Materials Science and Engineering C 50 (2015) 294–299

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

MWNT-hybrided supramolecular hydrogel for hydrophobic camptothecin delivery Shansong Mu a, Yuanyuan Liang b,c, Shuaijun Chen d, Liming Zhang ⁎,e, Tao Liu ⁎,d a

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China College of chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China d Department of Otolaryngology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China e Institute of Polymer Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China b c

a r t i c l e

i n f o

Article history: Received 3 January 2015 Received in revised form 29 January 2015 Accepted 11 February 2015 Available online 13 February 2015 Keywords: Supramolecular hydrogel Carbon nanotube Camptothecin High loading Sustained release

a b s t r a c t To encapsulate the hydrophobic camptothecin (CPT) into hydrogel matrix with a high loading amount, a supramolecular hydrogel hybrided with multi-walled carbon nanotubes (MWNTs) was developed by the host-guest interactions and used for loading and delivering CPT. Firstly, carboxylated MWNTs were modified by polyethylene glycol monomethyl ether (MPEG), which resulted in the water-dispersed MPEG-MWNTs. Then α-cyclodextrin (α-CD) was mixed with MPEG-MWNTs and the hybrid supramolecular hydrogel was fabricated by the inclusion interactions between α-CD and MPEG. The used MPEG not only dispersed MWNTs in aqueous solution, but also functioned as hydrogel matrix by interacting with α-CD. The gelation time for the sol–gel transition and rheological properties of the resultant hydrogels were studied. Due to the excellent application of MWNTs in drug delivery, hydrophobic CPT could be loaded into the hydrogel matrix by a higher amount compared with micelles. By in vitro release and cell viability tests, it was found that the encapsulated CPT could exhibit a controlled and sustained release behavior as well as sustained antitumor efficacy. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Conventional forms of drug delivery via long-term frequent oral dosing or injections, such as micelles, polymer conjugates, microspheres, electrospun fibers, hybrid nanoparticles and others, can be used to maintain a constant drug concentration in blood for a specific amount of time [1–5]. Among them, the supramolecular hydrogels based on the host-guest interactions between α-cyclodextrin (α-CD) and PEGylated polymers have attracted much attention for the application in drug delivery due to their high permeability to drugs, high loadings, controlled and sustained releases of drugs, good biocompatibility and so on [6–10]. A series of supramolecular hydrogels have been reported for the controlled release of drugs. For example, Simoes et al. encapsulated vancomycin hydrochloride into the supramolecular hydrogel based on α-CD and Pluronic F-127. The formed hydrogel could sustainedly release vancomycin hydrochloride to provide prolonged antibacterial effect [11]. Higashi et al. used the high molecular weight PEG to interact with α-CD directly, and lysozyme could be sustainedly release from the obtained hydrogel [12]. Li et al. synthesized a MPEG-PCL-PDMAEMA block copolymer to interact with α-CD, and the formed hydrogel could be used for gene delivery [13]. ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Zhang), [email protected] (T. Liu).

http://dx.doi.org/10.1016/j.msec.2015.02.016 0928-4931/© 2015 Elsevier B.V. All rights reserved.

As a hydrophilic matrix, most supramolecular hydrogels were used to deliver hydrophilic drugs, proteins or genes, while the applications in hydrophobic drug delivery were less concerned. Camptothecin (CPT) is known as topoisomerase-I inhibitors exhibiting high antitumor activity against a wide spectrum of human malignancies, such as lung, prostate, breast, colon, stomach, and ovarian carcinomas [14]. Ma et al. used the supramolecular hydrogel to load hydrophobic CPT. The amphiphilic Pluronic F-127 was used to encapsulate CPT into micelles and then interact with α-CD to form the hydrogel. However, the loading amount of CPT was low (0.134 mg/g) [15], which limits the application of these supramolecular hydrogels. Recently, the carbon nanotubes (CNTs) have attracted much attention in drug delivery due to their low cytotoxicity, good penetration ability and high drug loading amount [16]. Many works have been reported to use the CNTs to deliver hydrophobic drugs and showed the high loading amounts [17–21]. In this work, the multiwalled carbon nanotubes (MWNTs) were hybrided in supramolecular hydrogel to improve the hydrophobic drug loading amount of the hydrogel. MWNTs were grafted by MPEG, and then mixed with α-CD to form the hybrid hydrogel network by the inclusion interaction between α-CD and MPEG. The used MPEG could act not only as effective stabilizing agents for the MWNTs, but also as the guest molecule for the supramolecular self-assembly with α-CD. The gelation time for the sol–gel transition and rheological properties of the formed gel were studied. And the in vitro release and cell

S. Mu et al. / Materials Science and Engineering C 50 (2015) 294–299

viability of loaded CPT were also tested. Moreover, the hydrogel had good biocompatibility, indicating a promising application in sustained drug delivery.

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in water, its morphology was observed by a JEM-2100 transmission electron microscope (TEM, JEOL, Japan). TEM samples were prepared by dropping colloid on copper grids and dried overnight at room temperature.

2. Experiment section 2.4. CPT-loaded supramolecular hydrogel 2.1. Materials Poly(ethylene glycol) methyl ether with the molecular weight of 5000 (MPEG-5k) was purchased from Sigma and used after dried under vacuum at 45 °C for 24 h. α-Cyclodextrin (α-CD) was purchased from Tokyo Chemical Industry Company in Japan. Carboxylic Multiwalled carbon nanotubes (MWNTs-COOH) with 2.0 wt.% carboxyl were purchased from Chengdu Organic Chemicals Co. Ltd., Chinese Academy of Science. The MWNTs-COOH was dried at 45 °C for 24 h before use. Thionyl chloride (SOCl2) was purchased from Guangzhou Chemical Reagent Company and used directly. The other chemicals were of analytical grade and used without further purification. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS) and 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Invitrogen Corp. The human nasopharyngeal carcinoma HNE-1 cells were supplied by Southern Medical University. 2.2. Synthesis of MPEG-grafted MWNTs (MPEG-MWNTs) Carboxylated MWNTs (100 mg) were stirred in a mixture of SOCl2 (25 mL) and dimethylformamide (DMF, 5 mL) at 70 °C for 24 h. After centrifugation at 12,000 r/min for 20 min, the brown supernatant was removed and the remaining solid was washed with dried tetrahydrofuran. The solid was dried at room temperature under vacuum to obtain SWNTs-COCl2. SWNTs-COCl2 (100 mg) and MPEG-5 k (1000 mg) were mixed in DMF (20 mL) and stirred for 40 h at 80 °C under argon atmosphere. After that, to remove the unreacted MPEG, the resulted solution was filtrated with a Millipore PTFE membrane filter with a 450 nm pore size and the filter residue was repeatedly washed with distilled water. The black filter residue, MPEG-MWNTs, was retained after drying at vacuum. Its chemical structure was characterized by FT-IR (Nicolet/Nexus 670). For composition analysis, MPEG-MWNTs were test by thermogravimetric analysis from 50–700 °C under nitrogen with a heating rate of 10 °C/min.

For hydrogel formation, 8.0 wt.% CPT-loaded MPEG-MWNT dispersion was prepared firstly, and then mixed with an equal volume of aqueous α-CD solution with required concentrations. After mixed around adequately, the mixture was set aside for one night at room temperature for the formation of supramolecular hydrogels. To investigate the gelation kinetics of the aqueous MPEG-MWNTs/ α-CD system, time-sweep rheological analysis was performed by an Advanced Rheometric Extended System (ARES, TA Co.) in oscillatory mode with parallel plate geometry (25 mm diameter, 1.0 mm gap) at 25 °C. In this case, the samples were placed on the plate immediately after mixed together and the measurement began 2 min thereafter. The viscoelastic parameters were measured as a function of time within the linear region previously determined by a strain scan. To investigate the mechanical property of the resultant hydrogel, frequency sweep rheological analysis was conducted by the same ARES. In this case, the hydrogel sample was allowed to consolidate for 12 h before beginning the analysis. The frequency applied to hydrogel sample increased from 0.1 to 100 rad/s with a strain of 0.05%. In addition, X-ray diffraction (XRD) measurements were performed using a Rigaku D/max-2200 type X-ray diffractometer fitted with Ni-filtered Cu Kα radiation at a wavelength of 0.154 nm to confirm the inclusion complex formed from MPEG-MWNTs and α-CD. 2.5. In vitro drug release To study the in vitro CPT release behavior, 300 μL CPT-loaded hydrogel was immersed in 1000 μL phosphate buffer saline (PBS, 0.01 mol/L, pH = 7.4) at 37 °C containing 10% Span 80, and the release profiles were studied. At predetermined time points, 500 μL of the medium solution was taken out and 500 μL fresh PBS was added back to maintain the same total solution volume. The amount of the released CPT was determined according to a method reported previously using a UV spectrophotometer (S52, China) at the absorbance wavelength of 369 nm [15]. All release studies were carried out in triplicate. The standard curve equation was as follows:   2 Y ¼ 12:4  X þ 0:027 R ¼ 0:999

2.3. CPT loaded MPEG-MWNTs MPEG-MWNTs (1000 mg) were added into 50 mL aqueous solution and were treated with ultrasonic for 5 min. CPT (200 mg) was dissolved in methanol (50 mL) and was added into the MPEG-MWNT solution dropwise. The mixture was stirred at 37 °C for 12 h and dialyzed in distilled water for 3 days. The dialysate was centrifuged at 200 r/min for 5 min to remove the free CPT and then frozen to dry to obtain the MPEG-MWNTs/CPT. The loading amount of CPT was calculated by weighing the dried free CPT collected by centrifugation. For the MPEG-MWNTs/CPT dispersion

where Y was the absorbency at 369 nm, and X was the CPT concentration. 2.6. Cell viability assay MTT assay was carried out to evaluate the sustained antitumor activity of the CPT-loaded hydrogels. PBS and blank hydrogel were set as control. The human nasopharyngeal carcinoma HNE-1 cells were cultured onto a 24-well plate (2 × 10 5 cells/well) in DMEM

Scheme 1. Synthesis routes to MPEG-MWNTs.

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MWNTs

MPEG-MWNTs

Weight ratio (%)

Transmittance (a.u.)

MPEG

100

MWNTs

80

MPEG-MWNTs

60 40 20 MPEG

0 500 1000 1500 2000 2500 3000 3500 4000 -1

Wavenumber (cm )

100

200

300

400

500

600

700

o

Temperature ( C)

Fig. 1. FT-IR spectra of MPEG, MWNTs and MPEG-MWNTs. Fig. 3. TG curves of MPEG, MWNTs and MPEG-MWNTs.

(10% fetal bovine serum supplemented) culture medium in a humidified atmosphere of 5% CO2 at 37 °C for 24 h. The growth medium was replaced with 500 μL complete DMEM culture medium that contained freshly-prepared hydrogel samples, and five multiple wells were set for every sample. At predetermined time points, fresh DMEM culture medium was used and cell viability of five multiple wells was assayed by adding MTT (Sigma) solution (5 mg/mL). After incubation at 37 °C for another 4 h, the formed crystals were dissolved in 200 μL of DMSO. The absorbance that correlated with the number of viable cells in each well was measured by an MRX-Microplate Reader at a test wavelength of 570 nm. 3. Results and discussion 3.1. MPEG-MWNT synthesis and CPT loading MWNTs have attracted much attention in drug delivery due to their low cytotoxity, good penetration ability and high drug loading amount. However, their dispersion stability was usually poor, which limits their applications in biomedicine [22]. To stabilize MWNTs in aqueous solution, the hydrophilic MPEG was used to modify the surface of MWNTs by esterification (Scheme 1). Its chemical structure was characterized by FT-IR analysis shown in Fig. 1, which confirmed that the MPEG chains were chemically attached to MWNTs. A sharp peak at 1649 cm− 1

Fig. 2. TEM image for the morphology of MPEG-MWNTs.

corresponded to the C_O stretching mode of the carboxylic acid groups for SWNTs-COOH. For MPEG-MWNTs, the peak at 1756 cm−1 corresponded to the C_O stretching mode of the ester bonding and the peaks from 800–1500 cm−1 corresponded to the MPEG [23]. Consequently, it was confirmed that the MPEG segments were chemically attached to the MWNTs. After modified by MPEG, the obtained MPEG-MWNTs displayed a good stabilization in aqueous solution. Fig. 2 showed the typical TEM image of MPEG-MWNTs. It could be seen that the MPEG-MWNTs were dispersed well in water and appeared as tubular particles with the diameter of 10–20 nm. For the composition analysis, Fig. 3 showed the TG curves of MPEG, MWNTs and MPEG-MWNTs, and the content of MPEG in MPEGMWNTs could be calculated by the analysis of the residual mass [24]. Under the nitrogen atmosphere, the MWNTs remained 95.0% residue after heated to 700 °C, while that of the MPEG was only about 3.5%. The MPEG-MWNT copolymer began to decompose about 310 °C and remained 70.5% residue, from which the content of MPEG in MPEGMWNTs could be determined to be 26.8 wt.%. The obtained MPEG-MWNTs were used to load hydrophobic CPT in this work, which could improve the loading amount onto hydrophilic hydrogel matrix through the interactions between MWNTs and CPT [25]. It has been reported that carbon materials, such as carbon nanotubes or

Fig. 5. Wide-angle X-ray diffraction patterns of α-CD, MPEG-MWNTs and the supramolecular hybrid hydrogel.

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Fig. 4. Photographs for the formation of a supramolecular hydrogel when aqueous MPEG-MWNT dispersion was mixed with an equal volume of 14% α-CD solution; and the SEM image of the resultant hydrogel.

(G‴) were monitored as a function of time. Fig. 6 showed the time dependence of G′ and G‴ for three samples with different MWNT contents. For the system with 6.0% α-CD shown in Fig. 6C, it was found that the value of G″ was higher than G′ at the early time. In particular, a crossover point between G' and G″ was observed at the time of 12.2 min, which implied a sol–gel transition. Beyond the crossing, the G′ value becomes larger than the G″ value, indicating that the system becomes more elastic. The corresponding time of the crossover from a viscous behavior to an elastic response could be regarded as the gelation time [29]. The gelation time was recorded to be about 5.1 min in the case of 7.0% α-CD was doped into the system. And when 8.0% α-CD was used, the gelation process was further faster. In this case, the G′ value was observed to be larger than the G″ value in the time range investigated (Fig. 6A). It is clear that the gelation time decreases with the increase of the α-CD contents. This may be attributed to the enhanced inclusion complexation between α-CD and MPEG segments, which result in a faster gelation. After the MPEG-MWNT dispersion was mixed with an equal volume of α-CD solution and set aside over night, the supramolecular hydrogel was prepared at room temperature. The strength of hydrogel was studied by advanced rheometric extended system. Fig. 7 showed the effect of the α-CD content on the hydrogel strength by measuring G′ as a function of angular frequency. We found that the G′ value for all samples was independent of the frequency for the hydrogel samples investigated, which confirms that the resultant supramolecular hydrogels behave as a gel or “solid-like” fluid [30]. Moreover, a significant increase in G′ was observed with the increase of the α-CD contents, which indicated that α-CD could enhance the hydrogel strength.

grapheme, could load the hydrophobic drugs containing benzene ring structure with a high loading amount, because the carbon materials with large specific surface could interact with the drugs through π–π and hydrogen–bond interactions, which have been confirmed by UV, IR and fluorescence analysis [17,18]. Through UV analysis, CPT loading amount onto MPEG-MWNTs was determined as 22.6 mg/g, which was about 168 times as that employed Pluronic F-127 micelles [15]. 3.2. Supramolecular hydrogel formation For the colloidal MPEG-MWNT dispersion, it is interesting to find that a gelation could occur when it is mixed with α-CD solution at room temperature (Fig. 4). The SEM image of the resultant hydrogels obviously showed dispersed MWNTs with tubal diameter of 10–20 nm, and these particles showed clear figures and dispersed well in the hydrogel matrix (the bright dots in the SEM image were assigned to the MWNT particles). This phenomenon was attributed to the inclusion complex formation between MPEG and α-CD, which results in a supramolecular-structured hydrogel [26]. To confirm this, XRD measurements were conducted for pure α-CD, MPEG-MWNTs and hybrid hydrogel. From the XRD patterns shown in Fig. 5, it was found that samples of hydrogel had two characteristic diffraction peaks at 2θ = 19.8 (d = 4.44 Å) and 22.6° (d = 3.94 Å), which are assigned to the {210} and {300} reflections from the hexagonal lattice with a = 13.6 Å. The strong {210} reflection is a typical peak observed for polymer inclusion complexes with α-CD, which was obviously different from the peaks of other samples [27,28]. These results demonstrated that a supramolecular-structured hydrogel was formed by the inclusion between MPEG and α-CD. The peaks at 2θ = 10° and 32° were assigned to the crystal of salts in PBS. To explore the effect of α-CD on the supramolecular system, a time sweep measurement for the viscoelastic properties of each system was carried out, in which the storage modulus (G′) and loss modulus

3.3. In vitro CPT release As an injectable drug delivery system, the sustained release of CPT from the supramolecular hydrogel was investigated. Fig. 8 showed the

3000

(B) 7.0% α-CD

(A) 8.0% α-CD

(C) 6.0% α-CD

1200

800

400

0

G' G"

2000

1000

G',G" (Pa)

G' G"

G',G" (Pa)

G',G" (Pa)

1200

5.1 min

2

4

6

Time (min)

8

10

12.2 min 400

0

0 0

G' G"

800

0

5

10

Time (min)

15

0

5

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15

20

Time (min)

Fig. 6. The time dependence of storage modulus (G′) and loss modulus (G″) for the hybrid systems containing different amounts of α-CD (25 °C, 0.05% strain).

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6.0%

5

7.0%

8.0%

K

n

R2

Transport mechanism

6.0% α-CD 7.0% α-CD 8.0% α-CD

1.33 ± 0.06 1.21 ± 0.07 0.94 ± 0.04

0.29 ± 0.04 0.36 ± 0.06 0.43 ± 0.04

0.992 0.988 0.991

Pseudo-Fickian Pseudo-Fickian Pseudo-Fickian

4

10

3

10 0.1

1

10

100

Frequency (Hz) Fig. 7. Storage moduli as a function of frequency for supramolecular hydrogels with different amounts of α-CD (25 °C, 0.05% strain).

in vitro release profiles for CPT from the CPT-loaded supramolecular hydrogels with different α-CD concentrations in PBS at 37 °C. In all cases, CPT could be released simultaneity and no initial burst release was observed. Depending on α-CD concentration used for the hydrogel formation, various release rates were found for loaded CPT respectively. The CPT release rate decreased with the increase of α-CD concentration. To understand the release mechanism of loaded CPT, we fitted the accumulative release data using the following semi-empirical equation [31]: n

Mt =M ∞ ¼ K t ð for M t =M ∞ ≤0:6Þ where Mt and M∞ are the cumulative amount of the drug released at t and equilibrium respectively; K is the rate constant relating to the properties of the hydrogel matrix and the drug, and n is the release exponent characterizing the transport mechanism. According to this classification, there are four distinguishable modes of diffusion: (i) the value of n = 0.5 suggests Fickian or Case I transport behavior in which the relaxation coefficient is negligible during transient sorption; (ii) the value of n = 1 refers to a non-Fickian or Case II mode of transport where the morphological changes are abrupt; (iii) if 0.5 b n b 1, the transport process is anomalous, corresponding

Cumulative CPT Release (%)

Hydrogel composition

to Case III, and the structural relaxation is comparable to diffusion; (iv) a value of n b 0.5 indicates a pseudo-Fickian behavior of diffusion where sorption curves resemble Fickian curves, but the approach to final equilibrium is very slow. By plotting log(Mt/M∞) versus log (t), the n and K values as well as the corresponding determination coefficients (R2) were obtained, as listed in Table 1. The K values were found to decrease with the increase of α-CD concentration. This phenomenon could be explained by considering the greater gelation extent and the formation of denser hydrogel network in the case of higher α-CD concentration, which hindered the CPT release and avoided supramolecular hydrogel erosion [32]. In addition, the n values in all hydrogel cases were found to be in the range from 0.29 to 0.43, showing a pseudoFickian mechanism. 3.4. Cell viability Supramolecular hydrogels have been attracted much attention in biomedicine because they could be implanted into body through an injectable method with minimal surgical wounds, and release drugs sustainedly for a long time [33,34]. For this supramolecular system, it was expected that such an injectable hydrogel carrier has a sustained CPT release property and could inhibit cancer cells for a long time. Fig. 9 gave the MTT results of HNE-1 cells incubated with PBS, blank hydrogel and CPT-loaded hydrogel at various time points. It was seen that the number of cells incubated with PBS or blank hydrogel increased obviously with time, while CPT-loaded hydrogel group showed a lasting low viability within 7 days. This result indicated that such CPT-loaded hydrogel could sustainedly inhibit HNE-1 cells and therefore has a potential application for sustained cancer therapy [35]. Moreover, the blank hydrogel has no significant difference with PBS control, which indicated the good biocompatibility. Fig. 10 showed the photographs of HNE-1 cells incubated with PBS, blank hydrogel and CPT-loaded hydrogel after 7 days. It was seen that

100 8.0% 7.0% 6.0%

80 60 40 20 0

0

1

2

3 4 5 Time (day)

6

7

Fig. 8. In vitro CPT release profiles from supramolecular hydrogels with different α-CD concentrations (37 °C, PBS with 10% Span 80).

Absorbance at 570 nm

G' (Pa)

10

Table 1 Release characteristics of loaded CPT from the supramolecular hydrogels with different αCD concentration.

1.2 1.0

Control Blank hydrogel CPT-loaded hydrogel

0.8 0.6 0.4 0.2 0.0

1st D 2nd D 3rd D 5th D 7th D

Fig. 9. MTT results of HNE-1 cells incubated with PBS, blank hydrogel and CPT-loaded hydrogel at various time points (P b 0.05).

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Fig. 10. Photographs (×5) of HNE-1 cells incubated with PBS, blank hydrogel and CPT-loaded hydrogel after 7 days.

cells incubated with PBS showed a good attachment, spreading and morphology, the cells proliferated in 7 days with a high viability. The cells incubated with blank hydrogel also showed a good viability (there was no significant difference between PBS and blank hydrogel), indicating the non-toxicity of blank hydrogel. For CPT-loaded hydrogel, the cell growth has been inhibited and cell number decreased obviously compared to PBS or blank hydrogel group, and the cell viability sustainedly kept a low level [36].

4. Conclusion To improve the loading amount of hydrophobic drugs into the hydrophilic hydrogel matrix, MWNTs were hybrided into a supramolecular hydrogel. Firstly, carboxylated MWNTs were modified by polyethylene glycol monomethyl ether (MPEG), which resulted in water-dispersed MPEG-MWNTs. Then α-CD was mixed with MPEG-MWNTs and interacted with MPEG segments to form the hydrogel. The used MPEG not only dispersed MWNTs in aqueous solution, but also functioned as hydrogel matrix by interacting with α-CD. The gelation time for the sol–gel transition and the dynamic rheological properties of the resultant hydrogel could be modulated by the content of α-CD. With the increase of αCD content, the strength of hydrogel increased, and the gelation time decreased. By in vitro CPT release tests, CPT could be released sustainedly from the hydrogel matrix and the release rate depended on the α-CD amount. MTT results confirmed that this supramolecular hydrogel showed a sustained inhibition to HNE-1 cells. This MWNT-hybrided hydrogel provides a new strategy to construct sustained hydrophobic drug delivery systems and has a potential application as injectable hydrogel carrier for sustained drug delivery.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (51203134 and 81260406) as well as the China Postdoctoral Science Foundation grant (2014M552217).

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