Enhancement of Curcumin Bioavailability Using Nanocellulose ... - MDPI

polymers Article

Enhancement of Curcumin Bioavailability Using Nanocellulose Reinforced Chitosan Hydrogel Thennakoon M. Sampath Udeni Gunathilake 1 , Yern Chee Ching 1, * and Cheng Hock Chuah 2 1 2

*

Department of Chemical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia; [email protected] Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; [email protected] Correspondence: [email protected]; Tel.: +60-3-7967-4445; Fax: +60-3-7967-5317

Academic Editor: Antonio Pizzi Received: 14 January 2017; Accepted: 7 February 2017; Published: 15 February 2017

Abstract: A unique biodegradable, superporous, swellable and pH sensitive nanocellulose reinforced chitosan hydrogel with dynamic mechanical properties was prepared for oral administration of curcumin. Curcumin, a less water-soluble drug was used due to the fact that the fast swellable, superporous hydrogel could release a water-insoluble drug to a great extent. CO2 gas foaming was used to fabricate hydrogel as it eradicates using organic solvents. Field emission scanning electron microscope images revealed that the pore size significantly increased with the formation of widely interconnected porous structure in gas foamed hydrogels. The maximum compression of pure chitosan hydrogel was 25.9 ± 1 kPa and it increased to 38.4 ± 1 kPa with the introduction of 0.5% cellulose nanocrystals. In vitro degradation of hydrogels was found dependent on the swelling ratio and the amount of CNC of the hydrogel. All the hydrogels showed maximum swelling ratios greater than 300%. The 0.5% CNC-chitosan hydrogel showed the highest swelling ratio of 438% ± 11%. FTIR spectrum indicated that there is no interaction between drug and ingredients present in hydrogels. The drug release occurred in non-Fickian (anomalous) manner in simulated gastric medium. The drug release profiles of hydrogels are consistent with the data obtained from the swelling studies. After gas foaming of the hydrogel, the drug loading efficiency increased from 41% ± 2.4% to 50% ± 2.0% and release increased from 0.74 to 1.06 mg/L. The drug release data showed good fitting to Ritger-Peppas model. Moreover, the results revealed that the drug maintained its chemical activity after in vitro release. According to the results of this study, CNC reinforced chitosan hydrogel can be suggested to improve the bioavailability of curcumin for the absorption from stomach and upper intestinal tract. Keywords: biodegradable; chitosan; curcumin; nanocellulose; drug delivery; bioavailability

1. Introduction Hydrogels are highly swollen, hydrophilic, three-dimensional (3D) polymeric networks capable of absorbing large amounts of water without dissolving in water or aqueous solvent, yet are insoluble due to the presence of cross-links, entanglements, or crystalline regions [1]. Hydrogel can be formed by natural, synthetic or their hybrid polymers. Recently, attention has been focused on producing hydrogels using materials that are non-toxic and biocompatible in nature. Considering not only safety, but also biocompatibility and biodegradability, biopolymer-based hydrogels have been drawing increasing interest and attention as excellent candidates for biomedical applications, such as drug delivery, wound dressings, and tissue engineering matrices [2].

Polymers 2017, 9, 64; doi:10.3390/polym9020064

www.mdpi.com/journal/polymers

Polymers 2017, 9, 64

2 of 19

Biocompatible and biodegradable hydrogels are very important biomaterials used in drug delivery systems, which gained increasing attention of researchers. Chitosan is a highly swellable, pH responsive and biocompatible polymer which can be used to deliver drugs to specific sites of gastrointestinal tract. The pH-sensitive swelling and releasing behavior of hydrogels are useful for the design of oral drug delivery carrier. Chitosan is a linear ubiquitous polysaccharide and heteropolymer consist of glucosamine and N-acetyl-glucosamine units [3]. Amino groups of chitosan have a pKa value of around 6.5. At pH below this pKa value, the amino groups of chitosan get ionized and positively charged –NH3 + groups are distributed among hydrogel network. This leads to the repulsion of polymer chains within the hydrogel and allowing more water intake into the hydrogel network [4]. Referring to our previous study [5], chitosan hydrogel also exhibited a high swelling ratio in acidic medium than alkaline conditions. Cationic nature of chitosan in acidic medium increases the retention of the hydrogel at the site of application and readily binds to negatively charged surfaces such as mucosal membranes. Mucoadhesive ability could result in formulations containing chitosan being retained in the mucosal surface such as gastric mucosa due to the acidic environment of the stomach. Due to the high degree of swelling and mucoadhesive properties in acidic medium, chitosan-based hydrogels have been used as a carrier for stomach—specific drug delivery systems [6–11]. Adequate mechanical strength is desirable for hydrogels as drug delivery systems [12,13]. Improved mechanical strength can withstand the pressure during gastric contraction and prolong the gastric retention time [14]. Hydrogels with less mechanical strength can become fragmented after repetitive gastric contractions [15,16]. Recently, nanomaterials have been used as a reinforcing agent to improve the mechanical strength and stability of polymer hydrogels [17,18]. CNCs have received much attention as reinforcing agent because of their properties such as large surface area, high mechanical strength, low density, non-toxic nature, high aspect ratio, biocompatibility and biodegradability [18–23]. In our previous study [4], the compression strength of the chitosan hydrogel increased with physical reinforcement of cellulose nanocrystals. The maximum compression of chitosan hydrogel increased from 38.4 ± 1 kPa to 50.8 ± 3 kPa, with increasing CNC content from 0.5% to 2.5%. Nanocellulose reinforced chitosan hydrogel forms a semi-interpenetrating polymer network (semi-IPN) by diffusion of linear polymer chains into a preformed polymer network. Semi-IPN improves the mechanical properties and controls the swelling behavior of hydrogel [5,24]. Curcumin (diferuloylmethane), a natural polyphenolic nutraceutical, is a major component of turmeric (Curcuma longa) that has been associated with antioxidant, anticancer, anti-inflammatory, antiviral, and antibacterial activities as indicated by over 6000 citations and over one hundred clinical studies [25]. Recent studies showed that curcumin can block ethanol, indomethacin, stress-induced gastric ulcer and can prevent pylorus-ligation-induced acid secretion [26]. Helicobacter pylori (H. pylori) a gram-negative bacteria, is reported as etiologic factor in the development of the chronic gastritis, ulcers and gastric adenocarcinoma. Many studies highlighted the potential of curcumin as a promising antibacterial agent having property to restore and repair the gastric damage caused by H. pylori infection. The poor solubility and low bioavailability of curcumin have been highlighted as the major problem that can lead to loss of local therapeutic action in the stomach [27]. However, many attempts have been made to improve the pharmacotherapy of the stomach through local drug release, leading to high drug concentration at the gastric mucosa, making it possible to treat stomach and duodenal ulcer, gastritis and esophagitis [10,28–33]. CNC/chitosan hydrogels have been used for various drug delivery applications. Previous studies [34,35] had reported the use of CNC/chitosan hydrogels as potential drug carrier for procaine hydrochloride and formation of polyelectrolyte-macroion complexes. However, nanocellulose reinforced chitosan hydrogel is not yet investigated or reported for its application as drug delivery system for curcumin. Objective of this study is to improve the bioavailability of less water-soluble curcumin for the absorption from stomach and upper intestinal tract. In acidic medium, the protonated amino groups of chitosan will interact with sialic acid (N-acetylneuraminic) in gastric mucus by electrostatic interaction thus improve the gastric residence time and retaining the drug at


3 of 19

the absorption site for a prolonged period. A large extent of chitosan matrix swelling is found to occur in acidic medium, hence the drug molecules are expected to diffuse extensively through the swollen gel in to the exterior medium at gastric pH levels. Due to the fast swelling property the super porous hydrogels are widely used for delivery of poorly water-soluble drugs. Gas foaming is used to fabricate superporous hydrogel with large and widely interconnected pore structures. To sum-up, the mucoadhesiveness, stimuli sensitivity and faster swellability will make the hydrogel a suitable candidate for stomach specific drug delivery systems. 2. Materials and Methods 2.1. Materials Chitosan medium molecular weight (viscosity 200–500 cP, 0.5% acetic acid at 20 ◦ C), acetic acid glacial grade AR, methanol, hydrochloric acid, sodium chloride and sulfuric acid were purchased from Friendemann Schmidt Chemicals (Parkwood, Australia). Glutaraldehyde 25% was obtained from Thermo Fisher Scientific Inc. (Victoria, Australia). The drug curcumin was provided by Himedia Laboratories Pvt Ltd. (Mumbai, India). Microcrystalline cellulose and phosphate-buffered saline was obtained from R&M chemicals (Essex, UK). All chemicals were used as obtained. 2.2. Preparation of CNC–Chitosan Hydrogel CNCs were prepared from microcrystalline by sulfuric acid hydrolysis method reported in our previous study [36–38]. Chitosan was dissolved in 5% (v/v) aqueous acetic acid solution at room temperature and left overnight in the shaker with the rotation rate of 250 rpm to prepare a 2% (w/v) chitosan solution (High concentration of acetic acid induces lower viscosity and hence easier to dispersion of nanocrystals) [39,40]. The solution was then filtered through the filter paper to remove any insoluble matters. CNCs were homogenized using ultrasonic treatment for 10 min to obtain stable and uniformly distributed nanocrystals. To prepare chitosan hydrogel and CNC–chitosan hydrogel, CNCs in different concentrations were added (0%, 0.5%, 1%, 1.5%, 2%, 2.5%) to 2% (w/v) chitosan solution and stirred (250 rpm) for one hour. After that 0.2% (v/v) of glutaraldehyde (in the final mixture) was mixed with CNC–chitosan solution and stirred (350 rpm) for 1 min at room temperature. The mixture was then poured into the mold and allowed the hydrogel to solidify at room temperature for 24 h. Finally, hydrogels were rinsed several times with distilled water to remove any unreacted polymer or chemicals. 2.3. Equilibrium Swelling Study The swelling ratios were measured by the immersion of hydrogels in distilled water at 37 ◦ C. Before the swelling test, hydrogels were cut into disk shape pieces and dried in room temperature until they reached constant weight. Dried hydrogels were weighed before immersion into the distilled water. The weight of the swelled hydrogels after immersion in the distilled water was recorded at predetermined time intervals over 16 h. The swelling ratio of the hydrogels was calculated using Equation (1). −W0 (1) Swelling ratio (%) = W1W × 100 0 where W 1 is the weight of swollen hydrogel and W 0 is the initial dry weight of the hydrogel. 2.4. CO2 Gas Foaming of Hydrogels A 0.5% CNC–chitosan composition was used for the high pressure CO2 gas foaming process as it showed the highest swelling ratio from equilibrium swelling test. After mixing with the chitosan solution with glutaraldehyde and 0.5% CNC as described above, the mixture was poured in to the mold and placed inside the gas foaming apparatus. After that the apparatus was gradually pressurized with CO2 to predetermined pressure (10, 30 and 50 bar). The pressure was maintained to allow for


4 of 19

CO2 saturation and chitosan crosslinking for 48 h. The system was then depressurized at 1 bar/min, resulting in the generation of numerous gas bubbles which induce gas foaming. Swelling tests, drug loading and releasing tests were done for 0.5% CNC–chitosan hydrogel prepared at 10, 30 and 50 bar pressure and compared with the results of the hydrogel prepared at atmospheric condition. 2.5. Drug Loading Efficiency Curcumin was entrapped into hydrogels by swelling equilibrium method. Disk shaped hydrogels were immersed in 30 mg/L drug solution for 24 h at room temperature. Curcumin loaded hydrogels were removed from drug solution and rinsed with distilled water and washing was collected. Curcumin concentration remaining in the solution was determined by UV-2600, UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan) at 427 nm. The experiments were repeated three times and average values were taken. The drug encapsulation efficiency was calculated using Equation (2). Encapsulation efficiency % =

Amount of curcum in the hydrogel Initial drug amount in the solution

× 100

(2)

2.6. In Vitro Drug Release In vitro drug release from hydrogel networks with different drug loading contents, was investigated in simulated gastric condition (prepared by dissolving 2 g NaCl in 7.0 mL HCl and water up to 1000 mL) at 37 ◦ C. In order to study the release, 3 mL solution containing released drug was removed at predetermined time intervals and returned it back to the solution after the analysis. The concentration of released curcumin was measured at 427 nm using UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan). The experiments were performed triplicates and average values were taken. 2.7. FTIR Analysis FTIR studies of curcumin, hydrogel and curcumin loaded hydrogel were carried out using PerkinElmer spectrum 400 FTIR spectrometer (Shimadzu, Kyoto, Japan) over the range 3000–500 cm–1 . 2.8. Morphology Studies The morphology of the gas foamed hydrogel (at 50 bar) and hydrogels prepared at atmospheric condition was examined using (FE-SEM, SU8220) field emission scanning electron microscope (Hitachi, Tokyo, Japan). Hydrogels were freeze dried using freeze dryer to remove water without disturbing the morphology. After this, the hydrogels were coated with gold in order to prevent the charging effects at an accelerating voltage of 5 kV. 2.9. Mechanical Testing The mechanical properties of hydrogels were investigated by compression test using a (Shimadzu, AGS-X) universal/tensile tester (Shimadzu, Kyoto, Japan). The hydrogels were cut in to disk shapes (thickness (12 mm) and diameter (18 mm)) and allowed to equilibrate in pH 7.4 buffer solutions for 30 h. During the compressive strength tests, stress and strain responses were monitored under a load of 500 N and at a rate of 0.5 mm/min. Strain and stress were recorded using Trapezium Lite X software until maximum breaking strength was approached. Five replicates were tested to get the average and standard deviation.


5 of 19

2.10. In Vitro Degradation In vitro degradation studies of hydrogels were carried out in PBS solution for six weeks. The dried gels were kept in 50 mL of PBS. The PBS was renewed every week. At predetermined time intervals, samples were taken out, washed, blotted using filter paper and dried to constant weight at 45 ◦ C. Dry weight of the sample was measured and weight loss was determined using Equation (3). Weight loss % =

W0 − Wt × 100 W0

(3)

W 0 is the dry weight of the hydrogel taken initially and W t is the dry weight at time t. 3. Results and Discussion 3.1. Mechanical Properties Adequate mechanical strength is desirable for hydrogels as drug delivery systems. Improved mechanical strength can withstand the pressure during gastric contraction and prolong the gastric retention time. Hydrogels with less mechanical strength can become fragmented after repetitive gastric contractions [15,16]. Unmodified chitosan hydrogels exhibit relatively low mechanical strength uncontrollable porosity and degradability particularly in acidic aqueous solutions [41]. Thus many efforts have been made on modification of chitosan facilitated by its hydroxyl and amino groups [42]. Mechanical strength, can be improved by covalent crosslinking using chemical crosslinkers such as glutaraldehyde (GA), oxalic acid, formaldehyde, glyoxal, and genipine [43]. Previous studies revealed that the higher crosslinker concentrations are favorable from the mechanical stability point of view, but at the same time the decrease in porosity may cause lower drug release rates by diffusion through the porous media [44]. In general, a high concentration of crosslinking agent is required to improve the mechanical strength of a hydrogel [16]. In this research, we were able to improve the mechanical properties of chitosan hydrogel by physical reinforcement of CNC at a constant crosslinker concentration. Glutaraldehyde (0.2% (v/v)) was used to prepare both pure chitosan and 0.5% CNC reinforced chitosan hydrogel. Nanocellulose reinforced chitosan hydrogel forms a semi-interpenetrating polymer network (semi-IPN) via the diffusion of linear polymer chains into a preformed polymer network. A semi-IPN structure improves the mechanical properties of a hydrogel and controls its swelling behavior [30]. Several mechanisms are responsible for the interface reinforcement of polymer matrix by nanoparticles. The interaction between nanoparticles and the polymer matrix could form special microstructures such as finer scale lamellar structure result in improved mechanical properties; nanoparticles could effectively enhance their interaction with the matrix through chemical bonds (for instance, increase the crosslinking densities) or increase the physical interactions between polymer chains of the matrix. In this manner, nanoparticles can strongly influence to complement the poor mechanical and tribological performances of some polymer matrices [31]. In our previous study, the maximum compression of CNC–chitosan hydrogel increased from 25.9 ± 1 kPa to 50.8 ± 3 kPa with increasing CNC content ranging from 0% to 2.5%. In addition, the maximum compression did not increase significantly with the addition of over 2.5% CNCs to the chitosan hydrogel [4]. The results of previous study indicated a decrease in swelling ratios with increasing the CNC content of chitosan hydrogel. Within the CNC reinforced hydrogels, 0.5% CNC–chitosan hydrogel indicated the highest swelling ratio in distilled water. Therefore, 0.5% CNC–chitosan hydrogel was used for the gas foaming process and for the investigation of mechanical properties. As shown in Figure 1, the maximum compression of pure chitosan hydrogel was 25.9 ± 1 kPa and it increased to 38.4 ± 1 kPa with the introduction of 0.5% cellulose nanocrystals. The maximum compression of 0.5% CNC–chitosan hydrogel produced at atmospheric condition was 38.4 ± 1 kPa and it decreased with increasing the pressure of gas foaming process. This value for hydrogels


6 of 19

produced at 10, 30 and 50 bar was 25.7 ± 2, 17.0 ± 0.2 and 5.4 ± 0.2 kPa, respectively. The decrease Polymers 2017, 9, 64 6 of 19 in maximum compression is due to the increase of pore size and the pore interconnectivity of CNC–chitosan hydrogel produced gas foamingprocess. process.Several Several studies studies have chitosan hydrogel produced at at gas foaming have also alsoreported reportedthat thatthe the mechanical strength of hydrogels and scaffolds decreases with increase of the pore size and the pore mechanical strength of hydrogels and scaffolds decreases with increase of the pore size and the pore interconnectivity [46] reported that thethe compressive modulus of of glutaraldehyde interconnectivity[29,32,45]. [29,32,45].Ji Jietetal.al. [46] reported that compressive modulus glutaraldehyde crosslinked chitosan pressureof of60 60bar barwhen whencompare comparewith with crosslinked chitosanwas wasmore morethan thanthreefold threefoldlower lower at at CO2 pressure the the hydrogels produced at atmospheric condition. According to Ji, Annabi, Khademhosseini and hydrogels produced at atmospheric condition. According to Ji, Annabi, Khademhosseini and Dehghani [37] the maximum compression ofof gas foamed chitosan hydrogel is is comparatively greater Dehghani [37] the maximum compression gas foamed chitosan hydrogel comparatively greater than the results of of our study. This may bebe due to to thethe high concentration of of glutaraldehyde (0.5% v/v) than the results our study. This may due high concentration glutaraldehyde (0.5% v/v) used for their study. According to the results of Annabi et al. [47] the maximum compression of used for their study. According to the results of Annabi the maximum compression of αα-elastin hydrogelproduced producedat atdense densegas gasCO CO2 2(pressure (pressureatat6060bar) bar)was was ± 1.4 kPa; slightly lower elastin hydrogel 4.34.3 ± 1.4 kPa; slightly lower than than the results of our study, which is due to the nature of biopolymer used. the results of our study, which is due to the nature of biopolymer used. 0% cnc-chitosan 0.5% cnc-chitosan

40

Maximum compression (kPa)

35 30 25 20 15 10 5 0 P= 1 bar

P = 1 bar

P = 10 bar

P = 30 bar

P = 50 bar

Figure Maximum compression hydrogels formed different pressure conditions. Figure 1. 1. Maximum compression ofof hydrogels formed at at different pressure conditions.

3.2. FTIR Analysis 3.2. FTIR Analysis Figure 2 shows the FTIR spectra of pure chitosan hydrogel, 0.5% CNC–chitosan hydrogel, drug Figure 2 shows the FTIR spectra of pure chitosan hydrogel, 0.5% CNC–chitosan hydrogel, drug loaded 0.5% CNC–chitosan hydrogel and pure curcumin. Results of our previous study showed that loaded 0.5% CNC–chitosan hydrogel and pure curcumin. Results of our previous study showed that the FTIR spectra of CNC and chitosan are fairly similar due to the chemical similarity between the FTIR spectra of CNC and chitosan are fairly similar due to the chemical similarity between chitosan chitosan and cellulose [5]. In the FTIR spectrum of the CNC–chitosan hydrogel, peaks representing and cellulose [5]. In the FTIR spectrum of the CNC–chitosan hydrogel, peaks representing both the both the chitosan hydrogel and CNC were observed [5]. No change or new peak appeared in the chitosan hydrogel and CNC were observed [5]. No change or new peak appeared in the spectrum spectrum of 0.5% CNC–chitosan hydrogel, thereby indicating that the cellulose nanoparticles were of 0.5% CNC–chitosan hydrogel, thereby indicating that the cellulose nanoparticles were physically physically added to chitosan hydrogel. The spectrum of curcumin showed characteristic peaks at added to chitosan hydrogel. The spectrum of curcumin showed characteristic peaks at 1601, 1506, 1274, 1601, 1506, 1274, and 1152 cm–1, which corresponded to the stretching vibrations of the benzene ring, and 1152 cm–1 , which corresponded to the stretching vibrations of the benzene ring, C=C vibrations, C=C vibrations, aromatic C–O stretching, and C–O–C stretching modes, respectively [48,49]. Those aromatic C–O stretching, and C–O–C stretching modes, respectively [48,49]. Those characteristic peaks characteristic peaks are not shifted significantly in curcumin loaded 0.5% CNC–chitosan hydrogel are not shifted significantly in curcumin loaded 0.5% CNC–chitosan hydrogel revealed that there is revealed that there is no interaction between drug and ingredients present in hydrogels. Thus, there no interaction between drug and ingredients present in hydrogels. Thus, there is no change in the is no change in the chemical composition of the drug after the loading process. However, the intensity chemical composition of the drug after the loading process. However, the intensity of the bands in of the bands in curcumin and curcumin-loaded hydrogel are different. The decrease in peak intensity curcumin and curcumin-loaded hydrogel are different. The decrease in peak intensity is due to lower is due to lower concentration of curcumin in the hydrogel as its concentration was not 100%. Hence, concentration of curcumin in the hydrogel as its concentration was not 100%. Hence, FTIR analysis FTIR analysis provides significant evidence for the presence of curcumin in the drug-loaded provides significant evidence for the presence of curcumin in the drug-loaded hydrogel. hydrogel.

Polymers 2017, 9, 64 Polymers 2017, 9, 64

7 of 19 7 of 19 Pure curcumin 1274


1601

Pure curcumin

1506 1601

Curcumin loaded0.5% CNC-chitosan hydrogel

7 of 19

1152

1274 1152

1506

0.5% CNC-chitosan hydrogel Curcumin loaded0.5% CNC-chitosan hydrogel

0.5% CNC-chitosan hydrogel

Pure chitosan hydrogel

Pure chitosan hydrogel

3000 3000

2500

2000

2500

2000

1500

-1 Wave number (cm )

1500

1000 1000

500 500

-1 Figure2.2.FTIR FTIRspectra spectraofofpure purechitosan chitosan hydrogel, 0.5% CNC–chitosanhydrogel, hydrogel,curcumin curcuminloaded loaded Wave number (cm ) CNC–chitosan Figure hydrogel, 0.5% 0.5% CNC–chitosan hydrogel and pure curcumin. 0.5% CNC–chitosan hydrogel and pure curcumin. Figure 2. FTIR spectra of pure chitosan hydrogel, 0.5% CNC–chitosan hydrogel, curcumin loaded 0.5% CNC–chitosan hydrogel and pure curcumin.

3.3. Morphology Studies 3.3. Morphology Studies 3.3.Prior Morphology Studies to FESEM observation, hydrogels were freeze-dried to remove water to avoid disturbing Prior to FESEM observation, hydrogels were freeze-dried to remove water to avoid disturbing the morphology of hydrogels. After freeze-drying, hydrogelsto exhibited a porous network structure Prior to FESEM observation, hydrogels were freeze-dried remove awater to avoid disturbing the morphology of hydrogels. After freeze-drying, hydrogels exhibited porous network structure (as shown in Figure 3). After the gas foaming (Figure 3a), the pore size significantly increased with morphology hydrogels. freeze-drying, exhibited a porous network structure (asthe shown in Figureof3). After the After gas foaming (Figurehydrogels 3a), the pore size significantly increased with the the formation of widely interconnected porous structure. Gas foamed hydrogel exhibited rough cell (as shown Figureinterconnected 3). After the gas foaming (FigureGas 3a), foamed the porehydrogel size significantly increased with formation of in widely porous structure. exhibited rough cell wall wall which exposed more surface area to the drug molecules. Pore sizes of rough the hydrogel the structure formation widely interconnected porous structure. Gas foamed hydrogel cell structure which of exposed more surface area to the drug molecules. Pore sizes ofexhibited the hydrogel formed formed at atmospheric pressure (Figure 3b) was around 100 µm and it was more than 10 fold higher structurepressure which exposed surface area 100 to the Pore sizes of the hydrogel at wall atmospheric (Figuremore 3b) was around µmdrug and molecules. it was more than 10 fold higher in gas informed gas foamed hydrogel (at 50 bar) . at atmospheric pressure (Figure 3b) was around 100 µm and it was more than 10 fold higher foamed hydrogel (at 50 bar) . Due to sudden volume expansion, the original morphological features changed in the swollen in Due gas foamed hydrogel (at 50 bar). to sudden volume expansion, the original morphological features changed in the swollen hydrogel drug diffusion medium. In the gas foamed hydrogel, the large structure Due in to the sudden volume expansion, the original morphological features changed in pore the swollen hydrogel in the drug diffusion medium. In the gas foamed hydrogel, the large pore structure hydrogel inand the small drug interconnected diffusion medium. Informed the gasafter foamed hydrogel, the large structure disappeared pores the immersion in SGF (aspore shown in Figure disappeared and small interconnected pores formed after the immersion in SGF (as shown in Figure 3c). disappeared andhand, smallthe interconnected pores formed the immersion in SGF (as shown in Figure 3c). On the other initial porous structure ofafter the hydrogel prepared at atmospheric condition On the other hand, the initial porous structure of the hydrogel prepared at atmospheric condition was 3c).changed On the other theainitial porousstructure structure (Figure of the hydrogel at atmospheric condition was and hand, formed nonporous 3d). It isprepared clear that the interconnected pores changed and formed a nonporous structure (Figure 3d). It is clear that the interconnected pores in the was changed and formed a nonporous structure (Figure 3d). It is clear that the interconnected pores in the swollen state of gas foamed hydrogel promote the swellability, drug loading efficiency, and swollen state of gas foamed hydrogel promotepromote the swellability, drug loading efficiency, and release as in the as swollen state to of the gas hydrogel foamed hydrogel the swellability, release compared formed at atmospheric condition.drug loading efficiency, and compared to the hydrogel formed at atmospheric condition. release as compared to the hydrogel formed at atmospheric condition.

Figure3.3.Cont. Cont. Figure Figure 3. Cont.


8 of 19 8 of 19


8 of 19

Figure FESEMimages imagesfor forthe thecross crosssection section of: of: (a) gas at at Figure 3. 3. FESEM gas foamed foamed hydrogel; hydrogel;(b) (b)hydrogel hydrogelformed formed atmospheric condition;(c) (c)gas gasfoamed foamedhydrogel hydrogel after immersed formed at at atmospheric condition; immersedin inSGF; SGF;and and(d) (d)hydrogel hydrogel formed atmospheric condition afterimmersed immersedininSGF. SGF. atmospheric condition after Figure 3. FESEM images for the cross section of: (a) gas foamed hydrogel; (b) hydrogel formed at atmospheric Swelling Ratio condition; (c) gas foamed hydrogel after immersed in SGF; and (d) hydrogel formed at 3.4.3.4. Swelling Ratio atmospheric condition after immersed in SGF.

Preliminary swelling study is the indicative of the release mechanism of the drug from the Preliminary swelling study is the indicative of the release mechanism of the drug from the swollen swollen polymeric 3.4. Swelling Ratio hydrogel [50]. In this study, swelling experiments were carried out in distilled polymeric hydrogel [50]. In this study, swelling experiments were carried out in distilled water for water for chitosan hydrogels prepared with different percentage of CNCs. As shown in Figure 4, Preliminary swelling study is the indicative release of the 4, drug from the chitosan hydrogels prepared with different percentageofofthe CNCs. Asmechanism shown in Figure swelling increases swelling increases with time, [50]. first In rapidly and swelling then slowly, reaching a plateau. Allinthe hydrogel swollen polymeric hydrogel this study, experiments were carried out distilled with time, first rapidly and then slowly, reaching a plateau. All the hydrogel formulations showed more formulations showedhydrogels more thanprepared 100% swelling ratios within first 15CNCs. min. In our previous study, we water for chitosan with different percentage shown in Figure than 100% swelling ratiossizes within first 15hydrogels min. In our previous study,of we foundAsthat the pore sizes 4, of these found that the pore of these are in the range of several hundred micrometers. swelling increases with time, first rapidly and then slowly, reaching a plateau. All the hydrogel hydrogels areto inGanji the range of several hundredpossessing micrometers. to Ganji et al. micrometers [51] the hydrogels According et al.more [51] the poreAccording sizes of min. several hundred formulations showed thanhydrogels 100% swelling ratios within first 15 In our previous study, we are possessing pore sizes of several hundred micrometers are classified as super-porous hydrogels (SPHs) classified as super-porous (SPHs) which as range a capillary system causing a rapid water found that the pore sizeshydrogels of these hydrogels are inactthe of several hundred micrometers. which act as a capillary system causing a rapid water uptake into the porous structure. Such According to Ganji et al. [51] the hydrogels possessing sizes of of several hundred are a fast uptake into the porous structure. Such a fast swellingpore is because absorption ofmicrometers water by capillary swelling is because of absorption of water by capillary than by simple classified as super-porous hydrogels (SPHs) which actforce as a rather capillary system causingabsorption. a rapid water force rather than by simple absorption. uptake into the porous structure. Such a fast swelling is because of absorption of water byancapillary After the swelling stage, theswelling swelling ratio increased slowly to reach equilibrium After therapid rapid swelling stage, the ratio increased slowly to reach an equilibrium state. force rather than by simple absorption. state. During the process of hydrogel swelling, against the favorable osmotic force, there is an During the process of hydrogel swelling, against the favorable osmotic force, there is an opposite After the rapid swelling stage, the swelling ratio increased slowly to reach an equilibrium state. elasticity force, which balances the stretching of the of network and prevents its deformation. At opposite elasticity force, which balances the stretching the network and prevents its deformation. During the process of hydrogel swelling, against the favorable osmotic force, there is an opposite forces areare balanced andand prevent additional swelling [51,52]. At equilibrium, equilibrium,elasticity elasticityand andosmotic osmotic forces balanced prevent additional swelling [51,52]. elasticity force, which balances the stretching of the network and prevents its deformation. At Referring to our previous study, allhydrogels the hydrogels showed highest swelling at medium acidic Referring to our previous study, all the highest swelling ratio [51,52]. atratio acidic equilibrium, elasticity and osmotic forces are balancedshowed and prevent additional swelling medium (pH 4.01). Results of the swelling test of this study showed that the hydrogels swelled more (pH 4.01). Referring Results oftothe swelling of this showed showed that the highest hydrogels swelled more in distilled our previoustest study, all study the hydrogels swelling ratio at acidic in distilled water than those in buffer solutions pH 7 and pHswelled 10.01). Annabi, water than those swelled in buffer (pH 4.01, pH showed 7(pH and4.01, pH Annabi, Mithieux, Weiss medium (pH 4.01). Results of swelled the solutions swelling test of this study that10.01). the hydrogels more Mithieux, Weiss and Dehghani [47] described that the reason for lowering of swelling ratio of elastin in distilled water than those in buffer solutions 4.01, pH and pH 10.01). Annabi, and Dehghani [47] described that swelled the reason for lowering of(pH swelling ratio7 of elastin hydrogels in buffer hydrogels in Weiss buffer solutions isofdue the presence of salt in buffer solutions resulting aofcontraction Mithieux, and Dehghani [47] that the reason for lowering of swelling ratio elastin due solutions is due to the presence salttodescribed in buffer solutions resulting in a contraction ofin the material of the material tosolutions water expulsion. hydrogels in due buffer is due to the presence of salt in buffer solutions resulting in a contraction to water expulsion. of the material due to water expulsion. 450 450

400

400

350

350 300

0% CNC-Chitosan

250

Swelling (%)

Swelling (%)

300 0% CNC-Chitosan

250

0.5% CNC-chitosan 0.5% CNC-chitosan

200

1.0% CNC-chitosan

200

1.0% CNC-chitosan

150

1.5% CNC-chitosan

150

1.5% CNC-chitosan

2% CNC-chitosan

100 100

2% CNC-chitosan

2.5% CNC-chitosan 2.5% CNC-chitosan

5050 00 00

200 200

400 400

600 600

800 800

1000 1000

Time Time (minute) (minute)

Figure4.4.Equilibrium Equilibrium swelling swelling ratio with varying CNC. Figure ratioof ofhydrogels hydrogels Figure 4. Equilibrium swelling ratio of hydrogelswith withvarying varyingCNC. CNC.


9 of 19 9 of 19

Out types hydrogels, 0.5% CNC–chitosan hydrogel showed highest equilibrium Out of of allall types of of hydrogels, thethe 0.5% CNC–chitosan hydrogel showed thethe highest equilibrium swelling ratio (438% ± 11%), followed by pure chitosan hydrogel (407% ± 13%) and swelling ratio (438% ± 11%), followed by pure chitosan hydrogel (407% ± 13%) and then thethen 1.0%the 1.0% CNC–chitosan hydrogel ± 14%). swelling is because of water molecules forming CNC–chitosan hydrogel (395% (395% ± 14%). InitialInitial swelling is because of water molecules forming hydrogen bonds with hydrophilic functional groups (amine (–NH ) and hydroxyl groups (–OH)) hydrogen bonds with hydrophilic functional groups (amine (–NH 2) 2 and hydroxyl groups (–OH)) present in the chitosan chains. More water molecules then orientate around the bound water to form present in the chitosan chains. More water molecules then orientate around the bound water to form cage like structures. Finally, excess water enters freely into hydrogel network resulting more cage like structures. Finally, excess water enters freely into thethe hydrogel network resulting in in more swelling [53]. All CNC–chitosan hydrogels were expected to show increased swelling ratios due to swelling [53]. All CNC–chitosan hydrogels were expected increased swelling ratios due tothe property of the CNC. However, our results revealed that the 0.5% hydrogel thehydrophilic hydrophilic property of the CNC. However, our results revealed that theCNC–chitosan 0.5% CNC–chitosan achieved the highest swelling ratio. Swelling decreased with further increasing hydrogel achieved the equilibrium highest equilibrium swelling ratio. ratio Swelling ratio decreased with further of CNC. Previous studies have studies described thatdescribed the decrease swelling occurred with increase of CNC increasing of CNC. Previous have thatofthe decrease of swelling occurred with in the hydrogel due the filling space by CNCs [54]. increase of CNCisin thetohydrogel is up dueoftothe thefree filling upof ofhydrogel the free space of hydrogel by CNCs [54]. The CNC–chitosan hydrogel was used CO gas foaming process as it showed The 0.50.5 CNC–chitosan hydrogel was used forfor thethe CO 2 gas foaming process as it showed thethe 2 highest swelling properties in previous swelling studies. As shown in Figure 5, equilibrium swelling highest swelling properties in previous swelling studies. As shown in Figure 5, equilibrium swelling ratio foamed hydrogel studied varying the2 pressure CO2 pressure gas foaming process ratio of of gasgas foamed hydrogel waswas studied withwith varying the CO of gasoffoaming process (10, (10, 30 and 50 bar). The experiments were repeated three times and average values were taken. 30 and 50 bar). The experiments were repeated three times and average values were taken. Compared Compared with the hydrogels formed at condition, atmospheric thehydrogels gas foamed hydrogels with the hydrogels formed at atmospheric thecondition, gas foamed swelled fasterswelled and faster and reached to the stage equilibrium stage in a of short period of time (around 350 min). reached to the equilibrium in a short period time (around 350 min). However, theHowever, maximumthe maximum of each hydrogel was with not varied with the processing Rapid is swelling swelling ratioswelling of eachratio hydrogel was not varied the processing pressure. pressure. Rapid swelling due due to the formation larger and interconnected pore network of gas foamed super porous hydrogel to is the formation of largerofand interconnected pore network of gas foamed super porous hydrogel (as (as shown in Figure 3). Open capillary channels in super porous hydrogels absorb water capillary shown in Figure 3). Open capillary channels in super porous hydrogels absorb water byby capillary force rather than simple absorption resulting faster swelling behavior [55]. force rather than byby simple absorption resulting inin faster swelling behavior [55]. 500

Swelling (%)

400

300 50 bar 30 bar 10 bar

200

1 bar

100

0 0

200

400

600

800

1000

Time (minute)

Figure 5. Equilibrium swelling ratio of hydrogels formed at different pressure conditions. Figure 5. Equilibrium swelling ratio of hydrogels formed at different pressure conditions.

3.5. Drug Encapsulation Efficiency 3.5. Drug Encapsulation Efficiency For the curcumin molecule, intramolecular hydrogen bond is the primary interaction which For the curcumin molecule, intramolecular hydrogen bond is the primary interaction which consist of two hydroxyl groups (–OH) in the benzene ring and the hydroxyl near keto group (C=O) consist of two hydroxyl groups (–OH) in the benzene ring and the hydroxyl near keto group (C=O) [56]. [56]. Probable interactions between curcumin and chitosan molecules are shown in Figure 6. The Probable interactions between curcumin and chitosan molecules are shown in Figure 6. The oxygen of oxygen of hydroxyl on the benzene ring is an important binding site for chitosan molecules. One hydroxyl on the benzene ring is an important binding site for chitosan molecules. One hydrogen bond hydrogen bond can be formed by free hydroxyl groups of glucosamine and the other can be formed can be formed by free hydroxyl groups of glucosamine and the other can be formed by the free amino by the free amino group of glucosamine. The modeling studies of Liu et al. [57] showed that the lower group of glucosamine. The modeling studies of Liu et al. [57] showed that the lower total energy of total energy of curcumin loaded chitosan compared to the total energy of individual molecules curcumin loaded chitosan compared to the total energy of individual molecules proved the possibility proved the possibility of these interactions in the drug loaded system. Thus, it is possible that an of these interactions in the drug loaded system. Thus, it is possible that an interaction will occur interaction will occur between drug and polymer molecules through hydrogen bonding. Results from between drug and polymer molecules through hydrogen bonding. Results from our previous study our previous study showed that the degree of cross linking of chitosan hydrogel was 83.6%. This showed that the degree of cross linking of chitosan hydrogel was 83.6%. This reveals that more amine reveals that more amine groups present in chitosan backbone are occupied by the crosslinking and formation of Schiff base. Therefore, it will limit the availability of free amine groups to bind with drug molecules.


10 of 19

groups present in chitosan backbone are occupied by the crosslinking and formation of Schiff base. Therefore, Polymers 2017,it9,will 64 limit the availability of free amine groups to bind with drug molecules. 10 of 19 H HO

O

O

O

H2N

O

H

O

H

H

O

HO

O

O

Curcumin

O

OH O

H

N

O

H

n n

Chitosan

Chitosan

Figure molecules. Figure 6. 6. Probable Probable interactions interactions between between curcumin curcumin and and chitosan chitosan molecules.

From the data tabulated in Table 1, the maximum drug loading efficiency occurred for the 0.5% From the data tabulated in Table 1, the maximum drug loading efficiency occurred for the CNC–chitosan hydrogel. This result was followed by chitosan hydrogel, the 1% CNC–chitosan 0.5% CNC–chitosan hydrogel. This result was followed by chitosan hydrogel, the 1% CNC–chitosan hydrogel, and so on. This observed trend was similar to the swelling test pattern of the hydrogels as hydrogel, and so on. This observed trend was similar to the swelling test pattern of the hydrogels as indicated in Figure 4. When the hydrogel swelling ratio is high, a large amount of drug solution can indicated in Figure 4. When the hydrogel swelling ratio is high, a large amount of drug solution can be be absorbed and retained in the hydrogel network, leading to an incremental increase in the drug absorbed and retained in the hydrogel network, leading to an incremental increase in the drug loading loading efficiency. Therefore, we can conclude that the efficiency of drug loading in the hydrogel is efficiency. Therefore, we can conclude that the efficiency of drug loading in the hydrogel is dependent dependent upon the swelling of the hydrogel in water. upon the swelling of the hydrogel in water. Table 1. Encapsulation efficiency of curcumin for different hydrogels. Table 1. Encapsulation efficiency of curcumin for different hydrogels. Hydrogel 0% Hydrogel CNC–chitosan 0.5% CNC–chitosan 0% CNC–chitosan 1% CNC–chitosan 0.5% 1.5% CNC–chitosan 1% CNC–chitosan 1.5% 2% CNC–chitosan 2% CNC–chitosan 2.5% CNC–chitosan 2.5% CNC–chitosan

Encapsulation efficiency (%) Encapsulation 37 ± 0.8Efficiency (%) 4137 ± 2.4 ± 0.8 3641 ± 1.8 ± 2.4 3436 ± 0.5 ± 1.8 ± 0.5 3334 ± 1.9 ± 1.9 3033 ± 2.8 30 ± 2.8

As more CNC was added into the chitosan network, the voids in the chitosan network are gradually filled by CNC, forming a rigid structure and creating a barrier that would network prevent the As more CNC was added into the chitosan network, the voids in the chitosan are penetration of drug in to thea hydrogel, which and would cause aa decrease in the drug prevent loading gradually filled by solution CNC, forming rigid structure creating barrier that would efficiency. the penetration of drug solution in to the hydrogel, which would cause a decrease in the drug loading efficiency. 3.6. In Vitro Drug Release 3.6. In Vitro Drug Release The objective of this study is to improve the bioavailability of curcumin to increase absorption objective ofin this study is tomanner, improvethereby the bioavailability of curcumin to increase absorption fromThe acidic medium a controlled avoiding wastage of the drug and achieving the from acidic medium in a controlled manner, thereby avoiding wastage of the drug and achieving the desired treatment effect. As shown in Figure 7, the drug release pattern for all hydrogels investigated desired treatment shown Figure 7, the drug release pattern fordiffusion-controlled all hydrogels investigated can be divided in effect. to twoAs phases: anininitial burst release and a prolonged phase can beRapid divided in to two phases: an initial release andbe a prolonged phase [58]. drug release in phase one ofburst the test could due to thediffusion-controlled presence of the drug on [58]. the Rapid drug release in phase one of the test could be due to the presence of the drug on the surface of surface of the hydrogel and the higher drug concentration gradient present at the beginning of the hydrogel theconcentration higher drug concentration gradient present at the beginning the release test. This higher test. This and higher gradient could act as the driving force for the of drug from the concentration gradient could act as the driving force for the drug release from the hydrogel matrix. hydrogel matrix. After the burst release, the releasing rates decline steadily with time, which may be After release, decline steadily with time, which may be due to the thickness due tothe theburst thickness ofthe thereleasing hydrogelrates acting as a diffusion barrier [59]. of the hydrogel acting as a diffusion barrier [59].


11 of 19 11 of 19

Curcumin release (mg/L)

0.8

0.6

0.4

0% CNC-chitosan 0.5% CNC-chitosan 1% CNC-chitosan 1.5% CNC-chitosan 2% CNC-chitosan 2.5% CNC-chitosan

0.2

0.0 0

50

100

150

200

250

300

350

Time (min)

Figure 7. Curcumin release from different types of hydrogels. Figure 7. Curcumin release from different types of hydrogels.

In general, the rate of drug release from hydrogel matrix is depend on the interaction between In general, the rate of drug release from hydrogel matrix is depend on the interaction between the drug and the polymer molecules, the solubility of the drug, and hydrogel swelling in aqueous the drug and the polymer molecules, the solubility of the drug, and hydrogel swelling in aqueous media [60]. However, in this study, there is no indication for the presence of interaction between media [60]. However, in this study, there is no indication for the presence of interaction between hydrogel and drug. Therefore, the rate of drug release in this test depended only on the solubility of hydrogel and drug. Therefore, the rate of drug release in this test depended only on the solubility of the the drug and the swelling ratio of the hydrogel in aqueous media. It is clear that the drug release drug and the swelling ratio of the hydrogel in aqueous media. It is clear that the drug release profiles profiles of hydrogels are consistent with the data obtained from the swelling studies. The hydrogel of hydrogels are consistent with the data obtained from the swelling studies. The hydrogel with the with the higher swelling ratio achieved higher drug release percentages. In this study, the 0.5% CNC– higher swelling ratio achieved higher drug release percentages. In this study, the 0.5% CNC–chitosan chitosan hydrogel had the highest drug release percentage, whereas the 2.5% CNC–chitosan hydrogel hydrogel had the highest drug release percentage, whereas the 2.5% CNC–chitosan hydrogel produced produced the lowest drug release percentage. A lower concentration of curcumin release from these the lowest drug release percentage. A lower concentration of curcumin release from these hydrogels hydrogels is attributable to the fact that curcumin has very poor solubility in water. It has been is attributable to the fact that curcumin has very poor solubility in water. It has been reported that reported that curcumin has a solubility of around 2.67 µg/mL at pH 7.3 [61]. curcumin has a solubility of around 2.67 µg/mL at pH 7.3 [61]. It is reported that the average gastric emptying times for healthy individuals at 1, 2, and 4 h are It is reported that the average gastric emptying times for healthy individuals at 1, 2, and 4 h are >90%, 60% and 10% respectively [62]. Our results showed that all the hydrogels reached to a >90%, 60% and 10% respectively [62]. Our results showed that all the hydrogels reached to a prolonged prolonged diffusion-controlled phase around 120 min. According to the drug release pattern of diffusion-controlled phase around 120 min. According to the drug release pattern of hydrogels, this hydrogels, this drug delivery system can be suggested to improve the bioavailability of curcumin for drug delivery system can be suggested to improve the bioavailability of curcumin for the absorption the absorption from acidic medium during gastric residential time. from acidic medium during gastric residential time. 3.7. Drug Release Kinetics 3.7. Drug Release Kinetics Curcumin release kinetics was investigated using Ritger–Peppas model (Equation (4)). Curcumin release kinetics was investigated using Ritger–Peppas model (Equation (4)). FD =

Mt = Kt n M ∞ Mt

(4) n

= Kt (4) M∞ where Mt/M∞ is the ratio of curcumin release at time t to the equilibrium swollen state. K is the kinetic where M /M∞ is the ratio ofofcurcumin release at time t parameters to the equilibrium swollen state. K is the kinetic constant totmeasure velocity release and geometrical corresponding to drug–polymer constant measure velocity of release andtogeometrical parameters corresponding to drug–polymer system. n istothe diffusion exponent related transport mechanism. If n < 0.5, it indicates Fickian system. or n drug is therelease diffusion related to transport mechanism. If n < 0.5, it the indicates Fickian diffusion, thatexponent is diffusion-controlled and penetration of solvent into hydrogel is diffusion, or drug release that is diffusion-controlled and penetration of solvent into the hydrogel much faster than the polymer chain relaxation. When 0.5 < n < 1, diffusion and release of drug occur faster than the polymer chain relaxation. When 0.5release < n < 1,followed diffusionboth anddiffusion release ofand drug in is a much non-Fickian (anomalous) manner. This means that drug occur in a non-Fickian (anomalous) manner. This means that drug release followed both diffusion erosion controlled mechanisms [63]. If n = 1, it indicates case II transport, where the release rate is and erosion controlledby mechanisms [63]. If n =The 1, it values indicates transport, thestudy releaseare rate constant and controlled polymer relaxation. of case n, k II and R2 of thewhere present 2 of the present study are is constant and controlled polymer The values of n,released k and Rin shown in Table 2. The n valuesbyrange fromrelaxation. 0.61–0.76 when curcumin SGF and drug release shown in non-Fickian Table 2. The (anomalous) n values range from 0.61–0.76 curcumin insolvent SGF and drug release occurred manner [64]. If nwhen is close to unity,released it means penetration occurredby in relaxation non-Fickian manner If diffusion n is close [65]. to unity, it means solvent penetration controlled of (anomalous) polymer chains rather[64]. than It appears that n value for 0% controlled byhydrogel relaxation of polymer chains thanwith diffusion It appears that be n value CNC–chitosan is closer to unity whenrather compared other [65]. hydrogels. This may due tofor FD =

the fact that the 0% CNC–chitosan sample does not contain cellulose nanocrystals within the chitosan matrix. As the hydrogel does not contain the cellulose nanocrystals, there may be some relaxation of


12 of 19

0% CNC–chitosan hydrogel is closer to unity when compared with other hydrogels. This may be due to the fact that the 0% CNC–chitosan sample does not contain cellulose nanocrystals within the chitosan matrix. As the hydrogel does not contain the cellulose nanocrystals, there may be some relaxation of chitosan segments within the matrix. In this way, the curcumin release is not totally diffusion controlled but it is partly controlled by chain relaxation process also. On the other hand, there may probably be physical crosslinks between the surface hydroxyls of cellulose crystals and –OH groups of chitosan molecules. The presence of H-bonding interactions between chitosan and cellulose restricts the relaxation of chitosan chains. Therefore, the n values for CNC reinforced chitosan hydrogels is lower than the n value of 0% CNC–chitosan hydrogel and drug release depends more on the diffusion rather than relaxation of polymer chains. Furthermore, a significant weight loss was not observed for the hydrogels when immersed in the SGF for 12 h. It may be due to the high degree of cross linking (83.6%) of these hydrogels. This will further clarify that drug release is driven by diffusion driven, rather than erosion of the hydrogel. Table 2. Release kinetics of hydrogel in SGF. Hydrogel

n

R2

K

0% CNC–chitosan 0.5% CNC–chitosan 1% CNC–chitosan 1.5% CNC–chitosan 2% CNC–chitosan 2.5% CNC–chitosan

0.76 0.61 0.68 0.70 0.67 0.66

0.98 0.98 0.98 0.99 0.96 0.96

0.46 0.52 0.60 0.57 0.57 0.57

The difference in k values indicates the difference in physical properties of hydrogel and difference in drug polymer interaction. In addition, the k values are smaller due to the less interaction between drug and polymers. In this study, Ritger–Peppas model was used to analyze the drug release kinetics. By using this model, regression coefficient (R2 ) was found very close to unity, and it suggested the release data best fit to Ritger–Peppas model. The release of the drug is usually controlled by diffusion and so the release profile can be easily modified by changing the material properties such as pore size or the overall pore surface area, pore connectivity and pore geometry [66]. Larger pore size is suitable to load a high dose of drug molecules [67]. Due to the large pore size of hydrogels, a rapid initial burst release is typically observed. In this study, large-scale macroporous hydrogel with wide interconnected pores and large accessible surface area was obtained with using carbon dioxide gas foaming process. The 0.5% CNC–chitosan hydrogel was used for the gas foaming as it showed the highest swelling ratio and maximum drug release in the previous experiments. As shown in Figure 3, the pore size of hydrogel increased by more than tenfold after the gas foaming process. The increased pore size and pore interconnectivity act as a capillary system causing a rapid diffusion of drug solution through the hydrogel matrix. The drug encapsulation efficiency increased from 41% to 50% with the gas foaming of 0.5% CNC–chitosan hydrogel at 50 bar (as shown in Table 3). In addition, a rapid release of drug and high amount of drug release was observed in the gas foamed hydrogels as were typical of diffusion- controlled systems. As shown in Figure 8, the drug release was increased to 1.06 mg/L in the gas foamed 0.5% CNC–chitosan hydrogel at 50 bar, when compared to the drug release (0.74 mg/L) of 0.5% CNC–chitosan hydrogel formed at atmospheric condition.


13 of 19

Table 3. Encapsulation efficiency of curcumin for hydrogels formed at different pressure conditions. Pressure of Hydrogel Formation

Encapsulation Efficiency (%)

1 bar 10 bar 30 bar 50 bar

41 ± 2.4 43 ± 0.7 45 ± 0.9 50 ± 2.0


13 of 19

1.2

Curcumin release (mg/L)

1.0

0.8

0.6 1 bar

0.4

10 bar 30 bar 50 bar

0.2

0.0 0

50

100

150

200

250

300

350

Time (min)

Figure 8. Curcumin release from hydrogel formed at different pressure conditions. Figure 8. Curcumin release from hydrogel formed at different pressure conditions.

The basic steps inin the gas foaming process are: (1)(1) plasticization due toto CO into 2 diffusion Thethree three basic steps the gas foaming process are: plasticization due CO 2 diffusion into the polymer matrix at high pressure; (2) nucleation due to supersaturated gas and depressurization; the polymer matrix at high pressure; (2) nucleation due to supersaturated gas and depressurization; and bubbles toto the gas diffusion from the surrounding polymer [68]. and(3)(3)gas gas bubblesgrowth growthdue due the gas diffusion from the surrounding polymer [68].Skin Skinlayer layer formation and poor pore interconnectivity are common issues in porous fabrication technique. formation and poor pore interconnectivity are common issues in porous fabrication technique. However, these cancan be overcome by fabrication of polymer matrices using gas foaming method [69,70]. However, these be overcome by fabrication of polymer matrices using gas foaming method By[69,70]. tuning By the process conditions such as operating pressure, depressurization rate and temperature, tuning the process conditions such as operating pressure, depressurization rate the and final structure thefinal product can be of modified as required. results of previousThe studies showed temperature,ofthe structure the product can beThe modified asthe required. results of the that the solubility CO2 dramatically increases as the pressure isincreases increasedas(0.077 mol CO2is/kg H2 O previous studiesof showed that the solubility of CO 2 dramatically the pressure increased solubility at 1 bar increases to around 30-fold at 50 bar). On the other hand, the solubility of CO 2 is (0.077 mol CO2/kg H2O solubility at 1 bar increases to around 30-fold at 50 bar). On the other hand, decreased by increasing the temperature at each pressure [46]. the solubility of CO2 is decreased by increasing the temperature at each pressure [46]. InIn this study, wewe fabricated the the 0.5% CNC–chitosan hydrogel usingusing high high pressure CO2 atCO 10,2 at 30 10, and30 this study, fabricated 0.5% CNC–chitosan hydrogel pressure 50and bar in room temperature for two days. The operating temperature was not increased (maintained at 50 bar in room temperature for two days. The operating temperature was not increased room temperature) due to the liquefaction of hydrogel at elevated temperatures. Slow depressurization (maintained at room temperature) due to the liquefaction of hydrogel at elevated temperatures. Slow (1depressurization bar/min) was used as it helps to obtain poretosizes in the hydrogel matrix. (1 bar/min) was used aslarge it helps obtain large pore sizes in the hydrogel matrix. 3.8. Drug Activity 3.8. Drug Activity After Afterreleasing, releasing,the thechemical chemicalreactivity reactivityand andbiological biologicalactivity activityofofthe thedrug drugare arethe themost mostcritical critical parameters inin drug delivery systems [71]. drug delivery systems, drugs deteriorate due toto parameters drug delivery systems [71].For Forsome some drug delivery systems, drugs deteriorate due the denaturation reactions with carrier and cause some detrimental effects after release. In order to the denaturation reactions with carrier and cause some detrimental effects after release. In order to prevent this thethe carrier should not interact with the drug able to be delivered into the body prevent this carrier should not interact with theand drug and able to be delivered into without the body any chemical transformation. In this study, the activity of curcumin before loading and after and release without any chemical transformation. In this study, the activity of curcumin before loading after was observed by comparison of UV visible spectra. The spectra of pure curcumin and in the release release was observed by comparison of UV visible spectra. The spectra of pure curcumin and in the medium were recorded. As shownAs in shown Figure 9, significant differencedifference was not observed both release (SGF) medium (SGF) were recorded. in aFigure 9, a significant was not for observed spectra. This shows that the drug retained its chemical structure after release from the hydrogel. for both spectra. This shows that the drug retained its chemical structure after release from the

hydrogel.


14 of 19 14 of 19 Pure drug

0.10

Released drug

0.09 0.08

Absorbance

0.07 0.06 0.05 0.04 0.03 0.02 0.01 400

450

500

550

600

650

Wave length (nm)

Figure9.9.Chemical Chemicalactivity activityofofpure purecurcumin curcuminand andcurcumin curcuminafter afterrelease. release. Figure

3.9. In Vitro Degradation 3.9. In Vitro Degradation For oral drug administration, it is important to study the degradation of hydrogel in conditions For oral drug administration, it is important to study the degradation of hydrogel in conditions similar to living cells over long period of time. Degradation studies were carried out in PBS buffer at similar to living cells over long period of time. Degradation studies were carried out in PBS buffer 37 °C for six weeks. The results of this study are presented in Figure 10. The hydrogels showed at 37 ◦ C for six weeks. The results of this study are presented in Figure 10. The hydrogels showed degradation due to the presence of −OH and −NH2 groups in the chitosan backbone. These groups degradation due to the presence of −OH and −NH2 groups in the chitosan backbone. These groups have the ability to interact with water. The hydrogel matrix became loose due to swelling property. have the ability to interact with water. The hydrogel matrix became loose due to swelling property. Gels with low CNC content had greater ability to absorb the solvent and high swelling ability due to Gels with low CNC content had greater ability to absorb the solvent and high swelling ability due the presence of more void space in the hydrogel and loose network [71]. On the other hand, there to the presence of more void space in the hydrogel and loose network [71]. On the other hand, there may be physical crosslinks between the surface hydroxyls of cellulose crystals and –OH groups of may be physical crosslinks between the surface hydroxyls of cellulose crystals and –OH groups of chitosan molecules. The presence of H-bonding interactions between chitosan and cellulose restricts chitosan molecules. The presence of H-bonding interactions between chitosan and cellulose restricts the relaxation of chitosan segments within the matrix. Besides that, the crystallinity is an important the relaxation of chitosan segments within the matrix. Besides that, the crystallinity is an important factor that influences the degradation of hydrogel due to the different chain packing arrangements factor that influences the degradation of hydrogel due to the different chain packing arrangements of each polymer. Amorphous structure allows more water to penetrate in to its polymer matrix of each polymer. Amorphous structure allows more water to penetrate in to its polymer matrix resulting faster degradation rates. In the crystalline regions, polymer chains are closely packed and resulting faster degradation rates. In the crystalline regions, polymer chains are closely packed and resist the penetration of water molecules within the regions. CNCs are high crystalline particles with resist the penetration of water molecules within the regions. CNCs are high crystalline particles with highly ordered and closely packed chains. The X-ray diffraction patters of our previous study also highly ordered and closely packed chains. The X-ray diffraction patters of our previous study also indicated that the addition of CNCs will induce a combination of crystalline and amorphous regions indicated that the addition of CNCs will induce a combination of crystalline and amorphous regions in the nanocelulose reinforced chitosan hydrogel. Several studies have also demonstrated that a small in the nanocelulose reinforced chitosan hydrogel. Several studies have also demonstrated that a amount of CNC could lead to a remarkable decrease in the degradation rate of the composite matrix small amount of CNC could lead to a remarkable decrease in the degradation rate of the composite [72,73]. According to the results, 2.5% CNC–chitosan showed 69% weight loss as it contained high matrix [72,73]. According to the results, 2.5% CNC–chitosan showed 69% weight loss as it contained amount of CNCs, while chitosan hydrogel lost its 84% weight because it does not contain CNCs. high amount of CNCs, while chitosan hydrogel lost its 84% weight because it does not contain CNCs.


15 of 19 15 of 19 100 90 80

Weight loss (%)

70 60 50 0% CNC-chitosan

40

0.5% CNC-chitosan 1% CNC-chitosan

30

1.5% CNC-chitosan

20

2% CNC-chitosan 2.5% CNC-chitosan

10 0 0

1

2

3

4

5

6

7

Degradation time (week)

Figure Figure 10. 10. In In vitro vitrodegradation degradationstudy studyofofthe thehydrogels. hydrogels.

4. Conclusions 4. Conclusions In this study, CNC reinforced chitosan hydrogels were synthesized using chemical crosslinking In this study, CNC reinforced chitosan hydrogels were synthesized using chemical crosslinking method. It was observed that compression strength of chitosan hydrogel increased from 25.9 ± 1 kPa method. It was observed that compression strength of chitosan hydrogel increased from 25.9 ± 1 kPa to 38.4 ± 1 kPa with the introduction of 0.5% cellulose nanocrystals. Maximum compression of to 38.4 ± decreased 1 kPa with theincreasing introduction of 0.5% of cellulose nanocrystals. Maximum compression hydrogel with the pressure gas foaming process due to the formation of large of hydrogel with increasing thestructures. pressure of gas foaming thepores formation pores anddecreased widely interconnected pore FESEM images process showed due thattothe of the of large pores and widely interconnected pore structures. FESEM images showed that the pores of the hydrogel are around hundred micrometers and it increased more than tenfold after the gas foaming hydrogel are50around hundred micrometers and it increased more tenfold after and the gas foaming process (at bar). Further, it showed a highly interconnected open than 3-D pore network rough cell process (at 50 bar). Further, it showed a highly interconnected open 3-D pore network and rough wall structures. Maximum swelling ratio was obtained for 0.5% CNC–chitosan hydrogel and it cell wall structures. Maximum swelling ratioThis wasmay obtained forto0.5% hydrogel and decreased with increasing the CNC content. be due the CNC–chitosan filling up the free space of ithydrogel decreased with increasing the CNC content. This may be duehydrogels to the filling up the freethan space with CNCs as indicated by previous studies. Gas foamed swelled faster the of hydrogel CNCs asatindicated by previous studies. Gas revealed foamed hydrogels faster than the hydrogelswith prepared atmospheric condition. Results that drugswelled loading efficiency hydrogels at atmospheric condition. Results revealed thatHigher drug loading efficiency decreased decreasedprepared with increasing the percentage of CNC in the hydrogel. drug loading ability was with increasing percentage ofwhen CNCcompared in the hydrogel. Higher drugatloading ability was observed observed in gas the foamed hydrogel to hydrogel formed atmospheric condition. The in gasencapsulation foamed hydrogel whenofcompared hydrogelatformed at atmospheric drug drug efficiency gas foamedtohydrogels 10, 30 and 50 bar is 43%condition. ± 0.7%, 45%The ± 0.9% encapsulation efficiency of gasand foamed hydrogels and 50 bar is 43% ± 0.7%, 45% condition. ± 0.9% and and 50% ± 2.0%, respectively, it is 41% ± 2.4% at for10, the30hydrogel formed at atmospheric The ± in vitro release studies were±investigated in hydrogel SGF. The drug release profiles of hydrogels 50% 2.0%,curcumin respectively, and it is 41% 2.4% for the formed at atmospheric condition. were with the data studies obtainedwere frominvestigated the swellinginstudies. Highest release wasofindicated The inconsistent vitro curcumin release SGF. The drug drug release profiles hydrogels by 0.5% CNC–chitosan hydrogel (0.74 ± 0.03 mg/L) and it was lowest in 2.5% CNC–chitosan hydrogel were consistent with the data obtained from the swelling studies. Highest drug release was indicated (0.54 ± 0.04 mg/L). Thehydrogel kinetic parameters that the drug diffusion through the hydrogel by 0.5% CNC–chitosan (0.74 ± 0.03indicated mg/L) and it was lowest in 2.5% CNC–chitosan hydrogel were±in0.04 non-Fickian (anomalous) manner. The amount of drug increased through and faster release of (0.54 mg/L). The kinetic parameters indicated that therelease drug diffusion the hydrogel drug was observed in the gas foamed hydrogel compared to the hydrogel formed at atmospheric were in non-Fickian (anomalous) manner. The amount of drug release increased and faster release condition. itsfoamed structural integrity after release, is critical requirement for of drug wasCurcumin observed retained in the gas hydrogel compared to thewhich hydrogel formed at atmospheric preservingCurcumin drug activity. In vitro degradation of hydrogels was found dependent the swellingfor condition. retained its structural integrity after release, which is criticalonrequirement ratio and the amount of CNC of the hydrogel. Because the pH sensitive CNC-reinforced preserving drug activity. In vitro degradation of hydrogels was found dependent ongas thefoamed swelling hydrogel exhibited efficient drug carrier properties, it can be suggested as promising candidate for ratio and the amount of CNC of the hydrogel. Because the pH sensitive CNC-reinforced gas foamed stomach specific drug delivery. hydrogel exhibited efficient drug carrier properties, it can be suggested as promising candidate for

stomach specific drug delivery.

Acknowledgments: The authors would like to acknowledge the financial support from the Ministry of Education Malaysia (FP030-2013A and FP053-2015A), and University Malaya research grant (RU022A-2014, Acknowledgments: The authors would like to acknowledge the financial support from the Ministry of Education RP011A-13AET, PG160-2016A and RU018I-2016) for the success of this project. Malaysia (FP030-2013A and FP053-2015A), and University Malaya research grant (RU022A-2014, RP011A-13AET, PG160-2016A and RU018I-2016) for the success of this project. Author Contributions: Thennakoon M. Sampath Udeni Gunathilake wrote the paper; Thennakoon M. Sampath Author Contributions: M. Sampath Udeni Gunathilake wrote Thennakoon M. Sampath Udeni Gunathilake and Thennakoon Yern Chee Ching initiated and contributed to the scopethe of paper; the manuscript; and Yern Chee Udeni Yern Cheecritically Ching initiated andthe contributed to the scope of the manuscript; and Yern Chee Ching ChingGunathilake and Cheng and Hock Chuah reviewed manuscript. and Cheng Hock Chuah critically reviewed the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest.


16 of 19

References 1. 2.

3.

4. 5. 6. 7. 8.

9. 10.

11. 12.

13. 14. 15. 16. 17. 18. 19. 20.

21. 22.

Mahkam, M. New pH-sensitive glycopolymers for colon-specific drug delivery. Drug Deliv. 2007, 14, 147–153. [CrossRef] [PubMed] Shen, X.; Shamshina, J.L.; Berton, P.; Bandomir, J.; Wang, H.; Gurau, G.; Rogers, R.D. Comparison of hydrogels prepared with ionic-liquid-isolated vs commercial chitin and cellulose. ACS Sustain. Chem. Eng. 2015, 4, 471–480. [CrossRef] Rubentheren, V.; Ward, T.A.; Chee, C.Y.; Tang, C.K. Processing and analysis of chitosan nanocomposites reinforced with chitin whiskers and tannic acid as a crosslinker. Carbohydr. Polym. 2015, 115, 379–387. [CrossRef] [PubMed] Szymanska, ´ E.; Winnicka, K. Stability of chitosan—A challenge for pharmaceutical and biomedical applications. Mar. Drugs 2015, 13, 1819–1846. [CrossRef] [PubMed] Gunathilake, T.M.S.U.; Ching, Y.C.; Chuah, C.H. Synthesis and characterization of nanocellulose reinforced semi-interpenetrating polymer network of chitosan hydrogel. Cellulose (under review). Dubey, J.; Verma, A.; Verma, N. Evaluation of chitosan based polymeric matrices for sustained stomach specific delivery of propranolol hydrochloride. Indian J. Mater. Sci. 2015, 2015, 1–5. [CrossRef] [PubMed] Majithiya, R.J.; Murthy, R.S. Chitosan-based mucoadhesive microspheres of clarithromycin as a delivery system for antibiotic to stomach. Curr. Drug Deliv. 2005, 2, 235–242. [CrossRef] [PubMed] Patel, V.R.; Amiji, M.M. Preparation and characterization of freeze-dried chitosan-poly(ethylene oxide) hydrogels for site-specific antibiotic delivery in the stomach. Pharm. Res. 1996, 13, 588–593. [CrossRef] [PubMed] Hejazi, R.; Amiji, M. Stomach-specific anti-H. pylori therapy. I: Preparation and characterization of tetracyline-loaded chitosan microspheres. Int. J. Pharm. 2002, 235, 87–94. [CrossRef] Ali, M.S.; Pandit, V.; Jain, M.; Dhar, K.L. Mucoadhesive microparticulate drug delivery system of curcumin against Helicobacter pylori infection: Design, development and optimization. J. Adv. Pharm. Technol. Res. 2014, 5, 48–56. [CrossRef] [PubMed] Thakur, A.; Monga, S.; Wanchoo, R. Sorption and drug release studies from semi-interpenetrating polymer networks of chitosan and xanthan gum. Chem. Biochem. Eng. Q. 2014, 28, 105–115. Wen, Y.; Li, F.; Li, C.; Yin, Y.; Li, J. High mechanical strength chitosan-based hydrogels cross-linked with poly(ethylene glycol)/polycaprolactone micelles for the controlled release of drugs/growth factors. J. Mater. Chem. B 2017, 5, 961–971. [CrossRef] Amin, S.; Rajabnezhad, S.; Kohli, K. Hydrogels as potential drug delivery systems. Sci. Res. Essays 2009, 4, 1175–1183. Gupta, G.; Singh, A. A short review on stomach specific drug delivery system. Int. J. PharmTech Res. 2012, 4, 1527–1545. Mastropietro, D.J.; Omidian, H.; Park, K. Drug delivery applications for superporous hydrogels. Expert Opin. Drug Deliv. 2012, 9, 71–89. [CrossRef] [PubMed] Chavda, H.; Patel, C. Effect of crosslinker concentration on characteristics of superporous hydrogel. Int. J. Pharm. Investig. 2011, 1, 17–21. [CrossRef] [PubMed] Chang, C.W.; van Spreeuwel, A.; Zhang, C.; Varghese, S. PEG/clay nanocomposite hydrogel: A mechanically robust tissue engineering scaffold. Soft Matter 2010, 6, 5157–5164. [CrossRef] Park, M.; Lee, D.; Hyun, J. Nanocellulose-alginate hydrogel for cell encapsulation. Carbohydr. Polym. 2015, 116, 223–228. [CrossRef] [PubMed] Mohd, A.C.M.; Ching, Y.C.; Luqman, C.A.; Poh, S.C.; Chuah, C.H. Review of bionanocomposite coating films and their applications. Polymers 2016, 8, 246–253. Ching, Y.C.; Ershad, A.; Luqman, C.A.; Choo, K.W.; Yong, C.K.; Sabariah, J.J.; Chuah, C.H.; Liou, N.S. Rheological properties of cellulose nanocrystal-embedded polymer composites: A review. Cellulose 2016, 23, 1011–1030. [CrossRef] Choo, K.W.; Ching, Y.C.; Chuah, C.H.; Sabariah, J.; Liou, N.S. Preparation and characterization of polyvinyl alcohol-chitosan composite films reinforced with cellulose nanofiber. Materials 2016, 9, 644–652. [CrossRef] Yang, J.; Han, C.R.; Duan, J.F.; Ma, M.G.; Zhang, X.M.; Xu, F.; Sun, R.C.; Xie, X.M. Studies on the properties and formation mechanism of flexible nanocomposite hydrogels from cellulose nanocrystals and poly(acrylic acid). J. Mater. Chem. 2012, 22, 22467–22480. [CrossRef]


23.

24. 25. 26.

27. 28. 29. 30. 31. 32. 33.

34. 35. 36.

37.

38.

39.

40. 41.

42.

43.

17 of 19

Yang, X.; Bakaic, E.; Hoare, T.; Cranston, E.D. Injectable polysaccharide hydrogels reinforced with cellulose nanocrystals: Morphology, rheology, degradation, and cytotoxicity. Biomacromolecules 2013, 14, 4447–4455. [CrossRef] [PubMed] Ahmed, E.M. Hydrogel: Preparation, characterization, and applications: A review. J. Adv. Res. 2015, 6, 105–121. [CrossRef] [PubMed] Das, N. Preparation methods and properties of hydrogel: A review. Int. J. Pharm. Pharm. Sci. 2013, 5, 112–117. Prasad, S.; Tyagi, A.K.; Aggarwal, B.B. Recent developments in delivery, bioavailability, absorption and metabolism of curcumin: The golden pigment from golden spice. Cancer Res. Treat. 2014, 46, 2–18. [CrossRef] [PubMed] Mahattanadul, S. Floating gellan gum-based in situ gels containing curcumin for specific delivery to the stomach. Thai J. Pharm. Sci. 2016, 40, 33–36. Ridhima, D.; Shweta, P.; Upendra, K. Formulation and evaluation of floating microspheres of curcumin. Int. J. Pharm. Pharm. Sci 2012, 4, 334–336. Treesinchai, S.; Puttipipatkhachorn, S.; Pitaksuteepong, T.; Sungthongjeen, S. Development of curcumin floating tablets based on low density foam powder. Asian J. Pharm. Sci. 2016, 11, 130–131. [CrossRef] Shivashankar, M.; Mandal, B.K. A review on interpenetrating polymer network. Int. J. Phram. Phram. Sci 2012, 4, 1–7. Guo, D.; Xie, G.; Luo, J. Mechanical properties of nanoparticles: Basics and applications. J. Phys. D 2013, 47, 1–13. [CrossRef] Han, K.S.; Song, J.E.; Tripathy, N.; Kim, H.; Moon, B.M.; Park, C.H.; Khang, G. Effect of pore sizes of silk scaffolds for cartilage tissue engineering. Macromol. Res. 2015, 23, 1091–1097. [CrossRef] Chiu, Y.C.; Kocagöz, S.; Larson, J.C.; Brey, E.M. Evaluation of physical and mechanical properties of porous poly(ethylene glycol)-co-(L-lactic acid) hydrogels during degradation. PLoS ONE 2013, 8, 607–628. [CrossRef] [PubMed] Wang, H.; Roman, M. Formation and properties of chitosan-cellulose nanocrystal polyelectrolyte—Macroion complexes for drug delivery applications. Biomacromolecules 2011, 12, 1585–1593. [CrossRef] [PubMed] Akhlaghi, S.P.; Berry, R.C.; Tam, K.C. Surface modification of cellulose nanocrystal with chitosan oligosaccharide for drug delivery applications. Cellulose 2013, 20, 1747–1764. [CrossRef] Ng, T.S.; Ching, Y.C.; Awanis, N.; Ishenny, N.; Rahman, M.R. Effect of bleaching condition on thermal properties and UV-transmittance of PVA/cellulose biocomposites. Mater. Res. Innov. 2014, 18, 400–404. [CrossRef] Goh, K.Y.; Ching, Y.C.; Chuah, C.H.; Luqman, C.A.; Liou, N.S. Individualization of microfibrillated celluloses from oil palm empty fruit bunch: Comparative studies between acid hydrolysis and ammonium persulfate oxidation. Cellulose 2016, 23, 379–390. [CrossRef] Ching, Y.C.; Rahman, A.; Ching, K.Y.; Sukiman, N.L.; Cheng, H.C. Preparation and characterization of polyvinyl alcohol-based composite reinforced with nanocellulose and nanosilica. BioResources 2015, 10, 3364–3377. [CrossRef] Rubentheren, V.; Thomas, A.W.; Ching, Y.C.; Praveena, N.; Erfan, S.; Christopher, F. Effects of heat treatment on chitosan nanocomposite film reinforced with nanocrystalline cellulose and tannic acid. Carbohydr. Polym. 2016, 140, 202–208. [CrossRef] [PubMed] Rubentheren, V.; Ward, T.A.; Chee, C.Y.; Nair, P. Physical and chemical reinforcement of chitosan film using nanocrystalline cellulose and tannic acid. Cellulose 2015, 22, 2529–2541. [CrossRef] Mak, A.F.T.; Sun, S. Chapter 18 intelligent chitosan-based hydrogels as multifunctional materials. In Intelligent Materials, 1st ed.; Hilgers, P., Riechers, A., König, B., Eds.; The Royal Society of Chemistry: Cambridge, UK, 2008; Volume 47, pp. 447–463. Giri, T.K.; Thakur, A.; Alexander, A.; Badwaik, H.; Tripathi, D.K. Modified chitosan hydrogels as drug delivery and tissue engineering systems: Present status and applications. Acta Pharm. Sin. B 2012, 2, 439–449. [CrossRef] Berger, J.; Reist, M.; Mayer, J.M.; Felt, O.; Peppas, N.; Gurny, R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. Eur. J. Pharm. Biopharm. 2004, 57, 19–34. [CrossRef]


44. 45.

46. 47. 48.

49.

50. 51. 52.

53. 54.

55. 56.

57. 58. 59. 60. 61. 62.

63. 64. 65.

18 of 19

Hooda, A. Gastroretentive drug delivery systems: A review of formulation approaches. Pharm. Innov. 2012, 1, 79–88. Annabi, N.; Nichol, J.W.; Zhong, X.; Ji, C.; Koshy, S.; Khademhosseini, A.; Dehghani, F. Controlling the porosity and microarchitecture of hydrogels for tissue engineering. Tissue Eng. Part B 2010, 16, 371–383. [CrossRef] [PubMed] Ji, C.; Annabi, N.; Khademhosseini, A.; Dehghani, F. Fabrication of porous chitosan scaffolds for soft tissue engineering using dense gas CO2 . Acta Biomater. 2011, 7, 1653–1664. [CrossRef] [PubMed] Annabi, N.; Mithieux, S.M.; Weiss, A.S.; Dehghani, F. The fabrication of elastin-based hydrogels using high pressure CO2 . Biomaterials 2009, 30, 1–7. [CrossRef] [PubMed] Zhao, Z.; Xie, M.; Li, Y.; Chen, A.; Li, G.; Zhang, J.; Hu, H.; Wang, X.; Li, S. Formation of curcumin nanoparticles via solution-enhanced dispersion by supercritical CO2 . Int. J. Nanomed. 2015, 10, 3171–3183. [CrossRef] [PubMed] Pawar, H.; Karde, M.; Mundle, N.; Jadhav, P.; Mehra, K. Phytochemical evaluation and curcumin content determination of turmeric rhizomes collected from bhandara district of maharashtra (India). Med. Chem. 2014, 4, 588–591. [CrossRef] Sim, S.; Figueiras, A.; Veiga, F. Modular hydrogels for drug delivery. J. Biomater. Nanobiotechnol. 2012, 3, 185–199. Ganji, F.; Vasheghani-Farahani, S.; Vasheghani-Farahani, E. Theoretical description of hydrogel swelling: A review. Iran Polym. J. 2010, 19, 375–398. Saber-Samandari, S.; Gazi, M. Pullulan based porous semi-IPN hydrogel: Synthesis, characterization and its application in the removal of mercury from aqueous solution. J. Taiwan Inst. Chem. E 2015, 51, 143–151. [CrossRef] Khurma, J.R.; Rohindra, D.R.; Nand, A.V. Synthesis and properties of hydrogels based on chitosan and poly(vinyl alcohol) crosslinked by genipin. J. Macromol. Sci. Part A 2006, 43, 749–758. [CrossRef] Ooi, S.Y.; Ahmad, I.; Amin, M.C.I.M. Cellulose nanocrystals extracted from rice husks as a reinforcing material in gelatin hydrogels for use in controlled drug delivery systems. Ind. Crop. Prod. 2016, 93, 227–234. [CrossRef] Gemeinhart, R.A.; Park, H.; Park, K. Pore structure of superporous hydrogels. Polym. Adv.Technol. 2000, 11, 617–625. [CrossRef] Wegiel, L.A.; Zhao, Y.; Mauer, L.J.; Edgar, K.J.; Taylor, L.S. Curcumin amorphous solid dispersions: The influence of intra and intermolecular bonding on physical stability. Pharm. Dev. Technol. 2014, 19, 976–986. [CrossRef] [PubMed] Liu, Y.; Cai, Y.; Jiang, X.; Wu, J.; Le, X. Molecular interactions, characterization and antimicrobial activity of curcumin–chitosan blend films. Food Hydrocoll. 2016, 52, 564–572. [CrossRef] Huang, X.; Brazel, C.S. On the importance and mechanisms of burst release in matrix-controlled drug delivery systems. J. Control. Releas. 2001, 73, 121–136. [CrossRef] Fu, Y.; Kao, W.J. Drug release kinetics and transport mechanisms of non-degradable and degradable polymeric delivery systems. Expert Opin. Drug Deliv. 2010, 7, 429–444. [CrossRef] [PubMed] Kim, S.W.; Bae, Y.H.; Okano, T. Hydrogels: Swelling, drug loading, and release. Pharm. Res. 1992, 9, 283–290. [CrossRef] [PubMed] Bajpai, S.; Chand, N.; Ahuja, S. Investigation of curcumin release from chitosan/cellulose micro crystals (CMC) antimicrobial films. Int. Biol. Macromol. 2015, 79, 440–448. [CrossRef] [PubMed] Jobe, B.A.; Richter, J.E.; Hoppo, T.; Peters, J.H.; Bell, R.; Dengler, W.C.; DeVault, K.; Fass, R.; Gyawali, C.P.; Kahrilas, P.J. Preoperative diagnostic workup before antireflux surgery: An evidence and experience-based consensus of the esophageal diagnostic advisory panel. J. Am. Coll. Surg. 2013, 217, 586–597. [CrossRef] [PubMed] Emami, J.; Tajeddin, M.; Ahmadi, F. Preparation and in vitro evaluation of sustained-release matrix tablets of flutamide using synthetic and naturally occurring polymers. Iran. J. Pharm. Res. 2010, 7, 247–257. Chime, S.; Onunkwo, G.; Onyishi, I. Kinetics and mechanisms of drug release from swellable and non swellable matrices: A review. Res. J. Pharm. Biol. Chem. Sci. 2013, 4, 97–103. Agnihotri, S.A.; Aminabhavi, T.M. Novel interpenetrating network chitosan-poly(ethylene oxide-g-acrylamide) hydrogel microspheres for the controlled release of capecitabine. Int. J. Pharm. 2006, 324, 103–115. [CrossRef] [PubMed]


66. 67. 68. 69. 70. 71.

72. 73.

19 of 19

Gonzalez, G.; Sagarzazu, A.; Zoltan, T. Infuence of microstructure in drug release behavior of silica nanocapsules. J. Drug Deliv. 2013, 2013, 585–593. [CrossRef] [PubMed] Kwon, S.; Singh, R.K.; Perez, R.A.; Neel, E.A.A.; Kim, H.W.; Chrzanowski, W. Silica-based mesoporous nanoparticles for controlled drug delivery. J. Tissue Eng. 2013, 4, 82–86. [CrossRef] [PubMed] Annabi, N.; Fathi, A.; Mithieux, S.M.; Weiss, A.S.; Dehghani, F. Fabrication of porous PCL/elastin composite scaffolds for tissue engineering applications. J. Supercrit. Fluids 2011, 59, 157–167. [CrossRef] Dehghani, F.; Annabi, N. Engineering porous scaffolds using gas-based techniques. Curr. Opin. Biotechnol. 2011, 22, 661–666. [CrossRef] [PubMed] Sampath, U.G.; Ching, Y.C.; Chuah, C.H.; Sabariah, J.J.; Lin, P.C. Fabrication of porous materials from natural/synthetic biopolymers and their composites. Materials 2016, 9, 991–996. [CrossRef] Bashir, S.; Teo, Y.Y.; Ramesh, S.; Ramesh, K. Synthesis, characterization, properties of n-succinyl chitosan-g-poly(methacrylic acid) hydrogels and in vitro release of theophylline. Polymer 2016, 92, 36–49. [CrossRef] Abou-Zeid, R.E.; Hassan, E.A.; Bettaieb, F.; Khiari, R.; Hassan, M.L. Use of cellulose and oxidized cellulose nanocrystals from olive stones in chitosan bionanocomposites. J. Nanomater. 2015, 16, 172–177. [CrossRef] De Paula, E.L.; Mano, V.; Duek, E.A.R.; Pereira, F.V. Hydrolytic degradation behavior of PLLA nanocomposites reinforced with modified cellulose nanocrystals. Química Nova 2015, 38, 1014–1020. [CrossRef] © 2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).