Engineering biocompatible hydrogels from bicomponent natural

Tel: +86-020-87112841. E-mail address: [email protected]. Address: Wushan Road 381, Tianhe, Guangzhou, P. R. China 510006 ..... hydrogels, and the r...
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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

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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

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Abstract

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Natural hydrogels have attracted extensive research interests and recently showed great

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

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

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

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

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

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morphological, mechanical and swelling properties were comprehensively investigated.

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

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

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

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drug release percentage.

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Keywords

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Self-assembly; Biomaterial; Hydrogel; Drug delivery

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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

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

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

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

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

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

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

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

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materials to fabrication methods are still demanded for creating multifunctional products

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with desirable features for drug delivery in some specific conditions.

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

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

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

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

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

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

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In

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

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

recent

years,

cellulose

nanofibers

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based

on

TEMPO

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

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effect28.

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

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

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

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

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

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

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

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

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

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

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

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adopted for the preparation of α-chitin nanofibrils.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Many previous studies have shown promising advantages by using natural

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

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

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

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

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

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

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design hydrogels18,

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

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

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

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

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

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

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

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

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

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and evaluated in phosphate buffer saline (PBS).

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MATERIALS AND METHODS

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

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

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

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

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

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

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

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

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Ltd.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Preparation of aqueous TOCNF dispersions. Cellulose nanofibers were isolated from

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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stored at 4 oC and equilibrated for 30 min.

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

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

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

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

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

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hydrogels was evaluated using the expression42 in the below:

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SR =

ms − md md

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

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md is the weight of dried hydrogels.

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

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

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

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

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

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

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were conducted in the linear viscoelastic range.

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

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

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

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an atomic force microscope (AFM)

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

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

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

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

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

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

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

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

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

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

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bleached eucalyptus pulp and TOCNF were investigated by fourier transform infrared

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

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

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

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

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

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

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

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

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

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capillary cells at 25 oC. Zeta potential was calculated from

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

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generate error bar.

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

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

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

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

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electrophoretic mobility via

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

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

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

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

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

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M 5− FU = c 0ν 0 - c1ν 1

(2)

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LE 5− FU =

M 5- FU M total -5- FU

(3)

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

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

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

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

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

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

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

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

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

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

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by the following equation44: n −1

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CPDR% =

V0 ∑1 C i + V0 C n M CPDS

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

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

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

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was sucked out.

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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negatively-charged

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

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

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

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

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

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

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

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the protonation of amino groups of chitin in acidic conditions31.

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

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

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

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

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

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

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

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

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

TOCNF

suspension,

the

α-DECHN/TOCNF

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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degree45, 46.

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

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

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

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

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

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

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

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

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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

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

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

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hydrogels.

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

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

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

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

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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

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

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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

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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

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

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

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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

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

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

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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

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

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

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

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

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

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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

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

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

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

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

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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

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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

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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

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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

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

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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].

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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

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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.

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Release profiles of

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Figure Graphics Figure 1

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