Zein-Paclitaxel Prodrug Nanoparticles for Redox-Triggered Drug

Oct 19, 2018 - Departments of Radiation Oncology, Xiangya Hospital, Central South ... Center for Biomedical Materials and Interfaces, Shenzhen Institu...
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Zein-paclitaxel prodrug nanoparticles for redox-triggered drug delivery and enhanced therapeutic efficiency Heting Hou, Dong Zhang, Jiewen Lin, Yingying Zhang, Chengyong Li, Zhe Wang, Jiaoyan Ren, Maojin Yao, Ka Hing Wong, and Yi Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04627 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Journal of Agricultural and Food Chemistry

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Zein-paclitaxel prodrug nanoparticles for redox-triggered drug delivery and enhanced

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therapeutic efficiency

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Heting Hou

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Jiaoyan Ren •, Maojin Yao ⁞, Ka-hing Wong ‡,*, Yi Wang †,‡,*

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Shenzhen Key Laboratory of Food Biological Safety Control, Shenzhen Research Institute of

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Hong Kong Polytechnic University, Shenzhen 518057, China

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9

University, Hong Hum, Kowloon, Hong Kong Special Administrative Region

†,⊥,

Dong Zhang

‡,⊥,

Jiewen Lin †, Yingying Zhang §, Chengyong Li ¶, Zhe Wang ‖,

State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation) and

Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic

10

§

11

Hunan 410008, China

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School of Chemistry and Environment, Guangdong Ocean University, Zhanjiang 524088, China

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Center for Biomedical Materials and Interfaces, Shenzhen Institutes of Advanced Technology,

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Chinese Academy of Sciences, Shenzhen 518055, China

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16

TianHe District, Guangzhou, China

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Yan Jiang Xi Road, Guangzhou, Guangdong, 510120, China

Departments of Radiation Oncology, Xiangya Hospital, Central South University, Changsha,

School of Food Science and Engineering, South China University of Technology, Wushan RD.,

Department of Anesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. 107

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These authors contributed equally to this work

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* Corresponding authors

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Journal of Agricultural and Food Chemistry

Abstract

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Prodrug, in which the inactive parent drug with good bioavailability is metabolized into an

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active drug in body, is one of the main strategies to target the disease site to improve the drug

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efficiency and reduce the adverse effects of chemotherapy. Because of the good capability of

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chemical modification, zein, a plant derived protein, and drugs can be conjugated through

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environmentally sensitive links to form prodrugs capable of triggered drug release. In this study, a

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novel prodrug was synthesized using paclitaxel (PTX), zein, and a disulfide linker, and

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nanoparticles were formed by self-assembly of the prodrug. An effective in vitro triggered release,

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80-90% in 5 min, of the prodrug based nanoparticles (zein-S-S-PTX_NP) was successfully

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approached. The cytotoxicity of zein-S-S-PTX_NP as well as the zein encapsulation of PTX

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(zein_PTX_NP) and pure PTX on HeLa cells and NIH/3T3 fibroblast cells was tested using MTS

47

assay. It showed that, after the treatment of zein-S-S-PTX_NP at the equivalent PTX

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concentrations of 0.1, 0.5, 1, and 5 µg/ml, respectively, zein-S-S-PTX_NP had zero damage to

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normal cells but a similar cytotoxicity to cancer cells as pure PTX. In the animal study, the tumor

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was 50% of the original size after the treatment of zein-S-S-PTX_NP for 9 days with 3 doses. This

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study suggested that the novel prodrug based nanoparticle zein-S-S-PTX_NP could be a promising

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approach in chemotherapy with targeted delivery, improved efficacy, and reduced side effects.

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Keywords: prodrug; self-assembly; redox-responsive; zein; triggered release; paclitaxel. 3

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Introduction

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Cancer is the second leading cause of death worldwide, exceeded only by cardiovascular

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diseases. Current chemotherapy for cancer has such problems including the low effective tumor

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uptake of the drug, the indiscriminate destruction of normal cells, the development of multidrug

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resistance, and the severe side effects.1 All of these support the need of the development of new

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generation of drug delivery platforms with good biocompatibility, multi-functional and

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stimuli-responsive properties, and high drug loading capacity.

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The most substantial research field of drug delivery is to develop site-specific drug delivery

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system (SSDDS), which uses carriers to deliver drugs to the target sites and release them. SSDDS

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can be divided into two types: the passive targeting systems, which primarily takes the advantages

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of the enhanced permeability and retention effect, and the active targeting systems, which

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transport the drugs to the disease site via an interaction between the targeting ligand conjugated on

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the system and the receptors that over-expressed on the membrane of the target cells.2 Studies

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reported that SSDDS could improve the delivery efficacy,3-4 reduce drug toxicity,5 and achieve

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precise drug release 6-7.

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Prodrug, in which the inactive parent drug with good bioavailability is metabolized into an

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active drug in body, is one of the main strategies to target the disease site to improve the drug

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efficiency and reduce the adverse effects of chemotherapy.8 The prodrugs, chemically modified

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from the conventional drugs using drug carriers, such as liposomes, proteins, polymeric micelles, 4

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and nanoparticles (NPs),9 provide possibilities to overcome various obstacles, such as poor

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aqueous solubility,10 chemical instability, insufficient oral absorption, pre-systemic metabolism,

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poor blood brain barrier penetration, unwanted toxicity, and local irritation. A robust

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redox-responsive disulfide bond, which can be cleaved by glutathione (GSH), is widely used as the

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linkage and the trigger in the prodrug.11-12 As a thiol-containing endogenous tripeptide, GSH is an

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important antioxidant in plants and animals.13 The intracellular GSH concentration of the

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proliferating tumor cells is higher than that of normal cells.14 Therefore, GSH is applied as a target

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of prodrugs for treatment of cancer.

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Over the last few years, NPs have gained a lot of attentions in the field of drug delivery

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because of their desirable physiochemical and biological properties.15 It is a versatile approach to

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combine the advantages of both prodrug and NPs.16 The polymer-drug conjugate, as a prodrug,

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can be controlled to form self-assembled NPs. It was reported that biodegradable nanoparticle

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formulations and have shown comparable activity to traditional formulations and much faster

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administration.17 In addition, polymeric NPs have also been extensively explored for various

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prodrugs by integrating with stimuli-responsive units, which could respond to numerous factors,

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including pH, temperature, light, enzyme, and bio-reducible environment. The drug release from

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the prodrug specifically into the disease site can be triggered by the mentioned factors belonging to

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the local disease microenvironment through the stimuli-responsive unit.

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Plant-based proteins are inexpensive, abundant, biodegradable, and biocompatible.18 Many 5

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of them have been consumed as an integral part of human diet. Compared to synthetic polymers

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and animal proteins, such as gelatin and albumin, the transfer of zein based drug carriers to clinic

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can be more direct and rapid. Because of the good biodegradability, they undergo enzymatic or

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hydrolytic degradation in the biological environment into non-toxic byproducts.19 Over the past

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few decades, zein, the major protein of corn, has been investigated as drug and nutrient carriers in

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the forms of nano/microcapsules, films, nanofibers, and hydrogels.20 Zein is insoluble in water but

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readily disperses in ethanol-water mixtures.21 Zein contains primarily hydrophobic and neutral

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amino acids but also polar amino acids.22 Thus, as an amphiphile, zein can self-assemble into

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various structures, including spheres, sponges, and films.21 Self-assembled particles made by

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amphiphiles, including zein, have the ability to transfer through cell membranes,23 and they also

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have better stability than normal particles because of their unique surface properties. In vivo, zein

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NPs are beneficial for prolonged blood residence of a 7.2-fold increase.24 Because of the good

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capability of chemical modification, zein and drugs can be conjugated through environmentally

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sensitive links to form prodrugs capable of triggered drug release.

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Paclitaxel (PTX) is a chemotherapy drug used to treat a number of types of cancer, including

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ovarian cancer25, breast cancer26, non-small cell lung cancer27, cervical cancer28, and brain

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cancer29. PTX is the only drug that can promote microtubule polymerization and stabilization.

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However, the application of PTX has significant obstacles, including low aqueous solubility, low

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permeability,

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myelosuppression, neutropenia, and neurotoxicity30-32. Approaches on the development of drug

and

undesirable

side

effects,

which

include

severe

hypersensitivity,

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delivery systems to improve the efficacy and, at the same time, reduce the side effects of PTX are

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being sought.

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In this study, a novel prodrug zein-S-S-PTX was synthesized using PTX, zein, and a disulfide

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linker to improve the selectivity of PTX on cancer cells and reduce its toxicity to normal cells for

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chemotherapy. Nanoparticles zein-S-S-PTX_NP were formed by the self-assembly of

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zein-S-S-PTX. In vitro triggered release using GSH was conducted. In vitro cytotoxicity to cancer

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cells and the selectivity over normal cells were investigated. In vivo antitumor activity was

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evaluated using tumor xenograft animal model.

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Materials and Methods

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Materials. Zein was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).

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Ethanol (96% v/v) was obtained from Guangdong Guanghua Sci-Tech Co., Ltd (Guangzhou,

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China).

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[3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]

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(MTS) was purchased from Promega Corporation (Madison, USA). Phenazine methosulfate (PMS)

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was

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N,N-dicyclohexylcarbodiimide (DCC), 4-Dimethylminopyridine (DMAP), PTX, and GSH were

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purchased from Aladdin Industrial Corporation (Shanghai, China). Bis (2-hydroxyethyl) disulfide

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and dialysis bag was obtained from Alfa Aesar Chemicals Co., Ltd. (Shanghai, China). Dimethyl

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sulfoxide-d6 (DMSO-d6) was purchased from Xilong scientific Co., Ltd (Guangzhou, China).

obtained

from

Sigma-Aldrich

Chemical

Co.,

Ltd.

(St.

Louis,

MO).

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Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS),

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penicillin and streptomycin, trypsin, and phosphate buffer saline (PBS) were all purchased from

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Life Technologies (Grand Island, NY, USA).

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Synthesis of zein-S-S-PTX. As illustrated in part I. Chemical synthesis of the schematic

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diagram Figure 1, zein, DCC, DMAP, and bis(2-hydroxyethyl) disulfide were added in solution to

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form the intermediate zein-S-S-C2H4OH. And, then, PTX was added to the mixed solution to form

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zein-S-S-PTX.

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Formation of zein-S-S-PTX_NP and zein_PTX_NP. Zein-S-S-PTX_NP was prepared from

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zein-S-S-PTX solution using the phase separation method. Zein-S-S-PTX_NP was obtained by

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freeze drying and stored at 4 °C for further analysis. The molecular organization was illustrated in

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part II. Self-assembly of the schematic diagram Figure 1. Zein_PTX_NP was obtained following

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the same procedure using zein and PTX.

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The drug loading efficiency (LE) and encapsulation efficiency (EE). To measure the LE of

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PTX in zein-S-S-PTX_NP, the prepared sample of a certain weight was re-dispersed and GSH was

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added. The PTX released was analyzed using high performance liquid chromatography (HPLC),

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which was equipped with a C18 column and an ultraviolet-visible (UV-vis) detector. PTX was

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detected at a wavelength of 227 nm. Acetonitrile were chosen as mobile phase at a flow rate of 0.3

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ml/min. The total running time was set as 20 min.

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The EE and LE of PTX in zein_PTX_NP was determined using HPLC. Freeze-dried 8

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nanoparticles of a certain weight were re-dispersed and ultracentrifuged for 30 min. The PTX

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released in the supernatant was measured. Acetonitrile was used as the mobile phase at a flow rate

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of 1 ml/min. A calibration curve was obtained by measuring a series of solutions with different

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PTX concentrations. All measurements were conducted in triplicate.

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The LE of PTX in zein-S-S-PTX_NP and the EE and LE of PTX in zein_PTX_NP were calculated using the following formulas: total  PTX  free  PTX  100% total  PTX mass  of  PTX  in  nanoparticles( g ) LE  mass  of  nanoparticles( g )

EE  157

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Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance

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spectroscopy (NMR). The chemical structures of the samples were characterized using FTIR

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spectrometer (Thermo Scientific, Madison, WI). The powdered samples of PTX, zein,

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zein-S-S-C2H4OH, and zein-S-S-PTX, respectively, were mixed with bits of potassium bromide

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and pressed into a disk for spectra collection. The collection region was set to 400-4000 cm-1 and

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the resolution was 4 cm-1. 1H nuclear magnetic resonance (1H NMR) spectra of zein-S-S-C2H4OH

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and zein-S-S-PTX, respectively, were recorded on a Bruker AV400 NMR spectrometer. Chemical

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shifts were reported as δ (ppm).

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Dynamic light scattering (DLS) and scanning electron microscope (SEM). The particle

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size and zeta potential were measured using DLS instrument, Malvern ZETASIZER 3000HSA 9

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(London, England), at room temperature (25 °C). The prepared zein-S-S-PTX_NP sample was

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re-dispersed in PBS and the pH of the suspension was adjusted to 7.4. The particle size,

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polydispersity index (PDI), and zeta-potential were measured and calculated by the DLS

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instrument and the quantitative results were given. All measurements were conducted in triplicate,

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and the data are shown as mean ± standard deviation (SD).

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The morphology of the zein-S-S-PTX_NP and zein_PTX_NP, respectively, were observed

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using a JEOL JSM-6490 SEM (Tokyo, Japan). The samples were gold coated (300 Å) using an

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Edwards S150B sputter coater to help improve the electrical conductivity before the SEM

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observation.

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In vitro drug release. The in vitro release and stability of zein-S-S-PTX_NP and

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zein_PTX_NP, respectively, were studied. Zein-S-S-PTX_NP and Zein_PTX_NP were

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re-dispersed in PBS buffer solution with and without 5% serum, respectively. The suspension was

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then put into a membrane dialysis bag in a pool of the same PBS or PBS with 5% serum solution.

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At predetermined time intervals, sample solution was collected from the release medium and fresh

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PBS or PBS with 5% serum replenished.

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To study the triggered release, Zein-S-S-PTX_NP was re-dispersed in PBS with and without

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the addition of 10 mM GSH, respectively. The suspension was then put into a membrane dialysis

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bag in a pool of PBS. The zein-S-S-PTX_NP suspension with no GSH addition was set as the

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control group. At predetermined time intervals, sample solution was collected from the release 10

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medium and fresh PBS replenished. The concentration of PTX in the collected sample was

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determined using HPLC.

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Cell culture. HeLa cells were incubated in the RPMI 1640 medium supplemented with 10%

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(v/v) FBS, 100 mg/ml penicillin, and 100 mg/ml streptomycin. NIH 3T3 cells were cultured in

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DMEM medium containing 10% equine serum and 1% penicillin-streptomycin. The cells were

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supplemented with 10% FBS, and maintained in a humidified incubator at 37 °C in an

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environment with 5% CO2. Then, the cells were trypsinized using a 0.25% trypsin solution in PBS

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buffer for 5 min and were re-suspended in the complete culture medium for revival and subculture.

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MTS assay. The cytotoxicity of zein-S-S-PTX_NP to HeLa cells was evaluated using MTS

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assay. The HeLa cells were cultured in the growth medium in the 96-well plates at a density of

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7000 cells per well for 24 hr for cell attachment. The cells were incubated with zein-S-S-PTX_NP,

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zein_PTX_NP, and pure PTX, respectively, at various equivalent PTX concentrations at 37 °C for

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24 hr. The group with no drug treatment was set as control. After the incubation, the solution with

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MTS and PMS was added to each cell and incubated at 37 °C for another 2 hr. Then, the

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absorbance at 227 nm (A490), which is directly proportional to cell viability, of the cells was

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recorded with a microplate spectrophotometer (Bio-Rad, Hercules, CA, USA). Results were

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expressed as mean cell viability (%) ± SD, where the cell viability (%) represents the percentage

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ratio of the A490 of the samples to the A490 of the control. The measurements were conducted in

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triplicates. 11

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Confocal laser scanning microscope (CLSM). The cellular uptake of zein-S-S-PTX_NP

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was investigated using CLSM. Coumarin 6 was used as the fluorescent dye, which could label zein

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in the zein-S-S-PTX_NP. HeLa cells (4×104 cells/well) were seeded onto sterile micro-scope

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slides in a 6-well plate and allowed to attach for 24 hr. The cells were then incubated with

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coumarin 6 labeled zein-S-S-PTX_NP at a PTX concentration of 40 μg/ml for 30 min. Then, the

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treated cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min.

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The fixed cells were stained by 1 μg/ml DAPI for 20 min and washed three times with PBS. The

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prepared cell samples were examined using a confocal microscope (Leica, Germany).

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In vivo antitumor efficiency. All experiments were conducted strictly in accordance with the

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guidelines of the Chinese Association for the Accreditation of Laboratory Animals Care

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(CAALAC). Tumor xenograft animal model was established by subcutaneously injecting 5×105

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HeLa cells into the right flank of each female BALB/c nude mouse (average bodyweight 17 g, 7

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weeks old). Tumors were allowed to grow up to 400 mm3 before the subsequent experiments.

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Tumor xenograft mice were randomly divided into three groups (n=10). Zein-S-S-PTX_NP,

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zein_PTX_NP, and pure PTX, respectively, in the dose of equivalent 2 mg PTX/kg, were

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intravenously injected to the tumor xenografted mice every 3 days. The tumor volume and body

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weight of each mouse were measured daily.

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Biodistribution. Tumor xenograft mice were randomly divided into three groups (n=10).

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Zein-S-S-PTX_NP, zein_PTX_NP, and pure PTX, respectively, in the dose of equivalent 2 mg 12

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PTX/kg, were intravenously administered to mice via tail vein. The mice were sacrificed 4h after

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the administrations and the major organs, including kidney, liver, spleen, lungs, tumor, and heart,

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and blood samples were obtained. The PTX contents in the samples were determined using

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HPLC-MS.

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Results and Discussion

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Particle characterizations. The morphology of prepared zein-S-S-PTX_NP and

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zein_PTX_NP were characterized using SEM (Figure 2). For zein-S-S-PTX_NP (Figure 2A), the

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spherical particles had smooth surfaces and uniform particle sizes ranging from 200 to 300 nm.

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While, for zein_PTX_NP (Figure 2B), both large and small particles were shown in the image and

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the particle size distribution was wider compared to that of zein-S-S-PTX_NP. For drug delivery,

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the size of the NPs is important because it affects the drug release kinetics, biodistribution, and

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clearance from the organs.

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The particle sizes and zeta potentials of zein-S-S-PTX_NP and zein_PTX_NP were measured

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using dynamic light scattering (DLS) and the results were shown in Table 1. The hydrodynamic

239

sizes of zein-S-S-PTX_NP and zein_PTX_NP were 229.9±0.3 nm and 186.9±0.6 nm, respectively.

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It is reported that NPs smaller than 250 nm actively accumulate in tumor tissue due to the EPR

241

effect.33 The DLS results also showed that the bioconjugation of zein and PTX through the

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disulfide bond had an effect on the size of the self-assembled nanoparticles. After the

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bioconjugation of PTX on zein, the molecular size increased and resulted in a larger size of 13

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zein-S-S-PTX_NP than zein_PTX_NP after the molecular self-assembly. The PDI values of

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zein-S-S-PTX_NP and zein_PTX_NP were 0.200±0.011 and 0.134±0.005, respectively, which

246

indicated that the size distributions of both the zein-S-S-PTX_NP and zein_PTX_NP particles

247

were narrow. The zeta-potentials of zein-S-S-PTX_NP and zein_PTX_NP were -40.5±0.4 and

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-38.3±0.4, respectively. The zeta-potential of a high absolute value above 40 indicated that the

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formed NPs had good stability and were clear and stayed still from aggregation, while the

250

zeta-potential of an absolute value between 30 and 40 means moderate stability. So

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zein-S-S-PTX_NP suspension had a good stability while zein_PTX_NP suspension had a

252

moderate stability. It was also obtained using the HPLC that, the LE of zein-S-S-PTX_NP was

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6.96 mg/g, while the EE and LE of zein_PTX_NP were 98.3% and 16.4 mg/g, respectively.

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FTIR spectroscopy. The differences in chemical structures among zein, zein-S-S-C2H4OH,

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zein-S-S-PTX_NP, and PTX were studied and compared using FTIR spectroscopy. In Figure 3,

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the fingerprints of zein were found at 3408 cm-1, 2958 cm-1, 1653 cm-1, and 1540 cm-1, which were

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attributed to the vibration stretching of -OH group, the amide A’, the C=O stretching of the amide

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I, and the C-N stretching of the amide II, respectively.34-35 The fingerprints of PTX were found at

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1734 and 1712 cm-1, 1646 cm-1, 1245 cm-1, and 1074 cm-1, which were corresponding to the C=O

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stretching, C=C stretching, C-N stretching, and C=O stretching, respectively.36 Comparing the

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spectra of zein and zein-S-S-C2H4OH, it was observed that the peaks at 1017 cm-1, 1045 cm-1, and

262

1066 cm-1, which were all corresponding to the symmetric stretching of S-O bond,37-39 were shown

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in the spectra of zein-S-S-C2H4OH but not shown in that of zein. It was also observed that the peak

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at 3408 cm-1 in zein spectra, which was attributed to the -OH group, had a peak at 3300 cm-1 with

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higher intensity showed on its shoulder in the spectra of zein-S-S-C2H4OH. The peak at 3300 cm-1

266

was attributed to the -OH of ethanol like groups, such as -C2H4OH.40-41 It indicated that the

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disulfide HO-C2H4-S-S-C2H4-OH was successfully conjugated with zein and formed

268

zein-S-S-C2H4OH. Comparing the spectra of PTX and zein-S-S-PTX, it was observed that the

269

fingerprints of PTX, which were the peaks at 1735 cm-1, 1541 cm-1, and 1073 cm-1, were also

270

shown in the spectra of zein-S-S-PTX with tiny shifts to 1738 cm-1, 1550 cm-1, and 1082 cm-1,

271

respectively, after the synthesis. It was also observed that the peak at 3300 cm-1 in the spectra of

272

zein-S-S-C2H4OH, which was attributed to the -OH of ethanol like groups including -C2H4OH,

273

disappeared and the peak at 3408 cm-1, which was originally in the zein spectra and was attributed

274

to the -OH group, dominated again. It indicated that PTX was successfully conjugated with

275

zein-S-S-C2H4OH and formed zein-S-S-PTX.

276

1H

NMR of zein-S-S-PTX_NP. 1H NMR spectrum of zein-S-S-PTX_NP was recorded using

277

DMSO-d6 solvent at room temperature and the results were shown in Figure 4. The chemical

278

shifts (δ, ppm) of the protons on PTX were ascribed as follows: δ ~8.15 (Ar-H, 2H), 7.65 (Ar-H,

279

1H), 7.40 (Ar-H, 10H), 4.90 (peak n, 1H, -CH-O-), 4.80 (peak f, -C2′H-OH), 4.5 (peak 7H,

280

-C7H-OH), 4.39 (peak q, -C7H-OH), 4.30 and 4.18 (peak m, 2H, -CH2-), 3.70 (peak j, 1H, -CH-),

281

2.54 (peak p, 1H, -CHH-), 2.38 (peak t, 3H, -(C=O)-CH3), 2.30 (peak h, 2H, -CH2-), 2.24 (peak k,

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3H, -(C=O)-CH3), 1.88 (peak p′, 1H, -CHH-), 1.79 (peak y, 3H, =C-CH3), 1.68 (peak r, 3H,

283

-C-CH3), 1.24 (peak u, 3H, -C-CH3), and 1.14 (peak v, 3H, -C-CH3).42-43 While, the chemical

284

shifts (δ, ppm) of the protons on zein were ascribed as follows: δ ~ 9.16-9.14, 7.97 (amide I), and

285

0.84 (leucine).44 In Figure 4, the characteristic peaks of both PTX and zein were found in the

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spectrum of zein-S-S-PTX. Therefore, based on 1H NMR result, zein-S-S-PTX_NP was

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successfully synthesized, which was consistent with the FTIR result.

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In vitro release and stability of zein-S-S-PTX_NP and zein_PTX_NP. The in vitro release

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and stability of zein-S-S-PTX_NP and zein_PTX_NP, respectively, in PBS solution (pH 7.4) at 37

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◦C

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Figure 5. As shown, there was no release of PTX from zein-S-S-PTX_NP in either PBS or PBS

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with 5% serum up to 24 hr, while there was no release of PTX from zein_PTX_NP in PBS and

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only 2.5% PTX release from PBS with 5% serum in 24 hr. It was showed that zein-S-S-PTX_NP

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as well as zein_PTX_NP had a good stability in both PBS and serum solutions.

with and without 5% serum were studied, and the release profiles were obtained and shown in

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In vitro triggered release of PTX from zein-S-S-PTX_NP. It has been reported that the

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concentration of GSH in cancer cells is higher than that of normal cells and the disulfide link could

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be cleaved by the GSH at such concentration in cancer cells. Thus, the PTX can be triggered

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released from zein-S-S-PTX-NP by the GSH in cancer cells. The in vitro triggered release profiles

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of PTX from zein-S-S-PTX_NP in PBS solution (pH 7.4) at 37 ◦C with and without GSH,

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respectively, were obtained and the results were shown in Figure 6. As seen, there was no release 16

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of PTX from zein-S-S-PTX_NP when no GSH was added. With GSH, the PTX showed an initial

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burst release of 80-90% in the first 5 min. From 5 to 20 min, the cumulative release amount of PTX

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vibrated between 80-90% because of the unsteady distribution of the PTX in the dialysis bag in

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such a short time. After 30 min, the cumulative release amount of PTX was stabilized at 90% and

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gradually increased to 98.2% after 120 min. The result indicated that the addition of GSH triggered

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the release of PTX from zein-S-S-PTX_NP. As mentioned above, the concentration of GSH in

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normal cells is not high enough to cleave the disulfide bond, while the concentration of GSH in

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tumor cells is nearly 4 times higher than that of normal cells. The zero release of PTX from

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zein-S-S-PTX_NP in normal cells and fast release in cancer cells can maintain the drug molecules

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protected in the NPs during blood circulation, reduce the cytotoxicity to the normal cells, and

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enhance the targeted delivery and therapeutic effect on the tumor cells.

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In vitro cytotoxicity and selectivity of zein-S-S-PTX_NP. The in vitro cytotoxicity of

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zein-S-S-PTX_NP, zein_PTX_NP, and pure PTX, respectively, on HeLa cells was investigated

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and the results were shown in Figure 7A. At low equivalent PTX concentrations of 0.1 and 0.5

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µg/ml, zein_PTX_NP showed less cytotoxicity to HeLa cells than zein-S-S-PTX_NP and pure

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PTX, respectively, while zein-S-S-PTX_NP and pure PTX had similar cytotoxicity. It indicated

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that, considering on keeping the efficacy of PTX in zein based drug delivery systems, the

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encapsulation nanoparticles were not performed as well as the prodrug nanoparticles. The reason

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was considered to be the difference in the drug release mechanism. For the release of PTX from the

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low concentrations of zein-S-S-PTX_NP and zein_PTX_NP, respectively, the hydrolysis of

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disulfide bond of zein-S-S-PTX_NP by GSH in cancer cells was more efficient than the hydrolysis

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of the zein protein of zein_PTX_NP by specific proteases. When the equivalent PTX

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concentration increased to 1, 5, 10, 15, and 20 µg/ml, zein-S-S-PTX_NP, zein_PTX_NP, and pure

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PTX showed similar cytotoxicity to the HeLa cells. It indicated that, after the chemical and

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structural modifications on PTX, at the same equivalent PTX concentration, zein-S-S-PTX_NP

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had similar in vitro antitumor effect to HeLa cells as pure PTX.

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To evaluate the selectivity of zein-S-S-PTX_NP on cancer cells over normal cells, the

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cytotoxicity of zein-S-S-PTX_NP, zein_PTX_NP, and pure PTX, respectively, on normal NIH

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3T3 cells was investigated and the results were shown in Figure 7B. It showed that, at the low

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equivalent PTX concentrations of 0.1, 0.5, 1, and 5 µg/ml, zein-S-S-PTX_NP had no toxicity to

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the normal cells and the cell viability was 100% or over. It was considered that the higher cell

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viability than control was because of the nutritive effect of zein on normal cells. While with the

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same concentrations of 0.1, 0.5, 1, and 5 µg/ml, zein_PTX_NP and pure PTX showed toxicity to

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the normal cells, and the toxicity increased when the concentration increased. Pure PTX decreased

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the cell viability to 63.6%, 45.9%, 40.2%, and 39.8%, respectively, at the equivalent PTX

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concentrations of 0.1, 0.5, 1, and 5 µg/ml. The cell viability of NIH 3T3 cells after the treatment of

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zein-S-S-PTX_NP showed significant differences to that of pure PTX at a significant level of

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p