected]. Author Contributions †These authors contributed equally to this paper.

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant No. 81171446,

81371679),

(KQCX201405201541150), (GJHS20140610151856702,

Shenzhen Shenzhen

Overseas Science

Outstanding and

CXZZ20130506140505859),

Professional Technology

Guangdong

leading

Talent Program talents

program (Antibody/Protein Drugs for Major Diseases), Shenzhen Peacock Next-generation 32 ACS Paragon Plus Environment

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Monoclonal Antibody Drug research and development program

(KQTD201210), Dongguan

project on Social science and Technology Development (2015108101019), Guangdong medical university fund(2XB14015).

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