Designing a biocompatible hydrogel for the delivery of mesalamine

G Model

IJP 14978 1–10 International Journal of Pharmaceutics xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

1

Pharmaceutical nanotechnology

2

Designing a biocompatible hydrogel for the delivery of mesalamine

3 Q1

Lena Neufeld, Havazelet Bianco-Peled*

4

Department of Chemical Engineering, Technion–Israel Institute of Technology, Haifa 32000, Israel

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 March 2015 Received in revised form 15 June 2015 Accepted 18 June 2015 Available online xxx

A new design for nanocomposite hydrogels based on cross-linked chitosan for the delivery of mesalamine is presented. To enhance drug loading in chitosan, the mineral montmorillonite was incorporated into the matrix. The exfoliated silica montmorillonite nanosheets form interactions with both chitosan and mesalamine, which affect the hydrogel’s drug release mechanism and swelling properties. The impact of montmorillonite and glutaraldehyde concentrations on the hydrogel properties was investigated. In vitro drug-release studies detected slower release over short times when montmorillonite was introduced into the matrix. This study is the first to evaluate the influence of pH during mixing and on mixing duration. It was shown that lowering the pH during mixing delayed the release since the positively charged drug was better introduced between the montmorillonite layers, as confirmed by differential scanning calorimetry (DSC) and fourier transform infrared spectroscopy (FTIR) analysis. All hydrogels showed prolonged sustained release of mesalamine over 24 h in simulated colonic fluid (pH 7.4). When modeled, the mesalamine release profile suggests a complex release mechanism, involving adsorption of the drug to the montmorillonite and its diffusion. The results imply that chitosan– montmorillonite hydrogels can serve as potential drug carriers for controlled-release applications. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Chitosan Mesalamine (5-Aminosalicylic acid (5-ASA)) Montmorillonite Hydrogels

5

1. Introduction

6

Mesalamine (5-Aminosalicylic acid, 5-ASA) is a drug used extensively for long-term maintenance therapy in patients with mild to moderate inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis (Ritland et al., 1999). 5-ASA may additionally provide protection against the development of colorectal cancer. Numerous in vitro studies found that the beneficial effects of 5-ASA are related to its anti-inflammatory and anti-oxidant properties within the inflamed gut (Clemett and Markham, 2000). One major drawback of 5-ASA is its fast absorbance in the upper gastrointestinal tract, leading to a relatively small amount reaching the colon (Layer et al., 1995). Additionally, this treatment has gastrointestinal, hematological and general side effects. Agranulocytosis, toxic epidermal necrosis, paresthesia, hepatotoxicity, pancreatitis, pulmonary disease and male infertility have been reported (Omwancha et al., 2013). In order to maximize the efficacy of the drug and avoid adverse effects associated with systemic absorption, targeted release of 5ASA at the site of action, namely small bowel and/or colon, would be beneficial (Baumgart and Sandborn 2007). Many approaches aimed at the delivery of 5-ASA to the colon and circumventing the negative effects were suggested. Among these strategies, the use of

7 8 9 10 11 12 13 14 15 16 17 18 19 20 Q2 21 22 23 24 25 26

* Corresponding author. Fax: +972 4 829 672.

bacterial degradable polymers that exploit metabolism by the colonic microflora to release the drug (Davaran et al., 1999) seems to be suitable as a site-specific approach for colonic drug delivery (Sinha and Kumria, 2001). An example of such a bacterial degradable polymer is chitosan, a polysaccharide derived from the N-deacetylation and de-polymerization of chitin. Chitosan has many useful characteristics including nontoxicity, good biocompatibility and non-antigenicity, that make them good candidates for clinical use (Ravi Kumar 2000). Furthermore, chitosan has the potential to be used as an absorption enhancer across intestinal epithelial cells due to its mucoadhesive property and its utility as a permeability enhancer (Borchard et al., 1996). The latter characteristic arises from chitosan’s ability to open tight junctions between epithelial cells. The main limitation of chitosan as a carrier for 5-ASA is its short release times. As previously reported (Aguzzi et al., 2011), the release of 5-ASA from spray dried microspheres of chitosan was completed after one h in distilled water. Researchers have proposed nanocomposites based on montmorillonite (MMT) and chitosan as a way to combine the benefits of both as drug carriers (Aguzzi et al., 2010; Banik et al., 2012; Cojocariu et al., 2012; Hua et al., 2010; Liu et al., 2008; Wang et al., 2008). MMT is a clay commonly used as both an excipient and an active substance in pharmaceutical products due to its adsorption and drug-carrying capabilities (Joshi et al., 2009). MMT has negatively charged layers that allow small positively charged

http://dx.doi.org/10.1016/j.ijpharm.2015.06.026 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

G Model

IJP 14978 1–10 2 53

L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx

89

molecules to intercalate between the exfoliated layers (Sinha Ray and Okamoto, 2003). There are several mechanisms that may be involved in the interaction between clay minerals and organic molecules such as hydrophobic interactions, Van der Waals forces, hydrogen bonding, ionic interactions and water bridging (Aguzzi et al., 2007). When chitosan and MMT are mixed, the exfoliated silica clay nanosheets act as a cross-linker for chitosan, which, as a result, forms a network structure (Liu et al., 2008). In addition, the interactions between the clay and the drug may improve the encapsulation efficiency and sustain its release (Wang et al., 2008). Chitosan/MMT nanocomposites were shown to provide slower 5ASA release rates compared to chitosan alone. Yet, their ability to sustain the release of 5-ASA is still limited; drug release from compressed samples was completed within about 2 h in an acidic medium (Aguzzi et al., 2010). In light of potential advantages of chitosan/MMT nanocomposites, the aim of this study was to develop an approach that produce longer release times. The underlying hypothesis was that none of the mechanisms described above, which are responsible for sustained release of 5-ASA, namely, physical cross-linking of the polymer and drug encapsulation within MMT, are efficient enough. It is further assumed that since 5-ASA is a hydrophilic molecule, regulating its release solely based on electrostatic interactions with MMT is difficult. Moreover, the efficacy of crosslinking chitosan with MMT alone could be limited. Therefore, this research explores another approach, where chitosan is crosslinked with glutaraldehyde (GA), the most common cross-linking agent for this polymer (Muzzarelli and Pariser 1978). A systematic analysis of this new approach is presented. The effect of cross-linking density, MMT concentrations, pH of mixing and mixing duration before cross-linking on the potential of these hydrogels as a drug delivery system were investigated. It is demonstrated that sustained release over weeks can be obtained using these hydrogels. The design of biomedical hydrogels that are both biodegradable and offer sustained release could open the door to a new generation of cross-linked functional hydrogels.

90

2. Materials and methods

54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

91

2.1. Materials

92

106

5-Aminosalicylic acid (95% purity), montmorillonite K-10 and glutaraldehyde (GA) stock solution (50% (w/v)) were purchased from Sigma–Aldrich. Acetic acid (99.7% A.R.) was purchased from Gadot Biochemical Industries Ltd., Israel. Sodium hydroxide pearls and hydrochloric acid (32% A.R.) were purchased from Bio-Lab Ltd., Israel. Na2HPO4
12H2O and NaH2PO4
H2O were purchased from Merck KGaA, NaCl was purchased from Frutarom Ltd., Israel. All chemicals were of analytical grade and were used as delivered, without further purification. Double-distilled water (DDW) was used in all aqueous solutions. Low molecular weight chitosan was purchased from Sigma– Aldrich. The degree of deacetylation of the batch used in this study was determined in our previous work using FTIR and found to be 77%. The average molecular weight as determined by static light scattering was 207,000 g/mol (Keren Delmar 2015).

107

2.2. Hydrogel preparation

93 94 95 96 97 98 99 100 101 102 103 104 105

108 109 110 111 112 113

Chitosan hydrogels were prepared at a constant chitosan concentration and different MMT and GA concentrations. 0.5% (w/ v) chitosan solution in 2% (v/v) acetic acid was mixed with 0.5% (w/ v) 5-ASA solution. Different amounts of MMT powder were added to the mixture to obtain a concentration of 0.5% (w/v) or 1.5% (w/v) MMT. The acetic acid pH was adjusted to 5 with 5 M NaOH or to

1.4 with 1 M HCl. Next, 2.5 ml of acetic acid were poured into 8 ml glass vials and stirred at room temperature using a magnetic stirrer for 24 h or 2 h, depending on the experiment design. All solutions were freshly prepared, light-protected and nitrogen sealed to prevent the oxidative self-coupling of 5-ASA moieties (Jensen et al., 1992). Finally, the solution was mixed with 0.4% (v/v) or 0.2% (v/v) GA in order to achieve chemical cross-linking of chitosan. 300 ml of the final mixture were transferred into a PLA circular mold (diameter 14 mm, height 2 mm) to form hydrogels at room temperature.

114

2.3. Characterization of the hydrogels

124

2.3.1. Calibration curve of 5-ASA Known concentrations of 5-ASA in PBS 130 mM pH 7.4 were scanned in the range 200–400 nm by a UV–vis spectrophotometer (aSynergyTM HTBioTek1) (Cui et al., 2008). A sharp peak was noticed at 330 nm; however, this peak was only observed at the concentration range of 0–0.2344 mg/ml. Therefore, the peak at 350 nm, where 5-ASA can be detected at the broader concentration range of 0–0.3125 mg/ml, was used. The linear calibration curve was y = 5.8618  +0.0173 (R2 = 0.9995). Unknown concentrations of 5-ASA in drug release experiments were obtained by measuring the absorbance value at 350 nm.

125

2.3.2. Study of the drug-release rate from the hydrogels

136

2.3.2.1. Preparation of phosphate buffer saline (PBS). A phosphate buffer solution simulating intestinal fluids was prepared by dissolving 15.585 g Na2HPO4
12H2O (di-sodium hydrogen phosphate dodeca-hydrate) and 1.166 g NaH2PO4
H2O (sodium di-hydrogen phosphate) in 100 ml double-distilled water (DDW). 20 ml of this solution were mixed with 8.9 g NaCl (sodium chloride), the volume was raised to 1 L using DDW, and the pH was adjusted to 7.4 using 5 M NaOH.

137

2.3.2.2. In vitro drug release. Unless otherwise stated, releases experiments were performed by submerging as-prepared wet hydrogels in 20 ml glass vials containing 4.8 ml PBS buffer with ionic strength of 130 mM and pH 7.4. This release medium was selected in order to simulate intestinal fluids and maintain the average pH of the colon (Fadda et al., 2010). Release studies were performed at physiological temperature of 37  C in a bath shaken at 100 rpm. A pinch of the release medium (200 ml) was withdrawn at scheduled time intervals in order to determine the 5-ASA concentration using the UV–vis spectrophotometer reading at 350 nm and a calibration curve. 200 ml of PBS were returned to the vial to maintain a constant volume. All drug release experiments were performed in triplets and the data is shown as the average  standard deviation (SD). The in vitro drug release data were analyzed by fitting it to the Peppas model (Ritger and Peppas 1987):

145

Mt ¼ atn M1

115 116 117 118 119 120 121 122 123

126 127 128 129 130 131 132 133 134 135

138 139 140 141 142 143 144

146 147 148 149 150 151 152 153 154 155 156 157 158 159

Q3 160 161

(1)

where Mt is the drug release at time t, M1 is the maximal drug release, a is the release rate constant correlated to the diffusion coefficient and n is the release exponent, indicative of the drug release mechanism.

163 162 164

2.3.3. Thermal analysis via differential scanning calorimetry (DSC) DSC analysis was performed using a Q10, TA instrument. Physical mixtures of the compounds were mixed for 24 h in 2% acetic acid at pH 5, then dried overnight and ground to a powder. The samples (10–14 mg) were scanned in sealed aluminum pans.

167

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

165 166

168 169 170 171

G Model

IJP 14978 1–10 L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193

DSC termograms were obtained by scanning during the first heating cycle at 10  C/min in a temperature range of 50–350  C. 2.3.4. Fourier transform infra red spectroscopy (FT-IR) study FT-IR spectra were recorded using a Nicolet 6700 FTIR (Thermo Scientific) coupled to a liquid nitrogen cooled mercury–cadmium– telluride (MCT) detector, in attenuated total reflectance (ATR) mode. Physical mixtures were mixed for 24 h in 2% acetic acid pH 5, then dried overnight and ground to a powder. Spectra were collected in the range of 4000–600 cm1. 2.3.5. Morphological studies 1.5% (w/v) MMT were mixed with 2.5 ml 2% acetic acid pH 5 in 8 ml glass vials and stirred using a magnetic stirrer for 2 h or for 24 h at room temperature. Visualization of MMT particles was performed using an optical microscope system Nikon eclipse TS100 with S Plan Flour lenses microscope system. Image analysis of digital phase-contrast micrographs was performed by digital sight DS-U3 and NIS Elements v.4.10 software. 2.3.6. Molecular weight determination The average molecular weight of chitosan was calculated from its intrinsic viscosity using the Mark–Houwink equation (Stephen and Phillips 2010): ½h ¼ 8:43  103 Mw0:92

195 194 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211

where Mw is the viscosity average molecular weight of the polymer and [h] is the intrinsic viscosity. A Ubbelohde viscometer was used to determine the specific viscosity [hsp] and the relative viscosity [hr] of dilute chitosan solutions in 2% acetic acid at 25  C. The intrinsic viscosity was determined by extrapolating the [hsp]/C vs. C curve and the ln ([hr])/C vs. C curve to zero, and averaging the value of the intercept.

215 216 217 218 219

223 224 225 226 227

228

2.4. Statistical analysis

234

Data from independent experiments were analyzed for each variable using Microsoftã Excel. Comparisons between two treatments were made with Student’s t-test analysis using a two-tail distribution. A p-value of <0.05 was considered to be statistically significant and set at probabilities of *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

235

3. Results and discussion

241

3.1. Drug-release studies

242

3.1.1. Effect of cross-linker concentration Fig. 1 demonstrates the effect of cross-linker concentration and inclusion of MMT in the formulation on 5-ASA release patterns at pH 7.4. Since the maximal time a drug can stay in the colon is 24 h (Philip and Philip 2010), only the first day is presented; the data were normalized to the release at the end of day one. Without cross-linker or MMT, 90% of the drug is released immediately, and complete release is achieved after 1 h. These results are in line with the findings of Aguzzi et al. (2011). For both hydrogels that contain MMT (Fig. 1B) and hydrogels that do not (Fig. 1A), the release rate was reduced considerably and the release within short times (first 15 min) was smaller when the chitosan was cross-linked with GA.

243

100%ðw  w0 Þ w0

(3)

where w0 is the initial weight of the hydrogel before swelling and w is the weight of the hydrogel at any other time t. All swelling experiments were performed in quartets and the data is shown as the average  SD. 2.3.8. Mechanical characterization A Lloyd mechanical testing machine was used to determine the hydrogel’s Young’s modulus E, defined as: E¼

221 220 222

determined by recording the location of the sample holders at the zero strain, which is the initial point at which the force values start to increase during compressive loading. The Young’s modulus was calculated from the linear region of the stress–strain curve, as the slope between 1–10% strain (Krupa et al., 2010). Experiments were performed in quadruplicate.

2.3.7. Swelling and weight loss investigation Swelling studies were performed in a covered Petri dish containing 15 ml of PBS buffer with ionic strength of 130 mM and pH 7.4. Hydrogels were placed on a metal net and submerged in the buffer. Water uptake was determined by measuring the change in the hydrogel weight at scheduled time intervals after gently staggering excess water. The hydrogels were returned immediately to the petri dish. The swelling percentage (%Q) was calculated using the relation: %Q ¼

213 212 214

(2)

3

s e

(4)

where s is the stress applied on the sample and e is the strain in the linear region of the stress–strain curve. In practice, the Young’s modulus was experimentally determined by measuring the slope of the compressive stress–strain curve in the linear region. Sample preparation was the same as described in Section 2.2. The samples were compressed at a rate of 1 mm/min with a compressive displacement of up to 1.5 mm. The initial length of the sample was

Fig. 1. Comparison between 5-ASA release from (A) chitosan (B) chitosan hydrogels containing 1.5% (w/v) MMT cross-linked with different GA concentrations (^) 0.4%, (&) 0.2% and (~) 0%. All samples were mixed for 24 h prior to cross-linking.

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

229 230 231 232 233

236 237 238 239 240

244 245 246 247 248 249 250 251 252 253 254

G Model

IJP 14978 1–10 4 255 256 257 258 259

L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx

These results support the hypothesis that cross-linking the chitosan will reduce the release rates. Hydrogels cross-linked using the higher GA concentrations show a slower release rate and a smaller release at short times when MMT was combined.

283

3.1.2. Effect of MMT concentration Fig. 2 compares 5-ASA release patterns from chitosan hydrogels cross-linked with either 0.2% or 0.4% GA. When 0.2% GA is used, 5ASA release reaches a plateau after about 2.5 h (Fig. 2A). The release does not stabilize during the first 24 h at the higher cross-linking density of 0.4% GA (Fig. 2B). After 8 h, all hydrogels cross-linked with 0.2% GA released approximately 65% of the drug, while hydrogels cross-linked with 0.4% GA released approximately 45% of the drug. The longer release times at higher GA content can be attributed to the denser chitosan network that limits the drug diffusion. For hydrogels cross-linked with 0.2% GA, the release at short times is smaller when 0.5% (w/v) MMT is included in the formulation; however, the percentage of 5-ASA released after 24 h does not seem to depend on MMT presence or its concentration. Similarly, for hydrogels cross-linked with 0.4% GA, at the end of the first day, the release patterns obtained for the chitosan alone, chitosan with 0.5% (w/v) MMT or 1.5% (w/v) MMT are similar. Previous studies found that including MMT in the formulation decreased drug release rates (Aguzzi et al., 2010; Hua et al., 2010; Liu et al., 2008). One possible explanation for the low sensitivity to MMT concentration is limited interaction between MMT and 5ASA. This scenario will be discussed further in Section 3.1.3. Even though we present only the release on the first day (Fig. 2), the release continued slowly over almost 21 days (see Supplementary data).

284

3.1.3. The effect of the pre-cross-linking mixing conditions

285

3.1.3.1. The effect of pH. The interactions between 5-ASA and MMT can strongly affect the release profiles. According to Sinha Ray and Okamoto (2003), small, positively charged molecules such as 5-

260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282

286 Q4 287

Fig. 2. Comparison between chitosan and chitosan/MMT hydrogels cross-linked with different GA concentrations and mixed for 24 h prior to cross-linking of (A) 0.4% GA (B) 0.2% GA. (^) chitosan hydrogels (&) chitosan hydrogels containing 0.5% (w/v) MMT and (~) chitosan hydrogels containing 1.5% (w/v) MMT.

ASA can intercalate between the negatively charged MMT layers, leading to slower release. The charge carried by 5-ASA depends on its pKa1 (2.3  0.09) and pKa2 (5.69  0.04) values (French and Mauger, 1993) and the pH of the environment. The experiments described in Section 3.1.2 were performed with hydrogels prepared from chitosan/MMT/5-ASA mixtures in acetic acid at pH 5. This pH was chosen for the cross-linking reaction since GA only reacts with the primary amine groups (NH2) of chitosan (Genta et al., 1998; Mi et al., 2000) in acidic solutions at pH values from 4 to 5, close to the pKa of chitosan ranging, from 6.1 to 7 (Sorlier et al., 2001). Kildeeva et al. (2009) concluded that gel formation is faster at pH 5 since more charged amino (NH3+) groups turn into amine (NH2) groups at this pH value than in pH 4. At pH 5, 5-ASA is primary in its zwitterionic state, and probably is less easily intercalated between the MMT layers. Mixing 5-ASA with MMT at pH lower than its pKa’s is thus expected to enhance the positive charge carried by 5-ASA and can potentially lead to stronger ionic interaction between the drug and MMT. It can be further anticipated that when the hydrogel is transferred to a PBS solution with a pH of 7.4, 5-ASA will become negatively charged, allowing it to leave the MMT. In order to study the effect of pH during MMT and 5-ASA mixing and examine the above hypothesis, MMT and drug were added to a chitosan solution in acetic acid at a pH of either 1.4 or 5.0 and mixed for 24 h. For samples that were mixed at a pH of 1.4, the pH was adjusted to 5 immediately prior to cross-linking to allow the reaction to occur. Chitosan hydrogels without MMT were prepared using the same procedure, as a control. The release from hydrogels pre-mixed at pH 5 showed only a slight difference between chitosan—only hydrogels and those containing MMT. There was approximately 20% quickly, followed

Fig. 3. Comparison between 5-ASA release from (^) chitosan hydrogels crosslinked with 0.4% GA and (&) chitosan hydrogels containing 1.5% (w/v) MMT and cross-linked with 0.4% GA. All solutions were mixed for 24 h before cross-linking at (A) pH 5 (B) pH 1.4.

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

288

Q5 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317

G Model

IJP 14978 1–10 L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352

by a slower release rate (Fig. 3A), reaching approximately 50% after 24 h. However, release of 5-ASA was slower from hydrogels prepared after mixing with chitosan and MMT at pH 1.4 (Fig. 3B), reaching approximately 30% after 7 h. This slower release rate might suggest that 5-ASA has better intercalated between the MMT layers. The hydrogels demonstrated more sustained release since it is now harder for the drug to embark the MMT layers and the polymeric network. Thus manipulating the pH of the solution during mixing influences the release profiles. In order to further examine the above mechanism and gain insights into the interactions among 5-ASA, chitosan and MMT, DSC analysis of the pure components and their mixtures was performed (Fig. 4). The thermogram of 5-ASA in pH 1.4 is characterized by a sharp endotermic peak at 270  C, which corresponds to its melting point, accompanied by an exothermic peak related to drug decomposition (Aguzzi et al., 2011). This latter peak shifts to 280  C in pH 5, indicating possible hydrogen bonds between the drug’s charged amino groups and charged carboxylic group. MMT at pH 1.4 showed a single broad endotermic band from 70  C to 130  C, corresponding to the loss of free water molecules from the clay surface. This band is smaller in pH 5, indicating that a lower heat flow is required for the evaporation of water. The calorimetric curve of chitosan in pH 1.4 is typical of an amorphous hydrated compound (Mura et al., 2011), with a wide endothermic band from 60  C to 150  C, corresponding to the dehydration of the polymer, followed by an exothermic phenomenon around 220  C due to the decomposition of the polymeric chains. However, in pH = 5, the endothermic peak is relatively smaller, indicating that a lower heat flow is required for the dehydration process. This effect can be attributed to the lower amount of water adsorbed by the chitosan chains in pH 5 where it is less hydrophilic than in pH 1.4. The exothermic peak is larger in pH 1.4 than in pH 5, since polysaccharides such as chitosan tend to display accelerated decomposition in acidic media (Il’Ina and Varlamov, 2004).

Fig. 4. DSC thermograms of 5-ASA, MMT, chitosan and their mixtures at pH 1.4 and pH 5.

5

The melting peak of 5-ASA disappeared when 5-ASA was mixed with MMT or with chitosan in pH 1.4, indicating solid-state interactions between the drug and the MMT and between the drug and the chitosan (Mura et al., 2012). In pH 5, the melting peak shifts but does not disappear, indicating weaker interactions between the components at this pH. To further investigate the interactions between 5-ASA, chitosan and MMT, FT-IR spectra of the individual components and their mixtures were acquired (Fig. 5). The interactions between 5-ASA and MMT were previously investigated (Zou et al., 2007); however, the influence of pH on MMT/5-ASA and chitosan/MMT interactions was not explored. IR spectra of pure 5-ASA show a broad adsorption band at 2849 cm1, assigned to the stretching mode of OH groups (Fig. 5A). The two bands attributed to the antisymmetric and symmetric stretching modes of COOH groups are seen around 1634 cm1 and 1352 cm1 at pH 1.4, and around 1646 cm1 and 1376 cm1 at pH 5. The band around 1247 cm1 at pH 1.4 and around 1238 cm1 at pH 5 is due to the amide III (Orioli et al., 2004) bands (amide I and amide II bands were overlapped by the anti-symmetric stretching mode of COOH and by the deformation mode of interlayered water). IR spectra of MMT show an absorption band at 3360 cm1, which is attributed to the stretching vibration of Al OH (Fig. 5A). The symmetrical SiO Si band is characterized by the stretching band at 1028 cm1 at pH 1.4 and around 1015 cm1 at pH 5 (Salahuddin and Abdeen 2013). Fig. 5B shows the IR spectra of 5-ASA/MMT physical mixtures at pH 1.4 and pH 5 in the range of 700–1700 cm-1. The two bands of 5ASA around 1634 cm1 and 1352 cm1, which are associated with the anti-symmetric and symmetric stretching modes of COOH groups (Orioli et al., 2004), remain unchanged, indicating that there are no hydrogen bond interactions between COOH groups of the drug and MMT layers at pH 1.4. Moreover, there is no shift at the C¼C stretching modes of the aromatic ring in 5-ASA. However, the amide III band at 1247 cm1 shifts to a lower wave number of about 1210 cm1, implying that the amino group is involved in the hydrogen bond with the MMT layers. At pH 5, the anti-symmetric and symmetric stretching modes of COO shift to lower wave numbers of 1558 cm1 and 1346 cm1, suggesting that hydrogen bonds interact with 5-ASA and the hydroxyl groups of MMT layers. Moreover, there is a shift to a lower wave number at the C¼C stretching modes (1446 cm1 shifts to 1415 cm1), indicating the possible occurrence of interactions through p–p conjugation of the phenyl groups of interlayer 5-ASA ions with MMT layers. The band at 1238 cm1 (due to the amide III), however, shifts only slightly to a lower wave number of 1230 cm1. The smaller shift at pH 5 compared to pH 1.4 can be attributed to the fact that at pH 5, there are less protonated NH3+ groups carried by 5-ASA available for interaction with MMT. Thus, at pH 1.4 the interactions between 5-ASA and MMT are indeed stronger, presumably leading to the slower release rate observed when the components were mixed at this pH. Fig. 5B shows the IR spectra of 5-ASA/chitosan mixtures at pH 1.4 and pH 5. At pH 1.4, the two bands of 5-ASA associate with the anti-symmetric and symmetric stretching modes of COOH groups; the peak of C¼C stretching and the amine III peak remain unchanged, indicating that there are no hydrogen bond interactions between COOH groups of the drug and the chitosan at pH of 1.4. In contrast, at pH 5 there is a shift to lower wave numbers in the anti-symmetric stretching modes of COO (1556 cm1), indicating strong hydrogen bond interactions between 5-ASA and the chitosan. Further, the band at 1238 cm1 (due to the amide III) shifts slightly to a lower wave of 1230 cm1, implying that the amino group is involved in the hydrogen bond with the chitosan. The interaction between 5-ASA and chitosan at a pH of 5 may be

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417

G Model

IJP 14978 1–10 6

L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx

Fig. 5. FT-IR spectra of (A) 5-ASA, MMT and chitosan at pH 1.4 (B) 5-ASA + MMT and 5-ASA + chitosan at pH 1.4 and pH 5. 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

responsible in part for the very long release times (see Supplementary data). 3.1.3.2. The effect of mixing time. The kinetics of 5-ASA intercalation into MMT has not been studied in the past, and therefore, deciding how much time is required for MMT/drug contact is not straightforward. It can be assumed that short contact would not allow enough time for interaction between the components, thus leading to fast release. On the other hand, additional time after reaching equilibrium adsorption is not expected to have an effect on the drug release from chitosan/ MMT nanocomposites. To study the effect of mixing time, two sets of hydrogels were prepared; in one, solutions were pre-mixed for 2 h, and in the second, solutions were pre-mixed for 24 h prior to cross-linking. Each set contained hydrogels from chitosan alone, chitosan with 0.5% (w/v) MMT and chitosan with 1.5% (w/v) MMT. Surprisingly, the control chitosan hydrogels, which were premixed for only 2 h, released the drug more slowly than the ones pre-mixed for 24 h (Fig. 6A). The shorter mixing time led to maximal release of approximately 45% in 24 h—7% lower than the maximal release obtained from hydrogels pre-mixed for a longer time. One possible reason for the enhanced drug release rate is degradation of the chitosan due to hydrolysis, leading to shorter chains after long mixing, a less organized network structure and larger average mesh size. In order to test this suggestion, the average molecular weight of chitosan was determined. The molecular weight of chitosan mixed for 2 h was found to be 124  1.6 kDa, whereas after mixing for 24 h, the molecular weight decreased to 119  1.2 kDa. The reduction in the molecular weight is statistically significant (p < 0.05). In formulations containing chitosan and 0.5% (w/v) MMT, the drug release rate was not dependent on the mixing time (Fig. 6B). Since in this case the chitosan is also degraded, one would expect to see slower release in samples that were pre-mixed for 2 h as well. The experimental data show that this is not the case. Adding a mineral to the formulation that had been pre-mixed for a short time enhanced the release rate to a level similar to the one obtained for the formulation pre-mixed for 24 h. Similar and even more pronounced results were observed for samples containing

Fig. 6. Comparison between (A) chitosan hydrogels (B) chitosan hydrogels containing 0.5% (w/v) MMT (C) chitosan hydrogels containing 1.5% (w/v) MMT cross-linked with 0.4% GA after (^) 24 h of mixing and (&) 2 h of mixing.

1.5% (w/v) MMT (Fig. 6C) where the hydrogel pre-mixed for only 2 h displayed a greater release quickly and a faster overall release rate compared to the hydrogel that was pre-mixed for 24 h. Faster release after 2 h of pre-mixing may occur because of insufficient interactions between MMT and the drug, as initially

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

456 457 458 459 460

G Model

IJP 14978 1–10 L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476

suggested. Another mechanism that might be involved is interactions between the mineral and the polymer. As previously suggested (Liu et al., 2007; Liu et al., 2008), chitosan chains intercalate between MMT layers; these chains act as cross-linking points. It is possible that after a short mixing time, fewer polymer/ MMT connections form and the network is less dense, allowing faster release of the drug. Yet another effect may be the result of changes in the clay itself. Based on the appearance of the hydrogels, it was speculated that the size of MMT particles changes during the mixing procedure. Fig. 7 displays typical light microscopy images supporting this suggestion. It is evident that after long mixing, the MMT particles were smaller compared to their size after only 2 h of mixing. It is possible that the larger surface/area ratio resulting from the smaller particle size after long mixing contributes to more efficient drug/mineral interactions and slower release rates.

497

3.1.4. Effect of pH during in vitro release experiments Since chitosan is positively charged at low pH values, it is expected to swell and display considerably slower release rates in stomach conditions. To examine the release behavior under conditions mimicking transport of a dosage form in the GI track, experiments in which the hydrogel was immersed in stomach pH of 1.2 for 2 h and then transferred to PBS at pH 7.4 were performed. The results, presented in Fig. 8, confirm that the release in pH 1.2 was faster; 35.0% of the drug were released from chitosan hydrogels during the first 2 h, whereas 27.6% was released from hydrogels containing chitosan and 1.5% (w/v) MMT. The release curve plateaued after less than within 3 h for both formulations. The limitation of fast release in the stomach can be resolved by using polymers such as Eudragit1 S as protective coating (Kleinubing et al., 2014; Palugan et al., 2015; Tummala et al., 2015). This approach has been widely investigated and was shown to be efficient in in procuring site-specific release, as recently reviewed (Palugan et al., 2015). Coating will allow the pill to arrive intact to the colon where other benefits of the device, including sustained release in colon pH and chitosan’s ability to act as absorption enhancer across intestinal epithelial cells, could be realized.

498

3.2. Swelling and weight loss investigation

499

Swelling and collapse of a hydrogel network is one of its important characteristics. Swelling is driven by the favorable free energy of mixing with a solvent driven in part by the entropy of counterions wishing to expand the polymer phase to maximize the entropy (Sing et al., 2013). The swelling is resisted by the energy required to stretch network strands. The swelling reaches an

477 478 479 480 481 482 483 484 485 486 487 488 489 Q6 490 491 492 493 494 495 496

500 501 502 503 504

7

Fig. 8. Equilibrium de-swelling degree of hydrogels with 0.4% GA with and without 5-ASA at different MMT concentrations (A) 0% (B) 0.5% (C) 1.5% (w/v).

equilibrium state when the osmotic and elastic parts of the free energy balance (Bajpai and Giri 2003; Rubinstein and Colby 2003). The swelling ability of the hydrogels was examined in PBS pH 7.4 at room temperature. A kinetic study showed that most hydrogels reach an equilibrium state within approximately 4 h (data not shown). All hydrogels dehydrate; meaning that their weight at equilibrium was lower than their initial weight. This phenomenon may be referred to as de-swelling. Tanaka (2000) observed that cationic polyelectrolytes can undergo dramatic volume changes upon pH and ion concentration changes. The hydrogels under study were prepared in acetic acid 2% at pH 5, below chitosan’s pKa; therefore, the chains carried positively charged amino groups. The swelling experiments were performed at pH 7.4 in PBS solution in which the chitosan becomes uncharged and less soluble. The reduction in electrostatic repulsion shrinks the hydrogel. Note that de-swelling of chitosan in PBS was previously reported as a reversible phenomenon (López-León et al., 2005).

Fig. 7. Light microscopy images 10 mm of 1.5% (w/v) MMT in 2% acetic acid pH 5 after (A) 2 h of mixing (B) 24 h of mixing.

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521

G Model

IJP 14978 1–10 8

L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx Table 1 n, a and R2 values obtained from the release profiles. Sample CS CS + 0.5% MMT CS + 1.5% MMT

Fig. 9. Comparison between 5-ASA release from (^) chitosan hydrogels crosslinked with 0.4% GA and (&) chitosan hydrogels containing 1.5% (w/v) MMT and cross-linked with 0.4% GA. To simulate transport in the GI system, the release was performed in stomach pH of 1.2 for the first 2 h, and then continued at pH 7.4. 522

550

Interestingly, chitosan hydrogels containing 5-ASA dehydrated less than hydrogels without 5-ASA (Fig. 9A). The drug is negatively charged at pH 7.4; therefore, it is likely that it increases the osmotic pressure inside the hydrogel, limiting its dehydration compared to chitosan only hydrogels. Similarly, chitosan hydrogels containing 5-ASA and either 0.5% (w/v) MMT (Fig. 9B) or 1.5% (w/v) MMT (Fig. 9C) dehydrated less than identical hydrogels without the drug. This finding indicates that although part of the drug adsorbs on the mineral, some remains as solubilized molecules. These results are in line with the DSC and FTIR studies, implying that the drug interacts with MMT and chitosan. The effect of 5-ASA on dehydration is less pronounced when the mineral concentration is higher. This result is expected since a larger percentage of the drug is engaged with MMT. The influence of MMT on swelling is less straightforward. It has been suggested that MMT is capable of forming a chitosan network due to its interaction with the polymer (Liu et al., 2007; Liu et al., 2008). It can be expected that these additional cross-linking points will reduce the swelling at equilibrium, thus the dehydration is expected to be larger compared to the chitosan - only hydrogels. Further, de-swelling is expected to increase with MMT concentration. The experimental data shown in Fig. 9 deviate to some extent from this expectation. For chitosan hydrogels pre-mixed for 24 h, the dehydration is lowered by the addition of 0.5% (w/v) MMT but is not affected by the addition of 1.5% (w/v) MMT. For chitosan hydrogels pre-mixed for 2 h, the dehydration is not affected by the addition 0.5% (w/v) MMT and is lowered by the addition of 1.5% MMT. Nevertheless, increasing the concentration does lead to higher shrinkage, regardless of the pre-mixing time.

551

3.3. Quantitative analysis of the drug release kinetics

552

The in vitro drug release data presented in the previous sections were analyzed by fitting them to the Peppas model (Eq. (1)). For thin films, a value of n = 0.45 is correlated with Fickian release. n values obtained from all hydrogels were between 0.3 and

523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549

553 554 555

% GA Mixing time

0.4% GA n a

R2

0.2% GA n a

R2

24 h 2h 24 h 2h 24 h 2h

0.223 0.295 0.292 0.283 0.296 0.142

0.998 0.998 0.997 0.995 0.999 0.988

0.268 0.128 0.313 0.128 0.184 0.147

0.877 0.848 0.946 0.920 0.929 0.874

0.088 0.039 0.042 0.045 0.047 0.246

0.063 0.232 0.038 0.323 0.181 0.249

0.15 (Table 1), lower than 0.45, which is the value expected for classical Fickian behavior. Several reports found n values lower than 0.45 and attributed them to a diffusion controlled mechanism (Khamanga and Walker, 2012; Lavanya, et al., 2015; Mathew et al., 2007). However, since the drug in this study interacts with both the polymer and the mineral, it is suggested that the low value of n hints at a more complex release mechanism, involving adsorption of the drug to the MMT and diffusion. The values of the rate parameter a are shown in Fig. 10. The release from hydrogels cross-linked with 0.2% GA was fast, since the mesh size is higher when a low content of cross-linker is used. Hence, it is hard to claim that there were unambiguous differences between the release rates. In general, when the composites were mixed for only 2 h, highest release rates compared to samples premixed for 24 h were achieved. For hydrogels cross-linked with 0.4% GA, there is no inconclusive difference in a values between different mixing times, except with hydrogels that contained 1.5% (w/v) MMT and were mixed for 2 h. The high a value is due to the MMT particle size, which is larger in samples mixed for 2 h and smaller in samples mixed for 24 h. These findings are supported by morphological studies. The large MMT particles disrupt the crosslinking procedure and a high release rate was obtained even when a doubled GA concentration was used. This effect wears off when samples are mixed for 24 h.

Figure 10. Rate constant a obtained from the release profiles.

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579

G Model

IJP 14978 1–10 L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx 580

3.4. Mechanical properties

581

591

The compression modulus of control chitosan hydrogels with 5ASA cross-linked with 0.4% GA was found to be 5.2  0.7 kPa. Formulations with 0.5% (w/v) MMT had a modulus of 5.4  1.3 kPa, which is not statistically different than that of chitosan alone. However, addition of 1.5% (w/v) MMT resulted in an increased value of 7.6  2.2 kPa. The enhanced rigidity of this formulation can be attributed to the mineral acting both as filler and as an additional crosslinker. The observed compression modulus for both chitosan and chitosan/MMT hydrogels are comparable to those reported for other chitosan based hydrogels for drug delivery applications (Cheng et al., 2014; Tronci et al., 2014; Zan et al., 2006).

592

4. Conclusions

593

614

A new system for colonic delivery of 5-ASA, based on chitosan hydrogels cross-linked with glutaraldehyde (GA) in the presence of MMT, was investigated. In vitro drug-release studies showed that hydrogels cross-linked using higher GA concentrations showed a slower release rate owing to smaller mesh size. Slower release at short times resulted when MMT was introduced; however, the sensitivity of the release rate to MMT concentration was low. Lowering the pH during mixing delayed the release since the positively charged drug was better introduced between the MMT layers, as confirmed by DSC and FTIR analysis. The pre-mixing time prior to cross-linking affected the release rates as well. Faster release was observed after 2 h of pre-mixing, which may result from insufficient interactions between MMT and the drug, and the larger size of MMT particles. The drug release profiles were analyzed using the Peppas model for thin film. n values were lower than 0.45, which is the value expected for classical Fickian behavior. This finding hints at a more complex release mechanism, involving adsorption of the drug to the MMT and diffusion. A kinetic swelling study showed that the hydrogels dehydrated and reached an equilibrium state within approximately 4 h. It appears that increasing the MMT concentration leads to higher shrinkage, regardless of the pre-mixing time.

615 Q7

Uncited references

582 583 584 585 586 587 588 589 590

594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613

616

Morro and Müller (1988).

617

Acknowledgements

618 619

The authors wish to thank Dr. Shlomit Avidan and Mrs. Maayan Biton for their help with the mechanical characterization.

620

Appendix A. Supplementary data

621 623

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.06.026.

624

References

625 626 627 628 629 630 631 632 633 634 635 636

Aguzzi, C., Capra, P., Bonferoni, C., Cerezo, P., Salcedo, I., Sánchez, R., Caramella, C., Viseras, C., 2010. Chitosan–silicate biocomposites to be used in modified drug release of 5-aminosalicylic acid (5-ASA). Appl. Clay Sci. 50, 106–111. Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: possibilities and limitations. Appl. Clay Sci. 36, 22–36. Aguzzi, C., Ortega, A., Bonferoni, M., Sandri, G., Cerezo, P., Salcedo, I., Sánchez, R., Viseras, C., Caramella, C., 2011. Assessement of anti-inflammatory properties of microspheres prepared with chitosan and 5-amino salicylic acid over inflamed Caco-2 cells. Carbohydr. Polym. 85, 638–644. Bajpai, A., Giri, A., 2003. Water sorption behaviour of highly swelling (carboxy methylcellulose-g-polyacrylamide) hydrogels and release of potassium nitrate as agrochemical. Carbohydr. Polym. 53, 271–279.

622

9

637 Banik, N., Hussain, A., Ramteke, A., Sharma, H.K., Maji, T.K., 2012. Preparation and 638 evaluation of the effect of particle size on the properties of chitosan639 montmorillonite nanoparticles loaded with isoniazid. RSC Adv. 2, 10519–10528. 640 Baumgart, D.C., Sandborn, W.J., 2007. Inflammatory bowel disease: clinical aspects 641 and established and evolving therapies. Lancet 369, 1641–1657. 642 Borchard, G., Lueben, H.L., de Boer, A.G., Verhoef, J., Lehr, C., Junginger, H.E., 1996. 643 The potential of mucoadhesive polymers in enhancing intestinal peptide drug 644 absorption. III: Effects of chitosan-glutamate and carbomer on epithelial tight 645 junctions in vitro. J. Control. Release 39, 131–138. 646 Cheng, Y., Nada, A.A., Valmikinathan, C.M., Lee, P., Liang, D., Yu, X., Kumbar, S.G., 2014. In situ gelling polysaccharide-based hydrogel for cell and drug delivery in Q8 647 tissue engineering. J. Appl. Polym. Sci. 131. 648 Clemett, D., Markham, A., 2000. Prolonged-release mesalazine. Drugs 59, 929–956. 649 Cojocariu, A., Porfire, L., Cheaburu, C., Vasile, C., 2012. Chitosan/montmorillonite 650 composites as matrices for prolonged delivery of some novel nitric oxide donor 651 compounds based on theophylline and paracetamol. Cellul. Chem. Technol. 46, 652 35. 653 Cui, F., Qin, L., Li, F., Luo, H., 2008. Synchronous fluorescence determination and 654 molecular modeling of 5-aminosalicylic acid (5-ASA) interacted with human 655 serum albumin. J. Mol. Model. 14, 1111–1117. 656 Davaran, S., Hanaee, J., Khosravi, A., 1999. Release of 5-amino salicylic acid from 657 acrylic type polymeric prodrugs designed for colon-specific drug delivery. J. 658 Control. Release 58, 279–287. 659 Fadda, H., Sousa, T., Carlsson, A., Abrahamsson, B., Williams, J., Kumar, D., Basit, A., 2010. Drug solubility in luminal fluids from different regions of the small and 660 large intestine of humans. Mol. Pharmaceutics 7, 1527–1532. 661 French, D.L., Mauger, J.W., 1993. Evaluation of the physicochemical properties and 662 dissolution characteristics of mesalamine: Relevance to controlled intestinal 663 drug delivery. Pharm. Res. 10, 1285–1290. 664 Genta, I., Costantini, M., Asti, A., Conti, B., Montanari, L., 1998. Influence of 665 glutaraldehyde on drug release and mucoadhesive properties of chitosan 666 microspheres. Carbohydr. Polym. 36, 81–88. 667 Hua, S., Yang, H., Wang, W., Wang, A., 2010. Controlled release of ofloxacin from 668 chitosan–montmorillonite hydrogel. Appl. Clay Sci. 50, 112–117. 669 Il’Ina, A., Varlamov, V., 2004. Hydrolysis of chitosan in lactic acid. Appl. Biochem. 670 Microbiol. 40, 300–303. 671 Jensen, J., Cornett, C., Olsen, C.E., Tjornelund, J., Hansen, S.H., 1992. Identification of 672 major degradation products of 5-aminosalicylic acid formed in aqueous 673 solutions and in pharmaceuticals. Int. J. Pharm. 88, 177–187. 674 Joshi, G.V., Kevadiya, B.D., Patel, H.A., Bajaj, H.C., Jasra, R.V., 2009. Montmorillonite as 675 a drug delivery system: intercalation and in vitro release of timolol maleate. Int. 676 J. Pharm. 374, 53–57. Keren Delmar, H.B., 2015. The dramatic effect of small pH changes on the properties Q9 677 678 of chitosan hydrogels crosslinked with genipin. Carbohydr. Polym. (in press). Khamanga, S.M., Walker, R.B., 2012. In vitro dissolution kinetics of captopril from Q10 679 680 microspheres manufactured by solvent evaporation. Dissolut. Technol. 42–51. 681 Kildeeva, N., Perminov, P., Vladimirov, L., Novikov, V., Mikhailov, S., 2009. About 682 mechanism of chitosan cross-linking with glutaraldehyde. Russ. J. Bioorg. Chem. 683 35, 360–369. 684 Kleinubing, S.A., Seraphim, D.C., Vieira, M.G.A., Canevesi, R.L.S., da Silva, E.A., César, C.L., Mei, L.H.I., 2014. Gastro-resistant controlled release of OTC encapsulated in 685 alginate/chitosan matrix coated with acryl-EZE1 MP in fluidized bed. J. Appl. 686 Polym. Sci. 131. 687 Krupa, I., Nedelcev, T., Racko, D., Lacík, I., 2010. Mechanical properties of silica 688 hydrogels prepared and aged at physiological conditions: testing in the 689 compression mode. J. Sol. Gel. Sci. Technol. 53, 107–114. 690 Lavanya, T., Navaneetha, K., Haritha, P.N., Reddy, B.V., 2015. Formulation and 691 evaluation of zolmitriptan rapimelts. World J. Pharm. Res.. 692 Layer, P.H., Goebell, H., Keller, J., Dignass, A., Klotz, U., 1995. Delivery and fate of oral 693 mesalamine microgranules within the human small intestine. Gastroenterology 694 108, 1427–1433. 695 Liu, K., Liu, T., Chen, S., Liu, D., 2008. Drug release behavior of chitosan– 696 montmorillonite nanocomposite hydrogels following electrostimulation. Acta 697 Biomater. 4, 1038–1045. 698 Liu, K., Liu, T., Chen, S., Liu, D., 2007. Effect of clay content on electrostimulus 699 deformation and volume recovery behavior of a clay–chitosan hybrid 700 composite. Acta Biomater. 3, 919–926. 701 López-León, T., Carvalho, E., Seijo, B., Ortega-Vinuesa, J., Bastos-González, D., 2005. 702 Physicochemical characterization of chitosan nanoparticles: electrokinetic and 703 stability behavior. J. Colloid Interface Sci. 283, 344–351. 704 Mathew, S.T., Devi, S.G., Sandhya, K., 2007. Formulation and evaluation of ketorolac 705 tromethamine-loaded albumin microspheres for potential intramuscular 706 administration. AAPS PharmSciTech 8, E100–E108. 707 Mi, F., Kuan, C., Shyu, S., Lee, S., Chang, S., 2000. The study of gelation kinetics and 708 chain-relaxation properties of glutaraldehyde-cross-linked chitosan gel and 709 their effects on microspheres preparation and drug release. Carbohydr. Polym. 710 41, 389–396. 711 Morro, A., Müller, I., 1988. A study of swelling and collapse in polyelectrolyte gels. 712 Rheol. Acta 27, 44–51. 713 Mura, C., Nácher, A., Merino, V., Merino-Sanjuan, M., Carda, C., Ruiz, A., Manconi, M., 714 Loy, G., Fadda, A., Diez-Sales, O., 2011. N-Succinyl-chitosan systems for 5715 aminosalicylic acid colon delivery: In vivo study with TNBS-induced colitis 716 model in rats. Int. J. Pharm. 416, 145–154. 717 Mura, C., Nácher, A., Merino, V., Merino-Sanjuán, M., Manconi, M., Loy, G., Fadda, A., 718 Díez-Sales, O., 2012. Design, characterization and in vitro evaluation of 5-

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

G Model

IJP 14978 1–10 10 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746

L. Neufeld, H. Bianco-Peled / International Journal of Pharmaceutics xxx (2015) xxx–xxx

aminosalicylic acid loaded N-succinyl-chitosan microparticles for colon specific delivery. Colloids Surf. B Biointerfaces 94, 199–205. Muzzarelli, R.A., Pariser, E.R., 1978. Proceedings of the first international conference on chitin/chitosan. Mit Sea Grant Program. Massachusetts Institute of Technology, Cambridge, MA. Omwancha, W.S., Mallipeddi, R., Valle, B.L., Neau, S.H., 2013. Chitosan as a pore former in coated beads for colon specific drug delivery of 5-ASA. Int. J. Pharm. 441, 343–351. Orioli, M., Marinello, C., Cozzi, R., Piodi, L.P., Carini, M., 2004. LC-MS/MS and FT-IR analyses of stones from a patient with Crohn’s disease: A case report. J. Pharm. Biomed. Anal. 35, 1263–1272. Palugan, L., Cerea, M., Zema, L., Gazzaniga, A., Maroni, A., 2015. Coated pellets for oral colon delivery. J. Drug Delivery Sci. Tech. 25, 1–15. Philip, A.K., Philip, B., 2010. Colon targeted drug delivery systems: a review on primary and novel approaches. Oman Med. J. 25, 79–87. Ravi Kumar, M.N., 2000. A review of chitin and chitosan applications. React. Funct. Polym. 46, 1–27. Ritger, P.L., Peppas, N.A., 1987. A simple equation for description of solute release II. Fickian and anomalous release from swellable devices. J. Controlled Release 5, 37–42. Ritland, S.R., Leighton, J.A., Hirsch, R.E., Morrow, J.D., Weaver, A.L., Gendler, S.J., 1999. Evaluation of 5-aminosalicylic acid (5-ASA) for cancer chemoprevention: lack of efficacy against nascent adenomatous polyps in the Apc(Min) mouse. Clin. Cancer Res. 5, 855–863. Rubinstein, M., Colby, R., 2003.. Polymer Physics. Oxford University Press, New York. Salahuddin, N., Abdeen, R., 2013. Drug release behavior and antitumor efficiency of 5-ASA loaded chitosan-layered silicate nanocomposites. J. Inorg. Organomet. Polym. Mater. 23, 1078–1088.

Sing, C.E., Zwanikken, J.W., Olvera de la Cruz, Monica, 2013. Effect of ion–ion correlations on polyelectrolyte gel collapse and reentrant swelling. Macromolecules 46, 5053–5065. Sinha Ray, S., Okamoto, M., 2003. Polymer/layered silicate nanocomposites: a review from preparation to processing. Prog. Polym. Sci. 28, 1539–1641. Sinha, V., Kumria, R., 2001. Polysaccharides in colon-specific drug delivery. Int. J. Pharm. 224, 19–38. Sorlier, P., Denuzière, A., Viton, C., Domard, A., 2001. Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan. Biomacromolecules 2, 765–772. Stephen, A.M., Phillips, G.O., 2010. Food Polysaccharides and their Applications, 2nd ed. CRC Press. Tanaka, T., 2000. Experimental Methods in Polymer Science: Modern Methods in Polymer Research and Technology. Academic Press. Tronci, G., Ajiro, H., Russell, S.J., Wood, D.J., Akashi, M., 2014. Tunable drug-loading capability of chitosan hydrogels with varied network architectures. Acta Biomater. 10, 821–830. Tummala, S., Satish Kumar, M.N., Prakash, A., 2015. Formulation and characterization of 5-Fluorouracil enteric coated nanoparticles for sustained and localized release in treating colorectal cancer. Saudi Pharm. J. 23, 308–314. Wang, X., Du, Y., Luo, J., 2008. Biopolymer/montmorillonite nanocomposite: preparation, drug-controlled release property and cytotoxicity. Nanotechnology 19. Zan, J., Chen, H., Jiang, G., Lin, Y., Ding, F., 2006. Preparation and properties of crosslinked chitosan thermosensitive hydrogel for injectable drug delivery systems. J Appl. Polym. Sci. 101, 1892–1898. Zou, K., Zhang, H., Duan, X., 2007. Studies on the formation of 5-aminosalicylate intercalated Zn–Al layered double hydroxides as a function of Zn/Al molar ratios and synthesis routes. Chem. Eng. Sci. 62, 2022–2031.

Please cite this article in press as: Neufeld, L., Bianco-Peled, H., Designing a biocompatible hydrogel for the delivery of mesalamine. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.06.026

747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775