Acrylamide pH-Sensitive Smart Hydrogel

Article pubs.acs.org/molecularpharmaceutics

Bacterial Cellulose/Acrylamide pH-Sensitive Smart Hydrogel: Development, Characterization, and Toxicity Studies in ICR Mice Model Manisha Pandey, Najwa Mohamad, and Mohd Cairul Iqbal Mohd Amin* Centre for Drug Delivery Research, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz 50300, Kuala Lumpur, Malaysia S Supporting Information *

ABSTRACT: The objective of this study is to synthesize and evaluate acute toxicity of the bacterial cellulose (BC)/ acrylamide (Am) hydrogels as noncytotoxic and biocompatible oral drug delivery vehicles. A novel series of solubilized BC/ Am hydrogels were synthesized using a microwave irradiation method. The hydrogels were characterized by Fourier transform infrared spectroscopy (FTIR), swelling ratio, porosity, drug release, and in vitro and in vivo biocompatibility experiments. FTIR spectra revealed that the BC crystallinity and gel fraction decreased as the NaOH concentration increased from 2% to 10% w/v, whereas the optical transparency, pH sensitivity, and porosity were enhanced with increasing alkali concentration. Theophylline was used as a model drug for drug loading and release studies. The percentage of drug released was higher at pH 7.4 compared to pH 1.5. In vitro cytotoxicity and hemolytic tests indicated that the BC/Am hydrogel is noncytotoxic and hemocompatible. Results of acute oral toxicity tests on ICR mice suggested that the hydrogels are nontoxic up to 2000 mg/kg when administered orally, as no toxic response or histopathological changes were observed in comparison to control mice. The results of this study demonstrated that the pH-sensitive smart hydrogel makes it a possible safe carrier for oral drug delivery. KEYWORDS: acrylamide, acute oral toxicity, bacterial cellulose, cytotoxicity, hydrogel, microwave irradiation

1. INTRODUCTION Hydrogels are composed of a hydrophilic polymeric network capable of imbibing a large amount of biological fluid or water. Their elasticity, tissue mimicking properties, swelling and deswelling characteristics in response to environmental stimuli make them potential candidates for biomedical applications. Recently, hydrogels have been comprehensively used as smart drug delivery systems.1 For example, pH-sensitive hydrogels have been frequently used as a self-regulated carrier for oral delivery of lifesaving drugs because the acidic pH of the stomach is quite different from the neutral pH of the intestine. Therefore, pH-sensitive hydrogels can protect drugs from the hostile environment of the digestive system and also control the release of drugs in a predictable manner to match physiological needs.2,3 Biopolymers are gaining attention in hydrogel synthesis as a renewable resource with biocompatible and ecofriendly properties. Bacterial cellulose (BC) is a biopolymer, having hydrophilicity and biocompatibility as key features that are at the base of the vast applications of this amazing material.4 Micro- and nanofibers of BC as a reinforced material could enhance the thermal and mechanical properties of composite. The negative charge and large surface area of the BC nanofibers have been © 2014 American Chemical Society

suggested to govern the drug loading on the surface of composite. Moreover, the presence of hydroxyl groups in abundance on BC fibers enhancing its chemical modifying capacity with a range of chemical groups that can modulate the drug loading and release from the composite.5,6 By contrast, their low absorption capacity due to the presence of highly crystalline regions and variable physical stability limits their application.7−9 Recent studies have suggested that these limitations can be overcome with chemical modification of cellulose by graft copolymerization using synthetic10 or natural polymers.11 Previous studies have reported that bacterial cellulose (BC)-based hydrogels can be prepared by introducing a synthetic polymer solution within a cellulose network crosslinked with N,N′-methylenebis(acrylamide)10,12 using a convention-heating method, which may also help to overcome these disadvantages. An alternative method proposed by Halib et al. (2010) involves the synthesis of a BC/acrylic acid hydrogel through the stable dispersion of BC.13 Received: Revised: Accepted: Published: 3596

May 6, August August August

2014 21, 2014 26, 2014 26, 2014

dx.doi.org/10.1021/mp500337r | Mol. Pharmaceutics 2014, 11, 3596−3608

Molecular Pharmaceutics

Article

Table 1. Formulation Code, Gel Fraction, and Swelling Kinetics for BC/Am Hydrogel on the Basis of Solvent Composition for Bacterial Cellulose with Constant Concentration of Acrylamide, Initiator, and Crosslinker swelling kinetics formulation code for hydrogel

concentration of NaOH (% w/v)

concentration of urea (% w/v)

gel fraction of hydrogel (%)

na

Kb

R2c

H0−0 H0−2 H0−4 H0−6 H2−0 H2−2 H2−4 H2−6 H4−0 H4−2 H4−4 H4−6 H6−0 H6−2 H6−4 H6−6 H8−0 H8−2 H8−4 H8−6 H10−0 H10−2 H10−4 H10−6

0 0 0 0 2 2 2 2 4 4 4 4 6 6 6 6 8 8 8 8 10 10 10 10

0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6

83.32 ± 1.20 81.53 ± 1.02 82.95 ± 2.53 80.01 ± 1.04 78.32 ± 1.20 79.53 ± 1.02 76.95 ± 2.53 77.01 ± 1.04 74.09 ± 1.92 75.89 ± 1.73 76.44 ± 2.63 72.92 ± 1.83 71.78 ± 1.24 70.4 ± 2.01 73.06 ± 1.49 71.34 ± 0.94 69.56 ± 2.19 67.25 ± 0.92 68.96 ± 1.86 65.45 ± 0.72 68.23 ± 2.83 66.23 ± 1.02 67.23 ± 1.93 62.67 ± 1.95

0.334 0.351 0.357 0.317 0.693 0.536 0.449 0.549 0.604 0.669 0.656 0.651 0.663 0.683 0.681 0.850 0.892 0.872 0.686 0.765 0.651 0.670 0.695 0.725

0.396 0.309 0.330 0.298 0.575 0.534 0.520 0.534 0.555 0.558 0.598 0.615 0.633 0.720 0.687 0.789 0.797 0.805 0.595 0.639 0.628 0.657 0.674 0.692

0.991 0.998 0.984 0.996 0.994 0.994 0.997 0.989 0.714 0.988 0.989 0.985 0.998 0.988 0.994 0.975 0.997 0.990 0.988 0.991 0.990 0.993 0.991 0.989

a

n denotes the power law diffusion exponent. bK represents the rate constant. cR2 shows coefficient of determination.

lamide) hydrogels.20 The pore volume of hydrogels synthesized by microwave irradiation (0.45−2.7 × 10−2 cm3/g) was larger than those synthesized using the water bath method (0.05− 0.15 × 10−2 cm3/g). Moreover, microwave energy markedly accelerates the reaction rate constant of poly(acrylic acid) hydrogel formation from 32-fold to 43-fold compared to the conventional heating system,12 greatly shortening the synthesis time by lowering the activation energy of the reactants by up to 19%. Similarly, Kumar et al. (2009) grafted xanthan gum with polyacrylamide by using microwave irradiation, and observed increased grafting from 12.76% to 87.15% as the irradiation power increased from 40 to 100 W, resulting in the release of more model drug from the grafted polymer matrix compared to the ungrafted xanthan gum matrix.21 Here, we report the synthesis and characterization of BC/Am pH-sensitive smart hydrogels using solubilized BC in NaOH/ urea solution. The model drug theophylline was loaded in the hydrogel and assessed for various in vitro parameters. In addition, both in vitro and in vivo biocompatibility experiments were performed to evaluate the potential of the new hydrogels as noncytotoxic and biocompatible oral drug delivery vehicles.

Generally, two basic approaches are used for the synthesis of BC based composite that is in situ and ex situ.14 In the case of the in situ method, additional reinforcement materials are added in BC culture at the start of BC synthesis, so the material can entrap within the web type structure of BC. On the contrary, in the ex situ method, BC membrane is impregnated with reinforcement materials by dipping to produce composites.15 Previously, BC/polyacrylamide double network hydrogel were synthesized by introducing acrylamide within the BC matrix by conventional heating method.16−18 The main problem associated with this strategy is the nature and size of the reinforced material such as only nano and submicron particles can penetrate into BC matrix. Moreover, hydrophobic material cannot interact with BC fibrils and homogeneity of reinforce material is one of the main concern as BC web structure is not always uniform. Accordingly, there is a need to identify synthesis from solution of dissolved BC. In this study, solubilized BC was used to synthesize a BC/acrylamide (Am) hydrogel using an ecofriendly solvent system for cellulose dissolution. However, cellulose with a high degree of polymerization is more difficult to dissolve in single-component alkali solutions. The use of urea and thiourea together with NaOH has been reported to exhibit greater dissolution capability than NaOH alone.7 Most reported cases of graft copolymerization of BC by synthetic polymers were carried out by redox grafting18 and ionic irradiation.13,19 However, the use of accelerated microwave irradiation is gaining attention due to its low safety concerns, energy consumption, and production costs. Zhao et al. (2008) reported that microwave irradiation has specific advantages over conventional heating methods by comparing the two methods in the preparation of poly(N-isopropylacry-

2. EXPERIMENTAL SECTION 2.1. Materials. BC was isolated from nata de coco and further purified, lyophilized, characterized, and identified as described in British Pharmacopoeia (2010). Am, potassium persulfate (KPS), and N,N′-methylenebis(acrylamide) (MBA) were supplied by Sigma-Aldrich (St. Louis, MO, U. S. A.). Sodium hydroxide, sodium chloride, urea, and other reagents were of analytical grade quality and used without further purification. Phosphate buffered saline (pH 2, 5, 7, and 10) was prepared as described in British Pharmacopoeia (2010). 3597



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Chinese hamster lung fibroblasts (V79) purchased from the American Type Culture Collection (ATCC; Rockville, MD, U. S. A.) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS; Sigma). ICR mice (females) weighing 25 ± 2 g were used in the oral acute toxicity tests and histopathological observations. The animals were purchased from the Laboratory Animal Center of Universiti Kebangsaan Malaysia. The animals were housed at a controlled temperature of 20− 22 °C, relative humidity of 50−60%, and under 12-h light−dark cycles. Free access to food and water was allowed. All animals were quarantined for 1 week before treatment. 2.2. Dissolution of BC. The 2% (w/v) BC solution was prepared by dispersing microfibrillated BC in NaOH (0, 2, 4, 6, 8, 10% w/v) and urea at various concentrations (0, 2, 4, 6% w/ v), and maintained at −15 °C for 24 h. The frozen solution was thawed and stirred vigorously to obtain a homogeneous cellulose solution as described by Chang et al. (2010).22 2.3. Synthesis of BC/Am Hydrogels. Am (0.029 mol) was added to BC solution (20 mL) prepared from varying concentrations of NaOH/urea, followed by the addition of KPS (7.4 × 10−4 moles) as the initiator and MBA (1.29 × 10−3 moles) as a cross-linker. The resultant mixture was then irradiated with a modified microwave irradiator (MS2388K; LG Electronics; Seoul, South Korea) at 170 W power for 40 s to fabricate batches of hydrogels. The prepared hydrogels were soaked in distilled water for 120 h to remove unreacted monomers and water was changed twice daily. Then, extracted hydrogels were dried in an oven for 24 h at 60 °C. The prepared hydrogels were labeled on the basis of the NaOH/ urea concentration used to dissolve BC (Table 1). 2.4. Infrared (IR) Spectroscopic Analysis. The IR spectra of samples were recorded to analyze structural changes using FTIR Spectra 2000 (PerkinElmer; Waltham, MA, U. S. A.) at room temperature. The KBr disk method was adopted for the Fourier transform (FT)IR experiment, and samples were scanned over the range of 4000−400 cm−1. 2.5. 13C Solid State NMR. Solid-state cross-polarization/ magic angle spinning (CPMAS) 13C NMR spectra of the hydrogels were recorded using an NMR spectrometer (Avance 400; Bruker, Germany). Selected dried hydrogel was micronized using pulverizer (Pulverisette 14, Frisch, IdarOberstein, Germany), and 100 mg of samples were packed into a 4 mm inner diameter cylindrical zirconium oxide MAS rotor with an O-ring seal and end-cap. Chemical shifts were recorded in ppm with reference 29.50 ± 0.10 ppm (CH) and 38.56 ± 0.10 ppm (CH2) using adamantine as the external standard. 2.6. Thermogravimetric Analysis (TGA). TGA of the hydrogels was conducted using a PerkinElmer STA 6000 system. Approximately 10 mg of each sample was placed in a TGA sample pan and heated over a temperature range of 50− 700 °C. The heating rate for analysis was set at 10 °C/min with a constant nitrogen purge (20 mL/min). 2.7. Gel Fraction Determination. The gel fraction (GF) of hydrogel indicates the degree of grafting and cross-linking, as cross-linking prevents the hydrogel from solubilization and only enable the expansion of hydrogel in water during extraction. For the determination of GF, freshly prepared hydrogels were cut into the shape of a disc by using a 1.2 cm diameter mold and dried in an oven at 60 °C up to a constant weight (G0). The dried hydrogels were immersed in distilled water for 5 days at room temperature to extract the remaining reactants (BC and Am). The extracted hydrogels were then redried to a

constant weight (G1) at 60 °C. The percent gel fraction (% GF) was determined using the following equation: %GF = (G1/G0) × 100%

(1)

2.8. Morphological Analysis. The surface morphology and porous structure of freeze-dried BC/Am hydrogels were observed by scanning electron microscopy (SEM; LEO 1450 VP; Oberkochem-Zeiss; Germany). The samples were mounted on an aluminum stub and coated with gold in a sputter coater (SC500; BioRad; London, U. K.) under an argon atmosphere. 2.9. Swelling Studies. 2.9.1. Swelling Ratio. The swelling ratio of hydrogels was investigated under conditions of varying pH (2, 5, 7, and 10) and temperature (4 °C, 25 °C, 37 °C, and 45 °C). A known weight of dried extracted hydrogel (Gd) was immersed in a 50 mL volume of swelling media. Swollen samples were weighed (Gs) at fixed time intervals and excess media was removed by blotting with filter paper. The swelling ratio (% SR) of the hydrogel was calculated by using the following equation: %SR = (Gs − Gd /Gd) × 100%

(2)

2.9.2. Swelling Kinetics. Swelling kinetics of hydrogels at pH 7 containing different ratios of NaOH/urea was determined using the following equation: F = Wt /Weq = kt n

(3)

where Wt and Weq are the weight of water absorbed by the hydrogel at time t and at equilibrium, respectively, k denotes the rate constant, and n represents the power law diffusion exponent. The above equation is valid for the initial swelling state at a fraction water uptake (f) of 0.6 when the plot of ln f and ln t is linear. 2.10. Rheological Analysis. Rheological measurements were carried out using Bohlin Gemini II rheometer (Malvern Instrument, United Kingdom). Prior to that hydrogels with 200% swelling ratio were cut into cylindrical shape with diameter 20 mm with a mold and placed between two parallel plates before conducting the study. The oscillatory frequency sweep test was performed in the frequency range of 0.1 to 10 Hz with a constant strain of 0.1% using 20 mm parallel plates geometry with a gap of 2.4 mm at 30 °C. 2.11. Enzymatic Degradation Study. The degradation of hydrogels was determined in simulated intestinal fluid (SIF) with enzyme (pancreatin) at 37 °C for 5 weeks. The samples were prepared and a dry mass sample (100 mg) of the hydrogels was immersed in 10 mL of SIF. The incubation media (SIF) were refreshed daily in order to maintain enzymatic activity. At predetermined time periods, hydrogel samples were removed from the incubation media, washed gently with distilled water, and then lyophilized at −110 °C for 48 h to a constant weight. The degree of degradation was estimated from the weight loss of the hydrogel based on the equation given below. The average weight loss of three hydrogel specimens was measured %Weight Loss = [(W0 − Wt)/W0] × 100

(4)

Where W0 is the original weight of the dry hydrogel sample before immersion in SIF, and Wt is the dry weight of the hydrogel sample after time t of incubation. 2.12. Drug Loading. Theophylline was used as a model drug and loaded to the hydrogels (H6−4 and H8−4) using the 3598



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Next, hydrogel-treated cells were rinsed with PBS and stained with fluorescent dye (1 μM calcein AM and 2 μM ethidium homodimer-1 (EthD-1) per well) in the dark for 45 min. The cells were observed with a Floid cell imaging station (Molecular Probes Life Technology; France). The live cells could be distinguished by the green fluorescence produced from conversion of the nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein by the intracellular esterase enzyme, and the red fluorescence indicated dead cells due to binding of EthD-1 with the nucleic acids of damaged cells. 2.14.2. In Vitro Hemolytic Test. The hemolytic activity of the hydrogel was tested by using the direct contact method25 with the whole blood sample. Anticoagulated blood (200 μL) was added to 10 mL of hydrogel suspension samples in normal saline at different concentrations (20, 40, 60, 80 mg per 10 mL, respectively). Normal saline solution and distilled water were used as the negative and positive control, respectively. After gentle blending, the tubes were incubated in a water bath at 37 °C for 1 h. After incubation, the tubes were centrifuged at 1000 rpm for 10 min and absorbance of the supernatant (A) of each tube was analyzed with a spectrophotometer (UV-1601; Shimadzu; Kyoto, Japan) at 545 nm. The samples were run in triplicate and the rate of hemolysis was determined using the following equation:

swelling diffusion method. Dried and weighed hydrogel samples were soaked in a 20 mL volume of drug solution (10 mg/mL) in phosphate-buffered saline (PBS), pH 7.4, for 48 h. The swollen discs of hydrogels were removed from the solution, washed with distilled water, and dried at 37 °C to a constant weight.17 The concentration of drug remaining in the solution was determined spectrophotometrically at 272 nm using a spectrophotometer (UV-1601; Shimadzu; Kyoto, Japan). Drug entrapment efficiency and drug loading were calculated using the following equations: Drug entrapment efficiency = [(W0 − Wf )/W0] × 100 (5)

Drug loading = (Wdg /Wg) × 100

(6)

Where W0 and Wf are the total amounts of the drug in soaking solution before and after loading of the hydrogel, respectively, Wdg is the amount of drug entrapped in the hydrogel, and Wg is the weight of the dried hydrogel disc. 2.13. In Vitro Drug Release Study. The drug release profiles of the different hydrogel formulations were generated by submerging a drug-loaded hydrogel disc into PBS (100 mL; pH 1.5) for 2 h, followed by loading in a PBS of pH 7.4 for up to 48 h. The drug-loaded hydrogels were also submerged separately in the PBS of pH 7.4 alone in order to compare the drug release profiles between the two systems. The dissolution media were maintained at a fixed temperature (37 ± 2 °C) with constant agitation (50 rpm). Aliquots (4 mL) were withdrawn at defined intervals and replaced with fresh media. Drug concentrations were measured spectrophotometrically at 272 nm, and the results are presented as the cumulative percentage of release over time. In order to determine the drug release kinetics, the release rate data over 8 h was fitted into zero-order, first-order, and Korsmeyer−Peppas equations. The value of the release exponent of the Korsmeyer−Peppas equation (n) was used to describe the release mechanism of the drug from the hydrogel.23 2.14. Biocompatibility Test. 2.14.1. Cytotoxicity Study. 2.14.1.1. alamarBlue Cell Viability Assay. The Chinese hamster lung fibroblast (V79) cell lines were cultured in DMEM. Cultured cells maintained in DMEM were seeded in 96-well culture plates at 30 × 104 cells per well and incubated for 24 h. The cells was treated with a powdered hydrogel sample (prepared by crushing of freeze-dried H8−4) at concentrations ranging from 2 to 0.125 mg/mL, and incubated for an additional 24 and 48 h at 37 °C in a 5% CO2/95% air atmosphere. After incubation of the hydrogel at 37 °C, 20 μL of alamarBlue (Invitrogen; Carlsbad, CA, U. S. A.) reagent was added to the treated cells followed by 4 h incubation prior to analysis.24 The absorbance of each sample at 570 nm (A570) was measured on a microplate reader (Varioskan Flash; Thermo Scientific; Waltham, MA, U. S. A.). Cell viability was determined using the following equation:

Rate of hemolysis (%) ⎡ A test sample − A negative control ⎤ ⎥ × 100 =⎢ ⎣⎢ A positive control − A negative control ⎥⎦

A mean hemolysis value from the three test samples ≤5% was considered acceptable. 2.15. Acute Oral Toxicity Study. The acute oral toxicity of the BC/Am hydrogel (H8−4) was determined per the Organization for Economic Co-operation and Development (OECD) guidelines for the testing of chemicals 425. The study protocol received prior approval from the Animal Ethics Committee. Twelve nonpregnant 8-week-old female mice (ICR strain) were used for this study. The animals were assigned to two test groups: the hydrogel-treated group (hydrogel powder equivalent to 2000 mg/kg body weight in distilled water) and the control group (equivalent amount of normal saline). The hydrogel-treated group received BC-poly(acrylamide−sodium acrylate) hydrogel suspension three times at an interval of 4 h by gavage, at a total dose of 2000 mg/kg body weight and a total volume of 0.3 mL/10 g body weight. Accordingly, the control group was treated with the same volume of normal saline. The animals were given food again approximately 4 h after dosing. Thereafter, the animals were monitored for 48 h for clinical signs of toxicity or mortality. Next, an additional five animals in the test group received the same doses and were monitored for a further 14 days. Observations were conducted twice daily and mortality, injury, abnormal behavior, and the general condition (hair, activity, feces, behavioral patterns, and other clinical signs) were recorded. In addition, the body weight of all mice was recorded on study days 0, 3, 5 7, 9, 12, and 14. Upon completion of the study, the animals were sacrificed by cervical dislocation and necropsied to facilitate gross pathological examination of organs. 2.16. Statistical Analysis. All data are presented as the mean ± standard deviation (SD). Data were analyzed with the Student’s t-test and the Kruskal−Wallis test for pairwise differences between the treatment and control groups. Analysis

Cell viability (%) = (A570 of treated cells/A 570 of control cells) × 100

(8)

(7)

2.14.1.2. “Live/Dead” Assay. A “live/dead” assay was performed with the same cell lines in a 96-well plate using the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen). First, the cells were seeded at the same cell density and incubated for 24 h followed by hydrogel (H8−4) treatment for 24 and 48 h. 3599



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3.2. FTIR Analysis. The BC/Am hydrogels were synthesized by microwave irradiation using varying concentrations of NaOH/urea as a BC solvent. According to the IR data, two reactions may occur simultaneously. The first is the formation of cellulose derivatives (cellulose polyelectrolytes containing an acylamino and carboxyl group) and grafting, which was confirmed by comparing the relative intensity at 1568 and 1670 cm−1, corresponding to the asymmetrical stretching of  COO groups and amide I (CO stretching).26 The second reaction is the partial hydrolysis of polyacrylamide, leading to conversion of CONH2 into COO− and the concomitant liberation of ammonia gas. The hydrolysis of polyacrylamide was increased proportionally with the increase in NaOH concentration, as specified by the increased intensity of the 1568 cm−1 band and by another sharp peak appearing at 1408 cm−1 (attributed to the symmetric stretching mode of the carboxylate anion).27 The broad absorption band observed at 3438 cm−1 was due to the overlap of the OH stretching of BC and the NH stretching of Am. The strong absorption band observed at 2925 cm−1 was attributed to the overlapping of CH stretching of BC and Am (Figure 2a). By contrast, the spectra of the hydrogels displayed different urea concentrations in the solvent system of BC, suggesting no change in the crystallinity of BC (Figure 2b). These data indicated that NaOH is the primary agent in cleaving the inter- and intrahydrogen bonds in cellulose.28 As shown in Figure 2c, it was clear that urea alone was not sufficient to hydrolyze Am; however, in the presence of NaOH, urea promoted hydrolysis, as the peak intensity at 1568 and 1408 cm−1 increased with increasing urea concentration (0−6%) under constant NaOH concentration (8%). 3.3. 13C Solid State NMR. 13C solid state NMR of selected BC/Am hydrogel (H8−4) was carried out due to insolubility of hydrogel in the common solvents used for NMR analysis. The NMR spectra of the hydrogel (Figure 2d) showed a characteristic peak of acrylamide at δ = 181, which denotes the presence of CONH2 groups while the broad peak indicates the signal of COOH generated by hydrolysis.29 Moreover, the peak at 42 ppm stands for (CHCH2 CH)n represents the polymerization of acrylamide into polyacrylamide. The absence of characteristic peak of acrylamide at 130 and 133 ppm for sp2 hybridized carbon atoms (CH2CH) indicate the conversion of sp2 to sp3 hybridized carbon during polymerization. The NMR spectra also exhibited characteristic peak of BC at δ = 104 represents C1 of BC backbone while the broad peak at 72 corresponds to C2, C3, and C5. Furthermore, the peaks at 85 and 60 ppm showed an amorphous region of C4 and C6 of anhydroglucose unit in cellulose, respectively. The grafting of BC mainly occurs at the hydroxyl group on C6 of BC.30 The presence of characteristic peaks of BC and polyacrylamide in NMR spectra indicate the formation of grafted polymeric hydrogel. 3.4. TGA Analysis. The TGA of the Am/BC hydrogels was determined to evaluate the effect of grafting on the thermal degradation behavior of the hydrogels. Figure 3a represents the effect of NaOH concentration on thermal behavior of the hydrogels, and the effect of urea concentration on the TGA of hydrogels is presented in Figure 3b. The TGA curve of hydrogels without any solvent (H0−0) showed a slow initial weight loss (>7%) up to 230 °C followed by a two-stage degradation from 230−320 °C (an approximate 12% weight loss) and 320−500 °C (an approximate 40% weight loss). The weight loss observed up to 320 °C was attributed to

of variance (ANOVA), followed by posthoc Tukey’s analysis, was used to compare multiple groups with SPSS 19.0 software. P values less than 0.05 were considered to be statistically significant.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Hydrogels. The BC-g-poly(acrylamide− sodium acrylate) hydrogels were successfully synthesized by

Figure 1. Proposed reaction scheme of hydrogels synthesis.

graft polymerization of poly(acrylamide−sodium acrylate) onto BC by using KPS as an initiator and MBA as a cross-linker. The scheme of the proposed mechanism involved in the grafting and chemical cross-linking of the monomers is presented in Figure 1. The synthesis reaction was initiated by generation of sulfate ion radicals (SO4•−) using KPS as an initiator upon exposure to microwave irradiation. Upon reaction with water molecules, these radicals generated hydroxyl radicals (OH•), followed by reaction with other reactants, and produced an active center to initiate the polymerization of Am. Simultaneously, Am was hydrolyzed by NaOH, which was used as a solvent for BC.26,27 During this hydrolysis, ammonia gas was liberated and Am nonionic amide groups were converted to anionic carboxylate salts (sodium acrylates; Figure 1).27 The polymer molecules, which were in close vicinity to the reaction sites, became acceptors of the BC radicals, resulting in chain initiation, and subsequently served as free radical donors to neighboring molecules.13,14 Finally, MBA was added as a crosslinking agent to complete the cross-linking process of the polymers. 3600



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Figure 2. Comparative FTIR spectra of BC/Am hydrogels (a) containing different concentration of NaOH as a solvent for cellulose, (b) containing different concentration of urea without NaOH as a solvent for cellulose, (c) containing constant concentration of NaOH but different concentration of urea as a solvent for cellulose, (d) solid state 13C NMR spectra of BC/Am hydrogel (H8−4).

Figure 3. TGA curves of BC/Am hydrogels containing (a) different concentration of NaOH for cellulose dissolution, (b) different concentration of urea with constant concentration of NaOH.

dehydration of the entrapped moisture and elimination of

hydrogels indicated their higher thermal stability due to the grafting of individual components. Figure 3a shows that increasing the concentration of NaOH in the solvent significantly affected the thermal degradation behavior of the hydrogels. The TGA profile of H8−0 closely resembled that of the typical three-step TG curve of

ammonia gas from polyacrylamide (PAm),31 whereas the sharp weight loss observed from 320−400 °C was due to loss of acrylic moieties and BC degradation.19 The decreased intensity of the BC curve and the higher residual mass in the TG of 3601



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Figure 4. (a) Photograph of appearance of hydrogels containing different concentration of NaOH. (b) SEM images of BC/Am superporous hydrogels containing different concentration of NaOH for cellulose dissolution.

Figure 5. Swelling behavior of hydrogels at different (a) pHs and (b) temperatures.

poly(acrylic acid),32 signifying that hydrolysis of the PAm to sodium acrylates had occurred in the presence of NaOH. On the other hand, no significant difference in the TGA profile of the hydrogels was observed when the urea concentration was increased from 0 to 4%, suggesting that the presence of urea in the solvent system does not affect thermal degradation of the hydrogels. 3.5. Gel Fraction. The gel fraction of hydrogel decreased from 83.32% to 62.67%, as NaOH concentration increased from 0%−10% (Table 1). Marandi et al. (2008) reported similar observations when hydrolyzing a polyacrylamide hydrogel with NaOH.27 This observation was caused by an increase in alkali concentration, which in turn caused degradation of the cross-link points, leading to a reduction in cross-link density, thereby resulting in a reduced gel fraction. In fact, the polymeric chains easily separated, resulting in high sol content at high NaOH concentrations. By contrast, our data does not account for the effect of urea on the gel fraction,

which can be further improved by changing the microwave irradiation dose as well as the initiator and cross-linker amounts. 3.6. Appearance of Hydrogels. The hydrogel appeared more transparent at increasing NaOH concentrations (Figure 4a), with white color denoting less transparency and the background color denoting greater transparency. The nontransparent hydrogel, appearing at 2% and 4% alkali concentration, becomes transparent, as the concentration is increased from 6%−8%. This increased in transparency attributed to enhanced solubility of BC as alkali concentration increased in solvent. 3.7. Morphological Analysis. SEM analysis (150× magnification) of the surface of the hydrogels indicated that the porosity was increased with increasing alkali concentration (Figure 4b). This was caused by the ammonia gas liberated during hydrogel synthesis, which generated more pores on the surface. The amount of ammonia gas liberated was proportional 3602



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Figure 6. Oscillatory frequency sweep test of hydrogels containing different concentrations of NaOH (a) and urea (c). Elastic modulus G′ as a function of NaOH (b) and urea (d) concentration in hydrogel at fixed frequency (0.1 Hz).

Figure 7. Enzymatic degradation of BC/Am hydrogels containing (a) different concentrations of NaOH and (b) different concentrations of urea with constant concentration of NaOH.

Figure 8. In-vitro drug release profile of the superporous hydrogels (a) in buffer pH 1.5 followed by pH 7.4 (b) in phosphate buffer 7.4.

(approximately 80.3 to 255 μm) along with the small pores (25 to 5.5 μm), which is responsible for rapid swelling of hydrogel. 3.8. Swelling Studies. The percentage of swelling was dramatically increased upon increasing the NaOH concentration from 0 to 10% (Figure 5a). This observation was attributed to the hydrolysis of polyacrylamide, which is

to the alkali concentration, leading to a rougher surface and more spongy appearance. Previous studies have reported that interconnected pores and spongy structures allow greater diffusion of solvent molecules during swelling.13 It was clear from Figure 4 that hydrogel surface contained larger pores 3603



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Figure 9. Mean V79 fibroblast viability results obtained by the alamarBlue assay after incubation for 24 and 48 h with different concentrations of BC/Am hydrogel (2−0.125 mg/mL). Significant difference (P < 0.05) in cytotoxicity at 24 and 48 h are presented as a six-pointed star and double four-pointed stars, respectively.

amplified by increasing the alkali concentration and generating more carboxylate groups and ammonia gas,27 leading to a more porous structure (Figure 4). Conversely, urea alone did not affect swelling significantly (P > 0.05), but with the addition of NaOH, the swelling increased up to 4%. The pH sensitivity of the hydrogels was evaluated using various buffer solutions (pH 2, 5, 7, and 10). At pH 2, the %SR was increased up to 6 h, but subsequently decreased markedly up to 24 h. By contrast, at pH 5, 7, and 10, the %SR increased gradually up to 24 h. The swelling increased consistently in all hydrogels from pH 2−7, but decreased abruptly at pH 10, as displayed in Figure 5a(i) and (ii). This pattern may be attributed to the presence of carboxylate ions in the hydrolyzed hydrogel, whose pKa value is ≈4.7; at pH < 4.7, the carboxylate group becomes protonated, whereas at pH 5−7, deprotonation occurs, producing an electrostatic repulsive force and causing

Figure 11. Hemolytic test of BC/Am hydrogel where DW, NS, 20, 40, 80 mg/mL represents positive control of distilled water, negative control of normal saline, 20 mg/mL BC/Am hydrogel suspension, 40 mg/mL BC/Am hydrogel suspension, 80 mg/mL BC/Am hydrogel suspension in normal saline.

increased swelling. At pH 10, the sodium ion shields the charge of the carboxylate ion and reduces the effective repulsion force, resulting in decreased swelling.33−35 These observations may also be attributable to the reduction in the crystallinity of cellulose, as shown by the FTIR data. It is apparent that swelling increased rapidly from 4 to 25 °C but then increased gradually from 25 to 45 °C (Figure 5b(i) and (ii). This property can be attributed to an increase in the amount of bound fluid in the hydrogels. Comparable results were reported by Nizam El-Din et al. (2010)36 and Halib et al.

Figure 10. Live/dead staining of V79 fibroblast cell exposure to (a and c) control (DMEM) and (b and d) hydrogel for 24 h and 48 h, respectively. 3604



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Figure 12. (a) Mice body weight of control and hydrogel treated group during the observation period (n = 6). (b) Mice organs weight of control and hydrogel treated group (n = 6).

(2010),13 in which radiation polymerization was used to prepare hydrogels of Am/carboxymethylcellulose (CMC) and acrylic acid/BC, respectively. Nizam El-Din et al. (2010) reported that formulations containing derivatives of cellulose were responsible for thermosensitive behavior, and only the Am gel did not show temperature sensitivity.36 Therefore, the swelling behavior of our hydrogel may be attributable to the BC content, although this hypothesis requires further investigation. The swelling kinetics of hydrogels of different composition is shown in Table 1. For the cylindrical hydrogels with an n (power law diffusion exponent) value below 0.5, swelling showed diffusion control, whereas hydrogels with n values between 0.5 and 1 demonstrated an anomalous swelling mechanism.2 The R2 values revealed that the plots were linear (Table 1). On the other hand, the n values were all found to be above 0.5 in all hydrogels containing NaOH, indicating an anomalous swelling mechanism. However, swelling was diffusion-controlled in hydrogels without NaOH, as the n values were all below 0.5. Similarly, the rate constant of hydrogels containing NaOH was higher than those without NaOH in their reaction mixture. This may have been due to an increase in the repulsive forces between the carboxylate groups generated by hydrolysis (Figure 1) and the formation of a more relaxed network. 3.9. Rheological Analysis. Oscillatory frequency sweep test was performed at constant strain of 0.1% to determine the mechanical strength of hydrogels. Initially, to avoid overstrain which destroy the elastic structure of sample, stress sweep test was performed to determine the linear viscoelastic region (LVR). LVR for all hydrogels was found to be in between 0.01 to 1%. Considering this range, the strain was set to be at 0.1%, so that the physical structure of the hydrogel can be retained throughout the experiment. The mechanical spectra generated by frequency sweep test for all hydrogels (Figure 6a and c) showed that the storage (or elastic) modulus, G′ were always independent from loss (or viscous) modulus G″ and G′ is dominant over the entire frequency range (Figure 6a and c). Predominant elastic property of hydrogel is a typical behavior of strong gels.37 It was evident from Figure 6b, at frequency of 0.1 Hz, G′ decreased with increased in NaOH concentrations from 2% to 10% w/v in the reaction mixtures which indicate the reduction in mechanical stiffness of the hydrogels. In contrast, G′ remain unaltered in hydrogels containing various amount of urea (Figure 6d). As suggested by Marandi et al. (2008), this could be due to the decrease in cross-linking density in the hydrogel with the presence of alkali.27 3.10. Enzymatic Degradation. The in vitro degradation of hydrogels containing different ratios of NaOH/urea was

Figure 13. Photograph of the collected organs after oral administration of BC/Am hydrogel (2 g/kg b.w.). Mice liver, spleen, lung, stomach and kidneys photograph of control group (a, c, e, g, i,) and BC/Am hydrogel treated group (b, d, f, h, j), respectively (Scale bar = 200 μm).

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of the model drug via a swelling-controlled diffusion mechanism occurred concurrently. 3.12. Biocompatibility Test. For a polymer to be considered biocompatible it must be nonallergic, nontoxic, and noncarcinogenic. In vitro cytotoxicity and hemolytic tests are the main methods used for evaluating the biocompatibility of a polymer. On the basis of the results described above, hydrogel H8−4 was selected for further in vitro and in vivo biocompatibility tests. 3.12.1. Cytotoxicity Study. The cytotoxicity of the hydrogel was evaluated based on cell viability using V79 cells. As shown in Figure 9, the cell viability significantly decreased (P < 0.05) with increasing hydrogel concentration from 2 to 0.125 mg/ mL; conversely, cell viability increased with increase in incubation time with lower concentration (0.25 and 0.125 mg/mL) of hydrogel. Nonetheless, the cell viability was greater than 80% even when the input hydrogel concentration was increased from 2 to 0.125 mg/mL. This indicated that the hydrogel of Am grafted with BC was biocompatible with negligible cytotoxicity. Figure 10 depicts the viability and morphology of cells treated with a hydrogel sample and subsequently stained with calcein AM and EthD-1. There was no significant difference in cell morphology and viability in both the 24-h and 48-h hydrogel-treated cells compared to the control group. Most of the cells exhibited green fluorescence rather than red fluorescence (arrow indicates red fluorescence of dead cell), indicating that the hydrogel was nontoxic. 3.12.2. In Vitro Hemolytic Test. The hemocompatibility of the biomedical materials used for hemolysis was evaluated. As shown in Figure 11, the supernatant fraction of the hydrogel suspensions of different concentrations (20, 40, 80 mg/mL) showed slight differences in color compared with the negative control (normal saline). The mean hemolytic rate at the hydrogel concentration of 20, 40, and 80 mg/mL was 0.80 ± 0.013%, 0.98 ± 0.027%, and 1.22 ± 0.039%, respectively, which are all less than 2%, suggesting that the hydrogel suspension did not cause hemolysis at any concentration. 3.13. Acute Oral Toxicity. 3.13.1. General Conditions. During the period of acute oral toxicity, none of the animals of the hydrogel-treated group exhibited any sign of morbidity, and all animals survived during the observational period. The eyes, teeth, oral cavity, skin, and hair of mice were in normal condition. Similarly, the behavioral activities of the mice such as reaction to stimuli, breathing, movement, and secretion were also normal. In comparison to the control group, the treated mice showed the same reaction toward light, sound, and other stimulations. In addition, there was no sign of salivation or edema during the whole observation period. Furthermore, the feces from mice were normal without any signs of pus or blood. The food and water intake of treated mice was similar to the control group throughout the testing period, and there was no significant difference in body weights observed over 14 days between the treated and control mice (Figure 12a). The macroscopic appearance and weight (Figure 12b) of all organs evaluated were found to be normal. The acute oral toxicity test indicated that the BC-poly(acrylamide-sodium acrylate) hydrogel at a dosage of 2000 mg/kg did not cause any mortality and was therefore determined to be safe in vivo. 3.13.2. Histopathological Study. Organ samples were histopathologically observed under light microscopy, and no significant changes were detected between mice that received oral administration of hydrogel and controls. Figure 13a and b

investigated by weight loss in SIF with enzyme (pancreatin). The degradation patterns of hydrogels containing different concentrations of NaOH without urea are shown in Figure 7a, which illustrates that degradation was faster in the first week and proportional to the amount of NaOH added. The highest degradation rate (47.53%) was found in the hydrogel containing 10% w/v NaOH, and the lowest (29.40%) was observed in the hydrogel with 0% NaOH concentration in the fifth week. In general, the degradation of hydrogels depends on their cross-linking density and porosity. Densely cross-linked hydrogels resist the penetration of water or media (SIF) within the network, and hence degradation is reduced. Both the TGA and FTIR data confirmed that the hydrolysis of hydrogels increased with increasing alkali concentration, which caused a decrease in the cross-linking density. By contrast, increasing the urea concentration (from 0 to 6% w/v) increased the degradation of hydrogels, but not significantly, which is consistent with the TGA, gel fraction, and swelling data (Figure 7b). 3.11. In Vitro Drug Release. On the basis of appearance, homogeneity, and swelling, the hydrogels coded H6−4 and H8−4 were selected for further studies, whereas the hydrogel coded H10−4 was not selected due to its highly fragile nature after swelling. The entrapment efficiency of H6−4 and H8−4 was 40.4 ± 3.34% and 54.03 ± 4.80%, respectively, and the drug loading was 56.66 ± 4.35% and 81.56 ± 3.21%, respectively. These results indicated that the drug loading and entrapment efficiency depend on the porosity and swelling index of the gel. The in vitro release profile of the hydrogel principally depends on the percentage of swelling of the hydrogel in dissolution media, the solubility of the drug in media, and the interaction of the drug with the polymeric network. The release pattern was initially estimated in buffer of pH 1.5 for 2 h followed by pH 7.4, and then in buffer of pH 7.4 alone. The cumulative percentage release in phosphate buffer of pH 1.5 during the first 2 h in H8−4 and H6−4 was 13.32 ± 0.56% and 10.92 ± 0.83%, respectively, whereas the cumulative release in buffer of pH 7.4 for the first 2 h was 21.26 ± 0.67% and 19.42 ± 0.73%, respectively (Figure 8a, b). The same pattern was observed for total cumulative release, which is attributable to the swelling behavior of the hydrogel in varying pH. However, the total cumulative percentage release was lower in H6−4 than in H8− 4, which was expected due to the greater number of carboxylic groups and greater porosity. The drug release was characterized by a biphasic burst release pattern. This might have been due to leaching of the drug from the surface of the hydrogel followed by sustained release. Initially, rapid release was observed because the concentration gradient functioned as the driving force for release, followed by a reduced release rate due to depletion of the drug near the surface. The regression coefficient (R2) of the hydrogels (H6−4 and H8−4) was found to be 0.995 and 0.992, 0.921 and 0.894, and 0.940 and 0.942 for the zero-order, first-order, and Korsmeyer− Peppas models, respectively. The release kinetics of the hydrogels revealed that the release data of the hydrogels (H6−4 and H8−4) in buffer pH 1.5 fit best into the zero-order kinetic model, followed by the data for the pH 7.4 buffer. The release exponent values of the Korsmeyer−Peppas model (n) in H6−4 and H8−4 were 0.664 and 0.783, respectively, which is within the 0.45−0.89 range representing non-Fickian diffusion, indicating that absorption of the dissolution media and release 3606



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exhibit the light micrographs of livers treated without and with the hydrogel, respectively, and normal architecture of the liver was shown to be preserved. The central rein, sinusoids, and cords of hepatocytes and portal triads were also within normal limits. The microscopic image of the spleen showed extramedullary hemopoiesis but was otherwise within normal limits (Figure 13c and d). In addition, no ulceration or inflammatory cell infiltration was present in the lung (Figure 13f). The lung bronchus, bronchioles, and alveoli were within normal limits. Similarly, the stomach mucosa was intact and the muscular layer was normal (Figure 13g and h). The morphology of the kidney glomeruli was normal, and no basement membrane thickening or inflammatory cell infiltration was observed (Figure 13i and j). The tubules were normal and did not show any features of degeneration, although the interstitium was congested.

ASSOCIATED CONTENT

S Supporting Information *

Detail of 1H NMR spectra of hydrogel washing, to ensure the purity of hydrogel. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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4. CONCLUSIONS Here, we reported the characteristics and preliminary safety assessment of a newly synthesized hydrogel designed for application as an oral drug delivery carrier. The BC/Am hydrogel was synthesized by microwave irradiation using varying ratios of NaOH/urea to solvate BC, causing partial hydrolysis of polyacrylamide and assisting in the derivatization of BC, as confirmed by FTIR studies. The morphology of the hydrogel revealed greater transparency with increasing NaOH concentration. Swelling studies provided evidence of the pH and temperature sensitivity of the hydrogels. The drug release showed a biphasic burst release pattern due to leaching of the drug from the surface, followed by sustained release. The kinetics revealed that drug release followed non-Fickian diffusion, indicating that the absorption of the dissolution media and the release of the model drug via a swellingcontrolled diffusion mechanism occurred concurrently. In vitro cytotoxicity and hemolytic tests indicated that the BC/Am hydrogels are noncytotoxic and hemocompatible. The hydrogels appeared to be nontoxic up to 2000 mg/kg when administered orally, as no toxic response or histopathological changes were observed in hydrogel-treated mice in comparison to control mice. These results demonstrate that the developed pH-sensitive smart hydrogel is nontoxic and safe and may emerge as a better choice for oral drug delivery in the future.

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AUTHOR INFORMATION

Corresponding Author

*M. C. I. M. Amin. E-mail: [email protected]. Tel.: +603 9289 7690. Fax: +603 2698 3271. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Universiti Kebangsaan Malaysia (INOVASI-2013-005) and the Ministry of Higher Education, Malaysia (UKM-Farmasi-02-FRGS0192-2010) for their financial assistance and support. 3607



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