Engineering biocompatible hydrogels from bicomponent natural

Subscriber access provided by the University of Exeter

Article

Engineering biocompatible hydrogels from bicomponent natural nanofibers for anti-cancer drug delivery Junfei Xu, Shan Liu, Guangxue Chen, Ting Chen, Tao Song, Jing Wu, Congcan Shi, Minghui He, and Junfei Tian J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04210 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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 36

Journal of Agricultural and Food Chemistry

Engineering biocompatible hydrogels from bicomponent natural nanofibers for anti-cancer drug delivery Junfei Xu,† Shan Liu,‡ Guangxue Chen,† Ting Chen,† Tao Song,† Jing Wu,† Congcan Shi,† Minghui He,† Junfei Tian †,*

†

State Key Laboratory of Pulp and Paper Engineering, School of Light Industry and

Engineering, South China University of Technology, Guangzhou, 510640, P. R. China. ‡

School of Medicine, South China University of Technology, Guangzhou, 510006, P. R.

China.

*Corresponding author: Prof. Dr. Junfei Tian Fax: +86-020-87112841 Tel: +86-020-87112841 E-mail address: [email protected]. Address: Wushan Road 381, Tianhe, Guangzhou, P. R. China 510006

1 Environment ACS Paragon Plus


1

Abstract

2

Natural hydrogels have attracted extensive research interests and recently showed great

3

potential for many biomedical applications. In this study, a series of biocompatible

4

hydrogels were reported based on the self-assembly of positively-charged partially

5

deacetylated α-chitin nanofibers (α-DECHN) and negatively-charged TEMPO-oxidized

6

cellulose nanofibers (TOCNF) for anti-cancer drug delivery. The formation mechanisms of

7

the α-DECHN/TOCNF hydrogels with different mixing proportions were studied and their

8

morphological, mechanical and swelling properties were comprehensively investigated.

9

Additionally, the drug delivery performance of the hydrogels was compared via sustained

10

release test of an anti-cancer drug (5-fluorouracil). The results showed that the hydrogel

11

with higher physical cross-linking degree exhibited a higher drug loading efficiency and

12

drug release percentage.

13

Keywords

14

Self-assembly; Biomaterial; Hydrogel; Drug delivery

15 16 17 18 19 20


Page 2 of 36

Page 3 of 36

21


INTRODUCTION

22

Hydrogel is a kind of polymer with three-dimensional structure which is able to

23

absorb and maintain plenty of water without being dissolved1. Hydrogel products, including

24

synthetic polymers and natural polymers, have been widely utilized in tissue engineering,

25

biosensors and drug delivery, etc2-6. Many technical methods have been proposed to

26

fabricate hydrogels such as gas foaming7, phase separation8, 9, freeze drying10 and

27

cryogelation11. Based on these methods, continuous macroporous structure could be formed

28

during crosslinking and compose the unique network in a hydrogel11. Even though there

29

have been many advances regarding to hydrogels in recent years, new techniques from

30

materials to fabrication methods are still demanded for creating multifunctional products

31

with desirable features for drug delivery in some specific conditions.

32

Cellulose and chitin have been considered as the most and the second most abundant

33

natural polymers on the earth, respectively12, 13. The natural feature of the two polymers

34

enable them to be tailored to meet the emerging needs of biocompatible products. Among

35

them, cellulose and its derivatives are favorable materials for fabricating hydrogels14-25,

36

which are significant in a broad range of biomedical applications including wound healing,

37

tissue engineering scaffold, drug delivery, artificial skin, artificial neural, etc26.

38

In

39

(2,2,6,6-tetramethylpiperidine-1-oxyl) oxidation approach have attracted much attention

40

from scientists12, 27. The obtained nanofibers still kept original crystallinity of cellulose, and

recent

years,

cellulose

nanofibers


based

on

TEMPO


41

could be evenly dispersed in water because of electrostatic repulsive-force and osmotic

42

effect28.

43

As the other common biopolymer, chitin is also a desirable material for biomedical

44

utilizations due to its excellent biocompatibility and degradability29, 30. Normally, chitin

45

exists in many animals such as crab and shrimp, and can be can be classified as α- and

46

β-chitin allomorphs from different origins. Commonly, α-chitin existing crab or shrimp

47

shells and β-chitin existing squid pens or some special seaweeds. In general, α-chitin fibrils

48

have higher length-diameter ratio compared to β-chitin fibrils, therefore, are more favorable

49

for different applications due to the high-strength property31-35. Traditionally, α-chitin fibrils

50

can be obtained from acid treatment in conjunction with mechanical extrusion. Since the

51

structures of low lateral order were broken during the process, rod-shaped nanoparticles

52

were formed, which are so-called α-chitin nanofibrils. Apart from conventional acid

53

treatment approach, ionic liquid treatment36 and TEMPO-oxidation treatment29, 37 were also

54

adopted for the preparation of α-chitin nanofibrils.

55

In recent years, cancer has been a great threat to human health in the world. It has been

56

estimated that more than 13 million people will dead from cancer in 203038. In order to

57

improve cancer treatment, a variety of anti-cancer drugs were developed including

58

5-fluorouracil, aromasin tablet and avastin. However, some shortcomings such as the side

59

effects and low solubility limit the effective application of these drugs during cancer

60

treatment. Therefore, many drug carrier materials have been introduced to reduce the


Page 4 of 36

Page 5 of 36


61

disadvantages of anti-cancer drugs, and improve the treatment effect of cancer.

62

Five-fluorouracil (5-FU) is a typical anti-cancer drug, which shows effective inhibition

63

against a series of tumors from different organs. 5-FU has the capability to disrupt

64

nucleoside metabolism and interfere with nuclear molecules, resulting in cytotoxicity and

65

death of carcinoma cells39. To improve the therapeutic efficiency, the concentration of 5-FU

66

could be maintained at a certain high level in serum. As a common technique, intravenous

67

injection has been utilized to monitor the concentration of 5-FU. It can improve the

68

effectiveness of the medical treatment, but leads to many side effects at the same time.

69

Therefore, ideal drug vehicles such as oral administrated gels are needed since they are

70

biocompatible and biodegradable, and also can minimize these side effects38, 40.

71

Many previous studies have shown promising advantages by using natural

72

material-based hydrogels for biomedical applications. The study proposed in this paper is

73

still valuable since it provides an affordable and simple hydrogel delivery system with great

74

feasibility for practical fabrication and medical application. Moreover, considering the

75

inhibition of chitin to tumor colonization41, the α-DECHN was specially designed as one

76

major component in the hydrogels in order to achieve additional treatment against cancer

77

cells. Some paper have reported the use of TOCNF or α-DECHN as one component to

78

design hydrogels18,

79

nanofibers has not been proposed. The synthesized hydrogels consist of the two kinds of

80

nanofibers, which can not only reinforce the structure of hydrogels, but also promote slow

21

. However, to our best knowledge, a hydrogel based on the two



81

release capacity to avoiding burst drug release. Atomic force microscope (AFM) and

82

scanning electron microscopy (SEM) were utilized to compare the gelation properties of a

83

series of α-DECHN/TOCNF hydrogels in different proportions, and characterized their

84

structure and morphology. Subsequently, rheological and Zeta-potential tests were

85

performed to understand the gelation dynamics and formation mechanisms of the hydrogels.

86

The swelling behaviors and the sustained release behaviors of the hydrogels were studied

87

and evaluated in phosphate buffer saline (PBS).

88 89

MATERIALS AND METHODS

90

Materials. Bleached eucalyptus pulp (Arauco) was supplied by Jinan Silver Star Paper Co.

91

Ltd (China). Dried crab shell flakes (MFCD00466914) were purchased from Shanghai

92

Macklin Biochemical Co.,Ltd (China). Hydrochloric acid (HCl), sodium hydroxide

93

(NaOH), sodium chlorite (NaClO2), sodium hypochlorite with available chlorine of 6.32%

94

(NaClO), sodiumborohydride (NaBH4), sodiumbromide (NaBr), phosphate buffer saline

95

(PBS), and 2,2,6,6-Tetramethylpiperidiniyl-1-oxyl (TEMPO) were purchased from

96

Sigma-Aldrich. Deionized water was prepared in a Barnstead Easy pureRoDi purification

97

system (Barnstead, America). 5-FU was purchased from Mei Lan Industrial (Shanghai) Co.,

98

Ltd.

99

Preparation of aqueous α-DECHN dispersions. Crab shell flakes were demineralized in

100

1 M HCl by stirring with a mechanical mixer (LBJB-300, Changzhou LIAEBO Machinery


Page 6 of 36

Page 7 of 36


101

Equipment Technology Co.,Ltd, China) at room temperature for 2 h with a rotor speed of

102

600 rpm, and then washed with deionized water until pH value became 7. After that, the

103

sample was stirred in NaOH solution (1 M) with a rotor speed of 600 rpm for 1 h at 80 oC

104

in order to remove protein impurities. After the treatment, the sample was washed with

105

sufficient deionized water until pH value near neutrality. Then, bleaching was carried out

106

with 0.3% w/v NaClO2. The whole process was repeated several times until the sample was

107

completely cleaned. Subsequently, the treated α-chitin suspension was further treated in

108

NaOH solution (33% w/w) with a rotor speed of 600 rpm at 90 oC for 3 h. During the 3 h

109

treatment, 0.05% w/v NaBH4 was added to prevent depolymerization. The α-DECHN

110

slurry was then filtered using a Buchner funnel, and completely washed with deionized

111

water. After the treatment by a high-pressure homogenizer, the α-DECHN suspension was

112

adjusted to a proper pH value (3 ~ 3.5) by adding 1 M acetic acid.

113

Preparation of aqueous TOCNF dispersions. Cellulose nanofibers were isolated from

114

bleached eucalyptus pulp following a previously reported method12, 27. Briefly, bleached

115

eucalyptus pulp was dispersed in water using mechanical stirrer with a rotor speed of

116

600 rpm until a solid content of 1.7 wt% was achieved. Then, the desired amounts of

117

TEMPO (Ratio: 0.1 mmol to 1 gram of pure pulp) and NaBr (ratio: 1 mmol to 1 gram of

118

pure pulp) were dissolved into the slurry, and the oxidation was immediately initiated by

119

adding NaClO solution (ratio: 49.63 ml to 1 gram of pure pulp). During the reaction, the pH

120

value was controlled between 10 and 10.5 by NaOH (0.1 M) or HCl (0.1 M) solution.



Page 8 of 36

121

When pH value remained unchanged, the slurry was filtered and then repeatedly washed

122

with deionized water until the pH value of the filtrate achieved 7. After mechanical

123

disintegration by successively passing through a high-pressure homogenizer for three

124

cycles at a pressure of 1000 bar, TOCNF suspensions was obtained.

125

Preparation of hydrogels. The hydrogels of α-DECHN/TOCNF with different α-DECHN

126

content were prepared by adding α-DECHN dispersions into TOCNF suspension with

127

mechanical agitation (rotor speed: 80 rpm) at room temperature for 30 min.

128

Physical cross-linking was triggered between cationic α-DECHN and anionic TOCNF,

129

which led to formation of 3D network hydrogels. For comparison, pure α-DECHN and

130

TOCNF suspensions with a concentration of 1.7 wt% were also obtained at room

131

temperature. Finally, α-DECHN/TOCNF hydrogels, α-DECHN and TOCNF solutions were

132

stored at 4 oC and equilibrated for 30 min.

133

Swelling properties. Hydrogels of α-DECHN/TOCNF were examined through swelling

134

studies in deionized water. These hydrogels were soaked into deionized water at room

135

temperature and then removed and weighed every 3 hours during a day. The water was

136

changed before the subsequent immersion. When the equilibrium swelling was reached,

137

these hydrogels were dried at 40 oC for 3 h . The swelling ratio (SR) of α-DECHN/TOCNF

138

hydrogels was evaluated using the expression42 in the below:

139

SR =

ms − md md


(1)

Page 9 of 36


140

Where ms denotes the weight of α-DECHN/TOCNF hydrogels at its maximal swollen state,

141

md is the weight of dried hydrogels.

142

Rheological measurement. The elastic modulus (G') and the viscous modulus (G")

143

measurements were performed using an AR550 rheometer (TA Instruments, USA).

144

α-DECHN/TOCNF hydrogels were shaped to a specific size with a diameter of 30 mm and

145

a thickness of 2 mm. These hydrogels were measured in a parallel plate geometry (50 mm

146

diameter) under the oscillatory mode. During the frequency sweep, the tests were carried

147

out between 0.1 and 100 rad/s. A low strain of 10% was applied to make sure that the tests

148

were conducted in the linear viscoelastic range.

149

Morphology and structure characterization. In order to explore the dimensions of the

150

α-DECHN and TOCNF, samples were prepared by natural drying 0.01% TOCNF and

151

α-DECHN dispersions at room temperature. The morphology of nanofibers were studied by

152

an atomic force microscope (AFM)

153

nanofibers dispersion with a concentration of 0.001 wt% was placed on a piece of thin mica

154

sheet, which was dried at room temperature for 3 h for the AFM measurement. The

155

Nanoscope Analysis 1.7 software was applied to determine the diameter of nanofibers. The

156

inner morphologies of the TOCNF, α-DECH, and the α-DECHN/TOCNF hydrogels were

157

determined by a SEM instrument (JSM-6700F, JEOL, Japan). In order to obtain the

158

samples for SEM, the hydrogels were treated with liquid nitrogen first, and then

(NanoScope III, Digital Instruments, USA). A drop of



159

freeze-dried at the temperature of minus 50oC. After that, the dried samples were sputtered

160

with a layer of gold to improve electrical conductivity for SEM observation.

161

FTIR Spectroscopy. The changes of chemical structure of α-DECHN, crab shell flakes,

162

bleached eucalyptus pulp and TOCNF were investigated by fourier transform infrared

163

spectroscopy (FTIR). The trial samples were placed a disk, and dried in an oven (Shen Xian,

164

DHG-9030A, China) at 50 oC for 5 h before the FTIR experiments. These samples were

165

examined using an FTIR spectrometer (Nicolet iS5, Thermo Scientific, USA) from 4000 to

166

500 cm-1 based on 4 cm-1 resolution and 32 scans. These samples were grinded with

167

pre-dried KBr powder and then compressed into disks prior to the tests.

168

Zeta-potentials. Zeta-potentials of TOCNF, α-DECHN and α-DECHN/TOCNF hydrogels

169

were obtained from the dynamic light scattering (Zetasizer Nano, Malvern, UK) based on

170

the determination of the electrophoretic mobility of these hydrogels. Each sample

171

suspension with a concentration of 0.05 wt% was prepared and measured using folded

172

capillary cells at 25 oC. Zeta potential was calculated from

173

Smoluchowski’s formula43. For each sample, the measurement was repeated five times to

174

generate error bar.

175

Drug-loading studies. Five-fluorouracil (5-FU) which is a common anti-cancer drug was

176

selected for testing the hydrogels via a typical drug-loading process. Each kind of dried

177

hydrogel with weigh of 0.1 g was fully immersed in 20 mL (0.5 mg/mL) of the PBS

178

solutions at 25 oC for 30 h. Then, the swollen hydrogels were placed to a plate under


electrophoretic mobility via

Page 10 of 36

Page 11 of 36


179

vacuum drying at 40 oC for 24 h. Then, the absorbance of the remainder solution was

180

determined at 266 nm by an UV-Visible spectrophotometer (S3100, MAPADA, China). The

181

amount of 5-FU in the hydrogels were calculated based on the calibration curve

182

(absorbance vs. concentration). The loading amount (M5-FU) and efficiency (LE5-FU) of

183

5-FU in hydrogels were calculated by the following equations:

184

M 5− FU = c 0ν 0 - c1ν 1

(2)

185

LE 5− FU =

M 5- FU M total -5- FU

(3)

186

Here c0 represents initial concentration of 5-FU solution (0.5 mg/mL), and ν0 represents

187

initial volume (20 mL) of 5-FU solution. c1 represents the concentration of 5-FU solution in

188

the residual solution, and ν1 represent the volume of 5-FU solution in the residual solution.

189

Mtotal-5-FU is the total mass of 5-FU in the initial solution.

190

In Vitro Release Studies. Drug-loaded hydrogels were soaked in 50 mL of PBS buffer (pH

191

= 7.4, 37 °C) with magnetic stirring (150 rpm). At predetermined intervals of time (1 h, 3 h,

192

6 h, 12 h, 27 h, 39 h, 48 h), the absorbance value of the medium solution was determined.

193

For each measurement, 4 mL solution was sucked out from the beaker, which was

194

immediately refilled with the same volume of PBS solution to ensure the consistency of the

195

volume in the beaker. The cumulative percentage of drug release (CPDR%) was determined

196

by the following equation44: n −1

197

CPDR% =

V0 ∑1 C i + V0 C n M CPDS

, (M CPDS = M total-5-FU − M remainder-5-FU )


(4)


198

Where V0 represents the total volume of release solution (50 mL), MCPDR is the mass of

199

drug loading and Cn is the concentration of 5-FU solution in the beaker after the nth sample

200

was sucked out.

201 202

RESULTS AND DISCUSSION

203

Mechanisms of α-DECHN/TOCNF hydrogels formation. Zeta potential is widely

204

adopted for the determination of the numerical value of the electrical charge. The zeta

205

potentials of aqueous α-DECHN, α-DECHN/TOCNF and TOCNF with a concentration of

206

0.02% (w/v) solid content were tested at room temperature using a zeta potentials

207

measuring apparatus. As shown in Fig.1, pure α-DECHN dilute dispersions had positive

208

surface potential in distilled water, and the zeta potential reached +44.40 mv. Conversely,

209

pure TOCNF dilute dispersions had negative surface potential and displayed a relatively

210

lower zeta potential (-36.3 mV). When positively-charged α-DECHN dispersion and

211

negatively-charged TOCNF dispersion were mixed, their strong surface potential was

212

neutralized or partially neutralized, which triggered physical cross-linking between

213

α-DECHN and TOCNF. The zeta potential of a series of hydrogels with different

214

α-DECHN to α-DECHN/TOCNF ratio from 20% and 80% were determined. It is found

215

that the hydrogel with the ratio of 40% α-DECHN had the lowest zeta-potential. This result

216

predicts the hydrogel with the α-DECHN to TOCNF ratio of 40% to 60% may have the

217

highest cross-linking density comparing with other hydrogels with different mixed ratios.


Page 12 of 36

Page 13 of 36


218

Fig. 2A is the scheme which describes the formation process of the α-DECHN/TOCNF

219

hybrid hydrogels. When the positively-charged α-DECHN suspension were added into the

220

negatively-charged

221

formed by physically cross-linking of interfibers. Fig. 2B (1) and (2) explain the

222

mechanisms of the deacetylation of α-DECHN and the oxidation reaction of TOCNF,

223

respectively. The position of C6 carbon in molecular structure of cellulose was oxidized

224

using TEMPO, and the chains were modified by negatively-charged carboxyl groups.

225

Therefore, the pure TOCNF showed strongly negative surface potential. While, the

226

α-DECHN had a strong positive surface potential, which could be attributed to the

227

existence of the unsaturated positive charges (NH3+) on α-DECHN nanofibers because of

228

the protonation of amino groups of chitin in acidic conditions31.

229

AFM micrograph and FTIR spectroscopy. The dispersion state of TOCNF and

230

α-DECHN suspension could be very crucial to form excellent α-DECHN/TOCNF

231

hydrogels. Fig.3A shows the AFM image of eucalyptus-derived TOCNF dispersions. The

232

dispersions displayed the web-like structure consisting of many randomly entangled and

233

well-individualized fibers with the nano-sized dimension. Analysis from AFM image

234

indicated that most TOCNF showed the width between 10-20 nm and the length between

235

800 nm and 1.1 µm. This finding suggests that eucalyptus pulp fibers were successfully

236

nanofibrillated through the TEMPO-oxidation treatment integrated with high-pressure

237

homogenization. Fig. 3B demonstrates the AFM image of deprotonated α-DECHN, which

TOCNF

suspension,

the

α-DECHN/TOCNF


hydrogels

were


238

had a diameter of close to 12 nm, and its length was between 500-600 nm. Fig. 3C shows

239

the typical infrared absorption bands of TOCNF. The broad peak at 3418 cm-1 is attributed

240

to the stretching vibration of O-H vibration. The bands at 2911 cm-1 and 1258 cm-1 are

241

corresponding to the C-H2 and C-H stretching vibrations, respectively. The peak at 1042

242

cm-1 is assigned to the vibrations of the C-O-C bonds of glycosidic bridges. The peak at

243

1729 cm-1 which was found in the spectra of TOCNF represents the stretching vibration of

244

carboxylate groups. Due to the oxidation on C6 primary hydroxyl groups by TEMPO

245

treatment, the protonated carboxyl groups were generated, which results in the presence of

246

the 1729 cm−1 band. However, since there are only small amount of carboxylate groups in

247

the eucalyptus pulp, this band was hard to be detected by FTIR and could not be observed

248

from the spectra of dry pulp. Fig.3D shows the FTIR spectrum of α-DECHN. The peaks at

249

1565 cm-1 and 1602 cm-1 are ascribed to N-H bending (amide II), and the carbonyl

250

stretching (amide I), respectively. The degree of deacetylation can be reflected by the peak

251

at 1565 cm-1 of FTIR spectrum. Compared with the crab shell flakes, the α-DECHN shows

252

a lower peak at 1565 cm-1 in FTIR spectrum, which indicates the higher deacetylation

253

degree45, 46.

254

Microstructure observation. The observation of the structure of a hydrogel is necessary

255

since the structure is related to hydrogels’ water-retention capacity, which is highly

256

demanded in many applications especially in drug delivery systems. In order to estimate the

257

porous structure, freeze-dried hydrogels were prepared and examined by SEM. Fig. 4


Page 14 of 36

Page 15 of 36


258

shows the SEM images of the cross-section of these hydrogels with different ratios of

259

α-DECHN to TOCNF. It is interesting to find that the cross-section areas of

260

α-DECHN/TOCNF hydrogels were distinct from that of pure α-DECHN and pure TOCNF

261

hydrogels, and the ratio of α-DECHN to TOCNF affected the bulk structure of the obtained

262

hydrogels. As shown in Fig. 4, pure α-DECHN shows a dense structure, which is quite

263

distinguishing from that of pure TOCNF. While, with the increase of the proportion of

264

α-DECHN, a clear transition from mesh-like structure to dense structure could be observed.

265

Due to the contribution of α-DECHN, smaller pores appeared in the α-DECHN/TOCNF

266

hydrogels.

267

Rheological analysis. A series of rheological tests regarding to the viscoelastic behaviors

268

of pure TOCNF, pure α-DECHN and α-DECHN/TOCNF hydrogels were conducted under

269

oscillatory shear frequency from 0.1 to 100 rad/s. As shown in Fig. 5, the TOCNF shows

270

the lowest elastic modulus (G') and the viscous modulus (G") indicating the weak gel state

271

of itself. The α-DECHN/TOCNF hydrogels displayed larger values of elastic modulus and

272

the viscous modulus than both pure TOCNF and pure α-DECHN. This phenomena may be

273

attributed to the increased cross-linked networks caused by the attraction of opposite

274

charges between the two kinds of nanofibers. Comparing with other α-DECHN/TOCNF

275

hydrogels, the hydrogel containing 40% α-DECHN and 60% α-DECHN may have almost

276

neutralized surface potential and the strongest cross-linked structure, thus it showed the

277

largest elastic modulus and the viscous modulus illustrated in Fig. 5. However, Comparing



278

40% α-DECHN with 60% α-DECHN, the former has larger elastic modulus and the viscous

279

modulus is attributed to more neutralized surface potential. It was also observed that all

280

α-DECHN/TOCNF hydrogels showed the higher elastic modulus (G') and relatively lower

281

viscous modulus (G") corresponding the inherent property of hydrogels.

282

Swelling characteristics of α-DECHN/TOCNF hydrogels. The release rate of drugs

283

could be influenced by the swelling ratio, which reflects the ability of absorbing and

284

retaining water of hydrogels47. The water adsorption of hydrogels was affected by several

285

factors such as surface area, particle size and crosslink density48, 49. Fig. 6C shows the

286

dynamic change of swelling ratios of the six α-DECHN/TOCNF hydrogels after they were

287

immersed into deionized water at 25 °C. It can be seen that all the hydrogels swelled with

288

time until the equilibrium status was reached after around 15 h. It is also found that the

289

proportion of α-DECHN was an important factor which related to the swelling ratio of the

290

hydrogels. With the increase of the proportion of α-DECHN in the hydrogels (from pure

291

TOCNF to 60% α-DECHN/40% TOCNF), the equilibrium swelling ratio was increased.

292

The hydrogel with the α-DECHN to TOCNF proportion of 40% to 60% achieved the

293

highest equilibrium swelling ratio among the six hydrogels. This may be because

294

α-DECHN contains rich amine groups and hydroxyl groups which can form hydrogen

295

bonding with water molecules and lock certain amount of bound water in the nanofiber

296

networks. This deduction can be confirmed with experimental result displayed in Fig. 6C. It

297

has shown that pure α-DECHN hydrogel has even higher swelling ratio rather than pure


Page 16 of 36

Page 17 of 36


298

TOCNF hydrogel. Alternatively, extra porous networks performing were formed as water

299

molecules cages due to the interaction between the two kinds of fibers with opposite

300

charges. However, when the proportion of α-DECHN increased, the swelling ratio

301

decreased, indicating the reduced swelling of the hydrogels. It is probably that excessive

302

α-DECHN in hydrogels lead to the filling up of the free space of the hydrogels.

303

In Vitro Drug-Release.

304

hydrogel is proportional to its swelling capacity40, 50. The in vitro drug-release property of

305

α-DECHN/TOCNF hydrogels were evaluated in PBS solution at 37 oC. The drug loading

306

amount and efficiency of the hydrogels were evaluated in Fig.6A and Fig. 6B respectively.

307

The drug loading amount have high consistency with the drug loading efficiency. The

308

hydrogel (40% α-DECHN) with lowest surface charge exhibited the largest loading amount

309

(5331 µg per 0.1 g of hydrogel) and highest loading efficiency (53.31%) of 5-FU. No matter

310

the proportion of α-DECHN was increased or decreased, both drug loading capacity and

311

efficiency of a hydrogel were reduced. This trend was similar to the results of swelling

312

study illustrated in Fig. 6C. These results demonstrated that the loading efficiency was

313

associated with the swelling ratio. The hydrogels with higher swelling ratio can easily

314

absorb larger amount of drug solution into its porous networks and exhibit a higher drug

315

loading efficiency (Fig. 6B).

According to the literature, the drug-loading capacity of a

316

The drug release rate from a hydrogel may be effected by several factors such as the

317

interaction between the drug and components of the hydrogel, the solubility of the drug, as



318

well as the swelling behavior of the hydrogel in a certain media51. Fig.6D shows the release

319

behavior of 5-FU from a series of α-DECHN/TOCNF hydrogels in PBS solution at 37 °C.

320

The standard curve of 5-FU displayed in Fig. S1. Notably, the graph of drug release from

321

these hydrogels has great similarity with the chart of swelling behavior illustrated in Fig.

322

6C. When a hydrogel has a higher swelling ratio, it would exhibit a higher drug release

323

percent. As can be seen from Fig.6D, the highest amount of 5-FU was released from

324

preloaded hydrogel with 40% α-DECHN within 30 hours, and the lowest release percentage

325

of 5-FU was detected from the media solution containing preloaded hydrogel made of pure

326

chitin. All the α-DECHN/TOCNF hydrogels showed a similar trend during drug release

327

process which consists of the initial rapid release phase, the slow release phase and the

328

following plateaus phase. The rapid release may be attributed to the high gradient of drug

329

concentration which provided a driving force promoting the diffusion of drug molecules

330

from the hydrogels. With the increase of time, the release rate was slowed. Due to the

331

thickness of the hydrogels, the drug molecules absorbed in the inner hydrogel networks

332

were more difficult to be released into the media. A low-cost, simple and reproducible

333

method was proposed in this study to fabricate natural hydrogels for controlled drug release.

334

The hydrogels were synthesized via the self-assembling of anionic TOCNF and cationic

335

α-DECHN with adjustable mechanical and swelling features by varying the proportion of

336

the two kinds of nanofibers. The results showed that the hydrogel with 40% α-DECHN and

337

60% α-DECHN has the strongest cross-linked structure and largest swelling ratio. The


Page 18 of 36

Page 19 of 36


338

performance of α-DECHN/TOCNF hydrogels as anti-cancer drug delivery carriers was

339

investigated using 5-FU as a model drug. The results indicated that the drug delivery

340

behavior of the hydrogels was associated with their swelling property. The maximum

341

loading efficiency and drug release percentage were obtained when the hydrogel containing

342

40% of α-DECHN. When the proportion was changed, both the loading efficiency and drug

343

release percentage declined. This study indicated that the α-DECHN/TOCNF hydrogels

344

exhibited desirable properties for drug delivery, and were expected to be a promising

345

hydrogel concept for medical therapies.

346 347

ACKNOWLEDGEMENTS

348

The research was financially supported by the National Natural Science Foundation of

349

China [Grant number 81671780]; the Natural Science Foundation of Guangdong Province

350

[Grant number 2017A030313740]; the Fundamental Research Funds for the Central

351

Universities [Grant number 2017MS076].

352

REFERENCES

353

1. Chang, C. Y.; Zhang, L. Z.; Zhou, J. P.; Zhang, L. N.; Kennedy, J. F., Structure and

354

properties of hydrogels prepared from cellulose in NaOH/urea aqueous solutions.

355

Carbohydr. Polym. 2010, 82, 122-127.

356 357

2. Chang, C. Y.; Zhang, L. N., Cellulose-based hydrogels: Present status and application prospects. Carbohydr. Polym. 2011, 84, 40-53.



358

3. He, M.; Zhao, Y.; Duan, J.; Wang, Z.; Chen, Y.; Zhang, L., Fast Contact of Solid-Liquid

359

Interface Created High Strength Multi-Layered Cellulose Hydrogels with Controllable

360

Size. ACS Appl. Mater. Interfaces 2014, 6, 1872-1878.

361

4. Cao, X. F.; Peng, X. W.; Zhong, L. X.; Sun, R. C., Multiresponsive Hydrogels Based on

362

Xylan-Type Hemicelluloses and Photoisomerized Azobenzene Copolymer as Drug

363

Delivery Carrier. J. Agric. Food Chem. 2014, 62, 10000-10007.

364

5. Han, J. H.; Krochta, J. M.; Kurth, M. J.; Hsieh, Y. L., Lactitol-based poly(ether polyol)

365

hydrogels for controlled release chemical and drug delivery systems. J. Agric. Food

366

Chem. 2000, 48, 5278-5282.

367

6. Lin, N.; Geze, A.; Wouessidjewe, D.; Huang, J.; Dufresne, A., Biocompatible

368

Double-Membrane Hydrogels from Cationic Cellulose Nanocrystals and Anionic

369

Alginate as Complexing Drugs Codelivery. ACS Appl. Mater. Interfaces 2016, 8,

370

6880-6889.

371

7. Sannino, A.; Netti, P. A.; Madaghiele, M.; Coccoli, V.; Luciani, A.; Maffezzoli, A.;

372

NicolaiS, L., Synthesis and characterization of macroporous poly(ethylene glycol)-based

373

hydrogels for tissue engineering application. J. Biomed. Mater. Res. Part A 2006, 79A,

374

229-236.

375

8. Levesque, S. G.; Lim, R. M.; Shoichet, M. S., Macroporous interconnected dextran

376

scaffolds of controlled porosity for tissue-engineering applications. Biomaterials 2005,

377

26, 7436-7446.


Page 20 of 36

Page 21 of 36


378

9. Liu, Q.; Hedberg, E. L.; Liu, Z. W.; Bahulekar, R.; Meszlenyi, R. K.; Mikos, A. G.,

379

Preparation of macroporous poly(2-hydroxyethyl methacrylate) hydrogels by enhanced

380

phase separation. Biomaterials 2000, 21, 2163-2169.

381

10. Van Vlierberghe, S.; Cnudde, V.; Dubruel, P.; Masschaele, B.; Cosijns, A.; De Paepe, I.;

382

Jacobs, P. J. S.; Van Hoorebeke, L.; Remon, J. P.; Schacht, E., Porous gelatin hydrogels:

383

1. Cryogenic formation and structure analysis. Biomacromolecules 2007, 8, 331-337.

384

11. Plieva, F. M.; Ekstrom, P.; Galaev, I. Y.; Mattiasson, B., Monolithic cryogels with open

385

porous structure and unique double-continuous macroporous networks. Soft Matter 2008,

386

4, 2418-2428.

387

12. Fukuzumi, H.; Saito, T.; Wata, T.; Kumamoto, Y.; Isogai, A., Transparent and High Gas

388

Barrier Films of Cellulose Nanofibers Prepared by TEMPO-Mediated Oxidation.

389

Biomacromolecules 2009, 10, 162-165.

390

13. Fan, Y. M.; Fukuzumi, H.; Saito, T.; Isogai, A., Comparative characterization of

391

aqueous dispersions and cast films of different chitin nanowhiskers/nanofibers. Int. J.

392

Biol. Macromol. 2012, 50, 69-76.

393

14. Bhattacharya, M.; Malinen, M. M.; Lauren, P.; Lou, Y. R.; Kuisma, S. W.; Kanninen, L.;

394

Lille, M.; Corlu, A.; GuGuen-Guillouzo, C.; Ikkala, O.; Laukkanen, A.; Urtti, A.;

395

Yliperttula, M., Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell

396

culture. J. Control. Release 2012, 164, 291-298.



397 398

15. Cai, J.; Zhang, L., Unique gelation behavior of cellulose in NaOH/Urea aqueous solution. Biomacromolecules 2006, 7, 183-189.

399

16. Cai, X.; Yang, L. Q.; Zhang, L. M.; Wu, Q., Synthesis and anaerobic biodegradation of

400

indomethacin-conjugated cellulose ethers used for colon-specific drug delivery.

401

Bioresour. Technol. 2009, 100, 4164-4170.

402

17. Chang, C. Y.; Duan, B.; Cai, J.; Zhang, L. N., Superabsorbent hydrogels based on

403

cellulose for smart swelling and controllable delivery. Eur. Polym. J. 2010, 46, 92-100.

404

18. Dong, H.; Snyder, J. F.; Williams, K. S.; Andzelm, J. W., Cation-Induced Hydrogels of

405

Cellulose Nanofibrils with Tunable Moduli. Biomacromolecules 2013, 14, 3338-3345.

406

19. Klemm, D.; Kramer, F.; Moritz, S.; Lindstrom, T.; Ankerfors, M.; Gray, D.; Dorris, A.,

407

Nanocelluloses: A New Family of Nature-Based Materials. Angew. Chem.-Int. Edit. 2011,

408

50, 5438-5466.

409 410

20. Roy, C.; Budtova, T.; Navard, P., Rheological properties and gelation of aqueous cellulose-NaOH solutions. Biomacromolecules 2003, 4, 259-264.

411

21. Muller, A.; Wesarg, F.; Hessler, N.; Muller, F. A.; Kralisch, D.; Fischer, D., Loading of

412

bacterial nanocellulose hydrogels with proteins using a high-speed technique. Carbohydr.

413

Polym. 2014, 106, 410-413.

414 415

22. Saito, H.; Sakurai, A.; Sakakibara, M.; Saga, H., Preparation and properties of transparent cellulose hydrogels. J. Appl. Polym. Sci. 2003, 90, 3020-3025.


Page 22 of 36

Page 23 of 36


416

23. Sun, B. Z.; Duan, L.; Peng, G. G.; Li, X. X.; Xu, A. H., Efficient production of glucose

417

by microwave-assisted acid hydrolysis of cellulose hydrogel. Bioresour. Technol. 2015,

418

192, 253-256.

419

24. Yang, J.; Han, C., Mechanically Viscoelastic Properties of Cellulose Nanocrystals

420

Skeleton Reinforced Hierarchical Composite Hydrogels. ACS Appl. Mater. Interfaces

421

2016, 8, 25621-25630.

422

25. Dai, H. J.; Huang, H. H., Enhanced Swelling and Responsive Properties of Pineapple

423

Peel Carboxymethyl Cellulose-g-poly(acrylic acid-co-acrylamide) Superabsorbent

424

Hydrogel by the Introduction of Carclazyte. J. Agric. Food Chem. 2017, 65, 565-574.

425 426 427 428

26. Seliktar, D., Designing Cell-Compatible Hydrogels for Biomedical Applications.

Science 2012, 336, 1124-1128. 27. Isogai, A.; Saito, T.; Fukuzumi, H., TEMPO-oxidized cellulose nanofibers. Nanoscale

2011, 3, 71-85.

429

28. Su, Y.; Burger, C.; Hsiao, B. S.; Chu, B., Characterization of TEMPO-oxidized

430

cellulose nanofibers in aqueous suspension by small-angle X-ray scattering. J. Appl.

431

Crystallogr. 2014, 47, 788-798.

432 433

29. Fan, Y. M.; Saito, T.; Isogai, A., TEMPO-mediated oxidation of beta-chitin to prepare individual nanofibrils. Carbohydr. Polym. 2009, 77, 832-838.

434

30. Izumi, R.; Azuma, K.; Izawa, H.; Morimoto, M.; Nagashima, M.; Osaki, T.; Tsuka, T.;

435

Imagawa, T.; Ito, N.; Okamoto, Y.; Saimoto, H.; Ifuku, S., Chitin nanofibrils suppress



Page 24 of 36

436

skin inflammation in atopic dermatitis-like skin lesions in NC/Nga mice. Carbohydr.

437

Polym. 2016, 146, 320-327.

438

31. Junkasem,

J.;

Rujiravanit,

R.;

Supaphol,

P.,

Fabrication

of

alpha-chitin

439

whisker-reinforced poly(vinyl alcohol) nanocomposite nanofibres by electrospinning.

440

Nanotechnology 2006, 17, 4519-4528.

441

32. Qi, Z. D.; Fan, Y. M.; Saito, T.; Fukuzumi, H.; Tsutsumi, Y.; Isogai, A., Improvement of

442

nanofibrillation efficiency of alpha-chitin in water by selecting acid used for surface

443

cationisation. RSC Adv. 2013, 3, 2613-2619.

444

33. Serrano, C. V.; Leemreize, H.; Bar-On, B.; Barth, F. G.; Fratzl, P.; Zolotoyabko, E.;

445

Politi, Y., Ordering of protein and water molecules at their interfaces with chitin

446

nano-crystals. J. Struct. Biol. 2016, 193, 124-131.

447

34. Villa-Lerma, G.; Gonzalez-Marquez, H.; Gimeno, M.; Trombotto, S.; David, L.; Ifuku,

448

S.; Shirai, K., Enzymatic hydrolysis of chitin pretreated by rapid depressurization from

449

supercritical 1,1,1,2-tetrafluoroethane toward highly acetylated oligosaccharides.

450

Bioresour. Technol. 2016, 209, 180-186.

451

35. Zhang, Y.; Chen, Z. G.; Bian, W. Y.; Feng, L.; Wu, Z. W.; Wang, P.; Zeng, X. X.; Wu, T.,

452

Stabilizing oil-in-water emulsions with regenerated chitin nanofibers. Food Chem. 2015,

453

183, 115-121.


Page 25 of 36


454

36. Kadokawa, J.; Takegawa, A.; Mine, S.; Prasad, K., Preparation of chitin nanowhiskers

455

using an ionic liquid and their composite materials with poly(vinyl alcohol). Carbohydr.

456

Polym. 2011, 84, 1408-1412.

457 458

37. Kato, Y.; Kaminaga, J.; Matsuo, R.; Isogai, A., TEMPO-mediated oxidation of chitin, regenerated chitin and N-acetylated chitosan. Carbohydr. Polym. 2004, 58, 421-426.

459

38. Anirudhan, T. S.; Mohan, A. M., Novel pH switchable gelatin based hydrogel for the

460

controlled delivery of the anti cancer drug 5-fluorouracil. RSC Adv. 2014, 4,

461

12109-12118.

462

39. Faria, H. A. M.; de Queiroz, A. A. A., A novel drug delivery of 5-fluorouracil device

463

based on TiO2/ZnS nanotubes. Materials Science & Engineering C-Materials for

464

Biological Applications 2015, 56, 260-268.

465

40. Ha, W.; Yu, J.; Song, X.-y.; Zhang, Z.-j.; Liu, Y.-q.; Shi, Y.-p., Prodrugs forming

466

multifunctional supramolecular hydrogels for dual cancer drug delivery. Journal of

467

Materials Chemistry B 2013, 1, 5532-5538.

468

41. Murata, J.; Saiki, I.; Makabe, T.; Tsuta, Y.; Tokura, S.; Azuma, I., Inhibition of

469

tumor-induced angiogenesis by sulfated chitin derivatives. Cancer research 1991, 51,

470

22-6.

471 472

42. Dogu, Y.; Okay, O., Swelling-deswelling kinetics of poly(N-isopropylacrylamide) hydrogels formed in PEG solutions. J. Appl. Polym. Sci. 2006, 99, 37-44.



473

43. Kaszuba, M.; Corbett, J.; Watson, F. M.; Jones, A., High-concentration zeta potential

474

measurements using light-scattering techniques. Philos. Trans. R. Soc. A-Math. Phys.

475

Eng. Sci. 2010, 368, 4439-4451.

476

44. Wei, W.; Qi, X.; Li, J.; Zuo, G.; Sheng, W.; Zhang, J.; Dong, W., Smart Macroporous

477

Salecan/Poly(N,N-diethylacrylamide) Semi-IPN Hydrogel for Anti-Inflammatory Drug

478

Delivery. Acs Biomaterials Science & Engineering 2016, 2, 1386-1394.

479 480

45. Abdou, E. S.; Nagy, K. S. A.; Elsabee, M. Z., Extraction and characterization of chitin and chitosan from local sources. Bioresour. Technol. 2008, 99, 1359-1367.

481

46. Liu, S.; Sun, J.; Yu, L.; Zhang, C.; Bi, J.; Zhu, F.; Qu, M.; Jiang, C.; Yang, Q.,

482

Extraction and Characterization of Chitin from the Beetle Holotrichia parallela

483

Motschulsky. Molecules 2012, 17, 4604-4611.

484

47. Anirudhan, T. S.; Divya, P. L.; Nima, J., Synthesis and characterization of silane coated

485

magnetic nanoparticles/glycidylmethacrylate-grafted-maleated cyclodextrin composite

486

hydrogel as a drug carrier for the controlled delivery of 5-fluorouracil. Materials Science

487

& Engineering C-Materials for Biological Applications 2015, 55, 471-481.

488 489

48. Teng, Y.; Zhou, Q. X., Adsorption behavior of Sudan I-IV on a coastal soil and their forecasted biogeochemical cycles. Environ. Sci. Pollut. Res. 2017, 24, 10749-10758.

490

49. Guan, Y.; Qi, X. M.; Zhang, B.; Chen, G. G.; Peng, F.; Sun, R. C., Physically

491

Crosslinked Composite Hydrogels of Hemicelluloses with Poly(vinyl alcohol phosphate)

492

and Chitin Nanowhiskers. BioResources 2015, 10, 1378-1393.


Page 26 of 36

Page 27 of 36


493

50. Jayaramudu, T.; Raghavendra, G. M.; Varaprasad, K.; Raju, K. M.; Sadiku, E. R.; Kim,

494

J., 5-Fluorouracil encapsulated magnetic nanohydrogels for drug-delivery applications. J.

495

Appl. Polym. Sci. 2016, 133.

496

51. Ooi, S. Y.; Ahmad, I.; Amin, M., Cellulose nanocrystals extracted from rice husks as a

497

reinforcing material in gelatin hydrogels for use in controlled drug delivery systems. Ind.

498

Crop. Prod. 2016, 93, 227-234.

499 500 501 502 503 504 505 506 507 508 509 510 511 512



Figure captions Figure 1 Zeta-Potential of dilute dispersions of α-DECHN, TOCNF and α-DECHN/TOCNF at room temperature.

Figure 2 (A) The schematic process illustrating the formation of α-DECHN/TOCNF hybrid hydrogels. (B) Simplified reaction mechanisms about negatively-charged TOCNF and positively-charged α-DECHN.

Figure 3 Preparation of the TOCNF and α-DECHN. (A) AFM image of TOCNF. (B) AFM image of α-DECHN. (C) FTIR spectra of bleached eucalyptus pulp and TOCNF. (D) FTIR spectra of crab shell flakes and α-DECHN.

Figure 4 SEM pictures of α-DECHN/TOCNF hydrogels. (A) pure α-DECHN (B) pure TOCNF (C) 20% α-DECHN (D) 40% α-DECHN (E) 60% α-DECHN (F) 80% α-DECHN.

Figure 5


Page 28 of 36

Page 29 of 36


Elastic modulus (G') (A) and viscous modulus (G") (B) of α-DECHN hydrogel, TOCNF hydrogel and α-DECHN/TOCNF hydrogels during frequency sweep with a strain of 10%.

Figure 6 (A) Loading amount of the hydrogels. (B) Loading efficiency of the hydrogels. (C) Swelling behavior of the hydrogels in deionized water at 25 oC. (D) 5-FU from the hydrogels in PBS at 37 oC.


Release profiles of


Figure Graphics Figure 1


Page 30 of 36

Page 31 of 36


Figure 2



Figure 3


Page 32 of 36

Page 33 of 36


Figure 4



Figure 5


Page 34 of 36

Page 35 of 36


Figure 6



TOC Graphic


Page 36 of 36

Engineering biocompatible hydrogels from bicomponent natural

Recommend Documents