Coloaded Nanoparticles of Paclitaxel and Piperlongumine for Enhancing Synergistic Antitumor Activities and Reducing Toxicity

Accepted Manuscript Co-loaded nanoparticles of paclitaxel and piperlongumine for enhancing synergistic anti-tumor activities and reducing toxicity Qi Liu, Di Zhao, Xiaojie Zhu, Huili Chen, Yue Yang, Jiaqiu Xu, Qing Zhang, Ali Fan, Ning Li, Chaorui Guo, Ying Kong, Yang Lu, Xijing Chen PII:

S0022-3549(17)30384-2

DOI:

10.1016/j.xphs.2017.05.027

Reference:

XPHS 830

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 24 March 2017 Revised Date:

25 April 2017

Accepted Date: 16 May 2017

Please cite this article as: Liu Q, Zhao D, Zhu X, Chen H, Yang Y, Xu J, Zhang Q, Fan A, Li N, Guo C, Kong Y, Lu Y, Chen X, Co-loaded nanoparticles of paclitaxel and piperlongumine for enhancing synergistic anti-tumor activities and reducing toxicity, Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.xphs.2017.05.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title Page Title: Co-loaded nanoparticles of paclitaxel and piperlongumine for enhancing synergistic

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anti-tumor activities and reducing toxicity

Authors:

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Qi Liu1, , Di Zhao1, , Xiaojie Zhu1, Huili Chen2, Yue Yang1, Jiaqiu Xu3, Qing Zhang3,

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Ali Fan1, Ning Li3, Chaorui Guo1, Ying Kong1, Yang Lu1,*, Xijing Chen1,*

The two authors contribute equally to this work.

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Affiliation:

Clinical Pharmacokinetics Laboratory, School of Basic Medicine and Clinical

China

Department of Molecular and Cellular Biology, College of Science, The University

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2

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Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu province 211198,

of Arizona, Tucson, Arizona 85719, United States 3

Department of Pharmacology, China Pharmaceutical University, Nanjing, Jiangsu

province 211198, China

Suggested running header: Co-loaded paclitaxel and piperlongumine nanoparticles

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ACCEPTED MANUSCRIPT *The two authors were considered as co-corresponding authors: Prof. Dr. Xijing Chen and Dr. Yang Lu Clinical Pharmacokinetics Laboratory, School of Basic Medicine and Clinical

China Tel.: +86 25 8618 5379; Fax: +86 25 8618 5379

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Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu province 211198,

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E-mail address: [email protected] (Xijing Chen); [email protected]

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(Yang Lu)

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Abstract The purpose of this study was to develop a nano-carrier system for co-delivery of paclitaxel (PTX) and piperlongumine (PL) and investigate the therapeutic potential of

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improving efficacy and reducing toxicity. PTX and PL were formulated into poly lactic-co-glycolic acid (PLGA) and D-α-tocopheryl polyethylene glycol succinate (TPGS) via organic solvent evaporation method. The average diameter was 117.1 ±

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1.9 nm and the zeta potential was -43.25 ± 2.76 mV. PL facilitated the cellular uptake

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of PTX and the increased cytotoxicity was similarly displayed. The formulation with the PTX/PL concentration ratio at 1:200 showed the best anti-tumor activity, the IC50 of PTX were 5.10 ± 0.08 nM in HepG2 cells and 3.79 ± 1.01 nM in MCF-7 cells. Correspondingly, the combination index was 0.79 and 0.76. Furthermore, intracellular

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uptake of PTX towards HepG2 cells in co-encapsulated nanoparticles was significantly more than free solution. In addition, the antitumor effect of PTX/PL-PTNPs in the HepG2 xenograft tumor model suggested that the nanoparticles

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showed a higher antitumor efficacy with reduced toxicity to other tissues compared

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with free PTX. In summary, the results indicated that PTX/PL-PTNPs processed well characteristics and enhanced its therapeutic efficacy, thus this delivery system could be clinically effective for treatment of cancers.

Key words: paclitaxel; piperlongumine; nanoparticles; synergistic effect

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Abbreviations 5-fluorouracil

C6

Coumarin-6

Cmax

Maximum concentration

CD31

Cluster of differentiation 31

CI

Combination index

CLSM

Confocal laser scanning microscopy

DL

Drug loading

DMEM

Dulbecco’s Modified Eagle’s Medium

DMSO

Dimethyl sulfoxide

EE

Encapsulation efficiency

EPR

Enhanced permeability and retention

FBS

Fetal bovine serum

HBSS

Hank's Balanced Salt Solution

H&E

Hematoxylin & eosin

HPLC

High Performance Liquid Chromatography

IC50

Concentration inhibiting half of the cells

Ki-67

Antigen KI-67

LC-MS/MS

Liquid chromatograph-mass spectrometer tandem mass spectrometer

MDR

Multidrug resistance

MTT

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

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5-FU

NaN3

Sodium azide

NP

Nanoparticle

PBS

Phosphate buffer saline

PDI

Polydispersity index

P-gp

P-glycoprotein

PL

Piperlongumine

PLGA

Poly lactic-co-glycolic acid

PTX

Paclitaxel

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ACCEPTED MANUSCRIPT Reticuloendothelial system

ROS

Reactive oxygen species

RPMI-1640

Roswell Park Memorial Institute (RPMI) 1640

SD

Standard deviation

SRM

Selective reaction monitoring

TPGS

D-α-tocopheryl polyethylene glycol succinate

TUNEL

Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling

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RES

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Introduction Cancer is one of the most serious diseases in the world with high morbidity and mortality. Although the incidence of some major cancers, such as lung cancer, breast

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cancer and colorectal cancer appeared to be declined from 2003 to 2012, the rise of death rates among the liver cancer, pancreas cancer and anus cancer never stopped1. Chemotherapy is one of the main modalities to treat cancer in clinic. Paclitaxel (PTX,

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Fig. 1) is a first-line drug to treat many cancers, such as ovarian cancer, lung cancer

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and non-small cell lung cancer2-4. However, due to the poor water solubility of PTX, the clinical applications are usually accompanied with Cremophor EL and ethanol which in return result in several side effects, such as anaphylactic reactions, nephrotoxicity, hematological toxicity and neurotoxicity5. In addition, the increasing

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severity of multidrug resistance (MDR) has made it one of the biggest obstacles in the cancer treatment. Therefore, it is urgent to explore an effective modality to reduce

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these adverse reactions and maintain the efficacy of PTX.

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Natural products played important roles in the human history and have gained more and

more

attentions

nowadays.

Besides

PTX,

piperlongumine

(PL,

5,6-dihydro-1-(1-oxo-3-[3,4,5-trimethoxyphenyl]-2-propenyl)-2[1H]-pyridinone, Fig. 1), one natural alkaloid product first isolated in 1961 from the plant Piper longum and then structure determined by Atal and Banga in 19636-8 with two α, β-unsaturated imide functionalities, shows obvious electrophilicity, targeting the stress response to reactive oxygen species (ROS)9. Furthermore, PL selectively kills cancer cells but not

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ACCEPTED MANUSCRIPT normal cells9. In addition, it has reported that PL increases the cytotoxicity of 5-fluorouracil (5-FU) and curcumin in several tumor cell lines10,11, which shows that PL can not only exhibit cytotoxic effect, but also increase the anti-tumor activity even

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though the dosage of the chemotherapeutic drugs is reduced. Thus, PL could work as a promising anticancer agent as well as a combination with traditional antitumor drugs

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that would reduce the resistance in clinical cancer chemotherapy.

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Polymeric nanoparticle (NP) is a promising drug delivery system with several advantages comparing with traditional dosage-form of anti-tumor drugs12: 1) transferring anti-tumor drugs through enhanced permeability and retention (EPR) effect13 or obtaining active targeting by surface modification to achieve accumulation

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of drugs in the tumor site; 2) improving the stability of anti-tumor drugs in the blood in order to achieve long-circulating14; 3) achieving synergies and reducing toxicity in the form of combined multiple drugs; 4) regulating the release of anti-tumor drugs

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according to the change of biochemical level in vivo; 5) sustaining drug-release

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properties through prodrug strategy with drug combination15. In this study, poly lactic-co-glycolic acid (PLGA) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) were chosen for NPs preparation. PLGA is one of the most frequently used in biodegradable polymeric NPs due to its biocompatibility, ability for sustained drug release, and ease of parenteral administration16-19, which has been approved by US Food and Drug Administration (FDA). TPGS, a water-soluble derivative of natural Vitamin E, has been approved by US FDA as a safe

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ACCEPTED MANUSCRIPT pharmaceutical adjuvant in drug formulation and widely used as product carrier, emulsifier, additive, absorption enhancer and permeation enhancer20-22. Furthermore, TPGS can also inhibit P-glycoprotein (P-gp) to reverse MDR and increase the

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intracellular amount of anticancer drugs23. Despite of the materials selection, the formulation process condition plays an important role in size, shape and encapsulation

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efficacy of nanoparticles24.

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In this study, an organic solvent evaporation method was taken to prepare PLGA/TPGS polymeric nanoparticles (PTNPs). PTX and PL were co-loaded in the PTNPs (PTX/PL-PTNPs). The physicochemical properties, the release and clearance profiles, in vitro cytotoxicity of PTX/PL-PTNPs were investigated, providing novel

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and significant insight into the application of PTX/PL-PTNPs for the treatment of cancers. Moreover, in vivo tumor growth inhibition studies were also carried out using the tumor bearing mice with HepG2 cells. The results indicated that co-loaded

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delivery system of PTX and PL might be a promising therapy to treat cancer with

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low-dose PTX.

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Methods Materials PTX and PL were purchased from Shanghai Sunve Pharmaceutical Co. Ltd and

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Alligator Reagent (Nanjing, China), respectively. PLGA with a 75:25 monomer ratio (MW: 20000 g/mol) was bought from Jinan Daigang Biomaterial Co., Ltd (Shandong, China). TPGS was provided by Aike Reagent (Chengdu, China). Sodium azide (NaN3)

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was provided by Micxy Reagent (Chengdu, China). Chlorpromazine hydrochloride

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and amiloride hydrochloride hydrate were purchased from Aladdin® (Shanghai, China). Sucrose was from Beijing Yili Fine Chemicals Co., Ltd. (Beijing, China). Genistein was from National Institutes for Food and Drug Control (Beijing, China).

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The human liver cancer cell line (HepG2) and michigan cancer foundation-7 (MCF-7) cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. DMEM, RPMI-1640, fetal bovine serum (FBS),

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penicillin-streptomycin solution, PBS, trypsin and HBSS were provided by Hyclone®

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(GE Healthcare Life Sciences). Cell culture plates were obtained from Corning Costar (Cambridge, MA). Bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Coumarin-6 was provided by Sigma-Aldrich®

(Shanghai,

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

China). bromide

(MTT)

was

purchased from J&K Scientific Ltd. (Beijing, China). 4,6-diamidino-2-phenylindole (DAPI) was obtained from KeyGEN BioTECH Co., Ltd (Jiangsu, China). Dimethyl

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ACCEPTED MANUSCRIPT sulfoxide (DMSO), formic acid, methyl-t-butyl ether and sodium acetate were of analytical grade. High Performance Liquid Chromatography (HPLC)-grade

Preparation and characterization of nanoparticle

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acetonitrile and methanol were provided by Tedia (Fairfield, OH, USA).

PL was the main drug in this preparation, thus it was employed as the indicator for

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screening the optimized prescription. The nanoparticles were prepared by an organic

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solvent evaporation method. The ratio of PL to PLGA, TPGS, and the proportions of aqueous and organic phase were shown in Table S1. Briefly, PL and PLGA with different ratios were co-dissolved in different volume of acetone under mild stirring as the organic phase. Meanwhile, TPGS was dissolved in ethanol and added into

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different volume of water under magnetic stirring as the aqueous phase until the ethanol volatilized completely. With a syringe pump, the organic phase was added dropwise (4 mL/min) into the aqueous phase under magnetic stirring. After the end of

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the drop, stirring continued until the organic solvent (acetone) volatilized completely.

After the optimal prescription was determined, PTX and PL co-loaded nanoparticles (PTX/PL-PTNPs) were prepared as described above with the weight ratio of 1:20, 1:50, 1:100, 1:200 (PTX:PL, w/w). The fluorescent nanoparticles were prepared in the same way with PTX and PL replaced by coumarin-6 (C6). The preparation process was schematically shown in Fig. 2A.

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ACCEPTED MANUSCRIPT The nanoparticle size, polydispersity index (PDI) and ζ-potential of prepared nanoparticles were characterized by Laser scattering particle size analyzer and Zeta plus Zeta Potential Analyzer (Brookhaven, USA). Each sample was diluted to the

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appropriate concentration with purified water. All the results were presented as mean ± standard deviation (SD) based on three separate experiments.

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The drug loading (DL) and encapsulation efficiency (EE) of the nanoparticles were

DL % =

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calculated as following calculation equations:

The weight of entrapped drug

The total weight of the nanoparticles

The weight of entrapped drug The total weight of drug

× 100 %

(1)

(2)

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EE % =

× 100 %

The nanoparticles were dissolved in acetonitrile to determine the total weight of drug.

Then

the

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The free drug was separated from the nanoparticles using an ultrafiltration process. amount

of

total

and

free

drug

was

determined

by

Liquid

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chromatograph-mass spectrometer tandem mass spectrometer (LC-MS/MS).

In vitro drug release study The in vitro drug release from the nanoparticles was investigated using the dialysis bag diffusion technique. 1 mL of nanoparticles were placed in the dialysis bag (molecular mass cutoff is 3000 Da) and immersed into 50 mL of the phosphate buffer saline solution (pre-warmed to 37°C, pH = 5.0, 7.4) containing 0.1% (w/v) Tween 80.

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ACCEPTED MANUSCRIPT The entire system was kept at 37°C with continuous shaking at 100 rpm. At specific intervals, 1 mL of samples were withdrawn from the external medium and replaced with 1 mL of fresh buffer solution. The amount of drug in each sample was

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determined by LC-MS/MS. All diffusion experiments were valued in triplicate, and the mean values and standard deviations (mean ± SD) were calculated.

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Cell culture

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The HepG2 cells were cultured in DMEM, supplemented with 10% FBS, 1% penicillin-streptomycin. For MCF-7 cells, RPMI-1640 medium was used instead of DMEM. All these cells were incubated under 37°C in a humidified atmosphere with 5%

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CO2.

In vitro cytotoxic activity assay

PL and PTX were dissolved separately in DMSO with its volume added to the cell

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suspension being less than 0.1% (v/v). Cells were seeded into 96-well plates (5,000

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cells/well in 200 µL of medium) and incubated for 24 h at standard condition to reach exponential growth prior to the test. The cells were treated with graded concentrations of the drugs in the following day. After incubated for 48 h, the medium was discarded and the plates were washed twice with PBS. Then, the medium was replaced with 200 µL of fresh medium (without FBS) containing 0.5 mg/mL MTT. After another four hours’ incubation, the MTT medium was removed followed by adding 150 µL DMSO to dissolve formazan samples. The absorbance was measured at 490 nm using a

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ACCEPTED MANUSCRIPT microplate reader (Multiskan FC). The drug effects were expressed as the percentage of untreated control absorbance. MTT assay was repeated in six replicates for each

Abs t - Abs b Abs control - Abs b

× 100 %

(3)

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Cell viability (%) =

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Cell viability was calculated by the following equation:

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experiment.

Where Abs t is the absorbance of the cells incubated with different concentration of drugs, Abs

control

is that of the cells incubated with the culture medium only (positive

control), and Abs b is that of the culture medium without cells (negative control). The data was analyzed by GraphPad Prism 5 and the concentration inhibiting half of the

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cells (IC50) value was calculated.

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The combination index (CI) for the combination of PTX and PL in nanoparticles can

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be described by:

CI =

(D)1 (Dx)1

+

(D)2 (Dx)2

(4)

Where (Dx)1 and (Dx)2 are the IC50 of PTX and PL alone, respectively, and (D)1 and (D)2 are the IC50 of PTX and PL in the combination of the two drugs in nanoparticles, respectively. CI <1, =1, and >1 indicates synergism, additive effect, and antagonism, respectively25. 13

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Cellular uptake study of nanoparticles on HepG2 cells HepG2 cells at a density of 1×105 cell/well were seeded and cultured in 24-well plates

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with 1mL of culture medium under 5% CO2 at 37 °C. When the cells were grown to about 80% confluence, the culture medium was removed, washed twice with HBSS

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nanoparticles or free drugs was added into each well.

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and 1 mL of fresh FBS-free culture medium containing the different drug-loaded

For the experiment of time-dependent, the cells were incubated at various time intervals. For the concentration-dependent experiment, the cells were incubated with a series of concentrations of drugs. At the end of each experiment, the medium

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containing drug was removed followed by washing twice with cold HBSS, then added 1mL deionized water into each well and stored in -70°C until analysis. The BCA protein assay kit was used for quantifying the protein amount, while the drug

drug /

Con

pro)

was calculated, where Con

drug

and Con

pro

are represented

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uptake (Con

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concentration was determined by LC-MS/MS. The drug concentration of cellular

the concentration of drug in the cells and cell protein, respectively.

C6 was employed in nanoparticles to evaluate cellular uptake in HepG2 cells with confocal laser scanning microscopy (CLSM, Carl Zeiss, USA). Briefly, HepG2 cells were seeded onto glass bottom cell culture dishes (NEST®, USA) for 24 h under 5% CO2 at 37 °C. Media was changed with fresh FBS-free medium containing free C6

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ACCEPTED MANUSCRIPT and nanoparticles loaded with C6, measurement was taken at 1 h, 2 h and 4 h, respectively. Then the cells were washed thrice with PBS and the nuclei were stained with DAPI for another 15 min. The cells were rinsed with PBS for three times,

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followed by confocal microscopy examination.

Endocytosis pathway of nanoparticles

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With small particle size ranging from 100 nm to 1000 nm, nanoparticles are

endocytosis,

caveolar-mediated

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internalized though various pathways, including micropinocytosis, clathrin-mediated endocytosis

and

clathrin-independent

and

caveolar-independent endocytosis26. In order to study the endocytosis pathway of prepared nanoparticles, HepG2 cells were pre-incubated for 30 min with different

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endocytosis inhibitors, sucrose (154 mg/mL), sodium azide (NaN3, 1.5 mg/mL), chlorpromazine hydrochloride (5 µg/mL), amiloride hydrochloride hydrate (500 µM), genistein (50 µg/mL). Nanoparticles were then added into each well and incubated for

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another period of time. Drug concentration of cellular uptake was determined by

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LC-MS/MS and corresponding protein was determined using BCA methods. All experiments were carried out in triplicate.

Xenograft tumor model and in vivo tumor growth inhibition study BALB/c nude mice (male, 18~20 g) were purchased from KeyGEN BioTech (Jiangsu, China). The animal experiments were approved by Animal Care and Use Committee of China Pharmaceutical University. HepG2 cells (2×107 cell/mouse) were injected

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When the tumor volume reached more than 200 mm3, the mice were randomly

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divided into three groups (n=10). During a period of 15 days as per the schedule of 0, 3rd, 6th and 9th days, normal saline, free PTX and PTX/PL-PTNPs were intravenously injected via tail vein into each group of mice at an equivalent PTX dose of 1.5

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mg/kg27, respectively. Tumor volume and body weight were measured every three

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days for a total period of 15 days from the starting of the dosing. The tumor volumes were calculated using the formulation: V = (Length×Width2)/2, where length and width were the longest diameter and the shortest diameter of the tumor, respectively. At the end of the study, the mice were sacrificed and tumor tissue and vital organs

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including heart, liver, spleen and kidney from each group were harvested, fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (H&E) for pathological analysis. Moreover, terminal deoxynucleotidyl transferase-mediated dUTP nick-end

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labeling (TUNEL) staining assay, antigen KI-67 (Ki-67) and cluster of differentiation

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31 (CD31) staining were used to evaluate the apoptosis and proliferation of cancer cells, respectively.

Sample preparation and analysis by LC-MS/MS Cell samples were homogenized in deionized water. Firstly, 50 µL methanol containing diazepam (5 ng/mL) was evaporated completely in a vacuum concentrator (Labconcol, USA). 80 µL homogenate was added to redissolve it. Then 480 µL

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was injected into the LC-MS/MS system for the concentration determination.

The method of PTX determination was established in our laboratory before28. For PL,

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the method was described as follows: A Thermo Scientific TSQ Quantum MS/MS

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system equipped with electrospray ionization interface operated in the positive ion mode operation. The mass quantification was accomplished in selective reaction monitoring (SRM) by monitoring the transition of m/z 318.0→221.0 for PL and m/z 285.0→154.0 for diazepam (the internal standard). With the HPLC system (LC-20AD,

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Shimadzu), LC separation was performed on a BDS Hypersil C18 column (5µm particle size, 50 × 2.1 mm, Serial No. 1100808A5). The mobile phase consisted of methanol: water plus 0.1% formic acid. The gradient was 0.01-0.5 min, 10% B;

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0.5-5.0 min, linear from 10 to 90% B; 5.0-6.0 min, 90% B; 6.0-6.5 min, 90 to 10% B;

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6.5-8.0 min, 10% B. The flow rate was 0.3 mL min-1 and the temperature of column was 40 °C.

Data analysis

The experiments were carried out in triplicates and the values were expressed as the mean ± SD. All calculations were performed using SPSS 19.0 statistical software program. The p-value < 0.05 was considered statistically significant, and the p-value

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< 0.01 was considered highly significant.

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Results and discussion Characterization of nanoparticles Orthogonal experiment designed by SPSS was used to determine optimal prescription

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of five factors, including dosage of PLGA, TPGS, drug and volume of organic phase and aqueous phase (Table S1). According to the statistical analysis of the orthogonal experimental results (Table S2), the optimal formula was as follows: the ratios of

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organic phase and aqueous phase, drug and PLGA, PLGA and TPGS were 1:5, 1:4

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and 1:4, respectively. Then we investigated the physicochemical properties of the optimal prescription, the average size of the nanoparticles was 117.1 ± 1.9 nm (Fig. 2B) with a uniform size distribution (PDI was 0.157 ± 0.017, lower than 0.200), which represented a narrow distribution. With the lower PDI, the small particle size

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might be attributed to the excellent self-assembly process of PLGA-TPGS nanoparticles. The small size of particles is expected to reduce the chance of reticuloendothelial system (RES) uptake and to prolong the systemic circulation that

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could facilitate extravasation from leaky capillaries29. Moreover, the ζ potential of

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nanoparticles is of importance from a stability perspective. In the present study, the PTX/PL-PTNPs showed about -43.25 ± 2.76 mV potential (Fig. 2C), which is in the excellent range of dispersion stability and in vivo circulation. Furthermore, the results of EE (85.1% for PTX and 89.6% for PL) of PTX/PL-PTNPs were better, probably partly due to the effect of TPGS served as an emulsifier. All these results from this part suggested that nanoparticles may be able to overcome delivery limitations of free PTX and PL as well as facilitate co-therapy delivery.

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In vitro release of PTX and PL in nanoparticles To assess the profile of drug release from the nanoparticles, in vitro release

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experiment was performed with dialysis bag. As shown in Fig. 2D, the cumulative release of PTX in PTX/PL-PTNPs exhibited a sustained-release profile compared with free drug. Two pH (pH 7.4 and 5.0) were selected to represent the physiologic

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pH of the plasma and the acidic pH of tumor cell endosomes in this experiment. The

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PTX in the PTX/PL-PTNPs was completely released after 96 h, whereas that was completely released within 24 h in free solution. The release rate was slower than that in free solutions, indicated that drugs were not located on or near the surface of the nanoparticles. It has been reported that TPGS could affect the release behavior of

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nanoparticle drug delivery systems30. Thus, the sustained drug release profiles could be attributed to materials of TPGS. Furthermore, the cumulative release of PTX in

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PTX/PL-PTNPs were not affected by the change of pH (Fig. 2D).

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Cytotoxicity activity of PTX and PL Combination of natural compound with chemotherapy drugs will be a promising strategy to enhance the anti-tumor effect31,32. In order to evaluate the cytotoxicity of the PTX/PL-PTNPs with different weight ratios of PTX and PL after confirming the preparation of nanoparticles, MTT assays were performed to quantify the cell viability with various concentrations of PTX and PL. As the IC50 values were shown in Table 1 and Table 2, HepG2 and MCF-7 cells were unequally sensitive to free drugs and

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ACCEPTED MANUSCRIPT nanoparticles. The IC50 of free PTX and free PL were 24.63 ± 5.46 nM, 4.69 ± 0.17 µM in HepG2 cells, and 157.73 ± 32.61 nM, 2.78 ± 0.26 µM in MCF-7 cells, respectively, which were higher than PTX/PL-PTNPs. Compared with MCF-7 cells,

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each treatment group exhibited a higher cytotoxicity to HepG2 cells at the same dose (Fig. 3). Moreover, in both HepG2 and MCF-7 cells, the IC50 of nanoparticles were lower than that of free drugs. From the Table 1 and Table 2, it was noticed that the CI

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values of all the nanoparticles with different ratios were all less than 1.0, which

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indicated a synergistic effect combining with PL and PTX in each group. Considering of the reduction of PTX dosage and decrease of side effects, the PTX and PL ratio of 1:200 was selected for further study.

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Cellular uptake of PTX and PL

In consideration of enhanced cytotoxicity of nanoparticles, which is often associated with increased cellular accumulation, cellular uptake of free PTX, free PL and

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PTX/PL-PTNPs were performed in HepG2 cells at different time intervals and

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different concentrations. As shown in Fig. 4A, PTX concentration in cells increased over the incubation time, reached the maximum uptake rate at 2 h and saturated at about 8 h, suggesting that there existed time-dependent cellular uptake. At the same time, the data also suggested that the PTX accumulation in cells treated with PTX/PL-PTNPs was more than that in cells treated with free PTX. In addition, increasing differences between the two groups were observed as time went by. Besides, within the incubation time of 2 hours, both free PTX group and nanoparticles

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with different nanomaterial33,34. PTX is a known substrate of P-gp, and TPGS could be able to inhibit drug efflux transporter through allosteric regulation of the P-gp ATP enzyme and then blocked the energy source of P-gp23. Thus, the inhibition of PTX

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efflux would increase cellular concentration of PTX. In addition, it has been reported

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that PL may be an inhibitor of cytochrome P450 (CYP) 3A435 while PTX can be metabolized through 3A4 and 2C836,37, which could also be an explanation for the increasing cellular accumulation of PTX in PTX/PL-PTNPs compared with free

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solution.

In contrast, the cellular uptake behavior of PL was different from that of PTX in HepG2 cells. Fig. 4B showed that PL accumulation was increased at the beginning,

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but decreased after reaching maximum concentration (Cmax) at 15 min. In the

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dose-dependent experiments, as shown in Fig. 4D, both free PL and nanoparticles presented dose-dependent cellular uptake. Besides, the data from Fig. 4B and 4D displayed that cellular PL concentration treated with PTX/PL-PTNPs was less than that of free PL, which was in contrast to PTX. The similar phenomenon was observed in concentration-dependent experiments. The reason might be that free PL mainly through the passive diffusion into the cells without any transport media or energy. In turn, after loading drug into the nanoparticles, the process of transporting requires

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with the profile of PTX. The possible reasons for this phenomenon might be that HepG2 cells contained many CYP metabolic enzymes and PL could be metabolized by CYP 3A4 according to previous study38. Consequently, the time-dependent uptake

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experiment of PL showed that the initial period was uptake-oriented, followed by the

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metabolize-oriented profile. However, PL was still able to present a synergistic effect with PTX when incubated up to 48 h from previous results of MTT assay. Bazerra et al. advocated that two α, β-unsaturated carbonyl linkages are important structure of PL for cytotoxic activity10. Adams et al. further demonstrated that electrophilicity of

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C2-C3 olefin was critical for cellular toxicity and C7-C8 olefin enhances cellular toxicity39. Therefore, we tentatively put forward that the possible metabolic products

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of PL might contain the active groups.

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The qualitative cellular uptake was visually determined by CLSM images. C6, a fluorescence probe, was employed to reflect the PTX cellular uptake. Fluorescence images in Fig. 5 were used to compare the intracellular concentration of C6 after incubating C6-solution and C6-PTNPs with HepG2 cells for 1 h, 2 h and 4 h. It was clear that the fluorescence intensity of C6-solution was much lower than C6-PTNPs, which suggested that PTNPs could effectively facilitate PTX and PL into HepG2 cells. Furthermore, the cellular uptake of NPs by HepG2 cells was time dependent.

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These results suggested that the encapsulation of PTX and PL into nanoparticles could significantly increase the cellular uptake of PTX and then enhance the cytotoxic

Endocytosis mechanism of nanoparticles

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efficacy of PTX in HepG2 cells.

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Cellular uptake of nanoparticles may involve many forms of endocytosis. To

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understand the mechanism involved in the endocytosis, intracellular level of PTX and PL were determined in HepG2 cells with various inhibitors. After incubation of PTX/PL-PTNPs for 2 h followed by treated with inhibitors for 30 min, the level of PTX were all significantly decreased compared with corresponding control treatment

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(Fig. 4E, P < 0.05). The similar result was found in condition of 4 °C (P < 0.01). This was consistent with previous conclusion39 that the endocytosis pathway was in need of energy. At the same time, inhibitions of clathrin-mediated endocytosis with

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chlorpromazine hydrochloride, caveolae-mediated endocytosis with genistein and

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macropinocytosis pathway with amiloride respectively reduced the cellular uptake of PTX in PTX/PL-PTNPs by 43.5%, 31.0% and 56.1%. Thus, these results indicated that the macropinocytosis pathway played an important role in the uptake of PTX/PL-PTNPs while a minor percentage of the nanoparticles might be internalized through clathrin-mediated pathway and caveolae-mediated pathway.

In vivo antitumor efficacy and safety evaluation

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ACCEPTED MANUSCRIPT The body weight and tumor size were measured during in vivo experiment to monitor the antitumor efficacy and toxicity (Fig. 6). As shown in Fig. 6A, the PTX/PL-PTNPs significantly suppressed tumor growth compared with mice treated with saline. The

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sizes and weights of tumor in saline, PTX solution and PTX/PL-PTNPs groups were shown in Fig. 6B and 6C. For both PTX solution and PTX/PL-PTNPs, the weights of tumors were 1.033 ± 0.057 g and 0.766 ± 0.208 g, respectively, which were

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significantly lighter than saline group (1.700 ± 0.300 g, P < 0.05 and P < 0.01,

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respectively). The similar results were obtained from the H&E staining. As shown in Fig. 6F, tumor cells with normal shape were observed in the saline group, while different degrees of necrosis cells were observed in PTX solution group and PTX/PL-PTNPs group. Moreover, the nuclear shrank and the tumor cells lost their

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normal morphology in the PTX/PL-PTNPs group. What’s more, the group treated with PTX/PL-PTNPs showed the largest necrosis areas among the three groups, which indicated that PTX/PL-PTNPs exhibited better antitumor effects and it could be

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attributed to synergistic effects of PTX and PL.

The tissue and systemic toxicity of formulation were evaluated by the survival of mice and H&E staining. The survival rate of PTX/PL-PTNPs was higher than saline and PTX solution groups, suggesting that the prepared nanoparticles had little systemic toxicity. What’s more, the H&E results from Fig. 6F obviously illustrated histopathologic alterations in heart, liver and kidney treated with PTX/PL-PTNPs, PTX solution compared with saline. However, there was no pathological change in

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ACCEPTED MANUSCRIPT the spleen tissue. With some of the fibers appeared segmental coagulation and contraction, the myocardial tissue in the PTX solution group was more serious, while the muscle fibers in the PTX/PL-PTNPs group arranged in order and there was no

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pathological damage. Liver cells necrosis and infiltration of inflammatory cells found in the liver tissue of PTX solution group were more than that of PTX/PL-PTNPs group. In addition, there existed renal tubular lumen expansion, peripheral

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inflammatory cells exudation, interstitial edema and inflammatory cells infiltration in

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the PTX solution group. Nonetheless, the injury of the PTX/PL-PTNPs group was lighter, and the inflammatory cell infiltration was less than that of the PTX solution group. These results demonstrated that the nanoparticles could effectively reduce the

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toxicity.

The role of apoptosis in the reduction of tumor was evaluated by TUNEL staining. As shown in Fig. 6E, the results obviously demonstrated more apoptotic tumor cells with

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brown-stained nuclei were observed in the PTX/PL-PTNPs group compared to saline

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and PTX solution groups. Ki-67, a cell cycle-related marker with prognostic value in cancer patients, is used to determine the proliferation of cells, and CD31 immunostaining visualizes the formation of microvessel in the tumor tissue. Cells with strong or moderate brownish cytoplasmic staining were considered as positive, whereas cells with weakly stained or unstained cytoplasmic were considered as negative. The Ki-67 and CD31 in Fig. 6E were less expressed in the PTX/PL-PTNPs group compared to saline and PTX solution groups. Thus, these results demonstrated

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while reducing toxicity.

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Conclusion In conclusion, we have designed and prepared PLGA-TPGS-based nanoparticles to entrap PTX and PL. The prepared nanoparticles displayed a size of less than 120 nm,

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with a sustained drug release rate. Moreover, the nanoparticles exhibited increased cytotoxicity and cellular uptake in vitro compared with free PTX. In addition, the data we gathered in vivo further demonstrated that PTX/PL-PTNPs suppressed tumor

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growth more efficient and less toxicity than PTX solution. These results suggested

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that PTX/PL-PTNPs prepared in this study was a good form of chemotherapy and might be successfully utilized as an effective treatment for cancer. Besides, further studies will be carried out to ascertain the superior effect of PTX/PL-PTNPs

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nanoparticles.

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Acknowledgements This study was financially supported by the National Natural Science Foundation of China (No. 81473272, No. 81503148) and Graduate Program of Scientific Research

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and Innovation in Jiangsu Province (Grant No. KYLX15_0651). The authors are thankful to Xiaoyu Lu, Yu Dai, Han Xing, Xiaojiao Wang, Siqi Xue, Jiaqi Zhou for

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their helps in this study.

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ACCEPTED MANUSCRIPT Table 1 IC50 values of free PTX, free PL and PTX/PL-PTNPs with different ratios in HepG2 cells. (Data represent mean ± SD, n=6) free PTX

PTX : PL

PTX : PL

(1:20)

(1:50)

free PL

Group

PTX : PL

PTX : PL

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Treatment

(1:100)

(1:200)

24.63±5.46

-

10.06±1.11**

8.72±0.24**

8.01±0.80**

5.10±0.08**

PL(µM)

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4.69±0.17

0.54±0.06**

1.18±0.03**

2.16±0.22**

2.75±0.05**

CI

-

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0.52±0.08

0.61±0.07

0.79±0.13

0.79±0.05

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PTX(nM)

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(*P < 0.05 and **P < 0.01 versus free solution) Abbreviations: IC50, concentration inhibiting half of the cells; PTX, paclitaxel; PL, piperlongumine; PTNPs, poly lactic-co-glycolic acid (PLGA)/D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) polymeric nanoparticles; CI, combination index

ACCEPTED MANUSCRIPT Table 2 IC50 values of free PTX, free PL and PTX/PL-PTNPs with different ratios in MCF-7 cells. (Data represent mean ± SD, n=6) free PTX

PTX : PL

PTX : PL

(1:20)

(1:50)

free PL

Group

PTX : PL

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Treatment

PTX : PL

(1:100)

(1:200)

157.73±32.61

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15.22±5.40**

12.91±6.48**

5.76±0.79**

3.79±1.01**

PL(µM)

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2.78±0.26

0.82±0.29**

1.74±0.87*

1.55±0.21*

2.04±0.55

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-

-

0.39±0.20

0.71±0.28

0.59±0.12

0.76±0.26

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PTX(nM)

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(*P < 0.05 and **P < 0.01 versus free solution) Abbreviations: IC50, concentration inhibiting half of the cells; PTX, paclitaxel; PL, piperlongumine; PTNPs, poly lactic-co-glycolic acid (PLGA)/D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) polymeric nanoparticles; CI, combination index

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ACCEPTED MANUSCRIPT Legends Fig.1. Chemical structure of PL and PTX. (A) PL (B) PTX. Fig.2. Characterization and drug release profile of PTX/PL-PTNPs. (A) Preparation scheme of PTX/PL-PTNPs. (B) Particle size distribution of PTX/PL-PTNPs. (C) ζ

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potential of PTX/PL-PTNPs. (D) Conmulative release of PTX in PTX/PL-PTNPs at pH 7.4 and pH 5.0. (n=3)

Fig.3. Effects of different treatments on cell viabilities of HepG2 cells and MCF-7

cells for 48 h. The cell viability was determined by standed MTT assay. HepG2 cells

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were treated with concentrations of PTX (A) and PL (B). MCF-7 cells were treated

with concentrations of PTX (C) and PL (D). All data were presented as mean ± SD.

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(n=6)

Fig.4. Uptake and endocytosis mechanism of PTX/PL-PTNPs in HepG2 cells. (A) Time-dependent of PTX. (B) Time-dependent of PL. (C) Concentration-dependent of PTX. (D) Concentration-dependent of PL. (E) Effects of inhibitors on endocytosis in HepG2 cells. All data were presented as mean ± SD. (n=3, *P < 0.05 and **P < 0.01

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versus control group)

Fig.5. Cellular uptake of C6. CLSM images of C6-solution and C6-PTNPs in HepG2 cells for 1 h, 2 h and 4 h.

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Fig.6. In vivo antitumor effects of the PTX/PL-PTNPs against saline and PTX solution. (A) Relative tumor volume changes after treatment with saline, PTX solution or PTX/PL-PTNPs. (n=10, **P < 0.01) (B) The body weight changes of

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HepG2 tumor-bearing nude mice after treatment with saline, PTX solution or PTX/PL-PTNPs. (n=10) (C) Pictures of tumor tissue after treatment with saline, PTX solution or PTX/PL-PTNPs. (D) The tumor weight of HepG2 tumor-bearing nude mice after they were sacrificed. (n=10, *P < 0.05, **P < 0.01) (E) Survival curves of HepG2 tumor-bearing nude mice after treatment with saline, PTX solution or PTX/PL-PTNPs. (F) TUNEL, Ki-67 and CD31 immunohistochemical assays of HepG2 tumor tissues. (G) H&E staining assays of the vital organs and tumors of HepG2 tumor-bearing nude mice.