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Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker
G Model IJP 15260 No. of Pages 9
International Journal of Pharmaceutics xxx (2015) xxx–xxx
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker Suguna Selvakumarana , Ida Idayu Muhamada,b,* a b
Department of Bioprocess Engineering, Faculty of Chemical Engineering, Johor Bahru 81310, Johor, Malaysia IJN-UTM Cardiovascular Engineering Centre, V01 FBME, 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 3 June 2015 Received in revised form 17 September 2015 Accepted 3 October 2015 Available online xxx
Genipin, a natural and non-toxic cross linker, was used to prepare cross linked floating kappa carrageenan/sodium carboxymethyl cellulose hydrogels and the effect of genipin on hydrogels characterization was investigated. Calcium carbonates were employed as gas forming agents. Ranitidine hydrochloride was used as drug. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA) were carried out to study the changes in the characteristics of hydrogels. Furthermore, scanning electron microscope (SEM) was performed to study microstructure of hydrogels. The result showed that all formulated hydrogels had excellent floating behavior. It was discovered that the cross linking reaction showed significant effect on gel strength, porosity and swelling ratio compared to non-cross linked hydrogels. It was found that the drug release was slower and lesser after being cross linked. Microstructure study shows that cross linked hydrogels exhibited hard and rough surface. Therefore, genipin can be an interesting cross linking agent for controlled drug delivery in gastrointestinal tract. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Floating hydrogel Genipin cross linking Kappa carrageenan/sodium carboxymethyl cellulose Characterization
1. Introduction Hydrogel is in increasing demand in the biomedical and pharmaceutical applications due to their biocompatibility, biodegradability and non-toxic properties (Ratner et al., 1976; Ratner & Williams, 1981; Peppas, 1987; Muhamad et al., 2011). Hydrogel is a polymeric three dimensional network obtained from a class of synthetic and natural polymers which can absorb and retain significant amount of water (Rosiak and Yoshii, 1999). The hydrogel structure is created by the hydrophilic groups ( OH, COOH, NH2,CONH2, and SO3H) present in a polymeric network upon the hydration in an aqueous environment. Hydrogel is an excellent biomaterial that is capable of exhibiting significant volume changes in response to small changes in pH, temperature, electric field, and light (Nho et al., 2005). Hydrogel made from kappa carrageenan act as a good drug carrier for drug delivery system, especially in the gastrointestinal tract. Kappa carrageenan is a linear, sulfated polysaccharide, composed of repeating D-galactose and 3,6-anhydro-D-galactose units (Zhai et al., 2004). Kappa carrageenan hydrogel has its own specific advantages such
* Corresponding author at: Department of Bioprocess Engineering, Faculty of Chemical Engineering, Johor Bahru, 81310, Johor, Malaysia. Fax: +60 75558553. E-mail address: [email protected] (I.I. Muhamad).
as nontoxicity, easy availability, easy gelling properties, thermo reversibility of the gel network and appropriate viscoelastic properties (Liu et al., 2006) that enables it to undergo harsh condition. This behavior makes the kappa carrageenan hydrogel as an extraordinary carrier in the drug delivery system. Kappa carrageenan based floating hydrogel has currently gained wide attention among researchers. Incorporation of carbonates and bicarbonates salt into hydrogels allow the hydrogels to constantly float in the stomach and deliver drug in a controlled manner. In this work, calcium carbonates were used as gas forming agents. Compared to normal hydrogel, floating hydrogel has its own advantages as it can constantly float in the stomach for long period meanwhile normal hydrogel is removed via antrum due to peristaltic waves with poor drug release pattern. Floating hydrogels not only prolong the residence time of carrier but also maximize the amount of drugs reaching their absorption site in solution and hence ready for absorption (Dolas et al., 2011). In addition, floating hydrogel results in dissolution of drugs in the gastric fluid, this would then make them available for absorption in the small intestine after emptying of the stomach content. It is expected that the drugs will be fully absorbed from floating dosage forms if it remains in a solution form even at the alkaline pH of the intestine system (Mayavanshi and Gajjar, 2008; Chordiya et al., 2011).
http://dx.doi.org/10.1016/j.ijpharm.2015.10.005 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Selvakumaran, S., Muhamad, I.I., Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.005
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Fig. 1. Chemical structures of kappa carrageenan (a), sodium carboxymethyl cellulose (b), genipin (c) and mechanisms of genipin (d).
Please cite this article in press as: Selvakumaran, S., Muhamad, I.I., Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.005
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However, some modification is needed in order to enhance the physical and mechanical properties of floating kappa carrageenan hydrogels. Thus, it has to be cross linked with cross linking reagents. Glutaraldehyde, tripolyphosphate, ethylene glycol, diglycidyl ether and diisocyanate are examples of cross linking reagents. These reagents are relatively cytotoxic. An alternative, natural crosslinking reagent does exist and it is known as genipin. Genipin is a natural cross linker that is obtained from geniposide, a component of traditional chinese medicine and is isolated from the fruits of the plant, Gardenial jasminoides Ewith. Genipin has been was widely used in herbal medicine. Sung et al. (1999) reported that when comparing the cytotoxicity of genipin to glutaraldehyde through in vitro using 3T3 fibroblasts via MTT assay, they found that genipin was 10,000 times less cytotoxic than glutaraldehyde. In addition, according to Mi et al. (2002), chitosan microspheres cross-linked with genipin showed better biocompatibility and slower degradation rate than glutaraldehyde cross-linked microspheres. Thus, it was concluded that the compatibility of the genipin was superior to glutaraldehyde. Genipin has been used as a cross linker to control swelling ratio and mechanical properties (Chen et al., 2004; Jin et al., 2004; Mi et al., 2005, 2001; Hezaveh and Muhamad, 2012a). Besides that, genipin was also used to control drug release rate. A study conducted by Muhamad et al. (2011) incorporated the use of kappa carrageenan beads that were cross linked by genipin for b-carotene. It was discovered that the swelling ratio and the amount of b-carotene released from the genipin-cross-linked hydrogel were different at various pH. It was also found that the beads released beta-carotene slower and lesser after being crosslinked. According to Meena et al. (2007), cross-linking mechanisms improve the properties of polysaccharide as compared to nonmodified ones. The presence of genipin in polymer matric helps to control the drug release rate. This was described by (Hezaveh and Muhamad, 2013a) where in vitro of beta carotene was changed by the addition of genipin. The results indicated that genipin had caused a decrease in the release rate in acidic, neutral and alkaline medium. In neutral medium, the accumulative beta-carotene release of non-modified hydrogels was 0.2 mg/ml; however, it decreased to 0.048 mg/ml for genipin 1.0 mM. These results clearly suggest that the incorporation of genipin contributed to the improvement of the physical properties of hydrogels and managed to sustain the drug release. Due to its stability and biocompatibility, genipin can be an excellent cross linking agent to be cross linked with floating hydrogel in the drug delivery system. There are numerous studies on genipin cross linked kappa carrageenan hydrogels, but genipin cross linked of floating hydrogels have not been reported. The aim of this study was to prepare genipin cross linked floating kappa carrageenan hydrogel and to investigate the effect of genipin on physical, chemical and mechanical properties of hydrogel and in vitro release of Ranitidine hydrochloride as well. Fig. 1 shows the chemical structures of kappa carrageenan (a), sodium carboxymethyl cellulose (b), genipin (c), and mechanisms of genipin (d).
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2.2. Preparation of hydrogels 2.2.1. Preparation of floating hydrogel k-Carrageenan/NaCMC based floating hydrogels are formulated based on the kC:NaCMC at ratio of 80:20 with 0.5% of calcium carbonates (Selvakumaran and Muhamad, 2014, 2016). Briefly, for a typical hydrogel synthesis, 0.48 g of kappa carrageenan and 0.12 g NaCMC was dissolved in 20 ml of distillated water at 80 C before mixing with 0.15 g CaCO3 dissolved in 10 ml distillated water. NaCMC was blended with kappa carrageenan to improve the swelling properties of hydrogel. The solution was stir for 1 h to obtain a clear, viscous and homogenous solution with no bubble. Then, the resultant hot solution was poured into ceramic moulds to form the hardened hydrogel of a desired shape. Samples were equilibrated with ambient temperature (25 C) for 24 h prior to drying at 37 C in oven night. kC: NaCMM: CaCO3 was used to further modify with different concentration of genipin. 2.2.2. Genipin cross linking Genipin cross-linked hydrogels were prepared with different concentration of genipin varying 0.5 mM, 1.0 mM and 1.5 mM at 80 C under vigorous stirring. Then genipin in different concentrations of genipin stock solutions were used by dissolving genipin powder in 10% of water. Continuous stirring ensured that no air bubbles formed in the hydrogels. Homogeneous gels with different genipin concentrations were poured into separated molds and kept at room temperature (27 C) over night. Finally, hydrogel were dried at 37 C for 24 h in an oven. 2.3. Swelling ratio studies Prepared cross linked hydrogels were immersed in different pH buffer solution of pH 1.2 and pH 7.4 at room temperature (25 C). Synthesized gels were placed in a petri dish filled with 30 ml of each buffer solutions. Prior to weighting, filter paper was used to remove the surface water of swollen hydrogel. The test was conducted in triplicate. The swelling ratio (%) was then determined using Eq. (1) Wt W0 ð1Þ 100 Swelling ratio ð%Þ ¼ W0 where W0 is the initial weight of samples and Wt is the weight of swollen gels at predetermined time t. To allow hydrogels to reach their highest swelling ability, they were immersed in fresh buffer solution after weighting. 2.4. In vitro buoyancy study Floating properties of cross linked hydrogels were evaluated with 50 ml of 0.1 N HCl at 37 C 0.1 C. The time required for hydrogels to rise to the surface and float were measured by visual observation (Rosa et al., 1994). The test was conducted in triplicate. 2.5. Porosity measurement of hydrogel
2. Material and methods 2.1. Material Ranitidine hydrochloride from Pusat Kesihatan UTM, KappaCarrageenan (kC) Sigma–Aldrich (Malaysia), sodium carboxymethyl cellulose (NaCMC) (average molecular weight of 250,000) was purchased from Acros Organic (Malaysia), Calcium Carbonate (CaCO3) was purchased from Sigma–Aldrich (Malaysia). Genipin was purchased from Challenge Bioproducts Co., Ltd. (Taiwan). Distilled water was used in hydrogel synthesis and all chemical were used as received with no additional purification.
Wet hydrogels after reaction with 0.1 N HCl were weighed after the excess HCl on the surface was blotted. Then the dried hydrogels were weighed after the hydrogels were fully dried at room temperature. The test was conducted in triplicate. The porosity was calculated based on the following Eq. (2) Ww Wd ð2Þ Porosity : rV where Ww and Wd are mass of wet and dry hydrogel respectively; r is density of 0.1 N HCl and V is volume of the hydrogel.
Please cite this article in press as: Selvakumaran, S., Muhamad, I.I., Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.005
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2.6. Immobilization of ranitidine hydrochloride and in vitro release 150 mg of Ranitidine hydrochloride was dissolved in 5 ml of distilled water. Then it mixed with kC/NaCMC/CaCO3 solution with different amount of genipin. The solution was stirred until no air bubbles formed in the hydrogels solution. Homogeneous gel solution were poured into separated molds and kept at room temperature (27 C) over night. Finally, hydrogel were dried at 37 C for 24 h in an oven. The release test was performed immediately after hardening. The Ranitidine hydrochloride release was performed by immersing loaded hydrogels in beaker containing with 50 ml of 0.1 N HCl and placed in an incubator at 37 C 0.5 C. 20 ml of the sample was withdrawn at time intervals, filtered, diluted suitably and analyzed spectrophotometrically at 313 nm. Equal amount of fresh medium was replaced immediately after withdrawal of the test sample. The amount of Ranitidine hydrochloride was calculated by interpolation from the Ranitidine hydrochloride standard curve at 313 nm. 2.7. Instrumentation 2.7.1. Gel strength analysis The mechanical test (compression) of hydrogel was measured by using a texture analyzer (Brookfield Engineering Laboratories, USA, CT3) in accordance to ASTM F2900. The samples were placed on the platform and the probe was travelled on the surface of swollen hydrogel with speed of 0.5 mm/s; the probe was kept moving until it reached the base and the strength of the gel was calculated. The test was conducted in triplicate. 2.7.2. FTIR analysis of hydrogels FTIR analysis was performed for non-cross linked and cross linked floating hydrogels with different concentrations. Measurements were carried out within the range of 370–4000 cm1 on IRTracer-100 FTIR (Shimadzu, Japan).
2.7.3. Hydrogel microstructure Scanning electron microscope (SEM) test was performed to study the surface morphology of the hydrogels and the effect of cross linking on floating hydrogels using TM3000 Table Top SEM (Hitachi, German) operating at 15 kV accelerating voltage. Photomicrographs were then taken at 50x magnification. 2.7.4. X-ray diffraction (XRD) X-ray diffraction measurements of non-cross linked and cross linked kC/NaCMC/CaCO3 were carried out at room temperature using a Siemens Diffraktometer D5000 X-ray diffractometer with Cu Ka at 40 keV and 40 mA with a step length of 0.05 and a step time of 1 s. The diffraction angle (2u) was between 20 and 80. Hydrogel were dried in an oven and make powder before experiment. 2.7.5. Thermogravimetric analysis (TGA) Thermal analysis (thermogravimetric analysis (TGA)) of both non-cross linked and cross linked hydrogels was performed on a PerkinElmer TGA 4000 (USA) TGA System at the rate of 10 C min1, and a temperature range of 30–950 C for 65 min. 3. Result and discussion 3.1. FTIR analysis Fig. 2 illustrates the FTIR spectra of both non-cross linked and cross linked floating hydrogels in different concentrations. The broad band absorbed at 3381–3348 cm1 was attributed to the stretching of OH groups of kC/NaCMC. By increasing the genipin amount to 1.5 mM, this band shifted to 3348 cm1. Two important bands at 844 and 923 cm1 can be attributed to D-galactose-4sulfate and 3,6-anhydride-galactose, respectively. The absorption bands at 2922–2929 cm1 resulted from the stretching frequency of CH3 groups and a symmetrical CH bending appeared at 1419 cm1. The peaks at 1155 cm1 were assigned to vibrational
Fig. 2. FTIR spectra of non-cross linked (a) and cross linked [0.5 mM (b), 1.0 mM (c) and 1.5 mM (d)] floating hydrogels.
Please cite this article in press as: Selvakumaran, S., Muhamad, I.I., Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.005
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modes associated with saccharide units in kappa carrageenan (Butler et al., 2003). In addition, the band at 1230 cm1 and 1039 cm1 corresponded to ester sulphate and glycosidic linkage, respectively, of kappa carrageenan. However, these two bands shifted to 1228 cm1 and 1037 cm1 as the amount of genipin was increased. This was due to the cross linking reaction that caused the molecular structure of kappa carrageenan to change. The carbonates peak at 871 cm1 appeared in the spectrum of all hydrogels. This signified the existence of calcium carbonates in non-cross linked and cross linked floating hydrogels. It also showed that cross linking reaction was not affected the molecular structure of calcium carbonates. Thus, it was expected that the cross-linked hydrogels would give better floating behaviors, as discussed in later section. The primary amide peak formed at 1627–1624 cm1 indicated that amino group present in kappa carrageenan matrix. According to Meena et al. (2007), there is no nitrogen in the structure of kappa-carrageenan meanwhile amino acid group are present in kappa carrageenan matrices. This statement was proved by Palace et al. (1999). The analysis conducted by Palace et al. (1999) showed the amino acid concentration of glycine in kappa carrageenan polymeric matrix was 508 ng/mg (0.051%). Thus, it is expected that the cross linking reaction occurred between the protein present in the polymeric matrix of kappa carrageenan and genipin. This was supported by the colour of hydrogel which changed from transparent to dark bluish on treatment with genipin, showing that cross linking reaction had occurred (Butler et al., 2003; Mi et al., 2001). 3.2. X-ray diffraction analysis X-ray diffraction (XRD) results of non-cross linked and cross linked (0.5 mM, 1.0 mM and 1.5 mM) floating hydrogels are shown in Fig. 3(a–d). Based on the results, many sharp diffraction peaks could be observed in the XRD curve. In Fig. 3(a), the two main sharp peaks at 2U = 28.65 and 29.7 (1 0 4) were attributed to the diffraction plane of kappa carrageenan and calcium carbonates, respectively. The level of crystanility of kappa carrageenan gel was enhanced by the incorporation of calcium carbonate. This result is supported by a study conducted by Eirasa and Pessanb (2009). As depicted in Fig. 3(b–d), the diffraction plane of kappa carrageenan and calcium carbonates still existed and the peak intensity of kappa carrageenan and calcium carbonates showed a gradual increase and become sharper, indicating that genipin cross linking enhanced the crystanility properties of hydrogels. Genipin itself showed high crystallinity. This was in agreement with a previous study conducted by Zu et al. (2014) and Harris et al. (2010). There were several new peaks that were expected to belong to genipin. Therefore, other than calcium carbonates, incorporation of genipin also enhances the crystallinity properties of hydrogels. Hence, it is believed that the mechanical properties of hydrogels will be improved with high crystallinity properties, which will be discussed in the gel mechanical strength part. 3.3. Thermogravimetric analysis (TGA) The TGA curves for non-cross linked and cross linked (0.5 mM and 1.5 mM) floating hydrogels are shown in Fig. 4. The weight loss curves of non-cross-linked and cross linked hydrogels were shown in two stages. The first thermal loss for non-cross linked and cross linked hydrogels were occurred in the temperature range 30– 250 C, where weight loss for non-cross linked, 0.5 mM cross linked and 1.5 mM cross linked were 13.29%, 12.07% and 11.85%, respectively. This may correspond to the loss of absorbed and bound moisture in hydrogels network. A clear variation has been observed in TGA experiments that cross linked hydrogels exhibit excellent thermal stability than non-cross linked hydrogels. This is
Fig. 3. X-ray diffraction results of non-cross linked (a) and cross linked [0.5 mM (b), 1.0 mM (c) and 1.5 mM (d)] floating hydrogels.
due to, in second stage, non-cross linked hydrogels loss more mass (48.72%) in the temperature range 700–800 C meanwhile, 0.5 mM and 1.5 mM cross-linked hydrogels only loss 42.63% and 43.92% in temperature ranges of 750–800 C, respectively. When comparing genipin cross linked hydrogels, 0.5 mM hydrogels showed more thermal stability than 1.5 mM. By increasing the genipin content in
Please cite this article in press as: Selvakumaran, S., Muhamad, I.I., Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.005
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Fig. 4. TGA analysis of non-cross linked and cross linked [0.5 mM and 1.5 mM] floating hydrogels.
hydrogels to 1.5 mM, the mass loss increased 1.3%, indicating a slight change in hydrogel thermal properties. It seems that increasing genipin concentration to the network has caused less thermal stability (Hezaveh and Muhamad, 2013b).
3.4. Swelling measurement of hydrogels The result of swelling ratio of non-cross linked and genipin cross linked floating kappa carrageenan hydrogels in acidic (pH 1.2) and neutral (pH 7.4) media are presented in Fig. 5 respectively. Formation of blue pigment was observed in hydrogels, which indicated the spontaneous reaction of genipin with amino group in kappa carrageenan. As can be seen in the Fig. 5, swelling ratio for genipin cross linked hydrogels was much lower than the non-cross linked hydrogels. The swelling ratio decreased as the concentration of genipin was increased from 0.5 to 1.5 mM. Hydrogels cross-linked with 0.5 mM genipin recorded a swelling ratio of 602.94% and 1212.64% in pH 1.2 and 7.4 medium, respectively. In contrast, 1.5 mM cross-linked hydrogels showed the lowest percentage of swelling ratio, approximately 306.21% in pH 1.2 and 630.27% in pH 7.4 medium. This may be due to the presence of a high amount of genipin that could result to a great extent of chemical cross linking of the kC/NaCMC/CaCO3 chains. This confines the movement and hydration of the macromolecular chain in the beads and leads to less swelling in diameter (Muhamad et al., 2011). These results are similar to a previous report in which swelling of films decreased with the increase of genipin concentrations up to 2.5 mM but not at higher concentration (Mi et al., 2001). Compared to acidic medium, cross linked hydrogels showed highest swelling in neutral medium. At higher pH, the ionization of carboxylic acid group occurs. Electrostatic repulsion force caused by the breakdown of hydrogen bonds leads to more water penetrating into the network. Meanwhile, at pH 1.2, the carboxylic group on the cross linked hydrogels became progressively protonated. This caused the network to become more compact and retarded the movement of structure, therefore resulting in less swelling (Hezaveh and Muhamad, 2012b; Song et al., 2009). 3.5. In vitro buoyancy
Fig. 5. Swelling ratio of non-cross linked and genipin cross linked floating kappa carrageenan hydrogels in acidic (pH 1.2) and alkaline (pH 7.4) medium.
The results of the floating ability of non-cross linked and different concentrations of genipin cross linked hydrogels are presented in Table 1. All formulated hydrogels showed excellent floating behavior (100%). Floating ability is directly related to the gas content of the polymer matrix. From the result, there was no significance difference in the floating lag time. Therefore, it could
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Table 1 Gel strength, porosity and floating properties analysis of non-cross linked and genipin cross linked floating hydrogels (n = 3).
Non-cross linked Cross linked
Genipin concentration (mM)
Floating properties
Gel strength (g)
Porosity (%)
0 0.5 1.0 1.5
++ ++ ++ ++
361.33 495.33 533.67 846.00
73.07 68.38 67.27 67.10
* Completely sink; slightly float; +/ partially sink or float; ++ completely float; + slightly sink.
be concluded that the incorporation of cross linker did not affect the floating behavior of hydrogels. 3.6. Mechanical strength study The mechanical strength of non-cross linked and genipin cross linked floating hydrogels are shown in Table 1. Gel strength measurement for non-cross linked and genipin cross linked hydrogels were 361.33 g, 495.33 g, 533.67 g and 846 g, respectively. Cross linked hydrogels showed higher gel strength compared to non-cross linked hydrogels. It was observed that the gel became more stable and was strengthen as genipin amount was increased. Cross linking is an oxidation reaction between active chemical groups that could improve the mechanical strength by creating new chemical bonds between the polymers (Bi et al., 2011; Lund et al., 2008). Thus, creation of new bonds between genipin and kappa carrageenan hydrogel that was discussed in the FTIR analysis in earlier section caused the mechanical strength of the gel to be strengthen. Besides that, mechanical strength of gels is directly related to its porosity, which will be discussed later. It is expected that an increase in mechanical strength can be cause by a gradual decrease in porosity. For the cross linked samples, the enhanced mechanical strength due to chemical cross linking was stronger than the inhibition caused by the increasing pores size. Consequently, an increase in mechanical strength was achieved as the genipin concentration was increased.
(a)
3.7. Porosity Pore formation in hydrogel matrix is due to the effervescent reaction between CaCO3 and gastric fluid which releases CO2 that permeates pores with hydrogels network. The result of porosity measurement for non-cross linked and genipin cross linked floating hydrogels are given in Table 1. Porosity of non-cross hydrogel was higher (73.07%) than cross linked gels. Cross linked floating hydrogels with the highest amount of genipin (1.5 mM) showed least percentage of porosity (67.10%). Meanwhile, 0.5 mM cross linked hydrogels showed high porosity (68.38%). The result concluded that the porosity decreases as genipin concentration was increased. This is mainly due to the fact that the cross linking mechanisms cause the hydrogel matrix to become more compact and this reduces the pore formation as the molecular arrangement of the hydrogel matrix was changed after cross linked with genipin (Hezaveh and Muhamad, 2013a; Yin et al., 2007). The results from previous studies by Chen et al. 2009 and Yan et al. 2010 also showed that an increase in cross-linker concentration could result in a material with smaller pores which is similar to what occurs with glutaraldehyde and other types of genipin cross linking. 3.8. In vitro drug release Fig. 6(a) displays Ranitidine hydrochloride release of non-cross linked and cross linked floating hydrogel at 37 C in 0.1 N HCl. The release of Ranitidine hydrochloride from the non-cross linked hydrogels was faster than cross linked hydrogels. The drug release at t = 5 min from non-cross linked hydrogels was 37% while for
(b) Fig. 6. Ranitidine hydrochloride drug release of non- cross linked and cross linked floating hydrogel (a) and comparison of Ranitidine hydrochloride release from noncross linked and 1.5 mM genipin cross linked (b) under in vitro release condition.
0.5 mM cross linked hydrogels was only 23%. The amount of Ranitidine hydrochloride released from the cross linked hydrogels was found less than non-cross linked hydrogels. At lower amount of genipin used, larger amount of drug released. This result due to, as the concentration of cross linker decreases, the crosslinking degree will decrease. This makes the polymer network less dense. Consequently, as the available free space for drug diffusion increases, the rate of drug release also increases. Meanwhile, an increase of the degree of polymer cross-linking increases the polymer density and therefore decreases the available free space for drug diffusion which results in a decrease in drug release rates (Bachtsi and Kiparissides, 1995). In addition at t = 120 min the in vitro release from non-cross linked was 96% meanwhile drug release from 0.5 mM, 1.0 mM, 1.5 mM genipin cross linked floating hydrogels were 88%, 86% and 79%, respectively. In addition pore formation by effervescent reaction also influence the release of Ranitidine hydrochloride. Non-cross linked hydrogels shows high drug release rate due to its high porosity and pore size. As discussed in porosity study, floating hydrogels with higher amount of genipin leave fewer pores on hydrogel matrix; hence the drug was release in control manner as amount of cross linker increases.
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This is due to fewer pores makes the matrix highly compact and the drug diffuse slowly from the hydrogel matrix. From the result it can concluded that 1.5 mM genipin cross linked floating hydrogels showed the optimum drug release rate than other formulation. In addition, Fig 6(b) shows the comparison of Ranitidine hydrochloride release from non-cross linked and 1.5 mM genipin cross linked (optimum concentration). The result revealed that 1.5 mM cross linked hydrogels showed controlled drug release pattern than noncross linked hydrogels. 3.9. Hydrogels microstructure Fig. 7A (a–d) and B (a–d) illustrates the surface morphology and cross sectional image of non-cross linked and cross linked hydrogels with different concentrations at 50 magnification. It can be observed that non-cross linked hydrogels (Fig. 7A (a)) had smoother surface compared to genipin cross linked hydrogels (Fig. 7A (b–d)). As the amount of genipin increases, the surface became hard and rough. In addition, the result also showed that the pores were formed on the surface of non-cross linked and cross linked hydrogels. This clearly shows that incorporation of genipin did affect the pores formation as the pores were still intact on the surface even after cross linking reaction has occurred. However, it was obvious that the amount and size of pores became smaller as the concentration of genipin was increased. This was supported by the cross sectional image in Fig. 7B (a–d). Non-cross linked hydrogel (Fig. 7B (a)) produced larger pores compared to cross linked hydrogels. In contrast, 0.5 mM hydrogel (Fig. 7B (b)) formed large pores while smaller pores were observed in 1.5 mM hydrogels (Fig. 7B (d)). Changes in molecular structure during cross linking reaction reduced the pore formation in hydrogel network. This was in accordance with porosity results in Section 3.7. 4. Conclusion In conclusion, genipin cross linked floating kappa carrageenan hydrogel was successfully prepared using natural cross linker. The significantly higher level of mechanical strength, swelling capacity, good floating properties as well as sustained drug release makes the genipin cross linked floating kappa carrageenan hydrogel a suitable polymeric carrier for bioactive material in the gastrointestinal tract. Acknowledgements We would like to thanks the Department of Bioprocess Engineering, Faculty of Chemical Engineering, Cardiovascular Engineering, IJN-UTM, Grant Vot 4H023 from Research Management Centre and MyPhD Scholarship from Ministry of Higher Education (MOHE) for support of this study. References
Fig. 7. Surface morphology 6A (a–d) and cross sectional image 6B (a–d) of noncross linked and cross linked hydrogels with different concentrations at 50 magnification.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker Suguna Selvakumarana , Ida Idayu Muhamada,b,* a b
Department of Bioprocess Engineering, Faculty of Chemical Engineering, Johor Bahru 81310, Johor, Malaysia IJN-UTM Cardiovascular Engineering Centre, V01 FBME, 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 3 June 2015 Received in revised form 17 September 2015 Accepted 3 October 2015 Available online xxx
Genipin, a natural and non-toxic cross linker, was used to prepare cross linked floating kappa carrageenan/sodium carboxymethyl cellulose hydrogels and the effect of genipin on hydrogels characterization was investigated. Calcium carbonates were employed as gas forming agents. Ranitidine hydrochloride was used as drug. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA) were carried out to study the changes in the characteristics of hydrogels. Furthermore, scanning electron microscope (SEM) was performed to study microstructure of hydrogels. The result showed that all formulated hydrogels had excellent floating behavior. It was discovered that the cross linking reaction showed significant effect on gel strength, porosity and swelling ratio compared to non-cross linked hydrogels. It was found that the drug release was slower and lesser after being cross linked. Microstructure study shows that cross linked hydrogels exhibited hard and rough surface. Therefore, genipin can be an interesting cross linking agent for controlled drug delivery in gastrointestinal tract. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Floating hydrogel Genipin cross linking Kappa carrageenan/sodium carboxymethyl cellulose Characterization
1. Introduction Hydrogel is in increasing demand in the biomedical and pharmaceutical applications due to their biocompatibility, biodegradability and non-toxic properties (Ratner et al., 1976; Ratner & Williams, 1981; Peppas, 1987; Muhamad et al., 2011). Hydrogel is a polymeric three dimensional network obtained from a class of synthetic and natural polymers which can absorb and retain significant amount of water (Rosiak and Yoshii, 1999). The hydrogel structure is created by the hydrophilic groups ( OH, COOH, NH2,CONH2, and SO3H) present in a polymeric network upon the hydration in an aqueous environment. Hydrogel is an excellent biomaterial that is capable of exhibiting significant volume changes in response to small changes in pH, temperature, electric field, and light (Nho et al., 2005). Hydrogel made from kappa carrageenan act as a good drug carrier for drug delivery system, especially in the gastrointestinal tract. Kappa carrageenan is a linear, sulfated polysaccharide, composed of repeating D-galactose and 3,6-anhydro-D-galactose units (Zhai et al., 2004). Kappa carrageenan hydrogel has its own specific advantages such
* Corresponding author at: Department of Bioprocess Engineering, Faculty of Chemical Engineering, Johor Bahru, 81310, Johor, Malaysia. Fax: +60 75558553. E-mail address: [email protected] (I.I. Muhamad).
as nontoxicity, easy availability, easy gelling properties, thermo reversibility of the gel network and appropriate viscoelastic properties (Liu et al., 2006) that enables it to undergo harsh condition. This behavior makes the kappa carrageenan hydrogel as an extraordinary carrier in the drug delivery system. Kappa carrageenan based floating hydrogel has currently gained wide attention among researchers. Incorporation of carbonates and bicarbonates salt into hydrogels allow the hydrogels to constantly float in the stomach and deliver drug in a controlled manner. In this work, calcium carbonates were used as gas forming agents. Compared to normal hydrogel, floating hydrogel has its own advantages as it can constantly float in the stomach for long period meanwhile normal hydrogel is removed via antrum due to peristaltic waves with poor drug release pattern. Floating hydrogels not only prolong the residence time of carrier but also maximize the amount of drugs reaching their absorption site in solution and hence ready for absorption (Dolas et al., 2011). In addition, floating hydrogel results in dissolution of drugs in the gastric fluid, this would then make them available for absorption in the small intestine after emptying of the stomach content. It is expected that the drugs will be fully absorbed from floating dosage forms if it remains in a solution form even at the alkaline pH of the intestine system (Mayavanshi and Gajjar, 2008; Chordiya et al., 2011).
http://dx.doi.org/10.1016/j.ijpharm.2015.10.005 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structures of kappa carrageenan (a), sodium carboxymethyl cellulose (b), genipin (c) and mechanisms of genipin (d).
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However, some modification is needed in order to enhance the physical and mechanical properties of floating kappa carrageenan hydrogels. Thus, it has to be cross linked with cross linking reagents. Glutaraldehyde, tripolyphosphate, ethylene glycol, diglycidyl ether and diisocyanate are examples of cross linking reagents. These reagents are relatively cytotoxic. An alternative, natural crosslinking reagent does exist and it is known as genipin. Genipin is a natural cross linker that is obtained from geniposide, a component of traditional chinese medicine and is isolated from the fruits of the plant, Gardenial jasminoides Ewith. Genipin has been was widely used in herbal medicine. Sung et al. (1999) reported that when comparing the cytotoxicity of genipin to glutaraldehyde through in vitro using 3T3 fibroblasts via MTT assay, they found that genipin was 10,000 times less cytotoxic than glutaraldehyde. In addition, according to Mi et al. (2002), chitosan microspheres cross-linked with genipin showed better biocompatibility and slower degradation rate than glutaraldehyde cross-linked microspheres. Thus, it was concluded that the compatibility of the genipin was superior to glutaraldehyde. Genipin has been used as a cross linker to control swelling ratio and mechanical properties (Chen et al., 2004; Jin et al., 2004; Mi et al., 2005, 2001; Hezaveh and Muhamad, 2012a). Besides that, genipin was also used to control drug release rate. A study conducted by Muhamad et al. (2011) incorporated the use of kappa carrageenan beads that were cross linked by genipin for b-carotene. It was discovered that the swelling ratio and the amount of b-carotene released from the genipin-cross-linked hydrogel were different at various pH. It was also found that the beads released beta-carotene slower and lesser after being crosslinked. According to Meena et al. (2007), cross-linking mechanisms improve the properties of polysaccharide as compared to nonmodified ones. The presence of genipin in polymer matric helps to control the drug release rate. This was described by (Hezaveh and Muhamad, 2013a) where in vitro of beta carotene was changed by the addition of genipin. The results indicated that genipin had caused a decrease in the release rate in acidic, neutral and alkaline medium. In neutral medium, the accumulative beta-carotene release of non-modified hydrogels was 0.2 mg/ml; however, it decreased to 0.048 mg/ml for genipin 1.0 mM. These results clearly suggest that the incorporation of genipin contributed to the improvement of the physical properties of hydrogels and managed to sustain the drug release. Due to its stability and biocompatibility, genipin can be an excellent cross linking agent to be cross linked with floating hydrogel in the drug delivery system. There are numerous studies on genipin cross linked kappa carrageenan hydrogels, but genipin cross linked of floating hydrogels have not been reported. The aim of this study was to prepare genipin cross linked floating kappa carrageenan hydrogel and to investigate the effect of genipin on physical, chemical and mechanical properties of hydrogel and in vitro release of Ranitidine hydrochloride as well. Fig. 1 shows the chemical structures of kappa carrageenan (a), sodium carboxymethyl cellulose (b), genipin (c), and mechanisms of genipin (d).
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2.2. Preparation of hydrogels 2.2.1. Preparation of floating hydrogel k-Carrageenan/NaCMC based floating hydrogels are formulated based on the kC:NaCMC at ratio of 80:20 with 0.5% of calcium carbonates (Selvakumaran and Muhamad, 2014, 2016). Briefly, for a typical hydrogel synthesis, 0.48 g of kappa carrageenan and 0.12 g NaCMC was dissolved in 20 ml of distillated water at 80 C before mixing with 0.15 g CaCO3 dissolved in 10 ml distillated water. NaCMC was blended with kappa carrageenan to improve the swelling properties of hydrogel. The solution was stir for 1 h to obtain a clear, viscous and homogenous solution with no bubble. Then, the resultant hot solution was poured into ceramic moulds to form the hardened hydrogel of a desired shape. Samples were equilibrated with ambient temperature (25 C) for 24 h prior to drying at 37 C in oven night. kC: NaCMM: CaCO3 was used to further modify with different concentration of genipin. 2.2.2. Genipin cross linking Genipin cross-linked hydrogels were prepared with different concentration of genipin varying 0.5 mM, 1.0 mM and 1.5 mM at 80 C under vigorous stirring. Then genipin in different concentrations of genipin stock solutions were used by dissolving genipin powder in 10% of water. Continuous stirring ensured that no air bubbles formed in the hydrogels. Homogeneous gels with different genipin concentrations were poured into separated molds and kept at room temperature (27 C) over night. Finally, hydrogel were dried at 37 C for 24 h in an oven. 2.3. Swelling ratio studies Prepared cross linked hydrogels were immersed in different pH buffer solution of pH 1.2 and pH 7.4 at room temperature (25 C). Synthesized gels were placed in a petri dish filled with 30 ml of each buffer solutions. Prior to weighting, filter paper was used to remove the surface water of swollen hydrogel. The test was conducted in triplicate. The swelling ratio (%) was then determined using Eq. (1) Wt W0 ð1Þ 100 Swelling ratio ð%Þ ¼ W0 where W0 is the initial weight of samples and Wt is the weight of swollen gels at predetermined time t. To allow hydrogels to reach their highest swelling ability, they were immersed in fresh buffer solution after weighting. 2.4. In vitro buoyancy study Floating properties of cross linked hydrogels were evaluated with 50 ml of 0.1 N HCl at 37 C 0.1 C. The time required for hydrogels to rise to the surface and float were measured by visual observation (Rosa et al., 1994). The test was conducted in triplicate. 2.5. Porosity measurement of hydrogel
2. Material and methods 2.1. Material Ranitidine hydrochloride from Pusat Kesihatan UTM, KappaCarrageenan (kC) Sigma–Aldrich (Malaysia), sodium carboxymethyl cellulose (NaCMC) (average molecular weight of 250,000) was purchased from Acros Organic (Malaysia), Calcium Carbonate (CaCO3) was purchased from Sigma–Aldrich (Malaysia). Genipin was purchased from Challenge Bioproducts Co., Ltd. (Taiwan). Distilled water was used in hydrogel synthesis and all chemical were used as received with no additional purification.
Wet hydrogels after reaction with 0.1 N HCl were weighed after the excess HCl on the surface was blotted. Then the dried hydrogels were weighed after the hydrogels were fully dried at room temperature. The test was conducted in triplicate. The porosity was calculated based on the following Eq. (2) Ww Wd ð2Þ Porosity : rV where Ww and Wd are mass of wet and dry hydrogel respectively; r is density of 0.1 N HCl and V is volume of the hydrogel.
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2.6. Immobilization of ranitidine hydrochloride and in vitro release 150 mg of Ranitidine hydrochloride was dissolved in 5 ml of distilled water. Then it mixed with kC/NaCMC/CaCO3 solution with different amount of genipin. The solution was stirred until no air bubbles formed in the hydrogels solution. Homogeneous gel solution were poured into separated molds and kept at room temperature (27 C) over night. Finally, hydrogel were dried at 37 C for 24 h in an oven. The release test was performed immediately after hardening. The Ranitidine hydrochloride release was performed by immersing loaded hydrogels in beaker containing with 50 ml of 0.1 N HCl and placed in an incubator at 37 C 0.5 C. 20 ml of the sample was withdrawn at time intervals, filtered, diluted suitably and analyzed spectrophotometrically at 313 nm. Equal amount of fresh medium was replaced immediately after withdrawal of the test sample. The amount of Ranitidine hydrochloride was calculated by interpolation from the Ranitidine hydrochloride standard curve at 313 nm. 2.7. Instrumentation 2.7.1. Gel strength analysis The mechanical test (compression) of hydrogel was measured by using a texture analyzer (Brookfield Engineering Laboratories, USA, CT3) in accordance to ASTM F2900. The samples were placed on the platform and the probe was travelled on the surface of swollen hydrogel with speed of 0.5 mm/s; the probe was kept moving until it reached the base and the strength of the gel was calculated. The test was conducted in triplicate. 2.7.2. FTIR analysis of hydrogels FTIR analysis was performed for non-cross linked and cross linked floating hydrogels with different concentrations. Measurements were carried out within the range of 370–4000 cm1 on IRTracer-100 FTIR (Shimadzu, Japan).
2.7.3. Hydrogel microstructure Scanning electron microscope (SEM) test was performed to study the surface morphology of the hydrogels and the effect of cross linking on floating hydrogels using TM3000 Table Top SEM (Hitachi, German) operating at 15 kV accelerating voltage. Photomicrographs were then taken at 50x magnification. 2.7.4. X-ray diffraction (XRD) X-ray diffraction measurements of non-cross linked and cross linked kC/NaCMC/CaCO3 were carried out at room temperature using a Siemens Diffraktometer D5000 X-ray diffractometer with Cu Ka at 40 keV and 40 mA with a step length of 0.05 and a step time of 1 s. The diffraction angle (2u) was between 20 and 80. Hydrogel were dried in an oven and make powder before experiment. 2.7.5. Thermogravimetric analysis (TGA) Thermal analysis (thermogravimetric analysis (TGA)) of both non-cross linked and cross linked hydrogels was performed on a PerkinElmer TGA 4000 (USA) TGA System at the rate of 10 C min1, and a temperature range of 30–950 C for 65 min. 3. Result and discussion 3.1. FTIR analysis Fig. 2 illustrates the FTIR spectra of both non-cross linked and cross linked floating hydrogels in different concentrations. The broad band absorbed at 3381–3348 cm1 was attributed to the stretching of OH groups of kC/NaCMC. By increasing the genipin amount to 1.5 mM, this band shifted to 3348 cm1. Two important bands at 844 and 923 cm1 can be attributed to D-galactose-4sulfate and 3,6-anhydride-galactose, respectively. The absorption bands at 2922–2929 cm1 resulted from the stretching frequency of CH3 groups and a symmetrical CH bending appeared at 1419 cm1. The peaks at 1155 cm1 were assigned to vibrational
Fig. 2. FTIR spectra of non-cross linked (a) and cross linked [0.5 mM (b), 1.0 mM (c) and 1.5 mM (d)] floating hydrogels.
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modes associated with saccharide units in kappa carrageenan (Butler et al., 2003). In addition, the band at 1230 cm1 and 1039 cm1 corresponded to ester sulphate and glycosidic linkage, respectively, of kappa carrageenan. However, these two bands shifted to 1228 cm1 and 1037 cm1 as the amount of genipin was increased. This was due to the cross linking reaction that caused the molecular structure of kappa carrageenan to change. The carbonates peak at 871 cm1 appeared in the spectrum of all hydrogels. This signified the existence of calcium carbonates in non-cross linked and cross linked floating hydrogels. It also showed that cross linking reaction was not affected the molecular structure of calcium carbonates. Thus, it was expected that the cross-linked hydrogels would give better floating behaviors, as discussed in later section. The primary amide peak formed at 1627–1624 cm1 indicated that amino group present in kappa carrageenan matrix. According to Meena et al. (2007), there is no nitrogen in the structure of kappa-carrageenan meanwhile amino acid group are present in kappa carrageenan matrices. This statement was proved by Palace et al. (1999). The analysis conducted by Palace et al. (1999) showed the amino acid concentration of glycine in kappa carrageenan polymeric matrix was 508 ng/mg (0.051%). Thus, it is expected that the cross linking reaction occurred between the protein present in the polymeric matrix of kappa carrageenan and genipin. This was supported by the colour of hydrogel which changed from transparent to dark bluish on treatment with genipin, showing that cross linking reaction had occurred (Butler et al., 2003; Mi et al., 2001). 3.2. X-ray diffraction analysis X-ray diffraction (XRD) results of non-cross linked and cross linked (0.5 mM, 1.0 mM and 1.5 mM) floating hydrogels are shown in Fig. 3(a–d). Based on the results, many sharp diffraction peaks could be observed in the XRD curve. In Fig. 3(a), the two main sharp peaks at 2U = 28.65 and 29.7 (1 0 4) were attributed to the diffraction plane of kappa carrageenan and calcium carbonates, respectively. The level of crystanility of kappa carrageenan gel was enhanced by the incorporation of calcium carbonate. This result is supported by a study conducted by Eirasa and Pessanb (2009). As depicted in Fig. 3(b–d), the diffraction plane of kappa carrageenan and calcium carbonates still existed and the peak intensity of kappa carrageenan and calcium carbonates showed a gradual increase and become sharper, indicating that genipin cross linking enhanced the crystanility properties of hydrogels. Genipin itself showed high crystallinity. This was in agreement with a previous study conducted by Zu et al. (2014) and Harris et al. (2010). There were several new peaks that were expected to belong to genipin. Therefore, other than calcium carbonates, incorporation of genipin also enhances the crystallinity properties of hydrogels. Hence, it is believed that the mechanical properties of hydrogels will be improved with high crystallinity properties, which will be discussed in the gel mechanical strength part. 3.3. Thermogravimetric analysis (TGA) The TGA curves for non-cross linked and cross linked (0.5 mM and 1.5 mM) floating hydrogels are shown in Fig. 4. The weight loss curves of non-cross-linked and cross linked hydrogels were shown in two stages. The first thermal loss for non-cross linked and cross linked hydrogels were occurred in the temperature range 30– 250 C, where weight loss for non-cross linked, 0.5 mM cross linked and 1.5 mM cross linked were 13.29%, 12.07% and 11.85%, respectively. This may correspond to the loss of absorbed and bound moisture in hydrogels network. A clear variation has been observed in TGA experiments that cross linked hydrogels exhibit excellent thermal stability than non-cross linked hydrogels. This is
Fig. 3. X-ray diffraction results of non-cross linked (a) and cross linked [0.5 mM (b), 1.0 mM (c) and 1.5 mM (d)] floating hydrogels.
due to, in second stage, non-cross linked hydrogels loss more mass (48.72%) in the temperature range 700–800 C meanwhile, 0.5 mM and 1.5 mM cross-linked hydrogels only loss 42.63% and 43.92% in temperature ranges of 750–800 C, respectively. When comparing genipin cross linked hydrogels, 0.5 mM hydrogels showed more thermal stability than 1.5 mM. By increasing the genipin content in
Please cite this article in press as: Selvakumaran, S., Muhamad, I.I., Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.005
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Fig. 4. TGA analysis of non-cross linked and cross linked [0.5 mM and 1.5 mM] floating hydrogels.
hydrogels to 1.5 mM, the mass loss increased 1.3%, indicating a slight change in hydrogel thermal properties. It seems that increasing genipin concentration to the network has caused less thermal stability (Hezaveh and Muhamad, 2013b).
3.4. Swelling measurement of hydrogels The result of swelling ratio of non-cross linked and genipin cross linked floating kappa carrageenan hydrogels in acidic (pH 1.2) and neutral (pH 7.4) media are presented in Fig. 5 respectively. Formation of blue pigment was observed in hydrogels, which indicated the spontaneous reaction of genipin with amino group in kappa carrageenan. As can be seen in the Fig. 5, swelling ratio for genipin cross linked hydrogels was much lower than the non-cross linked hydrogels. The swelling ratio decreased as the concentration of genipin was increased from 0.5 to 1.5 mM. Hydrogels cross-linked with 0.5 mM genipin recorded a swelling ratio of 602.94% and 1212.64% in pH 1.2 and 7.4 medium, respectively. In contrast, 1.5 mM cross-linked hydrogels showed the lowest percentage of swelling ratio, approximately 306.21% in pH 1.2 and 630.27% in pH 7.4 medium. This may be due to the presence of a high amount of genipin that could result to a great extent of chemical cross linking of the kC/NaCMC/CaCO3 chains. This confines the movement and hydration of the macromolecular chain in the beads and leads to less swelling in diameter (Muhamad et al., 2011). These results are similar to a previous report in which swelling of films decreased with the increase of genipin concentrations up to 2.5 mM but not at higher concentration (Mi et al., 2001). Compared to acidic medium, cross linked hydrogels showed highest swelling in neutral medium. At higher pH, the ionization of carboxylic acid group occurs. Electrostatic repulsion force caused by the breakdown of hydrogen bonds leads to more water penetrating into the network. Meanwhile, at pH 1.2, the carboxylic group on the cross linked hydrogels became progressively protonated. This caused the network to become more compact and retarded the movement of structure, therefore resulting in less swelling (Hezaveh and Muhamad, 2012b; Song et al., 2009). 3.5. In vitro buoyancy
Fig. 5. Swelling ratio of non-cross linked and genipin cross linked floating kappa carrageenan hydrogels in acidic (pH 1.2) and alkaline (pH 7.4) medium.
The results of the floating ability of non-cross linked and different concentrations of genipin cross linked hydrogels are presented in Table 1. All formulated hydrogels showed excellent floating behavior (100%). Floating ability is directly related to the gas content of the polymer matrix. From the result, there was no significance difference in the floating lag time. Therefore, it could
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Table 1 Gel strength, porosity and floating properties analysis of non-cross linked and genipin cross linked floating hydrogels (n = 3).
Non-cross linked Cross linked
Genipin concentration (mM)
Floating properties
Gel strength (g)
Porosity (%)
0 0.5 1.0 1.5
++ ++ ++ ++
361.33 495.33 533.67 846.00
73.07 68.38 67.27 67.10
* Completely sink; slightly float; +/ partially sink or float; ++ completely float; + slightly sink.
be concluded that the incorporation of cross linker did not affect the floating behavior of hydrogels. 3.6. Mechanical strength study The mechanical strength of non-cross linked and genipin cross linked floating hydrogels are shown in Table 1. Gel strength measurement for non-cross linked and genipin cross linked hydrogels were 361.33 g, 495.33 g, 533.67 g and 846 g, respectively. Cross linked hydrogels showed higher gel strength compared to non-cross linked hydrogels. It was observed that the gel became more stable and was strengthen as genipin amount was increased. Cross linking is an oxidation reaction between active chemical groups that could improve the mechanical strength by creating new chemical bonds between the polymers (Bi et al., 2011; Lund et al., 2008). Thus, creation of new bonds between genipin and kappa carrageenan hydrogel that was discussed in the FTIR analysis in earlier section caused the mechanical strength of the gel to be strengthen. Besides that, mechanical strength of gels is directly related to its porosity, which will be discussed later. It is expected that an increase in mechanical strength can be cause by a gradual decrease in porosity. For the cross linked samples, the enhanced mechanical strength due to chemical cross linking was stronger than the inhibition caused by the increasing pores size. Consequently, an increase in mechanical strength was achieved as the genipin concentration was increased.
(a)
3.7. Porosity Pore formation in hydrogel matrix is due to the effervescent reaction between CaCO3 and gastric fluid which releases CO2 that permeates pores with hydrogels network. The result of porosity measurement for non-cross linked and genipin cross linked floating hydrogels are given in Table 1. Porosity of non-cross hydrogel was higher (73.07%) than cross linked gels. Cross linked floating hydrogels with the highest amount of genipin (1.5 mM) showed least percentage of porosity (67.10%). Meanwhile, 0.5 mM cross linked hydrogels showed high porosity (68.38%). The result concluded that the porosity decreases as genipin concentration was increased. This is mainly due to the fact that the cross linking mechanisms cause the hydrogel matrix to become more compact and this reduces the pore formation as the molecular arrangement of the hydrogel matrix was changed after cross linked with genipin (Hezaveh and Muhamad, 2013a; Yin et al., 2007). The results from previous studies by Chen et al. 2009 and Yan et al. 2010 also showed that an increase in cross-linker concentration could result in a material with smaller pores which is similar to what occurs with glutaraldehyde and other types of genipin cross linking. 3.8. In vitro drug release Fig. 6(a) displays Ranitidine hydrochloride release of non-cross linked and cross linked floating hydrogel at 37 C in 0.1 N HCl. The release of Ranitidine hydrochloride from the non-cross linked hydrogels was faster than cross linked hydrogels. The drug release at t = 5 min from non-cross linked hydrogels was 37% while for
(b) Fig. 6. Ranitidine hydrochloride drug release of non- cross linked and cross linked floating hydrogel (a) and comparison of Ranitidine hydrochloride release from noncross linked and 1.5 mM genipin cross linked (b) under in vitro release condition.
0.5 mM cross linked hydrogels was only 23%. The amount of Ranitidine hydrochloride released from the cross linked hydrogels was found less than non-cross linked hydrogels. At lower amount of genipin used, larger amount of drug released. This result due to, as the concentration of cross linker decreases, the crosslinking degree will decrease. This makes the polymer network less dense. Consequently, as the available free space for drug diffusion increases, the rate of drug release also increases. Meanwhile, an increase of the degree of polymer cross-linking increases the polymer density and therefore decreases the available free space for drug diffusion which results in a decrease in drug release rates (Bachtsi and Kiparissides, 1995). In addition at t = 120 min the in vitro release from non-cross linked was 96% meanwhile drug release from 0.5 mM, 1.0 mM, 1.5 mM genipin cross linked floating hydrogels were 88%, 86% and 79%, respectively. In addition pore formation by effervescent reaction also influence the release of Ranitidine hydrochloride. Non-cross linked hydrogels shows high drug release rate due to its high porosity and pore size. As discussed in porosity study, floating hydrogels with higher amount of genipin leave fewer pores on hydrogel matrix; hence the drug was release in control manner as amount of cross linker increases.
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This is due to fewer pores makes the matrix highly compact and the drug diffuse slowly from the hydrogel matrix. From the result it can concluded that 1.5 mM genipin cross linked floating hydrogels showed the optimum drug release rate than other formulation. In addition, Fig 6(b) shows the comparison of Ranitidine hydrochloride release from non-cross linked and 1.5 mM genipin cross linked (optimum concentration). The result revealed that 1.5 mM cross linked hydrogels showed controlled drug release pattern than noncross linked hydrogels. 3.9. Hydrogels microstructure Fig. 7A (a–d) and B (a–d) illustrates the surface morphology and cross sectional image of non-cross linked and cross linked hydrogels with different concentrations at 50 magnification. It can be observed that non-cross linked hydrogels (Fig. 7A (a)) had smoother surface compared to genipin cross linked hydrogels (Fig. 7A (b–d)). As the amount of genipin increases, the surface became hard and rough. In addition, the result also showed that the pores were formed on the surface of non-cross linked and cross linked hydrogels. This clearly shows that incorporation of genipin did affect the pores formation as the pores were still intact on the surface even after cross linking reaction has occurred. However, it was obvious that the amount and size of pores became smaller as the concentration of genipin was increased. This was supported by the cross sectional image in Fig. 7B (a–d). Non-cross linked hydrogel (Fig. 7B (a)) produced larger pores compared to cross linked hydrogels. In contrast, 0.5 mM hydrogel (Fig. 7B (b)) formed large pores while smaller pores were observed in 1.5 mM hydrogels (Fig. 7B (d)). Changes in molecular structure during cross linking reaction reduced the pore formation in hydrogel network. This was in accordance with porosity results in Section 3.7. 4. Conclusion In conclusion, genipin cross linked floating kappa carrageenan hydrogel was successfully prepared using natural cross linker. The significantly higher level of mechanical strength, swelling capacity, good floating properties as well as sustained drug release makes the genipin cross linked floating kappa carrageenan hydrogel a suitable polymeric carrier for bioactive material in the gastrointestinal tract. Acknowledgements We would like to thanks the Department of Bioprocess Engineering, Faculty of Chemical Engineering, Cardiovascular Engineering, IJN-UTM, Grant Vot 4H023 from Research Management Centre and MyPhD Scholarship from Ministry of Higher Education (MOHE) for support of this study. References
Fig. 7. Surface morphology 6A (a–d) and cross sectional image 6B (a–d) of noncross linked and cross linked hydrogels with different concentrations at 50 magnification.
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Please cite this article in press as: Selvakumaran, S., Muhamad, I.I., Evaluation of kappa carrageenan as potential carrier for floating drug delivery system: Effect of cross linker. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.005