In situ formation of chitosan–gold hybrid hydrogel and its application for drug delivery

Colloids and Surfaces B: Biointerfaces 97 (2012) 132–137

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

In situ formation of chitosan–gold hybrid hydrogel and its application for drug delivery Rui Chen b , Qi Chen b , Da Huo a , Yin Ding c , Yong Hu a,∗ , Xiqun Jiang b,∗∗ a

National Laboratory of Solid State Microstructure, Department of Material Science and Engineering, Nanjing University, Nanjing 210093, PR China Laboratory of Mesoscopic Chemistry and Department of Polymer Science and Engineering, College of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210093, PR China c State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China b

a r t i c l e

i n f o

Article history: Received 26 December 2011 Received in revised form 13 February 2012 Accepted 28 March 2012 Available online 27 April 2012 Keywords: Chitosan Gold Hydrogel Controlled drug delivery

a b s t r a c t A novel chitosan–gold (CS–Au) hybrid hydrogel was developed from chitosan and chloroauric acid in aqueous solution. Its physiochemical characteristics, including UV absorption, structure, morphology, swelling properties were studied. The CS–Au hybrid hydrogel exhibited an excellent water-absorbing property and could be applied as a drug delivery system for anticancer drug: doxorubicin (DOX) due to its high equilibrium water swelling content. The drug loading and release experiments elicited an efficient drug loading content and sustained drug release pattern. Moreover, DOX released from hydrogel which itself had no cytotoxicity was biological active similar as the free DOX, but lower cytotoxicity due to its controllable release. All proved it an ideal local drug delivery system indicating a promising potential future in medical or pharmaceutical area. © 2012 Elsevier B.V. All rights reserved.

1. Introductions Normally, hydrogels are three-dimensional networks of hydrophilic polymers with porous structure, which are not soluble in water but can absorb and retain a lot of water or other guest molecules inside the pores [1]. Because of their high water content, hydrogels have a “soft and wet” form just like biological material after water-absorbing. The soft and wet surface as well as the organization affinity greatly reduce the material’s irritation to the surrounding tissue, and mimic the extracellular matrix similar to the macromolecular-based components in the human body [2], providing good biocompatibility to the hydrogels, which offers their wide applications in biomedical field, including wound dressing [3] tissue engineering [4], and drug carriers [5]. Practically, they make a volume change with water filled in the cavity of networks or drained by the shrinkage of networks accompanying with the variation of the extra condition, such as pH [6], light [7], temperature [8] and ionic strength [9]. This stimuli-responsive property makes them having many potential applications, such as smart drug delivery systems, that release drugs by diffusion through their porous structure under specific stimulation [10]. Wide ranges of hydrophilic polymers have been examined as potential candidates

∗ Corresponding author. Tel.: +86 025 83594668; fax: +86 025 83594668. ∗∗ Corresponding author. Tel.: +86 025 83597138; fax: +86 025 83597138. E-mail addresses: [email protected] (Y. Hu), [email protected] (X. Jiang). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.03.027

to synthesize such hydrogels. Among them, natural born polymer, such as chitosan is thought to be the best candidate to synthesize these hydrogels. Chitosan is the deacetylated derivative of chitin (the natural polymer extracted from shrimp or crab shells), and has been extensively used in drug delivery systems, gene therapy, and tissue engineering [11–13]. This is not only because of its low price, excellent biocompatibility, biodegradability, low toxicity, but also its unique cationic property and facile functionalization with various molecules due to amino and hydroxide groups in chitosan. CS has positive charges in acidic solution and form noncovalently cross-linked interpenetrating or semi-interpenetrating network hydrogels through the hydrogen or static electronic interaction with polyanions, such as polyacrylic acid [9], polymethyl methacrylate [14], poly(vinyl alcohol) [15]. To increase the water solubility of hydrophobic drug inside the CS based hydrogels, carboxymethyl–hexanoyl chitosan mediated hydrogel were fabricated by modifying CS molecules with functional materials [6]. Although, these non-covalently cross-linked chitosan hydrogels showed good swelling ability in water, high loading ability for both hydrophilic and hydrophobic drugs, and potential application in drug delivery and tissue engineering, their lower mechanical strength and stability are not satisfied with the practical clinical applications [16]. Consequently, CS hydrogels covalently cross-linked by UV irradiation [17], or glutaraldehyde [18], with better stability were received increasing interests. These covalent cross-linked hydrogels have good mechanical strength because

R. Chen et al. / Colloids and Surfaces B: Biointerfaces 97 (2012) 132–137

of its irreversible chemical cross-linking. However, the majority of cross-linking agents used in these covalent cross-linked hydrogels, such as glutaraldehyde are a certain degree of toxicity and poor biocompatibility, and therefore to some extent limit the biological application of these hydrogels [19]. Furthermore, the cross-linking procedure of hydrogels greatly reduces their biodegradability with a dense cross-linking degree since these hydrogels are linked by irreversible bonds, accompanying with side effect after the administration of such systems in human being. Therefore, stimuli-responsive hydrogels with good biocompatibility, biodegradability, as well as good mechanical strength are highly desirable. Herein, regarding to the reduction of AuCl4 − ions by chitosan in situ, we develop a new type of hydrogels based on chitosan by a physical cross-linking method. With the injection of chloroauric acid into CS solution, chitosan–gold (CS–Au) semi-interpenetrating hybrid hydrogels were fabricated instantly upon the simultaneous formation of Au nanoparticles, which acted as physical crosslinking agents, without the additional chemical cross-linking agent or reducing agent. Considering the bio-inert, biocompatible and superior electronic and optic properties of gold nanoparticles, this physical cross-linking CS–Au hybrid hydrogel combines the merits of both CS and gold nanoparticles, which will great expand their application in biomedical field. 2. Materials and methods 2.1. Materials Chitosan (CS) (Nantong Shuanglin Biological Product Inc.) was refined twice as following procedure. Chitosan was first dissolved in dilute acetic acid solution (1%, wt/v) and the solution was filtered with paper filter (5 ␮m). Then, the filtered liquor was poured into excess aqueous NaOH solution to precipitate CS. The obtained precipitation was washed with distilled water three times. Finally, the CS was dried in vacuum at room temperature for use. The deacetylation degree of CS was 88% determined by acid–base titration method [20], and the average molecular weight of chitosan was 80 kDa determined by viscometric methods [21]. Acrylic acid (AA) (Shanghai Guanghua Chemical Company) was distilled under reduced pressure in nitrogen atmosphere. Doxorubicin hydrochloride (DOX·HCl) was purchased from Shanghai Aladdin Reagent Co. HAuCl4 (Shanghai Chemical Reagent Co.) and 3-[4,5-dimehyl2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma) were used as received. All other reagents are of analytic grade. 2.2. Preparation of CS–Au hybrid hydrogels 0.60 g CS and 0.33 g AA were dissolved in 20 mL distilled water at room temperature. After the CS was completely dissolved, this solution was heated to 70 ◦ C, and added with 1 mL of 1% (wt/v) of HAuCl4 solution. Immediately, a yellow hydrogel was obtained. 10 min later, the color shift from yellow to wine red, and the reaction temperature of the hydrogel was cooled to room temperature. 2.3. Hydrogel equilibrium swelling experiment Equilibrium swelling ratio of this hybrid hydrogel was examined by a classic weight measurement method. Firstly, the dried hydrogel was immerged in distilled water under room temperature and stored for more than 24 h to reach a swelling equilibrium. Then, this hydrogel was taken out from the water and the surface of this hydrogel was wiped with filter paper to eliminate the absorbed

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water on the surface of hydrogel. The equilibrium swelling ratio (SR) at equilibrium state was termed as following equation: SR =

Wwet × 100% Wdry

where Wwet and Wdry are the weights of the hydrogel after and before the swelling test. 2.4. Loading doxorubicin on hydrogel 15 mg freeze-dried hydrogel was soaked in 5 mL distilled water in vials and different amounts of DOX were dissolved in the vial respectively. These vials were stored under room temperature for 2 days to insure that the hybrid hydrogel reached the equilibrium state. The drug loading amount was determined by the difference between the initial feeding amount of DOX and the amount of the DOX in the solution. The concentration of DOX was measured with UV–vis spectroscopy at a wave length of 480 nm (Optizen 2120UV, Mecasys, China) and the amount of DOX was calculated from the standard curve. All processes were carried out under dark conditions. 2.5. Release of doxorubicin 5 mg drug-loaded hydrogel was placed into a dialysis bag with a molecular weight cut-off of 12,000 Da. The dialysis bag was immersed in 25 mL phosphate buffer solution (PBS, 0.1 M, pH 7.4) in the plastic tube and stirred continuously at 100 rpm in a vibrating incubator at 37 ◦ C. 5 mL release medium was periodically replaced by 5 mL fresh medium. These collected samples were assayed by spectrophotometry at 480 nm to measure the released amount of DOX based on the standard curves. 2.6. Cytotoxicity of the hydrogel MTT assay was performed to investigate the cytotoxicity of the hydrogel by the elution method according to the literature [6]. 100 ␮L C6 cells in Dulbecco’s modified Eagle medium (DMEM) with a concentration of 5.0 × 104 cells/mL−1 was added to each well in a 96-well plate. After incubation for 24 h in an incubator (37 ◦ C, 5% CO2 ), the medium was replaced with different amounts of extracted drug solution extracted fluids from the CS–Au hybrid hydrogel, and the mixture was further incubated for 24 h. The culture medium was replaced by fresh DMEM, and 10 ␮L of MTT solution (5 mg mL−1 ) was added. After incubation for 4 h, 110 ␮L of DMSO was added to each well and shaken at room temperature. The optical density (OD) was then measured at 570 nm using SAFIRE (XFLUOR4, TECAN). The viable rate was calculated after subtracting OD control of DMEM. Preparing of drug solution extracted from the CS–Au hybrid hydrogels: both the blank hydrogel and the DOX loaded hydrogel are soaked in fresh PBS for 48 h, and then the solution was filtrated through 0.22 ␮m cellulose acetate filter membrane to remove hydrogel network. The filtrate is diluted by PBS pH 7.4 in different drug concentration. 2.7. Characterization Freeze-dried hydrogel mixed with BaSO4 powder were pressed to thin film and pure BaSO4 powder was used for background correction. UV spectra were recorded on a Shimadsu UV-2401 spectrophotometer over the 350–700 nm. Freeze-dried hydrogels coated with gold were directly analyzed by scanning electron microscope (JEOL JSM-6700), to obtain the surface morphology of these CS–Au hybrid hydrogels. JEOL JEM-100S transmission electron microscope was used to observe the morphology of the

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0.8 532 nm

0.5% 1.0% 1.5% 2.0% 2.5%

0.7

Absorbance

0.6 0.5 0.4 0.3 0.2 0.1 0.0 350

400

450

500

550

600

650

700

Wavelength (nm) Fig. 2. Solid UV–Vis absorption spectra of CS–Au hydrogel.

too small to be seen in the SEM image mainly because of the large scale bar. 3.2. UV–vis spectrum analysis of the hydrogel

3. Results and discussion

Solid UV–vis absorption spectra were used to measure the UV spectra of these CS–Au hybrid hydrogels with different feeding amounts of HAuCl4 as depicted in Fig. 2. A strong absorption peak centered at 530 nm relative to the characteristic surface plasma resonance peak of gold nanoparticles is shown in Fig. 2. As the concentration of feeding HAuCl4 solution increases, a broadening and red shift band with stronger strength is clearly observed in the UV spectra, which indicates the formation of larger gold nanoparticles with broaden size distribution. These results confirmed the instant formation of gold nanoparticles inside the hydrogel due to the feeding amount of HAuCl4 .

3.1. Transition electron microscopy analysis

3.3. The mechanism of formation CS–Au hybrid hydrogel

In present work, chitosan was dissolved in acrylic acid solution, and was heated to about 70 ◦ C. After that, HAuCl4 solution with different concentration was injected into this solution rapidly. A wine red hydrogel was formed instantly in the solution. Fig. 1a shows the transmission electronic microscopy of freeze-dried CS–Au hydrogels with the addition of 1 mL of 1.0% HAuCl4 solution (if not stated, the CS–Au hydrogel was obtained with the addition of 1.0% HAuCl4 in following experiments). It is clear to see that this hydrogel presents a highly irregular porous structure with a pore size of 100 nm inside the hydrogel. These pores connect with each other and form a network structure. Inside these pores, small dark spots are clearly observed, related to the gold nanoparticles. The size of gold nanoparticles is about 40 nm and they locate at the edge of the pores. These gold nanoparticles functionalize as physical cross-link agents to strength this hydrogel. This network like structure provides functional channels for water molecules to facilitate them in and out from the hydrogel, leading a possibility of absorbing a large amount of water in the hydrogel. This characteristic porous structure was confirmed by examining the cross-section of the CS–Au hydrogel by the scanning electronic microscopy as shown in Fig. 1b. Although the pore size in the SEM image is bigger than that in the TEM, the morphology of the macroporous structure is homogeneous throughout the entire monolith section, exhibiting a cellular-patterned like structure in the SEM image. The size difference of pore size can be attributed to the different measurement methods. The gold nanoparticles were

Chitosan is widely used in biomedical fields, and it can form hydrogels with different materials or by itself in aqueous solution [22–24]. Different methods were developed to synthesize chitosan hydrogels with special functions. In this work, chitosan hydrogel was formed instantly after HAuCl4 was injected into the chitosan solution. In acrylic acid solution, chitosan was dissolved and its amino group was positively charged. When chloroauric acid was injected into this system, it provided lots of negatively charged AuCl4 − ions and the positively charged amino groups from chitosan were neutralized by the negatively charged AuCl4 − ions, resulting in a reduced water solubility of the chitosan. Additionally, the presence of AuCl4 − provided a strong static electronic interaction between chloroauric acid and chitosan. Combining these two factors, a viscous solution was obtained instantly. After that, the color of the mixture turned from yellow to wine red, and solid hydrogel appeared in the solution accompanying with the color change. This color change combined with the results of UV spectra clearly indicated the formation of zerovalent Au or Au nanoparticles inside the hydrogel. AuCl4 − ions could be reduced to zerovalent Au NPs by the chitosan itself in hot acidic solution [25,26]. In this system, some of the AuCl4 − ions were reduced to zerovalent Au domains instantly after the injection of chloroauric acid, acted as nucleation site to form big Au nanoparticles. The presence of deprotonated or neutralized amine groups in the CS molecules is important for stabilization of reduced gold due to the high affinity of gold to amine groups

Fig. 1. (a) TEM and (b) SEM of freeze-dried CS–Au hydrogel.

hydrogels samples. Hydrogel was directly coated on copper grids and dried in air before examination.

R. Chen et al. / Colloids and Surfaces B: Biointerfaces 97 (2012) 132–137

3.4. Swelling properties of hydrogels The swelling ability of Au–CS hybrid hydrogel was characterized with the equilibrium swelling ratio (SR) in the distilled water at room temperature. These hybrid hydrogels showed excellent water absorbing ability with a SR around 300 no matter with the preparation condition. This high water absorbing ability mainly attributed to the highly irregular porous structure with a large amount of pores inside the hydrogels. Besides, the chitosan is also highly hydrophilic, which greatly enhances the water absorbency for the hydrogel. The further study of the hydrogel swelling performance after drying is carried out by a re-swelling kinetic experiment. Fig. 3 illustrates the re-swelling kinetics of freeze-dried hydrogels in deionized water at 37 ◦ C. It shows that the water absorption rate rises fast during the first hour. After 10 h, it reaches 60% compared to the original one, and reaches 90% after 30 h incubation, very close to the initial water absorption value. During the re-swelling procedure, the water molecules penetrated into the hydrogel, and subsequently, the hydrogel network expanded because of the hydration and relaxation of CS molecules. The fast re-swelling rate of CS–Au hydrogel is mostly attributed to the porous interconnected structures, which can provide many entrance channels for the diffusion of water molecules. The reversible water absorption ability proves that the hybrid CS–Au hydrogel has good mechanical strength and is stable in aqueous solution.

100 90 80

Water Uptake (%)

[27]. Furthermore, the surfaces of gold nanoparticles are negatively charged, the remaining protonated amino groups also protected these Au domains through the static electronic interaction to separate out from the solution [28]. As the heating time extended, more and more AuCl4 − ions were reduced to zerovalent Au0 , and deposited on the former Au domains, resulting in a size enlargement to these Au domains and transformed to Au nanoparticles. Because of the strong interaction between Au nanoparticles and amino groups, CS–Au hydrogel was formed in the solution and Au nanoparticles worked as physical cross-linking points. The suggested mechanism of the formation of gold nanoparticles is presented in Scheme 1. Firstly, CS was dissolved in acrylic acid and dispersed homogeneously in the solution in molecular state. A yellow solution was obtained as shown in Scheme 1. After the injection of chloroauric acid, a brown soft hydrogel was obtained. In this stage, most of the AuCl4 − ions were neutralized by the protonated amino groups, and some of them were reduced to zerovalent Au0 domain, which acted as crosslinking point in the hydrogel. Further extending the reaction time made almost all the AuCl4 − ions reduce to zerovalent Au, resulting bigger Au nanoparticles, and the strong interaction between Au nanoparticles and amino groups conferred the good mechanical properties to this hydrogel. The color of CS–Au hybrid hydrogel changed from brown to wine-red due to the characteristic surface plasma resonance (SPR) effect of Au nanoparticles. During the preparation procedure, a minimum concentration of CS was required to successfully synthesize the CS–Au hybrid hydrogel. If the concentration of CS was lower than this value, no hydrogel could be obtained. This result indicated that only the concentration in the solution was high enough to ensure the neighboring CS molecular chains could entangle with each others, the formed Au nanoparticles could acted efficiently as physical crosslinking points, leading to the gelation phenomenon. Alternatively, when using acetic acid instead of acrylic acid dissolving CS, we could get the CS–Au hybrid hydrogel with the similar experimental condition. Therefore, the effect of polymerization of acrylic acid and the formation of CS and polyacrylic acid hydrogel can be ignored.

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70 60 50 40 30 20 10 0 0

5

10

15

20

25

30

Time (h) Fig. 3. Re-swelling experiment of freeze-dried CS–Au hydrogen.

3.5. Drug loading and in vitro cytotoxicity Hydrogel is a kind of soft biomaterial and has been widely used in biomedical fields. In this work, we proposed that this CS–Au hybrid hydrogel was a good candidate to be used as implant drug release system. For this end, doxorubicin hydrochloride (DOX·HCl), one of most wide used anticancer drug was selected as the model drug loaded inside this hydrogel, to investigate the encapsulation, release and in vitro cytotoxicity on C6 cells. Table 1 lists the drug-loading content of hydrogel with different drug feeding ratios. With increasing the feeding amount of DOX·HCl, the drugloading content increased while drug-loading efficiency decreased. The low drug loading efficiency of DOX·HCl is mainly because of its high water solubility. In this work, the high drug loading content (19.77%) sample was used for the drug release experiment. Fig. 4 shows the cumulative release of DOX from CS–Au hydrogels at 37 ◦ C in PBS solution. An initial burst release of doxorubicin is observed in the hydrogel system during the first release stage. About 20% of the loaded DOX inside the hydrogel is released into the medium in the first hour, and then another 20% of DOX is released from the hydrogel in the following 7 h. After that, the CS–Au hydrogel shows a sustained release profile within the next 80 h and reaches a flat, where about 60% of the loaded DOX releases to the medium. After that, no more DOX released from the CS–Au hydrogel. We thought the stabile gel network would retard the diffusion of the DOX through the deep gel pores into the release medium. Therefore, we increase the incubation temperature to destroy the hydrogel structure, aiming to accelerate the release rate (in our experiment, we found that the hydrogel would break to pieces when it was maintained at 70 ◦ C less than 1 h). However, the cumulative release did not exceed 65% after additional 30 h incubation, even though no integrate hydrogel was seen inside the release medium. So it was assumed there was strong interaction between DOX and the CS–Au hydrogel that retarded the release of DOX in the PBS solution. In the solution of pH 7.4, the CS is in the de-protonated state and it was negatively charged (pKa of 6.5), while for DOX, it Table 1 Drug-loading content and efficiency of hydrogel. Freeze-dried hydrogel (mg)

Doxorubicin content (mg)

Drug-loading content (wt.%)

Drug-loading efficiency (%)

15 15 15 15 15

0.2 1 2 5 10

0.72 3.57 5.97 12.46 19.77

54.09 53.50 44.76 37.37 29.66

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Scheme 1. CS–Au hydrogel formation mechanism.

is positively charged (pKa of 8.6). The CS and DOX can form complex through the static electronic interaction, which responds the incomplete release of DOX from the CS–Au hydrogel. Therefore, strong acidic condition was performed to destroy this static electronic interaction by incubating the DOX loaded hydrogel in PBS with pH 3.0. Surprising, a burst DOX release was observed when this hydrogel was immersed in the release medium. After 30 h (from

100

Cumulative Release (%)

80 60

b

a

40 20 0 0

50

100

150

200

250

Time (h) Fig. 4. Cumulative release of DOX·HCl from CS–Au hydrogel at 37 ◦ C and pH 7.4. Point a: increase temperature to 70 ◦ C for 1 h, then re-incubate the hydrogel in PBS at 37 ◦ C; Point b: the pH value of PBS was changed to 3.0.

point b) incubation, more than 90% of loaded DOX released into the medium, which confirmed that the static electronic interaction between CS and DOX resulted in the retarding release of DOX from the hydrogel. The above results indicate that CS–Au hydrogel is an ideal sustained drug release system, and the model drug release behavior is influenced by the external stimuli, such as temperature or pH. So this hydrogel might be used as smart drug release system. This kind of hydrogel was supposed to be candidate as drug carrier in biomedical fields. Therefore, its cytotoxicity was investigated by MTT assay. Fig. 5 shows the cytotoxicity of blank CS–Au hydrogel. During all the tested concentration of hydrogel, the C6 cells maintained their viability over 100%, which clearly indicated that the blank hydrogel has no apparent cytotoxicity on C6 cells, showing the good biocompatibility of this hydrogel. The cytotoxic results of free DOX and DOX loaded hydrogel against C6 cells are presented in Fig. 6. The free DOX and DOX released from the CS–Au hydrogel both have biological activities and show high cytotoxicity to the C6 cells. However, the DOX loaded hydrogel shows lower cytotoxicity compared to the free DOX with the same drug dose, which is because that the actual concentration of DOX in the medium released from DOX loaded CS–Au hydrogel is lower than the free DOX due to the sustained release behavior of CS–Au hydrogel. With the increase of initial drug concentration, the cytotoxicity of both free DOX and DOX loaded hydrogel enhances. This primary results of in vitro drug release and cytotoxicity results indicated the potential application of this CS–Au hydrogel in biomedical fields. Considering the stimuli responsibility and the positive charge properties of CS, this hydrogel may be extensively used as a smart drug delivery system.

R. Chen et al. / Colloids and Surfaces B: Biointerfaces 97 (2012) 132–137

days. Furthermore, the drug releasing property was responsible to the external pH stimuli, which showed its potential application for smart drug delivery. The in vitro cytotoxicity experiments confirmed that this hydrogel showed no toxicity to C6 cells and doxorubicin maintained its biological effect to kill the tumor cells when doxorubicin was released from the hydrogel.

120 100

Cell viabity (%)

80

Acknowledgements

60

This work is supported by the National Natural Science Foundation of China (Nos. 21074051, 51033002 and 20874042), Fundamental Research Funds for the Central Universities (1085021309), and the Provincial Natural Science Foundation of Jiangsu Province (BK2010301). Dr. Hu also thanks the support by Alexander von Humboldt Foundation.

40 20 0 25

12.5

100

50

200

Concentration( g/mL) Fig. 5. Cell viability of blank hydrogel.

100 Hydrogel loaded DOX Free DOX

90 80 70

Cell viabity (%)

137

60 50 40 30 20 10 0 1.25

2.5

5

10

20

40

Concentration of doxorubicin( g/mL) Fig. 6. Cell viability of DOX loaded hydrogel and free DOX.

4. Conclusions A new type of hybrid CS–Au hydrogel was simply fabricated in aqueous solution through in situ reduction of HAuCl4 in the CS solution. The hydrogel had irregular porous structure and high water-absorbing ability. Au nanoparticles existed in pores, acting as a role of physical cross-linking agents. This physical cross-linked hydrogel showed excellent drug loading ability when doxorubicin used as a model drug. The drug loading content could be controlled by the drug feeding content. The encapsulated doxorubicin could be released from the hydrogel with a sustained manner over 10

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