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Using casein and oxidized hyaluronic acid to form biocompatible composite hydrogels for controlled drug release
Materials Science and Engineering C 36 (2014) 287–293
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Using casein and oxidized hyaluronic acid to form biocompatible composite hydrogels for controlled drug release Nan-Nan Li, Chao-Ping Fu, Li-Ming Zhang ⁎ DSAPM Lab, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China PCFM Lab, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China
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Article history: Received 19 August 2013 Received in revised form 10 December 2013 Accepted 17 December 2013 Available online 27 December 2013 Keywords: Oxidized hyaluronic acid Casein protein Gelation Drug delivery Biocompatibility
a b s t r a c t To develop biocompatible polymeric hydrogels for the in-situ encapsulation and controlled release of hydrophilic drugs, the oxidized hyaluronic acid containing aldehyde groups was prepared by the reaction between hyaluronic acid and sodium periodate, and then used for the first time to crosslink casein protein in aqueous system. By changing its aldehyde group content or amount, we found that the gelation kinetics and the properties of resultant composite hydrogel could be modulated. Particularly, an increase of its aldehyde group content or amount was found to result in a shorten gelation time, an enhanced gel strength, a reduced swelling ratio and a prolonged drug release. In addition, the as obtained composite hydrogel was also evaluated for its in vitro cytotoxicity on L929 mouse fibroblast cells and was confirmed to have a good biocompatibility. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent years, there has been an increasing interest in using polysaccharide-based cross-linkers to prepare biocompatible polymeric hydrogels with wide biomedical applications for drug delivery, cell encapsulation and tissue engineering. In this context, some natural polysaccharides containing vicinal hydroxyl groups, including hyaluronic acid [1–6], sodium alginate [7], dextran [8], gum arabic [9] and chondroitin sulfate [10], were modified with periodate to form aldehyde groups, and then were used to crosslink amino group-containing polymers in aqueous systems by the Schiff's base reaction between amino and aldehyde groups. The resultant hydrogels could be formed in situ, and exhibit better biocompatibility when compared to those obtained from photo-crosslinking by a potentially toxic photosensitizer [11] or chemical crosslinking by potentially toxic carbodiimide, glutaraldehyde, and adipic dihydrazide [12–15]. As a kind of important biopolymers, caseins are the phosphoproteins that precipitate from raw skim milk by acidification, whose molecular weights have been reported to range from 19,007 to 25,230 Da [16]. Over 55% of the amino acids in casein proteins contain hydrophilic functional groups such as \COOH, \NH2 and \OH [17]. They possess a number of favorable characteristics suitable for the development of hydrogel biomaterials, such as high hydrophilicity, good biodegradation, lack of toxicity, and availability of reactive sites for chemical
⁎ Corresponding author. Tel./fax: +86 20 84112354. E-mail address: [email protected] (L.-M. Zhang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.025
modification [18]. Moreover, they are less expensive and more readily available when compared to collagen [19], silk fibroin [20], bovine serum albumin [21] and elastin-like polypeptides [22], which are usually used for the preparation of protein-based hydrogels. Nevertheless, little work was dealt with the chemical crosslinking of casein protein in aqueous systems except for our recent studies [23,24], in which casein was crosslinked by naturally occurring genipin or microbial transglutaminase to form biocompatible protein-based hydrogels for the controlled release of hydrophilic drugs. In the present work, oxidized hyaluronic acid containing reactive aldehyde groups was prepared and then used for the first time to crosslink casein protein in aqueous system. In particular, the gelation kinetics and the properties of resultant composite hydrogels were investigated with respect to the aldehyde group content and amount of the oxidized hyaluronic acid. In addition, the in vitro drug release behavior and cytotoxicity were also studied for the resultant composite hydrogels.
2. Experimental 2.1. Materials Hyaluronic acid (Mw = 50 kDa) was purchased from Guangzhou Reborn Chemical Company (China). Casein powder was purchased from Lanzhou Tongjian Biotechnology Company (China), which was made from the Yak milk with high quality. Sodium periodate was obtained from Guangzhou Guanghua Chemical Company (China). Salicylic
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acid was purchased from Acros Organics. Mouse fibroblasts of the cell line L929 were kindly provided by Second Affiliated Hospital of Sun Yat-sen University (China). All other chemicals and reagents were of analytical grade and used as received. 2.2. Preparation and characterization of oxidized hyaluronic acid Oxidized hyaluronic acid (O-HA) containing aldehyde groups was prepared by the reaction between hyaluronic acid and sodium periodate. According to previous reports [2,25], the vicinal hydroxyl groups on the sugar ring of hyaluronic acid could be oxidized by sodium periodate to form two aldehyde groups in each oxidized monomeric unit. Briefly, 1.0 g hyaluronic acid sodium was dissolved in 100 mL distilled water at a concentration of 10 mg/mL. An aqueous solution of sodium periodate (0.5 mol/mL, 5 mL) was added dropwise, and the reaction was continued for various time (2, 10 or 24 h) at room temperature in the dark under magnetic stirring. 1 mL ethylene glycol was then added to inactivate un-reacted sodium periodate. After that, the resultant solution was dialyzed exhaustively for 3 days against water and lyophilized to obtain O-HA product with a yield of about 75%. By changing the reaction time, three O-HA samples with different contents of the aldehyde groups were prepared, namely O-HA-1 in the case of 2 h, O-HA-2 in the case of 10 h and O-HA-3 in the case of 24 h. By the hydroxylamine hydrochloride method [26], the aldehyde group content was determined to be 1.16% for O-HA-1, 3.19% for O-HA-2 and 5.22% for O-HA-3, respectively. 1H NMR (300 MHz, D2 O): δ (ppm) 1.9 (acetamide protons, NH\CO\CH3), 3.0–3.9 (protons of sugar units, CH\O and CH 2 \O), 4.3 and 4.4 (anomeric protons, O\CH\O), 5.0–5.1 (hemiacetalic proton formed from aldehyde and adjacent hydroxyl groups). 2.3. Preparation and characterization of composite hydrogels For the formation of each composite hydrogel sample, aqueous casein solution was first prepared by dissolving casein powder in distilled
water, and its pH value was then adjusted to be about 8 by 2.0 mol/mL sodium hydroxide solution. After that, a required amount of oxidized hyaluronic acid (O-HA) (dissolved in distilled water) was added for the gelation of aqueous casein solution. The total concentration of casein in the mixed system was fixed to be 10% (w/w). In order to investigate the effects of O-HA amount on the gelation, 1.0, 3.0 and 5.0% of its concentrations (in mixed system) were used, respectively. The gelation process was monitored by a rheological method [27], and the viscoelastic parameters of resultant composite hydrogels (aged for 24 h) were measured by using an advanced rheometric extended system (ARES, TA Co.) in oscillatory mode with a Couette geometry (25 mm diameter). To ensure the rheological measurements within a linear viscoelastic region, a dynamic strain sweep was conducted prior to the frequency sweep, and the corresponding strain was determined to be 5.0%. The dried composite hydrogels were obtained by lyophilization for SEM observation. In this case, the composite hydrogel samples were sputtercoated with gold under vacuum and imaged using a Hitachi S-520 scanning electron microscope (Tokyo, Japan). The swelling tests were carried out in pH 7.4 phosphate buffered saline solutions for various composite hydrogel samples. The swelling ratio (SR) was calculated according to the following equation: SR ¼ ðW t −W 0 Þ=W 0
ð1Þ
where Wt is the weight of the swollen composite hydrogel at a given time t during swelling and W0 is the weight of the dried composite hydrogel. All swelling tests were carried out in triplicate. 2.4. In situ encapsulation and in vitro release tests for model drug For the in situ encapsulation of salicylic acid as a hydrophilic model drug, 0.2% salicylic acid was added into aqueous 10 wt.% casein dispersion under continuous stirring. After dissolution of the drug, required amount of O-HA was introduced to induce the gelation. To study the in vitro drug release profile, each composite hydrogel sample loaded
Fig. 1. Photographs for the composite hydrogel formation when 10.0% aqueous casein solution was mixed with 3.0% aqueous O-HA-3 solution and a graphical representation for Schiff's base crosslinking reaction.
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with 0.2% salicylic acid was immersed in 20 mL phosphate buffer saline (PBS, pH 7.4) at 37 °C. At predetermined time points, 3.0 mL of this solution was taken out and a 3.0 mL fresh buffer solution was added back to maintain the same total solution volume. The cumulative percentage of released salicylic acid drug was determined by means of a UV spectrophotometer (S52, China) at the absorbance wavelength of 296 nm. All release studies were carried out in triplicate. 2.5. In vitro cytotoxicity assay for composite hydrogel The in vitro cytotoxicity of the composite hydrogel formed from 10.0% casein and 3.0% O-HA-3 was evaluated on L 929 mouse fibroblast cells using a Cell Counting Kit-8 (CCK-8) with the experimental conditions recommended by the CCK-8 manual. For the comparative studies, aqueous casein solution, aqueous O-HA-3 solution and phosphate buffer saline were investigated for their in vitro cytotoxicity, respectively. All materials were sterilized by gamma irradiation with a dose of 25 kGy. Briefly, L 929 mouse fibroblast cells were seeded in 96-well plates at 3 × 103 cells per well and incubated at 37 °C under a 5% CO2 humidified atmosphere in Dulbecco's modified Eagle's medium (DMEM) for 24 h prior to the test. Then 20 μL of sterilized sample were added (for control group: 20 μL of PBS was added; for casein group: 20 μL of 10.0% casein was added; for O-HA-3 group: 20 μL of 5.0% O-HA-3 was added; for hydrogel group: 10 μL of 10.0% casein and 10 μL of 5.0% O-HA-3 were added by a dual barreled syringe) into each well of the plate which was placed for 24 h in an incubator (37 °C and 5% CO2). After that the cells were treated with the recommended dose of CCK-8 assay solution. Absorbance was measured at 450 nm by using an automated ELISA reader (Labsystems Dragon Wellscan MK-3, Finland). The cell viability was calculated as a percentage ratio of absorbance of the experiment group and PBS group. For microscopic observation, an inverted phase contrast microscope (IX70, Olympus, Tokyo, Japan) was used to obtain the microscopic images for the cells in direct contact with PBS and the cells in direct contact the casein/O-HA-3 composite hydrogel. 3. Results and discussion 3.1. Formation and characteristics of casein/oxidized hyaluronic acid composite hydrogels Due to the Schiff's base crosslinking reaction between the amino groups in casein and the aldehyde groups in oxidized hyaluronic acid (O-HA), aqueous casein solution (10.0%) could be transformed quickly
Fig. 3. Time dependence of elastic modulus (G′) and viscous modulus (G″) for aqueous mixed casein/O-HA system: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%. (Test conditions: 25 °C, 1.0 rad/s).
into the invertible hydrogel when aqueous solution (1.0–5.0%) of O-HA was introduced at room temperature in the absence of any extraneous crosslinking agent, as shown in Fig. 1. Similar gelation phenomena
Fig. 2. Photographs for aqueous mixed system composed of 10.0% casein and 3.0% unmodified hyaluronic acid before and after standing for 48 h.
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were also reported for aqueous mixed system composed of chitosan or hydroxypropyl chitosan and sodium alginate dialdehyde [28] as well as aqueous mixed system composed of N-carboxyethyl chitosan and periodate-oxidized dextran [29], in which Schiff's base formation between the available aldehyde and amino groups resulted in the in situ formed biodegradable composite hydrogels for corneal endothelium reconstruction and enhanced wound healing. Moreover, there is an obvious color change from light yellow to deep red when aqueous casein solution gelled in the presence of O-HA. This phenomenon could be attributed to the formation of Schiff's base links between free amino groups of casein and the aldehyde groups of O-HA, as described previously [9,30–32]. For a comparative study, we prepared aqueous mixed solution composed of 10.0% casein and 3.0% unmodified hyaluronic acid, and didn't observe any phase separation or precipitation. Moreover, such a mixed system didn't show perceptible viscosity change and retained its original flowability even after standing for 48 h, as shown in Fig. 2. These results confirm further the crosslinking reaction for aqueous casein/oxidized hyaluronic acid system. Dependent on the aldehyde group content and amount of used OHA, aqueous mixed casein/O-HA systems were observed to have various gelation rates. To illustrate this, time sweep measurements were carried out by means of an advanced rheometric extended system, in which the elastic modulus (G′) and viscous modulus (G″) were monitored as a function of time. Fig. 3 shows the time dependences of G′ and G″ for
aqueous mixed casein/O-HA systems with various compositions. In each case, a crossover point between G′ and G″ was observed, which implied that there was a sol–gel transition [27]. 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 [27]. From Fig. 3a, the gelation time was determined to be about 50.6 min in the case of 3.0% O-HA-1 (containing 1.16% aldehyde group), 29.7 min in the case of 3.0% O-HA-2 (containing 3.19% aldehyde group), and 11.1 min in the case of 3.0% O-HA-3 (containing 5.22% aldehyde group), respectively. From Fig. 3b, the gelation time was determined to be about 48.7 min in the case of 1.0% O-HA-3, 11.1 min in the case of 3.0% O-HA-3, and 3.4 min in the case of 5.0% O-HA-3, respectively. The higher the aldehyde group content or amount of used O-HA, the shorter the gelation time was. In other words, the in situ gelation took place more readily when a higher aldehyde group content or amount of O-HA was used. This may be attributed to enhanced Schiff's base crosslinking in these cases. For the resultant casein/O-HA composite hydrogels, their viscoelastic and swelling properties as well as their morphologies were investigated. Fig. 4 gives the G′ and G″ values as a function of frequency for the resultant composite hydrogels with various compositions. For each composite hydrogel sample, the G′ value was observed to be greater than the G″ value over the entire range of frequency. This indicates
Fig. 4. Elastic modulus (G′) and viscous modulus (G″) as a function of frequency for various casein/O-HA composite hydrogels: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%.
Fig. 5. Swelling kinetics of various casein/O-HA composite hydrogels at 37 °C in pH 7.4 phosphate buffered saline solutions: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%.
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that the resultant composite hydrogel could display a viscoelastic behavior with dominant elastic property [33,34]. Moreover, such a viscoelastic behavior was found to become more obvious and stronger for the composite hydrogel formed in the case of a higher aldehyde group content or a higher O-HA amount. Among all the composite hydrogels investigated, the greatest G′ value was observed for the composite hydrogel formed from aqueous mixed system composed of 10.0% casein and 5.0% O-HA-3. In this case, the highest aldehyde group content and the highest O-HA amount were used, which may result in the greatest extent of Schiff's base crosslinking. Fig. 5 shows the swelling kinetics of casein/O-HA composite hydrogels with various compositions at 37 °C in pH 7.4 phosphate buffered saline solutions. As seen, these hydrogels could attain equilibrium swelling in 60 min and hold large amount of water. This is due to the wicking action of solution through the hydrogel pores as well as the hydration action of some functional groups such as \OH, \NH2 and \COOH. In particular, the lowest swelling ratio was found for 10.0% casein/5.0% O-HA-3 composite hydrogel, which resulted from the greatest crosslinking extent when the highest amount (5.0%) of O-HA-3. In addition, the swelling ratio in the case of 5.0% O-HA-3 (green curve in Fig. 5b) had a slight decrease after 120 min. This may be attributed to the formation of more hydrogen bonds in the hydrogel network during the swelling, which induced the shrinkage of the hydrogel network. Similar phenomenon was observed by other investigators [35–37] when they studied the swelling
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behavior of other biopolymer-based hydrogels. With the help of SEM observation, we found that such a composite hydrogel was characteristic of a more compact network structure when compared to those of other hydrogels, as shown in Fig. 6. 3.2. Drug release behavior from casein/oxidized hyaluronic acid composite hydrogels To explore the possibility of casein/oxidized hyaluronic acid composite hydrogels for drug delivery application, 0.2% salicylic acid drug was encapsulated in situ in the hydrogel matrices during the gelation. Fig. 7 gives the in vitro release profiles of encapsulated salicylic acid by various composite hydrogels at 37 °C in pH 7.4 phosphate buffered saline solutions. As seen, there is a sustained release behavior for loaded salicylic acid, regardless of the hydrogel compositions. Depending on the aldehyde group content of O-HA or O-HA amount used for the hydrogel formation, various release rates were found for the encapsulated drug. To understand better the release characteristics and mechanism of encapsulated salicylic acid from resultant composite hydrogels with different compositions, we fitted the accumulative drug release data using the following semi-empirical equation [38]: Mt =M ∞ ¼ kt
n
ðMt =M∞ ≤0:6Þ
ð2Þ
Fig. 6. SEM images of various casein/O-HA composite hydrogels: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%.
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N.-N. Li et al. / Materials Science and Engineering C 36 (2014) 287–293 Table 1 Release characteristics and mechanism of encapsulated salicylic acid from resultant casein-based hydrogels with different compositions (pH 7.4, 37 °C). Hydrogel constitution
k
n
R
Transport mechanism
0.27 ± 0.01
0.991
0.38 ± 0.02
0.996
0.46 ± 0.02
0.997
Pseudo-Fickian diffusion Pseudo-Fickian diffusion Pseudo-Fickian diffusion
55.15 ± 2.75
0.44 ± 0.02
0.997
42.90 ± 2.14
0.46 ± 0.02
0.997
37.52 ± 1.87
0.48 ± 0.02
0.993
Effect of O-HA composition 3.0% O-HA-1 + 73.11 ± 3.65 10.0% casein 3.0% O-HA-2 + 53.30 ± 2.66 10.0% casein 3.0% O-HA-3 + 42.90 ± 2.14 10.0% casein Effect of O-HA amount 1.0% O-HA-3 + 10.0% casein 3.0% O-HA-3 + 10.0% casein 5.0% O-HA-3 + 10.0% casein
Pseudo-Fickian diffusion Pseudo-Fickian diffusion Pseudo-Fickian diffusion
3.3. In vitro cytotoxicity of casein/oxidized hyaluronic acid composite hydrogels
Fig. 7. In vitro release profiles of encapsulated salicylic acid drug from various casein/ O-HA composite hydrogels at 37 °C in pH 7.4 phosphate buffered saline solutions: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%.
where M∞ is the initial mass of drug loaded in situ in the composite hydrogel, Mt is the cumulative amount of the drug released at time t, k is the rate constant, 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 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 (R) were obtained, as listed in Table 1. The k values were found to decrease with the increase of O-HA aldehyde group content or O-HA amount. This phenomenon could be attributed to the formation of a denser hydrogel network in the case of a higher aldehyde group content of O-HA or O-HA amount, which hindered the release of encapsulated drug from the composite hydrogel matrices. In addition, the n values in all cases were found to be smaller than 0.5, showing a pseudo-Fickian diffusion release mechanism.
Further investigation was dealt with the in vitro cytotoxicity of casein/oxidized hyaluronic acid composite hydrogel. Fig. 8a gives the cell viabilities of L929 fibroblast cells cultured for 24 h in the media containing PBS (control group), freshly prepared casein/O-HA-3 hydrogel (casein, 10.0%; O-HA-3, 3.0%), aqueous casein solution (10.0%) and aqueous O-HA-3 solution (3.0%), respectively. Regardless of test media, all of the cells were almost viable. Fig. 8b gives the microscopic images of direct contact assay for L929 fibroblast cell monolayer and the L929 fibroblast cells in contact with the composite hydrogel sample for 24 h. As seen, the L929 fibroblast cells in contact with the composite hydrogel sample still maintained their characteristic spindle shaped morphology. These results confirm that the resultant casein/ oxidized hyaluronic acid composite hydrogel is non-cytotoxic and potentially suitable for biomedical applications.
4. Conclusions New composite hydrogels based on casein protein and oxidized hyaluronic acid (O-HA) were obtained at room temperature without the need of any small molecule cross-linker. Their mechanical, swelling and drug-release properties could be regulated by changing the aldehyde group content of O-HA or O-HA amount. In particular, a mechanically strong composite hydrogel with three-dimensional network could be formed in a short time span under the optimized preparation conditions. Furthermore, such a composite hydrogel was confirmed to be non-cytotoxic and biocompatible. Here we present a facile route to in situ forming composite hydrogel based on casein and O-HA, which may find new applications for the controlled release of bioactive molecules, the encapsulation of living cells and wound healing. Considering that the aldehyde groups of O-HA may react with amino groups of serum proteins, our next work will focus on how to shorten the gelation time as soon as possible and enhance the injectability by a dual barreled syringe for this dual-component system.
Acknowledgments This work is supported by the National Natural Science Foundation of China (21074152, 51273216, J1103305), the Doctoral Research Program of Education Ministry in China (20090171110023), the Key Project of Scientific and Technical Innovation for Universities in Guangdong Province (cxzd1102) and the National Natural Science Foundation of Guangdong Province in China (S2013010012549).
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Fig. 8. (a) The cell viabilities of L929 mouse fibroblast cells cultured for 24 h in the media containing PBS (control group), freshly prepared casein/O-HA-3 composite hydrogel (casein, 10.0%; O-HA-3, 3.0%), aqueous casein solution (10.0%) and aqueous O-HA-3 solution (3.0%), respectively; (b) microscopic images for the cells in direct contact with PBS and the cells in direct contact with the casein/O-HA-3 composite hydrogel.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
L. Weng, H. Pan, W. Chen, J. Biomed. Mater. Res. 85A (2008) 352–365. H. Tan, C.R. Chu, K.A. Payne, K.G. Marra, Biomaterials 30 (2009) 2499–2506. S. Nair, N.S. Remya, S. Remya, P.D. Nair, Carbohydr. Polym. 85 (2001) 838–844. G.D. Prestwich, J. Control. Release 155 (2001) 193–199. Y.C. Chen, W.Y. Su, S.H. Yang, A. Gefen, F.H. Lin, Acta Biomater. 9 (2013) 5181–5193. M.N. Collins, C. Birkinshaw, Carbohydr. Polym. 92 (2013) 1262–1279. Q. Wang, W. He, J. Huang, S. Liu, G. Wu, W. Teng, Q. Wang, Y. Dong, Macromol. Biosci. 11 (2011) 362–372. H. Zhang, A. Qadeer, W. Chen, Biomacromolecules 12 (2011) 1428–1437. K.K. Nishi, A. Jayakrishnan, Biomacromolecules 8 (2007) 84–90. S. Dawlee, A. Sugandhi, B. Balakrishnan, D. Labarre, A. Jayakrishnan, Biomacromolecules 6 (2005) 2040–2048. J.B. Leach, K.A. Bivens, C.N. Collins, C.E. Schmidt, J. Biomed. Mater. Res. 70A (2004) 74–82. K.H. Bouhadir, G.M. Kruger, K.Y. Lee, D.J. Mooney, J. Pharm. Sci. 89 (2000) 910–919. T.A. Holland, J.K. Tessmar, Y. Tabata, A.G. Mikos, J. Control. Release 94 (2004) 101–114. B. Balakrishnan, A. Jayakrishnan, Biomaterials 26 (2005) 3941–3951. J.A. Wieland, T.L. Houchin-Ray, L.D. Shea, J. Control. Release 120 (2007) 233–241. S. Morimoto, Ind. Eng. Chem. 2 (1970) 23–32. P.F. Fox, D.M. Mulvihill, Casein, in: P. Harris (Ed.), Food gels, Elsevier Applied Science, London, 1990, pp. 121–173. A.O. Elzoghby, W.S.A. El-Fotoh, N.A. Elgindy, J. Control. Release 153 (2011) 206–216. A.J.E. Farran, S.S. Teller, A.K. Jha, T. Jiao, R.A. Hule, R.J. Clifton, D.P. Pochan, R.L. Duncan, X. Jia, Tissue Eng. A 16 (2011) 1247–1261.
[20] J. Kundu, L.A. Poole-Warren, P. Martens, S.C. Kundu, Acta Biomater. 8 (2012) 1720–1729. [21] V. Abbate, X. Kong, S.S. Bansal, Enzym. Microb. Technol. 50 (2012) 130–136. [22] S.S. Amruthwar, A. V., J. Mater. Sci. Mater. Med, 23 (2012) 2903–2912. [23] F. Song, L.M. Zhang, J.F. Shi, N.N. Li, Colloids Surf. B: Biointerfaces 79 (2010) 142–148. [24] F. Song, L.M. Zhang, C. Yang, L. Yan, Int. J. Pharm. 373 (2009) 41–47. [25] T. Ito, Y. Yeo, C.B. Highley, E. Bellas, C.A. Benitez, D.S. Kohane, Biomaterials 28 (2007) 975–983. [26] H. Zhao, N.D. Heindel, Pharm. Res. 8 (1991) 400–402. [27] H.H. Winter, F. Chambon, J. Rheol. 30 (1986) 367–378. [28] Y. Liang, W. Liu, B. Han, C. Yang, Q. Ma, F. Song, Q. Bi, Colloids Surf. B: Biointerfaces 82 (2011) 1–7. [29] L. Weng, A. Romanov, J. Rooney, W. Chen, Biomaterials 29 (2008) 3905–3913. [30] L.S. Guinesi, E.T.G. Cavalheiro, Carbohydr. Polym. 65 (2006) 557–561. [31] R. Zhang, Z. Huang, M. Xue, J. Yang, T. Tan, Carbohydr. Polym. 85 (2011) 717–725. [32] J.E. Hodge, J. Agric. Food Chem. 1 (1953) 928–943. [33] F. Song, L.M. Zhang, N.N. Li, J.F. Shi, Biomacromolecules 10 (2009) 959–965. [34] J.J. Roberts, A. Earnshaw, V.L. Ferguson, S.J. Bryant, J. Biomed. Mater. Res. B 99 (2012) 158–169. [35] E.P. Azevedo, V. Kumar, Carbohydr. Polym. 90 (2012) 894–900. [36] Y. Fu, K. Xu, X. Zheng, A.J. Giacomin, A.W. Mix, W.J. Kao, Biomaterials 33 (2012) 48–58. [37] W. Lou, H. Zhang, J. Ma, D. Zhang, C. Liu, S. Wang, Z. Deng, H. Xu, J. Liu, Carbohydr. Polym. 90 (2012) 1024–1031. [38] P. Costa, L.J.M. Sousa, Eur. J. Pharm. Sci. 13 (2001) 123–133.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Using casein and oxidized hyaluronic acid to form biocompatible composite hydrogels for controlled drug release Nan-Nan Li, Chao-Ping Fu, Li-Ming Zhang ⁎ DSAPM Lab, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China PCFM Lab, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China
a r t i c l e
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Article history: Received 19 August 2013 Received in revised form 10 December 2013 Accepted 17 December 2013 Available online 27 December 2013 Keywords: Oxidized hyaluronic acid Casein protein Gelation Drug delivery Biocompatibility
a b s t r a c t To develop biocompatible polymeric hydrogels for the in-situ encapsulation and controlled release of hydrophilic drugs, the oxidized hyaluronic acid containing aldehyde groups was prepared by the reaction between hyaluronic acid and sodium periodate, and then used for the first time to crosslink casein protein in aqueous system. By changing its aldehyde group content or amount, we found that the gelation kinetics and the properties of resultant composite hydrogel could be modulated. Particularly, an increase of its aldehyde group content or amount was found to result in a shorten gelation time, an enhanced gel strength, a reduced swelling ratio and a prolonged drug release. In addition, the as obtained composite hydrogel was also evaluated for its in vitro cytotoxicity on L929 mouse fibroblast cells and was confirmed to have a good biocompatibility. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent years, there has been an increasing interest in using polysaccharide-based cross-linkers to prepare biocompatible polymeric hydrogels with wide biomedical applications for drug delivery, cell encapsulation and tissue engineering. In this context, some natural polysaccharides containing vicinal hydroxyl groups, including hyaluronic acid [1–6], sodium alginate [7], dextran [8], gum arabic [9] and chondroitin sulfate [10], were modified with periodate to form aldehyde groups, and then were used to crosslink amino group-containing polymers in aqueous systems by the Schiff's base reaction between amino and aldehyde groups. The resultant hydrogels could be formed in situ, and exhibit better biocompatibility when compared to those obtained from photo-crosslinking by a potentially toxic photosensitizer [11] or chemical crosslinking by potentially toxic carbodiimide, glutaraldehyde, and adipic dihydrazide [12–15]. As a kind of important biopolymers, caseins are the phosphoproteins that precipitate from raw skim milk by acidification, whose molecular weights have been reported to range from 19,007 to 25,230 Da [16]. Over 55% of the amino acids in casein proteins contain hydrophilic functional groups such as \COOH, \NH2 and \OH [17]. They possess a number of favorable characteristics suitable for the development of hydrogel biomaterials, such as high hydrophilicity, good biodegradation, lack of toxicity, and availability of reactive sites for chemical
⁎ Corresponding author. Tel./fax: +86 20 84112354. E-mail address: [email protected] (L.-M. Zhang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.025
modification [18]. Moreover, they are less expensive and more readily available when compared to collagen [19], silk fibroin [20], bovine serum albumin [21] and elastin-like polypeptides [22], which are usually used for the preparation of protein-based hydrogels. Nevertheless, little work was dealt with the chemical crosslinking of casein protein in aqueous systems except for our recent studies [23,24], in which casein was crosslinked by naturally occurring genipin or microbial transglutaminase to form biocompatible protein-based hydrogels for the controlled release of hydrophilic drugs. In the present work, oxidized hyaluronic acid containing reactive aldehyde groups was prepared and then used for the first time to crosslink casein protein in aqueous system. In particular, the gelation kinetics and the properties of resultant composite hydrogels were investigated with respect to the aldehyde group content and amount of the oxidized hyaluronic acid. In addition, the in vitro drug release behavior and cytotoxicity were also studied for the resultant composite hydrogels.
2. Experimental 2.1. Materials Hyaluronic acid (Mw = 50 kDa) was purchased from Guangzhou Reborn Chemical Company (China). Casein powder was purchased from Lanzhou Tongjian Biotechnology Company (China), which was made from the Yak milk with high quality. Sodium periodate was obtained from Guangzhou Guanghua Chemical Company (China). Salicylic
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acid was purchased from Acros Organics. Mouse fibroblasts of the cell line L929 were kindly provided by Second Affiliated Hospital of Sun Yat-sen University (China). All other chemicals and reagents were of analytical grade and used as received. 2.2. Preparation and characterization of oxidized hyaluronic acid Oxidized hyaluronic acid (O-HA) containing aldehyde groups was prepared by the reaction between hyaluronic acid and sodium periodate. According to previous reports [2,25], the vicinal hydroxyl groups on the sugar ring of hyaluronic acid could be oxidized by sodium periodate to form two aldehyde groups in each oxidized monomeric unit. Briefly, 1.0 g hyaluronic acid sodium was dissolved in 100 mL distilled water at a concentration of 10 mg/mL. An aqueous solution of sodium periodate (0.5 mol/mL, 5 mL) was added dropwise, and the reaction was continued for various time (2, 10 or 24 h) at room temperature in the dark under magnetic stirring. 1 mL ethylene glycol was then added to inactivate un-reacted sodium periodate. After that, the resultant solution was dialyzed exhaustively for 3 days against water and lyophilized to obtain O-HA product with a yield of about 75%. By changing the reaction time, three O-HA samples with different contents of the aldehyde groups were prepared, namely O-HA-1 in the case of 2 h, O-HA-2 in the case of 10 h and O-HA-3 in the case of 24 h. By the hydroxylamine hydrochloride method [26], the aldehyde group content was determined to be 1.16% for O-HA-1, 3.19% for O-HA-2 and 5.22% for O-HA-3, respectively. 1H NMR (300 MHz, D2 O): δ (ppm) 1.9 (acetamide protons, NH\CO\CH3), 3.0–3.9 (protons of sugar units, CH\O and CH 2 \O), 4.3 and 4.4 (anomeric protons, O\CH\O), 5.0–5.1 (hemiacetalic proton formed from aldehyde and adjacent hydroxyl groups). 2.3. Preparation and characterization of composite hydrogels For the formation of each composite hydrogel sample, aqueous casein solution was first prepared by dissolving casein powder in distilled
water, and its pH value was then adjusted to be about 8 by 2.0 mol/mL sodium hydroxide solution. After that, a required amount of oxidized hyaluronic acid (O-HA) (dissolved in distilled water) was added for the gelation of aqueous casein solution. The total concentration of casein in the mixed system was fixed to be 10% (w/w). In order to investigate the effects of O-HA amount on the gelation, 1.0, 3.0 and 5.0% of its concentrations (in mixed system) were used, respectively. The gelation process was monitored by a rheological method [27], and the viscoelastic parameters of resultant composite hydrogels (aged for 24 h) were measured by using an advanced rheometric extended system (ARES, TA Co.) in oscillatory mode with a Couette geometry (25 mm diameter). To ensure the rheological measurements within a linear viscoelastic region, a dynamic strain sweep was conducted prior to the frequency sweep, and the corresponding strain was determined to be 5.0%. The dried composite hydrogels were obtained by lyophilization for SEM observation. In this case, the composite hydrogel samples were sputtercoated with gold under vacuum and imaged using a Hitachi S-520 scanning electron microscope (Tokyo, Japan). The swelling tests were carried out in pH 7.4 phosphate buffered saline solutions for various composite hydrogel samples. The swelling ratio (SR) was calculated according to the following equation: SR ¼ ðW t −W 0 Þ=W 0
ð1Þ
where Wt is the weight of the swollen composite hydrogel at a given time t during swelling and W0 is the weight of the dried composite hydrogel. All swelling tests were carried out in triplicate. 2.4. In situ encapsulation and in vitro release tests for model drug For the in situ encapsulation of salicylic acid as a hydrophilic model drug, 0.2% salicylic acid was added into aqueous 10 wt.% casein dispersion under continuous stirring. After dissolution of the drug, required amount of O-HA was introduced to induce the gelation. To study the in vitro drug release profile, each composite hydrogel sample loaded
Fig. 1. Photographs for the composite hydrogel formation when 10.0% aqueous casein solution was mixed with 3.0% aqueous O-HA-3 solution and a graphical representation for Schiff's base crosslinking reaction.
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with 0.2% salicylic acid was immersed in 20 mL phosphate buffer saline (PBS, pH 7.4) at 37 °C. At predetermined time points, 3.0 mL of this solution was taken out and a 3.0 mL fresh buffer solution was added back to maintain the same total solution volume. The cumulative percentage of released salicylic acid drug was determined by means of a UV spectrophotometer (S52, China) at the absorbance wavelength of 296 nm. All release studies were carried out in triplicate. 2.5. In vitro cytotoxicity assay for composite hydrogel The in vitro cytotoxicity of the composite hydrogel formed from 10.0% casein and 3.0% O-HA-3 was evaluated on L 929 mouse fibroblast cells using a Cell Counting Kit-8 (CCK-8) with the experimental conditions recommended by the CCK-8 manual. For the comparative studies, aqueous casein solution, aqueous O-HA-3 solution and phosphate buffer saline were investigated for their in vitro cytotoxicity, respectively. All materials were sterilized by gamma irradiation with a dose of 25 kGy. Briefly, L 929 mouse fibroblast cells were seeded in 96-well plates at 3 × 103 cells per well and incubated at 37 °C under a 5% CO2 humidified atmosphere in Dulbecco's modified Eagle's medium (DMEM) for 24 h prior to the test. Then 20 μL of sterilized sample were added (for control group: 20 μL of PBS was added; for casein group: 20 μL of 10.0% casein was added; for O-HA-3 group: 20 μL of 5.0% O-HA-3 was added; for hydrogel group: 10 μL of 10.0% casein and 10 μL of 5.0% O-HA-3 were added by a dual barreled syringe) into each well of the plate which was placed for 24 h in an incubator (37 °C and 5% CO2). After that the cells were treated with the recommended dose of CCK-8 assay solution. Absorbance was measured at 450 nm by using an automated ELISA reader (Labsystems Dragon Wellscan MK-3, Finland). The cell viability was calculated as a percentage ratio of absorbance of the experiment group and PBS group. For microscopic observation, an inverted phase contrast microscope (IX70, Olympus, Tokyo, Japan) was used to obtain the microscopic images for the cells in direct contact with PBS and the cells in direct contact the casein/O-HA-3 composite hydrogel. 3. Results and discussion 3.1. Formation and characteristics of casein/oxidized hyaluronic acid composite hydrogels Due to the Schiff's base crosslinking reaction between the amino groups in casein and the aldehyde groups in oxidized hyaluronic acid (O-HA), aqueous casein solution (10.0%) could be transformed quickly
Fig. 3. Time dependence of elastic modulus (G′) and viscous modulus (G″) for aqueous mixed casein/O-HA system: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%. (Test conditions: 25 °C, 1.0 rad/s).
into the invertible hydrogel when aqueous solution (1.0–5.0%) of O-HA was introduced at room temperature in the absence of any extraneous crosslinking agent, as shown in Fig. 1. Similar gelation phenomena
Fig. 2. Photographs for aqueous mixed system composed of 10.0% casein and 3.0% unmodified hyaluronic acid before and after standing for 48 h.
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were also reported for aqueous mixed system composed of chitosan or hydroxypropyl chitosan and sodium alginate dialdehyde [28] as well as aqueous mixed system composed of N-carboxyethyl chitosan and periodate-oxidized dextran [29], in which Schiff's base formation between the available aldehyde and amino groups resulted in the in situ formed biodegradable composite hydrogels for corneal endothelium reconstruction and enhanced wound healing. Moreover, there is an obvious color change from light yellow to deep red when aqueous casein solution gelled in the presence of O-HA. This phenomenon could be attributed to the formation of Schiff's base links between free amino groups of casein and the aldehyde groups of O-HA, as described previously [9,30–32]. For a comparative study, we prepared aqueous mixed solution composed of 10.0% casein and 3.0% unmodified hyaluronic acid, and didn't observe any phase separation or precipitation. Moreover, such a mixed system didn't show perceptible viscosity change and retained its original flowability even after standing for 48 h, as shown in Fig. 2. These results confirm further the crosslinking reaction for aqueous casein/oxidized hyaluronic acid system. Dependent on the aldehyde group content and amount of used OHA, aqueous mixed casein/O-HA systems were observed to have various gelation rates. To illustrate this, time sweep measurements were carried out by means of an advanced rheometric extended system, in which the elastic modulus (G′) and viscous modulus (G″) were monitored as a function of time. Fig. 3 shows the time dependences of G′ and G″ for
aqueous mixed casein/O-HA systems with various compositions. In each case, a crossover point between G′ and G″ was observed, which implied that there was a sol–gel transition [27]. 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 [27]. From Fig. 3a, the gelation time was determined to be about 50.6 min in the case of 3.0% O-HA-1 (containing 1.16% aldehyde group), 29.7 min in the case of 3.0% O-HA-2 (containing 3.19% aldehyde group), and 11.1 min in the case of 3.0% O-HA-3 (containing 5.22% aldehyde group), respectively. From Fig. 3b, the gelation time was determined to be about 48.7 min in the case of 1.0% O-HA-3, 11.1 min in the case of 3.0% O-HA-3, and 3.4 min in the case of 5.0% O-HA-3, respectively. The higher the aldehyde group content or amount of used O-HA, the shorter the gelation time was. In other words, the in situ gelation took place more readily when a higher aldehyde group content or amount of O-HA was used. This may be attributed to enhanced Schiff's base crosslinking in these cases. For the resultant casein/O-HA composite hydrogels, their viscoelastic and swelling properties as well as their morphologies were investigated. Fig. 4 gives the G′ and G″ values as a function of frequency for the resultant composite hydrogels with various compositions. For each composite hydrogel sample, the G′ value was observed to be greater than the G″ value over the entire range of frequency. This indicates
Fig. 4. Elastic modulus (G′) and viscous modulus (G″) as a function of frequency for various casein/O-HA composite hydrogels: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%.
Fig. 5. Swelling kinetics of various casein/O-HA composite hydrogels at 37 °C in pH 7.4 phosphate buffered saline solutions: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%.
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that the resultant composite hydrogel could display a viscoelastic behavior with dominant elastic property [33,34]. Moreover, such a viscoelastic behavior was found to become more obvious and stronger for the composite hydrogel formed in the case of a higher aldehyde group content or a higher O-HA amount. Among all the composite hydrogels investigated, the greatest G′ value was observed for the composite hydrogel formed from aqueous mixed system composed of 10.0% casein and 5.0% O-HA-3. In this case, the highest aldehyde group content and the highest O-HA amount were used, which may result in the greatest extent of Schiff's base crosslinking. Fig. 5 shows the swelling kinetics of casein/O-HA composite hydrogels with various compositions at 37 °C in pH 7.4 phosphate buffered saline solutions. As seen, these hydrogels could attain equilibrium swelling in 60 min and hold large amount of water. This is due to the wicking action of solution through the hydrogel pores as well as the hydration action of some functional groups such as \OH, \NH2 and \COOH. In particular, the lowest swelling ratio was found for 10.0% casein/5.0% O-HA-3 composite hydrogel, which resulted from the greatest crosslinking extent when the highest amount (5.0%) of O-HA-3. In addition, the swelling ratio in the case of 5.0% O-HA-3 (green curve in Fig. 5b) had a slight decrease after 120 min. This may be attributed to the formation of more hydrogen bonds in the hydrogel network during the swelling, which induced the shrinkage of the hydrogel network. Similar phenomenon was observed by other investigators [35–37] when they studied the swelling
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behavior of other biopolymer-based hydrogels. With the help of SEM observation, we found that such a composite hydrogel was characteristic of a more compact network structure when compared to those of other hydrogels, as shown in Fig. 6. 3.2. Drug release behavior from casein/oxidized hyaluronic acid composite hydrogels To explore the possibility of casein/oxidized hyaluronic acid composite hydrogels for drug delivery application, 0.2% salicylic acid drug was encapsulated in situ in the hydrogel matrices during the gelation. Fig. 7 gives the in vitro release profiles of encapsulated salicylic acid by various composite hydrogels at 37 °C in pH 7.4 phosphate buffered saline solutions. As seen, there is a sustained release behavior for loaded salicylic acid, regardless of the hydrogel compositions. Depending on the aldehyde group content of O-HA or O-HA amount used for the hydrogel formation, various release rates were found for the encapsulated drug. To understand better the release characteristics and mechanism of encapsulated salicylic acid from resultant composite hydrogels with different compositions, we fitted the accumulative drug release data using the following semi-empirical equation [38]: Mt =M ∞ ¼ kt
n
ðMt =M∞ ≤0:6Þ
ð2Þ
Fig. 6. SEM images of various casein/O-HA composite hydrogels: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%.
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k
n
R
Transport mechanism
0.27 ± 0.01
0.991
0.38 ± 0.02
0.996
0.46 ± 0.02
0.997
Pseudo-Fickian diffusion Pseudo-Fickian diffusion Pseudo-Fickian diffusion
55.15 ± 2.75
0.44 ± 0.02
0.997
42.90 ± 2.14
0.46 ± 0.02
0.997
37.52 ± 1.87
0.48 ± 0.02
0.993
Effect of O-HA composition 3.0% O-HA-1 + 73.11 ± 3.65 10.0% casein 3.0% O-HA-2 + 53.30 ± 2.66 10.0% casein 3.0% O-HA-3 + 42.90 ± 2.14 10.0% casein Effect of O-HA amount 1.0% O-HA-3 + 10.0% casein 3.0% O-HA-3 + 10.0% casein 5.0% O-HA-3 + 10.0% casein
Pseudo-Fickian diffusion Pseudo-Fickian diffusion Pseudo-Fickian diffusion
3.3. In vitro cytotoxicity of casein/oxidized hyaluronic acid composite hydrogels
Fig. 7. In vitro release profiles of encapsulated salicylic acid drug from various casein/ O-HA composite hydrogels at 37 °C in pH 7.4 phosphate buffered saline solutions: (a) effect of O-HA composition (aldehyde group content: O-HA-1, 1.16%; O-HA-2, 3.19%; O-HA-3, 5.22%); (b) effect of O-HA-3 amount (1.0%, 3.0% and 5.0%). For the hydrogel preparation, the concentration of aqueous casein solution was fixed to be 10.0%.
where M∞ is the initial mass of drug loaded in situ in the composite hydrogel, Mt is the cumulative amount of the drug released at time t, k is the rate constant, 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 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 (R) were obtained, as listed in Table 1. The k values were found to decrease with the increase of O-HA aldehyde group content or O-HA amount. This phenomenon could be attributed to the formation of a denser hydrogel network in the case of a higher aldehyde group content of O-HA or O-HA amount, which hindered the release of encapsulated drug from the composite hydrogel matrices. In addition, the n values in all cases were found to be smaller than 0.5, showing a pseudo-Fickian diffusion release mechanism.
Further investigation was dealt with the in vitro cytotoxicity of casein/oxidized hyaluronic acid composite hydrogel. Fig. 8a gives the cell viabilities of L929 fibroblast cells cultured for 24 h in the media containing PBS (control group), freshly prepared casein/O-HA-3 hydrogel (casein, 10.0%; O-HA-3, 3.0%), aqueous casein solution (10.0%) and aqueous O-HA-3 solution (3.0%), respectively. Regardless of test media, all of the cells were almost viable. Fig. 8b gives the microscopic images of direct contact assay for L929 fibroblast cell monolayer and the L929 fibroblast cells in contact with the composite hydrogel sample for 24 h. As seen, the L929 fibroblast cells in contact with the composite hydrogel sample still maintained their characteristic spindle shaped morphology. These results confirm that the resultant casein/ oxidized hyaluronic acid composite hydrogel is non-cytotoxic and potentially suitable for biomedical applications.
4. Conclusions New composite hydrogels based on casein protein and oxidized hyaluronic acid (O-HA) were obtained at room temperature without the need of any small molecule cross-linker. Their mechanical, swelling and drug-release properties could be regulated by changing the aldehyde group content of O-HA or O-HA amount. In particular, a mechanically strong composite hydrogel with three-dimensional network could be formed in a short time span under the optimized preparation conditions. Furthermore, such a composite hydrogel was confirmed to be non-cytotoxic and biocompatible. Here we present a facile route to in situ forming composite hydrogel based on casein and O-HA, which may find new applications for the controlled release of bioactive molecules, the encapsulation of living cells and wound healing. Considering that the aldehyde groups of O-HA may react with amino groups of serum proteins, our next work will focus on how to shorten the gelation time as soon as possible and enhance the injectability by a dual barreled syringe for this dual-component system.
Acknowledgments This work is supported by the National Natural Science Foundation of China (21074152, 51273216, J1103305), the Doctoral Research Program of Education Ministry in China (20090171110023), the Key Project of Scientific and Technical Innovation for Universities in Guangdong Province (cxzd1102) and the National Natural Science Foundation of Guangdong Province in China (S2013010012549).
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Fig. 8. (a) The cell viabilities of L929 mouse fibroblast cells cultured for 24 h in the media containing PBS (control group), freshly prepared casein/O-HA-3 composite hydrogel (casein, 10.0%; O-HA-3, 3.0%), aqueous casein solution (10.0%) and aqueous O-HA-3 solution (3.0%), respectively; (b) microscopic images for the cells in direct contact with PBS and the cells in direct contact with the casein/O-HA-3 composite hydrogel.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
L. Weng, H. Pan, W. Chen, J. Biomed. Mater. Res. 85A (2008) 352–365. H. Tan, C.R. Chu, K.A. Payne, K.G. Marra, Biomaterials 30 (2009) 2499–2506. S. Nair, N.S. Remya, S. Remya, P.D. Nair, Carbohydr. Polym. 85 (2001) 838–844. G.D. Prestwich, J. Control. Release 155 (2001) 193–199. Y.C. Chen, W.Y. Su, S.H. Yang, A. Gefen, F.H. Lin, Acta Biomater. 9 (2013) 5181–5193. M.N. Collins, C. Birkinshaw, Carbohydr. Polym. 92 (2013) 1262–1279. Q. Wang, W. He, J. Huang, S. Liu, G. Wu, W. Teng, Q. Wang, Y. Dong, Macromol. Biosci. 11 (2011) 362–372. H. Zhang, A. Qadeer, W. Chen, Biomacromolecules 12 (2011) 1428–1437. K.K. Nishi, A. Jayakrishnan, Biomacromolecules 8 (2007) 84–90. S. Dawlee, A. Sugandhi, B. Balakrishnan, D. Labarre, A. Jayakrishnan, Biomacromolecules 6 (2005) 2040–2048. J.B. Leach, K.A. Bivens, C.N. Collins, C.E. Schmidt, J. Biomed. Mater. Res. 70A (2004) 74–82. K.H. Bouhadir, G.M. Kruger, K.Y. Lee, D.J. Mooney, J. Pharm. Sci. 89 (2000) 910–919. T.A. Holland, J.K. Tessmar, Y. Tabata, A.G. Mikos, J. Control. Release 94 (2004) 101–114. B. Balakrishnan, A. Jayakrishnan, Biomaterials 26 (2005) 3941–3951. J.A. Wieland, T.L. Houchin-Ray, L.D. Shea, J. Control. Release 120 (2007) 233–241. S. Morimoto, Ind. Eng. Chem. 2 (1970) 23–32. P.F. Fox, D.M. Mulvihill, Casein, in: P. Harris (Ed.), Food gels, Elsevier Applied Science, London, 1990, pp. 121–173. A.O. Elzoghby, W.S.A. El-Fotoh, N.A. Elgindy, J. Control. Release 153 (2011) 206–216. A.J.E. Farran, S.S. Teller, A.K. Jha, T. Jiao, R.A. Hule, R.J. Clifton, D.P. Pochan, R.L. Duncan, X. Jia, Tissue Eng. A 16 (2011) 1247–1261.
[20] J. Kundu, L.A. Poole-Warren, P. Martens, S.C. Kundu, Acta Biomater. 8 (2012) 1720–1729. [21] V. Abbate, X. Kong, S.S. Bansal, Enzym. Microb. Technol. 50 (2012) 130–136. [22] S.S. Amruthwar, A. V., J. Mater. Sci. Mater. Med, 23 (2012) 2903–2912. [23] F. Song, L.M. Zhang, J.F. Shi, N.N. Li, Colloids Surf. B: Biointerfaces 79 (2010) 142–148. [24] F. Song, L.M. Zhang, C. Yang, L. Yan, Int. J. Pharm. 373 (2009) 41–47. [25] T. Ito, Y. Yeo, C.B. Highley, E. Bellas, C.A. Benitez, D.S. Kohane, Biomaterials 28 (2007) 975–983. [26] H. Zhao, N.D. Heindel, Pharm. Res. 8 (1991) 400–402. [27] H.H. Winter, F. Chambon, J. Rheol. 30 (1986) 367–378. [28] Y. Liang, W. Liu, B. Han, C. Yang, Q. Ma, F. Song, Q. Bi, Colloids Surf. B: Biointerfaces 82 (2011) 1–7. [29] L. Weng, A. Romanov, J. Rooney, W. Chen, Biomaterials 29 (2008) 3905–3913. [30] L.S. Guinesi, E.T.G. Cavalheiro, Carbohydr. Polym. 65 (2006) 557–561. [31] R. Zhang, Z. Huang, M. Xue, J. Yang, T. Tan, Carbohydr. Polym. 85 (2011) 717–725. [32] J.E. Hodge, J. Agric. Food Chem. 1 (1953) 928–943. [33] F. Song, L.M. Zhang, N.N. Li, J.F. Shi, Biomacromolecules 10 (2009) 959–965. [34] J.J. Roberts, A. Earnshaw, V.L. Ferguson, S.J. Bryant, J. Biomed. Mater. Res. B 99 (2012) 158–169. [35] E.P. Azevedo, V. Kumar, Carbohydr. Polym. 90 (2012) 894–900. [36] Y. Fu, K. Xu, X. Zheng, A.J. Giacomin, A.W. Mix, W.J. Kao, Biomaterials 33 (2012) 48–58. [37] W. Lou, H. Zhang, J. Ma, D. Zhang, C. Liu, S. Wang, Z. Deng, H. Xu, J. Liu, Carbohydr. Polym. 90 (2012) 1024–1031. [38] P. Costa, L.J.M. Sousa, Eur. J. Pharm. Sci. 13 (2001) 123–133.