Injectable and body temperature sensitive hydrogels

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Carbohydrate Polymers 186 (2018) 82–90

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Injectable and body temperature sensitive hydrogels based on chitosan and hyaluronic acid for pH sensitive drug release

T



Wei Zhang, Xin Jin, Heng Li, Run-run Zhang, Cheng-wei Wu

State Key Laboratory of Structure Analysis for Industrial Equipment, Department of Engineering Mechanics, Dalian University of Technology, Dalian 116024, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Hyaluronic acid Chitosan Hydrogel Drug delivery Injectability

Hydrogels based on chitosan/hyaluronic acid/β-sodium glycerophosphate demonstrate injectability, body temperature sensitivity, pH sensitive drug release and adhesion to cancer cell. The drug (doxorubicin) loaded hydrogel precursor solutions are injectable and turn to hydrogels when the temperature is increased to body temperature. The acidic condition (pH 4.00) can trigger the release of drug and the cancer cell (Hela) can adhere to the surface of the hydrogels, which will be beneficial for tumor site-specific administration of drug. The mechanical strength, the gelation temperature, and the drug release behavior can be tuned by varying hyaluronic acid content. The mechanisms were characterized using dynamic mechanical analysis, Fourier transform infrared spectroscopy, scanning electron microscopy and fluorescence microscopy. The carboxyl group in hyaluronic acid can form the hydrogen bondings with the protonated amine in chitosan, which promotes the increase of mechanical strength of the hydrogels and depresses the initial burst release of drug from the hydrogel.

1. Introduction Hydrogels are widely used in vivo drug delivery system due to their low inter-facial energy, which limits both protein adsorption and inflammatory responses (Mateen & Hoare, 2014; Zhao, Xu, Mitomo, & Yoshii, 2006). The traditional hydrogels, however, lack in situ injectability and require surgical implantation, causing trauma and financial burden to patients. In response to this drawback, the injectable and body temperature (37 °C) sensitive hydrogels emerge. These hydrogels exist in a solution state at room temperature and can be injected into human body by simply using a syringe. When in physiological conditions, the hydrogels transform to a gel state owing to the increase of temperature to body temperature. Obviously, the application of these hydrogels can effectively reduce the trauma level. Aside from this, these hydrogels can form the desired shape as required by the surrounding tissues/organs, enabling the 3-D conformal therapy. These hydrogels can also be applied to the sites that are inaccessible by surgery (Phan, Thambi, Duong, & Lee, 2016; Liu et al., 2016; Bae, Wang, & Kurisawa, 2013; Huynh, Nguyen, & Lee, 2011). As such, the injectable and body temperature sensitive hydrogels have gained a lot of attention. On the other hand, one challenge remains common for chemotherapy is the severe cytotoxic side effects and the physical dependence incurred by chronic use of drug. Ideally, the drug should be delivered to the specific sites and released as demanded. When the injectable and body temperature sensitive hydrogels are loaded with



anti-cancer drugs and injected into human body, an in situ drug depot will be formed, offering the possibility of realizing localized delivery of drug and reducing toxic effects. Then specific molecules such as glucose and antigen, fields (magnetic or electric), light, pressure and pH can work as the trigger to induce the response of hydrogels and achieve the site-specific release of drug (Qiu & Park, 2001; Gupta, Vermani, & Garg, 2002). Among these approaches, the pH stimulated drug release is of particular interest since tumors and intracellular endosomal/lysomal compartments exhibit abnormally high local acidity due to Warburg effects (Chen et al., 2013; Lim et al., 2011). Apparently, hydrogels simultaneously possessing the merits of injectability, body temperature sensitivity and pH sensitivity are highly desired. In terms of components of the injectable and body temperature sensitive hydrogels, chitosan/β-glycerophosphate (GP) system is representative since they were first reported by Chenite et al., (2000) Nonetheless, this system has undesired properties, for instance, poor mechanical strength and long period of gelation time. To circumvent these, the chemical modification of chitosan, the addition of a second compound and/or polymer (Liu, Gao, Lu, & Zhou, 2016; Nguyen & Lee, 2010; Liu et al., 2014; Li et al., 2014) and the replacement of GP with alternative alkali (Liu, Gao, Lu, & Zhou, 2016; Supper et al., 2013; Liu, Tang, Wang, & Guo, 2011) have be attempted. Another disadvantage of chitosan/GP system is its poor pH sensitivity behavior (dissolved at acidic conditions) and thus is not suitable for tumor physical pH triggered drug release.

Corresponding author. E-mail address: [email protected] (C.-w. Wu).

https://doi.org/10.1016/j.carbpol.2018.01.008 Received 30 October 2017; Received in revised form 17 December 2017; Accepted 2 January 2018 Available online 03 January 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.

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solution for preventing water evaporation during the tests. To alleviate the damage to the gelling system, a dynamic oscillatory mode measurements were performed at the strain of 5%, which falls in the linear viscoelastic regime, ensuring that G' and G” are independent of the strain amplitude, as the strain sweep experiments demonstrates, see Fig. 1 in Data in Brief (Zhang, Jin, Li, Zhang, & Wu, 2017). In dynamic temperature sweep, the frequency was set as 10 rad/s and the temperature was increased from 10 °C to 45 °C with a rate of 2.5 °C/min. The sweep time was set as 1000 s. In dynamic time sweep, the sample plate was pre-heated to 37 °C. Then the CS/HA/GP solution was piped onto the plate and kept there for 30 s. Following this, the time sweep was conducted.

In this communication, the novel chitosan (CS)/hyaluronic acid (HA)/sodium glycerophosphate (GP) hydrogel systems are proposed. HA is a linear polysaccharide that consists of alternating units of a repeating disaccharide, β-1,4-D-glucuronic acid-β-1,3-N-acetyl-D-glucosamine. HA exists ubiquitous in human body, for instance, in connective tissues, skin and synovial joint fluids, and can be degraded via oxidative species and hyaluronidases (Borzacchiello, Russo, Malle, SchwachAbdellaoui, & Ambrosio, 2015). Owing to its bio-compatibility and native bio-functionality, HA have gained plenty of bio-medical and pharmaceutical applications for over thirty years (Kogan, Šoltés, Stern, & Gemeiner, 2007; Highley, Prestwich, & Burdick, 2016). HA is introduced here for the following considerations. (i) HA molecules bear plenty of hydroxyl, carboxyl and amino groups and this may enhance the interactions with chitosan molecules, improving the mechanical properties of the hydrogel. (ii) The introduction of a second polymer (HA) may influence the micro structure of hydrogel, possibly reducing pore size, and this may be utilized to regulate the drug release rate. (iii) Owing to lower pKa of HA than GP (Szymańska, Winnicka, Wieczorek, Sacha, & Tryniszewska, 2014; Brown & Jones, 2005), HA will take the priority to react with protonated amino group of chitosan, avoiding the abrupt release of drug initiated by the dissolution of hydrogel skeleton. (iv) High levels of CD44 are expressed in many cancers such as liver carcinoma and glioma, and HA is a principal ligand for the CD44 receptor. This indicates the incorporation of HA may facilitate the binding of hydrogel with tumor and increase the immobility of in situ formed hydrogel (Yin et al., 2016). To the best of our knowledge, no work has been conducted on the CS/HA/GP hydrogels. It is expected that these bespoke gelling systems demonstrate good temperature sensitivity, pH sensitive drug release behavior and adhesion to cancer cell without sacrificing the injectability.

2.3. Fourier transform infrared spectroscopy (FTIR) The attenuated total reflection (ATRR)-FTIR analyses were conducted on a Thermo Nicolet FTIR (Nexus-670) to characterize the functional groups and bonds of the hydrogel. FTIR spectra were recorded over the wavenumber range of 400–4000 cm−1 at a resolution of 4 cm−1. 2.4. Scanning electron microscopic observation The hydrogels (HG0, HG1, HG2, HG3) were frozen in liquid nitrogen for 20 min and then fractured to obtained the cross-section. The obtained sample was immediately transferred to a freeze drier (FD-1A50, China Shanghai Xinweng) and then lyophilized for 24 h. After coated with gold on a megnetron ion sputter metal coating device (Vacuum Device MSP-1s, Japan), the surface morphology was examined on a scanning electron microscopy (SEM, FEI Quanta 200, FEI, USA), operated at an accerating voltage of 20 kV.

2. Experimental section

2.5. In vitro drug release

2.1. Preparation of CS/HA/GP solution

Powered doxorubicin (DOX, 1 mg) were added into the 2 mL CS/ HA/GP solution and then incubated in 37 °C water bath for 5 min to induce the formation of hydrogel. Then 10 mL of phosphate buffer solutions (PBS, pH 4.00 and pH 6.86) were added to the vial containing the DOX loaded hydrogel. At predetermined collection times, 4 mL of medium was replaced and analyzed in a UV–vis-NIR spectrometer (Lambda 750s, PerkinElmer, USA) at 501 nm. Prior to characterizing the drug release quantitatively, the calibration curves of DOX in PBS solutions at different pHs were established, see Fig. 2 in Data in Brief (Zhang, Jin, Li, Zhang, & Wu, 2017).

3% (w/v) CS (Mw: 8.75 × 104, deacetylation degree: 81.3%, Sinopharm Chemical Reagent Co. Ltd., China) was added to acetate acid aqueous solution (0.1 mol/L) under stirring until the chitosan was dissolved completely. This chitosan solution was then kept at 4 °C for further use. 1%, 2%, and 3% (w/v) HA (Mw: 1.26 × 106, Shengjiade Bio Science and Technology Co. Ltd. China) and 60% (w/v) GP (Ruibio, Germany) were dissolved in di-water and then keep at 4 °C to form GPHA solution. Following this, 0.4 mL HA-GP solution was added into 1.6 mL chitosan solution in ice bath to form CS/GP/HA solution. The composition of CS/HA/GP hydrogels are given in Table 1.

2.6. Adhesion of cancer cell to hydrogel Hela cells were cultured in high glucose Dulbecco’s modified Eagle medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco) in a 5% CO2 incubator at 37 °C. Prior to the in vitro study, the hydrogels (HG0, HG3) were washed with ethanol and then sterilized under UV lights for 1 h. Subsequently, the Hela cells were trypsinzed, centrifuged and resuspended at the density of 1 × 105 cells/mL in the culture medium. 100 μL cell suspension was added into each well, coated with the prepared hydrogels, of the 96-well plate, which was incubated in 5% CO2 incubator for 1 h and 4 h. The morphology and cell adhesion of Hela cells were studied using SEM and fluorescence microscopy (FM, Olympus BX71, Japan). In SEM observations, following the co-incubation, the cells were washed with phosphate buffer saline (PBS, Hyclone) 3 times, fixed in 2.5% glutaraldehyde at 4 °C for 4 h, and then dehydrated by increasing concentrations of ethanol (30%, 50%, 75%, 90%, 100%, 100%) for 15 min each. The hydrogel co-incubated with cells was then freezed drying as described in Section 2.4, and the morphology was examined on a SEM (FEI Quanta 200, FEI, USA).

2.2. Rheological measurement The rheological properties were performed on a rotational rheometer (Anton Paar MCR302, Austria) fitted with a PP25 plate indenter – platform plate configuration. The CS/HA/GP were piped between the plates, and mineral oil was used to cover the marginal surface of the Table 1 Composition of CS/HA/GP hydrogels. Hydrogel name

HG0 HG1 HG2 HG3

CS solution

Aqueous mixture of GP and HA

CS content (w/v)

volume(mL)

GP content (w/v)

HA content (w/v)

volume(mL)

3% 3% 3% 3%

1.6 1.6 1.6 1.6

60% 60% 60% 60%

– 1% 2% 3%

0.4 0.4 0.4 0.4

83

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In the fluorescence microscopy, after washing with PBS for 3 times, the co-incubated cells were stained with Calcein-AM (Sigma, Mo, USA) and then incubated in 5% CO2 incubator for 15 min. After washing with PBS twice, the hydrogel incubated with cells was observed under a fluorescence microscope (FM). The cell amount was counted using the Image-Pro Plus 6.0 software. 3. Results 3.1. Rheological property To examine whether the hydrogels could be used in clinical applications, the viscoelastic properties of CS/HA/GP gelling systems with various HA contents were assessed by measuring the storage modulus (G'), representing the elastic behavior of the material, and loss modulus (G”), indicative of the viscous behavior. The hydrogel precursor solution behaves more like a solution and G' is relative small. When the gelation takes place, the G' will increase abruptly. For this reason. The gelation temperature is where the hydrogel precursor solution turns from a solution to a hydrogel and is usually defined as the temperature where G' equals to G”. The dynamic viscoelasticity of the CS/GP/HA gelling systems were tracked by G' and G” as a function of temperature and are shown in Fig. 1. Clearly, for all the systems investigated, there exists a temperature where G' is equal to G”. i.e. the gelation temperature, implying the thermo sensitivity of the gelling system. When the temperature is lower than this temperature, G' is smaller than G”. In the vicinity of this temperature, G' increases sharply, in particular over the gelation temperature. Fig. 1 demonstrates that the gelation temperature ranges from 31.2 °C to 37.2 °C, suggesting that for all the gelling systems investigated, body temperature can incur the transition from a solution to a gel. It can also be observed that in contrast to the CS/GP system (37.2 °C), the gelation temperature of CS/HA/GP drops and decreases gradually with the increase of HA content, namely, HG1: 37.1 °C, HG2: 36.4 °C and HG3: 31.2 °C. This indicates that the introduction of HA facilitates the formation of hydrogel.

Fig. 2. Plots of the elastic modulus (G') and viscous modulus (G”) of the hydrogel originating from gelling system with varying HA content verse time at a frequency of 10 rad/s.

The gelation time is referred to the time elapsed when the storage modulus of the gelling system turns out to be greater than the loss modulus at a given temperature. The gelation time can reflect the gelation rate and the strength of the hydrogel. To study the gelation process at body temperature (37 °C), the dynamic time sweep of the hydrogel precursor solution were carried out. To ensure the homogeneous heating, all the precursor solutions were pre-heated for 30 s at 37 °C before conducting the rheological measurements. As Fig. 2 exhibits, both G' and G” increase upon time. For HG0 and HG1, the gelation time is about 100 s and 30 s, respectively. For HG2 and HG3, the gelation time cannot be clearly observed. This means the precursor solution forms gel in the pre-heating and the gelation time is within 30 s. These results reveal that the introduction of HA can effectively shorten the gelation time. This is in agreement with the observed variation trend of the gelation temperature. As the time-temperature Fig. 1. The dynamic temperature sweep test of the hydrogel originating from gelling system with varying HA content: (a) HG0; (b) HG1; (c) HG2; (d) HG3.

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Fig. 3. SEM images focused on the cross-section of hydrogels originating from gelling system with varying HA content: (a) HG0; (b) HG1; (c) HG2; (d) HG3. Scale bar represents 30 μm.

accumulative release percent increases gradually and reaches 57% after 130 h, demonstrating good sustained drug release behavior.

superposition principle states (Li, 2000), a lower gelation temperature indicates a shorter gelation time at a given temperature above the gelation temperatures. As the strength is concerned, G' increases with the increasing HA content. After 1000 s, G' of HG0, HG1, HG2 and HG3 are 471 Pa, 617 Pa, 1130 Pa and 2430 Pa, respectively, indicating the introduction of HA improves the mechanical properties of hydrogel. Fig. 2 also illustrates that after the formation of hydrogel, G' is always greater than G”, indicating a stable gel state at body temperature.

3.4. Thermal sensitivity and injectability Fig. 5a briefly illustrates the procedure for the preparation of hydrogels and gives the representative photographs of the initial hydrogel precursor solution at 4 °C and the formed hydrogel at 37 °C, demonstrating the body temperature sensitivity. It is noted that the hydrogel become turbid in contrast to the initial precursor solution. Figs. 1 and 2 shows that the introduction of HA will enhance the mechanical strength of the formed hydrogel and this trend is strengthened with increasing HA content. This may lead to the worry that such a strong hydrogel may lose it injectability. To examine this issue, the HG3 precursor solution was sucked into a syringe and a liquid droplet can readily be ejected, see Fig. 5b–d, exhibiting the injectability of HA hydrogel. The hydrogel loaded with DOX also maintain its injectability, as Fig. 6a exhibits, suggesting the possible application in injectable drug delivery. When the drug DOX is involved, the hydrogel precursor solution still maintains its temperature sensitivity. The solution is at solution state at 4 °C and turns out to be a gel at 37 °C and no flow can be observed, see Fig. 6b.

3.2. Hydrogel morphology In order to acquire the information regarding microscopic structure, the hydrogels were lyophilized for morphology observation using scanning electron microscopy (SEM), see Fig. 3. It is apparent that all the hydrogels have porous structure. Compared with CS/GP hydrogel (Fig. 3a), the CS/HA/GP hydrogels have more condensed structure and the pore size decreases with the increasing HA content (Fig. 3b–d). Moreover, no obvious two-phase structure can be observed when HA is involved, revealing the good interactions between CS and HA molecular chains. 3.3. In vitro drug release

3.5. Adhesion of cancer cell to hydrogel

With DOX as an anti-cancer drug, accumulative release of DOX from the hydrogels in aqueous solutions with different pHs was shown in Fig. 4. As demonstrated, at pH 6.86, a burst release occurs in about 5 h for all the hydrogels and no apparent release can be observed thereafter. After 130 h, the accumulative release percentage is less than 30% for all the hydrogels. In contrast, more DOX can be released at pH 4.00, indicating the pH sensitive drug release of the hydrogels. It is also clear that at acidic condition, a burst release of DOX from HG0 can be observed and over 90% DOX is released within 20 h. After the introduction of HA, the initial burst release is depressed significantly and the release rate decreases with the increase of HA content. For HG3, the

The functionality of HA on programming cancer cell adhesion were assessed using human cervical cancer cells (Hela) for 1 h and 4 h. Hela cells were used as the demonstrating cancer cells, as CD44 are expressed for Hela cells (Li & Zhou, 2011). The in vitro response of Hela cells on the hydrogels reveals the remarkable influence of HA on modulating cancer cell adhesion. Comparing Fluorescence Microscopy (FM) images Fig. 7a–d, it can be found that in contrast to HG0, more cells adheres on the surface of HG3. As statistical results on cell counts in Fig. 7e present, the number of cells per unit area increases 85

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Fig. 4. Accumulative release percentage of DOX from hydrogels in aqueous solutions with pH 4.00 and pH 6.86 (a), photos of hydrogels after the release of drug for 130 h in aqueous solution with pH 4.00 (b) and 6.86 (c).

Fig. 5. (a) The preparation of hydrogel precursor solution (HG3) and the solution at low temperature of 4 °C and the formed hydrogel at 37 °C, (b–d) the HG3 precursor solution can be readily ejected to form droplets using a syringe.

the incorporation of HA can promote the adhesion of cancer cell to the hydrogels, which may be attributed to the specific binding of HA and CD44 (Yin et al., 2016).

dramatically on the hydrogel with HA. Comparisons of SEM images of Fig. 7f and g, Fig. 7h and i agree well with the results of FM, cancer cell adhesion is greatly promoted on the hydrogel modified with HA. The close SEM examinations indicates that for the hydrogel with HA, the cells adheres in organization with their numerous filopodia and actin filaments stretched along the hydrogel surface, as shown in Fig. 7k and m. On the contrary, the few cells that survived the repelling mechanism of the hydrogel without HA and attached to hydrogel are smaller and lack filopodia extensions, see Fig. 7j and l. FM and SEM results suggest

4. Discussion The rheological characterization, morphology observation and drug release analyses above have clearly demonstrated that the introduction of HA molecules plays important roles in the gelation and drug release Fig. 6. (a) the hydrogel (HG3)-DOX precursor solution can be readily ejected to form droplets using a syringe, (b) Hydrogel (HG3)-DOX precursor solution at low temperature of 4 °C and the formed hydrogel at 37 °C.

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Fig. 7. FM images of Hela cells cytoskeleton, stained in green, attached on the HG0 (a, c) and HG3 (b,d) after co-incubation of 1 h (a, b) and 4 h (c, d). Comparison between the number of cells attached to HG0 and HG3, four repetitions with ± SD (e). SEM images of Hela cells adhered to HG0 (f, h) and HG3 (g, i) after co-incubation of 1 h (f, g) and 4 h (h. i). j, k, l, m are the corresponding enlarged images of f, g, h, i, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

addition, the increase of temperature will reduce the apparent proton dissociation constant (pKa) of CS (Filion, Lavertu, & Buschmann, 2007) and leads to the poor ionization of CS. As a combined result of these effects, the solution turns to a hydrogel (Fig. 8c). It should be pointed out that at current stage it is still difficult to ascertain the individual contribution arising from each aspect and further work is still necesary. The rheological property measurements reveals that the introduction of HA can improve the mechanical strength of hydrogels and reduce the gelation temperature. SEM observation indicates that the introduction of HA can significantly influence the microstructure of the hydrogel and reduce pore size. These changes may result from the change of inner molecular interactions. To investigate the effect of HA, FT-IR spectra of HG0 and HG3 are analyzed, see Fig. 9. Two noticeable differences can be observed. For HG0, the in-plane bending vibration of −NH2 appears at 1656 cm−1 and the NH3+ signal resulting from the protonation of CS emerges at 2943 cm−1 (Li et al., 2014). In contrast, these two peaks in HG3 are presents at 1632 cm−1 and 2934 cm−1, shifting to low wavenumber by Δn 24 cm−1 and Δν 9 cm−1 respectively. This shift can be ascribed to the fact that the electrons are shared by the electron donor and the electron acceptor, lowering the vibrating frequency (Zhang, Dehghani-Sanij, & Blackburn, 2008), also known as red shift. The above results suggest the occurrence of hydrogen bonding between the carboxyl group in HA molecules and amino group in CS, more likely in format of HA-COO⋯H⋯NH2-CS, as illustrated in Fig. 8c. That is to say, the introduced HA molecules will cross-link with CS molecules. This may account for why the

processes. In this section, we attempt to obtain an understanding of these roles. 4.1. Formation of hydrogel Briefly, the gelation process includes two steps, as Fig. 5a depicts, namely the maintaining of the mixture of CS/GP/HA molecules in solution at low temperature and the formation of hydrogel upon the increase of temperature. The CS molecular chain bears amino group, which will become protonated in acidic aqueous medium. And the strong electrostatic repulsion forces endow the solubility of CS (Fig. 8a). When the mixing solution of GP and HA is introduced, the carboxyl group in HA molecule and the phosphate group will partially neutralize the protonated amino, reducing the repulsion between CS molecules. Nevertheless, the CS/HA/GP molecules are all rich of polar and hydrophilic groups such amino, hydroxyl, carbonyl and ether, which can form hydrogen bondings with water molecular. These surrounding water molecules can suspend the large organic molecules in solution (Fig. 8b). But this is only a temporary and metastable state. Upon the increase of temperature, the electrostatic attraction between NH3+ of CS and OPO32− of GP, NH3+ of CS and COO− of HA can reduce electrostatic repulsion force, inducing the attractive hydrophobic and hydrogen bondings between CS and HA chains (Liu et al., 2014). The structuring action of GP on water upon heating also promotes the hydrophobic interactions bewteen CS and HA chains, which in turn promotes the association of CS and HA chains (Chenite et al., 2000). In 87

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Fig. 8. Representation of the gelation mechanism of CS/HA/GP precursor solution.

are given in Table 2 (Nokhodchi, Raja, Patel, & Asare-Addo, 2012). It is clear that the drug release behavior of hydrogels in both acidic and basic solutions can be described using Ritger-Peppa, as indicated by the goodness of fit R2. Ritger-Peppas model is a semi-experimental exponential function equation considering diffusion of drug and erosion of polymer matrix (Ritger & Peppas, 1987a), wherein n is the diffusional exponent, indicative of the release mechanism. When n ≤ 0.45, Fickian diffusional release takes the dominant role. When 0.45 < n < 0.89, non-Fickian diffusional release prevails and the drug releases by dissolution, diffusion and erosion of matrix. Wherea n ≥ 0.89, the drug mainly releases through the erosion of polymer matrix (Ritger & Peppas, 1987b; Peppas & Sahlin, 1989). At pH 4.00, n of HG0 is 0.4661, indicating the non-Fickian release mechanism. For HG1, HG2 and HG3, n ranges from 0.236 to 0.274, much smaller than 0.45, suggesting the diffusion is largely responsible for the drug release. At pH 6.86, n is smaller than 0.002 for all hydrogels, implying the diffusional release mechanism. The above mechanism discussion is partly supported by the experimental observation that at pH 4.00, the volume of HG1, HG2 and HG3 shrinks to a certain level; in sharp contrast, the volume of HG0 shrinks considerably, indicating the erosion of polymer skeleton, see Fig. 4b. At pH 6.86, the volume of all drug loaded hydrogels is almost unchanged after drug release, see Fig. 4c. Fig. 4a exhibits that more drug can be released from hydrogels at acidic conditions, this can be ascribed to the fact that on this condition, the amine group in CS chain becomes protonated and the electronic repulsion between CS chains is enhanced, facilitating the release of drug from the hydrogel. Fig. 4a also exhibits that the introduction of HA can greatly depress the initial burst release. This can be ascribed to the difference of acid dissociation constant of GP and HA, and consequently

Fig. 9. FT-IR spectra of HG0 and HG3.

introduction of HA molecules can strengthen the hydrogel, shorten the gelation time, and reduce pore size, as discussed in Sections 3.1 and 3.2. 4.2. Drug release from hydrogel To investigate the drug release mechanism, Zero-order, first-order, Higuchi, Ritger-Peppas models were employed to fit the accumulative drug release curves presented in Fig. 4a and the fitted parameter values 88

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Table 2 The fitted parameter values of different models for DOX release at pH 4.00 and pH 6.86. Name of sample

HG0

HG1

HG2

HG3

Type of model

Zero-order First-order Higuchi Ritger-Peppas Zero-order First-order Higuchi Ritger-Peppas Zero-order First-order Higuchi Ritger-Peppas Zero-order First-order Higuchi Ritger-Peppas

Equations

Q = kt + b Q = 100-bexp(−kt) Q = kt1/2 + b Q = ktn + b Q = kt + b Q = 100-bexp(−kt) Q = kt1/2 + b Q = ktn + b Q = kt + b Q = 100-bexp(−kt) Q = kt1/2 + b Q = ktn + b Q = kt + b Q = 100-bexp(−kt) Q = kt1/2 + b Q = ktn + b

pH 4.00

pH 6.86

k

b

n

R2

k

b

n

R2

2.789 0.07563 17.62 20.17 0.6512 0.02416 8.263 29.88 0.5528 0.01594 7.044 29.45 0.4194 0.008214 5.296 24.48

20.71 90.11 −0.018 −2.807 29.08 85.41 9.91 −17.02 25.7 83.84 9.264 −18.13 18.73 85.35 6.514 −16.42

– – – 0.4661 – – – 0.274 – – – 0.2522

0.9171 0.9868 0.9923 0.9928 0.7892 0.9769 0.9522 0.983 0.7833 0.9364 0.9532 0.9889 0.8031 0.8957 0.9597 0.9811

0.139 0.002073 1.939 5212 0.1253 0.001717 1.75 3507 0.1307 0.001797 1.817 5296 0.1272 0.001726 1.757 1758

20.43 80.06 15.67 −5198 15.71 84.66 11.41 −3496 15.36 85.05 10.92 −5286 14.79 85.59 10.52 −1748

– – – 0.000785 – – – 0.0009985 – – – 0.0006988 – – – 0.001913

0.495 0.5262 0.7505 0.9653 0.503 0.5299 0.7651 0.9751 0.5248 0.5548 0.7903 0.9764 0.5437 0.5732 0.8087 0.9758

0.2362

References

the ability to form hydrogen bonding with the protonated amine group (NH3+). GP has a pKa of 6.34, whereas pKa of HA is about 3.0 21,22 This means HA molecules are prone to form hydrogen bonding with CS in format of HAeCOO⋯H⋯NH2eCS. This is partially evidenced by the FT-IR analyses above. In this respect, the pending carboxyl group in HA resembles a pH buffer and alleviate the burst release of drug incurred by the sudden protonation of a large quantity of amine group in CS. It is worth pointing out that the enhanced interactions between HA and CS also contribute to the strengthening of the mechanical strength of hydrogel, shortening of the gelation time and more dense microstructure of hydrogel, as reflected by Figs. 1–3, respectively.

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5. Conclusions Herein the novel CS/HA/GP hydrogels are developed. The hydrogels precursor solutions can be injected using a syringe and will change to the gels upon the increase of temperature to body temperature. Meanwhile, the anti-cancer drug loaded in the hydrogels can be released when stimulated by the acidic tumor condition, wherein the diffusion release mechanism prevails. The carboxyl groups in HA will form hydrogen bondings with the protonated amine group in CS, which contributes to the strengthening of mechanical strength of hydrogels, lowering of gelation temperature and depressing of the burst release of drug loaded in the hydrogels. This means the rheological properties of the hydrogels and the drug release behavior can be regulated by tuning the HA content. Moreover, when incubated with cancer cells, the CS/ HA/GP hydrogels demonstrates good affinity to cancer cell. This work shows that the novel hydrogels demonstrate good temperature sensitivity, pH sensitive drug release behavior and adhesion to cancer cell with injectability, indicating the potential use of these hydrogels in tumor site-specific administration of drug.

Funding This work was supported by the NSFC [11772086,51775541,11572080]; the NSFC of Liaoning Province [2015020198]; Fundamental Research Funds for the Central Universities [DUT17ZD229].

Competing financial interests The authors declare no competing financial interests. 89

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