Accepted Manuscript

Title: Self-healable Hydrogel on Tumour Cell as Drug

Delivery System for Localized and Effective Therapy

Author: Guanru Chang Yan Chen Yanjie Li Shikuo Li

Fangzhi Huang Yuhua Shen Anjian Xie

PII: S0144-8617(15)00022-3
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2014.12.077
Reference: CARP 9593

To appear in:

Received date: 10-8-2014

Revised date: 10-12-2014
Accepted date: 28-12-2014

Please cite this article as: Chang, G., Chen, Y., Li, Y., Li, S., Huang, F.,
Shen, Y., and Xie, A.,Self-healable Hydrogel on Tumour Cell as Drug Delivery
System for Localized and Effective Therapy, Carbohydrate Polymers (2015),
http://dx.doi.org/10.1016/j.carbpol.2014.12.077

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1 1. Self-healing hydrogel as an injectable drug carrier was facilely prepared.

2 2. The hydrogel shell could be easily formed on tumor cells, and its self-healing ability helps to

3 solve the catheter clogging in the process of injection, and could repair itself when damaged.

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4 3. The hydrogel has good biocompatibility, possesses good retention ability for 5-FU at pH 7.4

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5 preventing the loss of drug to normal cells, as well as shows pH sensitive continuous and

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6 controllable release at pH 5.0, with enhanced antitumor effect.

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 7 Self-healable Hydrogel on Tumour Cell as Drug Delivery System for
8 Localized and Effective Therapy

9 Guanru Chang a,b, Yan Chenc, Yanjie Lia, Shikuo Li a, Fangzhi Huang a, Yuhua Shen a *, and

10 Anjian Xie a *

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a
11 School of Chemistry and Chemical Engineering, Collaborative Innovation Center of Modern

12 Bio-Manufacture, Anhui university, Hefei 230601, P. R. China.

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b
13 School of Chemistry and Chemical Engineering, Huangshan University, Huangshan, 245041, P.

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14 R. China.

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c
15 Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei 230601, P. R.

16 China.
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17 * Corresponding Author E-mail: s_yuhua@163.com, anjx@163.com ; Fax: +86 0551 63861475;

18 Tel: +860551 63861475

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19 KEYWORDS: Self-healable hydrogel, Localized injection therapy, Controllable release, 5-FU,

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20 Hydrogel shell
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21 Chemical compounds studied in this article:

22 Chitosan (PubChem CID: 71853); Polyvinyl alcohol (PubChem CID: 11199);

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23 Fluorouracil (PubChem CID: 3385); Propidium iodide (PubChem CID:104981);

24 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (PubChem CID: 64965);

25 Dimethylsulfoxide (PubChem CID: 679); Glutaraldehyde (PubChem CID: 3485);

26 Hoechst 33342 (PubChem CID: 1464);

27

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28 ABSTRACT: A self-healable chitosan(CS)/polyvinyl alcohol (PVA) hydrogel as an injectable

29 drug carrier was firstly prepared in situ on tumor cells for effective and localized therapy. PVA

30 molecules have a synergistic effect on the formation and maintenance of 3D network

31 conformation of hydrogel. The hydrogel shows good biocompatibility and could be easily and

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32 rapidly formed. When loaded with fluorouracil (5-FU), the hydrogel possessed good drug

33 retention ability at pH 7.4, which can prevent the loss of drug to normal cells and reduce the side

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34 effect. As well, the hydrogel shows continuous and controllable drug release, with the final

35 cumulative releasing amount of 84.8% at pH 5.0. Therefore, the hydrogel not only could

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36 maintain a higher 5-FU concentration around tumor cells to enhance the antitumor effect, but

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37 also can achieve pH sensitive controllable drug release at the lesion site. Meantime, the attractive

38 self-healing ability of the CS/PVA hydrogel is firstly revealed in this study, which contributes to

39 the regeneration of its integral network from the broken fragments. The CS/PVA hydrogel may
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40 hold promise for better applications in anti-tumor therapy.
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41 1.Introduction
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42 Cancer is one of the most common causes of death in the world, and chemotherapy continues
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43 to be one of the basic treatment methods. However, the nonspecific distribution of anti-cancer
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44 drugs during the process of traditional intravenous injection remains a large obstacle to

45 enhancing therapeutic efficacy and reducing side effect. Therefore, the drug delivery systems
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46 with the features of sustained drug release (Peer, Karp, Hong, FaroKhzad, Margalit, ＆ Langer,

47 2007; Allen ＆ Cullis, 2004) and delivery of an adequate dose to the target site (Gai, Yang, ＆ Li,

48 2010), have attracted much attention. Hydrogel can act as a particular delivery system for

49 indissoluble anti-neoplastic drug with poor pharmaco kinetic profiles, and avoid repeat

50 administration to achieve a proper dose (Abulateefeh, Spain, Thurecht, Aylott, Chan, Garnetta,

51 ＆ Alexander, 2013). It could accommodate irregular shaped defects and remain at the desired

52 position for controlled release of encapsulated therapeutics (Ratner ＆ Bryant, 2004). However,

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53 these traditional hydrogels come cross some limitations, such as, drug loss and diffusion from the

54 targeting site because of the slow gelation (Gupta, Tator, ＆ Shoiche, 2006) , and delivery failure

55 due to catheter clogging in the process of extremely rapid gelation (Martens, Godier, Parks, Wan,

56 Koeckert, Eng, Hudson, ＆ Sherman, 2009). Different from traditional hydrogels, self-healing

t
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57 hydrogels based on chitosan possess automatic self-repair capability after damage and can be

58 used as injectable materials (Yang, Zhang, Zhang, Tao, Li, ＆Wei, 2012; Zhang, Tao, Li, Wei.

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59 2012), are becoming increasingly attractive due to their great potential in biomedical applications.

60 The self-healing hydrogel could homogeneously encapsulate pharmaceutical drugs/cells within

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61 the precursor solution prior to the injection (Yan ＆ Pochan, 2010; Guvendiren, Lu, ＆ Burdick,

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62 2012; Tang, Du, Hu, Shi,＆ Kennedy, 2007 ). After injection, the broken self-healing hydrogel

63 can regenerate the integral network and remain at the site of injection (Zhang, Xia, ＆ Zhao,

64 2012; Yan, Altunbas, Yucel, Nagarkar, Schneider,＆Pochan, 2010), avoiding the risk of catheter
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65 clogging. Therefore, self-healing hydrogel as new drug delivery system could enhance the

66 medicine delivery efficiency and improve the therapeutic effect, which opens a new avenue for
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67 application in minimally invasive and in situ-forming implants.

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68 As is known to all, hydrogel for biomedical applications must have good biocompatibility and
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69 nontoxicity. However, the designers generally put too much emphasis on their self-healing
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70 property while take no account of the biocompatibility and toxicity issues (Chen, Kushner,

71 Williams, ＆ Guan, 2012; Zheng ＆ McCarthy, 2012). Although some self-healable and
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72 biocompatible hydrogels have been successfully prepared by the self-assembly of amphiphilic

73 peptides (Hamley, 2011), the expensive and complicate preparation method hinders the large-

74 scale manufacture and counteracts the advantages of those self-healing hydrogels (Yan, ＆

75 Pochan, 2010). Chitosan, a natural amino functionalized polysaccharides, is a biocompatible

76 and biodegradable pH-dependent polymer (Chenite, Chaput, Wang, Combes, Buschmann,

77 Hoemann, Leroux, Atkinson, Binette, ＆ Selmani, 2000; Zhang, Tao, Li, ＆ Wei, 2012), and has

78 been used as bioadhesives and nonviral vectors to deliver genes (Calvo, Vila-Jato, ＆Alonso,

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 79 1997; Koping-Hoggard, Tubulekas, Guan, Edwards, Nilsson, Varum, ＆ Artursson, 2001). Due

80 to its lower toxicity toward mammalian cells and higher antibacterial activity, chitosan was also

81 extensively used as wound healing material (Archana, Singh, Dutta, Dutta, 2013; Archana, Singh,

82 Dutta, Dutta, 2014; Archana, Dutta, Dutta, 2013). The amino group in chitosan has a pKa value

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83 of ~6.4, thus chitosan solution can be prepared via protonation of its amine group in acidic

84 environment. Its innate defect, the formation of white precipitate at the physiological conditions,

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85 could be prevented by the addition of PVA (Tang, Du, Hu, Shi,＆ Kennedy, 2007 ), which is a

86 cheap, non-toxic and water soluble polyhydroxy polymer and is considered as one of the most

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87 suitable polymers for biomedical applications. Recently, some researchers also have paid

attention to the self-healing property of PVA hydrogel (Zhang, Xia, ＆ Zhao, 2012). The

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89 combination of chitosan and PVA is selected due to some synergistic interaction including

90 hydrogen bonding and transient hydrophobic interactions, which assist in forming pH reversible
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91 and self-healable hydrogel for control drug releases at the target site. Many chitosan-based

92 hydrogels have been prepared through covalent linkages (Bhattarai, Gunn, ＆ Zhang, 2010) or
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93 non-covalent interactions, such as polyelectrolyte ( Lin, Larsson,＆ Liu, 2011; Berger, Reist,
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94 Mayer, Felt, Gurny, 2004; Ladet, David, ＆ Domard, 2008), polyhydroxy polymer (Islam ＆
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95 Yasin, 2012) , interpolymer complex (Wang, Mynar, Yoshida, Lee, Lee, Okuro, Kinbara, ＆
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96 Aida, 2010) and proteins (Foo, Lee, Mulyasasmita, Parisi-Amon, ＆ Heilshorn, 2009; Austin,

97 Brine, Castle, ＆ Zikakis, 1981). The reversible and self-healable CS/PVA hydrogel, which
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98 could be easily and rapidly formed in situ on tumor cells for localized and effective therapy, has

99 never been reported.

100 Herein, the study aims to prepare the self-healable CS/PVA hydrogel for control drug release

101 at the target site. Fluorouracil is a thymidylate synthase inhibitor and an antineoplastic. And

102 fluorouracil injection is a sterile solution with pH approximately 9.2 for intravenous

103 administration. From its structure (in Scheme 1), the polar groups in 5-FU can form multiple

104 hydrogen bonding interaction with amino and hydroxyl in CS and PVA molecules, which could

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105 be used as controlled response to external stimuli, such as pH, temperature. Furthermore, the

106 formation of hydrogel shell on tumor cells in situ, its biocompatibility and self-healing property

107 are investigated. Controlled release of 5-FU from the CS/PVA hydrogel is performed at different

108 pH values as well. The results suggest the hydrogel shell rapidly formed on cancer cells could

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109 maintain a higher 5-FU concentration and prevent the drug from diffusing to normal cells.

110 Besides, the hydrogel possesses a good drug retention ability and biocompatibility, as well as

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111 achieves continuous and controllable release of 5-FU at pH 5.0. The self-healing characteristic

112 unveiled in this work endows the ordinary CS/PVA hydrogel with a new appealing property, and

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113 holds promise for practical biomedical applications.

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114 2. Experimental Section

115 2.1 Materials and Characterization

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116 Chitosan (CS, Mw: 5.5×105, a degree of deacetylation: ~92%, provided by Maypro Industries
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117 Inc., Purchase, NY). Fluorouracil (5-FU), glacial acetic acid (HAc), polyvinyl alcohol (PVA, Mw:
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118 1750±50), glutaraldehyde, sodium hydroxide (NaOH), sodium acetate(CH3COONa), potassium

119 dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4), sodium chloride
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120 (NaCl), potassium chloride (KCl) and dimethylsulfoxide (DMSO) were obtained from Aladdin
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121 reagent database Inc. (Shanghai, P. R. China). 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-

122 tetrazolium bromide (MTT), Hoechst 33342 and propidium iodide (PI) were from Sangon
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123 Company Limited (Shanghai, P. R. China). Dulbecco’s Modified Eagle’s Medium (DMEM),

124 Dulbecco's phosphate buffered saline (PBS) were obtained from Gibco-BRL (Invitrogen Corp.,

125 Carlsbad, USA). Hela cells, an immortal human cervical cancer cells used in scientific research

126 and derived from Henrietta Lacks. All reagents were used without further purification and double

127 distilled water was used throughout the experiment to prepare the solutions.

128 FTIR spectra were recorded using a NEXUS-870 spectrophotometer (Thermo Fisher, USA,

129 frequency range from 4000 to 500 cm-1) with the KBr pellet method. UV-Vis absorption spectra

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130 of samples were collected by UV-4100 spectrophotometer (Hitachi, Japan) in the range of 200-

131 900 nm. SEM images were obtained on a S4800 ESEM FEG scanning electron microscope, with

132 the lyophilized hydrogel sprayed with a fine layer of gold. The OD values of MTT assay were

133 obtained using a RT-2100C spectrophotometric microplate reader (Rayto, Shenzhen, and PRC).

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134 Fluorescence images were performed on a DMI3000B inverted fluorescence microscope (Leica,

135 German).

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136 2.2 Preparation of self-healing hydrogel

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137 A transparent CS solution was prepared by dissolving CS (200 mg) in HAc solution (10 mL,

138 0.1M) and chilled in an ice bath. 150mg of PVA and 20mg of NaOH were dissolved into

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139 deionized water (5 mL) to prepare transparent solution. Then, PVA (5 mL, 3 wt.%) - NaOH (0.1

140 M) solution was added dropwise to the CS solution, thus a homogeneous CS/PVA precursor
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141 solution (the mass ratio of CS/PVA=4:3) was obtained. The precursor was dialysed for 24h to

142 remove small molecules and deposited in the refrigerator for future use. The self-healing
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143 hydrogel can be formed in 10 min at 37 ℃ via the addition of glutaraldehyde (5 wt.%, 5 µL) into
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144 the precursor solution.

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145 2.3 Self-healing ability test of hydrogel

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146 A hole with diameter of ~1.0 cm was punched in the middle of the CS/PVA hydrogel or CS

147 hydrogel. The photographs at different time intervals were taken to record the self-healing
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148 process of the hole in the gel. In a control experiment, the CS/PVA hydrogel was replaced by CS

149 hydrogel prepared without PVA in the same condition.

150 2.4 Drug load and release

151 5-FU loaded CS/PA precursor was prepared by adding the mixture of fluorouracil injection

152 (3mL, 25 mg/ml) and PVA aqueous solution (2mL, 7.5 wt.% ) to 10 mL of CS (2 wt.%) - HAc

153 solution. The precursor turned into hydrogel at 37℃ via the addition of glutaraldehyde (5 wt.%,

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154 5 µL). The drug loaded hydrogels were immersed into 20 mL of PBS buffer with pH 7.4 or pH

155 5.0 at 37 ℃,which was used to simulate the slightly acidic environment of tumor cells and

156 normal cell environment respectively.An aliquot (2 mL) of dialysis solution was taken out at

157 predefined time interval (range from 1 to 8 days) and an equal volume of fresh PBS was added,

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158 maintaining the total volume constant. The absorption spectra of samples were measured by UV

159 spectrophotometer at 280 nm, and the cumulative release amount of 5-FU from hydrogel can be

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160 calculated according to the standard concentration-absorbency curve of 5-FU, whose linear

161 regression equation is A=0.0485C+0.0057. All experiments repeated three times.

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162 2.5 Cytotoxicities assay of hydrogel

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163 The biocompatibility of PVA solution, CS/PVA precursor and the CS/PVA hydrogel, which

164 were filtered to remove contaminants and pathogenic bacteria with a PVDF syringe filter, were
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165 investigated in vitro by MTT assay using Hela cells. In a typical procedure, Hela cells were

166 seeded into 96-well plates at a density of 5×103 cell per well and incubated in DMEM (Gibco,
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167 USA) for 48 h (37℃, 5% CO2). Fresh culture medium containing different concentrations of
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168 PVA (0, 2.0, 4.0, 6.0 and 8.0 mg/mL) and PVA/CS precursor ( 0, 4.7, 9.3, 14.0 and 18.7 mg/mL)

169 (according to the dosage of PVA as measurement standard) were prefabricated. To test the Hela
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170 cell viability in CS/PVA hydrogel, 0, 4, 8, 12 and 16 µL of glutaraldehyde (1 wt.%) was

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171 correspondingly added into 10 mL of the culture medium containing different concentrations of

172 PVA/CS precursor. After cell attachment, 100 ! L of the fresh culture medium above-mentioned
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173 were pipetted into each well, respectively. After incubation for 12 hours, the cells were washed

174 with PBS three times, the culture medium was replaced with 40! L of MTT solution (5 mg/mL)

175 and further cultured for 4 h. The culture medium was removed and 150 ! L of DMSO was added

176 to dissolve formazan crystals, which were formed during the reductive cleavage of MTT (a

177 yellow tetrazole) by mitochondrial succinate dehydrogenases of living cells. The number of

178 viable cells was quantified by a spectrophotometric microplate reader, which is in proportion to

179 the absorbance of formazan at 490 nm. The cells only treated with culture medium were served

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180 as a negative control group. The percent reduction of MTT dye was compared to controls, which

181 represented 100% reduction of MTT dye. The experiment was repeated three times and results

182 are presented as mean ± standard deviation (SD).

183 2.6 Formation of hydrogel shell on tumor cells

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184 Hela cells were seeded into 6-well plates at a density of 5×104 cell per well and incubated in

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185 DMEM (Gibco, USA) for 48h. After cell attachment, 1 mL of CS/PVA precursor and 0.5 mL of

186 fresh culture media were added to each well. Then glutaraldehyde (1 wt.%, 2 µL) was pipetted

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187 into each well and gently mixed to induce the formation of hydrogel. The formation of hydrogel

188 shell on tumor cells and cell encapsulation were investigated using fluorescence microscope.

189 2.7 Fluorescence images

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190 For fluorescence microscope imaging, Hela cells were seeded at a density of 5×104 cells per

191 well in 6-well plates and incubated for 24 h (37℃, 5% CO2). After cell attachment, 0.5 mL of
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192 fresh DMEM and 1mL of CS/PVA precursor (loaded with or without 5-FU) were added into
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193 each well. Afterward, the cells were incubated in the DMEM containing CS/PVA precursor for

194 30 min, and were stained with 0.5 mL of Hoechst 33342 (1 ! g/mL) and 0.5 mL of PI (1 ! g/mL)
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195 for 20 min in the darkness. Dual fluorescence-stained cells were washed with PBS and observed
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196 under an inverted fluorescence microscope. All experiments were carried out in triplicate.

197 3 Results and Discussion

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198 In the current work, we prepare CS/PVA a self-healing hydrogel via a facile and cheap method,

199 and focus on evaluating its application in controlled drug release, cells encapsulation and anti-

200 tumor. The flow chart of the experimental was summarized in Fig. 1.

201

202 Fig. 1. Schematic illustration of the preparation of CS/PVA hydrogel and its application in 5-FU delivery

203 and anti-tumor.

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204

205 3.1 Formation and self-healing ability of hydrogel

206 The formation, decomposition and regeneration of CS/PVA hydrogel were shown in Fig. 2A.

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207 It is observed that the prepared precursor is homogeneous and flowing due to hydrogen bonds

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208 among PVA, chitosan and water. When the temperature elevated, the intermolecular hydrogen

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209 bonds between water molecules and polymer are weakened, and water molecules surrounding

210 the CS and PVA chains are removed, creating the chances of contacts between the dewatered

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211 hydrophobic PVA and CS chains (Tang, Du, Hu, Shi, ＆ Kennedy, 2007). With the help of a

212 small amount of glutaraldehyde, the mobility of CS chains are downshifted and hydrophobic

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213 interaction among CS chains is further enhanced. Thus the CS/PVA hydrogel is facilely formed

214 at 37°C as shown in Fig. 2A. Therefore, hydrophobic interactions between polymer chains are
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215 supposed to be the main driving force to generate and maintain the network structure of

216 hydrogels. The hydrogel decomposed into a solution after the addition of HAc solution. It may
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217 be ascribed to electrostatic repulsion among the protonated CS molecules and hydrogen bonds
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218 between polymer and water. Subsequent addition of NaOH solution neutralized the acid, the

219 hydrogel can also regenerate at 37°C. These results indicate that electrostatic repulsion and
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220 hydrogen bonds between polymer chains are probably major influence factors to reversibly form
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221 the hydrogels.

222 By punching a hole with the diameter of ~1.0 cm in both CS/PVA and CS hydrogel, the self-
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223 healing ability of CS/PVA hydrogel is shown in Fig. 2B. The hole in the CS/PVA hydrogel

224 closed gradually, and finally disappeared within 1h, as shown in Fig. 2B (a). In contrast, the hole

225 still remains in the CS hydrogel for the time period of 1 h. The perforated CS/PVA hydrogel

226 regenerated automatically without any external stimuli, which demonstrates that the hydrogel

227 possesses excellent self-healing capacity. When the network structure of the hydrogel was

228 damaged, caused by the spontaneous diffusion of water molecules, PVA and CS chains on the

229 wound surfaces could rearrange to form intermolecular hydrogen bonds with water molecules to

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230 minimize the surface energy, which accelerates the mobility and diffusion of polymer chain.

231 Then, interchain hydrogen bonds between CS and PVA are re-formed and enhanced, which can

232 lead to the dissolution of chitosan chains and the formation of mobile phase across the wound

233 surface of hydrogel (Tang, Du, Hu, Shi, ＆ Kennedy, 2007; Zhang, Xia,＆ Zhao, 2012). It could

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234 be deduced that the moving and cross-linking of polymer chains on the wound surfaces cause the

235 healing of wound surfaces and the recovery of three-dimensional network of hydrogel.

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236

237 Fig. 2. (A) Digital photographs of the formation, decomposition and regeneration of the CS/PVA hydrogel; (B)

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238 Self-healing process of (a) CS/PVA hydrogels and (b) CS hydrogel.

239

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240 3.2 FTIR spectra and SEM images

241 FTIR spectra of CS, PVA and CS/PVA hydrogel are shown in Fig. 3. It is seen from Fig. 3a
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242 that the peaks around 893.6 and 1156.7 cm-1 correspond to saccharide structure in chitosan. The

243 broad peak at 1075.9 and 3445.4 cm-1 are attributed to the typical C-O and O-H stretching
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244 vibration, respectively. The FTIR spectrum of pure PVA is shown in Fig. 3b, peaks related to
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245 hydroxyl (3443.2 cm-1) and acetate groups (1635.4 cm-1 ) are observed. It is confirmed that there
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246 is not chemical reaction between CS and PVA, as evidenced by the lack of obvious peaks on the
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247 IR spectra of the CS/PVA hydrogel in Fig. 3c. Chitosan and PVA molecules are only physically

248 entangled inside the 3D networks of the hydrogel (Tang, Du, Hu, Shi, ＆ Kennedy, 2007).
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249 Therefore, it could be concluded that hydrogen bonding and transient hydrophobic interaction

250 between chitosan and PVA chains are essential for the formation and maintenance of 3D

251 structure.

252

253 Fig. 3. FTIR spectra of (a) CS, (b) PVA and (c) CS/PVA hydrogel

254

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255 The morphologies and interior microstructure of the lyophilized hydrogels are observed by

256 SEM. As shown in Fig. 4a-d, the samples present porous morphology and a sponge-like cross-

257 section structure. The pore diameter of the hydrogel is about 10-50 ! m. From Fig. 4b and 4d, it

258 could be clearly observed that the porous structure gets more compact and the pore size becomes

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259 smaller (about 10! m), which could be ascribed to the hydrogen bonding interaction between 5-

260 FU and PVA, CS molecules.

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262

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Fig. 4. SEM images of lyophilized hydrogel (a,b) CS/PVA, (c,d) CS/PVA loaded with 5-FU.

263

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264 3.3 Cell viability

265 MTT assay was used to examine the viability of Hela cells in the presence of PVA, CS/PVA
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266 precursor and CS/PVA hydrogel. The cells were incubated for 48 h and the cell survival rates are

267 shown in Fig. 5. No obvious decrease in cell survival rate is observed during the examination of
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268 cell viability. When the concentration of PVA is 2 mg/ml, survival rate of the cell cultured in
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269 PVA, CS/PVA precursor and CS/PVA hydrogel is 98.4%, 95.4%, and 93.4% respectively. As

270 expected, the cell survival rates exhibit a downward trend with increasing PVA concentration. A
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271 possible reason for the results may be that the corresponding amount of glutaraldehyde needed
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272 will rise with the concentration increase of PVA and CS. However, even the concentration of

273 PVA reaches up to 8 mg/mL, the corresponding cell viability still reaches 87.4%, 85.6% and
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274 81.9%. Compared with PVA/CS precursor, the addition of a little glutaraldehyde seems not to

275 bring about obvious cytotoxicity to the hydrogel system. The results confirm that both PVA/CS

276 precursor and PVA/CS hydrogel are biocompatible.

277 Fig. 5. Hela cell viability incubated in PVA, CS/PVA precursor and CS/PVA hydrogel

278

279 3.4 Drug release

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280 As shown in Fig. 6, the release curve of 5-FU from hydrogel at pH 7.4 PBS buffer shows a

281 burst release for the first day and a slow release over a period of 2-5 days, then enters platform

282 stage. The final releasing amount is only 39.2%, that is, 60.8% 5-FU is retained in the hydrogel.

283 In contrast, a continuously and controllable release of 5-FU over 1-6 days at pH 5.0 can be

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284 clearly observed, nearly 79.3 % of 5-FU molecules are released within 6 days, with the final

285 cumulative releasing amount of 84.8%. The simulation of drug releasing shows the acidic

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286 environment of tumor cells could promote drug release because in acidic condition hydrogel

287 would swell easily and turn gradually into liquid, which is conductive to the penetration of drugs

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288 into tumor cells. However, in normal cell environment, the hydrogel system could retain the drug

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289 for weakening side effect. Therefore, CS/PVA hydrogel as a pH-responsive system for 5-FU

290 delivery, not only maintains a higher concentration of drug around tumor cells, but also prevents

291 5-FU from entering and killing normal cells, which greatly reduces side effects and improves the
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292 effect of antitumor.

293 Fig. 6. Cumulative release profiles of 5-FU from hydrogel at pH 7.4 and pH 5.0 in PBS buffer at 37 ℃
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294
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295 3.5 Hydrogel shell on tumor cells

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296 Furthermore, the formation of hydrogel shell on the surface of tumor cells was investigated
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297 using fluorescence microscope. The images of the bare Hela cells and cells with hydrogel

298 coatings are presented in Fig. 7a and 7b, respectively. By comparison of both figures, it is easily
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299 observed that the surface of Hela cells is covered with a homogenous and continuous hydrogel

300 shell. As we know, chitosan can be adsorbed on the surface of cancer cells (Koping-Hoggard,

301 Tubulekas, Guan, Edwards, Nilsson, Varum, ＆ Artursson, 2001), so that a CS/PVA hydrogel

302 shell form successfully in situ on the Hela cells. This kind of shell contributes to preventing drug

303 from spreading to normal tissue and making sure a high concentration of drug on the lesion site

304 to enhance the antitumor effect.

305

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306 Fig. 7. Optical microscopic images of (a) bare Hela cells and (b) cells encapsulated in CS/PVA hydrogel

307

308 3.6 Fluorescence images

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309 Fluorescence images of Hela cells stained with fluorescein Hoechst 33342/PI are shown in

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310 Fig.8 to illustrate the antitumor activity of the CS/PVA/5-FU hydrogel. As we known, Hoechst

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311 33342 can pass cell membranes freely and stain nuclear DNA in blue. PI is impermeable to intact

312 membranes, and only enters necrotic cells and late-phase apoptotic cells to stain nuclear DNA in

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313 red (Kundu ＆ Kundu, 2012; J. Zhao, Huang, Song, Zhao, Jin, Wang, ＆ Huang, 2009). All the

314 cells incubated with CS/PVA precursor are dyed in blue by Hoechst 33342 (Fig. 8a) and none of

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315 nuclei are stained in red by PI (Fig. 8b), indicating CS/PVA precursor has nearly no toxicity. Fig.

316 8 (d-f) exhibits the cells encapsulated in CS/PVA hydrogel for 12 h, few cells appear to be
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317 stained by PI (only faint red fluorescence signals), which further confirms that CS/PVA hydrogel

318 has good biocompatibility. When Hela cells were cultured in CS/PVA/5-FU hydrogels, a strong
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319 red (Fig. 8h) and a purple (Fig. 8i) fluorescence images are observed, showing the tumor cells
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320 are nearly all dead. The experiment demonstrates that CS/PVA/5-FU hydrogel system has a good

321 killing effect on tumor cells.

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322
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323 Fig. 8. Fluorescence microscopy images of Hela cells (a-c) incubated with CS/PVA precursor, (d-f)

324 encapsulated in CS/PVA hydrogel, (g-i) encapsulated in CS/PVA/5-FU hydrogels.

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325

326 4 Conclusion

327 In summary, we have facilely prepared injectable hydrogels with good self-healing using

328 chitosan and PVA as basal components. The precursor was in the form of liquid at low

329 temperature, then turned into hydrogel and remained at the lesion site when injected into human

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330 body temperature environment. The hydrogel shows good biocompatibility, could easily and

331 rapidly formed in situ on tumor cells. When loaded with 5-FU, the hydrogel can achieve the

332 localized controllable release of drugs and improve the efficacy of anti-tumor. The simulation of

333 drug releasing shows the acidic environment of tumor cells could promote drug release. Also, in

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334 acidic condition, hydrogel would swell easily and turn into liquid, which is conductive to the

335 penetration of drugs into tumor cells. However, in normal body environment, the hydrogel

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336 system could prevent 5-FU from entering and killing normal cells indicated by our experiment

337 results. Considered the biocompatible component materials and the facile preparation approach,

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338 this self-healing hydrogel may hold promise for better applications in anti-tumor therapy.

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339 Acknowledgment

340 This work is supported by the National Nature Science Foundation of China (21171001,
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341 51372004, 51202001 and 21301001), Anhui Province Key Laboratory of Environment-friendly

342 Polymer Materials and Anhui provincial natural science research projects in Colleges and
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343 Universities (KJ2012Z387).

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425

426 List of Figure Captions

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427

428 1. Schematic illustrations of the preparation of CS/PVA hydrogels and their applications in 5-FU delivery and
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429 anti-tumor.
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430
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431 2. (A) Digital photographs of the formation, decomposition and regeneration of the CS/PVA hydrogel; (B)
432 Self-healing process of (a) CS/PVA hydrogels and (b) CS hydrogel.
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433
434
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435 3. FTIR spectra of (a) CS, (b) PVA and (c) CS/PVA hydrogel
436
437 4. SEM images of lyophilized hydrogel (a,b) CS/PVA, (c,d) CS/PVA loaded with 5-FU.

438

439 5. Hela cell viability incubated in PVA, CS/PVA solution and CS/PVA hydrogel.

440

441 6. Cumulative release profiles of 5-FU from hydrogel at pH 7.4 and pH 5.0 in PBS buffer at 37℃.

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442

443 7. Optical microscopic images of (a) bare Hela cells and (b) cells encapsulated in CS/PVA hydrogel.
444

445 8. Fluorescence microscopy images of Hela cells (a-c) incubated with CS/PVA precursor, (d-f) encapsulated in

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446 CS/PVA hydrogel, (g-i) encapsulated in CS/PVA/5-FU hydrogels.

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447

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448
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449 1. Schematic illustrations of the preparation of CS/PVA hydrogels and their applications in 5-FU delivery and
450 anti-tumor.
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451
452
453 2. (A) Digital photographs of the formation, decomposition and regeneration of the CS/PVA hydrogel; (B)

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454 Self-healing process of (a) CS/PVA hydrogels and (b) CS hydrogel.
455

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456

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457 3. FTIR spectra of (a) CS, (b) PVA and (c) CS/PVA hydrogel
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458
459 4. SEM images of lyophilized hydrogel (a,b) CS/PVA, (c,d) CS/PVA loaded with 5-FU.
460
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461
462
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5. Hela cell viability incubated in PVA, CS/PVA precursor and CS/PVA hydrogel (according to the dosage
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463 of PVA as measurement standard).
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464
465 6. Cumulative release profiles of 5-FU from hydrogel at pH 7.4 and pH 5.0 in PBS buffer at 37 ℃

466
467 7. Optical microscopic images of (a) bare Hela cells and (b) cells encapsulated in CS/PVA hydrogel.
468

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469
470
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8. Fluorescence microscopy images of Hela cells (a-c) incubated with CS/PVA precursor, (d-f) encapsulated in
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471 CS/PVA hydrogel, (g-i) encapsulated in CS/PVA/5-FU hydrogels.
472
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