Engineering Multifunctional Coatings on Nanoparticles Based on

3 hours ago - ... drug or DOX-loaded zein NPs without coatings, resulting from the pH-responsive release of loaded drug in extra/intra-cellular enviro...
0 downloads 0 Views 3MB Size
Subscriber access provided by Kaohsiung Medical University

Functional Structure/Activity Relationships

Engineering Multifunctional Coatings on Nanoparticles Based on Oxidative Coupling Assembly of Polyphenols for Stimuli-Responsive Drug Delivery Hongshan Liang, Bin Zhou, Jing Li, Xingnian Liu, Ziyu Deng, and Bin Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01208 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Journal of Agricultural and Food Chemistry

1

Engineering Multifunctional Coatings on Nanoparticles Based on Oxidative

2

Coupling Assembly of Polyphenols
 
for Stimuli-Responsive Drug Delivery

3

Hongshan Liang a,c, Bin Zhou b, Jing Li a,c, Xingnian Liu a,c, Ziyu Deng a,c, Bin Li a,c,d*

4

a

5

430070, China

6

b

7

Wuhan 430068, China

8

c

9

University), Ministry of Education, China

College of Food Science and Technology, Huazhong Agricultural University, Wuhan

School of Food and Biological Engineering, Hubei University of Technology,

Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural

10

d

11

China

12

*Corresponding author: Bin Li

13

E-mail address: [email protected]

Functional Food Enginnering & Technology Research Center of Hubei Province,

14 15 16 17 18 19 20 21 22

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

23

Abstract

24

In this study, zein nanoparticles (NPs) with novel multifunctional coatings based on

25

oxidative coupling assembly of polyphenols were synthesized for the first time. This

26

coating was formed by oxidative self-polymerization of the organic ligands

27

(polyphenols) in alkaline condition, which could be biodegraded by acidic pH, as a

28

result, impacting pH-responsive property of the system. More importantly, the high

29

level of intracellular glutathione (GSH) could induce the biodegradation of the

30

polyphenol coatings, resulting in a fast release of trapped anticancer drugs in the cells.

31

Based on confocal laser scanning microscopy (CLSM) and cytotoxicity experiments,

32

drug-loaded and polyphenol-coated zein NPs were shown to possess highly efficient

33

internalization and an apparent cytotoxic effect on HeLa cells. Notably, the CLSM

34

observation illustrated that coated zein NPs showed delayed drug release compared

35

with free drug or DOX-loaded zein NPs without coatings, resulting from the

36

pH-responsive release of loaded drug in extra/intra-cellular environment. Additionally,

37

the short-time cytotoxicity and morphology observation also confirmed the delayed

38

drug release behavior of coated NPs. These highly biocompatible and biodegradable

39

polyphenol-coated zein NPs may be promising vectors in the field of

40

controlled-release biomedical applications and cancer therapy.

41 42

Key words: polyphenol, self-polymerization, pH-responsive, redox dual-responsive

43 44

2

ACS Paragon Plus Environment

Page 2 of 37

Page 3 of 37

Journal of Agricultural and Food Chemistry

45

1. Introduction

46

Encapsulation of guest molecules within micro/nano-sized hosts provides a variety of

47

promising applications in the fields of catalysis 1, medical diagnostics 2, drug delivery

48

3, 4

49

emulsions,

50

nanoparticles (NPs) have attracted considerable attention as potential drug delivery

51

devices

52

light- 11 and temperature- 12 responsive, is now a key theme in the biomedical field for

53

effective loading and release of guest molecules with high specificity to targeted cells

54

in a controlled manner.

55

Protein-based systems represent a major class for drug and gene delivery due to their

56

enhanced properties of absorbability and low toxicity in the degradation of end

57

products

58

candidates for drugs or bioactives delivery

59

controlled release systems have been developed by employing different kinds of

60

coatings or stabilizers to improve the stability or loading capacity of bulk zein NPs

61

19-21

62

based on zein NPs or zein/quaternized chitosan NPs capped with metal

63

ion-polyphenol coatings

64

used as a stimuli-responsive mechanism to trigger drug release. Despite these

65

designable pH-responsive delivery systems, current zein-based drug delivery systems

66

still have a number of flaws, including for example low stimulus sensitivity especially

and materials science 5. Among the various hosts/carriers including micelles, hydrogel

and

inorganic

particles,

self-assembled

6-8

biodegradable

. Engineering stimuli-responsive functional NPs, e.g., pH- 9, redox-

10

,

13-15

. Of protein-based NPs, zein NPs have been highlighted as excellent 16-18

. Additionally, several zein-based

. In our previous work, we reported a bio-responsive controlled-release system

22, 23

. The metal ion-polyphenol coordination bonding was

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

67

in mild acidic, the relative slow release of the drug when entering the cell and

68

introducing potential toxic species. Therefore, the development of a green,

69

biocompatible, and effective drug delivery system with high stimulus sensitivity and

70

intracellular controlled-release features remains a desirable goal.

71

Plant polyphenols are widely distributed in plant tissues and plays a critical role in a

72

range of biological functions such as photosynthesis, structural support, oxygen

73

transportation and adhesion 24, 25. With the high content of dihydroxyphenyl (catechol)

74

and trihydroxyphenyl (gallic acid), plant polyphenols display versatile physical and

75

chemical properties, including absorption of UV radiation, radical scavenging, and

76

metal ion complexation

77

based on these properties of polyphenols in the field of chemistry and materials

78

science. Very recently, plant polyphenols have been investigated as precursors for the

79

formation of multifunctional coatings on different substrates 25, 26. Coating deposition

80

was most effective from alkaline condition (0.6M NaCl, pH 7.8) as compared to pure

81

water, resulting from the oxidation of plant polyphenols leading to the self-polymerize

82

27, 28

83

polyphenols 28.

84

Due to the unique properties of polyphenols, we engineered multifunctional coatings

85

on zein NPs based on oxidative coupling assembly of polyphenols. Tannic acid (TA),

86

which contains high amount of galloyl groups, was first used as the model polyphenol

87

precursor for the deposition of the coatings. The degradation of the coatings was

88

tailored by the pH conditions and intracellular glutathione (GSH) level, leading to

25

. Significant interest has been given to building blocks

. Notably, thiolysis reaction could lead to the degradation of polymeric

4

ACS Paragon Plus Environment

Page 4 of 37

Page 5 of 37

Journal of Agricultural and Food Chemistry

89

drug release and, hence, cellular apoptosis. This simple coating method without any

90

other organic reagents or cross-linkers met the requirements for a variety of

91

biomedical applications.

92

2. Materials and methods

93

2.1. Materials

94

Zein (Z0001) was purchased from Tokyo Chemistry Industry, Co., Ltd. (Tokyo,

95

Japan). Tannin acid (TA) was purchased from Aladdin Chemistry Co., Ltd.

96

(-)-epigallocatechin-3-gallate (EGCG) was purchased from Xi'an Natural Field

97

Bio-Technique Co., Ltd. (Xi’an China). Persimmon tannin (PT, 98.7% purity) was

98

kindly provided by Shanghai Ocean University composed of polymers ranging from 7

99

to 20 kDa. 3-(N-morpholino)-propanesulfonic acid (MOPS) was obtained from the

100

Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). Glutathione (GSH),

101

phosphate buffer solution (PBS) and MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl

102

tetrazolium bromide] were purchased from Sigma-Aldrich (St. Louis, MO, USA).

103

Dubelcco's modified Eagle's medium (DMEM), fetal bovine serum (FBS),

104

trypsin-EDTA and penicillin-streptomycin mixtures were from Gibco®BRL (Carlsbad,

105

CA, USA). Other chemicals used were of analytical grade. All the solutions used in

106

the experiments were prepared using ultrapure water through a Millipore (Millipore,

107

Milford, MA, USA) Milli-Q water purification system.

108

2.2. Preparation and characterization of polyphenol-coated zein NPs

109

Preparation of polyphenol-coated zein NPs: All solutions were freshly prepared for

110

immediate use. The standard preparation process was described as follows: zein was

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

111

dissolved in aqueous ethanol solutions (75% v/v) to obtain a stock solution with final

112

concentration of 10 mg/mL. Then 0.5 mL zein solution was added to 9.5 mL of

113

MOPS buffer (10 mM, pH 7.8). Next, different volume (40 µL, 60 µL, 80 µL or 100

114

µL) of TA solution (24 mM) was added and the dispersion was under vigorous stirring

115

for 0.5-2 h at room temperature. The product was then purified by successive dialysis

116

(MWCO 3500) against deionized water for 48 h to remove the free TA. Then 200 µL

117

of HAuCl4 aqueous solutions (10 mM) was added in the zein-TA solution to observe

118

the metallization of gold.

119

Dynamic laser scattering (DLS) and zeta potential: DLS and zeta potential

120

measurement were performed on a commercial laser light scattering instrument

121

(Nano-ZS90, Malvern, UK). The apparent Z-average hydrodynamic diameter and

122

polydispersity index (PDI) were obtained at 25°C with a fixed scattering angle of

123

173°. The zeta potential was calculated by the Dispersion Technology Software.

124

Transmission electron microscope (TEM): Images were taken on a JEM-2100F

125

(JEOL, Japan). The samples were prepared by dropping solution onto copper grids

126

coated with carbon and then dried naturally.

127

Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron

128

spectroscopy (XPS) analysis: FT-IR spectra were obtained with a Jasco 4100 series

129

with an attenuated total reflection cell (Jasco Inc., Easton, MO). All samples were

130

prepared as KBr pellets and were scanned against a blank KBr pellet background.

131

XPS observations were conducted on an axis ultra DLD apparatus (Kratos, U.K.).

132

2.3. DOX encapsulation

6

ACS Paragon Plus Environment

Page 6 of 37

Page 7 of 37

Journal of Agricultural and Food Chemistry

133

The stock of 4 mg/mL DOX prepared was completely dissolved in zein solution for

134

60 min. The formulation containing DOX was prepared by adding the above solution

135

dropwise to MOPS buffer (10 mM, pH 7.8) with magnetic stirring. Next different

136

volume (40 µL, 60 µL, 80 µL or 100 µL) of TA solution (24 mM) was added and the

137

dispersion was under vigorous stirring for 0.5-2 h at room temperature. The free DOX

138

was obtained by calculating the DOX content that were ultracentrifuged at 4000 × g

139

for 30 min in a refrigerated centrifuge (TGL-20000cR) with angle rotor through 10

140

kDa MWCO Amicon filter and determined by a UV-vis spectrophotometer (UV-1100,

141

MAPADA) at 480 nm. The encapsulation efficiency was defined as the drug content

142

that was entrapped into zein/TA NPs and calculated as follows:

EE (%) =

143

Total DOX - Free DOX × 100 % Total DOX

144

2.4. Release of DOX

145

Drug release from NPs and release kinetics study were carried out using dialysis

146

membrane tubing (MWCO = 3500). Briefly, an aliquot of drug loaded NPs was taken

147

into a dialysis bag and suspended in 100 mL release medium with different pH values

148

(pH = 4.0, 5.0, 6.2, 7.4) or different GSH concentrations at room temperature and

149

gently shaken at 100 rpm in a water bath (37.0 ± 0.5 °C). At predetermined intervals,

150

1 mL dissolution sample was collected and the concentration of DOX was measured.

151

After that, each 1mL aliquot was returned to the original solution for maintaining total

152

volume and total DOX amount.

153

2.5. In vitro cell toxicity assay

154

The cytotoxicity of free DOX and coated NPs was evaluated using the MTT assay. 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

155

Briefly, human cervical cancer cells (HeLa cells) were seeded in 96-well microplates

156

at the density of 1 × 104 cells/well and incubated at 37 °C under a 5% CO2 atmosphere

157

for 24 h to allow cell attachment. After incubation, the medium was replaced by the

158

fresh medium containing various NPs (either blank or DOX containing NPs) at DOX

159

concentrations ranging from 0.1 to 2.5 µg/mL for further incubation of 24 h. Then 20

160

µL MTT solution (5 mg/mL) was added to each well, and the cells were incubated for

161

another 4 h. After incubation, the culture solution was removed carefully, leaving the

162

precipitate. Subsequently, 100 µL of DMSO was added to each well to solubilize the

163

formazan crystals formed. The absorbance at 490 nm was measured by a multilabel

164

microplate reader (Victor X3, PerkinElmer 2030). Cell viability (%) was expressed by

165

the following equation:

166

Cell viability (%) =

Abs 490 nm of treated group × 100 % Abs 490 nm of control group

167

2.6. Intracellular uptake study

168

For quantitative study, HeLa cells were seeded in a 6-well plate at a density of 2×105

169

cell/well in 2 mL growth medium and the cells were incubated at 37 °C for 24 h to

170

allow cell attachment. Then the culture medium was replaced by 2 mL of fresh

171

medium containing DOX-loaded NPs and incubated for 1, 2, 4 or 6 h, respectively.

172

After incubation, the suspension was collected and the wells were washed three times

173

with cold PBS and harvested. The cellular uptake of NPs was measured by flow

174

cytometry analysis (Beckman Coulter, Miami, FL, USA).

175

For qualitative study, cells were seeded in a 35 mm petri dish at a density of 1×105

176

viable cells/well in 2 mL growth medium and the cells were incubated at 37 °C for 24 8

ACS Paragon Plus Environment

Page 8 of 37

Page 9 of 37

Journal of Agricultural and Food Chemistry

177

h to allow cell attachment. Cells were washed for three times after incubation for 1, 2,

178

4 or 6 h using DOX-loaded NPs and then fixed by 4% paraformaldehyde in PBS (pH

179

7.4) for 20 min. The cells were further washed twice with PBS. Fluorescence images

180

were collected using a CLSM (Zeiss LSM 710, Germany).

181

2.7. Morphology Observation

182

The morphology of the cells after incubation of 24 h with blank or DOX containing

183

NPs was evaluated by using optical microscopy with a 10×objective.

184

2.8. Statistics analysis

185

All of the data were expressed as the mean ± standard error. ANOVA analysis and

186

Student’s t-test were performed to compare the significant difference. A value of p