Development of Yam Dioscorin-Loaded ... - ACS Publications

Subscriber access provided by Gothenburg University Library

Article

Development of yam dioscorin-loaded nanoparticles for paracellular transport across human intestinal Caco-2 cell monolayers Hung-Ling Hsieh, Chia-Hung Lee, and Kuo-Chih Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04150 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 43

Journal of Agricultural and Food Chemistry

TOC Graphic

CH 2OH H O H H H H O OH H H H H H NH+3 HOH 2C O HO

H3C

CH3

N+ CH3 H

H O

H

O

H3C

H

-

P

CH 2OH O H H O OH H

O HO

O

P

O

TPP

HO

H3C

P O

HO

O -

N CH HOH 2C 3 H H OH H H O H H H O H H CH 2OH H

H

CH3

H

O H

HOH 2C

TMC

P O

P O O

O HO

H H

O

O HO

-

NH+3

H

CH3

N+

H

+

H3C

H O H

H O

H

NH+3

H3C

P O O

-

N+

CH3

HOH 2C

CH3

OH H

H

O H

H O

O H CH 2OH

H H

H

H

NH+3

N+(CH3)3

H

+ + + N (CH ) + + rDioscorin N (CH ) + + +N (CH )

N+(CH3)3

+

+

3 3

3 3

+

N+(CH3)3

ACS Paragon Plus Environment

3 3


1

Development of yam dioscorin-loaded nanoparticles for paracellular transport

2

across human intestinal Caco-2 cell monolayers

3 4

Hung-Ling Hsieh, Chia-Hung Lee, Kuo-Chih Lin*

5 6

Department of Life Science, National Dong Hwa University, Hualien County 974,

7

Taiwan

8 9 10 11

*Corresponding author; E-mail: [email protected].

12 13


Page 2 of 43

Page 3 of 43


14

ABSTRACT

15

Dioscorins, the major storage proteins of yam tubers, exert immunomodulatory

16

activities. To improve oral bioavailability of dioscorins in the intestine, recombinant

17

dioscorin (rDioscorin) was coated with N,N,N-trimethyl chitosan (TMC) and

18

tripolyphosphate (TPP), resulting in the formation of TMC-rDio-TPP nanoparticles

19

(NPs). The loading capacity and entrapment efficiency of rDioscorin in the NPs were

20

26+0.7% and 61+1.4%, respectively. The NPs demonstrated a substantial release

21

profile in the pH environment of the jejunum. The rDioscorin released from the NPs

22

stimulated proliferation and phagocytosis of the macrophage RAW264.7 and activated

23

the gene expression of IL-1β and IL-6. Incubation of the NPs in the Caco-2 cell

24

monolayer led to a 5.2-fold increase of Papp compared with rDioscorin alone,

25

suggesting that rDioscorin, with the assistance of TMC, can be promptly transported

26

across the intestinal epithelia. These results demonstrate that the TMC-rDio-TPP NPs

27

can be utilized for elucidating the immunopharmacological effects of dioscorins

28

through oral delivery.

29 30 31

Key words: Dioscorin; Trimethyl chitosan; Nanoparticles; Oral delivery; Caco-2

32



33

1. INTRODUCTION

34

Yams (Dioscorea spp.) are an important staple in Africa and are also widely

35

consumed in Asia.1 Dried slices of yam tubers have been used in Chinese herbal

36

medicines since the ancient times. It may strengthen the functions of the spleen,

37

kidney, liver, and stomach, reduce phlegm, and heal fatigue, chronic diarrhea, and

38

diabetes.2 Yam tubers comprise of approximately 6%–13% crude proteins on a dry

39

weight basis.3 Dioscorins, the major storage proteins of yam tubers, account for

40

approximately 85% of the total protein content in yam tubers. Several studies have

41

demonstrated that both native and recombinant dioscorins (rDioscorin) of several yam

42

species exhibit immunomodulatory activities by activating the Toll-like receptor 4

43

(TLR4) signaling pathway and upregulating the expression of cytokine genes

44

involved in pro-inflammatory, inflammatory, and anti-inflammatory responses.4–7

45

Furthermore, both native and recombinant dioscorins inhibit ovalbumin-induced

46

allergic reactions in the BALB/c mice by promoting T helper (Th) 1 cell responses

47

and thus modulating the Th1/Th2 imbalance.8,9 Oral administration of dioscorins from

48

D. alata to BALB/c mice may improve hypertension, stimulate splenocytes to secrete

49

cytokines such as IFN-γ, IL-4, and IL-10, increase the subpopulation in NK and B

50

cells and the numbers of Peyer’s patches, attenuate oxidative status and learning

51

dysfunction, and improve the metabolic syndrome of diabetes mice.10–13 The feasible

52

dosages for the dioscorins used in oral administration range from 50 to 80 mg/kg/dose.

53

However, for a person with a 60-kg body weight, the dosage is approximately 0.5–0.8

54

g/dose according to the body surface area scaling method for dose translation from

55

animal to human.14

56

Oral bioavailability of protein and peptide therapeutics has been demonstrated to

57

be very poor, mainly because of protease digestion in the gastrointestinal (GI) track,

58

the epithelial cells that prevent cellular uptake of hydrophilic macromolecules, and the


Page 4 of 43

Page 5 of 43


59

tight junctions between epithelial cells that inhibit paracellular transport of protein

60

and peptide therapeutics.15 To overcome these obstacles, many studies have focused

61

on using chitosan as a delivery carrier of biomacromolecules for oral administration to

62

enhance intestinal absorption and paracellular transport across mucosal epithelia to

63

improve the oral bioavailability of biological macromolecules.16–18 Chitosan is a low

64

toxic, biodegradable, and biocompatible muco-adhesive biopolymer. The US Food

65

and Drug Administration (FDA) has approved the use of chitosan in the hemostatic

66

ChitoFlex® PRO dressing (HemCon Medical Technologies Inc., OR, USA) for

67

hemorrhage control.19 However, the low solubility of chitosan at neutral pH values

68

restricts its use as an absorption enhancer at specific sites of the intestine, such as in

69

the jejunum and ileum.20 To solve this problem, N,N,N-trimethyl chitosan (TMC), a

70

quaternized derivative of chitosan, has been synthesized by adding methyl groups to

71

the amine groups of chitosan molecules.20–24 Synthesis of TMC may produce

72

O-methylation on C3 and C6 carbons, depending on the synthetic strategy used.25

73

O-methylation lowers the solubility of TMC in aqueous solution, while O-methylated

74

TMC displays reduced cytotoxicity comparing to O-methyl free TMC.23 TMC

75

exhibits a permeation-enhancing trait, which is attributed to its positively charged

76

ionic interaction with the tight junctions via the C-Jun NH2-terminal kinase-dependent

77

pathway.20,26

78

The TMC nanoparticles (NPs) have been widely explored for diverse

79

pharmaceutical and biomedical applications such as drug delivery, protein and peptide

80

delivery, gene delivery, vaccine delivery, absorption enhancement, diagnostic, and

81

tissue engineering applications.22,24 TMC NPs can be prepared through ionic

82

cross-linking with negatively charged multivalent anions or polyanions such as

83

tripolyphosphate (TPP), poly(lactide-co-glycoside), and poly(γ-glutamic acid) to



Page 6 of 43

84

improve the packaging of therapeutic macromolecules and the stability of the NPs.27

85

All of these cross-linking agents have been approved by the FDA for use in food or

86

clinical applications.27–29 Of these polyanions, the TMC-TPP has been applied for

87

encapsulating antigens such as bovine serum albumin and ovalbumin for nasal

88

vaccination,

89

neuroexcitation-associated diseases such as epilepsy and convulsions, vitamins as

90

nutritional supplements in certain foods, paclitaxel against several types of cancers,

91

diclofenac sodium for relieving ocular inflammation, and curcumin against tumor

92

cells.30–36 All these reports demonstrate that the TMC-TPP NPs have the potential

93

application in various pharmaceutical, biomedical, and biomaterials fields.

anti-neuroexcitation

peptide

for

the

treatment

of

94

To protect the rDioscorin from degradation in strong acidic environment in the

95

stomach, enhance intestinal absorption, and improve effective dose usage for

96

elucidating the immunopharmacological effects of dioscorins through oral delivery,

97

we aim to use TMC-TPP NPs for encapsulating rDioscorin and further evaluate the

98

immunomodulatory effects of the encapsulated rDioscorin and the potential of

99

TMC-rDio-TPP NPs for oral delivery. TMC was synthesized with a high degree of

100

quaternization and rDioscorin coated with TMC-TPP to make TMC-rDio-TPP NPs.

101

The quaternization of TMC on the formation of particles can increase the electrostatic

102

attractions between the protein and TPP molecules, which may reduce the amount of

103

protein-leaching that occurs during the delivery process and protect the rDioscorin

104

from acid degradation and protease digestion in the GI tract. Various physicochemical

105

properties of the TMC-rDio-TPP NPs such as particle size, zeta potential, loading

106

capacity, entrapment efficiency, in vitro release profiles, and paracellular permeability

107

were characterized and the immunomodulatory effects of released rDioscorin

108

investigated. Finally, the effect of TMC-rDio-TPP NPs on the improvement of

109

paracellular permeability of released rDioscorin was evaluated in vitro using Caco-2


Page 7 of 43


110

cell monolayers.

111



112

2. MATERIALS AND METHODS

113

Chitosan (50-80 kDa, 93% deacetylated), TPP, bovine serum albumin (BSA),

114

and the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

115

Sodium iodide (NaI) and 1-methyl-2-pyrrolidinone (NMP) were purchased from Alfa

116

Aesar (Heysham, UK) and methyl iodide (CH3I) from Kanto (Tokyo, Japan).

117

2.1 Preparation of recombinant dioscorin

118

The rDioscorin (pI 6.27) of Japanese yam was isolated from the cultures of E.

119

coli BL21 (pDj-dioA3) and purified according to our previous study Jheng et al.6

120

2.2 Preparation of TMC

121

TMC was obtained from chitosan according to the methodology published by

122

Sieval et al. with modification (Supplementary material).21

123

2.3 Determination of the degree of quaternization of TMC

124

We used nuclear magnetic resonance (NMR) to determine the degree of

125

quaternization of TMC according to the method described by Sieval et al.21 Briefly,

126

10 µg each of chitosan and TMC were lyophilized and subsequently dissolved in 500

127

µL D2O (Sigma, MO, USA). The 1H-NMR spectrum of TMC was measured using a

128

500 MHz spectrometer (Bruker-Spectro Spin 400 UltraShieldTM, Allemagne,

129

Germany). The degree of quaternization was calculated from the following equation:

130

Degree of quaternization = [(∫TM/∫H) x 1/9] x 100%

131

∫TM: is the integral of chemical shift of the hydrogens of -N+(CH3)3 peak at 3.3 ppm.

132

∫H is the integral of H1 peaks between 4.8 and 6.3 ppm, related to hydrogen atoms

133

bound to carbon 1 of the chitosan molecule, which is taken as the reference signal.

134

Fourier Transform Infrared Spectrometer (FTIR) analysis was performed by

135

using potassium bromide (KBr) pellet method.37 Ten micrograms each of chitosan and

136

TMC were lyophilized. One gram of KBr powder (Thermo, Waltham, USA) was

137

heated in oven at 100 oC for 24 h and used as a blank. The TMC samples were mixed


Page 8 of 43

Page 9 of 43


138

with KBr in a mass ratio of 1:99, compressed into a thin piece, and scanned for

139

percent transmittance in the range of 500–4000 cm-1 using FTIR spectrophotometer

140

(Alpha FT-IR Spectrometer, Bruker, USA).

141

2.4 Preparation of TMC-rDio-TPP NPs and calculation of loading capacity and

142

entrapment efficiency

143

TMC-rDio-TPP NPs were prepared by ionic gelation process according to the

144

methods described by Gan and Wang.38 Fifty micrograms of TMC were dissolved in

145

50 mL of ddH2O (1 mg/mL, pH 6.5). A solution of TPP at the concentration of 0.6

146

mg/mL was prepared with ddH2O (pH 6.5). Various amounts of rDioscorin were

147

separately mixed with 0.2 mL TPP (0.6 mg/mL) at mass ratio ranged from 0.2:1 to 2:1

148

for rDio:TPP, and the rDio-TPP mixtures were stored at 4 oC. The rDio-TPP mixtures

149

were separately added dropwise to the 0.5 mL TMC solution (1 mg/mL) to form

150

various ratios ranged from 4:0.2:1 to 4:2:1 (TMC:rDio:TPP). An opalescent

151

suspension was formed spontaneously under the aforementioned conditions. The

152

TMC-rDio-TPP NPs were separated by centrifugation at 20000 xg at 15 oC for 30 min.

153

The supernatant was collected, and the amount of unincorporated rDioscorin was

154

determined according to the absorbance at OD280. The pellet of TMC-rDio-TPP NPs

155

was collected followed by freeze drying and stored at 4 oC for further studies. Both

156

the loading capacity and entrapment efficiency were determined by indirect method:

157

loading capacity (%) = (protein in the NPs/weight of the NPs) x 100%。

158

entrapment efficiency (%) = (protein in the NPs/the feeding protein) x 100%。

159

2.5 Determination of particle size, zeta potential, and polydispersity index

160

The TMC-TPP NPs or TMC-rDio-TPP NPs were diluted to 0.1 mg/mL with

161

ddH2O and adjusted to pH 6.3–6.4 by 1.0 mM HCl. The particle diameter, zeta

162

potential, and polydispersity index (PDI) of NPs were determined by Laser diffraction

163

submicron particle size analyzer (Zetasizer 3000HS, Malvern Instruments, UK)



164

through the 90° scattering angle (Supplementary Figure). The samples were filled into

165

a plastic cuvette with an electrode for sample analysis. The zeta potential was

166

determined by the electrophoretic mobility of water-dispersed samples in the automatic

167

mode. The measured conditions were: laser wavelength at 532 nm, temperature at 25

168

°C, six consecutive measurements for the number of scan. Size distribution was

169

measured by intensity analysis. The samples were detected in triplicate, and a total of

170

three independent experiments were performed.

171

2.6 Examination of the nanoparticles by scanning electron microscopy and

172

atomic force microscope

173

The morphology of the TMC-rDio-TPP NPs was assessed by scanning electron

174

microscopy (SEM) and atomic force microscope (AFM). Accordingly, the

175

nanoparticles were prepared in solution as described above at pH 6.5 at 37 oC for 1 h.

176

The TMC-rDio-TPP NPs were washed with ddH2O (pH 6.5) for 4 times to eliminate

177

the ions and then lyophilized. The lyophilized nanoparticles were coated with a gold

178

layer under vacuum followed by SEM (S-3400N, HITACHI, Japan) and AFM

179

(Caliber-Peak Force Tapping, Bruker, USA) examination. The NPs size from AFM or

180

SEM analysis was obtained from the measurement of approximately 50 particles, and

181

the representative image was presented in Figure 3.

182

2.7 Evaluation of dioscorin released from nanoparticles in various pH

183

environments

184

Three hundred micrograms of TMC-rDio-TPP NPs (containing approximately 50

185

µg of rDioscorin) were incubated in 1 mL each of hydrochloride solution at pH 2.5

186

(simulating gastric fluid), 10 mM phosphate buffer saline (PBS) at pH 6.0 (simulating

187

duodenal fluid), or 10 mM PBS at pH 7.4 (simulating jejunal fluid) at 37 oC, 50 rpm,

188

and 150 µL of each sample was collected for various time intervals. The samples were

189

centrifuged at 20000 xg for 30 min at 15 oC. The release of rDioscorin in the


Page 10 of 43

Page 11 of 43


190

supernatant was determined according to the absorbance at OD280. The studies were

191

performed in triplicate, and the values were collected from three separate

192

experiments.

193

2.8 Proliferation assay of macrophage RAW264.7 in response to TMC-rDio-TPP

194

NPs

195

Detection of the proliferation of macrophage cell line RAW264.7 in response to

196

TMC-rDio-TPP NPs was performed according to our previous study.5 Briefly, the

197

macrophage RAW264.7 of 1 x 104 cells/100 µL/well were treated with 10 µL each of

198

10 mM PBS buffer (pH 7.4), lipopolysaccharide (LPS, from E. coli 0111:B4, Sigma,

199

MO, USA)(5 µg/mL), rDioscorin (50 µg/mL), TMC-TPP NPs (500 µg/mL), or

200

TMC-rDio-TPP NPs (600 µg/mL) (containing approximately 100 µg/mL rDioscorin).

201

To eliminate any possible LPS contamination in the recombinant dioscorin extracts, the

202

TMC-rDio-TPP NPs were predisposed to LPS inhibitor polymyxin B (10 µg/mL,

203

Sigma, Missouri, USA) for 20 min. After incubation at 37°C in a humidified incubator

204

containing 5% CO2 for 48 h, viability of the cells were determined by CellTiter 96

205

AQuenous Non-Radioactive Cell Proliferation Assay (Promega, WI, USA) following

206

the manufacture’s protocol. The values were calculated as relative intensity of the

207

absorbance as compared to the vehicle control with PBS. All the studies were

208

performed in triplicate, and the values were collected from three separate experiments.

209

2.9 Phagocytic effect of TMC-rDio-TPP NPs on macrophage RAW264.7

210

The effect of TMC-rDio-TPP NPs on phagocytosis of RAW264.7 was performed

211

according to our previous study.5 Briefly, the macrophage RAW264.7 (1 x 105

212

cells/100 µL/well) were cultured with 100 µL LPS (5 µg/mL), rDioscorin (50 µg/mL),

213

TMC-TPP NPs (500 µg/mL), TMC-rDio-TPP NPs (600 µg/mL), or 10 mM PBS

214

buffer (pH 7.4) as a control. The rDioscorin and TMC-rDio-TPP NPs were

215

predisposed to LPS inhibitor polymyxin B (10 µg/mL, Sigma, Missouri, USA) for 20



216

min to eliminate any possible LPS contamination in the recombinant dioscorin extracts.

217

After incubation at 37°C, 5% CO2 for 4 h, 100 µL of fluorescein isothiocyanate

218

(FITC)-labeled E. coli DH5α (1 x107 cfu/well) was added to each well to obtain an

219

effector : target ratio of 100:1 for phagocytosis analyses. The fluorescence intensity of

220

FITC was determined by using a microplate fluorometer (Fluoroskan Ascent FL,

221

Lsbsystems, Finland) with an excitation wavelength at 480 nm and emission

222

wavelength at 517 nm. The phagocytic effect of dioscorins on RAW264.7 is defined as

223

relative intensity of FITC fluorescence as compared to the control. Each treatment was

224

performed in triplicate, and the values were collected from three separate experiments.

225

2.10 Stimulation of cytokine gene expression by TMC-rDio-TPP NPs

226

The effect of TMC-rDio-TPP NPs on stimulating cytokine gene expression was

227

performed according to our previous study.7 Briefly, the macrophage RAW264.7 of 1

228

x 106 cells/500 µL/well were treated with 2 µL of 10 mM PBS buffer (pH 7.4), LPS (4

229

ng/mL), rDioscorin (0.2 µg/mL), TMC-TPP NPs (1.0 µg/mL), or TMC-rDio-TPP NPs

230

(1.2 µg/mL) (containing approximately 0.2 µg/mL rDioscorin) for 4 h. The rDioscorin

231

and TMC-rDio-TPP NPs were predisposed to 10 µg/mL LPS inhibitor polymyxin B

232

for 20 min to eliminate any possible LPS contamination in the recombinant dioscorin

233

extracts. Real-time quantitative PCR (RT-qPCR) amplification of the cDNA clones

234

were performed in thermocycling conditions of 95°C for 2 minutes followed by 39

235

cycles of 95°C for 15 sec, Tm°C for 1 min, and a final 1 min at 72°C on a CFX96TM

236

real-time PCR detection system (Bio-Rad, CA, USA). The amplified GAPDH gene

237

was used as an internal control. Relative gene expression levels were calculated with

238

the 2-△△Ct method. All the qPCR were performed in triplicate, and the values were

239

collected from three separate experiments.

240

2.11 Test of paracellular transport of TMC-rDio-TPP NPs


Page 12 of 43

Page 13 of 43


241

The transcellular investigation of TMC-Dio-TPP NPs through Caco-2 cell

242

monolayers were carried out by the method described by Arshad et al.39 Two hundred

243

microliters of Caco-2 cells (3x104 cells/insert) were seeded in the apical chamber

244

(PET membrane, 0.4 mm in pore size, diameter 6.5 mm, 0.33 cm2 of cell growth area)

245

of Costar Transwell 24 wells/plate (Corning Costar Corp, Cambridge, MA, USA)

246

which made the cell number to be 8.9x104 cells/cm2. In addition, 0.8 mL of medium

247

was added in the basolateral chamber. To test the confluent growth of Caco-2 cell

248

monolayer, the PBS in the apical chamber were replaced with 0.2 mL of PBS (pH 7.4)

249

containing 10 µg/mL of sodium fluorescein (Na-Flu; Sigma, MO, USA) as

250

paracellular marker with or without 0.2 mL of 1.5 mg/mL TMC-TPP NPs. One

251

hundred microliters of the solution was collected from the basolateral chamber at

252

various time points, and the removed solution was replenished with 100 µL of fresh

253

PBS. The amount of Na-Flu transported across the Caco-2 cell monolayer was

254

determined by an ELISA plate reader (Multiskan Go, Thermo, USA) with λex at 460

255

nm and λem at 515 nm. The Na-Flu of 0.01–5 µg/mL was used as a standard. The

256

results were collected from three separate experiments.

257

For transport study, the medium in the apical chamber were replaced with 0.2

258

mL of FITC-rDioscorin (300 µg/mL) or TMC-FITC-rDio-TPP NPs (1750 µg/mL)

259

which contains 300 µg/mL of FITC-rDioscorin based on the entrapment efficiency of

260

60%. At predetermined time intervals, 0.1 mL of samples were collected from the

261

basolateral chamber, and the removed solution was replenished with 0.1 mL of fresh

262

PBS. The amount of FITC-rDioscorin transported across the Caco-2 cell monolayer

263

was determined by an ELISA plate reader (Multiskan Go, Thermo, USA) with λex at

264

485 nm and λem at 535 nm. The amount of FITC-rDioscorin transported through the

265

cell monolayers was calculated according to the standard FITC-rDioscorin (0.01–0.5

266

µg/mL). The results were collected from three separate experiments. The cumulative



267

amount of transported FITC-rDioscorin were determined. The apparent permeability

268

coefficients (Papp, cm/s) was calculated according to the following equation:

269

Papp = dQ/dt x 1/A．C0

270

Where Q is the total amount of FITC-rDioscorin permeated (ng), t is the total time of

271

the experiment, A is the diffusion area of the cell monolayer (cm2), and C0 is the

272

initial concentration of the FITC-rDioscorin in the insert compartment (ng/cm3).

273

2.12 Statistical analysis

274

Statistical analysis for two groups was performed using Student's t-test.

275

Differences were considered to be statistically significant if P < 0.05 (*), P < 0.01

276

(**), or P < 0.001 (***). Comparison among multiple groups was performed by

277

one-way ANOVA followed by Tukey’s honest significant different (HSD) test using

278

Statistical Product and Service Solutions version 14.0 (SPSS, IL, USA).

279


Page 14 of 43

Page 15 of 43


280

3. RESULTS AN DISCUSSION

281

3.1 Preparation and characterization of TMC-rDio-TPP NPs

282

To improve the bioavailability of dioscorin in the intestine by oral administration,

283

avoid enzymatic (or acidic) degradation in the GI tract, and prevent poor GI

284

permeability, the TMC-rDio-TPP NPs were prepared through ionic gelation with the

285

positively charged TMC, the negatively charged TPP, and rDioscorin at pH 6.5. TPP

286

was used to cross-link and stabilize the NPs. The TMC used in this study was

287

synthesized by methylating chitosan according to the method described by Sieval et al.

288

with modification.21 The results of the NMR that was used to analyze the modification

289

of the TMC are depicted in Figure 1A. The 1H-NMR spectrum of the TMC is similar

290

to that reported by Sieval et al.21 The peak at 3.0–3.1 ppm assigned to the hydrogen

291

atoms of C2 in chitosan disappeared, while the peak at 3.35 ppm assigned to

292

-N(CH3)3+ in TMC increased substantially. In addition, the peaks at 3.6–3.9 ppm

293

assigned to the hydrogen atoms of C3-C6 in chitosan disappeared, and at 3.4–3.6 ppm,

294

new peaks appeared, which were assigned to the O-methylated site [3-OCH3] and

295

[6-OCH3] in TMC. The percentage of O-methylation on [3-OCH3] and [6-OCH3] is

296

32.99% and 37.14%, respectively. The integral for the peak at 3.3–3.4 ppm is 9.0,

297

while it is 1.5 for the peak at 4.8–6.3 ppm (hydrogen of the anomeric carbon). The

298

degree of quaternization is 67.0%, which is within the range of high degree of

299

quaternization (60%–80%).24 FTIR spectroscopy was also performed for functional

300

group identification of the TMC (Figure 1B). In the FTIR spectrum, the peak at 1580

301

cm−1 of -NH2 on chitosan decreased in TMC. By contrast, strong absorption peaks at

302

2890 cm−1 (C-H stretch) and 1489 cm−1 (-CH3 asymmetric angular deformation) for

303

TMC were detected, compared with those for chitosan, indicating that the -NH2

304

functional group of chitosan had been quaternized to -N(CH3)3+, resulting in the

305

formation of TMC.



306

To determine the optimal ratio of TMC to TPP for preparing NPs, different

307

combinations of the two were tested. The results presented in Table 1 reveal that with

308

a decreasing TMC:TPP ratio (from 6:1 to 2:1), the particle size first decreased

309

drastically from 428 nm to 235 nm and then increased to 304 nm. The TMC:TPP

310

mass ratio of 3:1 and 4:1 gave the smallest particle sizes (235–259 nm). With

311

increasing amounts of TPP in the NP preparation, the TPP anions bound to positively

312

charged TMC, resulting in an increase in the cross-linking density between TMC and

313

TPP, which may have compacted the NPs and therefore decreased the particle size.

314

However, when greater amounts of negatively charged TPP bound to TMC, it may

315

have neutralized the positive charge of TMC, decreasing the zeta potential, and

316

destabilizing the cross-linkage between TMC and TPP, resulting in the dilation of the

317

NPs. The results of this study are similar to those reported by Fan et al.,40 who studied

318

the effect of the mass ratio of chitosan to TPP on particle size and zeta potential and

319

found that with increasing mass ratio of TPP to chitosan, the particle size first

320

decreased and then increased drastically.

321

The zeta potential decreased from +24 mV to +14 mV in accordance with the

322

decreasing mass ratio of TMC to TPP (Table 1). The TMC:TPP mass ratios of 3:1 and

323

4:1 gave good zeta potential values (+19.7 mV to +23.3 mV). The polydispersity

324

index (PDI) of both ratios was less than 0.7. Since the TMC:TPP ratio of 4:1 gave the

325

smallest particle size (235 nm) and a high zeta potential (+23.3 mV), it was chosen as

326

the ratio for preparing the TMC-rDio-TPP NPs. Based on the 4:1 ratio, various

327

amounts of rDioscorin were tested for the NPs, ranging from 4:0.2:1 to 4:2:1 for the

328

TMC:rDio:TPP ratio. The particle sizes of the NPs for the various combinations were

329

between 250 nm and 284 nm (Figure 2A). The zeta potential decreased with

330

increasing amounts of rDioscorin added to the NPs (Figure 2B). The NPs with a

331

4:0.28:1 TMC:rDio:TPP ratio had the highest zeta potential (+33 mV). The more


Page 16 of 43

Page 17 of 43


332

rDioscorin that was added, the greater the quantity of proteins that was loaded into the

333

NPs (Figure 2C). The highest loading capacity was 26%, obtained from the NPs with

334

4:2:1 ratio. The entrapment efficiency ranged from 59% to 72% for the various

335

combinations, with the highest entrapment efficiency for NPs being at the 4:0.8:1

336

ratio (Figure 2D). The loading capacity and entrapment efficiency of TMC-rDio-TPP

337

NPs are in the range of those of TMC NPs encapsulated with various drugs or

338

proteins.22,24 Because the ratio of 4:2:1 gave the maximal loading capacity at 26% and

339

high entrapment efficiency at 61%, with similar particle size and zeta potential

340

compared with those of other ratios, the ratio of 4:2:1 for TMC:rDio:TPP was used for

341

preparing the NPs for the subsequent study experiments.

342

The morphology of the TMC-rDio-TPP NPs was examined by SEM and AFM

343

(Figure 3). The results of SEM and AFM reveals that the TMC-TPP NPs exhibited a

344

particle size in the range of 100–150 nm and 107–136 nm, respectively, while the

345

TMC-rDio-TPP NPs exhibited a spherical shape and a slightly larger particle size in

346

the range of 150–200 nm for SEM and of 158–235 nm for AFM. The particle sizes of

347

the TMC-rDio-TPP NPs analyzed by SEM coincided well with those determined by

348

AFM, ranging from 150 nm to 235 nm. However, the size of the TMC-rDio-TPP NPs

349

determined by a particle size analyzer was 284 + 6.6 nm, which was larger than that

350

determined by SEM and AFM. This discrepancy in the size of the TMC-rDio-TPP

351

NPs between the particle size analyzer and SEM and AFM could be due to the

352

swelling of the TMC-rDio-TPP NPs in aqueous media, which yields the

353

hydrodynamic diameter of nanoparticles, whereas SEM and AFM give the actual

354

diameter of nanoparticles in a dry state. Similar results were also observed for the

355

TMC-insulin polyelectrolyte complexes by Mao et al., QCS/AL NPs by Li et al., and

356

TMC-TPP NPs by Fan et al.40–42 Recently, Facchi et al. reported the use of

357

water/benzyl alcohol microemulsion method for preparing TMC-TPP NPs with an



358

average particle size of 98.8 nm and a zeta potential of 47.2 mV, suggesting that

359

microemulsion method provides a potential approach for synthesizing ultrafine

360

TMC-TPP NPs.36

361

3.2 In vitro release of rDioscorin from TMC-rDio-TPP NPs

362

To establish an in vitro–in vivo correlation and evaluate the in vitro release

363

profiles of rDioscorin from the TMC-rDio-TPP NPs in the GI tract, the

364

TMC-rDio-TPP NPs were incubated in buffers of pH 2.5, pH 6.0, or pH 7.4, which

365

reflect the pH environments of the stomach, duodenum, and jejunum, respectively,

366

and the amounts of rDioscorin released from the NPs were determined. The particles

367

sizes, zeta potential, and PDI of the TMC-rDio-TPP NPs in the various pH buffer

368

environments were examined. The results of Table 2 reveals that the particle sizes of

369

the TMC-rDio-TPP NPs in buffers of pH 2.5 and pH 6.0 were 237.7 nm and 231.3 nm,

370

respectively, whereas in the buffer at pH 7.4, they swelled to 457.3 nm. The zeta

371

potential of the TMC-rDio-TPP NPs at pH 2.5 and pH 6.0 were +18.5 mV and +13.1

372

mV, respectively, whereas at pH 7.4, it sharply decreased to +1.0 mV. The PDI were

373

similar among the TMC-rDio-TPP NPs in the different pH buffers.

374

The zeta potential decreased in both pH 2.5 and pH 6.0 solution, while the

375

particle size of TMC-rDio-TPP NPs in both the pH 2.5 and pH 6.0 solution was

376

similar to that of the TMC-rDio-TPP NPs prepared at pH 6.5 (Figure 2), suggesting

377

that the TMC-rDio-TPP NPs remain intact in both pH environments, although the

378

charge density over NP surface was affected in both pH solutions. By contrast, the

379

particle size of TMC-rDio-TPP NPs in the pH 7.4 solution swelled to almost twice its

380

size in both pH 2.5 and pH 6.0 solution, and the zeta potential sharply decreased to

381

+1.0 mV, indicating that the charge of the TMC-rDio-TPP NPs was neutralized at pH

382

7.4, thus leading to the dissociation of the ionic bonds among TMC, rDioscorin, and

383

TPP and subsequently, the swell of TMC-rDio-TPP NPs.43 The neutralization of


Page 18 of 43

Page 19 of 43


384

charge might be due to the following factors. First, with the increase of solution pH

385

from acidic to neutral condition, the negative charge numbers of TPP increase

386

accordingly.44 Secondly, in addition to TMC, N-monomethyl chitosan and

387

N,N-dimethyl chitosan may exist in the TMC polymers. With increasing pH value,

388

monomethylated and dimethylated amines of the TMC polymers could be

389

deprotonated.36 Thirdly, the rDioscorin, with a pI of 6.27, may become negatively

390

charged at pH 7.4.6 All these factors may contribute to the decrease of zeta potential

391

at pH 7.4 and lead to the intermolecular electrostatic repulsion, resulting in the swell

392

of the TMC-rDio-TPP NPs.

393

The results of in vitro release profiles of rDioscorin from the TMC-rDio-TPP

394

NPs reveals that in pH 2.5 buffer, 34.7% of the rDioscorin was released by 15 min,

395

and the release reached 48.6% at 8 h after treatment (Figure 4). In pH 6.0 buffer,

396

20.7% of the rDioscorin was released by 15 min, and the release gradually increased

397

to 38.7% at 8 h. By contrast, in pH 7.4 buffer, 19.0% of rDioscorin was released by 15

398

min, and the release increased to 65.0% at 8 h after treatment. A significant amount of

399

the rDioscorin (60.7%–65.0%) was released from the TMC-rDio-TPP NPs at pH 7.4

400

at 1–8 h after incubation compared with those at pH 2.5 (42.0%–48.6%) and pH 6.0

401

(30.0%–38.7%), indicating that the rDioscorin may release more efficiently from the

402

TMC-rDio-TPP NPs in jejunum.

403

Significant amount of rDioscorin released from the TMC-rDio-TPP NPs in the

404

pH environment of the jejunum demonstrated that the TMC-rDio-TPP NPs can be

405

used for studying the immunopharmacological effects of the oral administration of

406

dioscorins. However, at 15 min after treatment, 34.7% and 20.7% of the rDioscorin

407

had been released at pH 2.5 and pH 6.0, respectively (Figure 4). Because the particle

408

size did not vary substantially in these pH environments (Table 2), the burst release of

409

the rDioscorin from TMC-rDio-TPP NPs at pH 2.5 and pH 6.0 during the early period



Page 20 of 43

410

of treatment may be attributed to the diffusion of the rDioscorin that was absorbed on

411

the external surfaces of the TMC-rDio-TPP NPs rather than packaged inside the NPs

412

during NP preparation. The change in pH may have affected the electrostatic

413

interaction between the loosely bonded rDioscorin and the NPs, resulting in the quick

414

release of rDioscorin. Similar phenomena have been observed in TMC-TPP loaded

415

with bovine serum albumin or bovine hemoglobin, TMC-TPP loaded with paclitaxel,

416

a

417

methacrylamide copolymer) or TMC-cysteine encapsulated with insulin.34,45–47 In

418

these reports, an initial burst release of 20%–50% is observed in the first 15–30 min.

419

Chen et al. attempted to use alginate-modified TMC NPs to reduce burst release

420

without getting a satisfactory result.45

classical

microtubule

inhibitor,

and

TMC-HPMA

(N-(2-hydroxypropyl)

421

The amount of rDioscorin released from TMC-rDio-TPP NPs at pH 2.5

422

(42.0%–48.6%) and pH 6.0 (30.0%–38.7%) at 1–8 h after incubation remain high

423

(Figure 4). Further efforts must be taken to minimize the release of rDioscorin from

424

NPs at pH 2.5 and pH 6.0. Enteric-coated gelatin capsules or polymethacrylate-based

425

copolymers such as Eudragit® could be utilized to load such NPs to prevent the

426

release of rDioscorin from NPs in the gastric environment.48

427

3.3 Bioactivity assay of rDioscorin released from TMC-rDio-TPP NPs

428

Previous studies have demonstrated that both native and recombinant dioscorins

429

have stimulation effects on immune cell proliferation, phagocytic activity, and

430

expression of cytokine genes, such as IL-1β and IL-6.5 –7 To elucidate whether the

431

rDioscorin released from TMC-rDio-TPP NPs are bioactive and have a stimulation

432

effect on the immune cell proliferation, the macrophage cell line RAW264.7 was

433

treated with LPS, rDioscorin alone, TMC-TPP NPs, or TMC-rDio-TPP NPs for 48 h,

434

and cell proliferation was subsequently measured. The results of Figure 5 shows that

435

the rDioscorin released from TMC-rDio-TPP NPs had a similar effect on stimulation


Page 21 of 43


436

of RAW264.7 proliferation as LPS and rDioscorin alone. By contrast, TMC-TPP NPs

437

that did not contain rDioscorin did not stimulate cell proliferation. RAW264.7 cells

438

were then treated with LPS, rDioscorin alone, TMC-TPP NPs, or TMC-rDio-TPP NPs

439

for 4 h and the phagocytic activity of RAW264.7 macrophages against E. coli was

440

evaluated. The results of Figure 6 reveals that the rDioscorin released from the

441

TMC-rDio-TPP NPs, LPS, and rDioscorin alone stimulated RAW264.7 phagocytosis

442

2 to 3 times more than the control or TMC-TPP NPs. The effect of the rDioscorin

443

released from TMC-rDio-TPP NPs on stimulating cytokine gene expression was also

444

evaluated. RAW264.7 cells were treated with LPS, rDioscorin alone, TMC-TPP NPs,

445

or TMC-rDio-TPP NPs for 4 h, and the expression of IL-1β and IL-6 genes was

446

evaluated. As shown in Figure 7, similar to LPS and rDioscorin alone, the rDioscorin

447

released from the TMC-rDio-TPP NPs upregulated the expressions of the IL-1β and

448

IL-6 genes. Overall, this study demonstrated that the rDioscorin released from the

449

TMC-rDio-TPP NPs stimulated proliferation and phagocytic activity of RAW264.7

450

macrophages and activated cytokine gene expression (Figures 5, 6, 7), indicating that

451

the rDioscorin released from the TMC-rDio-TPP NPs remains bioactive.

452

The methodology used for TMC preparation might lead to the formation of

453

surface ionic pairs between TMC NPs and NaCl, resulting in shielding the NPs by

454

NaCl and preventing the interaction of TMC NPs with cell membranes.49 The surface

455

ionic pairs promote a Cl- excess on the TMC structure which may increase

456

cytotoxicity of the TMC NPs. The shielding effect can be eliminated by dialysis

457

process. Therefore, removal of surface ionic pairs by dialysis in future TMC

458

preparation may increase drug delivery performance by providing effective

459

interaction of TMC-rDio-TPP NPs with cells.

460

3.4 Paracellular transport of rDioscorin released from TMC-rDio-TPP NPs

461

across Caco-2 cells



462

Chitosan and TMC have been demonstrated to assist the transport of target

463

proteins across intestinal epithelium cells by opening the tight junctions.20 To evaluate

464

the effect of TMC-rDio-TPP NPs on the improvement of paracellular permeability of

465

released rDioscorin, transport of rDioscorin across a Caco-2 cell monolayer, a cell

466

line that has been widely used to determine drug permeability of intestinal epithelium

467

cells, was investigated.50 The monolayer integrity was evaluated by testing the

468

permeability of sodium fluorescein (Na-Flu), a paracellular marker. Caco-2 cells were

469

incubated with Na-Flu or Na-Flu plus TMC-TPP NPs for various time periods. The

470

results of Figure 8A reveals that the cumulative amount of Na-Flu transported across

471

the Caco-2 cell monolayer was 0.01 µg at 0.5 h and increased to only 0.06 µg at 3 h

472

after treatment. The apparent permeability coefficient (Papp) value for the

473

permeability of Na-Flu was only 1.63 + 0.2 × 10−6 cm/s at 3 h after incubation (Figure

474

8C). The low cumulative amount and Papp value for the permeability of Na-Flu

475

indicated that the Caco-2 cell monolayer was intact, and only a small amount of

476

Na-Flu was transported across the Caco-2 cell monolayer. However, in the presence

477

of TMC-TPP NPs, the cumulative amount of Na-Flu transported across the Caco-2

478

cell monolayer was significantly increased to 0.12 µg at 3 h after treatment, which is

479

2-fold higher than that of Na-Flu only. The Papp value also significantly increased to

480

3.37 + 0.08 × 10−6 cm/s in the presence of TMC-TPP NPs, which is more than 2-fold

481

faster than that of the Na-Flu only, suggesting that TMC-TPP caused the tight

482

junctions to open and assisted in the transport of Na-Flu across the Caco-2 cell

483

monolayer.

484

Next, the Caco-2 cells were incubated with FITC-rDioscorin (FITC-rDio) or

485

TMC-FITC-rDio-TPP NPs and the permeability of FITC-rDio across the Caco-2 cell

486

monolayer was tested. The cumulative amount of FITC-rDio transported across the

487

Caco-2 cell monolayer was 0.43 µg at 0.5 h and increased to 1.0 µg at 3 h after


Page 22 of 43

Page 23 of 43


488

treatment (Figure 8B). The Papp value for the permeability of FITC-rDio was only

489

0.97 + 0.36 × 10−6 cm/s at 3 h after incubation. By contrast, the cumulative amount of

490

FITC-rDio released from the TMC-FITC-rDio-TPP NPs and transported across the

491

Caco-2 cell monolayer was 1.86 µg at 0.5 h and increased to 5.52 µg at 3 h after

492

treatment, which was 5-fold more than that of the Caco-2 cell monolayer incubated

493

with FITC-rDio alone. The Papp value for the permeability of FITC-rDio released

494

from TMC-rDio-TPP NPs was 5.16 + 0.76 × 10−6 cm/s at 3 h after incubation, which

495

is more than 5-fold faster than that of the Caco-2 cell monolayer incubated with

496

FITC-rDio alone and is faster than the Papp values for the insulin released from the

497

TMC-cysteine-insulin NPs (4.0 × 10−6 cm/s), the TMC-insulin-HPMA NPs (3.7 ×

498

10−6 cm/s), and the TMC-insulin-PLGA NPs (4.48 × 10−6 cm/s).46,47,51

499

Overall, the results of this study indicate that TMC-TPP NPs can efficiently

500

encapsulate rDioscorin and, with the assistance of TMC, rDioscorin can be promptly

501

transported across the mucosal epithelia of the intestine and enter the bloodstream

502

through a paracellular pathway to improve the oral bioavailability. Therefore, the

503

TMC-rDio-TPP NPs can be orally administered and used for elucidating the

504

immunopharmacological effects of dioscorins in vivo.

505



506

ACKNOWLEDGEMENT

507

We are indebted to Dr. Chin-Piao Chen and Mr. Ting-Horng Cho of Department of

508

Chemistry of National Dong Hwa University for their assistance in operating NMR

509

spectrometer and Dr. Shien-Der Tzeng and Mr. I-Chih Ni of Department of Physics of

510

National Dong Hwa University in operating SEM and AFM. We thank Drs.

511

Ching-Feng Weng and Jung-Hsin Hsu of Department of Life Science of National

512

Dong Hwa University for kindly providing RAW264.7 and Caco-2 cells, and cell

513

culture facility, respectively. This project was funded by Ministry of Science and

514

Technology (MOST 103-2514-S-259 -003), Taiwan.

515


Page 24 of 43

Page 25 of 43


516

REFERENCES

517

1. FAO. FAOSTAT Agriculture data. Food and Agriculture Organization of the

518

United Nations. 2014, URL (http://faostat.fao.org)

519

2. Li, S. C. Pen-T’sao Kang Mu (Systematic Pharmacopoeia). 1596, Nanjing, China.

520

3. Harvey, P. J.; Boulter, D. Isolation and characterization of the storage protein of

521

yam tubers (Dioscorea rotundata). Phytochem. 1983, 11, 1687–1693.

522

4. Fu, S. L.; Hsu, Y. H.; Lee, P. Y.; Hou, W. C.; Hung, L. C.; Lin, C. H.; Chen, C. M.;

523

Huang, Y. J. Dioscorin isolated from Dioscorea alata activates TLR4-signaling

524

pathways and induces cytokine expression in macrophages. Biochem. Biophys.

525

Res. Commun. 2006, 339, 137–144.

526

5. Lin, P. L.; Lin, K. W.; Weng, C. F.; Lin, K. C. Yam storage protein dioscorins from

527

Dioscorea alata and Dioscorea japonica exhibit distinct immunomodulatory

528

activities in mice. J. Agric. Food Chem. 2009, 57, 4606–4613.

529

6. Jheng, Y. J.; Tsai, W. Y.; Chen, K. H.; Lin, K. W.; Chyan, C. L.; Yang, C. C.; Lin,

530

K. C. Recombinant yam storage protein dioscorins expressed in Escherichia coli

531

exhibit antioxidant and immunomodulatory activities. Protein Expr. Purif. 2012,

532

85, 77–85.

533

7. Hsu, Y. J.; Hsu, J. H.; Lin, K. C. Yam storage protein dioscorins modulate

534

cytokine gene expression in BALB/c and C57BL/6 lymphocytes. Food Agric.

535

Immunol. 2015, 26, 909–923.

536

8. Hsu, Y. J.; Weng, C. F.; Lin, K. W.; Lin, K. C. Suppression of allergic reactions in

537

ovalbumin-sensitized mice by yam storage proteins dioscorins. J. Agric. Food

538

Chem. 2013, 61, 11460–11467.

539

9. Yang, C. C.; Lin, K. C. Class A dioscorins of various yam species suppress

540

ovalbumin-induced allergic reactions. Immunopharmacol. Immunotoxicol. 2014,

541

36, 242–249.



542

10. Lin, C. L.; Lin, S. Y.; Lin, Y. H.; Hou, W. C. Effects of tuber storage protein of

543

yam (Dioscorea alata cv. Tainong No. 1) and its peptic hydrolyzates on

544

spontaneously hypertensive rats. J. Sci. Food Agric. 2006, 86, 1489–1494.

545

11. Liu, Y. W.; Liu, J. C.; Huang, C. Y.; Wang, C. K.; Shang, H. F.; Hou, W. C.

546

Effects of oral administration of yam tuber storage protein, dioscorin, to BALB/c

547

mice for 21-days on immune responses. J. Agric. Food Chem. 2009, 57,

548

9274–9279.

549

12. Han, C. H.; Lin, Y. F.; Lin, Y. S.; Lee, T. L.; Huang, W. J.; Lin, S. Y.; Hou, W. C.

550

Effects of yam tuber protein, dioscorin, on attenuating oxidative status and

551

learning dysfunction in D-galactose-induced BALB/c mice. Food Chem. Toxicol.

552

2014, 65, 356–363.

553

13. Shih, S. L.; Lin, Y. S.; Lin, S. Y.; Hou, W. C. Effects of yam dioscorin

554

interventions on improvements of the metabolic syndrome in high-fat diet-induced

555

obese rats. Bot. Stud. 2015, DOI 10.1186/s40529-015-0084-8.

556 557 558 559 560 561

14. Reagan-Shaw, S.; Nihal, M.; Ahmad, N. Dose translation from animal to human studies revisited. FASEB J. 2007, 22, 659–661 15. Goldberg, M.; Gomez-Orellana, I. Challenges for the oral delivery of macromolecules. Nat. Rev. Drug Discov. 2003, 2, 289–295. 16. Hejazi, R.; Amiji, M. Chitosan-based gastrointestinal delivery systems. J. Control. Release 2003, 89, 151–165.

562

17. Sonjae, K.; Lin, K. J.; Tseng, M. T.; Wey, S. P.; Su, F. Y.; Chuang, E. Y.; Hsu, C.

563

W.; Chen, C. T.; Sung, H. W. Effects of chitosan-nanoparticle-mediated tight

564

junction opening on the oral absorption of endotoxins. Biomaterials 2011, 32,

565

8712–8721.

566

18. Chen, M. C.; Mi, F. L.; Liao, Z. X.; Hsiao, C. W.; Sonaje, K.; Chung, M. F.; Hsu,

567

L. W.; Sung, H. W. Recent advances in chitosan-based nanoparticles for oral


Page 26 of 43

Page 27 of 43


568

delivery of macromolecules. Adv. Drug Deliv. Rev. 2013, 65, 865–879.

569

19. Wedmore, I.; McManus, J. G.; Pusateri, A. E.; Holcomb, J. B. A special report on

570

the chitosan-based hemostatic dressing: experience in current combat operations. J.

571

Trauma 2006, 60, 655–658.

572 573

20. Thanou, M.; Verhoef, J. C.; Junginger, H. E. Chitosan and its derivatives as intestinal absorption enhancers. Adv. Drug Deliv. Rev. 2001, 50, S91–S101.

574

21. Sieval, A. B.; Thanou, M.; Kotzé, A. F.; Verhoef, J. C.; Brussee, J.; Junginger, H.

575

E. Preparation and NMR characterization of highly substituted N-trimethyl

576

chitosan chloride. Carbohydr. Polym. 1998, 36, 157–165.

577 578

22. Sonia, T. A.; Sharma, C. P. Chitosan and its derivatives for drug delivery perspective. Adv. Polym. Sci. 2011, 243, 23–54.

579

23. Verheul, R. J.; Amidi, M.; van der Wal, S.; van Riet, E.; Jiskoot, W.; Hennink, W.

580

E. Synthesis, characterization and in vitro biological properties of O-methyl free

581

N,N,N-trimethylated chitosan. Biomaterials 2008, 29, 3642–3649.

582

24. Kulkarni, A. D.; Patel, H. M.; Surana, S. J.; Vanjari, Y. H.; Belgamwar, V. S.;

583

Pardeshi, C. V. N,N,N-trimethyl chitosan: an advanced polymer with myriad of

584

opportunities in nanomedicine. Carbohydr. Polym. 2017, 157, 875–902.

585

25. Benediktsdóttir, B. E.; Baldursson, Ó.; Másson, M. Challenges in evaluation of

586

chitosan and trimethylated chitosan (TMC) as mucosal permeation enhancers:

587

From synthesis to in vitro application. J. Control. Release 2014, 173, 18–31.

588

26. Zhang, J.; Zhe, X.; Jin, Y.; Shan, W.; Huang, Y. Mechanism study of cellular

589

uptake and tight junction opening mediated by goblet cell-specific trimethyl

590

chitosan nanoparticles. Mol. Pharm. 2014, 11, 1520−1532.

591

27. Chen, M. C.; Sonaje, K.; Sung, H. W. A review of the prospects for polymeric

592

nanoparticle platforms in oral insulin delivery. Biomaterials 2011, 32, 9826–9838.



593

28. FDA

CFR,

21CFR182.1810,

Sodium

Page 28 of 43

tripolyphosphate,

URL

594

(https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=182.

595

1810) (8/14/2017)

596

29. FDA

GRAS

Notices,

GRN

No.339,

Poly(γ-glutamic

acid),

URL

597

(https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=3

598

39) (9/30/2017)

599

30. Sayın, B.; Somavarapu, S.; Li, X. W.; Thanou, M.; Sesardic, D.; Alpar, H. O.;

600

Şenel, S. Mono-N-carboxymethyl chitosan (MCC) and N-trimethyl chitosan

601

(TMC) nanoparticles for non-invasive vaccine delivery. Int. J. Pharm. 2008, 363,

602

139–148.

603

31. Slütter, B.; Jiskoot, W. Dual role of CpG as immune modulator and physical

604

crosslinker in ovalbumin loaded N-trimethyl chitosan (TMC) nanoparticles for

605

nasal vaccination. J. Control. Release 2010, 148, 117–121.

606

32. Wang, S.; Jiang, T.; Ma, M.; Hu, Y.; Zhang, J. Preparation and evaluation of

607

anti-neuroexcitation peptide (ANEP) loaded N-trimethyl chitosan chloride

608

nanoparticles for brain-targeting. Int. J. Pharm. 2010, 386, 249–255.

609

33. de Britto, D.; de Moura, M. R.; Aouada, F. A.; Mattoso, A. L. H. C.; Assis, O. B.

610

G. N,N,N-trimethyl chitosan nanoparticles as a vitamin carrier system. Food

611

Hydrocolloids 2012, 27, 487–493.

612

34. Song, R. F.; Li, X. J.; Cheng, X. L.; Fu, A. R.; Wang, Y. H.; Feng, Y. J.; Xiong, Y.

613

Paclitaxel-loaded trimethyl chitosan-based polymeric nanoparticle for the

614

effective treatment of gastroenteric tumors. Oncol. Rep. 2014, 32, 481–1488.

615

35. Asasutjarit, R.; Theerachayanan, T.; Kewsuwan, P.; Veeranodha, S.; Fuongfuchat,

616

A.; Ritthidej, G. C. Development and evaluation of diclofenac sodium

617

loaded-N-trimethyl

618

PharmSciTech. 2015, 16, 1031–1024.

chitosan

nanoparticles

for


ophthalmic

use.

AAPS

Page 29 of 43


619

36. Facchi, S. P.; Scariot, D. B.; Bueno, P. V. A.; Souza, P. R.; Figueiredo, L. C.;

620

Follmann, H. D. M.; Nunes, C. S.; Monteiro, J. P.; Bonafé, E. G.; Nakamura, C. V.;

621

Muniz, E. C.; Martins, A. F. Preparation and cytotoxicity of N-modified chitosan

622

nanoparticles applied in curcumin delivery. Int. J. Biol. Macromol. 2016, 87,

623

237–245.

624

37. Bayat, A.; Sadeghi, A. M. M.; Avadi, M. R.; Amini, M.; Rafiee-Tehrani, M.;

625

Shafiee, A.; Majlesi, R.; Junginger, H. E. Synthesis of N, N-dimethyl N-ethyl

626

chitosan as a carrier for oral delivery of peptide drugs. J. Bioactive Compatible

627

Polym. 2006, 21, 433–444.

628

38. Gan, Q.; Wang, T. Chitosan nanoparticle as protein delivery carrier-systematic

629

examination of fabrication conditions for efficient loading and release. Colloids

630

Surf. B Biointerfaces 2007, 59, 24–34.

631

39. Arshad, M.; Sonja, B.; Flavia, L.; Muhammad, I.; Gintare, L.; Andreas, B. S. An

632

in-vitro exploration of permeation enhancement by novel polysulfonate thiomers.

633

Int. J. Pharm. 2015, 496, 304–313.

634

40. Fan, W.; Yan, W.; Xu, Z.; Ni, H. Formation mechanism of monodisperse, low

635

molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Surf.

636

B Biointerfaces 2012, 90, 21–27.

637

41. Mao, S.; Bakowsky, U.; Jintapattanakit, A.; Kissel, T. Self-assembled

638

polyelectrolyte nanocomplexes between chitosan derivatives and insulin. J. Pharm.

639

Sci. 2006, 95, 1035–1048.

640 641

42. Li, T.; Shi, X. W.; Su, Y. M.; Tang, Y. F. Quaternized chitosan/alginate nanoparticles for protein delivery. J. Biomed. Mater. Res. 2007, 83, 383–390.

642

43. Bhumkar, D. R.; Pokharkar, V. B. Studies on effect of pH on cross-linking of

643

chitosan with sodium tripolyphosphate: a technical note. AAPS PharmSciTech.

644

2006, 7, E1–E6.



645

44. Shu, X. Z.; Zhu, K. J. The influence of multivalent phosphate structure on the

646

properties of ionically cross-linked chitosan films for controlled drug release.

647

Euro. J. Pharm. Biopharm. 2002, 54, 235–243.

648

45. Chen, F.; Zhang, Z. R.; Huang, Y. Evaluation and modification of N-trimethyl

649

chitosan chloride nanoparticles as protein carriers. Int. J. Pharm. 2007, 336,

650

166–173.

651

46. Yin, L.; Ding, J.; He, C.; Cui, L.; Tang, C.; Yin, C. Drug permeability and

652

mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral

653

insulin delivery. Biomaterials 2009, 30, 5691–5700.

654

47. Liu, M.; Zhang, J.; Zhu, X.; Shan, W.; Li, L.; Zhong, J.; Zhang, Z.; Huang, Y.

655

Efficient mucus permeation and tight junction opening by dissociable

656

"mucus-inert" agent coated trimethyl chitosan nanoparticles for oral insulin

657

delivery. J. Control. Release 2016, 222, 67–77.

658 659

48. Thakral, S.; Thakral, N. K.; Majumdar, D. K. Eudragit: a technology evaluation. Expert Opin. Drug Deliv. 2013, 10, 131–149.

660

49. Martins, A. F.; Facchi, S. P.; Follmann, H. D. M.; Gerola, A. P.; Rubira, A. F.;

661

Muniz, E. C. Shielding effect of ‘surface ion pairs’ on physiochemical and

662

bactericidal properties of N,N,N-trimethyl chitosan salts. Carbohydr. Res. 2015,

663

402, 252–260.

664

50. Hubatsch, I.; Ragnarsson, E. G. E.; Artursson, P. Determination of drug

665

permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc.

666

2007, 2, 2111–2119.

667

51. Sheng, J.; Han, L.; Qin, J.; Ru, G.; Li, R.; Wu, L.; Cui, D.; Yang, P.; He, Y.;

668

Wang, J. N-trimethyl chitosan chloride-coated PLGA nanoparticles overcoming

669

multiple barriers to oral insulin absorption. Appl. Mater. Interfaces 2015, 7,

670

15430–15441.


Page 30 of 43

Page 31 of 43


671

Figure legends

672

Figure 1. 1H-NMR (A) and FTIR (B) spectra of chitosan (CS) and TMC.

673 674

Figure 2. Effect of mass ratio of TMC/rDio/TPP on particle size (A), Zeta potential

675

(B), loading capacity (C), and entrapment efficiency (D). The column not sharing a

676

letter in common differs significantly (P < 0.05) according to Tukey's HSD test.

677 678

Figure 3. SEM and AFM images of TMC-TPP (A) and TMC-rDio-TPP (B) NPs at pH

679

6.5. The scale bar in SEM image (left panel) is 200 nm.

680 681

Figure 4. Cumulative in vitro release profiles of rDioscorin from TMC-rDio-TPP NPs

682

at pH 2.5, 6.0, and 7.4. The column not sharing a letter in common differs

683

significantly (P < 0.05) according to Tukey's HSD test.

684 685

Figure 5. The effect of the rDioscorin released from TMC-rDio-TPP NPs on the

686

proliferation of macrophage RAW264.7. The proliferation index of each treatment

687

was calculated as compared to that in PBS treatment (control group). Asterisk (*)

688

represented a significant difference of the proliferation index between the

689

experimental groups and the control group if P < 0.05 (*) or P < 0.01 (**).

690 691

Figure 6. The effect of the rDioscorin released from TMC-rDio-TPP NPs on the

692

phagocytosis of macrophage RAW264.7. The phagocytosis index of each treatment

693

was calculated as compared to that in PBS treatment (control group). Asterisk (*)

694

represented a significant difference of the phagocytosis index between the

695

experimental groups and the control group if P < 0.05 (*) or P < 0.01 (**).

696



697

Figure 7. RT-qPCR amplification assay of the effect of the rDioscorin released from

698

TMC-rDio-TPP NPs on the stimulation of cytokine gene expression. The relative fold

699

increase of each gene was calculated as compared to that in PBS treatment (control

700

group). Asterisk (*) represented a significant difference of the relative expression

701

level of cytokine genes between the experimental groups and the control group if P

Development of Yam Dioscorin-Loaded ... - ACS Publications

Recommend Documents