Zein-paclitaxel prodrug nanoparticles for redox-triggered drug delivery

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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 †,‡,*

5

†

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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

8

‡

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

12

¶

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

13

‖

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

14

Chinese Academy of Sciences, Shenzhen 518055, China

15

•

16

TianHe District, Guangzhou, China

17

⁞

18

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

1

ACS Paragon Plus Environment


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These authors contributed equally to this work

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⊥

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* Corresponding authors

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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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

(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

48

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

49

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

59

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

60

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

61

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

66

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

67

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

68

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

69

precise drug release 6-7.

70

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

71

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

72

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

78

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

79

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

81

of prodrugs for treatment of cancer.

82

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

83

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,

89

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

111

permeability,

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

and

undesirable

side

effects,

which

include

severe

hypersensitivity,

6


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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

116

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,

124

China).

125

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

126

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

127

was

128

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

129

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

130

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

131

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

obtained

from

Sigma-Aldrich

Chemical

Co.,

Ltd.

(St.

Louis,

MO).

7



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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

136

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

141

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

143

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

145

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),

147

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

148

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

149

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

152

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

153

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

154

PTX concentrations. All measurements were conducted in triplicate.

155 156

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

158

Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance

159

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

160

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

161

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

162

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

163

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

164

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

165

shifts were reported as δ (ppm).

166

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

167

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

observation.

177

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

178

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

179

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

180

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

181

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

182

PBS or PBS with 5% serum replenished.

183

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

184

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

185

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

186

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

188

determined using HPLC.

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

triplicates. 11



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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

weight of each mouse were measured daily.

223

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

224

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

226

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

227

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

228

HPLC-MS.

229

Results and Discussion

230

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

231

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

232

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

233

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

234

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

235

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

236

clearance from the organs.

237

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

238

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.

240

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

242

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

243

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

245

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

248

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

249

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

251

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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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263

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

264

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

265

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

267

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,

15



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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

286

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

287

successfully synthesized, which was consistent with the FTIR result.

288

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

289

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

290

◦C

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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


Page 17 of 42


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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

303

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

304

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

305

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

306

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

307

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

308

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

310

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.

312

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

314

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

315

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

316

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

317

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

318

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

319

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

17



Page 18 of 42

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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

323

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

325

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.

327

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

328

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

330

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

332

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

335

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

336

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

337

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

338

p

Zein-paclitaxel prodrug nanoparticles for redox-triggered drug delivery

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