Preparation and characterization of electrospun colon-specific delivery

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Agricultural and Environmental Chemistry

Preparation and characterization of electrospun colon-specific delivery system for quercetin and its anti-proliferative effect on cancer cells Peng Wen, Minhua Zong, Teng-gen Hu, Lin Li, and Hong Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02614 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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Journal of Agricultural and Food Chemistry

Preparation and characterization of electrospun colon-specific delivery system for quercetin and its anti-proliferative effect on cancer cells

Peng Wen1, Min-Hua Zong1, Teng-Gen Hu1, Lin Li2, Hong Wu1,3*

1

School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China 2

School of Chemical Engineering and Energy Technology, Dongguan University of Technology, Dongguan 523808, China

3

Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, Guangzhou 510640, China

To whom correspondence should be addressed. Tel.: +86-20-22236669; E-mail: [email protected] (H. Wu)

1

ACS Paragon Plus Environment


1

ABSTRACT

2

To improve the oral bioavailability of quercetin (Q) and achieve colon-specific

3

release, a core-sheath electrospun fiber mat containing Q-loaded chitosan nanoparticle

4

(Q-loaded EFM) was developed in this study. The nanoparticle was first fabricated

5

and its antioxidant activity was as effective as free Q. Then, the uniform Q-loaded

6

EFM was obtained using response surface methodology optimization, and its

7

core-sheath structure was characterized by confocal laser scanning microscopy. In

8

vitro release kinetics confirmed the colon targeting profile, and the release rate of Q

9

varied inversely with fiber diameter. The data of Cell Counting Kit-8 suggested

10

Q-loaded EFM inhibited the proliferation of Caco-2 cells in a dose- and

11

time-dependent manner, with an IC50 of 4.36, 2.81 and 2.01 mg/mL after 24, 48 and

12

72 h, respectively, and it was caused by arresting cell cycle on G0/G1 phase and

13

triggering apoptotic cell death. This study suggests that the Q-loaded EFM represents

14

a promising form in the oral therapy of colon disorders.

15 16 17 18

Keywords: Coaxial electrospinning, Core-sheath nanostructure, Colon-specific

19

delivery, Quercetin, Bioactivity

20 21 22 23 24 25

2


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26

INTRODUCTION

27

Recently, the utilization of bioactive compounds have been extensively

28

studied to reduce the risks of chronic diseases, including certain forms of cancer and

29

other age related diseases, as a cause of prevention instead of cure. 1-2 Quercetin (Q), a

30

natural bioflavonoid with many health promoting effects, has been applied in the

31

functional foods and pharmaceutical industries. In particular, it exhibits a crucial role

32

in the treatment of colon disorders since it was demonstrated that Q has the inhibition

33

effects on colon cancer cell lines in vitro.3 However, studies have shown that Q

34

undergoes first-pass metabolism following oral administration and its antioxidant

35

capacity decreases dramatically when exposed to the upper gastro-intestinal tract

36

(GIT),

37

Consequently, it is necessary to construct an effective delivery approach that can

38

encapsulate, preserve bioactivity and achieve colon targeting of Q.

4

these factors limit the biological activity and potential health benefits.

39

The colon-specific delivery system is able to protect the loaded compounds from

40

degradation in the upper portion of GIT and subsequently ensuring controlled release

41

in the colon.5-7 Especially, the polysaccharides’ inability of being digested by upper

42

GIT’s enzyme are considered to be the preferable and promising matrixes for

43

developing a colon-specific delivery system.8 Using natural polysaccharides, the issue

44

of toxicity is minimized in comparison with the Eudragit polymers.9 Studies have

45

been conducted to prepare the polysaccharides-based colon targeting delivery system

46

for Q.10-11 In this work, sodium alginate (SA) and chitosan (CS) were employed for

47

the preparation of colon-specific delivery system of Q, since SA is hydrophilic in

48

nature and swells in gastric environment, forming viscous colloidal or sols,

49

which retards the release of Q from the dosage form. Meanwhile, CS can’t

50

dissolve in small intestine and is susceptible to be degradation in the colonic 3



51

environment. Hence, the combination of both can be used for the formulation of

52

colon targeting delivery system for Q.

53

Efforts have been dedicated to the colon-targeted delivery system through

54

traditional methods on solid dosage forms, including multi-layered films, tablets and

55

hydrogels etc.12-13 However, these types of delivery systems may have poor site

56

specificity, complex manufacture procedure or use toxicity solvent. Electrospinning,

57

is presently booming due to the ease of implementation and cost-effectiveness of

58

process for the encapsulation of bioactive compounds.14-16 Previous studies showed

59

that electrospun fibers can aid in increasing the solubility and bioavailability of

60

the poor water soluble compounds.17-19 Moreover, traditional and modified coaxial

61

electrospinning has been investigated to prepare nanofibers in protecting the bioactive

62

compounds from the stress of environment and achieving a well-defined release

63

profile.

64

electrospinning is based on the reasonable selection of matrix, while the modified

65

coaxial electrospinning relies on the use of the unspinnable organic solvents as sheath

66

fluids, which can eliminate several problems associated with the single-fluid process.

67

22

68

developing site-specific delivery system, for example, the study of Wang et al

69

revealed the capacity of coaxial electrospinning for the colon targeting release of

70

ferulic acid.23 Herein, traditional coaxial electrospinning was used for the construction

71

of Q-loaded core-sheath structured colon-specific delivery system.

20-21

The differences between them are that the traditional coaxial

In the literatures, traditional coaxial electrospinning technique has been applied for

72

Thus, this study was to design a colon-specific delivery system for Q to

73

increase its oral bioavailability. Firstly, Q was encapsulated into chitosan

74

nanoparticle (QCNP) and its physiochemical characterization was assessed.

75

Then, co-axial electrospinning was employed to fabricate the Q-loaded 4


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electrospun fiber mat (Q-loaded EFM) using SA as shell part for retard release

77

of Q in gastric acid and the QCNP as the core part for the controlled and

78

targeted release of Q. The process parameters on the fiber morphology were

79

investigated using response surface methodology (RSM) based on scanning

80

electron microscopy (SEM). We tested the release behaviour of Q from the

81

core-sheath Q-loaded EFM in vitro and analyzed the colon-specific release

82

mechanism by establishing corresponding mathematics model. Moreover, the

83

effects of Q-loaded EFM on the prevention of colon cancer cell growth and

84

apoptosis on Caco-2 cells were examined.

85

MATERIALS AND METHODS Materials. Q,SA, sodium tripolyphosphate (TPP) and β-glucosidase (≥6 U/mg)

86 87

were

purchased

88

na);CS (160 kDa, DD was 87%) was obtained from Dacheng Biotech. Co. Ltd.

89

(Weifang, China); 1,1-diphenyl-2-picrylhydrazyl (DPPH) was provided by

90

Shanghai Yuanye Bio-Technology Co. Ltd. (Shanghai, China); polyoxyethylene

91

(PEO, 100 kDa), trypsin, pluronic F127 and pepsin were obtained from Aladdin

92

biological technology Co., Ltd. (Shanghai, China); Fetal bovine serum (FBS),

93

penicillin/streptomycin, trypsin-EDTA (0.25%) and dulbecco’s modified eagle’s

94

medium (DMEM) were provided by Gibco Life TechnologiesPaisley, UK).

95

Propidium iodide (PI), dihydrochloride fluoropure grade (DAPI), trypsin-EDTA and

96

annexin V Apoptosis Detection Kit FITC/PI were purchased from Beyotime

97

Biotechnology (Shanghai, China). Polyvinyl alcohol (PVA, Mw: 85000-124000)

98

was

99

China). CCC-HIE-2 was donated by Guangdong Academy of Agricultural Sciences

100

(Guangzhou, China). Caco-2 cell was provided by the Cell library of Chinese

obtained

from

from

Sigma-Aldrich

Tianma

fine

company

chemical

5


(Shanghai,

factory

Chi

(Guangzhou,


101

academy of sciences (Shanghai, China).

102

Preparation and characterization of QCNP. 9 mL of 4 mg/mL CS solution

103

(pH 5.3) that dissolved in 1% acetic acid was mixed with 0.2 g pluronic F127

104

(F127) and 1 mL of 3.3 µmol/mL Q solution (dissolved in ethanol). The Q-loaded

105

CS nanoparticle (QCNP) was formed by adding the 1 mg/mL TPP solution

106

(dissolved in water) into the above Q-CS solution to obtain a mass ratio of 5:1 for CS:

107

TPP. The obtained nanoparticles were stirred for 60 min before further analysis.

108

Transmission electron microscopy (TEM, JEOL, Japan) was used to observe the

109

morphology of singe QCNP. The polydispersity index (PDI), zeta potential and

110

hydrodynamic diameter of QCNP were determined by Zetasizer Nano ZS

111

(Malvern Instruments, UK). Fourier transform infrared spectroscopy (FTIR)

112

spectrophotometer (Bruker Co., Ettlingen, Germany) was used to investigate the

113

interaction between the components. Encapsulation efficiency (EE) and loading

114

content (LC) of Q in QCNP was determined according to the method of Ha et al with

115

slight modifications.

116

poorly water soluble Q. Mixtures were then ultra-centrifuged by L-100XP

117

centrifuge (Beckman Coulter Inc., USA) at 20,000 rpm (10oC, 20 min) to obtain

118

supernatant (unencapsulated Q). The HPLC anaylsis of Q amount was carried out as

119

following: the system was composed of agilent technologies 1260 pumps, viasampler,

120

and VWD detector. The analytical column used was the agilent ZORBAX C-18 (250

121

mm×4.6 mm, i.d. 5 µm) and the temperature was 25 oC. The mobile phase was set

122

at 0.5mL/min and comprised water and acetonitrile in a ratio of 60:40. The

123

wavelength of VWD detector was maintained at 374 nm. The calibration curve of Q

124

was linear (r2=0.999) within range 0–1 mg/mL (Figure S1). The EE and LC of Q were

125

determined using the following equation:

24

5 mL of QCNP was mixed with 5 mL of ethanol to solubilize

6


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126

EE% = (total Q-free Q in supernatant)/total Q

127

LC%= (total Q-free Q in supernatant)/total QCNP

128

Antioxidant activity analysis. Antioxidant activity of free Q and QCNP was

129

determined via DPPH radical scavenging assay. A certain amount of QCNP and Q

130

were immersed in 3 mL of 10–4 mol/L DPPH solution prepared in ethanol/water

131

(50:50) solution. Then, the mixed solution was kept in the dark for 30 min. After

132

that, the absorbance at 517 nm was recorded by UV–V is spectroscopy. The

133

scavenging rate by the sample was calculated as follows:

134

% of DPPH scavenging rate=100*(AB- AS)/AB

135

where AB and AS are the absorption of the blank and the sample, respectively.

136

Preparation and characterization of Q-loaded EFM. Then, the Q-loaded

137

EFM was prepared by co-axial electrospinning. The core solution was

138

composed of the above QCNP suspension and PVA (10% w/w) in a volume

139

ratio of 1:1, whilst the SA and PEO (total polymer 9%, 80:20, w/w) solution

140

that dissolved in water/pure ethanol (40:60, v/v) comprised the shell solution.

141

RSM was employed and the parameters were varied at three levels: distance 12, 15,

142

18 cm, applied voltage 14, 17, 19 kV; flow rate 0.1, 0.3, 0.5 mL/h. The electrospun

143

fiber mat without Q (EFM) under the same conditions was prepared and used as

144

control. Cover slip (14 mm) was put on the collector to collect fibers for the

145

biocompatibility investigation. Then, the electrospun fiber scaffold was

146

crosslinked by placing the fiber mat loaded cover slip in a sealed desiccator

147

containing 25% glutaraldehyde aqueous solution for 24 h. The crosslinked films

148

were then washed several times with PBS to remove the unreacted

149

glutaraldehyde. After that, the samples were put in the vacuum oven (25oC).

150

SEM (Zeiss EVO 18, Carl Zeiss Jena, Germany) was used to observe the 7



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151

morphology of nanofiber, and the core-sheath structure of Q-loaded EFM was

152

examined using confocal laser scanning microscopy (CLSM, LSM 510 META, Carl

153

Zeiss Inc. USA) by labeling SA and CS with fluorescein isothiocynate and

154

Rhodamine, respectively.25

155

In-vitro release test. The release behavior of Q from Q-loaded EFM under

156

artificial digestive fluids was carried out as the method described by Wen et al.25

157

The Q content in the medium sample was calculated by the above calibration curve of

158

Q. The release data of Q were fitted using different kinetics models to investigate the

159

release mechanism.

160

161

162

Higuchi: Weibull:

ெ౪ ୑ಮ ெ౪ ୑ಮ

=kt1/2

(1)

=1-exp (-atb)

(2)

Ritger-peppas:

ெ౪ ୑ಮ

=ktn

(3)

163

(Mt/M∞ represents the accumulated fraction of Q in time t; k and n are the

164

release rate constant and the release exponent, respectively).

165

Cell culture. The DMEM medium supplemented with 10 % FBS, penicillin

166

(50 IU/mL) and streptomycin (50 IU/mL) was used to culture cells. The pH of

167

the growth media was adjusted to physiological pH (7.4). Cells were cultured at 37 oC

168

in a 5% CO2 incubator. Fresh medium was replaced every other day to maintain

169

cell growth. At specified time, cells were washed with PBS and treated with

170

0.25% trypsin-EDTA to detach them from the culture plate. The collected cells

171

were then re-suspended in fresh medium for further analysis.

172

Biocompatibility of EFM. Biocompatibility of EFM was conducted by

173

evaluation the proliferation ability of normal mucosal cells CCC-HIE-2 on the EFM

174

through SEM. Before testing, the EFM was crosslinked using glutaraldehyde. Then, 8


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175

it was sterilized using UV light followed by soaking it in DMEM culture

176

medium for 12 h to facilitate cell attachment onto the EFM surface. Thereafter,

177

the EFM was cut into discs and placed at the bottom of a 24-well plates.

178

CCC-HIE-2 cells were then seeded on the EFM (1 × 105 cells/well). The medium was

179

replaced every other day. At each specified seeding time, fiber mat were washed

180

with PBS and fixed with 3% glutaraldehyde for 2 h, followed by dehydrating

181

through different alcohol solutions (30%, 50%, 70%, 80%, 90%, 95%, 100%),

182

and finally dried overnight. The morphology of samples was observed by SEM.

183

Anti-proliferation activity. The inhibitory effect of Q-loaded EFM on colon

184

cancer Caco-2 cells was determined via CCK-8 assay. Cells were cultured and

185

treated as the above steps and the densities were adjusted to 1×104 cells/well.

186

Meanwhile, one hundred microlitres of cell suspension was placed into each well of

187

96-well plates and incubated for 24 h. Then, various concentrations of Q-loaded EFM

188

was immersed into the continuous simulated GIT fluid (SGF for 2 h, and then

189

transferred to SIF for 4 h, afterwards, to SCF for 16 h) to obtain the Q medium that

190

released from the EFM. One hundred microlitres of Q medium in SCF was added to

191

each control well, whilst 100 µL of fresh SCF medium was as control. After

192

incubating for 24, 48, and 72 h, the IC 50 (inhibitory concentrations leading to 50%

193

reduction in cell number) value was also calculated.

194

Cell cycle and apoptosis analysis. 2 × 105 Caco-2 cells were seeded to each

195

well of 24-well plates. After incubation for 24 h, different concentrations of

196

medium containing the released Q were added. The cells were treated as the

197

above step to get the Q-loaded EFM treated cells. For cell cycle analysis, the

198

treated cells were fixed with 80% ice-cold ethanol for 2 h, and then centrifuged

199

to remove fixative followed by washing with PBS. After treatment with 0.25% 9



200

Triton X-100 in an ice bath for 5 min, cells were re-suspended in PBS

201

containing 4 µg/mL RNase A and 40 µg/mL PI. After incubation in a dark

202

room for 20 min, the cell cycle distribution was analyzed using a flow

203

cytometer (Becton Dickinson, Mountain View, CA, USA). For apoptosis

204

analysis, the detached cells were centrifuged for 5 min at 1500 rpm. Then, 0.1

205

mL binding buffer was added to re-suspend cells, and the cells were stained

206

with 5 µL of annexin V-FITC and 5 µL of PI. After reacting at room

207

temperature for 15 min in the dark, the cells were also analyzed by flow

208

cytometer immediately after staining.

209

Statistical analysis. All experiments were performed in triplicate, and each

210

data was presented as mean±standard deviation (SD). The statistics were

211

analyzed using the one-way analysis of variance (ANOVA) and Duncan’s t-test

212

was used for comparing the statistically significant differences (SPPS version

213

13.0; SPSS, Inc., Chicago, IL, USA). P ≤ 0.05 was considered significant.

214

RESULTS AND DISCUSSIONS

215

Preparation and characterization of QCNP. QCNP was prepared based on the

216

ionic gelation of CS with TPP before the fabrication of Q-loaded EFM. The

217

physicochemical properties of QCNP were determined using TEM and DLS. TEM

218

was used to observe the morphology of single QCNP. As can be seen in Figure 1a, the

219

single QCNP had a spherical structure and the size of QCNP was 31.2±2.8 nm. DLS

220

measurement showed the mean diameter of QCNP solutuion was about 188.3 nm with

221

a polydispersity index (PDI) of 0.187 (Figure 1c), and the zeta potential of QCNP was

222

33.2 mV (Figure 1d). Results from DLS and TEM indicated that the size of QCNP in

223

solution was apparently larger than dry QCNP, which was owing to the amphiphilic

224

property of polymer in aqueous solution. Additionally, determined by HPLC (Figure 10


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225

1b) and calculated using the calibration equation, the LC and EE of QCNP were 11.53%

226

and 92.2%, respectively. After 1 month storage, the diameter of QCNP was increased

227

to 218.9 nm, while the ζ-potential, LC and EE decreased to 29.8 mV, 9.67% and 83.4%

228

respectively (Figure 1e and f). This reduction may be that the Q molecules close to the

229

surface of QCNP was oxidized by the presence of some oxygen. Overall, the results

230

indicated that QCNP had relatively good physical stability during storage and were

231

able to preserve the stability of the encapsulated Q.

232

FTIR analysis was conducted to investigate the interaction between Q and CS

233

and the results were depicted in figure 2. The typical molecular peaks of free Q were

234

listed as follows: 1381 cm−1 (C–OH), 1264 cm−1 (C–O–C), 1610 cm

235

1662 cm−1 (C=O). The characteristic bands of 1,650 and 1,590 cm−1 in QCNP

236

spectrum attributed to NH-bending units of glucosamine of CS, whilst the exhibited

237

bands at 1051 cm−1 and 1080 cm−1 are related to CS’s saccharide structure. No

238

characteristic peaks of Q were observed in the spectrum of QNP, indicating that Q was

239

encapsulated into NP. This interaction probably caused by the hydrophobic

240

interactions or hydrogen bonds. Similar results of the characteristic peaks of Q after

241

encapsulation was also observed by Marthyna et al.26

−1

(C=C) and

242

Antioxidant activity. Previous studies confirmed that Q has high antioxidant

243

activity. 27 Herein, DPPH was applied to study the proton scavenging activity of the Q,

244

empty chitosan nanoparticle (CNP) and QCNP at varying concentrations. As shown in

245

Figure 3, QCNP can change the color of the test solution from yellow color to

246

blue and the ability relied on the concentration of QCNP. Therefore, it can be

247

concluded that Q was effectively encapsulated in the CNP maintaining its antioxidant

248

activity. Furthermore, the inhibition concentration of 50% of DPPH (IC50) obtained

249

in the present study to QCNP was 56.24 µg/mL (the loading content of Q is 6.48 11



250

µg/mL (21.45 µM) and IC50 of free Q was 6.27 µg/mL (20.76 µM), indicating that

251

antioxidant activity of Q was not interfered by CNP.

252

Preparation and characterization of Q-loaded EFM. During electrospinning,

253

some process parameters may influence the spinning ability and fiber morphology.

254

RSM has been applied for investigating the effects of key parameters in the

255

electrospinning process on the response variables.28,29 In this study, the effects of

256

electrospinning parameters, including applied voltage, distance, and feed rate on the

257

morphology (standard deviation of the fiber diameter(STDEV)) and average diameter

258

of the obtained nanofibers were studied using RSM to obtain the uniform and

259

bead-free fibers. The levels of the three experimental factors were summarized in

260

Table S1, and 17 experimental runs were performed (Table S2). Variance analysis of

261

the experimental response was conducted to evaluate the quadratic response surface

262

models. A factor has a significant impact on the response when P is less than 0.05.

263

Another important factor is R2. It describes the proportion of the total variability that

264

can be explained by the regression model.

265

quadratic models for average diameter and STDEV were desirable and significant

266

with an F-value of 334.21 and 2823.80, respectively. The values of lack-of-fit were

267

not significant, indicating that the developed models were valid. The ANOVA results

268

also showed that all of the independent variables have a significant impact on average

269

diameter and STDEV (P< 0.05). The R-squared values obtained were 0.9977 and

270

0.9997 for average diameter and STDEV, respectively, which means 99.77% and

271

99.97% of the variations on the response of the model could be explained. Final

272

equations in terms of actual factors are:

273

Average diamter=1775.03472 – 98.61111 * Distance – 72.05556 * Voltage

274

-601.66667 * Feed rate + 0.55556 * Distance * Voltage-16.66667 * Distance *

28

As shown in table S3 and S4, the

12


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2

275

Feed rate + 20.83333* Voltage * Feed rate + 2.80556 * Distance + 1.97222 *

276

Voltage + 1193.75000 * Feed rate

277

STDEV=228.16278 - 5.79611 * Distance - 14.87994 * Voltage - 66.00333 *

278

Feed rate + 0.017222 * Distance * Voltage + 0.22083 * Distance * Feed rate -

279

0.17083 * Voltage * Feed rate + 0.17950 * Distance + 0.43228 * Voltage +

280

111.70000 * Feed rate

2

2

2

2

2

281

In addition, design-expert software can predict of the optimum conditions for

282

response variables. Table S5 presented the optimum solution for average diameter and

283

STDEV by setting average diameter in range and the response variable STDEV as

284

minimization. The suggestion can give thinner fiber and uniform fiber morphology.

285

The optimized electrospinning conditions were: distance = 15.15 cm, voltage = 16.97

286

kV and flow rate = 0.29 mL/h. Then, confirmation runs were also performed to verify

287

the adequacy of the estimated model, and the SEM image and diameter distribution of

288

obtained fibers under optimized parameters were shown in Figure 4a and 4b. The

289

predicted and the actual experimental values of average diameter and STDEV

290

(368.889 nm and 48.33 vs 390 nm and 49.73, respectively) were compared and the

291

percentage error was calculated. Results indicated that the experimental values are

292

consisted with the predicted responses (percentage variation

Preparation and characterization of electrospun colon-specific delivery

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