Impact of metal oxide nanoparticles on oral release properties of pH-sensitive hydrogel nanocomposites

International Journal of Biological Macromolecules 50 (2012) 1334–1340

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Impact of metal oxide nanoparticles on oral release properties of pH-sensitive hydrogel nanocomposites 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

Article history: Received 22 February 2012 Received in revised form 18 March 2012 Accepted 21 March 2012 Available online 30 March 2012 Keywords: Nanocomposite hydrogel Modified ␬-carrageenan Nanofillers Targeted release Gastrointestinal tract

a b s t r a c t In this article, modified ␬-carrageenan hydrogel nanocomposites were synthesized to increase the release ability of carrageenan hydrogels under gastrointestinal conditions. The effect of MgO nanoparticle loading in a model drug (methylene blue) release is investigated. Characterization of hydrogels were carried out using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Field Emission Scanning Electron Microscope (FESEM) and Differential Scanning Calorimetry (DSC). Genipin was used to increase the delivery performance in gastrointestinal tract delivery by decreasing release in simulated stomach conditions and increasing release in simulated intestine conditions. It is shown that the amount of methylene blue released from genipin-cross-linked nanocomposites can be 67.5% higher in intestine medium and 56% lower in the stomach compared to ␬-carrageenan hydrogel. It was found that by changing the nanoparticle loading and genipin concentration in the composite, the amount of drug released can be monitored. Therefore, applying nanoparticles appears to be a potential strategy to develop controlled drug delivery especially in gastrointestinal tract studies. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Recently, a great deal of research has focused on the application of nanotechnology in drug delivery as it provides a suitable means for both time and site-specific controlled delivery of drugs and bioactive agents [1–8]. Applying nanoparticles (NPs) in pharmaceutical studies offer many advantages providing targeted delivery of drugs, improving bioavailability and stability of therapeutic agents against degradation, and extending drug effect in target tissue [9]. Since nanoparticles have high specific surface area and unique physicochemical characteristics, they have been employed extensively in medical applications [10–13]. Hydrogels are intelligent materials capable of exhibiting significant volume changes in response to small changes in pH, temperature and other environmental stimuli [13,14]. Hydrogels have a wide range of applications in controlled drug delivery systems, tissue engineering, artificial organs in biotechnology, the recognition of certain bio-molecules and so on [15,16]. These materials can absorb large amounts of water and swell due to the existence of the hydrophilic groups (–OH, –COOH, –NH2 , –CONH2 , and –SO3 H) in their structures [16,17]. In addition, hydrogels can be modified with new functional groups or prepared as

∗ Corresponding author. Tel.: +607 5535577; fax: +607 5536163. E-mail addresses: [email protected] (H. Hezaveh), [email protected] (I.I. Muhamad). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.03.017

composites to increase their chemical/physical properties [18]. Guiseley [19] proposed the synthesis of ␬-carrageenan associated with hydroxyalkyl groups. Hydrogels prepared from hydroxyalkyl ␬-carrageenan derivatives showed a decrease in syneresis (liquid extraction from a gel) and could be used in a wide range of industrial fields. Among inorganic materials, metal oxides such as MgO are of particular interest as they are stable under harsh process conditions and are known to be essential minerals for human health [20–23]. Recently, the application of MgO nano- and micro-sized particles has attracted attention due to its biomedical applications [24–27]. However, the toxicity of the MgO nanoparticles to cells and organs remains fairly undiscovered. Ge et al. [28] reported on the cytotoxicity study of MgO nanoparticles on human umbilical vein endothelial cells (HUVECs) in vitro using the transmission electron microscope (TEM) and nanoparticle size analyser. Their results from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 htetrazolium bromide) assay, 4,6-diamidino-2-phenylindole (DAPI) staining analysis, NO release and total antioxidation competence (T-AOC) assay showed that most the MgO nanoparticles significantly enhanced the NO release and T-AOC content of the HUVECs. The testing results indicated that low concentration of MgO nanoparticles (below 200 g/ml) exhibited non-cytotoxicity. However, once the concentration of MgO nanoparticles was higher than 500 g/ml, the relative growth rate was lower than the control. Meanwhile Genipin, a naturally occurring cross-linking agent, is used in many biological research applications [29–31]. It is an

H. Hezaveh, I.I. Muhamad / International Journal of Biological Macromolecules 50 (2012) 1334–1340

effective cross-linker for polymers containing amino groups [32] with less cytotoxicity than other conventional cross-linkers such as formaldehyde and glutaraldehyde [33]. Genipin has been widely used in herbal medicine due to its anti-inflammatory, diuretic, choleretic and hemostatic properties [29]. The aim of this study is to improve the performance of carrageenan hydrogels as drug delivery vehicles in gastrointestinal conditions using metal oxide nanoparticles. Characteristics of biomaterials composites are also studied. Genipin is used to control and improve the amount of drug released in the intestine. 2. Materials and methods

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pH 4, pH 6.5 and pH 8 at room temperature (25 ◦ C). Synthesized gels were placed in a Petri dish filled with 50 ml of each buffer solution. Prior to weighting, filter paper was used to remove the surface water of swollen hydrogels. The swelling ratio (%) was then determined using Eq. (1). Swelling ratio (%) =

w − w  t 0 w0

× 100

(1)

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. The test was conducted in triplicate and reported as mean values to maximize accuracy.

2.1. Materials 2.4. Instrumentation ␬-Carrageenan (␬C) (Sigma–Aldrich), sodium carboxymethyl cellulose (NaCMC) (average molecular weight of 250,000, Acros Organic), methylene blue (C16 H18 ClN3 S·3H2 O) (Riedel-deHaën), magnesium oxide nanopowder (MgO) (<50 nm (BET), Sigma–Aldrich), genipin (GN) (Challenge Bioproducts Co., Ltd. Taiwan), HCl (Qrec Grad AR), NaOH (Qrec Grad AR). Distilled water is used in hydrogel synthesis and all chemicals are used as received with no additional purification. 2.2. Preparation of -carrageenan nanocomposites 2.2.1. Preparation of modified -carrageenan hydrogel Clearly, when hydrogel swelling is increased, its ability to release higher amounts of encapsulated drug will also increase. Therefore ␬-carrageenan hydrogel was blended with NaCMC to improve the swelling properties of carrageenan. Different concentrations of NaCMC were prepared and their swelling ability was tested to find the most suitable blend to be loaded with MgO nanofillers. Briefly, for a typical hydrogel synthesis, 0.48 g of ␬C was dissolved in 20 ml of distillated water at 80 ◦ C before mixing with 0.12 g NaCMC dissolved in 10 ml distillated water. One hour gentle stirring provided a clear, viscous and homogenous solution with no bubbles and the reflux kept the water content of the system constant. 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 an oven over night. Swelling test was carried out immediately after the drying process. Non-modified ␬C hydrogel was also prepared using the same method. Hydrogels will be referenced using their carrageenan concentration in the blend. For example 90:10 (kC:NaCMC) will be referred as kC90 and so on. 2.2.2. Preparation of methylene blue loaded MgO/-carrageenan nanocomposites MgO/␬-carrageenan nanocomposites were prepared by blending the MgO NPs with the polymer matrix as follows: 0.1 ml of methylene blue (MB) 1% containing MgO NPs in different concentrations were prepared using ultra sonication and rigorous stirring. Then 10 ml of well-dispersed solution was added to 20 ml of hot modified ␬C solution under gentle stirring after further intense sonication of hot solution in a Brandson (USA) ultrasonic processor at 80 ◦ C. Afterwards, hydrogel nanocomposite was poured into ceramic moulds and immediately put in a fridge to be cooled at −10 ◦ C for 10 min. The final disc was approximately 3.5 cm in diameter and a thickness of 1.0 cm. Table 1 lists the synthesis conditions for the different hydrogels synthesized in this experiment. 2.3. Measuring the swelling ratio To study the swelling behavior of hydrogels, modified and nonmodified gels were immersed in different pH buffer solutions of

2.4.1. FTIR analysis of hydrogel Blank and nanocomposite hydrogels were dried and 3 mg quantities were grounded and then mixed with 10 times as much KBr powder. 500 kg/cm2 pressure with hydraulic press formed the sample pellets. Prepared samples were analyzed using a Fourier transform infrared spectroscope (FTIR) (Nicolet 670 FTIR, USA) with 16 scan per sample in the region of 370–4000 cm−1 at 1.0 cm−1 intervals and a resolution of 4. 2.4.2. X-ray diffraction (XRD) Nanocomposites X-ray diffraction measurements were conducted at room temperature using a Siemens D5000 X-ray diffractometer with Cu K␣ at 40 keV and 40 mA and step length of 0.05◦ with step time of 1 s. The diffraction angle (2) was set between 20 and 80. Nanocomposites were dried in an oven before experimentation to reach constant weight. Disc thickness was approximately 0.55 ± 0.05 mm. 2.4.3. Hydrogel nanocomposites microstructure Field emission scanning electron microscope (FESEM) (Gemini Supra 35VP) was used to investigate the micro-structural changes in nanocomposite hydrogels. Prior to observation, samples were coated with gold using gold sputter coater Bio Rad Polaran Division (E6700, USA) under vacuum. Samples were studied at accelerating voltage of 10 kV and magnification of 1000× and 5000×. Samples were lyophilized before measurement and were left in liquid nitrogen to keep the hydrogels pores intact for imaging. 2.4.4. Differential Scanning Calorimetry (DSC) Thermal properties of hydrogels were determined using Differential Scanning Calorimetry (DSC822 METTLER TOLEDO, Switzerland) of the dried hydrogel samples. 5 ± 0.5 mg of each hydrogel sample was sealed in an aluminum pan and then heated from room temperature to 350 ◦ C at a heating rate of 5 ◦ C/min. The flow rate of N2 was maintained at 50 ml/min. 2.5. Release study of encapsulated methylene blue in gastrointestinal conditions As a model drug, methylene blue (MB) was used to study the gastrointestinal tract (GIT) release behavior of nanocomposite hydrogels. The MB loading process into the nanocomposites is as described in Section 2.2.2. Prepared samples were exposed to 60 ml of the medium solutions in Petri dishes placed in a thermostatic Kuhner Climo-Shaker (ISF1-X) at 37 ◦ C and 120 rpm. For the first 10 min the pH of the medium was kept at 0.1 N HCl then changed to buffer solution pH 6.6 for another 10 min and finally pH 7.4 up to 30 min. At regular intervals, 1.0 ml aliquot of the buffer solutions was removed to determine its concentration using Shelton UV/Vis spectroscopy (ct 06484 USA). The same amount of fresh solution

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Table 1 Synthesis conditions of modified ␬C hydrogels, hydrogel nanocomposites and cross-linked hydrogel nanocomposites. Sample designation

␬C (g)

NaCMC (g)

Nanofiller (g)

Genipin (mM)

Water (ml)

kC60 kC70 kC80 kC90 kC100 MgO-0.10 MgO-0.15 MgO-0.20 MgO-0.2/GN0.5 MgO-0.2/GN1.5

0.36 0.42 0.48 0.54 0.60 0.48 0.48 0.48 0.48 0.48

0.24 0.18 0.12 0.06 0.00 0.12 0.12 0.12 0.12 0.12

– – – – – 0.10 0.15 0.20 0.20 0.20

– – – – – – – – 0.5 1.5

30 30 30 30 30 30 30 30 30 30

was replaced to keep the original volume after each withdrawal. Experiments were performed in triplicate. Regarding Daniel-da-Silva et al. [34], in order to ensure that there is no interaction between the MB released and ␬-carrageenan, the withdrawn solution was diluted with KCl 1 M (dilution ratio 1:6). The MB released concentration was then measured at  = 663 nm which is the wavelength of maximum absorbance for MB [34]. Finally, the cumulative amount of MB released was determined from interpolation of standard calibration curve at  = 663 nm.

nanocomposite does not differ from unloaded hydrogel nanocomposite, indicating that there is no interaction between the model drug loaded and the hydrogel network.

3.1.2. XRD analysis The XRD diffractogram (Fig. 2) of the nanocomposite is consistent with the presence of metal oxide (MgO) as the main crystalline phase. Since kC based hydrogel is an amorphous material [35] it is not expected to affect the crystalline behavior of nanocomposites. The diffraction peaks at 37◦ , 43◦ and 62◦ are attributed to (1 1 1), (2 0 0) and (2 2 0) MgO, respectively. Clearly, by increasing MgO to the amorphous structure of carrageenan, the crystallinity is increased.

3. Results and discussion 3.1. Characterization of modified -carrageenan hydrogel nanocomposites

3.1.3. Field emission scanning electron microscope FESEM was used to study the surface structure of nanocomposite hydrogels. As can be seen from Fig. 3, the addition of MgO nanoparticles has changed the surface morphology of kC80 in such a way that the surface bulge is reduced, resulting in a flatter surface. Moreover, it seems that the porosity of nanocomposite is increased. From Fig. 3 it is also clear that the MgO nanoparticles are finely dispersed in the matrix and a uniform surface is produced. By cross-linking the network with genipin, as shown in our previous study (Hezaveh et al. [43]), the surface of cross-linked hydrogel becomes more compact as the cross-linker is added. This change in surface topography could be due to the change in molecular arrangement after cross-linking with genipin. This compact structure can hinder the diffusion of material in/out of the matrix.

3.1.1. Fourier transform infrared spectroscopy analysis FTIR analysis of blank, MgO-0.2 and loaded MgO-0.2 hydrogel nanocomposite are presented in Fig. 1. The bands observed at 847, 925 and 1258 cm−1 are attributed to d-galactose-4-sulfate, 3, 6 anhydrod-galactose and ester sulfate stretching, respectively. The broad band in the range of 3422–3585 cm−1 is due to –OH symmetric stretching vibrations of the kC/NaCMC hydrogel. This peak in both loaded and unloaded nanocomposite appears at 3422 indicating the interaction between the reduced MgO nanoparticles and the network. When the nanofillers are added to the network, a peak of 1158 cm−1 is formed for both loaded and unloaded nanocomposites. The peak which appeared at 1636 cm−1 is attributed to the asymmetric stretching band of –COOH of kC/NaCMC hydrogel. From Fig. 1 it is also clear that the FTIR spectrum of MB loaded

a 847.55 970.62 925.87 1258.74

2918.76

1121.671096.50

1636.36 3585.43 3422.96

b

970.62 847.55 925.87 1158.04 1124.47 1071.32 1258.74

%T

2918.76

1636.36

c

3422.96

970.62 847.55 925.87 1158.04 1124.47 1258.74 1071.32

2918.76 3422.96

4000.0

3600

1636.36

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

cm-1 Fig. 1. FTIR analysis of (a) kC80 (b) MgO-0.2 and (c) loaded MgO-0.2.

600

400.0

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Fig. 2. X-ray diffraction patterns of MgO nanocomposite. The Miller indices corresponding to the most intense reflection peaks are indicated in brackets.

3.1.4. DSC Characterization In the DSC graph, the existence of Tg and Tc indicates the crystalline behavior of nanocomposite material. As can be seen in Fig. 4, by increasing the MgO nanoparticles, Tg increases. kC80 showed

glass transition temperature at 85 ◦ C and by increasing the MgO in MgO-0.1 and MgO-0.2 hydrogel nanocomposites, the temperature increases to 100 and 110 ◦ C, respectively. Also, nanocomposites showed more crystallinity than kC80 hydrogel as the surface under

Fig. 3. FESEM micrograph of (a) kC80 (b) MgO-0.2 and (c) MgO-0.2/GN1.5, left images with magnification of 1000× and right images 5000×.

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(a)

200

kC100

kC80

160

10

MgO -0.1

140

MgO -0.2

120

12

Swelling (%)

Heat Flow (mW)

180

100 80

kC90 kC80

8

kC70

6

kC60

4

60 40

2

20 0

0

0

50

100

150

200

250

300

350

400

0

50

100

Temperature (C) Fig. 4. DSC analysis for kC80, MgO-0.1 and MgO-0.2 hydrogel nanocomposites.

(b)

200

20 kC100

Swelling (%)

18

the crystallinity peak has increased. This is in agreement with XRD pattern explained in Section 3.1.2. The exothermic peak at 250 and 255 ◦ C corresponding to crystalline presence of MgO-0.1 and MgO0.2 nanocomposites along with transition temperature changes show that the strength of hydrogel has increased by increasing MgO nanofillers.

150

Time (min)

kC90

16

kC80

14

kC70

12

kC60

10 8 6 4

3.2. Modification of -carrageenan

3.3. Effect of modification on pH-sensitivity of carrageenan-based hydrogel kC is a natural polyelectrolyte and NaCMC has carboxylic acid groups in its molecular structure. The degree of dissociation of carboxylic acid groups is related to the pH value of the solution, which results in pH-sensitivity of the hydrogel [39]. Fig. 6 shows the pH sensitivity of both kC100 and kC80 hydrogels. It was found that

0 0

100

200

300

400

300

400

Time (min)

(c)

25

kC100 kC90 kC80 kC70 kC60

Swelling (%)

20 15 10 5 0

100

0

200

Time (min) Fig. 5. Swelling behavior of kappa-carrageenan and modified kappa-carrageenan with different NaCMC concentrations in (a) pH4 (b) pH 6.5 and (c) pH 8.

by increasing the medium pH, swelling ability of both modified and non-modified hydrogels increases. It was notable that kC80 swelling in alkaline medium was more than 10% higher than that of acidic medium, while in alkaline medium kC100 swelled 5% more 25 pH 4

20

Swelling (%)

The purpose of modification was to increase the capacity of hydrogel to release more of loaded drug under gastrointestinal conditions. Since more swelling results in more drug release in hydrogels, kC was blended with sodium carboxymethylcellulose (NaCMC) to increase its swelling ability in different buffer solutions. NaCMC is a polyelectrolyte of a smart cellulose derivative that shows both pH-sensitivity and ionic-strength variations. Moreover, it has exhibited very good swelling capability [36,44]. The swelling behavior of both native and modified hydrogels in pH 4, 6.5 and 8 are shown in Fig. 5. As can be seen from Fig. 5, kC80 blend shows the most swelling (%) compared to other blends and non-blended kC hydrogel. This can be due to more hydrophilic chains or hydration of functional groups on the polymeric chains (–OH) and (–COOCH3 Na) [37,38]. It was observed that higher kC content of hydrogels (more than 80%) causes harder gel structures that hinder the swelling rate, whereby more NaCMC content results in less physically stable hydrogels due to matrix erosion causing drug carriers to be less suitable for oral drug delivery applications since they cannot survive the harsh stomach environment. It was found that in kC70 and kC60 hydrogels had lower physical stability. kC60, for example, could not swell properly due to erosion of the hydrogel network. It was found that kC80 hydrogel possesses both structural strength and the highest swelling among other hydrogels so it served as an optimum blend ratio for blending. It is desired that the maximum interaction occur in the developed polymer matrices; stabilization achieved due to the hydrophilic–hydrophobic interaction resulted in an optimum network and porosity that is kC80 with the ratio of 80:20 (k-carrageenan: NaCMC). Hence it is expected that the network formed in kC80 is the most stable gel structure to carry out further processing.

2

pH 6.5 pH 8

15 10 5 0 kC

Modified kC

Fig. 6. pH sensitivity of kC100 and kC80 hydrogels in different pH.

beta carotene release (mg/ml)

H. Hezaveh, I.I. Muhamad / International Journal of Biological Macromolecules 50 (2012) 1334–1340

Beta carotene released (mg/ml)

0.3 Blank

0.25

MgO - 0.1 MgO - 0.15

0.2

MgO - 0.2

0.15 0.1 0.05 0 0

5

10

15

20

25

30

35

Time (min) Fig. 7. Release profile of blank and MgO hydrogel nanocomposites under gastrointestinal conditions.

than acidic medium. This shows that modification has significantly affected pH-sensitivity of kC hydrogels and modified hydrogels were more sensitive to pH compared to non-modified hydrogel. In acidic medium, kC80 swelled up to 9.52%; however, the swelling (%) in pH 6.5 and 8 was 16.63 and 19.61%, respectively. The protonation of carboxylic groups results in the formation of more hydrogen bonds between the carboxylic acid groups in kC hydroxyl groups. A more compact hydrogel structure with restricted movement and relaxed structure resulted in formations that decrease the swelling ability, the time to reach the equilibrium swelling and cause less flexibility of network in this medium. As synthesized hydrogels are prepared in pH 6.25 ± 0.25 (pH of distilled water), and the pKa of carboxylic acid in the polysaccharide is ∼4.6, the ionization carboxylic acid groups is expected [39]. Electrostatic repulsion force caused by the breakdown of hydrogen bonds leads to more water penetrating into the network. In higher pH, this ionization of carboxylic acid groups becomes more pronounced and polymer chains expand more allowing more water to penetrate into the hydrogel matrix. In this experiment, kC80 exhibited the best swelling ability in all pH mediums and was selected for loading with metal oxide nanoparticles. In all cases in this article, kC:NaCMC 80:20 (kC80) blend ratio is referred to as “blank hydrogel”. 3.4. Effect of MgO loading on GIT release of MB Fig. 7 shows the release profile of modeled drug in simulated gastrointestinal environment for nanocomposite hydrogels with different MgO loadings. It can be seen that by increasing the MgO content of nanocomposites, MB release is significantly increased. By increasing NPs concentration from 0.1 g to 0.2 g, the maximum MB release increases from 0.174 to 0.267 mg/ml. Also, compared to blank hydrogel, the addition of MgO NPs has increased the cumulative release up to 52%, which means that more MB release is achieved. It is believed that NPs within the network can keep the loaded MB in their interphase with nanocomposite structure. This has been also observed by other researchers [40–42]. Studies by Durme et al. [40] show that the nano-sized SiO2 particles within the PNIPAM matrix act as nano-sized reservoirs that lead to dramatic changes in the diffusion characteristics of water molecules through the nanocomposite hydrogels. When nanocomposites are in contact with medium solution, these drug nano-sized reservoirs release the entrapped MB and act as MB releasing channels for the interior MB within the hydrogel network. Therefore, the release in nanocomposite hydrogels is higher than that of blank gels. All hydrogels with different concentrations of MgO NPs had almost the same profile in acidic stomach environments and 8 min after release in this medium, changes in the release profile have been observed. This suggests that the behavior of the

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0.3 MgO-0.2

0.25

MgO-0.2/GN1.5 0.2

MgO-0.2/GN0.5

0.15 0.1 0.05 0 0

5

10

15

20

25

30

35

Time (min) Fig. 8. Methylene blue release behavior of MgO-0.2 nanocomposite and genipincross-linked MgO-0.2 nanocomposites under gastrointestinal conditions.

nanocomposites is controlled by the kC/NaCMC network in lower pH. This can be explained by more compact structural network in this pH as discussed before. At low pH, a compact network results in blocking the interphase region between the nanoparticles and the network in the nanocomposite, resulting less MB release as observed. It is worthwhile to note that the release profile of the MgO0.1 nanocomposite hydrogel is very similar to the profile observed for blank hydrogel in stomach and intestine mediums; however, in the Duodenum area, MgO loaded hydrogels displayed more release than that of kC/NaCMC. Since the concentration of MgO NPs in MgO0.1 hydrogel nanocomposite is low, after releasing almost all the MB entrapped in the interphase reservoirs in pH 6.6, the amount of MB release in intestine medium becomes almost the same as kC80 hydrogel. This suggests that the release behavior of nanocomposites is controlled by both nano-sized reservoir and network pores. 3.5. Controlling drug release via cross-linking the hydrogel nanocomposites Since controlling the amount of release is essential in many medical, genipin cross-linking was used to obtain greater control over the amount of drug released. Cross-linking can change the release properties of hydrogels by affecting the porosity and structure of hydrogel network, which can also improve the physical stability of hydrogels. As the amount of cross-linker changes, the pore size will vary, causing the swelling ratio and drug release to change; therefore the drug release can be regulated to a desired level. As shown in Fig. 8, by increasing the genipin concentration of hydrogel nanocomposites, the total amount of MB released is decreased. Noticeable achievements resulting from changes in genipin increment were the increase in MB release in intestinal environments and reduced release in acidic medium. After a period of 10 min, non-cross-linked nanocomposite in acidic medium released 0.1617 mg/ml MB; however, MgO-0.2/GN0.5 and MgO-0.2/GN1.5 nanocomposite hydrogels released only 0.074 and 0.037 mg/ml MB in this medium, respectively. On the other hand the amount of drug released in intestinal medium (pH 7.4) by MgO-0.2 nanocomposite hydrogel was 0.044 mg/ml and the MB released for MgO-0.2/GN0.5 and MgO-0.2/GN1.5 nanocomposite hydrogels was 0.055 and 0.057 mg/ml, respectively. Table 2 shows the amount of MB released in each area. Since genipin can cause significant changes in hydrogel networks (see Fig. 2) it can be proposed that its incorporation in the network affects the nano-sized reservoirs by changing the network structure as was clearly shown in FESEM images. By increasing the genipin content of nanocomposite, carboxylic acid groups within the matrix also increase; therefore, protonation of hydrogel network in acidic medium

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Table 2 The performance of blank, MgO-0.2 and cross-linked hydrogel nanocomposites under GIT conditions.

Blank kC MgO-0.2 MgO-0.2/GN0.5 MgO-0.2/GN1.5

Stomach (pH 1.2) (mg/ml)

Duodenum (pH 6.6) (mg/ml)

Intestine (pH 7.4) (mg/ml)

0.085 0.161 0.074 0.037

0.056 0.062 0.068 0.064

0.034 0.044 0.055 0.057

increases, the resulting in a more compact network in this medium and a consequent reduction in the amount of MB. On the other hand, by changing pH 1.2–6.6 and 7.4, this ionization of carboxylic acid groups is more than that of the non-cross-linked hydrogel, and electrostatic repulsion forces cause more material to diffuse out. Therefore by changing the amount of MgO NPs and genipin concentration in the system, the amount of MB released can be monitored.

from Research Management Centre UTM for support of this study. References [1] [2] [3] [4]

4. Conclusion

[5]

In this study, ␬-carrageenan hydrogel was modified to synthesize hydrogel nanocomposites with MgO nanoparticles to be used in gastrointestinal tract study. It was found that by increasing MgO nanoparticles MB release increases noticeably. Different hydrogel nanocomposites were formulated and their performance under gastrointestinal conditions was studied. Genipin was used to increase the delivery performance by decreasing MB release in stomach pH and increasing it in intestine pH; therefore, the hydrogel nanocomposites performance was improved. The study revealed that in cross-linked nanocomposites the MB released was 67% more in intestines and 56% less in stomach compared to blank hydrogels. FTIR spectrum revealed that MB does not have any significant effect on the matrix since the IR spectrum for both loaded and unloaded nanocomposites were the same. XRD pattern also indicates that by increasing the MgO nanofillers, the crystallinity of hydrogel increases. Surface morphology of blank, MgO nanocomposite and cross-linked nanocomposites show that MgO nanoparticles have a noticeable impact on the hydrogel microstructure. By increasing the genipin content of the network, a more compact network will be formed as a result of changing the molecular arrangement after cross-linking with genipin (a natural cross-linker). This compact structure can hinder the material penetration in/out of the hydrogels. DSC analysis shows that biomaterial strength and crystallinity can be increased by increasing nanofiller in the composite. It is suggested that MgO nanofillers can act as nano-sized reservoirs for methylene blue and when hydrogels come into contact with the release medium, these hydrophilic reservoirs can serve as releasing channels for interior MB within the network. The interphase between the nanoparticles and the network can control the release properties. Comparing the performance of cross-linked and non-crosslinked hydrogel nanocomposites with blank hydrogels, it can be concluded that the incorporation of genipin and MgO nanoparticles in kC/NaCMC hydrogels can significantly improve drug release under GIT conditions. Also, control over drug release can be achieved by varying the nanoparticle loading and genipin concentration in the composite. Incorporation of nanoparticles seems to be a potential strategy for developing nanocomposite materials in the design of novel biomaterials for controlled drug delivery systems.

[6] [7]

Acknowledgment

[8] [9] [10] [11]

[12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44]

We would like to thank the Food and Biomaterial Engineering lab, Bioprocess Engineering technicians and RUGrant

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