Reduction-sensitive Paclitaxel Prodrug Self-assembled Nanoparticles

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Reduction-sensitive Paclitaxel Prodrug Self-assembled Nanoparticles with Tetrandrine Effectively Promote Synergistic Therapy Against Drug-sensitive and Multidrug-resistant Breast Cancer Mengjuan Jiang, Ruoshi Zhang, Yingli Wang, Wenna Jing, Ying Liu, Yan Ma, Bingjun Sun, Menglin Wang, Peizhuo Chen, Hongzhuo Liu, and Zhonggui He Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00381 • Publication Date (Web): 12 Sep 2017 Downloaded from http://pubs.acs.org on September 13, 2017

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Molecular Pharmaceutics

Reduction-sensitive Paclitaxel Prodrug Self-assembled Nanoparticles with Tetrandrine Effectively Promote Synergistic Therapy Against Drug-sensitive and Multidrug-resistant Breast Cancer

Mengjuan Jiang,§ Ruoshi Zhang,ǁ Yingli Wang,ǁ Wenna Jing,ǁ Ying Liu,‡ Yan Ma,⊥ Bingjun Sun,ǁ Menglin Wang,ǁ Peizhuo Chen,§ Hongzhuo Liu,*, § Zhonggui Heǁ

§

School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,

Shenyang, 110016, China. ǁ

Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua

Road, Shenyang, 110016, China. ‡

National Institute for Food and Drug Control, No.2 Tiantanxili, Beijing, 100050,

China. ⊥

School of Chinese Materia Medica, Guangzhou University of Chinese Medicine,

Guangzhou, 510405, China.

*Corresponding author: Hongzhuo Liu, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua

Road,

Shenyang,

110016,

China.

Tel:

+86-24-23986325; E-mail: [email protected]

ACS Paragon Plus Environment

+86-24-23986325;

Fax:


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ABSTRACT Co-delivery of multiple drugs with complementary anticancer mechanisms by nano-carriers offers an effective strategy to treat cancers. Herein, conjugation (PTX-SS-VE) of paclitaxel (PTX) to vitamin E succinate (VE) self-assembled nanoparticles were used to load tetrandrine (TET) for combinational treatment against breast carcinoma. The ratio of PTX-SS-VE and TET was optimized. Compared with PTX, the TET/PTX-SS-VE co-loaded nanoparticles (TPNPs) demonstrated superior cytotoxicity against both MCF-7 cells and MCF-7/Adr cells. TPNPs were facilitated to release PTX and TET under highly reductive environment in tumor cells through the in vitro simulative release study. Cell apoptosis study and western blotting analysis exhibited TPNPs could significantly increase the cell apoptosis via modulating the levels of Bcl-2 protein and Caspase-3, which might be triggered by excess cellular reactive oxygen species (ROS) production through intracellular ROS detection test. Cellular uptake study showed that TET could increase PTX accumulation in MCF-7/Adr cells but not in MCF-7 cells, which explained stronger synergetic efficacy of TPNPs on MCF-7/Adr cells. Overall, encapsulation of hydrophobic drugs, such as TET, in reduction-sensitive PTX-SS-VE nanoparticles provides a prospective strategy to effectively overcome the multidrug resistance of tumor cells in a synergistic manner. Such a uniquely small molecular weight prodrug-nanocarrier

opens

up

new

perspectives

for

the

development

of

nanomedicines. Keywords: paclitaxel, tetrandrine, combination therapy, reduction-sensitive, breast cancer

1. INTRODUCTION Conventional chemotherapy can cause drug resistance over therapy if ineffective in eradicating cancer cells.1, 2 To enhance the drug efficacy and overcome potential drug resistance against cancers, combination chemotherapy has been extensively studied and developed.3, 4 Multiple agents with their own targets to modulate cellular signal pathways carry out combinational attacks against cancer cells which are hard to


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tolerate. Commonly, combination therapy of multiple agents is expected to achieve synergistic therapeutic effect or to overcome drug resistance of tumor cells, which can minimize side effects as well as drug resistance rate.5,

6

However, conventional

combination chemotherapy suffers certain limitations, such as uncorrelated pharmaceutics and biodistribution of each agent, which may compromise the desired therapeutic efficacy.7 Co-formulation of multiple agents within the same particulate system can improve the limitation through similar pharmacokinetic behavior and careful adjustment of drugs for synergistic or reversing drug resistance ratios.8 Paclitaxel (PTX) is clinically an indispensable anti-tumor drug to treat breast carcinoma.9,

10

However, occurrences of drug resistance in tumor cells limit the

further application of PTX, which are mainly correlated with overexpressed P-glycoprotein (P-gp), changes in apoptosis regulatory genes or mutation of tubulins.11 The increased antioxidant capacity was also reported to get involved in the resistance of tumor cells to PTX.12, 13 In addition, antioxidants could significantly decrease the cytotoxicity of PTX, which could be enhanced through inhibiting the antioxidant capacity.14,

15

Therefore, it can be a prospective method to increase

toxicity of PTX by modulating cellular reactive oxygen species (ROS) level. Tetrandrine (TET) as a natural compound has been utilized to treat hypertension, silicosis and inflammatory pulmonary diseases.16 It was reported that TET had anti-tumor capacity by inducing the apoptosis of tumor cells or reversing multidrug resistance (MDR).17-20 Recent studies found TET could effectively induce ROS production to trigger the apoptosis pathways of tumor cells and activate Caspase-3.21, 22

As a type of natural product with biologically friendly potential, TET can be a

prospective agent to treat tumors. However, the application of TET is also challenged for its low water solubility and bioavailability. Vitamin E succinate which was reported to exhibit anti-tumor activity and reverse MDR has been applied to the treatment of tumors.23-25 In our previous studies, PTX was coupled to vitamin E succinate with disulfide bond inserted to form hydrophobic prodrug (PTX-SS-VE, Fig. 1A), which could self-assemble into nanoparticles with PTX loading efficiency up to about 60 wt%. Furthermore,



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PTX-SS-VE self-assembled nanoparticles could be easily PEGylated to reduce clearance by the reticuloendothelial system (RES) and showed significant growth inhibition of the KB-3-1 tumors in mice compared with taxol.26 With the prominent anti-tumor activity and good druggability of PTX-SS-VE, in our current study, we used PTX-SS-VE as the nanoparticulate carrier to load TET (Fig. 1B) for synergistic therapy against MCF-7 cells and MCF-7/Adr cells. PTX-SS-VE co-assembled with TET to form unanimous nanoparticles with high physical stability and encapsulation efficiency. The in vitro anti-tumor activity demonstrated that TET/PTX-SS-VE co-loaded nanoparticles exhibited a potent synergistic effect through increasing ROS production or reversing MDR to enhance cell apoptosis.

2. MATERIALS AND METHODS 2.1 Materials. PTX-SS-VE was synthesized in our laboratory. DSPE-PEG-2000 was purchased from Shanghai Advanced Vehicle Technology Pharmaceutical Co., Ltd. Paclitaxel was obtained from Beijing Maisuo Chemical Technology Co. Ltd. Tetrandrine was provided by Shanghai Yuanye Biotechnology Co., Ltd. MCF-7 cells and MCF-7/Adr cells were provided by the cell bank of Chinese Academy of Sciences (Beiijng, China). All other reagents used in this work were of analytical grade.

2.2 Preparation of nanoparticles TET/PTX-SS-VE co-loaded nanoparticles (TPNPs) were prepared according to the previous nanoprecipitation method.26 Briefly, 300 µl of ethanol solution containing PTX-SS-VE (17.2 mg/mL) and TET (1.7 mg/mL) was dropwise added to 3 mL of deionized water containing 20% DSPE-PEG-2000 (w/w) under a continuous stirring rate of 800 rpm. Consequently, TET was spontaneously loaded in the prodrug self-assembled nanoparticles. Finally, it was essential to evaporate the remaining ethanol under vacuum. The obtained concentration of TPNPs was 1.72 mg/mL of PTX-SS-VE (equivalent to 1 mg/mL PTX). The preparation procedure of PTX-SS-VE self-assembled nanoparticles (PNPs) was the same as described above


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except that only the ethanol solution of PTX-SS-VE was added to deionized water.

2.3 Characterization of nanoparticles The size distribution and surface potential of self-assembled nanoparticles were measured in triplicate by a ZetaSizer (Malvern Co., UK). Transmission electron microscopy (TEM, JEM2100, JEOL, Japan) observation was conducted according to the previous report.27 The encapsulation efficiency (EE) of TPNPs was determined by centrifugation method. Briefly, TPNPs suspension was allowed to centrifuge at 3500 rpm for 15 min to isolate the nonincorporated drugs from nanoparticles. After that, the supernatant containing nanoparticles was diluted ten times with acetonitrile to destroy nanoparticles. Then the concentrations of PTX-SS-VE and TET in the diluted supernatant were measured by HPLC system using a Welchrom C8 column (200 mm × 4. 5 mm, 5 µm) thermostated at T = 30 oC. The flow rate was set as 1 mL/min. Use 95% (v/v) of acetonitrile as the mobile phase to determine PTX-SS-VE with a UV wavelength of 227 nm. TET was determined through using the mobile phase consisting of methanol/water/triethylamine (80/20/0.03, v/v/v) at 282 nm. EE was determined on three independent samples and calculated as the following equation: EE % =

WS WT

× 100%

where WS indicates the weight of PTX-SS-VE or TET in nanoparticles and WT indicates the weight of feeding PTX-SS-VE or TET. The drug loading contents (DL) of PTX and TET were calculated as the following equation: DL % ( PTX ) =

WPTX × 100% WP + WT + WD

DL % (TET ) =

WT × 100% WP + WT + WD

where WP, WT and WD indicate the weight of PTX-SS-VE, TET and DSPE-PEG-2000,



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respectively, and WPTX indicates the occupied weight of PTX in PTX-SS-VE based on the molecular weight ratio.

2.4 Optimization of formulation ratio in nanoparticles The formulation ratio of PTX-SS-VE and TET was optimized by examining the physical stability, drug loading contents and anti-tumor effect of TPNPs on MCF-7/Adr cells. Firstly, TPNPs with various weight ratios of PTX-SS-VE and TET (10:1, 5:1, 2.5:1) were placed at 37 oC for 12 days to investigate the particle size variations by dynamic light scattering (DLS). Subsequently, we selected formulations with high physical stability to explore the encapsulation efficiency and drug loading contents of the nanoparticles. Finally, TPNPs were evaluated by MTT assay for the cytotoxicity and the combination index (CI) as the following procedures. 8 × 103 MCF-7/Adr cells were seeded in each well in 96-well plates with complete RPMI 1640 cultured medium (Gibco, USA). After incubated for 24 h, the cells were exposed to blank medium (control group) and series of concentrations of TET, PNPs and TPNPs in fresh cultured medium (treated group) for 48 h. Next, MTT (Gibco, USA) was added to each well for 4 h incubation at 37 oC. Then remove the medium in the plates and add DMSO (Sigma, USA) to dissolve the formed formazan crystals. The absorbance at 570 nm was selected to measure the optical density (OD) of each well by a microplate reader (Thermo, USA). The cytotoxicity was expressed by using % cell viability (OD of treated cells/OD of control cells × 100). The combination index (CI) of PTX-SS-VE and TET was evaluated by using the Chou-Talalay method with the following equation: 28 CIx = (D)1/(Dx)1 + (D)2/(Dx)2 where Dx represents the required dose of PTX-SS-VE or TET to produce x% inhibition rate alone, while (D)1 and (D)2 respectively represent the doses of PTX-SS-VE and TET required to achieve the same inhibition effect in combination. CI value > 1 reveals antagonism, CI value < 1 reveals synergy, and CI = 1 reveals additivity.


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2.5 Physical stability of TPNPs The aqueous suspension of TPNPs (equivalent to 1 mg/mL PTX) was stored at room temperature and 4 °C for one month to investigate the physical stability. In addition, nanoparticles were also suspended in complete RPMI 1640 cultured medium under a continuous shaking rate of 100 rpm at 37 oC for 24 h. The size distribution of TET/PTX-SS-VE co-loaded nanoparticles was measured at predetermined intervals by DLS determination.

2.6 The in vitro release study of TPNPs The release profiles of PTX and TET from TPNPs were studied by utilizing ethanol-containing PBS (pH 7.4, 30% ethanol (v/v)) with 10 mM or 10 µM of Dithiothreitol (DTT) as the release medium. The nanoparticulate suspension (equivalent to 600 µg PTX) in preswelled dialysis bags with 14000 Da molecular weight cutoff were incubated in 20 mL release medium under the shaking of 100 rpm at 37 °C. At pre-determined time intervals, withdraw 1 mL of solution for HPLC analysis and then supplement immediately an equal volume of fresh release medium. The concentrations of free PTX and TET released from TPNPs were measured by HPLC. Use 55% of acetonitrile as the mobile phase with a determination wavelength of 227 nm to detect PTX.

2.7 Synergistic anti-tumor efficacy study The in vitro cytotoxicities of PTX solution, TET solution, PNPs, TPNPs and the mixtures of PNPs and free TET (PNPs + TET, the same ratio as TPNPs) were evaluated in MCF-7 cells and MCF-7/Adr cells by MTT assay. The interaction nature of PTX-SS-VE and TET in the two types of cells was investigated by using combination index. The CI-FA (fraction affected) curves were obtained through CompuSyn software.29, 30

2.8 Cell apoptosis study Annexin V-FITC/PI Detection Kit (Genview Scientific Inc., USA) and flow



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cytometer (Becton Dickinson, USA) were employed to analyze cell apoptosis. 2 mL of complete RPMI 1640 cultured medium containing 2.5 × 105 MCF-7 cells or 7.5 × 105 MCF-7/Adr cells were added to each well in 6-well plates for incubation overnight. After adhering to plates, cells were incubated in fresh medium containing TPNPs and PNPs (0.3 µM and 5 µM PTX-SS-VE for MCF-7 cells, 4 µM and 6 µM PTX-SS-VE for MCF-7/Adr cells) and free TET (0.07 µM and 1.2 µM TET for MCF-7 cells, 0.9 µM and 1.4 µM TET for MCF-7/Adr cells) for 48 h, respectively. The

cells

were

digested

by

0.25%

trypsin

(Gibco,

USA)

and

then centrifugated at 2000 rpm for 5 min. After washed with 4 oC PBS twice, cell pellets were resuspended with 100 µL binding buffer. Thereafter, the cell solution was mixed with Annexin V-FITC (5 µL) and of PI (5 µL). The mixtures were then allowed to stand for 15 min away from light. In the end, the stained cells were further diluted by 400 µL binding buffer and the obtained solution was analyzed by flow cytometry.

2.9 Western blotting analysis After treated with TPNPs and PNPs (0.3 µM PTX-SS-VE for MCF-7 cells, 4 µM PTX-SS-VE for MCF-7/Adr cells) and free TET (0.07 µM TET for MCF-7 cells, 0.95 µM TET for MCF-7/Adr cells) for 48 h, the cells were collected and the total protein was extracted. Firstly, BCA Protein Assay Kit (Meilunbio, China) was used to determine the concentration of protein to assure that the equivalent amount of protein samples were fractionated on an appropriate concentration of SDS-PAGE gel. Subsequently, polyvinylidene difluoride membranes (Millipore Corporation, USA) were covered with the proteins from the gels by electrophoresis. After blocked by 5% skimmed milk, the membranes were immunoblotted with primary and secondary antibodies (Santa Cruz Biotechnology, USA) in turn through incubation. Primary antibodies including anti-Mdr-1, Bcl-2, Caspase-3 and β-actin were respectively diluted with Tris-Buffered-Saline with Tween (TBST) to the ratio of 1:1000. Peroxidase–conjugated secondary antibody was diluted to the ratio of 1:2000. Finally, the protein bands on the membranes were observed by utilizing ECL reagents


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(Thermo Scientific).

2.10 Cellular uptake of nanoparticles MCF-7 cells and the multidrug-resistant MCF-7/Adr cells were respectively cultured on 24-well plates at a density of 2.5 × 105 cells/1 mL/well and 7.5 × 105 cells/1 mL/well. After adhering to plates, the cells were exposed to TPNPs and PNPs (equivalent to 10 µg/mL PTX) in fresh medium for 3 h and 5 h. Then the cells were collected and disrupted by ultrasonication. 100 µL cell samples were diluted with 300 µL acetonitrile. The obtained mixtures were centrifuged with a speed of 13000 rpm for 10 min. The supernatant was sampled for the determination of PTX by UPLC-MS/MS analysis.

2.11 Detection of intracellular ROS Intracellular ROS levels were evaluated using flow cytometer. Briefly, 6-well plates were employed to culture cells with the same densities mentioned above. After incubated for 48 h with TPNPs and PNPs (0.1 µM and 0.3 µM PTX-SS-VE for MCF-7 cells, 3 µM and 4 µM PTX-SS-VE for MCF-7/Adr cells) and free TET (0.02 µM and 0.07 µM TET for MCF-7 cells, 0.7 µM and 0.9 µM TET for MCF-7/Adr cells), the cells were collected and were suspended in serum-free RPMI 1640 medium containing 10 µM DCFH-DA for 30 min at 37°C. Afterwards, the cells were washed with serum-free medium and then the intracellular ROS levels were analyzed by flow cytometer.

2.13 Statistical Analysis The differences of the groups were analyzed by utilizing student’s t-test and one-way analysis of variance (ANOVA). When P value is lower than 0.05, the result is considered to be statistically significant.

3. RESULTS AND DISCUSSION 3.1 Preparation of nanoparticles



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TET/PTX-SS-VE co-loaded nanoparticles were prepared according to the previous nanoprecipitation method.26 The previous study reported that the self-assembly of PTX-SS-VE was possibly driven by intermolecular nonbonded hydrophobic interaction and π-π stacking for more energetically favorable conformations. The insertion of disulfide bond in the prodrug contributed to inhibiting crystallization and facilitating the self-assembly process.26, 31 The structure of TET is characterized by four phenyl rings, which possibly promot the π-π stacking with the planar structures of PTX-SS-VE. As a result, the intermolecular interaction between TET and PTX-SS-VE facilitated the encapsulation of TET in PTX-SS-VE self-assembled nanoparticles.

3.2 Optimization of formulation ratio in nanoparticles The weight ratio of the co-assembled drugs has a significant influence on therapeutic efficacy.7 Herein, we optimized the formulation ratio of PTX-SS-VE and TET by examining the physical stability, drug loading contents and cytotoxicity of TPNPs against MCF-7/Adr cells. DLS measurements suggested that TPNPs with the ratios of 10:1 and 5:1 could remain relatively stable, while obvious increases in particle size and polydispersity index (PDI) were observed in TPNPs with the ratio of 2.5:1 (Fig. 2). Subsequently, we evaluated the encapsulation efficiency and drug loading contents of TPNPs with the ratios of 10:1 and 5:1, which both exhibited high encapsulation efficiency (Table 1). In addition, the less TET was loaded, the higher drug loading contents of PTX was. The cytotoxicity (IC50) and the combination index (CI50) of TPNPs in MCF-7/Adr cells indicated that TPNPs with the ratio of 10:1 exhibited a stronger cytotoxicity and synergistic effect than nanoparticles with the ratio of 5:1. Therefore, TET/PTX-SS-VE co-loaded nanoparticles with the ratio of 10:1 was selected for further study. Table 1. Encapsulation efficiency and drug loading contents of TPNPs (10:1, 5:1) and their IC50 and CI50 against MCF-7/Adr cells. Samples

IC50 (µM)

CI50

EE (%)


DL (%)

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PTX-SS-VE

TET

PNPs

241.088

-

-

-

-

TET

-

6.252

-

-

-

98.9 ± 0.6 (TET)

7.6 ± 0.1 (TET)

TPNPs (10:1)

3.520

0.832

0.148 97.9 ± 1.9 (PTX)

44.6 ± 0.1 (PTX)

96.4 ± 1.9 (TET)

13.8 ± 0.2 (TET)

98.3 ± 0.9 (PTX)

41.6 ± 0.1 (PTX)

TPNPs (5:1)

3.843

1.821

0.307

3.3 Particle size and morphology of nanopaticles Table 2. The particle size, zeta potential and PDI of TPNPs and PNPs (mean ± SD, n = 3).

Samples

Size (nm)

PDI

Zeta potential (mV)

TPNPs (10:1)

107.2 ± 1.4

0.18 ± 0.03

-18.5 ± 1.0

PNPs

95.3 ± 0.9

0.17 ± 0.02

-14.4 ± 3.4

DLS measurements demonstrated the average diameters of TPNPs and PNPs were 107.2 ± 1.4 nm and 95.3 ± 0.9 nm with low PDI below 0.2, respectively (Table

2, Fig. 3B). The larger size of TPNPs might be attributed to the encapsulation of TET in PTX-SS-VE nanoparticles. TPNPs had lower zeta potential than PNPs, which contributed to the physical stability. TEM observation showed that the morphology of TPNPs was spherical with a diameter of approximately 100 nm, which corresponded with the DLS measurement (Fig. 3A).

3.4 Physical stability of TPNPs The physical stability of TPNPs for storage was investigated. As shown in Fig.

3C, TPNPs presented considerable storage stability at room temperature and 4 oC for one month. To investigate the plasma stability, TPNPs were incubated in complete RPMI 1640 cultured medium (pH 7.4) under shaking (100 rpm) for 24 h at 37 oC, which showed that nanoparticles remained nearly unchanged in the particle size and PDI (Fig. 3D). The results indicated the satisfied physical stability of TPNPs.



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3.5 The in vitro release profiles of TPNPs Dithiothreitol (DTT) as a prevailing GSH simulatant was utilized to investigate the reductive responsiveness of TPNPs. The in vitro release study of TPNPs was tested in PBS containing 30% ethanol (v/v) with 10 mM or 10 µM DTT. As shown in

Fig. 4, we found TPNPs released 84.1% of PTX after incubated for 24 h in PBS with 10 mM DTT, while less than 5% of PTX was released from TPNPs in PBS containing 10 µM DTT. TET also exhibited faster release in PBS containing 10 mM DTT than that containing 10 µM DTT. The results indicated TPNPs have significant reduction-responsive drug release profiles and will be facilitated to release PTX and TET under highly reductive environment in tumor cells.

3.6 Synergistic anti-tumor efficacy study Table 3. The IC50 (µM) of PTX solution, TET solution, PNPs, PNPs + TET, TPNPs on MCF-7 cells and MCF-7/Adr cells. Increased rate of cytotoxicity Samples

MCF-7

(vs. PNPs)

MCF-7/Adr

MCF7

MCF-7/Adr

PTX solution

0.930

22.186

-

-

TET solution

5.068

6.252

-

-

PNPs

3.872

241.088

-

-

Mixtures

0.776

4.675

5.0

51.6

TPNPs

0.331

3.520

11.7

68.5

In order to verify the enhanced anti-tumor effect of TET/PTX-SS-VE co-loaded nanoparticles, the in vitro cytotoxicity of PTX solution, TET solution, PNPs, TPNPs and PNPs + TET for 48 h were firstly performed by MTT assay against MCF-7 cells, respectively. All drug treatments demonstrated dose-dependent effects on MCF-7 cells in Fig. 5A. PNPs decreased the cell viability as the drug concentration increased


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from 0.02 µM to 12 µM, resulting in an IC50 of 3.872 µM. In contrast, PNPs were less effective in killing tumor cells in vitro than PTX solution with an IC50 of 0.930 µM (Table 3), which might be attributed to the delayed release of the active PTX molecule from PTX-SS-VE conjugate. TET exhibited an IC50 of 5.068 µM and the cell viability was over 90% when using TET concentration < 2 µM. Compared with single drug treatment, combinational treatment in the mixture form of PNPs and free TET caused relatively lower cell viability. The IC50 of PNPs was reduced to 0.776 µM when administered in combination with 10% (w/w) of TET with negligible cytotoxocity. Furthermore, the cytotoxicity of TPNPs was further enhanced with an IC50 of 0.331 µM, 2.3 fold and 2.8 fold lower than those of PNPs + TET and PTX solution, respectively. The increased efficacy of TPNPs could be attributed to the following reasons. TET co-loaded in prodrug nanoparticles was endocytosed more efficiently by cells compared with the free TET. Besides, the two cytotoxic agents were simultaneously internalized into the cells, which allowed them to accentuate their individual anti-tumor effect each other.32, 33 To elucidate the nature of the combinational interaction, the Chou-Talalay method analysis was performed for evaluating the degree of synergy. As shown in Fig.

5C, whether PNPs and TET were administered by combination in the mixture form or nanoparticles, the CI values were approximately lower than 0.4 when Fa was between 0.2 and 0.75, demonstrating a strong synergistic anti-proliferative efficacy. Furthermore, it was noteworthy that the combination in nanoparticles caused lower CI values than that in mixtures. Notably, concurrent delivery of PTX-SS-VE with TET has produced a clearly synergistic cytotoxicity. The relatively profound synergistic efficacy of TPNPs was consistent with the cytotoxicity results. MCF-7/Adr cells express excess P-gp, which is the main cause to induce multidrug resistance (Fig. 7A). As depicted in Table 3, compared with PTX solution, PNPs showed weaker anti-tumor effect on MCF-7/Adr cells with an IC50 value of 241.088 µM, 62.3 fold higher than that on MCF-7 cells. In addition, an enhanced anti-tumor effect was observed in the mixtures of PNPs and free TET, resulting in an IC50 value of 4.675 µM, which was 51.6 and 4.7 fold lower than those of PNPs and



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PTX treatment, respectively. Similar with MCF-7 cells, the combinational treatment effect of PNPs + TET on MCF-7/Adr cells was found to be synergistic with CI values below 0.3 at Fa 0.25-0.90 (Fig. 5D). In contrast, the efficacy of the combinational treatment against MCF-7/Adr cells was further improved when TET was loaded in the PTX-SS-VE nanoparticles with lower IC50 value and CI values. Furthermore, the increased rate of cytotoxicity of TPNPs (vs. PNPs) against MCF-7/Adr cells was higher than that against MCF-7 cells, which also indicated the stronger synergistic effect of TPNPs on the multidrug-resistant cells. It could be due to the probability that TET and vitamin E could reverse the multidrug resistance.19, 34 The results above demonstrated the combinational administration of PTX-SS-VE and TET in nanoparticles generated a potent synergistic effect on MCF-7 cells, especially MCF-7/Adr cells, leading to stronger anti-tumor effects than single drug, mixture, or PTX treatment.

3.7 Cell apoptosis study Generally, mechanisms inducing cell death include apoptosis, autophagy and necrosis.35 To verify the hypothesis that tetrandrine synergistically enhanced the anti-tumor efficacy of PTX-SS-VE by inducing apoptosis, the cells were exposed to different concentrations of TPNPs, TET and PNPs followed by double staining for flow cytometric analysis. In MCF-7 cells, in comparison to the treatment of PNPs, the combination of PTX-SS-VE with TET in TPNPs significantly enhanced the apoptosis rate from 22.73% to 41.60% at a concentration of 0.3 µM and from 47.86% to 54.17% at a higher concentration of 5 µM (Fig. 6). In addition, TET solution scarcely induced cell apoptosis compared with the control group, which might be due to the too low concentration. In MCF-7/Adr cells, TET solution and PNPs hardly induced cell apoptosis. However, significant dose-dependent cell apoptosis occurred when cells were exposed to TPNPs. These results demonstrated the synergistic anti-tumor efficacy of TET/PTX-SS-VE co-loaded nanoparticles might be due to a synergistic effect on cell apoptosis.


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3.8 Western blotting analysis We further investigated the influence about the combination therapy of PTX-SS-VE with TET on the expression of Bcl-2 and the activity of Caspase-3, which had been researched to be involved in the PTX-induced apoptosis.36, 37 As shown in Fig. 7C, TET hardly caused any quantitative alterations of Bcl-2 protein and Caspase-3 in both MCF-7 cells and MCF-7/Adr cells, which accorded with the apoptosis results by flow cytometry. However, TPNPs could obviously reduce the level of Bcl-2 protein and enhance the activity of Caspase-3 compared with PNPs. The results demonstrated that TET co-loaded in PTX-SS-VE self-assembled nanoparticles could significantly enhance cell apoptosis induced by PTX-SS-VE.

3.9 Cellular uptake of nanoparticles The cytotoxicity of PTX-SS-VE was closely related to the accumulation of the released PTX in tumor cells. To elucidate the mechanism of enhanced anti-tumor effect of TPNPs, we examined the influence of TET on the cellular accumulation of PTX by quantifying PTX after tumor cells were incubated with PNPs or TPNPs for 3 h and 5 h. Fig. 8 illustrated cellular accumulation of PTX was time-dependent. In addition, the cellular concentration of PTX in MCF-7/Adr cells was much lower than that in MCF-7 cells when treated with the same concentration of PNPs or TPNPs. However, compared with PNPs, TPNPs obviously improved PTX content in MCF-7/Adr cells. There was no significant difference about the content of PTX in MCF-7 cells after exposed to PNPs and TPNPs, respectively. Therefore, TET co-loaded in PTX-SS-VE nanoparticles enhanced significantly the accumulation of the released PTX in MCF-7/Adr cells but not in MCF-7 cells, which indicated synergistic anti-tumor efficacy of TPNPs might be induced by other mechanisms. The western blotting results in Fig. 7A showed MCF-7/Adr cells overexpressed P-gp, which could promote PTX efflux to decrease the intracellular accumulation. It was reported that TET, as a potent and selective MDR modulator, could reverse multidrug resistance mediated by P-gp in MDR cells.38, 39 In our experiments, expression of P-gp wasn`t affected by TET (Fig. 7B). Thus, the increased PTX concentration in



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MCF-7/Adr cells by TPNPs treatment was likely ascribed to the reason that TET inhibited the function of P-gp, reduced the efflux of PTX and enhanced the intracellular accumulation of PTX.39

3.10 Detection of intracellular ROS ROS has been demonstrated to be a crucial event in anti-tumor drug-induced cell death.14 Oxidative stress was also investigated to be correlated with the down-regulation of Bcl-2 to induce apoptosis.40 Studies found that paclitaxel could increase cellular ROS level, which played an important role in modulating paclitaxel anti-tumor activity.14, 15 TET was also found to increase the cellular accumulation of ROS to induce cell apoptosis.41,

42

Therefore, to investigate whether synergistic

therapy was associated with ROS production, we detected the ROS levels by DCFH-DA assay in MCF-7 and MCF-7/Adr cells after treated with TPNPs, PNPs and TET solution. As shown in Fig. 9, different concentrations of TPNPs treatment could obviously increase the percentage of cells with increased ROS level compared with PNPs or TET treatment alone. It suggested that TET/PTX-SS-VE co-loaded nanoparticles could induce more ROS production in the cells in comparison to PNPs or TET. Besides, the intracellular ROS levels were also elevated by PNPs and TET treatment alone, with the exception that PNPs hardly changed the ROS level in MCF-7/Adr cells due to the cell resistance to PTX. The results indicated TET co-loaded in PTX-SS-VE self-assembled nanoparticles was likely to increase the sensitivity of tumor cells to PTX from PTX-SS-VE through increasing the production of intracellular ROS. As a consequence, the related apoptosis pathways in tumor cells were triggered to induce cell death.21

CONCLUSION To enhance the therapeutic effect and minimize the drug resistance rate, in this study, we successfully loaded TET in PTX-SS-VE self-assembled nanoparticles with high physical stability. TPNPs were facilitated to release PTX and TET under highly reductive environment in tumor cells. Importantly, compared with PTX solution,


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TET/PTX-SS-VE co-loaded nanoparticles demonstrated superior cytotoxicity against both MCF-7 cells and MCF-7/Adr cells due to the potent synergistic therapy and the possible reversal of MDR by TET. The mechanism researches of synergistic effect demonstrated TPNPs could enhance the cellular apoptosis by reducing the expression of Bcl-2 and enhancing the activity of Caspase-3. Furthermore, the cellular ROS levels were increased in response to TPNPs treatment, which might be involved in the synergistic effect of PTX-SS-VE and TET. Cellular uptake study showed that PTX accumulation could be significantly increased in MCF-7/Adr cells after TPNPs treatment, but not in MCF-7 cells, which further enhanced the anti-tumor effect of TPNPs on the multidrug resistant cells for the possible inhibition of P-gp efflux. Thus, PTX-SS-VE conjugate constructed an efficient nanocarrier system to load a hydrophobic natural drug by one-step facile fabrication, which demonstrated a potent synergistic therapeutic effect on breast cancer cells, and especially the multidrug-resistant cells.

Conflict of Interest No competing financial interest gets involved in this study.

Acknowledgment This study was supported by Nature Science Foundation of Guangdong Province (No. 2016A020217017).

Figure captions Abstract Graphic: Tetrandrine loaded in reduction-sensitive paclitaxel prodrug self-assembled nanoparticles synergistically promote apoptosis of cancer cells.

Fig. 1. The chemistry structure schemes of PTX-SS-VE and TET.

Fig. 2. The particle size and PDI variations of TPNPs with different ratios of



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PTX-SS-VE and TET (10:1, 5:1, 2.5:1) at 37 oC for 12 days.

Fig. 3. TEM image of TPNPs (A), size distribution of TPNPs and PNPs (B), particle size and PDI variations of TPNPs at room temperature and 4 oC for one month(C), particle size and PDI variations of TPNPs at 37 oC in RPMI 1640 containing 10% FBS (D).

Fig. 4. The cumulative release of PTX (A) and TET (B) in phosphate buffer solution containing 10 mM and 10 µM of DTT.

Fig. 5. Dose-response curves of PTX solution, TET solution, PNPs, PNPs + TET and TPNPs to MCF-7 cells (A) and MCF-7/Adr cells (B); CI-FA curves of PNPs + TET and TPNPs to MCF-7 cells (C) and MCF-7/Adr cells (D).

Fig. 6. Flow cytometric analysis for apoptosis of MCF-7 cells (A) and MCF-7/Adr cells (B) after treated with blank cultured medium, low and high concentrations of TET solution, PNPs and TPNPs for 48 h.

Fig. 7. Expression of P-gp in MCF-7 cells and MCF-7/Adr cells (A) and levels of P-gp in MCF-7/Adr cells after treated with different agents for 48 h (B). The levels of Bcl-2 protein and caspase-3 in MCF-7 cells and MCF-7/Adr cells treated with different agents for 48 h. Lower panel: semiquantitative analysis of Bcl-2 protein, * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, vs the control groups, n = 3.

Fig. 8. Cellular levels of PTX in MCF-7 cells (A) and MCF-7/Adr cells (B) after incubated with PNPs or TPNPs for 3 h and 5 h. * represents p ≤ 0.05 vs PNPs group, n = 3.

Fig. 9. Flow cytometric analysis for intracellular ROS of MCF-7 cells (A) and


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MCF-7/Adr cells (B) after treated with blank cultured medium, low and high concentrations of TET solution, PNPs and TPNPs.

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Fig. 1. The chemistry structure schemes of PTX-SS-VE and TET. 70x34mm (300 x 300 DPI)



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Fig. 2. The particle size and PDI variations of TPNPs with different ratios of PTX-SS-VE and TET (10:1, 5:1, 2.5:1) at 37 oC for 12 days. 45x34mm (300 x 300 DPI)


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Fig. 3. TEM image of TPNPs (A), size distribution of TPNPs and PNPs (B), particle size and PDI variations of TPNPs at room temperature and 4 oC for one month(C), particle size and PDI variations of TPNPs at 37 oC in RPMI 1640 containing 10% FBS (D). 52x34mm (300 x 300 DPI)



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Fig. 4. The cumulative release of PTX (A) and TET (B) in phosphate buffer solution containing 10 mM and 10 µM of DTT. 80x31mm (300 x 300 DPI)


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Fig. 5. Dose-response curves of PTX solution, TET solution, PNPs, PNPs + TET and TPNPs to MCF-7 cells (A) and MCF-7/Adr cells (B); CI-FA curves of PNPs + TET and TPNPs to MCF-7 cells (C) and MCF-7/Adr cells (D). 80x61mm (300 x 300 DPI)



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Fig. 6. Flow cytometric analysis for apoptosis of MCF-7 cells (A) and MCF-7/Adr cells (B) after treated with blank cultured medium, low and high concentrations of TET solution, PNPs and TPNPs for 48 h. 80x57mm (300 x 300 DPI)


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Fig. 7. Expression of P-gp in MCF-7 cells and MCF-7/Adr cells (A) and levels of P-gp in MCF-7/Adr cells after treated with different agents for 48 h (B). The levels of Bcl-2 protein and caspase-3 in MCF-7 cells and MCF7/Adr cells treated with different agents for 48 h. Lower panel: semiquantitative analysis of Bcl-2 protein, * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, vs the control groups, n = 3. 80x73mm (300 x 300 DPI)



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Fig. 8. Cellular levels of PTX in MCF-7 cells (A) and MCF-7/Adr cells (B) after incubated with PNPs or TPNPs for 3 h and 5 h. * represents p ≤ 0.05 vs PNPs group, n = 3. 80x32mm (300 x 300 DPI)


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Fig. 9. Flow cytometric analysis for intracellular ROS of MCF-7 cells (A) and MCF-7/Adr cells (B) after treated with blank cultured medium, low and high concentrations of TET solution, PNPs and TPNPs. 80x64mm (300 x 300 DPI)



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Abstract Graphic: Tetrandrine loaded in reduction-sensitive paclitaxel prodrug self-assembled nanoparticles synergistically promote apoptosis of cancer cells. 64x34mm (300 x 300 DPI)


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Reduction-sensitive Paclitaxel Prodrug Self-assembled Nanoparticles

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