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Preparation and characterization of proteinloaded electrospun fiber mat and its release kinetics Peng Wen, Yan Wen, Xiao Huang, Minhua Zong, and Hong Wu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 24, 2017

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Journal of Agricultural and Food Chemistry

1

Preparation and characterization of protein-loaded electrospun fiber

2

mat and its release kinetics

3

Peng Wen1#, Yan Wen1#, Xiao Huang1, Min-Hua Zong1, Hong Wu1,2* 1

4

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

5 2

6

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

7 8 9 10 11 12 13 14 15 16 17 18

*To whom correspondence should be addressed. Tel.: +86-20-22236669;

19

E-mail: [email protected] (H. Wu)

20

#

This authors have the same contribution and are co-first author

21 22 1

ACS Paragon Plus Environment


23

ABSTRACT

24

For the enhancement of protein’s bioavailability, a specific delivery system was

25

developed by coaxial electrospinning. Bovine serum albumin (BSA) was used as

26

protein model and the core-sheath fiber mat was fabricated using sodium alginate as

27

shell layer and the BSA-loaded chitosan nanoparticle that prepared previously as core

28

layer. By optimizing electrospinning parameters, uniform fibers with diameters ranging

29

from 200 nm to 600 nm were obtained, and transmission electron microscopy and

30

confocal laser scanning microscopy revealed its core-sheath structure. Fourier

31

transform infrared spectroscopy (FTIR) analysis demonstrated that there existed

32

molecular interaction between components, which enhanced the mat’s thermal stability

33

and mechanic property. It was found that the predominant release mechanism of BSA

34

from fiber mat was erosion, and little change was occurred in the secondary structure

35

of encapsulated BSA indicated by FTIR and circular dichroism analysis. The study

36

shows that the obtained fiber mat is a potential delivery system for protein.

37 38 39 40 41 42

KEYWORDS: coaxial electrospinning, core-sheath fiber, protein, release kinetics,

43

stability

44 2


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INTRODUCTION

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Recently, with the increased demands for improving human health through

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diet, bioactive proteins have gained tremendous attention for their health

48

benefits.1,2 However, their oral bioavailability are still very low due to the

49

significant barriers in the upper gastrointestinal tract (GIT), including

50

acid-catalyzed degradation in the stomach, proteolytic degradation by digestive

51

enzymes in the small intestine, and poor intestinal membrane permeability etc.3

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Hence, an effective delivery approach that can enhance bioavailability and

53

preserve biological activity of protein is desirable.

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Increased progress have been made over the years to improve the

55

bioavailability of protein, including the use of protease inhibitors or absorption

56

enhancers, muco-adhesive polymeric delivery systems, chemical modification of

57

macromolecules, nano/micro-particulate systems and the site-specific delivery to

58

the colon etc.4 Among them, the colon-specific delivery of protein is more

59

attractive since colon offers a near neutral pH, prolonged transit time, reduced

60

digestive enzymatic activity, abundant microflora (1011-1012 CFU/mL), and

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higher receptiveness of protein through colonic epithelia,5 which is desired not

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only for improving the oral bioavailability of protein, but also for treating colon

63

diseases. Especially, the inability of upper GIT enzymes to digest certain

64

polysaccharides (such as pectin, dextran, chitosan etc) is taken advantage of

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developing a colon-specific delivery system. The protein can be encapsulated

66

into these polysaccharides, as expected, the hydrolytic enzymes that secreted by 3



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microflora are responsible for degradation of carriers, which leads to the release

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of protein in the colon.6 Hence, efforts have been made to prepare

69

polysaccharide-based colonic delivery system for protein.6-8

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Regarding the methods for constructing delivery system, electrospinning, a

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simple and cost-effective technique, has been recognized as a promising

72

approach

73

demonstrated that electrospun fibers can improve the encapsulation efficiency,

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bioactivity and oral bioavailability of bioactive compounds.12,13 Moreover, a

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number of publications have revealed the developments in modified

76

electrospinning, such as the tri-axial electrospinning,14 the modified coaxial

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electrospinning,15 and the side-by-side electrospinning 16 techniques et al., which

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expand the capability of electrospinning in generating nano-products and could

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further

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electrospinning process by confining it into the core.17 Owing to these merits,

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electrospinning has been well established for small molecule drug delivery in the

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pharmaceutical fields,18 however, its application in food industry is less

83

investigated.19-21

to

encapsulate

protect

the

bioactive

fragile

compounds.9-11

bioactive

compounds

Previous

from

the

study

stress

has

of

84

Sodium alginate (SA) can resist gastric acid, and chitosan (CS) is insoluble in

85

small intestine and can be degraded by microbial enzymes in colon, thus, the

86

combination of both can potentially be used to prepare colon-specific delivery

87

system. Hence, in this study, bovine serum albumin (BSA) was chosen as the

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protein model and it was encapsulated into CS nanoparticle by ionic gelation. 4


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Then, the electrospun fiber mat loaded with BSA was fabricated by coaxial

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electrospinning using SA as shell layer and the BSA-loaded CS nanoparticle as

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core layer to increase the oral availability of protein. The preparation process

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was systematically studied by investigating the influence of solution property

93

and processing parameters on the nanofiber morphology through scanning

94

electron microscopy (SEM). Moreover, the electrospun solution property, and

95

the morphologies, structures, functional performance of final core-sheath

96

product, as well as the bioactivity and release profile of BSA from the obtained

97

fiber mat were characterized in details.

98

MATERIALS AND METHODS

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Materials. Sodium alginate (SA, from brown algae), β-glucosidase (≥6 U/mg) and

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sodium tripolyphosphate (TPP) were purchased from Sigma-Aldrich company

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(Shanghai, China); Chitosan (CS, 160 kDa, DD was 87%) was provided by Dacheng

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Biotech. Co. Ltd. (Weifang, China); polyoxyethylene (PEO, 100 kDa), pepsin (1:3000)

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and trypsin (1:250) were obtained from Aladdin biological technology Co., Ltd.

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(Shanghai, China); polyvinyl alcohol (PVA, Mw: 85000-124000) was purchased from

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Tianma fine chemical factory (Guangzhou, China). BSA was obtained from Shanghai

106

biological technology Co. Ltd. (Shanghai, China). Folin−ciocalteu reagent was

107

purchased from Qiyun biological technology Co., Ltd. (Guangzhou, China). Deionized

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water was used to prepare all the solutions.

109

Preparation and characterization of electrospinning solution. A series

110

concentrations of shell solution was prepared by dissolving a certain amount of SA and 5



111

PEO in water/pure ethanol (90:10, v/v) under constant stirring for 24 h. The viscosity

112

and conductivity of the above solution was measured by Brookfield viscometer (Model

113

DV-II + Pro, Brookfield Engineering Laboratories Inc., Stouchton, USA) and a

114

conductivity meter (DDS-11A, Leici Instrument Co., Shanghai, China), respectively.

115

Surface tension was determined using a pendant drop analyzer from SEO Co. Ltd

116

(model Phoenix 300, Lathes, South Korea). Rheological experiment was performed

117

with a stress-controlled rheometer (AR2000, TA Instruments Inc., New Castle, USA).

118

For core solution, BSA-loaded CS nanoparticle was firstly prepared using ionic

119

gelation with some modifications.22 9 mL of CS solution (3 mg/mL, dissolved in 1%

120

acetic acid) that adjusted for pH 5.3 previously was mixed with 1 mL of BSA solution

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(1.5 mg/mL, dissolved in 1% water). The nanoparticle was formed as the TPP solution

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(1 mg/mL in water) was added into the above BSA-CS solution in a rate of 400 µL/min)

123

to achieve a mass ratio of 4:1 for CS:TPP. Then, a mixed solution composed of the

124

above BSA-loaded CS nanoparticle suspension and PVA (6% w/w, dissolved in water)

125

in different volume ratios comprised the core solution.

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Electrospinning process. Electrospinning was conducted by applying a voltage

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varying from 14 to 20 kV with a power supply (ES50P-5W/DAM, Gamma, USA).

128

Two syringe pumps (NE-300, New Era Pump Systems Inc., USA) and a homemade

129

concentric spinneret (a 17 gauge for shell and 21 gauge for core) were adopted to give

130

the shell fluid and core fluid flow rate ranging from 0.2 mL/h to 0.6 mL/h, respectively.

131

A grounded collecting plate was used for fiber deposition. The distance between needle

132

tip and collector was varied from 14 cm to 18 cm. During the electrospinning, the 6


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temperature was 25±1oC and the relative humidity was around 35~40%. The obtained

134

fiber mat was dried in a desiccator before analysis.

135

Characterization of electrospun fiber mat. The morphology of nanofiber was

136

observed by SEM (Zeiss EVO 18, Carl Zeiss Jena, Germany), and the fiber diameter

137

distribution was calculated by analysis of around 50 fibers from the SEM image. The

138

surface morphology of the fiber mat was also characterized using AFM (Nanoscope

139

IIIa, Digital Instruments Inc., Santa Barbara, USA). The scanned area was 8 µm × 8

140

µm and the image was processed using the Nanoscope software. For the observation of

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core-sheath structure of fibers by TEM (JEM-2010, JEOL Ltd., Tokyo, Japan), a very

142

thin layer of electrospun fibers on the grid was prepared by putting the copper grid

143

onto the collector, and then the fibers were directly deposited on the grid during the

144

electrospinning process.

145

To study the distribution of each component, SA and CS was labeled with

146

fluorescein isothiocynate (SA-FITC) and Rhodamine (CS-RhB), respectively. These

147

labeled polymers were mixed with neat polymers to prepare electrospun fiber mat and

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the core-sheath structure was examined using CLSM (LSM 510 META, Carl Zeiss Inc.

149

USA). Briefly, CS-RhB was prepared by dissolving 100 mg of CS in 10 ml of

150

0.1mol/L acetic acid, and then 10 ml of methanol and 3.25 ml of 2 mg/mL RhB in

151

methanol were added into the above CS solution followed by reacting overnight.

152

SA-FITC was prepared by mixing 100 ml of 17.6 mg/mL SA with 10 mL of 1.77

153

mg/mL FITC in methanol at 4oC for 1 d. CS-RhB and SA-FITC were purified via

154

dialysis, freeze dried and stored at 4oC until use, respectively. 7



155

The interaction between the components was investigated using FTIR. All samples

156

were recorded on a Bruker Model Equinox 55 FTIR spectrophotometer (Bruker Co.,

157

Ettlingen, Germany). For electrospun fibers, ATR was used. While for the released

158

BSA, KBr disk was adopted. The FTIR was performed in the mean infrared region

159

with a wavenumber range of 4000-500 cm -1 and spectral resolution of 4 cm -1.

160

The thermal stability of different sample was evaluated by TGA. It was performed

161

from room temperature to 700oC, at a heating rate of 10oC/min and under nitrogen

162

atmosphere (Q500, TA Instruments Inc., New Castle, USA).

163

The physical form of components in the fiber was conducted using XRD (model D8

164

Advance, Bruker Co., Germany) with Cu Kα radiation (λ = 0.15406nm) accelerated at

165

voltage and current of 40 kV and 40 mA, respectively. The XRD pattern was collected

166

from 5° to 60° at an increment of 0.02° per 0.5 s.

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The mechanical property of electrospun fiber mat was determined at 25 °C using a

168

universal testing machine (INSTRON 5565, Instron Co., Canton, USA) according to

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ASTM standard method D882. Film was cut into strip (10 × 2.5 cm) and conditioned

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in desiccator with saturated NaBr solution (58% relative humidity) for 48 h prior to the

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test. The thickness of film was measured by a micrometer. The strip was mounted

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between the corrugated tensile grip of the instrument and the initial grip spacing and

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cross-head speed were set at 50 mm and 0.5 mm/s, respectively. A strain rate of 2

174

mm/min was used throughout the experiment. Three replications of each sample were

175

performed and average value of the measurement was used.

8


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BSA release profile and the diffusion coefficient. The release studies of BSA from

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fiber mat under simulated digestive fluids were performed using a dissolution rate test

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apparatus (RCZ-8B, Tianda Tianfa Co., Ltd., Tianjing, China) (100 rpm, 37°C)

179

according to the USP23 method. Simulated gastric fluid containing 10 mg/mL pepsin

180

(SGF, pH 1.2) was prepared by 0.1 mol/L HCl; simulated intestinal fluid containing 10

181

mg/mL trypsin (SIF, pH 6.8) and simulated colonic fluid containing β-glucosidase

182

(SCF, pH 7.4) were prepared by phosphate buffer solution. At predetermined time

183

interval, 1 mL of medium was sampled and replaced by an equal volume of fresh

184

medium. The BSA content in the sample was calculated using different calibration

185

curve of BSA that previously determined and standardized in different dissolution

186

medium (Figure S1).

187 188

The release profiles were fitted according to Higuchi and Ritger–Peppas model (equation 1 and 2) to describe the release mechanism.

==kt1/2

189

Higuchi:

190

Ritger-peppas:

(1)

==ktn

(2)

191

(where Mt/M∞ is the accumulated fraction of BSA release in time t;

k is the

192

release rate constant; n is the release exponent indicating release mechanism).

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Additionally, the diffusion coefficients of BSA from fiber mat under different

194

simulated digestive fluids were also calculated using the short-time and long-time

195

approximation equations (equation 3 and 4), respectively.23

196

short time:

197

long time:

= ；

= exp (−

(3)

);

(4) 9



198

(where D is the diffusion coefficient, L0 is the mat’s thickness).

199

The stability of BSA. Secondary structure characterization of BSA was performed

200

by FTIR and CD. Before the test, the lyophilized powder of BSA loaded CS

201

nanoparticle and the obtained electrospun fiber mat were immersed into incubation

202

mixture (0.1 mol/L phosphate buffer solution, pH6.8) for 48 h to obtain released BSA.

203

For FTIR measurement, the incubation supernatant containing BSA was dropped onto

204

the thin piece of KBr. The secondary structure of BSA was estimated quantitatively

205

from the amide I band (1700-1600 cm-1) by a curve-fitting program. The CD

206

measurement of BSA was recorded in the range 190–260 nm at a protein concentration

207

of 0.1 mg/mL, using a Chirascan spectropolarimeter (Applied Photophysics, UK). A

208

quartz cylindrical cell of 1 cm path length was used for the measurement. The native

209

BSA was used as control. To calculate the secondary structural contents of BSA, the

210

CD spectra were analyzed by a curve fitting programme of software CD Pro using

211

SELCON method, as described by Sreerama and Woody.24

212

Statistical analysis. Data was obtained at least in triplicate (n = 3), and was

213

expressed as mean±standard deviation (SD). Statistical analysis was performed

214

using one way analysis of variance (ANOVA). A value of P ≤ 0.05 was

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considered statistically significant.

216

RESULTS AND DISCUSSION

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Preparation of electrospun fiber mat. In order to achieve colon-specific delivery

218

of protein, core-sheath structured electrospun fiber mat was prepared by using SA as

219

the shell layer to resist gastric acid in SGF and BSA-loaded CS nanoparticle as core 10


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layer that can be degraded by microbial enzymes in SCF. Unfortunately,

221

electrospinning of SA was unsuccessful due to its rigid structure, a surfactant and

222

second material was added to improve the electrospinnability of SA.25 In this study, the

223

impacts of surfactant and PEO on the solution properties and the morphology of

224

obtained nanofibers were investigated (Table 1 and Figure 1). As shown in Figure 1, it

225

was observed that the addition of surfactant led to the morphological transmission from

226

beaded fibers to uniform fibers under the same mass ratio of SA to PEO, especially for

227

2% Pluronic F127. This was because the addition of surfactant could increase the

228

viscosity, and reduce the conductivity and surface tension of SA solution, which was in

229

favor of forming fibers (Table 1). However, the use of toxic surfactant Trition X-100

230

limited its application, thus, Pluronic F127, an FDA-approved surfactant, was selected

231

as the appropriate surfactant. It was also observed that the more PEO content, the less

232

beaded fibers. In order to obtain uniform fibers and maximize the content of SA, the

233

mass ratio of SA to PEO was chosen as 80:20.

234

Besides surfactant and mass ratio of SA to PEO, the impact of shell polymer’s

235

concentration on fiber morphology was also investigated (Figure 2). It showed that, to

236

some extent, the increase of concentration (from 7% to 9%, w/w) was favorable for the

237

formation of uniform fibers, whereas higher concentration (10%, w/w) resulted in

238

larger fiber diameter distribution. The optimized concentration of shell polymer was 9%

239

(SA: PEO=80:20). That was because PEO can interact with SA by forming

240

inter-molecular hydrogen bonding, leading to the improved chain entanglement and

241

flexibility, while higher concentration (more chain entanglement) caused less 11



242

stretching of the jet thus producing larger nanofibers. Rheological measurement was

243

further applied to study the effect of polymer concentration on the chain

244

conformation.25 As shown in Figure 3a, storage modulus (G’) and loss modulus (G’’)

245

had a tendency to approach each other at higher concentration, suggesting that the

246

chain entanglement between SA and PEO was enhanced.26 Additionally, the apparent

247

flexibility of SA chain was increased as the shear-thinning behavior of shell solution

248

enhanced considerably at higher concentration in the shear rate of 0.1–100 s−1 (Figure

249

3b). Therefore, the rheological measurements suggested that the beaded fibers

250

produced by shell solution with concentration < 9% can be due to the insufficient chain

251

entanglement.27

252

As described above, the combination of SA as shell layer and CS as core layer were

253

used to achieve the colon-specific delivery of protein. However, similar with SA, the

254

electrospinning of CS is difficult due to its high conductivity, hence, PVA was added to

255

improve the electrospinnability of CS and the composition of core solution was

256

optimized to obtain uniform fibers. As depicted in Figure 4, beads were observed in the

257

electrospun CS/PVA fibers under volume ratios of 70:30 and 40:60, while uniform

258

nanofiber was obtained at ratios of 50:50 and 40:60. In order to obtain uniform fibers

259

and maximize CS content, the volume ratio of 50:50 for CS to PVA was adopted.

260

Apart from the solution composition, the impacts of electrospinning parameters on

261

the fiber morphology, including distance, voltage and flow rate, were also investigated

262

and the results were presented in Figure S2-4. From the SEM images and the

263

calculated diameter distribution, the optimal electrospinning conditions were voltage 12


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17 kV, distance 16 cm and flow rate 0.3 mL/h, and the diameter distributions of

265

resulted core-sheath fiber mat were in the range of 200-600 nm.

266

Characterization of electrospun fiber mat. AFM was employed to characterize the

267

surface morphology of obtained electrospun fiber mat. As shown in Figure 5, the 2D

268

image revealed the fiber diameter ranging from 200 to 600 nm, which was consistent

269

with the previous SEM result. The 3D image exhibited the relatively smooth surface

270

with clear fibril structure.

271

The core-sheath structure of obtained fibers was then investigated by CLSM. As

272

shown in Figure 6, the shell fiber and core fiber exhibited green color and red color,

273

respectively, revealing the successful preparation of FITC-labeled SA and Rh

274

B-labeled CS. For the CLSM image of coaxial electrospun fibers, it emitted both green

275

and red fluorescence, which is an indication of the presence of FITC-labeled SA and

276

Rh B-labeled CS in the coaxial electrospun fibers. TEM was further conducted to

277

provide direct evidence that CS labeled with RhB was indeed encapsulated within the

278

shell material of SA labeled with FITC. As expected, a core-sheath structure fiber was

279

successfully constructed (Figure 6d), and the fibrous morphology without beads was

280

also consistent with SEM observation. It can be seen that both TEM and CLSM

281

revealed the core-sheath structure of obtained fibers.

282

FTIR was further carried out to investigate the peak shifts that could be attributed to

283

the interaction between components, such as hydrogen bonding (Figure 7). The

284

characteristic band of PEO at 1343 cm-1, 1122 cm-1 and 843 cm-1 was due to the -CH2

285

bond stretching, C–O–C asymmetric stretching and bending vibrations, respectively. 13



286

However, for SA/PEO electrospun mat, the band of hydroxyl stretching (3342 cm-1)

287

became much broader and the absorption band at 1122 cm-1 for original PEO shifted to

288

a lower wavenumber (1116 cm-1). This phenomenon indicated that the interaction

289

between SA and PEO was through hydrogen bonding, which was formed by hydroxyl

290

groups of SA and ether oxygen groups of PEO. Regarding the CS/PVA system, the

291

band at 3350 cm-1 (the OH stretching vibrations) of PVA had shifted toward lower

292

value (3338 cm-1) in CS/PVA electrospun fiber mat, suggesting the formation of

293

hydrogen bond between PVA and CS chains. In addition, the wavenumber of 1560

294

cm-1 observed in CS was owing to the N-H vibration, and it was shifted toward a lower

295

value (1543 cm-1) with the addition of PVA. This was another indication of hydrogen

296

bond between –OH of PVA and –NH of CS chains. Hence, the FTIR spectra of

297

SA/PEO and CS/PVA fiber mats demonstrated that there exist interactions between

298

different components.

299

XRD analyses were performed to monitor a possible change of crystallinity of

300

obtained electrospun fiber mat and assess the compatibility of different components.

301

As shown in Figure 8, the XRD patterns of SA showed broad peak, indicating its

302

amorphous nature, while the two sharp characteristic peaks of PEO powder at 18.9o

303

and 23.2o revealed its crystalline form. PVA had a strong peak at 19.6o with a small

304

shoulder at 22.4o and CS possessed two major peaks at 10.9o and 20.4o, respectively. It

305

was noted that after electrospinning, the PVA and PEO elctrospun fiber mats exhibited

306

a considerable reduction of crystallinity compared to the pure PEO and PVA powder,

307

respectively, indicating that crystalline structure can be influenced by electrospinning. 14


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The depression of crystallization in electrospun fiber mat is commonly explained by

309

the

310

electrospinning that limited the development of crystallinity, similar result was also

311

observed by others.28 For SA/PEO electrospun fiber mat, besides the effect of

312

electrospinning process, the reduction of crystallinity was also resulted from the

313

diluting effect of amorphous SA. Additionally, for crystalline substances, the XRD

314

pattern is generally expressed as simply mixed patterns of different components if

315

there is no interaction between molecules or they have low compatibility as described

316

by other literatures.29,30 For the electrospun CS/PVA fiber mat, it exhibited a

317

considerable reduction of crystallinity as a decrease of peak intensity approximately at

318

10° and 20° was observed, which was probably caused by the reduction of

319

intramolecular hydrogen bonding of PVA and the formation of intermolecular

320

hydrogen bonding between PVA and CS.31,32 Nevertheless, the obtained core-sheath

321

fiber mat was still in crystalline nature. Since it had been reported that the folding of

322

polymer chains during crystallization can be interrupted by another polymer chains

323

when the two types of chains are miscible and interact closely,33 it can be inferred that

324

good compatibility was existed among different components.

polymer

dissolution

and

subsequently

rapid

solidification

process

of

325

Thermal Property. TGA was used to evaluate the thermal stability of polymeric

326

materials.34 The weight loss and its derivate curve were presented in Figure 9. As

327

decipted in Figure 9, SA/PEO electrospun fiber mat degraded in three steps and it was

328

evidenced by the distinct reaction peaks at around 63 oC, 240 oC and 408 oC in the

329

DTG curves.They were attributed to the evaporation of absorbed water, and the 15



330

thermal degradation of SA and PEO, respectively. Additionally, the thermal

331

degradation temperature of the fiber mat was slightly increased with increasing of PEO

332

content, as shown in Figure 9c and 9d. This may be correspond to the formation of

333

hydrogen bonding from the ether oxygen of PEO and the hydroxyl groups of SA.

334

Simialar results were reported by others.35 The thermal degradation of CS/PVA

335

electrospun mat was also presented in three stages, the second weight loss step at

336

200-350 oC was due to the thermal degradation of PVA and chitosan, and the third

337

weight loss step at 400-500 oC was owing to the degradation of the by-products

338

generated by PVA.36,37 In the range between 200 and 350 oC, CS showed more thermal

339

stability (lower weight loss) than PVA. Consequently, as the CS ratio increased in the

340

blend, the residual mass increased from 9% to 11% (Figure 9e). Similar results were

341

reported by Bonilla et al 38. Further, it was found that the addition of CS resulted in an

342

increase of decomposition temperature, indicating the interaction between PVA and CS

343

(Figure 9f). Similar results were observed by Lewandowska 36 and Peesan et al.39

344

Tensile property. Since film may be subjected to various kinds of stress being used,

345

the study of mechanical property is of primary importance for the performance of

346

materials.40 In this study, a stress-strain behaviour of electrospun fiber mat was

347

investigated by tensile test, and the corresponding data of tensile strength (TS),

348

Young's modulus (Y) and elongation at break (E) were calculated and shown in Figure

349

10. It can be seen that the obtained core-sheath electrosun fiber mat had a TS of 6.65

350

MPa, Y of 90.51 MPa and E of 132.3 %. TS accounts for the film mechanical

351

resistance due to the cohesion between the chains, Y indicates the stiff degree of film, 16


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352

whereas E is a measure of toughness and stretch ability prior to breakage.41 For the

353

SA/PEO fiber mat, it was noted that the presence of PEO increased the TS and Y of SA

354

films, and as the ratio of PEO increased from 10% to 30%, the values of TS and Y

355

were enhanced from 2.97 MPa and 71.07 MPa to 4.45 MPa and 90.26 MPa,

356

respectively. Such a large increase in TS and Y could be attributed to the good

357

compatibility and also the formation of inter-molecular hydrogen bonds between the

358

hydroxyl groups of SA and oxygen atoms of PEO, which consequently restricts the

359

motion of the matrix and promotes rigidity. This observation was also supported by in

360

the FTIR and TG analysis. Similar phenomenon had been reported by Çaykara et al.35

361

Generally, an increase in TS is usually coupled with a decrease in E, however, no

362

significant decrease (P>0.5) on E was observed with the incorporation of PEO to SA in

363

spite of the increasing TS values in this study. This may be due to plasticizing effect of

364

the water absorbed and the plasticizer of F127 used.42 Regarding the CS/PVA system,

365

with the ratio of PVA increasing from 40% to 60%, TS and Y decreased from 8.37MPa

366

and 104.83 MPa to 7.28 MPa and 87.95 MPa, while E increased from 137.2 % to

367

171.5%, respectively. The reason was likely that CS had a high molecular weight and

368

hard backbones compared with PVA, similar results were also confirmed by Zhang et

369

al.43 Conversely, adding PVA into CS could greatly modify film’s flexibility and thus

370

leading to the increased E values, similar behavior was reported in other literature.44 It

371

had been reported that the mechanical property of composites can be affected by the

372

degree of crystallinity of the matrix,45 in this study, the good performance of tensile

373

strength, elongation, and modulus of obtained electrospun fiber mat can be associated 17



374

with its crystalline nature that demonstrated in XRD section.

375

Release profile and diffusion coefficient. In vitro release study was

376

performed to investigate the release profile of BSA from electrospun fiber mat

377

by mimicking mouth to colon transit. The results showed that a small percentage

378

of BSA (0.99), and the release exponent n was 1.043,

392

indicating the release mechanism was Super Case II transport, in which the

393

erosion of polymer matrix was dominant.18 This phenomenon was owing to the

394

gradual degradation of chitosan by β-glucosidase in SCF. Hence, it can be

18


Page 18 of 46

Page 19 of 46


395

concluded that the release of BSA from the fiber mat in SCF is a complex

396

process, in which the erosion was dominant.

397

Furthermore, the diffusion coefficients (D) of BSA from electrospun fiber mat in SGF,

398

SIF and SCF were also calculated. As described before, the release experimental data

399

can be divided into two regions: (1) the region of “short time” is valid for the first 60%

400

of the total released substance, and (2) the region of “long time” is corresponding to the

401

late state of release.23 Figure 11 showed the fitting curves of release data according to

402

equation 3 and 4, and the corresponding D values. It was noticed that the electrospun

403

fiber mat exhibited a lower D values in SGF and SIF. This can be attributed to the

404

alginate’s insolubility in SGF and the core component of chitosan that retarded BSA

405

release in SIF, hence, less amount of BSA would diffuse from the matrix. In SCF, larger

406

porosity was produced due to the degradation of chitosan by β-glucosidase, as respected,

407

a higher D was obtained. These data demonstrated that the degradation of matrix led to

408

the release of BSA, which was in accordance with the above research results that the

409

erosion was the dominant release mechanism.

410

Stability of BSA. Protein conformation plays an important role in determining the

411

bioactivity of protein. In this study, FTIR, a widely used technique for determining

412

secondary structure of protein, was applied to investigate the conformational changes

413

of BSA during the preparation of electrospun fiber mat. Common features of protein’s

414

FTIR spectra are the amide bands, among which, the amide I band (1700–1600 cm−1)

415

is the most useful one in the analysis of protein secondary structure.46 Generally, the

416

peaks between 1660 and 1650 cm−1 are considered to be characteristics of α-helical 19



417

structure; peaks range in 1640-1610 cm−1 represents β-sheet; the peaks at 1660-1700

418

cm−1 and 1640−1650 cm−1 are assigned to β-turns and random coil conformation,

419

respectively.46 By adding the peak areas assigned to particular secondary structure

420

elements, it was found that the native BSA contained 42.5% α-helix, 11.3% β-sheet,

421

21.4% β-turn and 24.8% random coil structures, suggesting that the secondary

422

structures of BSA were dominated by α-helix conformation

423

to the native BSA, the α-helix content of BSA that encapsulated in CS nanoparticle and

424

core-sheath electrospun fiber mat was decreased to be 38.6% and 36.8%, respectively,

425

while the β-sheets, turns, and random coil structures were all increased. It meant that a

426

part of α-helix was transformed into other second structures, which may be related to

427

the formation of intermolecular hydrogen bonding during the nanoparticle preparation.

428

Still, the encapsulated BSA was still regarded as native form according to the previous

429

definition.48 A similar reduction in the α-helix content of the released BSA from

430

nanoparticle was also observed by Kunda et al and Vakilian et al.49,50 Interestingly,

431

there was no significant difference in the α-helix content of BSA encapsulated in the

432

nanoparticle and fiber mat (P > 0.05), which was due to the protection of the

433

core-sheath structure of coaxial electrospinning. Yang et al also reported that the

434

core-sheath structured electrospun fibers protected the structural integrity of

435

encapsulated protein.51

47

(Figure 12). Compared

436

Besides FTIR, far-UV CD was also performed on the measurement of secondary

437

structure of BSA. As listed in Table 3, the control BSA contained 52.4% of α-helix, 8.7%

438

of β-strand, 15.9% of β-turn and 23.0% of random coil. The more content of α-helix 20


Page 20 of 46

Page 21 of 46


439

calculated from CD spectrum was owing to its sensitivity to α-helix. In line with the

440

aforementioned FTIR results, no significant difference in the α-helix content of BSA

441

encapsulated in the nanoparticle (47.6%) and fiber mat (46.1%) (P > 0.05). In

442

summary, FTIR and CD analysis clearly indicated that the encapsulated BSA in the

443

nanoparticle and electrospun fiber mat still kept their structural integrity, which was

444

favorable for the maintenance of protein bioactivity. The above results suggest that the

445

obtained electrospun fiber mat is a potential colon-specific controlled release delivery

446

system for bioactive protein and thus promising in functional foods.

447

ABBREVIATIONS USED

448

GIT, gastrointestinal tract; SA, sodium alginate; CS, chitosan; BSA, bovine serum

449

albumin; PEO, polyoxyethylene; PVA, polyvinyl alcohol; TPP, sodium

450

tripolyphosphate; SEM, scanning electron microscope; TEM,

451

transmission electron microscopy; AFM, atomic force microscope; CLSM, confocal

452

laser scanning microscopy; FTIR, fourier transform infrared spectroscopy; XRD,

453

X-Ray diffraction; TGA, thermogravimetric analysis; CD, circular dichroism; SGF,

454

simulated gastric fluid; SIF, simulated intestinal fluid; SCF, simulated colonic fluid.

455

ACKNOWLEDGEMENT

456

We acknowledge the National Natural Science Foundation of China (No.

457

31671852), the Natural Science Foundation of Guangdong Province (No.

458

2015A030313217), and the National Natural Science Foundation of China

459

(NSFC)-Guangdong Joint Foundation Key Project (No. U1501214) for financial

460

support. 21



461

REFERENCES

462

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614 28


Page 28 of 46

Page 29 of 46


615

Figure captions

616

Figure 1. Effect of surfactant and mass ratio of SA to PEO on the fiber mat’s

617

morphology (other conditions: total polymer 8%, feed rate 0.25 mL/h, distance 15 cm,

618

voltage 18 kV)

619 620

Figure 2. Effect of the concentration of shell polymer on the fiber mat’s morphology

621

(other conditions: SA: PEO=80:20, feed rate 0.25 mL/h, distance 15 cm, voltage 18

622

kv)

623 624

Figure 3. Rheological properties of different shell solution. a) dynamical modulus

625

change and b) viscosity variation of shell solution with different concentration .

626 627

Figure 4. Effect of volume ratio of CS to PVA on the fiber mat’s morphology (other

628

conditions: feed rate 0.25 mL/h, distance 15 cm, voltage 18 kv)

629 630

Figure 5. AFM images of electrospun fiber mat (a) 2D, (b) 3D.

631 632

Figure 6. CLSM images of (a) shell layer labeled with FITC; (b) core layer labeled

633

with Rh B; (c) the merge of core-sheath nanofiber and TEM image (d) of electrospun

634

fiber mat.

635 636

Figure 7. FTIR spectra of different sample. 29



Page 30 of 46

637 638

Figure 8. X-ray diffraction patterns of different sample.

639 640

Figure 9. TG (a, c, e) and DTG (b, d, f) curves of different samples.

641 642

Figure 10. Tensile strength (a), Young’s modulus (b) and elongation at break (c)

643

curves of different sample (P1S9-PEO/SA (10:90); P2S8-PEO/SA (20:80);

644

P3S7-PEO/SA

645

P6C4-PVA/CS (60:40); CN-coaxial electrospun fiber mat).

(30:70);

P4C6-PVA/CS

(40:60);

P5C5-PVA/CS

(50:50);

646 647

Figure 11. The fitted curves and diffusion coefficients of BSA in different medium.

648 649

Figure 12. Secondary structure contents of different sample from deconvoluted

650

FTIR spectra. (1-native BSA, 2-BSA released from nanoparticle; 3-2-BSA released

651

from electrospun fiber mat).

652 653 654 655 656 657 658 30


Page 31 of 46


Table 1 Physical properties of electrospun solutions

659 Solution

SA

PEO

Surfactant and solvent Triton

F127

DMSO

Conductivity (mS/cm)

Viscosity (cP)

Surface tension (mN/m)

SA:PEO =90:10

8.1% 8.1% 8.1% 8.1%

0.9% 0.9% 0.9% 0.9%

0 1% 0 0

0 0 2% 2%

0 0 0 5%

11.34±0.22 a 11.12±0.077 a 11.02±0.23 a 9.22±0.040 b

2075.0±35.4 b 2079.0±29.7 b 2234.0±36.7 a 2352.0±128.6 a

52.688±0.164 a 33.224±0.0544 c 39.474±0.132 b 39.958±0.474 b

SA:PEO =80:20

7.2% 7.2% 7.2% 7.2%

1.8% 1.8% 1.8% 1.8%

0 1% 0 0

0 0 2% 2%

0 0 0 5%

10.04±0.18 a 9.89±0.11 a 9.45±0.10 b 8.01±0.060 c

2976.0±199.4 a 2996.5±45.8 a 3018.0±66.5 a 3140.0±63.6 a

49.886±0.515 a 33.501±0.159 c 39.351±0.255 b 39.278±0.295 b

SA:PEO =70:30

6.3% 6.3% 6.3% 6.3%

2.7% 2.7% 2.7% 2.7%

0 1% 0 0

0 0 2% 2%

0 0 0 5%

9.06±0.077 a 8.79±0.041 b 8.49±0.078 c 8.01±0.060 d

3656.5±284.9 b 3839.0±79.2 b 4700.0±141.4 a 3140.0±63.6 b

40.370±0.123 a 33.149±0.103 c 38.891±0.364 b 39.278±0.295 a

660 661 662 663

Note: different letters in the individual column of same mass ratio of SA to PEO indicate the significant difference (P

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