halloysite nanocomposite hydrogels as potential drug delivery systems

Journal of the Taiwan Institute of Chemical Engineers 67 (2016) 426–434

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Kappa-carrageenan/halloysite nanocomposite hydrogels as potential drug delivery systems Ghorbanali Sharifzadeh a, Mat Uzir Wahit b,∗, Mohammad Soheilmoghaddam c, Wong Tuck Whye d, Pooria Pasbakhsh e a

Department of Polymer Engineering, Faculty of Chemical Engineering, UniversitiTeknologi Malaysia, 81310 Johor, Malaysia Center for Composites, Institute of Vehicle System and Engineering (IVeSE), UniversitiTeknologi Malaysia (UTM), 81310 Skudai, Johor, Malaysia Tissue Engineering and Microfluidics Laboratory, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Building 75, Corner of College and Cooper Roads, St Lucia Brisbane, QLD 4072 Australia d Medical Devices and Technology Group, Faculty of Biosciences and Medical Engineering, UniversitiTeknologi Malaysia, Johor, Malaysia e Mechanical Engineering Discipline, School of Engineering, Monash University, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor Darul Ehsan, Malaysia b c

a r t i c l e

i n f o

Article history: Received 21 March 2016 Revised 11 June 2016 Accepted 22 July 2016

Keywords: Hydrogels Nanocomposites Kappa-carrageenan Halloysite nanotube In vitro drug release

a b s t r a c t Novel kappa-carrageenan (Kc)/halloysite nanotube (HNT) nanocomposite hydrogels were synthesized via physical crosslinking for the gastro-intestinal tract (GIT) release. The influence of HNT nanoparticle content on Kc/HNT hydrogel properties such as thermal, swelling, drug loading and in vitro release was examined. Thermal results revealed that the incorporation of HNT nanoparticles enhanced the thermal stability of the nanocomposite hydrogels. Also, the nanocomposite hydrogels showed higher swelling, drug loading and release behavior compared to the pure Kc hydrogel. In vitro release from the Kc-HNT hydrogels exhibited that rhodamine B (RB), a cationic model drug, released higher than orange G (OG), an anionic model drug. RB in vitro release from the nanocomposite hydrogels reached to approximately 72%, while 54% of OG was released. Finally, in vitro cytotoxicity test revealed that both Kc and Kc-HNT hydrogels are biocompatible. Taking together, it was shown that Kc-HNT hydrogels may have a great potential applications in oral drug delivery systems. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction Hydrogels are defined as synthetic or natural crosslinked polymeric materials with an ability to absorb high quantities of water. These hydrophilic polymer networks swell in aqueous or biological mediums without being dissolved due to physical or/and chemical crosslinks [1]. The desirable properties of hydrogels such as low surface tension, suitable mechanical strength, tunable biological, chemical and physical characteristics, high water content, high biocompatibility, and their similarity to natural living tissue make them as valuable candidates in the field of biomedical and pharmaceutical applications [2–4]. Due to these promising properties, hydrogels have been enormously applied in a broad range of biomedical applications including cell encapsulation [5], diagnostic biomedical biosensors [6,7], drug delivery [8–10], and regenerative medicine [11–13].

∗

Corresponding author. Fax +60 7 5536165. E-mail addresses: [email protected] (G. Sharifzadeh), [email protected] (M.U. Wahit).

In recent years, naturally-derived hydrogels such as alginate, chitosan, collagen and hyaluronic acid have drawn significant attention as they are non-toxic, biodegradable, largely available in nature and renewable [1]. Among these natural polymers, carrageenan has emerged as suitable materials in pharmaceutical fields containing immunomodulatory, anticancer, anticoagulant studies [14,15]. Carrageenan belongs to sulfated and linear hydrophilic polysaccharides, consisted of anhydrogalactose and galactose units [16,17]. It is mainly used as gelling, thickening and emulsifier agent in food and pharmaceutical studies [18]. One of the most common types of carrageenan is Kc with repeating units of D -galactose and 3,6 anhydrogalactose which generally formed the strongest gel in the family of carrageenan. The gelation of Kc is a thermoreversible process which contains a coil to helix conformational transition succeeded by helix aggregation. The process can be implied upon cooling through suitable salt conditions via ionic interactions and hydrogen bonds [19]. Because of its gelling and biocompatibility characteristic, Kc has been extensively studied in tissue engineering [20,21] and drug delivery system [22–24]. However, the thermal stability, mechanical properties and drug release behavior of Kc should be further enhanced in order to extend its applications in biomedical areas.

http://dx.doi.org/10.1016/j.jtice.2016.07.027 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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Table 1 Fabrication conditions for the pure Kc and Kc-HNT nanocomposite hydrogels and drug loading (%). Sample

Kc (g)

HNT (g)

H2 O (ml)

RB-drug loading (%)

OG-drug loading (%)

Kc HNT5 HNT7 HNT10

0.5 0.5 0.5 0.5

0 0.05 0.075 0.1

30 30 30 30

0.22 3.33 7.53 14.13

1.90 5.19 6.85 7.70

Over the past few years, clay nanoparticles have been extensively studied in the medical fields due to their excellent biological, mechanical and thermal properties with high surface area, drug loading and absorption capabilities [25,26]. Within the clay family, halloysite nanotube (HNT) with a hollow tubular network and unique strength has gained attention. The inner and outer diameters of tubes are 10–30 nm and 40–70 nm, respectively, whereas the length of tubes is 0.2–1.5 μm [27]. Also, the positively (inner) and negatively (outer) charged surfaces of HNT can easily form electrostatic interaction with charged molecules or drugs [28]. It is expected that the porosity and hollow lumen microstructures of HNT enhance the loading and absorption capability of drug molecules, thus, causes high drug release behavior. However, the toxicity of HNT within hydrogel networks may arise as the major concern for biomedical applications. In a notable example, Vergaro et al. reported the cytocompatibility of HNT using different cells including MCF-7, Hela and cervical andenocarcinoma [29]. The results revealed that HNT is much less toxic than normal salt (NaCl) and showed no toxicity to these cells. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide) assay using swine cartilage chondrocytes also confirmed the cytocompatibility of HNT in PEG hydrogels in which both control and nanocomposite hydrogels exhibited similar cytotoxicity [30]. Compared with other nanoparticles such as carbon nanotube (CNT), HNT is highly hydrophilic, biocompatible, abundantly available and relatively cheap, hence, it has been extensively used in a broad range of biomedical applications including tissue engineering [31,32] and drug delivery [33,34]. It has also been introduced into hydrogels to improve their thermal and mechanical characteristics [35,36]. These remarkable properties of HNT motivate us to explore their applications in drug delivery systems. We hypothesize that the incorporation of HNT within Kc hydrogel network not only enhance the thermal and release characteristics but also enhance the biocompatibility of hydrogel. To the best of our knowledge, the addition of HNT nanoparticles into the Kc hydrogel networks has not been explored. Additionally, the effect of HNT on properties of Kc hydrogels such as thermal stability, swelling and release behavior has also remained undiscovered. The aim of this research is to design and fabricate Kc-HNT hybrid hydrogels as the new drug delivery systems. The effect of HNT nanoparticles on swelling and release properties of RB and OG as drug models in physiological conditions was investigated in details. Moreover, cell viability was also carried out in order to evaluate the effect of HNT on Kc biocompatibility.

2. Materials and methods 2.1. Materials K-carrageenan (30 0,0 0 0 g/mol) and halloysite nanotube (HNT) with a tube dimension of 30–70 nm × 1–4 μm were provided by Aldrich. Orange G (C16 H10 N2 Na2 O7 S2 ) with molecular weight of 452.37 g/mol and rhodamine B (C28 H31 CIN2 O3 ) possessing molecular weight of 479.01 g/mol were supplied by Sigma-Aldrich. All materials are used as received without further purification.

2.2. Preparation of nanocomposite hydrogels The nanocomposite hydrogels were synthesized by incorporating the HNT nanoparticles with the Kc as follows. Typically, 0.5 g of Kc was dissolved in 20 ml of distilled water and heated to 80 °C. This hot solution was stirred for 30 min to ensure complete dissolution of Kc without formation of bubbles. Then 10 ml of calculated HNT powder in distilled water was stirred for 1 h and added into the Kc solution with gentle stirring for 2 h and under reflux to ensure no solvent evaporation and good dispersion of HNT into the Kc/water solution. The solutions were poured into ceramic mold with the diameter of 3 cm. Finally, the hydrogels were formed by sealing the samples for 24 h at room temperature (25 °C). Pure Kc hydrogel was also fabricated in the same procedures, but without addition of HNT nanoparticle. The pure and nanocomposite hydrogels with different concentration of HNT were denoted as Kc, HNT5, HNT7 and HNT10. For example, the HNT5 stood for 0.5 g Kc with 0.05 g HNT powder. All the resultant hydrogels were washed with distilled water to remove unreacted monomers before experiment. Although all the nanocomposite hydrogels were milky, the pure gel was transparent. The preparation procedures for all hydrogels are summarized in Table 1.

2.3. Swelling studies All samples were dried overnight at 37 °C prior to swelling tests. The dried Kc and Kc-HNT hydrogels were immersed in 20 ml of two different pH mediums (pH 1.2 and pH 7.4) at 25 °C. At specified intervals, the hydrogels were taken out from solutions and wiped with filter paper to remove the extra surface water before weighing. The swelling ratio (%) of hydrogel was calculated by the following Eq. (1)

Swelling ratio (% ) =

W1 − W0 × 100 W0

(1)

where, W1 is the weight of the swollen hydrogel and Wo is the weight of the dried hydrogel at time t. To maximize the accuracy, the swelling test was performed in triplicate.

2.4. Drug loading and in vitro release studies RB and OG were chosen as the model drugs for release study of hydrogels through a swelling equilibrium procedure. 1 mg/ml of RB and OG solutions were separately mixed in distilled water, and loaded into the gel samples via placing the dried Kc and Kc-HNT hydrogels into the above media at 25 °C for 2 h to achieve equilibrium swelling. The samples were fetched out and washed with distilled water to remove the model drug molecules which were loosely attached to the hydrogels. Then they were dried at 37 °C in an oven overnight. The drug release experiments were performed in pH 7.4 phosphate buffer solution (PBS) at 37 °C. The in vitro release was carried out by introducing the above drug-loaded hydrogels in glass beakers containing PBS. After predetermined intervals, aliquots of the solutions were collected and analyzed by UV– vis spectroscopy at 543 nm for RB and 476 nm for OG. The drug

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loading (%) of hydrogels were measured by using Eq. (2)

Drug loading (% ) =

M × 100 Mt

2.6. Cytotoxicity test (2)

And then the cumulative release (%) of RB and OG were determined using Eq. (3)

Cumulative release (% ) =

M1 × 100 M

(3)

where M and M1 indicate the initial mass of loaded and cumulative amount of drugs released at time t, respectively. Mt is the initial mass of hydrogel for drug loading. The drug loading (%) of all the hydrogels is summarized in Table 1. The release tests were carried out in triplicate to maximize the accuracy. 2.5. Characterization of hydrogel 2.5.1. UV–vis spectroscopy (UV–vis) The UV–vis absorption spectra of the aliquots were measured with a Shelton UV–vis spectroscopy (ct 06484 USA). 2.5.2. Fourier Transform Infrared spectroscopy (FTIR) The FTIR spectra of pure and nanocomposite hydrogels were analyzed by a Fourier transform infrared spectroscopy (FTIR) instrument (Nicolet 670 FTIR, USA) sample in the region of 370– 40 0 0 cm−1 with a resolution of 4.

Cell cytotoxicity of pure Kc and Kc-HNT hydrogels was evaluated by dimethylthiazol diphenyl tetrazolium bromide (MTT) assay on human skin fibroblasts (HSF 1184). All the samples were sterilized by exposure to UV light for 1 h and incubated in PBS solution for 24 h. The cells were plated at a density of 2× 105 cells/well on the hydrogels in 96-well plates and incubated at 37 ºC in 5% CO2 atmosphere for 24 h. Next, 20 μl of MTT solution (5 mg/ml) was added to each well, and the plates were incubated for another 4 h. The medium was removed, and 200 μl of dimethylsulfoxide (DMSO) was added into each well. The absorbance of each well was measured at 570 nm on a microplate reader (BioRad,Model 680, USA). The cells inoculated directly on tissueculturepolystyrene plate (TCPS) were regarded as a negative control. Measurements were done in triplicates. 2.7. Statistical analysis One-way analysis of variance (ANOVA) with the post “Tukey’s post hoc test” for multiple comparisons was performed using SPSS software (Statistical Package for the Social Science, version 18). A p value less than 0.05 was considered statistically significant. 3. Results and discussion 3.1. Fourier transformed infrared spectroscopy (FTIR)

2.5.3. Thermal Gravimetric Analysis (TGA) The thermal behavior of the pure Kc, HNT and Kc-HNT nanocomposite hydrogels was evaluated by using Q50 TGA (Q50 TGA instruments, USA). The temperature ranged from 30–700 °C with a heating rate of 10 °C min−1 under nitrogen. 2.5.4. X-Ray Diffraction (XRD) X-ray diffraction of nanocomposite hydrogels was performed using a Bruker AXS D8 Advance equipped with Cu Kα radiation (λ = 0.15406 nm) source at voltage of 40 kV and current of 40 mA with a step size of 0.04° and step time of 1 s. Patterns were recorded in the region of 2θ ranging from 2° to 70°. The average crytalline size was evaluated by using Scherrer’s Eq. (4)

D=

Kλ β cos θ

(4)

where D is the average crystallite size, K, a Scherrer constant related to the crystallite shape and is approximately 0.9 for Kc, λ the wavelength of X-ray radiation, β the full width of the peak at half of the maximum intensity (in radians) and θ is the Bragg angle. 2.5.5. Field emission scanning electron microscopy (FESEM) A field emission scanning electron microscopy (FESEM) (Gemini Supra 35VP) was used to investigate the topographic changes in the 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 an accelerating voltage of 10 kV and three different magnifications. Samples were lyophilized before measurements and were left in liquid nitrogen to keep the hydrogels pores intact for imaging. 2.5.6. Transmission electronic microscopy (TEM) The TEM samples were cut into small slices with the thickness of approximately 100 nm from nanocomposite hydrogel imbedded into an epoxy resin via ultramicrotome (Power Tome XL). The hydrogel was positioned on a copper grid and was viewed using Philips CM12.

FTIR spectroscopy was used to analyze the chemical structure of Kc and nanocomposite hydrogels (Fig. S1). The broad absorption bands at 3436 cm−1 in Kc and Kc-HNT nanocomposite hydrogels are assigned to the O ̶ H stretching. A peak observed at 2966 cm−1 is attributed to stretching frequency of –CH3 group. The absorption bands at 1646 cm−1 that appeared in the spectrum of both Kc and Kc-HNT hydrogels are produced from the absorbed water ‘bending’ vibration and amide I band [37,38]. The bands observed at 847, 927 and 1262 cm−1 are attributed to D -galactose-4sulfate, 3,6-anhydro D -galactose, glycosidic linkage and ester sulfate stretching of Kc backbone, respectively [39]. The characteristic absorption peaks in the Kc-HNT nanocomposite hydrogels are approximately similar to the Kc hydrogel. However, the band at 380 0–310 0 cm−1 in the nanocomposite hydrogels is sharpened due to the hydroxyl groups of HNT, in comparison with pure Kc hydrogel [40]. Also, the intensity ratio of the absorbance at 1646 cm−1 for the nanocomposite hydrogels decreased compared to pure Kc because of the electrostatic interactions between Kc and HNT. This weak interaction might be related to the coulombic repulsion between HNT surface and polymer chain. The small shift of the peak at 3425 cm−1 due to the presence of Al ̶ OH also verified the formation of physical bonding inside the polymeric network. These results confirm the formation of physical interaction between Kc and HNT naoparticles in the hydrogels. 3.2. Thermal Gravimetric Analysis (TGA) Thermal stability of Kc, HNT and Kc-HNT nanocomposite hydrogels was analyzed by TGA, and the results of TGA thermograms are shown in Fig. 1. The thermal decomposition of the Kc and Kc-HNT nanocomposite hydrogels indicated a two-step decomposition. The first and second stages of thermal decomposition for pure Kc were attributed to the evaporation of moisture, and destruction of polymer network which were observed in the range of 30–130 ºC and 20 0–70 0 ºC, respectively [41,42]. However, these steps for nanocomposite hydrogels were associated with the polymer destruction, thermal dehydration of HNT in structural layer, as well as its dehydroxylation [43]. The 10 wt% weight loss temperature (T10 ),

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Fig. 2. XRD patterns of pure hydrogel (Kc), HNT and nanocomposite hydrogels (KcHNT). Fig. 1. TGA thermograms of Kc, HNT and Kc-HNT hydrogels.

30 wt% weight loss temperature (T30 ) and 50 wt% weight loss temperature (T50 ) of samples are presented in Table S1. From Fig. 1, the thermal behavior of nanocomposite hydrogels was significantly affected by HNT concentration. The addition of HNT shifted the thermal stability of hydrogel to a higher temperature because the HNT nanoparticles act as heat barriers and decreased the diffusion of volatile products [44]. Also, the lumen of the HNT can entrap the polymer chains, delaying the degradation of the Kc polymer and improving its thermal stability. For example, the T10 , T30 , and T50 of pure Kc hydrogel were 80, 200 and 400 ºC respectively, whereas these values increased to 120, 220 and 530 ºC respectively for Kc/HNT5 hydrogel. In addition, it can be seen that the char yields for the nanocomposite hydrogels improved with HNT addition into the nanocomposite hydrogels. The char yield of Kc was 43.4% at 650 ºC, while at the same temperature it increased to 49.8% for the HNT10 nanocomposite hydrogel. However, the residual increase is not proportional to the HNT content. This improvement in char yields and thermal stability of the nanocomposite hydrogels is attributed to the entrapment of volatile Kc products inside the lumen structure of HNT. Thus, this thermal improvement which hinder the polymer decomposition can be related to interaction between Kc and HNT. Previous researches on nanocomposites also reported the higher thermal stability of the nanocomposites due to interaction between the polymer and the HNT [38]. 3.3. X-Ray Diffraction (XRD) The presence of HNT nanoparticles within hydrogel network was confirmed by XRD diffractogram. The XRD patterns of Kc, HNT and nanocomposite hydrogels are shown in Fig. 2. The pure Kc hydrogel exhibited a peak at 2θ =28° which is attributed to the amorphous nature of Kc [45,46]. As seen in Fig. 2, the addition of HNT nanoparticles increased the diffraction intensity of nanocomposite hydrogels at 2θ =28°, 2θ =41°, 2θ =50° and 2θ =59°. This change in crystallinity of Kc in nanocomposites was attributed to the structural properties of HNT nanoparticles which acted as heterogeneous nucleation points, thus increased the crystallization in the nanocomposite hydrogels [47]. It can also be observed that the peak at 2θ =30° for HNT was shifted to 2θ =29° in nanocomposites with higher HNT contents (HNT7 and HNT 10) which might be due to the good dispersion of HNT within the nanocomposites. The crystalline size of pure Kc and nanocomposite hydrogels were calculated. Based on the results, the crystalline sizes of Kc-HNT hydrogels were higher than the pure Kc hydrogel. The data was found

to be 26.99 nm for pure Kc, while it increased to 38.71, 40.93 and 42.43 nm for HNT5, HNT7 and HNT10, respectively. 3.4. Morphology of Kc-HNT hydrogels The effect of HNT nanoparticles on the dry microstructure of polymeric networks was analyzed using field emission scanning electron microscopy (FESEM). Based on the above results, HNT10 exhibited higher thermal stability and crystalline size compared to other nanocomposite hydrogels, thus regarded as optimum value. Both cross sectional and surface FESEM images of pure Kc and HNT10 freeze-dried hydrogels are shown in Fig. 3. A relatively smooth cross sectional images of Kc (Fig. 3c, e) were shown to be completely changed by the incorporation of HNT (Fig. 3d, f–h). In the enlarged images of nanocomposite hydrogel (Fig. 3h), it is obvious that HNT is embedded in the Kc matrix, suggesting their good interfacial adhesion between HNT and Kc due to their electrostatic interaction. These images clearly indicated that the introduction of HNT nanoparticles within hydrogels induced structural changes at a microscopic level. The surface morphology of HNT10 (Fig. 3b) exhibited a rougher network structure compared with pure Kc which could be attributed to the improvement in crosslinking density of hydrogels containing HNT nanoparticles. This is in consistent with TGA result which was discussed in the previous sections and confirmed the structural reinforcement of hydrogels upon addition of HNT. Also, the formation of nanoparticles can significantly alter the porosity of hydrogels which further affect the swelling and release behavior of Rb and OG from nanocomposite hydrogels. Fig. 4 exhibited the transmission electron microscopy (TEM) images of the HNT10 nanocomposite hydrogel. The TEM result clearly confirmed the presence of HNT with tubular structure in the Kc hydrogel. This is in agreement with the FESEM findings shown in Fig. 3. 3.5. Swelling studies The swelling capability is one of the key properties of hydrogels which allows them to manipulate the drug release and the rate of water sorption. It is mainly controlled by pore size within the hydrogel network. The swelling ratio of Kc hydrogels in the presence and absence of HNT nanoparticles was investigated in physiological media (pH 1.2 and 7.4) (Fig. 5). The swelling ratio of Kc was found to be the lowest compared to those Kc-HNT hydrogels in the both media. As shown in Fig. 5a, Kc indicated a declining trend of swelling with time in pH 1.2 because of its minimum crosslinking ratio which made the hydrogels structure weaker, thus

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Fig. 3. Surface FESEM micrographs of freeze-dried (a) Kc and (b) HNT10 hydrogels. Cross sectional images of (c)–(e) Kc and (d),(f)–(h) HNT10 nanocomposite hydrogels.

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Fig. 4. TEM images of HNT10 nanocomposite hydrogel at (a) 50k and (b) 25k.

it swelled less than Kc-HNT hydrogels. Surprisingly, hydrogels with higher amount of HNT exhibited higher swelling ratio than that of pure hydrogel. As earlier demonstrated by TGA test, the stability of Kc hydrogels was improved upon addition of HNT nanoparticles, therefore lower swelling ratio would be expected. However, in our research stronger hydrogels swelled more than pure hydrogel which could be attributed to the surface charge of HNT and viscoelastic characteristics of Kc network. The nanocomposite hydrogels consist of polyelectrolyte polymers such as Kc with surface charged HNT nanoparticles forming an afflux of water to balance the osmotic pressure build-up, that results in higher hydrogel swelling. These results are in line with previous researches on nanocomposite hydrogels using magnetic and gold nanoparticles [23,24]. In addition, the tubular microstructure of HNT may lead to the absorption of high amount of water and causes higher swelling than pure hydrogel. From Fig. 5, it has been found that all pure and nanocomposite hydrogels were pH-dependent which showed higher swelling ratio in pH 7.4 (Fig. 5b). The swelling ratio of all samples in pH 7.4 increased to a greater amount than that at pH 1.2 as function of time. For example, the swelling ratio of HNT5 hydrogel in pH 1.2 was found to be 6.30% while it reached to 17.58% in pH 7.4. In addition, Fig. 5c clearly shows the maximum swelling difference of pure Kc and Kc-HNT hydrogels at 25 ºC in pH 1.2 and 7.4 in which both HNT nanoparticles and pH of medium altered the hydrogel swelling ratios. This observation could be explained by the fact that the hydrophilic groups of hydrogels were protonated in pH 1.2 (acidic medium) which restricted them to form hydrogen bonding with medium in pH 1.2, hence swell less. In pH 7.4, however, the neutral medium easily form hydrogen bonding with hydrophilic groups which results in high water absorption and therefore higher swelling ratio in comparison with pH 1.2 [1]. 3.6. Drug loading and in vitro release studies Hydrogels as soft biomaterials have been widely used for controlled release studies due to their controllable swelling ratio as well as good biocompatibility which allow them to solute [48]. To determine the drug loading and controlled release behavior of hydrogel, a cationic drug, RB and an anionic drug, OG, were chosen

as two hydrophilic model drugs. The loading capacity of RB and OG in Kc, HNT5, HNT7 and HNT10 hydrogels were 0.22%, 3.33%, 7.53%, 14.13% and 1.90%, 5.19%, 6.85%, 7.70%, respectively. It was observed that the loading capacity of both RB and OG was significantly improved through the introduction of HNT nanoparticles within hybrid hydrogels. This is understandable since addition of HNT with Kc hydrogel causes higher entrapment of RB and OG into the layers of HNT. Thereby, HNT nanoparticles can be considered as desirable drug nanocarriers. The cumulative release of RB and OG from Kc and Kc-HNT hydrogels at 37 ºC in PBS solution is shown in Fig. 6. It was observed that the addition of HNT into hydrogel network was improved the release of both RB and OG. It is worth mentioning that the swelling ratio of hydrogels is significantly related to the drug release, particularly, higher swelling ratio induces greater drug release by providing larger surface area [49]. Briefly, water soluble drugs such as RB and OG can only be released after water penetration within the hydrogel causing the swelling, dissolution and diffusion of drugs, respectively [49]. From Fig. 6, the drug release for nanocomposite hydrogels was higher than pure hydrogel due to their greater swelling properties. All samples showed similar release behavior of the encapsulated RB during 1 h in PBS. About 20% of the RB inside the hydrogels was released in the first 10 min, and then another 20% of the RB was released in the following 15 min. The highest release was observed for HNT10 hydrogel with 72.76%, while for HNT7, HNT5 and pure Kc hydrogel the released amount was 68.27%, 59.21% and 54.81%, respectively. The RB release from HNT10 nanocomposite hydrogel indicates an interesting difference in comparison with other hydrogels. At the initial stage (first 20 min), the HNT10 hydrogel release was slower than Kc hydrogel which then increased to higher release at later stage. This phenomenon is identified as bimodal or sigmoidal release, which might be attributed to the formation of denser gel networks upon HNT addition [22,50]. The formation of rougher polymeric network in nanocomposite hydrogels was previously demonstrated by FESEM micrographs. The more rapid RB cumulative releases of nanocomposite hydrogels at later stages are in association with their greater swelling ratio (Fig. 6a), which are strongly consistent with the previous findings [22,24].

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Fig. 6. In vitro drug release of (a) RB and (b) OG from the pure Kc and Kc-HNT nanocomposite hydrogels in PBS solution at 37 ºC.

Fig. 5. Swelling ratio of Kc and Kc-HNT nanocomposite hydrogels in (a) pH 1.2 and (b) pH 7.4 at 25 ºC. (c) The effect of HNT nanoparticles and pH medium in the swelling behavior of Kc pure hydrogel and Kc-HNT hydrogels.

Fig. 6b shows the effect of HNT nanoparticles on the cumulative release of OG. As shown, the HNT concentration can control the drug release of Kc-HNT nanocomposite hydrogels. By increasing the concentration of HNT in the Kc hydrogel, higher OG release was obtained. The OG cumulative release was 17.74% after 75 min which increased to 25.86%, 26.99% and 35.21% when HNT5, HNT7 and HNT10 were introduced, respectively. The in vitro OG release from HNT10 hydrogel experienced a gradual increase and reached to 54.63% after 90 min. As shown, higher drug release was observed upon the addition of HNT nanoparticles which could be related to the lumens and surfaces of HNT. The higher release rate of nanocomposite hydrogels was also attributed to the formation of water afflux between polyelectrolyte Kc with surface charged HNT nanoparticles which caused higher swelling in Kc-HNT hydrogels despite having stronger networks and is in line with the previous works [51,52]. Besides, the charge of drug molecules as well as their interactions with Kc and HNT nanoparticles play a significant role in the release behavior of hydrogels. More specifically,

the negatively charged OG was loaded into HNT via electrostatic interaction, while due to cationic character of RB, electrostatic repulsion was occurred with HNT. As a result, the electrostatic repulsion between RB and HNT accelerated the release of RB from nanocomposite hydrogels, leading to higher release rate compared to OG model drug. Clearly, this finding confirmed the influence of electrostatic interaction on the release of model drugs from hydrogels (Fig. 6). The same result has been reported for the release of ofloxacin [35]. In order to investigate the release mechanism of RB and OG from Kc and nanocomposite hydrogels, the in vitro drug release data were evaluated by Ritger–Peppas model, a semi-empirical power law using Eq. (5) [53] :

Mt = kt n M∞

(5)

where Mt and M∞ are referred to the weight of the hydrogels at time t and infinite time, respectively, k is the release rate coefficient and n is the diffusion exponent. The release mechanism of hydrogels is analyzed by n values. For cylindrical-shaped hydrogels, when n ≤ 0.45, it shows that the release is a controlled Fickian diffusion, 0.45≤ n ≤ 0.89 indicate non-Fickian diffusion (anomalous) which involves the combination of both Fickian and relaxation transport, and n ≥ 0.89 follows the relaxation controlled (Case II) transport which the relaxation of Kc polymer occurred slower than the diffusion process [54]. The n value was found to be 0.68, which demonstrated that the RB release from Kc hydrogel followed anomalous mechanism, thus the release kinetic controlled by both diffusion and relaxation transport [55]. However, by incorporation of HNT nanoparticles within hydrogel, this value increased to 1.09 and 1.18 for HNT5 and HNT7, respectively, exhibiting the Case II transport. Further increase in HNT

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Fig. 7. Cytotoxicity assay of Kc and Kc-HNT hydrogels on HSF 1184 cell. Data represented as means ± SD (n = 3).

concentration was significantly reduced the n value to 0.78, indicating the anomalous diffusion mechanism controlled the release of RB from HNT10. It is proposed that the addition of HNT nanoparticles induced tighter structure, therefore, affecting the drug release behavior. Compared with RB, the release mechanism of OG from hydrogels is completely different. The n values for all hydrogels were greater than 0.89, indicating the release of OG in Kc and Kc/HNT hydrogels were mainly controlled by relaxation and erosion mechanism. It should be pointed out that the faster drug release at a later stage of hydrogels with Case II transport mechanisms is possibly related to the permeation of water within their polymeric networks [22,56]. Based on the above experiments, the introduction of HNT within Kc hydrogel not only improved the drug loading but also provided a faster release rate for encapsulated drugs.

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and characterized. The addition of HNT nanoparticles was found to tune the thermal, swelling, drug loading and in vitro drug release characteristics of the Kc hydrogel. The swelling ratios of Kc-HNT hydrogels were significantly higher than pure Kc in pH 1.2 and 7.4, despite having stronger structures. This could be related to the encapsulation of the surface charged HNT nanoparticles into Kc hydrogel that caused a water afflux to balance the osmotic pressure build-up and resulted in greater hydrogel swelling. In vitro studies also exhibited enhanced release of RB and OG for the nanocomposite hydrogels in comparison with pure hydrogel where OG showed less release. The higher release of RB and OG from nanocomposite hydrogels is mainly attributed to the improved plasticity as a result of water permeation in the gels. It is suggested that, the higher release of RB and OG from nanocomposite hydrogels are mainly controlled by dispersion of HNT nanoparticles within hydrogel network which improved the strength of the hydrogel network and caused greater swelling ratio. The RB release from hydrogels showed both anomalous and Case II diffusion, whereas the OG release mechanism for all hydrogels followed Case II transport. From cytotoxicity test, Kc and Kc-HNT hydrogels showed good biocompatibility on HSF 1184 cells. Overall results demonstrate that these advanced Kc-HNT nanocomposite hydrogels have many potential applications for drug delivery system. Acknowledgments The authors wish to acknowledge Fundamental Research Grant Scheme (FRGS) Vote no: R.J130 0 0 0.7809.4F546 by Universiti Teknologi Malaysia from the Ministry of Education Malaysia. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jtice.2016.07.027.

3.7. Cytotoxicity assay

References

For biomedical applications, Kc and Kc-HNT nanocomposite hydrogels must be biocompatible and non-toxic. In this study, the cell cytotoxicity of the Kc and Kc-HNT nanocomposite hydrogels was evaluated by MTT assay using human skin fibroblasts (HSF 1184). According to the results shown in Fig. 7, no significant difference between Kc and HNT5 hydrogel was observed (p ˃ 0.05 one way ANOVA Tukey’s post hoc test). Consistent with previous researches, the interaction of the HNT with various cells has shown that HNT is non-toxic, thus it is considered biocompatible [29,30]. The relative cell viability percentage was 91.37 ± 2.13%, 88.41 ± 1.26% and 82.67 ± 1.54% for Kc, HNT5 and HNT7, respectively. This percentage was reduced to 80.23 ± 2.62% for HNT10 hydrogel. As shown in Fig. 7, with increasing the concentration of HNT, the viability for all nanocomposite hydrogels was decreased. This possibly may be attributed to the cells death due to the high concentration of nanoparticles which blocked most of the channels on the cell membrane. From the MTT assay, it is obvious that HNT has slight effect on the cytocompatibility of Kc hydrogel. FESEM results also indicated rougher polymeric surfaces in nanocomposite hydrogels due to incorporation of HNT. It is worth mentioning that the increased surface roughness of polymeric hydrogels by nanoparticles improve the cells growth and adhesion in comparison with smooth surfaces [47,57,58]. Therefore, it can be concluded that the Kc-HNT nanocomposite hydrogels are biocompatibe materials and they can be used safely as potential drug delivery systems.

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4. Conclusion Novel nanocomposites drug delivery carriers consisted of Rb and OG-loaded Kc-HNT hydrogels were successfully synthesized

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