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Modification and swelling kinetic study of kappa-carrageenan-based hydrogel for controlled release study
Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 182–191
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Modification and swelling kinetic study of kappa-carrageenan-based hydrogel for controlled release study Hadi Hezaveh, Ida Idayu Muhamad * Faculty of Chemical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Johor, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 May 2012 Received in revised form 3 October 2012 Accepted 23 October 2012 Available online 20 December 2012
In this article, the physical stability of carrageenan hydrogels was improved for in vitro controlled drug delivery in different pH mediums. Physical property changes were studied using various characterization tests. The effect of modification on swelling behavior of hydrogels as well as swelling kinetics of hydrogels was also investigated. It was found that the addition of genipin to the hydrogels can increase the physical properties of carrageenan hydrogels. An optimum genipin concentration in which molecular structure of hydrogel can be in its most stable form is also determined. The study of swelling kinetics revealed that the modification has resulted in the relaxation of carageenan molecules and the swelling mechanism from a diffusion-controlled mechanism is changed to a polymer relaxationcontrolled mechanism. Release study shows that by changing the genipin concentration in the hydrogel drug release can be controlled. ß 2012 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Hydrogel Modified kappa-carrageenan Genipin cross-linking Characterization Controlled delivery
1. Introduction Controlled drug delivery systems (DDS) have provided an alternative approach to mobilize the bioavailability of therapeutic agents. In DDSs, an active therapeutic is encapsulated into a polymeric network so that the drug is released from the material in desired conditions [1,2]. Hydrogels have recently attracted a great deal of interest in biomedical and pharmaceutical systems and have been utilized in various applications including biomedicine (wound dressings and tissue engineering), infant care (diapers), pharmaceuticals (drug delivery) and agriculture (water retention during drought) [3,4]. Hydrogels are synthetic polymers that can swell or degrade in response to internal or external elements. In industrial applications, biomaterials are being substituted for synthetic polymers and improving the biocompatibility of these polymers [4]. Recent advances in hydrogel technology have been focused more on the use of biocompatible and non-toxic materials for pharmaceutical and biomedical applications [5]. Among all these hydrogels, intelligent thermoand pH-sensitive systems are more promising, as temperature and the pH are key environmental parameters in the human body. Also, some diseases show themselves by a change in temperature and/or pH [6,7]. Kappa-carrageenan (kC) is mostly used in the food industry as gelling, stabilizing agent and thickener because of its high
* Corresponding author. E-mail address: [email protected] (I.I. Muhamad).
hydrophilicity, mechanical strength, biocompatibility and biodegradability [8]. Kappa-carrageenan would reduce or eliminate toxicity in biomedical applications. For this reason, kC has been applied for immobilizing protein and controlled drug delivery systems [3,9,10]. Palace et al. [3] reported that the estimated amino acid concentration in the polymeric matrix of kC is 4870 ng/ mg (0.487%). Many researches have been conducted to modify the physical and chemical properties of carrageenan [11,12]. Tari and Pekcan [13] reported the association of kappa-carrageenan with CaCl2 to change the swelling properties of carrageenan gels. Genipin is known as an iridoid glucoside which participates in both short- and long-range covalent cross-linking of e-amino groups in amine-containing polymers [4]. Genipin has been used widely in Chinese medicine as well as food dye production, in which genipincross-linked amine groups in the presence of oxygen, form blue pigments [14]. It is shown that using genipin in protein interaction can improve the adsorption capacity of chitosan spheres [15]. Genipin, a natural cross-linker is found to be 10,000 times less toxicity than the common used cross linking agent, glutaraldehyde is utilized to cross-link the hydrogels with minimum cytotoxic effect [9]. Kuo and Wang [16] successfully synthesized genipin-crosslinked chitosan/gelatin scaffolds and investigated the influence of BPE on the generation of cartilaginous components. Hydroxyethyl cellulose (HEC) with high amounts of –OH groups is a biocompatible martial with good solubility in water. Application of HEC in biotechnological and biophysical areas as well as many industrial fields has attracted the attention of researcher’s to its properties [17].
1876-1070/$ – see front matter ß 2012 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jtice.2012.10.011
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Fig. 1. Chemical structure of the b-carotene [17].
b-Carotene (Fig. 1) is known as an important derivative of the carotenoids family and is found in many fruits and vegetables [18]. b-Carotene consists of eight isopropene units as a basic molecular structure, containing forty atoms of carbon and two rings at the end of its conjugated double bond chain. b-Carotene with high conversion rate comprises substantial proportion of vitamin A in the human diet [19] and has been reported to protect against cancer in humans [20] and also cardiovascular diseases [21]. For these reasons, b-carotene and other carotenoids have attracted much attention as functional ingredients in industry. Mathematical modeling of drug delivery systems and predictability of drug release is a field of steadily increasing academic and industrial importance with enormous future potential [22]. Many mathematical theories have been proposed to discuss the swelling kinetic in the references [23–26]; however, there is no general model that can be applied to all types of drug delivery systems. The aim of this study is to modify the carrageenan hydrogel for controlled b-carotene release using natural cross-liner. Different characterization tests were carried out to study the physical changes in hydrogels. Modified hydrogels were used as a drug carrier to release b-carotene. The swelling kinetics of hydrogels is also investigated.
then was added to kC/HEC solution to formulate 0.1, 0.2, 0.3, 0.5 and 1.0 mM genipin cross-linked hydrogels at 80 8C. The homogeneous viscous solutions were maintained at room temperature (25 8C) over night and then were dried in an oven at 37 8C for nearly 24 h. The aim of the modification was to improve the physical properties of kC for in vitro release studies and control the amount of b-carotene released from kC hydrogels. From now, kC/HEC blended hydrogels will be referred to using the kC percentage and cross-linked hydrogels with their genipin concentration. For example, kC/HEC ratio of 90:10 will be referred to as kC90 and 1.0 mM genipin cross-linked hydrogels will be referred to as GN1.0. 2.3. Instrumentation 2.3.1. FTIR analysis of hydrogel To study the structure of hydrogels, 4 mg of the dried samples of both non-modified and modified hydrogels were ground and mixed with KBr powder (with the ratio of 1:10). Hydraulic press was used to form the sample pellets under 500 kg/cm2 pressure. Then samples were analyzed using a Fourier transform infrared spectroscope (FTIR) (Nicolet 670 FTIR, USA). 16 scans per sample were conducted with a resolution of 4 in the region between 370 and 4000 cm1 with 1.0 cm1 interval.
2. Experimental 2.1. Material Kappa-carrageenan (kC) (Sigma–Aldrich), hydroxyethyl cellulose (HEC) (Fluka Chemicals), genipin (GN) (Challenge Bioproducts Co., Ltd. Taiwan), b-carotene (CALBIOCHEM, La Jolla, CA, USA), HCl (Qrec Grad AR), NaOH (Qrec Grad AR). Distilled water was also used in synthesizing hydrogel. All chemicals are used as received with no additional purification. 2.2. Preparation of modified hydrogels In order to modify kC hydrogels, they are first blended with HEC in different concentrations to increase physical stability of hydrogels and then cross-linked with natural cross-linker genipin (the concentration of genipin varied from 0.1 mM to 1.0 mM). kC/ HEC blends were prepared by mixing HEC and kC in 30 ml distillated water at 80 8C in different ratios. The experiment was carried out under reflux to ensure that the water content in the system does not change. Gentle stirring prevented air bubble formation in the gels. The hot solutions were then poured into ceramic moulds to allow the cooling process to take place for about 24 h at ambient temperature (25 8C). Hydrogel samples with an approximate diameter of 5.5 cm and a thickness of 1.5 cm were made. Finally, samples were dried in an oven overnight at 37 8C to form hydrogels ready to swell test. The most suitable hydrogel was then chosen for further modification with genipin. To do this, different genipin concentrations of stock solutions were prepared by dissolving genipin powder in 10% hot water under rigorous stirring. Genipin solution
2.3.2. Thermogravimetric analysis (TGA) Thermogravimetric analysis (TGA) of non-modified and modified gels was determined on a SDTA 85 METTLER (TOLEDO, Switzerland) TGA System. The rate was set at 10 8C min1 in the temperature range of 30–700 8C for 65 min under nitrogen flow. 2.3.3. Differential scanning calorimetry (DSC) The thermal properties of kC/HEC hydrogels were examined using a DSC822 METTLER TOLEDO (Switzerland). Approximately 5.0 mg of kC/HEC samples were weighed into a sealed aluminum pan and heated at 5 8C min1 from 30 8C to 200 8C. The nitrogen flow rate purging to the system was maintained at 50 ml min1. 2.3.4. Hydrogel microstructure study Microstructure of hydrogels was studied using field emission scanning electron microscope (FESEM) on Gemini Supra 35 VP FESEM. Dried samples were mounted on the sample holder and coated with gold using Bio Rad Polaran Division (E6700, USA) gold sputter coater under vacuum before observation. Samples photomicrographs were studied on FESEM at the accelerating voltage of 10 kV and 500 magnification. 2.4. Swelling studies Swelling studies of hydrogels were carried out in buffer solutions of pH 1.2, 7 and 12 at room temperature. Samples were immersed in Petri dish filled with 30 ml of the each buffer solution. At different intervals, hydrogels were moved out, and the surface water of gels was wiped using filter paper and weighed. Fresh medium was used for further swelling study. The swelling
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10 9
where Wt is the weight of swollen gels at time t and W0 is the initial weight of samples. Swelling experiment was performed till hydrogels reached at their equilibrium condition. Tests were conducted in triplicate to minimize error and are reported as a mean value. Samples reached at equilibrium were ready for deswelling test. Fully hydrated gels were put in Petri dishes and placed in an oven at 37 8C. The samples were removed from the oven after predetermined time intervals. The dry weight of the samples was measured using a Sartorius scale at room temperature. In the final step, the dried samples were placed into separated Petri dishes filled with buffer solution of pH 7 to study the reswelling. The experiment was carried out at room temperature in triplicate to increase the accuracy. The water uptake (Wu) of the hydrogels was calculated using Eq. (2) [27] Wt W0 (2) 100 We W0 where Wt is the weight of the swollen hydrogel after given time t, W0 and We are the hydrogel weight before swelling and at equilibrium swelling, respectively [28,29].
kC60
8
(1)
kC70
7 Swelling (%)
percentage of hydrogels is then calculated: Wt W0 swelling ratio ð%Þ ¼ 100 W0
kC90
6
kC100
5 4 3 2 1 0
0
18
kC90
12
kC100
100
120
140
10 8 6
To encapsulate the b-carotene, 1.0 mg/ml of b-carotene was dissolved in ethanol and then added to kC/HEC hot solution under stirring using dripping method before adding genipin with mentioned concentrations. The solutions were kept at 80 8C. The cross-linked b-carotene loaded gels were then poured into ceramic moulds to harden and the release study was performed immediately after hardening. Samples were immersed in Petri dishes filled with 50 ml of medium solutions and placed in an incubator at 37 8C. Every 10 min, 4.0 ml aliquot of the solution was removed and the concentration of b-carotene released was determined using a UV–vis spectrophotometer. 4 ml of fresh buffer was replaced to maintain the original volume after each withdrawal. The amount of b-carotene was calculated by interpolation from the b-carotene standard curve at l = 446 nm [9].
2 0
0
10
20
30
40
Time (hr) Fig. 3. Non-cross-linked hydrogels swelling in pH 7 at room temperature.
and therefore less swelling. In neutral medium, hydrogels swelled more and kC100 and kC90 swelled up to 15.43 and 13.12%, respectively. Therefore, kC90 has exhibited a larger swelling increment in the neutral medium than kC100. kC70 and kC60 also swelled 11% and 6.91% in neutral medium, respectively. In alkaline medium, both kC100 and kC90 blend ratios exhibited more swelling than in acidic and neutral. This can be explained as electrostatic repulsion of polymer chains due to the ionization of carboxylic acid groups that breaks the hydrogen bonds and consequently, pushes the network and expands the structure to allow more water to diffuse in [31,32]. After 16 h, blends in neutral and alkaline medium reached equilibrium. 25
kC60 20 Swelling (%)
3.1.1. Effect of blending on swelling behavior Swelling (%) of various kC/HEC blends were performed in triplicate. Results in acidic (pH 1.2, 0.1N HCl), neutral (pH 7) and alkaline (pH 12, 0.1 M NaOH) environment are illustrated in Figs. 2–4, respectively. In all mediums, swelling rate was high at the beginning of the swelling process as ionic chemical potential difference was controlling the swelling. After that, internal and external ions reached their equilibrium state and interactions between polymer and solvent, which is less than ionic chemical potential difference, dominated the process, and consequently swelling rate decreased [30]. In acidic environment, hydrogels reached at equilibrium after almost 2 h. The highest swelling percentage was for kC100 ratio that swelled up to 8.92% followed by kC90 at 5.06%. Low swelling in this medium can be due to the protonation of carboxylic groups and creation of more hydrogen bonds in kC hydroxyl groups resulting in more compact networks
60 80 Time (min)
kC70
14
4
3.1. Swelling studies
40
kC60
16
2.5. Encapsulation and the in vitro release study of b-carotene
3. Results and discussion
20
Fig. 2. Non-cross-linked hydrogels swelling in pH 1.2 at room temperature.
Swelling (%)
184
kC70 kC90
15
kC100
10 5 0
0
5
10
Time (hr)
15
20
Fig. 4. Non-cross-linked hydrogels swelling in pH 12 at room temperature.
25
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100 90
pH 1.2
20
80
pH 7
70
pH 12
15
Wu (%)
Maximum Swelling
25
10 5 0
50
60
70
80 90 kC Concetration (%)
100
kC60
50
kC70
40 30
kC90
20
kC100
0
110
kC70 and kC60 blend ratios swelled at neutral environment more than alkaline. This is because of less cross-linking in neutral medium than alkaline medium [33]. Fig. 5 shows the equilibrium degree of swelling (EDS) of blended hydrogels vs. kC content, in all mediums. Clearly, EDS is strongly depended on the kC concentration. The swelling ratio increases as the hydrophilicity of hydrogels increases due to the presence of carrageenan. This has also been observed in our previous researches [34–37] and also reported by Meena et al. [38]. Hydrogels in neutral environment showed more stabile compared to acidic and alkaline environment. In the neutral and alkaline mediums, for 18 and 6 h, respectively, no changes in hydrogel weight were observed. During the experiment the stability of kC90, especially in alkaline medium, was more than other blends. It is worthwhile to note that kC60 blend ratio in both mediums reached its equilibrium after 2 h. Deswelling of hydrogels, shown in Fig. 6, was carried out in the oven at 37 8C over 92 h. It was found that by increasing the kC, hydrogels dehydrate at a lower rate compared to those having more HEC. This can be attributed to the formation of a thick layer on the surface of hydrogels with more kC as a result of shrinkage that prevents water to escape from the network; however as HEC increases these layers weaken and new channels also form in the gel network that helps the water within the hydrogel to diffuse out [27]. Fig. 7 shows the reswelling of hydrogels in pH 7 at room temperature. After 240 h, hyderogels reached their equilibrium, although they did not regain their previous weight during the reswelling. It is assumed that the water present in the gel network filled the space and larger pores in the network [39]. When
80
100 150 Time (hr)
200
250
3.1.2. Effect of modification on hydrogels swelling properties Figs. 8–10 show that the hydrogel swelling ratio decreases when it is modified with genipin in all mediums. This has also been observed in our previous studies [9,34]. It is clear that by increasing the genipin concentration in hydrogels, swelling decreases noticeably. In GN0.1 hydrogel the EDS was 6.18%; however, in GN1.0 it was only 0.48%. In pH 12 this amount of swelling was even less (0.14%) meaning hydrogel swelling has almost stopped. During the experiment it was found that the physical stability of modified hydrogels increased by adding genipin to the blend that was observed in GN0.1, GN0.2 and GN0.3. Less swelling in these hydrogels can be due to the fact that increasing genipin can result in the creation of vast chains, therefore limiting the mobility of the matrix causing less swelling. More than these concentrations, the physical stability of gels decreased during swelling. This can be attributed to the breakdown of some bonds and the formation of weaker bonds which will be discussed in more detail in coming sections. Also hydrogels in neutral medium had more swelling ratio in comparison with alkaline medium. This means that the addition
6 kC 90
kC70
5
kC90
4
Swelling (%)
100
50
dehydration begins, water diffuses out and during reswelling water penetrating into the network creates an elastic reaction force till the matrix reaches its equilibrium [27]. Considering the enhanced physical stability of kC90 blend ratio compared to other blends in all mediums (in alkaline medium this physical stability was more pronounced), this blend was chosen to be modified with different genipin concentrations: 0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM and 1.0 mM to find the most stable hydrogel for in vitro release study.
kC60
120
0
Fig. 7. Non-cross-linked hydrogels reswelling with different concentration of kappa-carrageenan and hydroxyethyl cellulose at 37 8C.
140
Deswelling (%)
60
10
Fig. 5. Equilibrium degree of swelling (EDS) of non-cross-linked hydrogels vs. kC content in different pH.
kC100
60 40
0.1 mM 0.2 mM 0.3 mM 0.5 mM
3
1.0 mM
2 1
20 0
185
0
20
40
Time (hr)
60
80
100
Fig. 6. Non-cross-linked hydrogels deswelling with different concentration of kappa-carrageenan and hydroxyethyl cellulose at 37 8C.
0
0
20
40
60 80 Time (min)
100
120
140
Fig. 8. Swelling behavior of modified gels with different genipin concentrations at room temperature in pH 1.2.
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16 14
Swelling (%)
12
kC90 0.1 mM 0.2 mM 0.3 mM 0.5 mM 1.0 mM
10 8 6 4 2 0
10
0
20 Time (hr)
30
40
Fig. 9. Swelling behavior of modified gels with different genipin concentrations at room temperature in pH 7.
of cross-linker to the gels has altered swelling ability in different buffer solutions. As discussed before, blended hydrogels exhibited more swelling in alkaline environment than neutral; however, by increasing the cross-linker concentration, the swelling capacity in neutral medium increased so that genipin cross-linked hydrogels could swell more in pH 7 than pH 12. Obviously, swelling ability depends on molecular structure and these results suggest that genipin had a noticeable impact on molecular structure. 3.2. Characterization of synthesized hydrogels 3.2.1. FTIR Analysis Fourier transform infrared spectroscope (FTIR) of modified and non-modified hydrogels is presented in Fig. 11. At 3424 cm1, the broad band absorption is attributed to the stretching of –OH groups of kC. The absorption band at 2923 cm1 results from the stretching frequency of –CH2 groups. A typical amide I peak at 1644 cm1 appears in the spectrum of all modified hydrogels. In 0.5 mM and 1.0 mM concentration, a new absorption band appeared at 1354 cm1 and 1442 cm1 which is assigned to C– OH and symmetrical stretching vibration of –COO groups, respectively. By increasing the genipin content of hydrogels, bands at 1642 and 1126 cm1 seems to shift to 1626 and 1110 cm1, respectively. For GN0.5 and GN1.0 hydrogels the intensity of absorption band at 1257 cm1, that is attributed to strong C–O stretch bond in the 20
kC90
18
GN0.1
16
GN0.2
Swelling (%)
14
GN0.3
12
GN0.5
10
GN1.0
8 6 4 2 0
0
5
10
15
20
25
Time (hr) Fig. 10. Swelling behavior of modified gels with different genipin concentrations at room temperature in pH 12.
main chain, is almost disappeared and the less intense peaks of 1229, 1153 and 1112 cm1 in lower regions (consistent with lower energy) are appeared. This indicates that the main chain of kC molecular structure is seriously affected. As a result, the strength of kC as main monomer in the gel is reduced. This has been also observed in previous studies by other researches [40,41] and explains the less stability of 0.5 and especially 1.0 mM genipin cross-linked hydogels during the test [40]. Also, at 1376 absorption band (symmetrical C–H bending) for GN0.2, GN0.3 and GN0.5 is intensified after modification. At 1044 cm1 a new absorption peak is attributed to carbonyl stretching which is formed during modification. Also, when crosslinking occurs, bands at 796, 768 and 733 cm1, are intensified. The bands observed at 844, 927 are assigned to D-galactose-4sulfate, 3,6-anhydrod-galactose, respectively. All the characteristic absorptions of non-modified hydrogel are found in modified gels. Thus, it can be concluded that first, the genipin cross-linked kC/ HEC hydrogel is successfully prepared and second, the addition of cross-linker from 0.1 mM to 0.3 mM concentration can result in the formation of more stable hydrogel structure (as it was optically observed during the test). 3.2.2. Thermal analysis of modified and non-modified hydrogels Fig. 12 indicates the differential scanning calorimetry (DSC) for kC90 and modified gels. At near 100 8C the endothermic peak represents water evaporation from the network. As the concentration of cross-linker increases, the peak decreases due to hydrophilic hydroxyl side-groups that are involved in the cross-linking reaction so molecules can retain more water and less amounts of water can evaporate from the hydrogel network [42]. Clearly, for GN1.0 the physical stability of the network decreases this peak increases again and water evaporation increases. Fig. 12 clearly shows that the genipin increment also results in increasing the glass transition temperature (Tg). The glass transition for non-modified, GN0.1 and GN1.0 are 118, 120 and 124 8C, respectively. The exothermic peak which appeared at 180 8C (for nonmodified films) is related to the polymer’s thermal decomposition [43]. By increasing the genipin, this temperature shifts to higher temperature (round 185 8C); however, at 1.0 mM concentration of cross-linker, this temperature again decreases to 164 8C, indicating the loss network strength as mentioned before. The DSC results along with FTIR data demonstrate that the addition of genipin up to 0.5 mM, can improve the physical properties of kC/HEC hydrogel. Fig. 13 shows TG analysis for non-modified and modified (GN0.1 and GN1.0) hydrogels. kC90, GN0.1 and GN1.0, have lost 45.26, 42.67 and 48.48% of their weight below 200 8C, indicating that thermal stability of GN0.1 is higher than others [44–46]. The weight lost at 700 8C for non-modified, GN 0.1 and GN 1.0 was 58.92, 56.66 and 49.84%, respectively. 3.2.3. Hydrogels microstructure The surface morphology of hydrogels is presented in Fig. 14. By increasing the cross-linker concentration in the network, morphology of the polymer changes noticeably. It is believed that these new chains were aggregates (fibers formed by kC and/or HEC) probably resulted from genipin-crosslink. Increasing genipin concentration resulted in the formation of more fibers with low aspect ratio. This also affects the swelling properties and can explain less swelling in cross-linked gels. These chains in the network form a hardly penetrable surface that does not let water diffuses in. This along with less physical stability as explained before result in low swelling in GN1.0 (see Fig. 14).
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Fig. 11. FTIR results for (a) kC90, (b) GN0.1, (c) GN0.2, (d) GN0.3, (e) GN0.5, and (f) GN1.0.
3.3. pH-response of modified kappa-carrageenan/hydroxyethyl cellulose hydrogels Polymers with ionizable functional groups that respond to pH changes are called pH-sensitive polymers. In these polymers, the electrostatic repulsion force causes an increase in the hydrodynamic volume of the polymer, as charges along the polymer backbone generated [47]. Fig. 15 shows hydrogel swelling versus genipin concentrations in all mediums. Both modified and non-modified hydrogels showed less swelling ability in acidic environment. This can be attributed to both carboxylic group protonation and also degradation of hydrogels in this pH. In neutral medium, however, non-modified hydrogel exhibited better swelling ability and
swelled up to 13.12%. The addition of genipin decreased the swelling and reduced it up to 0.48% for GN1.0 (almost stopped). In pH 12, non-modified hydrogel swelled the most (17.6%), but when cross-linked, hydrogels swelling decreased noticeably. Clearly, modification has changed the swelling behavior of hydrogels. For GN0.1 hydrogels swelled more in alkaline than other mediums, but for GN0.2 almost the same swelling percentage in alkaline and neutral mediums was observed. By further increasing the genipin, the swelling ability of hydrogels in alkaline medium decreased so that GN0.3, GN0.5 and GN1.0 had higher swelling ratios in neutral mediums than in alkaline medium. For GN1.0, the swelling ratios of the hydrogels in acidic, neutral and alkaline mediums were 0.34, 0.14 and 0.48%, respectively. Generally, modified hydrogels exhibited more
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188
Fig. 12. DSC thermograms recorded during heating of cross-linked and kC90 hydrogels.
swelling and stability in neutral mediums than other mediums, as was also observed by Meena et al. [33]. 3.4. Swelling kinetic study The mechanism that water diffuses into the hyderogels network is an important issue due to the unique applications of the hydrogels. To determine the nature of water diffusion into the hydrogels network power law was applied [48]: Mt ¼ ktn M1
(3)
where Mt and M1 are the absolute cumulative water penetrated into the network at time t, and infinite time, respectively. k is a constant indicating the structural and geometric properties of the matrix, and n is the diffusion exponent that indicates the transport mechanism of water taken up. This equation is a simple semi-empirical model with two different physically realistic meanings for n = 0.5 and n = 1.0. When n = 0.5 it represents Fickian or Case I transport behavior in which the relaxation coefficient is negligible and the pure drug diffusion release is considered. When the release rate is constant in time n takes the value of 1.0, known as zero-order release kinetics. In this case the drug release follows a polymer chain relaxation mechanism [23,49]. Values of n between 0.5 and 1.0 indicate non-Fickian or anomalous transport mechanism where structural relaxation is comparable to diffusion. When the n value becomes less than 0.5, the mechanism is known as pseudo-Fickian behavior 120 Fig. 14. FESEM images of (a) kC90, (b) GN0.1 and (c) GN1.0.
Nave
100
GN 0.1 Mass loss (%)
80
GN 1.0
60 40 20 0
0
100
200
300 400 Tempareture (C)
500
600
Fig. 13. TGA analysis of modified and kC90 hydrogels.
700
which means that the sorption curves are like Fickian curves, although the final equilibrium is very slow [23,49]. For a radial diffusion, however, different n values have been derived. n = 0.45 and n = 0.89 show diffusion-controlled mechanism and case II transport, respectively. n and k parameters can be determined by plotting ln(Mt/M1) vs. ln(t) and calculating the slope and intercept of the line. In Table 1 computed values of n, k and the corresponding determination coefficients (R2) for swelling of hydrogels in pH 7.0 (Fig. 9) are listed. For kC90 hydrogel, n = 0.63 and for cross-linked hydrogels it varies from 0.68 to 0.9. The n value for cross-linked hydrogels
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beta caroten release (mg/ml)
(a) 0.016
3.5. Immobilization and in vitro release
b-Carotene, one of the most important food additives, is widely used as a colorant in the food and beverage industry [51]. In this work, b-carotene as a bioactive material was used to study the in vitro release of modified kC/HEC hydrogels in pH of 1.2, 7 and pH 12. It is shown that the existence of cations like K+, Na+, or Ca2+ can induce conformational changes in hydrogel networks [52,53]. Due to the instantaneous ionic gelation properties, the immobilization of drugs in carrageenan is possible [53,54]. pH is a key factor in the drug delivery which determines the release of drug in pH-sensitive hydrogels. The effect of pH on the b-carotene release rate from the modified and non-modified hydrogels is shown in Fig. 16. As Fig. 16a clearly shows, when pH of the medium is 1.2 the concentration of b-carotene released from the native hydrogel is 0.013 mg/ml after 120 min. At pH 7, the drug concentration released is 0.043 mg/ml after 120 min which means that the release amount has increased almost 230%. In the alkaline medium, this increase after 120 min is almost 423% (0.068 mg/ ml). For GN0.3 hydrogel, the b-carotene release in pH of 1.2 was 0.008 mg/ml which was increased in neutral and alkaline mediums. It is well known that release profile of hydrogels is strongly dependent on swelling properties. kC90, GN0.1 and GN0.2 hydrogels showed the same release trend observed in swelling test,
GN0.2
0.01
GN0.3
0.008
GN0.5 GN1.0
0.006 0.004 0.002
beta caroten release (mg/ml)
20
0
(b) 0.08
40 60 Time (min)
80
100
kC90
0.07
GN0.1
0.06
GN0.2
0.05
GN0.3 GN0.5
0.04
GN1.0
0.03 0.02 0.01 0
0
20
40
60 80 Time (min)
100
120
140
60 80 Time (min)
100
120
140
(c) 0.05 beta caroten release (mg/ml)
(GN0.1, GN 0.2 and GN 0.3) indicates the non-Fickian or anomalous transport mechanism. It means that the swelling is controlled by polymer relaxation which is in good agreement with the fact that the strength gels increase with increasing genipin concentrations in these hydrogels [50]. Since the n value for GN0.5 and GN 1.0 is 0.8 and 0.9, the mechanism becomes a case II transport mechanism in which the swelling is controlled by polymer chain relaxation. These results suggest that when the polymer backbone is broken in these gels, the relaxation of polymer network is facilitated. In conclusion, the addition of genipin to the network has caused the swelling mechanism to shift from a diffusion-controlled towards polymer relaxation-controlled mechanisms.
GN0.1
0.012
0
Fig. 15. Maximum swelling of cross-linked hydrogels vs. genipin concentration in three different mediums.
kC90
0.014
0.045
kC90
0.04
GN0.1
0.035
GN0.2
0.03
GN0.3
0.025
GN0.5
0.02
GN1.0
0.015 0.01 0.005 0
0
20
40
Fig. 16. Concentration of b-carotene released in (a) acidic, (b) neutral and (c) alkaline medium.
i.e., the release was more in alkaline than in neutral and acidic mediums. But for GN 0.3, GN0.5 and GN1.0, the release profile was not the same as swelling trend suggesting that the b-carotene has influenced the hydrogels structure. During the experiment, all hydrogels which encapsulated the b-carotene seemed stronger than un-loaded gels. Results shown in Fig. 16 also indicate that the addition of genipin has caused a decrease in the release rate in all mediums. In acidic medium, hydrogels reached the their maximum b-carotene release after 90 min; however, in neutral and alkaline it took
Table 1 Different n and k values of power law model for native gel and various concentrations of genipin. Samples
n k R2 Transport mechanism
Native hydrogel
0.1 mM genipin
0.2 mM genipin
0.3 mM genipin
0.5 mM genipin
1.0 mM genipin
0.63 0.4481 0.98 Anomalous
0.68 0.3214 0.98 Anomalous
0.7 0.2931 0.96 Anomalous
0.73 0.2472 0.95 Anomalous
0.8 0.2035 0.98 Case II
0.9 0.1956 0.96 Case II
190
H. Hezaveh, I.I. Muhamad / Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 182–191
120 min to reach at maximum release. This can be attributed to more compact networks as a result of the protonation of carboxylic groups which hinders the drug release. In neutral medium, the accumulative b-carotene release of non-modified hydrogels was 0.2 mg/ml; however, it decreased to 0.048 mg/ml for GN1.0. Increasing cross-linker density causes less available free space for drug diffusion and consequently the release rate decreases [55]. 4. Conclusion In this research, carrageenan hydrogels were modified to improve their physical properties, enhanced their application in drug delivery systems. The effect of modification on swelling properties, swelling kinetic mechanism and bio active material release was investigated. Using different characterization techniques, the physical property changes of modified carrageenanbased hydrogels was studied. The optimum concentration of crosslinker in which hydrogel structure can be in its most stable form is also proposed. It was found that the modification has changed the swelling behavior of hydrogels. GN0.1 swelled more in alkaline than other mediums; however by increasing genipin content, the swelling ability in alkaline medium decreased in the way that GN0.3, GN0.5 and GN 1.0 had more swelling in neutral than in alkaline medium as result of the high concentration of the counter ions of COO in highly cross-linked hydrogels. The modification has changed the pH-sensitivity of hydrogel in the way that by increasing the genipin content of hydrogels, hydrogels swelled more in neutral medium than alkaline, however non-modified hydrogels showed reverse behavior. Swelling kinetic study shows that increasing the genipin content of matrix; increases the relaxation of carageenan molecules and the swelling mechanism from diffusion-controlled changes to polymer relaxation-controlled mechanisms. Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscope (FTIR) data has proven that the addition of genipin up to 0.5 mM, can improve the physical properties of polymeric network. Beyond this concentration, the main chain of kC molecular structure is seriously affected. Also FESEM test showed that the surface roughness of non-modified polymer is significantly changed when genipin is added to hydrogel. Release study has proven the capability of modified hydrogels to carry and release encapsulated drugs in various pH. By changing the concentration of genipin, it is possible to control in vitro drug release in different mediums.
Acknowledgments We would like to thank the Food and Biomaterial Engineering Lab, Bioprocess Engineering technicians and RUGrant from Research Management Centre UTM for support of this study. References [1] Wise DL. Hand book of pharmaceutical controlled release technology. New York: Marcel Dekker; 2000. [2] Jogani VV, Shah PJ, Mishra P, Mishra AK, Misra AR. Intranasal mucoadhesive microemulsion of tacrine to improve brain targeting. Alzheimer Dis Assoc Disord 2008;22:116–24. [3] Palace GP, Fitzpatrick R, Tran KV, Phoebe Jr HC, Norton K. Determination of amino acids in diverse polymeric matrices using HPLC, with emphasis on agars and agaroses. Biochim Biophys Acta 1999;509:1472. [4] Nickerson MT, Paulson AT, Wagar E, Farnworth R, Hodge SM, Rousseau D. Some physical properties of crosslinked gelatin–maltodextrin hydrogels. Food Hydrocolloid 2006;20:1072–9. [5] Song F, Zhang Li M, Yang C, Yan L. Genipin-cross-linked casein hydrogels for controlled drug delivery. Int J Pharm 2009;373:41–7.
[6] Ju HK, Kim SY, Kim SJ, Lee YM. pH/temperature-responsive semi-IPN hydrogels composed of alginate and poly(N-isopropylacrylamide). J Appl Polym Sci 2002;83:1128–39. [7] Wei L, Jianli W, Lizhuang Z, Shuqua Z. Synthesis and characteristic of the thermo- and pH-sensitive hydrogel and microporous hydrogel induced by the NP-10 aqueous two-phase system. Eur Polym J 2008;44:3688–99. [8] Guiseley KB, Stanley NF, Whitehous PA. Carrageenan in handbook of watersoluble gums and resins. New York: McGraw-hill; 1980. [9] Muhamad II, Fen LS, Hui HNg, Mustapha NA. Genipin-cross-linked kappa carrageenan/carboxymethyl cellulose beads and effects on beta-carotene release. Carbohydr Polym 2011;83:1207–12. [10] Leong KH, Chung LY, Noordin MI, Mohamad K, Nishikawa M, Onuki Y, et al. Carboxymethylation of kappa-carrageenan for intestinal-targeted delivery of bioactive macromolecules. Carbohydr Polym 2011;83:1507–15. [11] Guiseley KB. Modified kappa-carrageenan. US Patent Office 1978, Patent No. 4096327. [12] Hosseinzadeh H, Pourjavadi A, Mahdavinia GR, Zohuriaan-Mehr MJ. Salt- and pH-resisting collagen-based highly porous hydrogel. J Bioact Compat Polym 2005;20:475–90. [13] Tari O, Pekcan O. e-Polymers (online).
H. Hezaveh, I.I. Muhamad / Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 182–191 [38] Meena R, Prasad K, Siddhanta AK. Development of a stable hydrogel network based on agar–kappa-carrageenan blend cross-linked with genipin. Food Hydrocolloid 2009;23:497–509. [39] Hoffman A. Hydrogels for biomedical applications. J Adv Drug Deliv 2002;43:3–12. [40] Jing R, Hongfei H. Study on interpenetrating polymer network hydrogel of diallyldimethylammonium chloride with kappa-carrageenan by UV irradiation. Euro Poly J 2001;37:3413–7. [41] Zhai Maolina, Ha Hongfei, Yoshii F, Makuuchi K. Effect of kappa-carrageenan on the properties of poly(N-vinyl pyrrolidone)/kappa-carrageenan blend hydrogel synthesized by g-radiation technology. Radiat Phys Chem 2000;57:459–64. [42] Gorgieva S, Kokol V. Synthesis and application of new temperature-responsive hydrogels based on carboxymethyl and hydroxyethyl cellulose derivatives for the functional finishing of cotton knitwear. Carbohydr Polym 2011;85:664–73. [43] Han FQ, Shao B, Wang QW, Guo CG, Liu YX. Synthesis and characterization of carboxymethylcellulose and methyl methacrylate graft copolymers. Pigm Resin Technol 2010;39:156–62. [44] Vimala K, Samba Sivudu K, Murali Mohan Y, Sreedhar B, Mohana Raju K. Controlled silver nanoparticles synthesis in semi-hydrogel networks of poly(acrylamide) and carbohydrates: a rational methodology for antibacterial application. Carbohydr Polym 2009;75:463–71. [45] Park S, Murthy PSK, Park S, Mohan YM, Koh WG. Preparation of silver nanoparticle-containing semi-interpenetrating network hydrogels composed of pluronic and poly(acrylamide) with antibacterial property. J Ind Eng Chem 2011;17:293–7.
191
[46] Murthy PSK, Mohan YM, Varaprasad K, Sreedhar B, Mohana Raju K. First successful design of semi-IPN hydrogel–silver nanocomposites: a facile approach for antibacterial application. J Colloid Interface Sci 2008;318:217–24. [47] Sun JX, Luo YF, Peng H, Han ZW. Design, synthesis and characterization of a novel pH-sensitive hydrogel. Chin Chem Lett 2008;19:1475–8. [48] Peppas NA. Analysis of Fickian and non-Fickian drug release from polymers. Pharm Acta Helv 1985;60:110–1. [49] Fu Y, Kao WJ. Drug release kinetics and transport mechanisms of non-degradable and degradable polymeric delivery systems. Expert Opin Drug Del 2010;7:429–44. [50] Piculell L. In: Stephen AM, editor. Food polysaccharides and their applications. New York: Marcel Dekker; 1995. [51] Faure H, Galabert G, Le MG, Nabet F. Carotenoids: metabolism and physiology. Ann Biol Clin 1999;57:169–83. [52] MacArtain P, Jacquier JC, Dawson KA. Physical characteristics of calcium induced k-carrageenan networks. Carbohydr Polym 2003;53:395–400. [53] Naim S, Samuel B, Chauhan B, Paradkar A. Effect of potassium chloride and cationic drug on swelling, erosion and release from k-carrageenan matrices. AAPS Pharm SciTech 2004;5:1–8. [54] Sipahigil O, Dortunc B. Preparation and in vitro evaluation of verapamil HCl and ibuprofen containing carrageenan beads. Int J Pharm 2001;228: 119–28. [55] Bachtsi AR, Kiparissides C. Synthesis and release studies of oil-containing poly (vinyl alcohol) microcapsules prepared by coacervation. J Control Release 1996;38:49.
Contents lists available at SciVerse ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Modification and swelling kinetic study of kappa-carrageenan-based hydrogel for controlled release study Hadi Hezaveh, Ida Idayu Muhamad * Faculty of Chemical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Johor, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 May 2012 Received in revised form 3 October 2012 Accepted 23 October 2012 Available online 20 December 2012
In this article, the physical stability of carrageenan hydrogels was improved for in vitro controlled drug delivery in different pH mediums. Physical property changes were studied using various characterization tests. The effect of modification on swelling behavior of hydrogels as well as swelling kinetics of hydrogels was also investigated. It was found that the addition of genipin to the hydrogels can increase the physical properties of carrageenan hydrogels. An optimum genipin concentration in which molecular structure of hydrogel can be in its most stable form is also determined. The study of swelling kinetics revealed that the modification has resulted in the relaxation of carageenan molecules and the swelling mechanism from a diffusion-controlled mechanism is changed to a polymer relaxationcontrolled mechanism. Release study shows that by changing the genipin concentration in the hydrogel drug release can be controlled. ß 2012 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Hydrogel Modified kappa-carrageenan Genipin cross-linking Characterization Controlled delivery
1. Introduction Controlled drug delivery systems (DDS) have provided an alternative approach to mobilize the bioavailability of therapeutic agents. In DDSs, an active therapeutic is encapsulated into a polymeric network so that the drug is released from the material in desired conditions [1,2]. Hydrogels have recently attracted a great deal of interest in biomedical and pharmaceutical systems and have been utilized in various applications including biomedicine (wound dressings and tissue engineering), infant care (diapers), pharmaceuticals (drug delivery) and agriculture (water retention during drought) [3,4]. Hydrogels are synthetic polymers that can swell or degrade in response to internal or external elements. In industrial applications, biomaterials are being substituted for synthetic polymers and improving the biocompatibility of these polymers [4]. Recent advances in hydrogel technology have been focused more on the use of biocompatible and non-toxic materials for pharmaceutical and biomedical applications [5]. Among all these hydrogels, intelligent thermoand pH-sensitive systems are more promising, as temperature and the pH are key environmental parameters in the human body. Also, some diseases show themselves by a change in temperature and/or pH [6,7]. Kappa-carrageenan (kC) is mostly used in the food industry as gelling, stabilizing agent and thickener because of its high
* Corresponding author. E-mail address: [email protected] (I.I. Muhamad).
hydrophilicity, mechanical strength, biocompatibility and biodegradability [8]. Kappa-carrageenan would reduce or eliminate toxicity in biomedical applications. For this reason, kC has been applied for immobilizing protein and controlled drug delivery systems [3,9,10]. Palace et al. [3] reported that the estimated amino acid concentration in the polymeric matrix of kC is 4870 ng/ mg (0.487%). Many researches have been conducted to modify the physical and chemical properties of carrageenan [11,12]. Tari and Pekcan [13] reported the association of kappa-carrageenan with CaCl2 to change the swelling properties of carrageenan gels. Genipin is known as an iridoid glucoside which participates in both short- and long-range covalent cross-linking of e-amino groups in amine-containing polymers [4]. Genipin has been used widely in Chinese medicine as well as food dye production, in which genipincross-linked amine groups in the presence of oxygen, form blue pigments [14]. It is shown that using genipin in protein interaction can improve the adsorption capacity of chitosan spheres [15]. Genipin, a natural cross-linker is found to be 10,000 times less toxicity than the common used cross linking agent, glutaraldehyde is utilized to cross-link the hydrogels with minimum cytotoxic effect [9]. Kuo and Wang [16] successfully synthesized genipin-crosslinked chitosan/gelatin scaffolds and investigated the influence of BPE on the generation of cartilaginous components. Hydroxyethyl cellulose (HEC) with high amounts of –OH groups is a biocompatible martial with good solubility in water. Application of HEC in biotechnological and biophysical areas as well as many industrial fields has attracted the attention of researcher’s to its properties [17].
1876-1070/$ – see front matter ß 2012 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jtice.2012.10.011
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183
Fig. 1. Chemical structure of the b-carotene [17].
b-Carotene (Fig. 1) is known as an important derivative of the carotenoids family and is found in many fruits and vegetables [18]. b-Carotene consists of eight isopropene units as a basic molecular structure, containing forty atoms of carbon and two rings at the end of its conjugated double bond chain. b-Carotene with high conversion rate comprises substantial proportion of vitamin A in the human diet [19] and has been reported to protect against cancer in humans [20] and also cardiovascular diseases [21]. For these reasons, b-carotene and other carotenoids have attracted much attention as functional ingredients in industry. Mathematical modeling of drug delivery systems and predictability of drug release is a field of steadily increasing academic and industrial importance with enormous future potential [22]. Many mathematical theories have been proposed to discuss the swelling kinetic in the references [23–26]; however, there is no general model that can be applied to all types of drug delivery systems. The aim of this study is to modify the carrageenan hydrogel for controlled b-carotene release using natural cross-liner. Different characterization tests were carried out to study the physical changes in hydrogels. Modified hydrogels were used as a drug carrier to release b-carotene. The swelling kinetics of hydrogels is also investigated.
then was added to kC/HEC solution to formulate 0.1, 0.2, 0.3, 0.5 and 1.0 mM genipin cross-linked hydrogels at 80 8C. The homogeneous viscous solutions were maintained at room temperature (25 8C) over night and then were dried in an oven at 37 8C for nearly 24 h. The aim of the modification was to improve the physical properties of kC for in vitro release studies and control the amount of b-carotene released from kC hydrogels. From now, kC/HEC blended hydrogels will be referred to using the kC percentage and cross-linked hydrogels with their genipin concentration. For example, kC/HEC ratio of 90:10 will be referred to as kC90 and 1.0 mM genipin cross-linked hydrogels will be referred to as GN1.0. 2.3. Instrumentation 2.3.1. FTIR analysis of hydrogel To study the structure of hydrogels, 4 mg of the dried samples of both non-modified and modified hydrogels were ground and mixed with KBr powder (with the ratio of 1:10). Hydraulic press was used to form the sample pellets under 500 kg/cm2 pressure. Then samples were analyzed using a Fourier transform infrared spectroscope (FTIR) (Nicolet 670 FTIR, USA). 16 scans per sample were conducted with a resolution of 4 in the region between 370 and 4000 cm1 with 1.0 cm1 interval.
2. Experimental 2.1. Material Kappa-carrageenan (kC) (Sigma–Aldrich), hydroxyethyl cellulose (HEC) (Fluka Chemicals), genipin (GN) (Challenge Bioproducts Co., Ltd. Taiwan), b-carotene (CALBIOCHEM, La Jolla, CA, USA), HCl (Qrec Grad AR), NaOH (Qrec Grad AR). Distilled water was also used in synthesizing hydrogel. All chemicals are used as received with no additional purification. 2.2. Preparation of modified hydrogels In order to modify kC hydrogels, they are first blended with HEC in different concentrations to increase physical stability of hydrogels and then cross-linked with natural cross-linker genipin (the concentration of genipin varied from 0.1 mM to 1.0 mM). kC/ HEC blends were prepared by mixing HEC and kC in 30 ml distillated water at 80 8C in different ratios. The experiment was carried out under reflux to ensure that the water content in the system does not change. Gentle stirring prevented air bubble formation in the gels. The hot solutions were then poured into ceramic moulds to allow the cooling process to take place for about 24 h at ambient temperature (25 8C). Hydrogel samples with an approximate diameter of 5.5 cm and a thickness of 1.5 cm were made. Finally, samples were dried in an oven overnight at 37 8C to form hydrogels ready to swell test. The most suitable hydrogel was then chosen for further modification with genipin. To do this, different genipin concentrations of stock solutions were prepared by dissolving genipin powder in 10% hot water under rigorous stirring. Genipin solution
2.3.2. Thermogravimetric analysis (TGA) Thermogravimetric analysis (TGA) of non-modified and modified gels was determined on a SDTA 85 METTLER (TOLEDO, Switzerland) TGA System. The rate was set at 10 8C min1 in the temperature range of 30–700 8C for 65 min under nitrogen flow. 2.3.3. Differential scanning calorimetry (DSC) The thermal properties of kC/HEC hydrogels were examined using a DSC822 METTLER TOLEDO (Switzerland). Approximately 5.0 mg of kC/HEC samples were weighed into a sealed aluminum pan and heated at 5 8C min1 from 30 8C to 200 8C. The nitrogen flow rate purging to the system was maintained at 50 ml min1. 2.3.4. Hydrogel microstructure study Microstructure of hydrogels was studied using field emission scanning electron microscope (FESEM) on Gemini Supra 35 VP FESEM. Dried samples were mounted on the sample holder and coated with gold using Bio Rad Polaran Division (E6700, USA) gold sputter coater under vacuum before observation. Samples photomicrographs were studied on FESEM at the accelerating voltage of 10 kV and 500 magnification. 2.4. Swelling studies Swelling studies of hydrogels were carried out in buffer solutions of pH 1.2, 7 and 12 at room temperature. Samples were immersed in Petri dish filled with 30 ml of the each buffer solution. At different intervals, hydrogels were moved out, and the surface water of gels was wiped using filter paper and weighed. Fresh medium was used for further swelling study. The swelling
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10 9
where Wt is the weight of swollen gels at time t and W0 is the initial weight of samples. Swelling experiment was performed till hydrogels reached at their equilibrium condition. Tests were conducted in triplicate to minimize error and are reported as a mean value. Samples reached at equilibrium were ready for deswelling test. Fully hydrated gels were put in Petri dishes and placed in an oven at 37 8C. The samples were removed from the oven after predetermined time intervals. The dry weight of the samples was measured using a Sartorius scale at room temperature. In the final step, the dried samples were placed into separated Petri dishes filled with buffer solution of pH 7 to study the reswelling. The experiment was carried out at room temperature in triplicate to increase the accuracy. The water uptake (Wu) of the hydrogels was calculated using Eq. (2) [27] Wt W0 (2) 100 We W0 where Wt is the weight of the swollen hydrogel after given time t, W0 and We are the hydrogel weight before swelling and at equilibrium swelling, respectively [28,29].
kC60
8
(1)
kC70
7 Swelling (%)
percentage of hydrogels is then calculated: Wt W0 swelling ratio ð%Þ ¼ 100 W0
kC90
6
kC100
5 4 3 2 1 0
0
18
kC90
12
kC100
100
120
140
10 8 6
To encapsulate the b-carotene, 1.0 mg/ml of b-carotene was dissolved in ethanol and then added to kC/HEC hot solution under stirring using dripping method before adding genipin with mentioned concentrations. The solutions were kept at 80 8C. The cross-linked b-carotene loaded gels were then poured into ceramic moulds to harden and the release study was performed immediately after hardening. Samples were immersed in Petri dishes filled with 50 ml of medium solutions and placed in an incubator at 37 8C. Every 10 min, 4.0 ml aliquot of the solution was removed and the concentration of b-carotene released was determined using a UV–vis spectrophotometer. 4 ml of fresh buffer was replaced to maintain the original volume after each withdrawal. The amount of b-carotene was calculated by interpolation from the b-carotene standard curve at l = 446 nm [9].
2 0
0
10
20
30
40
Time (hr) Fig. 3. Non-cross-linked hydrogels swelling in pH 7 at room temperature.
and therefore less swelling. In neutral medium, hydrogels swelled more and kC100 and kC90 swelled up to 15.43 and 13.12%, respectively. Therefore, kC90 has exhibited a larger swelling increment in the neutral medium than kC100. kC70 and kC60 also swelled 11% and 6.91% in neutral medium, respectively. In alkaline medium, both kC100 and kC90 blend ratios exhibited more swelling than in acidic and neutral. This can be explained as electrostatic repulsion of polymer chains due to the ionization of carboxylic acid groups that breaks the hydrogen bonds and consequently, pushes the network and expands the structure to allow more water to diffuse in [31,32]. After 16 h, blends in neutral and alkaline medium reached equilibrium. 25
kC60 20 Swelling (%)
3.1.1. Effect of blending on swelling behavior Swelling (%) of various kC/HEC blends were performed in triplicate. Results in acidic (pH 1.2, 0.1N HCl), neutral (pH 7) and alkaline (pH 12, 0.1 M NaOH) environment are illustrated in Figs. 2–4, respectively. In all mediums, swelling rate was high at the beginning of the swelling process as ionic chemical potential difference was controlling the swelling. After that, internal and external ions reached their equilibrium state and interactions between polymer and solvent, which is less than ionic chemical potential difference, dominated the process, and consequently swelling rate decreased [30]. In acidic environment, hydrogels reached at equilibrium after almost 2 h. The highest swelling percentage was for kC100 ratio that swelled up to 8.92% followed by kC90 at 5.06%. Low swelling in this medium can be due to the protonation of carboxylic groups and creation of more hydrogen bonds in kC hydroxyl groups resulting in more compact networks
60 80 Time (min)
kC70
14
4
3.1. Swelling studies
40
kC60
16
2.5. Encapsulation and the in vitro release study of b-carotene
3. Results and discussion
20
Fig. 2. Non-cross-linked hydrogels swelling in pH 1.2 at room temperature.
Swelling (%)
184
kC70 kC90
15
kC100
10 5 0
0
5
10
Time (hr)
15
20
Fig. 4. Non-cross-linked hydrogels swelling in pH 12 at room temperature.
25
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100 90
pH 1.2
20
80
pH 7
70
pH 12
15
Wu (%)
Maximum Swelling
25
10 5 0
50
60
70
80 90 kC Concetration (%)
100
kC60
50
kC70
40 30
kC90
20
kC100
0
110
kC70 and kC60 blend ratios swelled at neutral environment more than alkaline. This is because of less cross-linking in neutral medium than alkaline medium [33]. Fig. 5 shows the equilibrium degree of swelling (EDS) of blended hydrogels vs. kC content, in all mediums. Clearly, EDS is strongly depended on the kC concentration. The swelling ratio increases as the hydrophilicity of hydrogels increases due to the presence of carrageenan. This has also been observed in our previous researches [34–37] and also reported by Meena et al. [38]. Hydrogels in neutral environment showed more stabile compared to acidic and alkaline environment. In the neutral and alkaline mediums, for 18 and 6 h, respectively, no changes in hydrogel weight were observed. During the experiment the stability of kC90, especially in alkaline medium, was more than other blends. It is worthwhile to note that kC60 blend ratio in both mediums reached its equilibrium after 2 h. Deswelling of hydrogels, shown in Fig. 6, was carried out in the oven at 37 8C over 92 h. It was found that by increasing the kC, hydrogels dehydrate at a lower rate compared to those having more HEC. This can be attributed to the formation of a thick layer on the surface of hydrogels with more kC as a result of shrinkage that prevents water to escape from the network; however as HEC increases these layers weaken and new channels also form in the gel network that helps the water within the hydrogel to diffuse out [27]. Fig. 7 shows the reswelling of hydrogels in pH 7 at room temperature. After 240 h, hyderogels reached their equilibrium, although they did not regain their previous weight during the reswelling. It is assumed that the water present in the gel network filled the space and larger pores in the network [39]. When
80
100 150 Time (hr)
200
250
3.1.2. Effect of modification on hydrogels swelling properties Figs. 8–10 show that the hydrogel swelling ratio decreases when it is modified with genipin in all mediums. This has also been observed in our previous studies [9,34]. It is clear that by increasing the genipin concentration in hydrogels, swelling decreases noticeably. In GN0.1 hydrogel the EDS was 6.18%; however, in GN1.0 it was only 0.48%. In pH 12 this amount of swelling was even less (0.14%) meaning hydrogel swelling has almost stopped. During the experiment it was found that the physical stability of modified hydrogels increased by adding genipin to the blend that was observed in GN0.1, GN0.2 and GN0.3. Less swelling in these hydrogels can be due to the fact that increasing genipin can result in the creation of vast chains, therefore limiting the mobility of the matrix causing less swelling. More than these concentrations, the physical stability of gels decreased during swelling. This can be attributed to the breakdown of some bonds and the formation of weaker bonds which will be discussed in more detail in coming sections. Also hydrogels in neutral medium had more swelling ratio in comparison with alkaline medium. This means that the addition
6 kC 90
kC70
5
kC90
4
Swelling (%)
100
50
dehydration begins, water diffuses out and during reswelling water penetrating into the network creates an elastic reaction force till the matrix reaches its equilibrium [27]. Considering the enhanced physical stability of kC90 blend ratio compared to other blends in all mediums (in alkaline medium this physical stability was more pronounced), this blend was chosen to be modified with different genipin concentrations: 0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM and 1.0 mM to find the most stable hydrogel for in vitro release study.
kC60
120
0
Fig. 7. Non-cross-linked hydrogels reswelling with different concentration of kappa-carrageenan and hydroxyethyl cellulose at 37 8C.
140
Deswelling (%)
60
10
Fig. 5. Equilibrium degree of swelling (EDS) of non-cross-linked hydrogels vs. kC content in different pH.
kC100
60 40
0.1 mM 0.2 mM 0.3 mM 0.5 mM
3
1.0 mM
2 1
20 0
185
0
20
40
Time (hr)
60
80
100
Fig. 6. Non-cross-linked hydrogels deswelling with different concentration of kappa-carrageenan and hydroxyethyl cellulose at 37 8C.
0
0
20
40
60 80 Time (min)
100
120
140
Fig. 8. Swelling behavior of modified gels with different genipin concentrations at room temperature in pH 1.2.
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186
16 14
Swelling (%)
12
kC90 0.1 mM 0.2 mM 0.3 mM 0.5 mM 1.0 mM
10 8 6 4 2 0
10
0
20 Time (hr)
30
40
Fig. 9. Swelling behavior of modified gels with different genipin concentrations at room temperature in pH 7.
of cross-linker to the gels has altered swelling ability in different buffer solutions. As discussed before, blended hydrogels exhibited more swelling in alkaline environment than neutral; however, by increasing the cross-linker concentration, the swelling capacity in neutral medium increased so that genipin cross-linked hydrogels could swell more in pH 7 than pH 12. Obviously, swelling ability depends on molecular structure and these results suggest that genipin had a noticeable impact on molecular structure. 3.2. Characterization of synthesized hydrogels 3.2.1. FTIR Analysis Fourier transform infrared spectroscope (FTIR) of modified and non-modified hydrogels is presented in Fig. 11. At 3424 cm1, the broad band absorption is attributed to the stretching of –OH groups of kC. The absorption band at 2923 cm1 results from the stretching frequency of –CH2 groups. A typical amide I peak at 1644 cm1 appears in the spectrum of all modified hydrogels. In 0.5 mM and 1.0 mM concentration, a new absorption band appeared at 1354 cm1 and 1442 cm1 which is assigned to C– OH and symmetrical stretching vibration of –COO groups, respectively. By increasing the genipin content of hydrogels, bands at 1642 and 1126 cm1 seems to shift to 1626 and 1110 cm1, respectively. For GN0.5 and GN1.0 hydrogels the intensity of absorption band at 1257 cm1, that is attributed to strong C–O stretch bond in the 20
kC90
18
GN0.1
16
GN0.2
Swelling (%)
14
GN0.3
12
GN0.5
10
GN1.0
8 6 4 2 0
0
5
10
15
20
25
Time (hr) Fig. 10. Swelling behavior of modified gels with different genipin concentrations at room temperature in pH 12.
main chain, is almost disappeared and the less intense peaks of 1229, 1153 and 1112 cm1 in lower regions (consistent with lower energy) are appeared. This indicates that the main chain of kC molecular structure is seriously affected. As a result, the strength of kC as main monomer in the gel is reduced. This has been also observed in previous studies by other researches [40,41] and explains the less stability of 0.5 and especially 1.0 mM genipin cross-linked hydogels during the test [40]. Also, at 1376 absorption band (symmetrical C–H bending) for GN0.2, GN0.3 and GN0.5 is intensified after modification. At 1044 cm1 a new absorption peak is attributed to carbonyl stretching which is formed during modification. Also, when crosslinking occurs, bands at 796, 768 and 733 cm1, are intensified. The bands observed at 844, 927 are assigned to D-galactose-4sulfate, 3,6-anhydrod-galactose, respectively. All the characteristic absorptions of non-modified hydrogel are found in modified gels. Thus, it can be concluded that first, the genipin cross-linked kC/ HEC hydrogel is successfully prepared and second, the addition of cross-linker from 0.1 mM to 0.3 mM concentration can result in the formation of more stable hydrogel structure (as it was optically observed during the test). 3.2.2. Thermal analysis of modified and non-modified hydrogels Fig. 12 indicates the differential scanning calorimetry (DSC) for kC90 and modified gels. At near 100 8C the endothermic peak represents water evaporation from the network. As the concentration of cross-linker increases, the peak decreases due to hydrophilic hydroxyl side-groups that are involved in the cross-linking reaction so molecules can retain more water and less amounts of water can evaporate from the hydrogel network [42]. Clearly, for GN1.0 the physical stability of the network decreases this peak increases again and water evaporation increases. Fig. 12 clearly shows that the genipin increment also results in increasing the glass transition temperature (Tg). The glass transition for non-modified, GN0.1 and GN1.0 are 118, 120 and 124 8C, respectively. The exothermic peak which appeared at 180 8C (for nonmodified films) is related to the polymer’s thermal decomposition [43]. By increasing the genipin, this temperature shifts to higher temperature (round 185 8C); however, at 1.0 mM concentration of cross-linker, this temperature again decreases to 164 8C, indicating the loss network strength as mentioned before. The DSC results along with FTIR data demonstrate that the addition of genipin up to 0.5 mM, can improve the physical properties of kC/HEC hydrogel. Fig. 13 shows TG analysis for non-modified and modified (GN0.1 and GN1.0) hydrogels. kC90, GN0.1 and GN1.0, have lost 45.26, 42.67 and 48.48% of their weight below 200 8C, indicating that thermal stability of GN0.1 is higher than others [44–46]. The weight lost at 700 8C for non-modified, GN 0.1 and GN 1.0 was 58.92, 56.66 and 49.84%, respectively. 3.2.3. Hydrogels microstructure The surface morphology of hydrogels is presented in Fig. 14. By increasing the cross-linker concentration in the network, morphology of the polymer changes noticeably. It is believed that these new chains were aggregates (fibers formed by kC and/or HEC) probably resulted from genipin-crosslink. Increasing genipin concentration resulted in the formation of more fibers with low aspect ratio. This also affects the swelling properties and can explain less swelling in cross-linked gels. These chains in the network form a hardly penetrable surface that does not let water diffuses in. This along with less physical stability as explained before result in low swelling in GN1.0 (see Fig. 14).
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187
Fig. 11. FTIR results for (a) kC90, (b) GN0.1, (c) GN0.2, (d) GN0.3, (e) GN0.5, and (f) GN1.0.
3.3. pH-response of modified kappa-carrageenan/hydroxyethyl cellulose hydrogels Polymers with ionizable functional groups that respond to pH changes are called pH-sensitive polymers. In these polymers, the electrostatic repulsion force causes an increase in the hydrodynamic volume of the polymer, as charges along the polymer backbone generated [47]. Fig. 15 shows hydrogel swelling versus genipin concentrations in all mediums. Both modified and non-modified hydrogels showed less swelling ability in acidic environment. This can be attributed to both carboxylic group protonation and also degradation of hydrogels in this pH. In neutral medium, however, non-modified hydrogel exhibited better swelling ability and
swelled up to 13.12%. The addition of genipin decreased the swelling and reduced it up to 0.48% for GN1.0 (almost stopped). In pH 12, non-modified hydrogel swelled the most (17.6%), but when cross-linked, hydrogels swelling decreased noticeably. Clearly, modification has changed the swelling behavior of hydrogels. For GN0.1 hydrogels swelled more in alkaline than other mediums, but for GN0.2 almost the same swelling percentage in alkaline and neutral mediums was observed. By further increasing the genipin, the swelling ability of hydrogels in alkaline medium decreased so that GN0.3, GN0.5 and GN1.0 had higher swelling ratios in neutral mediums than in alkaline medium. For GN1.0, the swelling ratios of the hydrogels in acidic, neutral and alkaline mediums were 0.34, 0.14 and 0.48%, respectively. Generally, modified hydrogels exhibited more
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188
Fig. 12. DSC thermograms recorded during heating of cross-linked and kC90 hydrogels.
swelling and stability in neutral mediums than other mediums, as was also observed by Meena et al. [33]. 3.4. Swelling kinetic study The mechanism that water diffuses into the hyderogels network is an important issue due to the unique applications of the hydrogels. To determine the nature of water diffusion into the hydrogels network power law was applied [48]: Mt ¼ ktn M1
(3)
where Mt and M1 are the absolute cumulative water penetrated into the network at time t, and infinite time, respectively. k is a constant indicating the structural and geometric properties of the matrix, and n is the diffusion exponent that indicates the transport mechanism of water taken up. This equation is a simple semi-empirical model with two different physically realistic meanings for n = 0.5 and n = 1.0. When n = 0.5 it represents Fickian or Case I transport behavior in which the relaxation coefficient is negligible and the pure drug diffusion release is considered. When the release rate is constant in time n takes the value of 1.0, known as zero-order release kinetics. In this case the drug release follows a polymer chain relaxation mechanism [23,49]. Values of n between 0.5 and 1.0 indicate non-Fickian or anomalous transport mechanism where structural relaxation is comparable to diffusion. When the n value becomes less than 0.5, the mechanism is known as pseudo-Fickian behavior 120 Fig. 14. FESEM images of (a) kC90, (b) GN0.1 and (c) GN1.0.
Nave
100
GN 0.1 Mass loss (%)
80
GN 1.0
60 40 20 0
0
100
200
300 400 Tempareture (C)
500
600
Fig. 13. TGA analysis of modified and kC90 hydrogels.
700
which means that the sorption curves are like Fickian curves, although the final equilibrium is very slow [23,49]. For a radial diffusion, however, different n values have been derived. n = 0.45 and n = 0.89 show diffusion-controlled mechanism and case II transport, respectively. n and k parameters can be determined by plotting ln(Mt/M1) vs. ln(t) and calculating the slope and intercept of the line. In Table 1 computed values of n, k and the corresponding determination coefficients (R2) for swelling of hydrogels in pH 7.0 (Fig. 9) are listed. For kC90 hydrogel, n = 0.63 and for cross-linked hydrogels it varies from 0.68 to 0.9. The n value for cross-linked hydrogels
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189
beta caroten release (mg/ml)
(a) 0.016
3.5. Immobilization and in vitro release
b-Carotene, one of the most important food additives, is widely used as a colorant in the food and beverage industry [51]. In this work, b-carotene as a bioactive material was used to study the in vitro release of modified kC/HEC hydrogels in pH of 1.2, 7 and pH 12. It is shown that the existence of cations like K+, Na+, or Ca2+ can induce conformational changes in hydrogel networks [52,53]. Due to the instantaneous ionic gelation properties, the immobilization of drugs in carrageenan is possible [53,54]. pH is a key factor in the drug delivery which determines the release of drug in pH-sensitive hydrogels. The effect of pH on the b-carotene release rate from the modified and non-modified hydrogels is shown in Fig. 16. As Fig. 16a clearly shows, when pH of the medium is 1.2 the concentration of b-carotene released from the native hydrogel is 0.013 mg/ml after 120 min. At pH 7, the drug concentration released is 0.043 mg/ml after 120 min which means that the release amount has increased almost 230%. In the alkaline medium, this increase after 120 min is almost 423% (0.068 mg/ ml). For GN0.3 hydrogel, the b-carotene release in pH of 1.2 was 0.008 mg/ml which was increased in neutral and alkaline mediums. It is well known that release profile of hydrogels is strongly dependent on swelling properties. kC90, GN0.1 and GN0.2 hydrogels showed the same release trend observed in swelling test,
GN0.2
0.01
GN0.3
0.008
GN0.5 GN1.0
0.006 0.004 0.002
beta caroten release (mg/ml)
20
0
(b) 0.08
40 60 Time (min)
80
100
kC90
0.07
GN0.1
0.06
GN0.2
0.05
GN0.3 GN0.5
0.04
GN1.0
0.03 0.02 0.01 0
0
20
40
60 80 Time (min)
100
120
140
60 80 Time (min)
100
120
140
(c) 0.05 beta caroten release (mg/ml)
(GN0.1, GN 0.2 and GN 0.3) indicates the non-Fickian or anomalous transport mechanism. It means that the swelling is controlled by polymer relaxation which is in good agreement with the fact that the strength gels increase with increasing genipin concentrations in these hydrogels [50]. Since the n value for GN0.5 and GN 1.0 is 0.8 and 0.9, the mechanism becomes a case II transport mechanism in which the swelling is controlled by polymer chain relaxation. These results suggest that when the polymer backbone is broken in these gels, the relaxation of polymer network is facilitated. In conclusion, the addition of genipin to the network has caused the swelling mechanism to shift from a diffusion-controlled towards polymer relaxation-controlled mechanisms.
GN0.1
0.012
0
Fig. 15. Maximum swelling of cross-linked hydrogels vs. genipin concentration in three different mediums.
kC90
0.014
0.045
kC90
0.04
GN0.1
0.035
GN0.2
0.03
GN0.3
0.025
GN0.5
0.02
GN1.0
0.015 0.01 0.005 0
0
20
40
Fig. 16. Concentration of b-carotene released in (a) acidic, (b) neutral and (c) alkaline medium.
i.e., the release was more in alkaline than in neutral and acidic mediums. But for GN 0.3, GN0.5 and GN1.0, the release profile was not the same as swelling trend suggesting that the b-carotene has influenced the hydrogels structure. During the experiment, all hydrogels which encapsulated the b-carotene seemed stronger than un-loaded gels. Results shown in Fig. 16 also indicate that the addition of genipin has caused a decrease in the release rate in all mediums. In acidic medium, hydrogels reached the their maximum b-carotene release after 90 min; however, in neutral and alkaline it took
Table 1 Different n and k values of power law model for native gel and various concentrations of genipin. Samples
n k R2 Transport mechanism
Native hydrogel
0.1 mM genipin
0.2 mM genipin
0.3 mM genipin
0.5 mM genipin
1.0 mM genipin
0.63 0.4481 0.98 Anomalous
0.68 0.3214 0.98 Anomalous
0.7 0.2931 0.96 Anomalous
0.73 0.2472 0.95 Anomalous
0.8 0.2035 0.98 Case II
0.9 0.1956 0.96 Case II
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120 min to reach at maximum release. This can be attributed to more compact networks as a result of the protonation of carboxylic groups which hinders the drug release. In neutral medium, the accumulative b-carotene release of non-modified hydrogels was 0.2 mg/ml; however, it decreased to 0.048 mg/ml for GN1.0. Increasing cross-linker density causes less available free space for drug diffusion and consequently the release rate decreases [55]. 4. Conclusion In this research, carrageenan hydrogels were modified to improve their physical properties, enhanced their application in drug delivery systems. The effect of modification on swelling properties, swelling kinetic mechanism and bio active material release was investigated. Using different characterization techniques, the physical property changes of modified carrageenanbased hydrogels was studied. The optimum concentration of crosslinker in which hydrogel structure can be in its most stable form is also proposed. It was found that the modification has changed the swelling behavior of hydrogels. GN0.1 swelled more in alkaline than other mediums; however by increasing genipin content, the swelling ability in alkaline medium decreased in the way that GN0.3, GN0.5 and GN 1.0 had more swelling in neutral than in alkaline medium as result of the high concentration of the counter ions of COO in highly cross-linked hydrogels. The modification has changed the pH-sensitivity of hydrogel in the way that by increasing the genipin content of hydrogels, hydrogels swelled more in neutral medium than alkaline, however non-modified hydrogels showed reverse behavior. Swelling kinetic study shows that increasing the genipin content of matrix; increases the relaxation of carageenan molecules and the swelling mechanism from diffusion-controlled changes to polymer relaxation-controlled mechanisms. Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscope (FTIR) data has proven that the addition of genipin up to 0.5 mM, can improve the physical properties of polymeric network. Beyond this concentration, the main chain of kC molecular structure is seriously affected. Also FESEM test showed that the surface roughness of non-modified polymer is significantly changed when genipin is added to hydrogel. Release study has proven the capability of modified hydrogels to carry and release encapsulated drugs in various pH. By changing the concentration of genipin, it is possible to control in vitro drug release in different mediums.
Acknowledgments We would like to thank the Food and Biomaterial Engineering Lab, Bioprocess Engineering technicians and RUGrant from Research Management Centre UTM for support of this study. References [1] Wise DL. Hand book of pharmaceutical controlled release technology. New York: Marcel Dekker; 2000. [2] Jogani VV, Shah PJ, Mishra P, Mishra AK, Misra AR. Intranasal mucoadhesive microemulsion of tacrine to improve brain targeting. Alzheimer Dis Assoc Disord 2008;22:116–24. [3] Palace GP, Fitzpatrick R, Tran KV, Phoebe Jr HC, Norton K. Determination of amino acids in diverse polymeric matrices using HPLC, with emphasis on agars and agaroses. Biochim Biophys Acta 1999;509:1472. [4] Nickerson MT, Paulson AT, Wagar E, Farnworth R, Hodge SM, Rousseau D. Some physical properties of crosslinked gelatin–maltodextrin hydrogels. Food Hydrocolloid 2006;20:1072–9. [5] Song F, Zhang Li M, Yang C, Yan L. Genipin-cross-linked casein hydrogels for controlled drug delivery. Int J Pharm 2009;373:41–7.
[6] Ju HK, Kim SY, Kim SJ, Lee YM. pH/temperature-responsive semi-IPN hydrogels composed of alginate and poly(N-isopropylacrylamide). J Appl Polym Sci 2002;83:1128–39. [7] Wei L, Jianli W, Lizhuang Z, Shuqua Z. Synthesis and characteristic of the thermo- and pH-sensitive hydrogel and microporous hydrogel induced by the NP-10 aqueous two-phase system. Eur Polym J 2008;44:3688–99. [8] Guiseley KB, Stanley NF, Whitehous PA. Carrageenan in handbook of watersoluble gums and resins. New York: McGraw-hill; 1980. [9] Muhamad II, Fen LS, Hui HNg, Mustapha NA. Genipin-cross-linked kappa carrageenan/carboxymethyl cellulose beads and effects on beta-carotene release. Carbohydr Polym 2011;83:1207–12. [10] Leong KH, Chung LY, Noordin MI, Mohamad K, Nishikawa M, Onuki Y, et al. Carboxymethylation of kappa-carrageenan for intestinal-targeted delivery of bioactive macromolecules. Carbohydr Polym 2011;83:1507–15. [11] Guiseley KB. Modified kappa-carrageenan. US Patent Office 1978, Patent No. 4096327. [12] Hosseinzadeh H, Pourjavadi A, Mahdavinia GR, Zohuriaan-Mehr MJ. Salt- and pH-resisting collagen-based highly porous hydrogel. J Bioact Compat Polym 2005;20:475–90. [13] Tari O, Pekcan O. e-Polymers (online).
H. Hezaveh, I.I. Muhamad / Journal of the Taiwan Institute of Chemical Engineers 44 (2013) 182–191 [38] Meena R, Prasad K, Siddhanta AK. Development of a stable hydrogel network based on agar–kappa-carrageenan blend cross-linked with genipin. Food Hydrocolloid 2009;23:497–509. [39] Hoffman A. Hydrogels for biomedical applications. J Adv Drug Deliv 2002;43:3–12. [40] Jing R, Hongfei H. Study on interpenetrating polymer network hydrogel of diallyldimethylammonium chloride with kappa-carrageenan by UV irradiation. Euro Poly J 2001;37:3413–7. [41] Zhai Maolina, Ha Hongfei, Yoshii F, Makuuchi K. Effect of kappa-carrageenan on the properties of poly(N-vinyl pyrrolidone)/kappa-carrageenan blend hydrogel synthesized by g-radiation technology. Radiat Phys Chem 2000;57:459–64. [42] Gorgieva S, Kokol V. Synthesis and application of new temperature-responsive hydrogels based on carboxymethyl and hydroxyethyl cellulose derivatives for the functional finishing of cotton knitwear. Carbohydr Polym 2011;85:664–73. [43] Han FQ, Shao B, Wang QW, Guo CG, Liu YX. Synthesis and characterization of carboxymethylcellulose and methyl methacrylate graft copolymers. Pigm Resin Technol 2010;39:156–62. [44] Vimala K, Samba Sivudu K, Murali Mohan Y, Sreedhar B, Mohana Raju K. Controlled silver nanoparticles synthesis in semi-hydrogel networks of poly(acrylamide) and carbohydrates: a rational methodology for antibacterial application. Carbohydr Polym 2009;75:463–71. [45] Park S, Murthy PSK, Park S, Mohan YM, Koh WG. Preparation of silver nanoparticle-containing semi-interpenetrating network hydrogels composed of pluronic and poly(acrylamide) with antibacterial property. J Ind Eng Chem 2011;17:293–7.
191
[46] Murthy PSK, Mohan YM, Varaprasad K, Sreedhar B, Mohana Raju K. First successful design of semi-IPN hydrogel–silver nanocomposites: a facile approach for antibacterial application. J Colloid Interface Sci 2008;318:217–24. [47] Sun JX, Luo YF, Peng H, Han ZW. Design, synthesis and characterization of a novel pH-sensitive hydrogel. Chin Chem Lett 2008;19:1475–8. [48] Peppas NA. Analysis of Fickian and non-Fickian drug release from polymers. Pharm Acta Helv 1985;60:110–1. [49] Fu Y, Kao WJ. Drug release kinetics and transport mechanisms of non-degradable and degradable polymeric delivery systems. Expert Opin Drug Del 2010;7:429–44. [50] Piculell L. In: Stephen AM, editor. Food polysaccharides and their applications. New York: Marcel Dekker; 1995. [51] Faure H, Galabert G, Le MG, Nabet F. Carotenoids: metabolism and physiology. Ann Biol Clin 1999;57:169–83. [52] MacArtain P, Jacquier JC, Dawson KA. Physical characteristics of calcium induced k-carrageenan networks. Carbohydr Polym 2003;53:395–400. [53] Naim S, Samuel B, Chauhan B, Paradkar A. Effect of potassium chloride and cationic drug on swelling, erosion and release from k-carrageenan matrices. AAPS Pharm SciTech 2004;5:1–8. [54] Sipahigil O, Dortunc B. Preparation and in vitro evaluation of verapamil HCl and ibuprofen containing carrageenan beads. Int J Pharm 2001;228: 119–28. [55] Bachtsi AR, Kiparissides C. Synthesis and release studies of oil-containing poly (vinyl alcohol) microcapsules prepared by coacervation. J Control Release 1996;38:49.