International Journal of Pharmaceutics 463 (2014) 1–9

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Pectin-coated chitosan–LDH bionanocomposite beads as potential systems for colon-targeted drug delivery Lígia N.M. Ribeiro a,b , Ana C.S. Alcântara a , Margarita Darder a , Pilar Aranda a , Fernando M. Araújo-Moreira b , Eduardo Ruiz-Hitzky a,∗ a b

Instituto de Ciencia de Materiales de Madrid, CSIC, Cantoblanco, 28049 Madrid, Spain Universidade Federal de São Carlos, Monjolinho, 13565-905 São Carlos, SP Brazil

a r t i c l e

i n f o

Article history: Received 14 November 2013 Received in revised form 13 December 2013 Accepted 17 December 2013 Available online 27 December 2013 Keywords: Drug delivery systems Bionanocomposites Layered double hydroxide Chitosan Pectin 5-aminosalicylic acid Chemical compounds studied in this article: Chitosan from crab shells (PubChemCID: 71853) pectin from citrus fruits (PubChemCID: 441476) mucin from porcine stomach (PubChemCID: 124890) 5-aminosalicylic acid (PubChemCID: 4075) AlCl3 ·6H2 O (PubChemCID: 24564) MgCl2 ·6H2 O (PubChemCID: 24644) CaCl2 ·2H2 O (PubChemCID: 24854).

a b s t r a c t This work introduces results on a new drug delivery system (DDS) based on the use of chitosan/layered double hydroxide (LDH) biohybrid beads coated with pectin for controlled release in the treatment of colon diseases. Thus, the 5-aminosalicylic acid (5ASA), the most used non-steroid-anti-inflammatory drug (NSAID) in the treatment of ulcerative colitis and Crohn’s disease, was chosen as model drug aiming to a controlled and selective delivery in the colon. The pure 5ASA drug and the hybrid material prepared by intercalation in a layered double hydroxide of Mg2 Al using the co-precipitation method, were incorporated in a chitosan matrix in order to profit from its mucoadhesiveness. These compounds processed as beads were further treated with the polysaccharide pectin to create a protective coating that ensures the stability of both chitosan and layered double hydroxide at the acid pH of the gastric fluid. The resulting composite beads presenting the pectin coating are stable to water swelling and procure a controlled release of the drug along their passage through the simulated gastrointestinal tract in in vitro experiments, due to their resistance to pH changes. Based on these results, the pectin@chitosan/LDH-5ASA bionanocomposite beads could be proposed as promising candidates for the colon-targeted delivery of 5ASA, with the aim of acting only in the focus of the disease and minimizing side effects. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The incidence of chronic intestinal diseases such as ulcerative colitis and Chron’s disease is continually increasing in different regions of the world (Latella and Papi, 2012) and the complications can account for almost 15% of all deaths (Munkholm, 2003). 5-Aminosalicylic acid (5ASA) is an anti-inflammatory drug used in the treatment of intestinal diseases, which has been considered as a potential chemopreventive agent (Velayos et al., 2005). The major limitation for its oral administration is the instability under the stomach conditions, which leads to low efficiency and other

∗ Corresponding author. Tel.: +34 913349000; fax: +34 913720623. E-mail address: [email protected] (E. Ruiz-Hitzky). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.12.035

serious side effects (Schroeder, 2002). Colonic drug delivery systems have been widely studied using certain polymers as protective coating of diverse drugs, such as the synthetic polymer Eudragit® S (Ashford et al., 1993), and hydroxypropylmethylcellulose (HPMC) (Gazzaniga et al., 1994). These systems showed to be more efficient in regulating the speed of drug liberation and more precise in procuring specific site release than the traditional oral formulations of this drug. Bio-hybrids are also interesting alternative systems for controlled drug delivery. These nanostructured materials are composed by an organic compound of natural origin conjugated with nanometric inorganic counterparts (Darder et al., 2007; RuizHitzky et al., 2008a, 2010a, 2011). Among this group of materials, bionanocomposites refer to those bio-hybrids involving a biopolymer assembled to an inorganic solid that shows at least one

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dimension at the nanometer scale (Darder et al., 2007; RuizHitzky et al., 2008b, 2010b). Layered and fibrous clay minerals together with layered double hydroxides (LDH) are probably the most widely employed solids for this purpose (Ruiz-Hitzky and Fernandes, 2013). The crystal structure of LDH consists of brucitelike layers in which the positions of the Mg2+ ions are not only occupied by divalent metal cations but also by trivalent metal cations, which results in a positive charge in the layers that is compensated by anions in the interlayer space (Forano et al., 2013). Thus, several specific negatively charged organic molecules, including polymers and biopolymers, have been intercalated within LDH structures, such as drug molecules (Costantino et al., 2008, 2009; Wang et al., 2009; Zhang et al., 2009), DNA (Choy et al., 2000), polysaccharides (Darder et al., 2005) or amino acids (Aisawa et al., 2004), for multipurpose applications. A remarkable number of studies have reported LDH compounds to be useful for diverse applications, such as ion-exchange materials, catalysts, or drug and biopolymers carriers (Guo et al., 2010; Leroux, 2012). Due to biocompatibility and biodegradability properties (Choy et al., 2007), and high versatility of synthesis, which included numerous methods such as direct ion-exchange, co-precipitation and reconstruction from previous calcined LDH, they are especially suitable for development of drug delivery systems (DDS) based on LDH (Costantino et al., 2008; Gunawan and Xu, 2008; Wang et al., 2009). However, due to the high sensitiveness of LDH to acid conditions, this inorganic solid alone is not suitable as DDS, since it results in the complete release of the drug in the stomach media (Forano and Prévot, 2008), before reaching the intestine. In this way, different authors have incorporated LDH-intercalated drug systems in diverse polymers and biopolymers with the aim to protect the drug and to have a more controlled release, which could be especially relevant for oral drugs that should be adsorbed in the intestinal tract (Alcântara et al., 2010; Ambrogi et al., 2008; Aranda et al., 2012; Costantino et al., 2012; Khan et al., 2001; Li et al., 2004; Ribeiro et al., 2009; Tammaro et al., 2007, 2009). This protective effect is especially relevant when the drug is specific for treatment of colonic diseases, as in this case it is crucial that the drug reaches the point of disease at the end of the intestinal tract. Chitosan and pectin are polysaccharides widely employed in food industries and biomedicine area as scaffolds, wound dressings or DDS (Boateng et al., 2008; George and Abraham, 2006; Shahidi et al., 1999; Simkovic, 2013). Chitosan is a cationic biopolymer mainly extracted from the exoskeleton of crustaceans, that consists of poly[␤-(1-4)-2-amino-2-deoxy-d-glucopyranose], with hydroxyl groups at the C2 positions of the glucose rings. Given the well known mucoadhesive properties of chitosan (George and Abraham, 2006), this biopolymer is used as carrier in systems where the drug needs to have a residence time in a specific mucous such as buccal or intestinal. However, chitosan does not resist in the stomach, being this fact a disadvantage for the release of a drug that needs to be delivered in the intestine, for example. In this sense, many modifications or complexes formation with other polymers have been proposed, in order to improve the stability of the chitosan in the stomach, its mucoadhesion properties and others (Alves and Mano, 2008; George and Abraham, 2006; Kafedjiiski et al., 2005). On the other hand, pectin is an anionic polysaccharide extracted from citrus fruits, consisting of (1,4)-␣-d-galacturonic acid residues (Mohnen, 2008). In contrast to chitosan, pectin is resistant to low pH conditions and to digestion by proteases and amylases present in the stomach, but at the same time it is sensitive to pectinases present in the colon, been totally digested in the intestinal tract (Itoh et al., 2007). These properties are desirable for colonic drug delivery systems, because an efficient drug release in the intestinal tract is expected. Thus, the combination of chitosan and pectin appears as a great opportunity to simultaneously profit from the properties of both biopolymers. In this way, some recent

approaches addressed the incorporation of the specific drug in DDS based on the formation of a polyelectrolyte complex through electrostatic interactions (Coimbra et al., 2011) or the preparation of pectin beads cross-linked with Ca2+ ions that are externally coated by chitosan as carriers of dietary compounds with health benefits (de Souza et al., 2009). In this work, we propose a new drug delivery system based on the use of 5ASA intercalated in a Mg–Al LDH which is incorporated in chitosan beads further coated with pectin, forming a drug-loaded bionanocomposite system (Scheme 1). These materials were expected to be far more efficient than the LDH or the biopolymers separately, for oral administration due to the following two factors: (i) pectin-coated chitosan beads provide resistance in conditions simulating the gastric passage (Semdé et al., 2000), and (ii) chitosan can play an important role on mucoadhesiveness improvement, and consequently should have greater efficiency on drug release in the colon (Illum et al., 1994; Lehr et al., 1992; Robinson et al., 1987; Shimoda et al., 2001; Vasir et al., 2003). Moreover, the presence of the 5ASA drug intercalated in the Mg–Al LDH can contribute to a more controlled release of the drug, as it has been proved in other DDS systems such as ibuprofen-LDH/zeinalginate bionanocomposites (Alcântara et al., 2010). In this way, the final goal of this work is to reach a DDS in which it was possible to tune properties in order to achieve the optimal release of the 5ASA in conditions simulating those in the intestinal colon, i.e., the site of interest in the action of this specific drug. 2. Materials and methods 2.1. Starting reagents Chitosan (CHT) of low (% deacetylation: 75–85, average molecular weight: 120 kDa), medium (% deacetylation: 75–85, average molecular weight: 250 kDa) and high molecular weight (% deacetylation: >75, average molecular weight: 340 kDa), from crab shells; pectin (PCT) from citrus fruits; mucin from porcine stomach (type II, bound sialic acid ∼1%); 5-aminosalicylic acid (5ASA); Schiff’s reagent 1% basic fuchsin; periodic acid and sodium metabisulfite were purchased from Sigma–Aldrich. Absolute ethanol and acetic acid were supplied by Panreac and HCl (37%, P.A.) was supplied by Carlo Erba. Deionized water (resistivity of 18.2 M cm) was obtained with a Maxima Ultrapure Water from Elga. Aqueous solutions were prepared from chemicals of analytical reagent grade: AlCl3 ·6H2 O (>99%, Fluka); MgCl2 ·6H2 O (Panreac); NaOH (98%, Fluka); NaCl (>99%, Sigma–Aldrich); Na2 CO3 (>99%, Merck); NaH2 PO4 ·H2 O (>99%, (Sigma–Aldrich) and CaCl2 ·2H2 O (>99%, Sigma–Aldrich). 2.2. Synthesis of the LDH-5ASA intercalation compound Mg2 Al-chloride LDH ([Mg0.67 Al0.33 (OH)2 ] Cl0.33 ·nH2 O) was prepared by co-precipitation reaction at constant pH, according to the procedure described by Constantino and Pinnavaia (1995). A mixture of MgCl2 ·6H2 O (18 mmol) and AlCl3 ·6H2 O (9 mmol) was dissolved on 400 mL of decarbonated bidistilled water. This aqueous solution and 1 M NaOH were added simultaneously and dropwise to 100 mL of 50% hydroalcoholic solution containing 5ASA (8 mmol) using a DOSINO pH module 800 (Metrohm). This system allowed a controlled addition of solutions in order to keep a constant pH of 9 during the synthesis. The resulting suspension was kept under nitrogen flow to remove CO2 and magnetically stirred for 48 h at 60 ◦ C. The solid product was isolated by centrifugation, washed five times with bidistilled water, and dried overnight at 60 ◦ C. The resulting hybrid material was denoted as LDH-5ASA. For comparison purposes, a pristine LDH was prepared following the same procedure, but replacing the hydroalcoholic solution that

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Scheme 1. Pectin-coated chitosan bead incorporating 5-aminosalicylic acid (5ASA) intercalated in Mg2 Al layered double hydroxide (LDH) as a new drug delivery system.

contains 5ASA by decarbonated bidistilled water. This material will be denoted as LDH or Mg2 Al LDH. 2.3. Preparation of pectin@chitosan/LDH-5ASA bionanocomposite beads The bionanocomposites based on the LDH-5ASA hybrid were conformed as beads according to the following procedure: an aqueous solution of 1% (w/v) chitosan (medium molecular weight) was prepared by dissolving 1 g of chitosan in 100 mL of 0.01 M HCl and its pH was raised by addition of NaOH (final pH ≈ 6). Next, the chitosan solution was magnetically stirred until homogenization with 0.2 g of pure 5ASA or with the appropriate amount of LDH-5ASA hybrid that affords 0.2 g of 5ASA. The resulting gel was introduced in a burette and then slowly poured as small droplets into a solution of 2 M NaOH. The resulting beads were filtered and washed with abundant doubly distilled water to remove non-entrapped 5ASA and residual Na+ ions. Subsequently, the CHT/5ASA or CHT/LDH5ASA beads were dispersed in pectin aqueous solutions of different concentration (Table 1), filtered and cross-linked in 10% (w/v) CaCl2 aqueous solution, where they were kept for about 15 min. The resulting PCT@CHT/5ASA or PCT@CHT/LDH5ASA bionanocomposite beads were filtered, washed with bidistilled water, frozen at −20 ◦ C and lyophilized in a freeze-dryer (Cryodos, Telstar) for later use. 2.4. Characterization Powder X-ray diffraction (XRD) data were collected in a BRUKER D8-ADVANCE diffractometer using a Cu-K␣ source, with a scan step of 2◦ min−1 between 2 values of 2 and 70◦ . Fourier Transform Infra Red (FTIR) spectra were recorded with a FTIR spectrophotometer BRUKER IFS 66 v/S. Each sample was placed in the sample holder as KBr pellets and scanned from 4000 to 250 cm−1 with 2 cm−1 resolution. The amount of organic matter in the LDH intercalation compound was determined by CHN elemental chemical microanalysis in a PerkinElmer 2400 series II CHNS/O elemental analyzer. The thermal behavior of the different prepared materials was analyzed from the simultaneously recorded thermogravimetric (TG) and differential thermal analysis (DTA) curves in a SEIKO SSC/5200 equipment, in experiments carried out under air atmosphere (flux of 100 ml min−1 ) from room temperature to 1000 ◦ C

at 10 ◦ C min−1 heating rate. Surface morphology was observed in a ZEISS DSM-960 microscope working at 15 kV and FE-SEM equipment FEI-NOVA NanoSEM 230 equipped with an EDAX-Ametek detector that allowed semi-quantitative analysis of elements in some samples. Sample preparations were performed by adhering particle samples on a carbon tape without requiring a gold conductive coating on the surface. 2.5. In vitro mucin mucoadhesion assay The mucoadhesive properties of chitosan were evaluated by its interaction with mucin (He et al., 1998). A Schiff reagent solution was prepared by mixing 30 mL of Schiff reagent with 6 mL of 1 M HCl aqueous solution. Sodium metabisulphite (0.1 g) was added to 6 mL of the solution mentioned above, and the resultant mixture was incubated at 37 ◦ C until it became colorless or pale yellow. The periodic acid reagent was freshly prepared with 10 ␮L of 50% periodic acid solution and 7 mL of 7% acetic acid solution. Standard calibration curves were obtained from 2 mL of mucin standard solutions (0.125, 0.25, 0.375 and 0.5 mg/mL), after addition of 0.2 mL of periodic acid reagent. The samples were incubated at 37 ◦ C for 2 h in a water bath. After this time, an aliquot of Schiff reagent (0.2 mL) was added at room temperature and the absorbance of the solution was measured 30 min later in a UV spectrophotometer (Shimadzu UV1201) at 555 nm (He et al., 1998). Samples were determined with the same procedure, 10 mg of each type of chitosan were dispersed in the mucin solutions, shaken and centrifuged at 4000 rpm for 5 min and the supernatant was used for the measurement of free mucin content estimated from the standard curve calibration. 2.6. Estimation of the 5ASA loading and the encapsulation efficiency Weighed PCT@CHT/5ASA or PCT@CHT/LDH-5ASA bionanocomposite beads (0.1 g) were immersed in 50 mL phosphate buffer at pH 6.8 for 12 h. Subsequently, the pH was adjusted to 1.2 by addition of 1 M HCl and the beads were kept in this solution for 12 h more. Then, the solution was filtered, and the 5ASA content was calculated from the absorbance  = 303 nm measured by UV spectrophotometry (Moharana et al., 2011). The estimation of drug

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Table 1 Different concentrations (w/v) of chitosan (CHT) and pectin (PCT) employed in the preparation of the bionanocomposite beads. Composition of samples

Nomenclature of samples

CHT (%)

PCT (%)

Chitosan encapsulating 5ASA Chitosan encapsulating LDH-intercalated 5ASA Pectin encapsulating 5ASA Pectin encapsulating LDH-intercalated 5ASA Pectin-coated chitosan encapsulating 5ASA Pectin-coated chitosan encapsulating LDH-intercalated 5ASA

CHT/5ASA CHT/LDH-5ASA PCT/5ASA PCT/LDH-5ASA PCT@CHT-5ASA PCT0.5%@CHT/LDH-5ASA PCT1%@CHT/LDH-5ASA PCT1.5%@CHT/LDH-5ASA

1 1 0 0 1 1 1 1

0 0 1 1 1 0.5 1 1.5

percentage loading and the encapsulation efficiency were obtained using equations (1) and (2) (Ambrogi et al., 2008): %drug loading =

amount of drug in bead × 100 amount of bead

% encapsulation efficiency =

drug loading × 100 theoretical loading

(1)

(2)

2.7. Water uptake studies The water uptake properties of the bionanocomposites beads were measured by the following procedure: aliquots of each system of bionanocomposite beads (0.05 g) were placed in Petri dishes, immersed in bidistilled water, phosphate buffer at pH 6.8 or 0.06 M HCl containing 0.1 wt.% NaCl (pH 1.2), and shaken eventually at room temperature. After a predetermined time interval, the beads were withdrawn, weighed on an analytical balance after removing the excess of water. The water uptake (mg H2 O/mg beads) was calculated from the equation (3): Water uptake (mg/mg) =

W2 − W1 W1

(3)

where W2 and W1 are the wet and initial mass of beads, respectively. 2.8. In vitro 5ASA cumulative release studies A given amount of bionanocomposite beads (0.1 g) was added to 50 mL of release medium and kept in a thermostatic bath at 37 ◦ C. The release study was carried out for 8 h in order to simulate the sequential pH changes that occur during the in vivo process. The beads were kept for 2 h in a 0.06 M HCl solution (pH 1.2) containing 0.1 wt.% NaCl, in order to simulate the gastric tract. Then, they were kept for 2 h at pH 6.8 (by adding 0.03 g NaOH, 0.40 g NaH2 PO4 ·H2 O and 0.62 g NaCl to the pH 1.2 solution), to simulate the first zone of intestinal fluid, and finally for 4 h at pH 7.4 (prepared by adding 1 M NaOH to the previous solution of pH 6.8), mimicking the colon intestinal zone. At certain time intervals, 5 mL of each medium were withdrawn in order to analyze the amount of 5ASA released from the drug-loaded beads by UV spectrophotometry ( = 303 nm), and the volume was kept constant by adding the measured sample back to the solution. All the experiments were carried out in triplicate.

method, according to previous results reported by Zhang et al. (2005). Thus, the intercalation of 5ASA progresses during the in situ formation of the LDH, i.e. the layers of the inorganic solid were formed in the presence of the drug molecules in its anionic form which act as their charge compensating anions. The X-ray diffraction pattern of LDH-5ASA together with the diffractogram of pristine LDH are shown in Fig. 1, where it is possible to appreciate the typical reflections of the pristine inorganic solid ((0 0 3), (0 0 6), (0 1 2), (0 1 5), (0 1 8), (1 1 0) and (1 1 3) planes) (Cavani et al., 1991) (Fig. 1a). For the LDH-5ASA hybrid compound, the shift of the 0 0 3 reflection towards lower 2 value (Fig. 1b) indicates a new basal spacing of 2.08 nm probing that the drug is intercalated within the LDH layers. Taking into account the thickness of brucite-like layers (0.48 nm) (Miyata, 1980), the interlayer spacing of the LDH-5ASA was estimated as 1.60 nm, which was higher than the 5ASA dimensions estimated by ViewerLite software (0.72 nm × 0.67 nm × 0.48 nm), suggesting a possible arrangement of the intercalated 5ASA in a molecular bilayer disposition, as already reported (Zhang et al., 2005). From the CHN analyses of LDH-5ASA, a drug content of 57.7 g 5ASA/100 g LDH is determined in the resulting material, which represents around 190 mEq/100 g LDH. Given that the anion-exchange capacity of Mg2 Al LDH is about 330 mEq/100 g, this result suggests the presence of other anions, chloride ions, in the interlayer space together with the intercalated drug molecules, both contributing to compensate the charge defect of the inorganic layers. FTIR spectroscopy also corroborates the formation of the LDH5ASA hybrids. The characteristic bands of the pristine 5ASA (Fig. 2a) can be attributed as follows: 1654 cm−1 assigned to C O stretching vibrations in free 5ASA, 1580 cm−1 assigned to N–H bending vibrations and 1449 cm−1 to C–N stretching vibrations (Zhang et al., 2005). The vibration band at 1654 cm−1 , which indicates the presence of the 5ASA in its anionic form, is shifted towards lower wavenumber values (1628 cm−1 ) after assembly with the

3. Results and discussion 3.1. Synthesis of LDH-5ASA Amongst the diversity of methods for the preparation of layered double hydroxides (LDH)-based materials described in the literature, such as co-precipitation, reconstruction and ion-exchange (Crepaldi and Valim, 1998; Roy et al., 1992), the intercalation of the anti-inflammatory drug 5-aminosalicylic acid (5ASA) in a LDH material was only successful by following the co-precipitation

Fig. 1. XRD patterns of (a) Mg2 Al LDH and (b) LHD-5ASA intercalation compound.

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Fig. 3. Mucin adsorption on three types of chitosan with different molecular weight.

3.3. Pectin@chitosan/LDH-5ASA bionanocomposite systems Fig. 2. FTIR spectra (4000–400 cm−1 region) of (a) 5ASA, (b) LDH-5ASA and (c) Mg2 Al LDH.

LDH (Fig. 2b). This shift is due to the intercalation of 5ASA in the LDH, corroborating the drug loading into the LDH matrix. The typical IR absorption bands of brucite-like layers are observed in the 600–400 cm−1 region of the spectra of both pristine LDH and LDH-5ASA material (Fig. 2b and c, respectively). These bands are attributed to the O–M–O bending vibrations of the LDH structural sheets (Velu et al., 1999), confirming the conservation of the LDH structure in the resulting hybrid material. The thermal behavior of both materials was analyzed by means of TG and DTA techniques. A detailed examination of the thermal events taking place when the samples are heated from room temperature to 900 ◦ C (Fig. S1), shows significant differences on the thermal behavior of the drug once intercalated. The DTA curve of the free drug exhibits an endothermic peak at 280 ◦ C, corresponding to its melting point (Mura et al., 2011), which did not occur in the LDH-5ASA material, suggesting that the drug is present in a different aggregation state when intercalated between the LDH layers.

3.2. In vitro mucin mucoadhesion assay The interaction of chitosan with mucin has been previously used to evaluate the mucoadhesive properties of this polysaccharide (He et al., 1998). In the current study, the mucin assay has been carried out with the aim to determine the most appropriate type of chitosan for the preparation of the 5ASA DDS. This assay, which simulates the conditions that take place in vivo in the intestinal tissue, was performed using three types of commercial chitosan with different molecular weights. As shown in Fig. 3, the three types of chitosan were able to adsorb mucin, which is attributed to the interaction between the negatively charged mucin and the positively charged chitosan (He et al., 1998). From these results, it is deduced that chitosan of medium molecular weight afforded the best mucoadhesive properties, adsorbing the greatest amounts of mucin (65–80%) from different starting concentrations. This result could be due to the different deacetylation degree of the samples, which is slightly higher for low and medium molecular weight chitosans. Thus, medium molecular weight chitosan was chosen to form the DDS involving the LDH-5ASA hybrid material.

The new DDS were prepared by dispersing the intercalation compound LDH-5ASA within a matrix of medium molecular weight chitosan, being this bionanocomposite processed as beads by dropping the mixture into a NaOH solution. The resultant beads were then covered with pectin, at several concentrations, as a gastroresistant coating. For comparison, diverse LDH-5ASA-loaded materials were prepared using only one of the two biopolymers, as well as with both biopolymers incorporating the free drug instead (Table 1). The amounts of the 5ASA loaded in different batches, and the encapsulation efficiency of each system are listed in Table 2. Systems that incorporate pure drug presented lower encapsulation efficiency than those loaded with the LDH-5ASA hybrid, most likely due to a protective effect of the inorganic counterpart against drug leaching during recovery and washing of the beads. The high value of drug loading in the PCT/LDH-5ASA material compared to the same system based on chitosan may be associated with the alkaline pH of the starting pectin solution, which could reduce the leaching of the drug from the LDH layers in the bead formation. In the case of the ternary systems, it is observed that the amount of 5ASA incorporated as LDH-5ASA into chitosan increases as the concentration of pectin used as coating of the CHT/LDH-5ASA system increases. Thus, for 1% pectin content or higher, it is possible to observe very similar values of encapsulation efficiency compared to the CHT/LDH-5ASA starting systems, which suggests the formation of stable bionanocomposites, where the amount of drug encapsulated is maintained throughout the coating process. The coating of the beads by pectin could be confirmed by FESEM images (Fig. 4), in which it is possible to clearly differentiate the coated and uncoated beads. Thus, uncoated CHT/LDH-5ASA Table 2 Encapsulation efficiency and amount of 5ASA loaded, either as pure drug or as LDH5/ASA, in different polymer systems (Data are mean ± S.D., n = 3). Formulation

5-ASA loaded (%)

Encapsulation efficiency (%)

CHT/LDH-5ASA CHT-5ASA PCT/LDH-5ASA PCT-5ASA PCT1%@CHT-5ASA PCT0.5%@CHT/LDH-5ASA PCT1%@CHT/LDH-5ASA PCT1.5%@CHT/LDH-5ASA

4.86 ± 0.17 3.80 ± 0.11 7.26 ± 0.13 1.49 ± 0.09 2.49 ± 0.05 2.58 ± 0.03 4.98 ± 0.12 4.72 ± 0.26

24.4 ± 1.10 19.0 ± 0.54 36.3 ± 0.63 7.46 ± 0.47 12.5 ± 0.22 12.9 ± 0.14 25.0 ± 1.04 23.7 ± 1.32

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Fig. 5. FTIR spectra (4000–400 cm−1 region) of pure CHT (a), CHT-Na (b), CHT/LDH5ASA (c), PCT1%@CHT/LDH-5ASA (d), PCT-Ca (e) and pure PCT (f).

Fig. 4. FE-SEM images of the external surface of CHT/LDH-5ASA (a) and PCT1%@CHT/LDH-5ASA (b) and a cross-section of bionanocomposite beads (c).

beads (Fig. 4a) present a roughened and porous texture, being the LDH crystals well integrated as it is not possible to distinguish them within the polymeric matrix. However, the images of the pectin-coated CHT/LDH-5ASA beads, for instance the PCT1%@CHT/LDH-5ASA sample (Fig. 4b and c), show a homogeneous coating of a less porous material at the external surface of the bead, with a thickness of around 3 ␮m (Fig. 4c). The different morphologies revealed before and after the pectin coating in the beads may result in different release profiles of 5ASA in each one of the bionanocomposite systems. The FTIR spectra of pure biopolymers and their respective bionanocomposite beads with or without pectin coating are shown in Fig. 5. For comparison, the spectra of pure chitosan and pure pectin beads (i.e., synthesized without the LDH-5ASA compound), CHT-Na and PCT-Ca, respectively, are also included in Fig. 5. The character-

istic bands of amide I and amine groups appearing in the spectrum of pure chitosan at 1654 and 1593 cm−1 , respectively, cannot be appreciated in that of CHT-Na beads, appearing only a band at 1453 cm−1 which is related to ıNH2 vibration modes (Pretsch et al., 1980). These observations point out to an almost complete deprotonation of the amine groups of chitosan during the beads formation process. However, in the spectrum of the CHT/LDH-5ASA beads, these bands assigned to C O of amide and ıNH3 vibration modes are shifted toward lower frequencies, appearing at 1640 and 1587 cm−1 , respectively, in the bionanocomposite beads. This fact is ascribed to a partial deprotonation of chitosan during the beads formation, in a similar way to that observed in CHT-Na beads. In addition, the CHT/LDH-5ASA spectrum also shows bands in the 500–800 cm−1 region, which are ascribed to the characteristic vibration modes of the LDH structure (Velu et al., 1999), confirming the preservation of the inorganic solid structure during the beads formation. In the case of the chitosan/LDH-5ASA beads coated with pectin, their corresponding spectrum (Fig. 5d) shows a very similar profile than the one of the PCT-Ca beads (Fig. 5e). In this case the bands in the pristine pectin appearing at 1745 and 1622 cm−1 , ascribed to the C O vibration mode of ester carbonyl groups and to the asymmetric stretching vibration of –COO− groups, respectively (Kim et al., 2003), appear in the PCT1%@CHT/LDH-5ASA bionanocomposite at 1730 and 1644 cm−1 , respectively, indicating a certain degree of interaction of the free carboxylate groups of pectin with a protonated amine groups from chitosan, as occurs in the formation of polyelectrolyte systems (Sharma and Ahuja, 2011). In fact, from the TG-DTA data (Fig. S2), it is possible to observe differences in the temperature of the exothermic processes ascribed to the pectin and chitosan decompositions, appearing as a unique process at 560 ◦ C, and corroborating the existence of a possible cross-linking between the two biopolymers. Taking into account that the swelling degree is a crucial factor in the release of the drug from the delivery systems, water uptake of the various types of bionanocomposite beads was investigated in different media (pH 1.2 and 6.8). Fig. 6 shows the evolution of the water content in diverse beads with time of exposure to the aqueous media. From these results, it is clearly observed that swelling behavior of each material depends on the pH in the medium. In this way, chitosan beads incorporating either 5ASA directly or encapsulated in a LDH matrix, swelled to a great extent in acidic medium (Fig. 6a), which drives in both cases to their final disaggregation

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Fig. 6. Water uptake properties of different chitosan bionanocomposite beads exposed to water media at (a) pH 1.2 and (b) in the presence of a phosphate buffer at pH 6.8. Each value is the mean ± S.D. n = 3.

after approximately 30 min of exposure. This behavior is explained considering that the amino groups of chitosan are readily ionized at low pH, causing the incorporation of water molecules and formation of a hydrogel matrix which leads at the end to the complete dissolution of the material. When these beads are protected by pectin (i.e., PCT@CHT systems), this type of disintegration is not observed at low pH, indicating a better stability of these coated beads in acid media clearly ascribed to the protective effect of pectin. The extent of swelling in these coated beads is strongly dependent on the concentration of pectin used in the preparation of the coating, showing the PCT0.5%@CHT/LDH-5ASA sample values of water uptake three times higher than the PCT1%@CHT/LDH5ASA (Fig. 6a). This behavior again corroborates the protective effect of pectin and the possibility to tune this property if necessary for a given application. The effect of a higher pH and the presence of salts in the medium were also evaluated by performing a swelling study of the beads exposed to a phosphate buffer solution of pH 6.8 (Fig. 6b). In this case, the chitosan beads show a considerable resistance to swell at this pH value, presenting the CHT/5ASA and CHT/LDH-5ASA beads a very similar swelling profile. However, the effect of phosphate ions strongly affect the systems protected by the pectin coating, presenting these last ones higher values of water absorption in this solution compared to those in acid media. This behavior can be related to the presence of phosphate ions in the medium, which may favor the release of Ca2+ ions acting as cross-linking points of the pectin chains resulting in the increase ˜ of water uptake by these systems (Remunan-López and Bodmeier, 1997). This study points out that the role of the pectin coating is

Fig. 7. Profiles of in vitro 5ASA release from beads based on CHT and PCT biopolymers (a) and from beads based on PCT@CHT systems (b) in conditions that simulate the gastrointestinal tract passage (pH and time) at 37 ◦ C.

more relevant for the systems in acidic medium when phosphate ions are not present, being possible to preserve the bead structure after 6 h of assay when the chitosan beads are coated with pectin. 3.4. In vitro release of 5ASA from pectin@chitosan-based beads The protective effect of the pectin coating on the chitosan-based beads was evaluated (in vitro experiments) following the 5ASA release under pH conditions that simulate the sequence of the drug passage in the human body, i.e., pH initially kept at 1.2 for 2 h, then at 6.8 for 2 h, and finally at 7.4 for 4 h (Fig. 7). In this way, chitosan and pectin systems that incorporate the 5ASA as pure drug or encapsulated in a LDH inorganic matrix were also evaluated for comparative purposes (Fig. 7a). The pure chitosan beads release almost 100% of the drug in the first 2 h, i.e. when the system is at pH 1.2. This occurs independently of the way in which the drug was incorporated into the chitosan matrix, reaching around 95% and 90% of 5ASA release in CHT/5ASA and CHT/LDH-5ASA, respectively. This behavior was expected since these systems present a high solubility and elevated degree of swelling at this pH, as observed in the water uptake study (vide supra). Thus, these results suggest that the beads would not resist the stomach environment during an in vivo application, releasing practically all drug encapsulated during their passage through the first step in the gastrointestinal tract.

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Conversely, pectin beads containing pure 5ASA or incorporated in the LDH matrix show a significant stability in those conditions that simulate the stomach media, releasing less than 10% of 5ASA. However, an elevated release is achieved when beads containing pure 5ASA are then exposed to pH 6.8, being the release even higher at pH 7.4. In contrast, a slower release is observed for PCT beads incorporating LDH under such basic conditions (Fig. 7a), as the encapsulated drug requires additional time for diffusion out of the inorganic host which is stable in basic media (Alcântara et al., 2010). In spite of this apparently satisfactory result from these in vitro tests, these beads may be not good enough for the intended use as DDS in the colon as the drug release has to be also controlled during its passage through the intestinal tract. The cumulative 5ASA release from the PCT@CHT systems in the in vitro experiment is presented in Fig. 7b. All of these bead systems show a high stability at pH 1.2, corroborating the protective role of the pectin coating in acid media. The PCT@CHT/5ASA system shows a slower release and high resistance during the simulated stomach passage, but a fast release of the drug occurs when the pH increases, being possible to reach almost the 100% in the simulated intestinal media in a very short time. Conversely, the PCT@CHT beads containing the LDH-intercalated drug show a more controlled release, especially those with the highest content of pectin in the external coating of the bead. In this way, the PCT0.5@CHT/LDH-5ASA beads show a faster release at pH 7.4 than those coated with 1% and 1.5% of pectin, which can be due to the high solubility of pectin in these simulated conditions. Thus, it is possible to change the release kinetics and the maximum amount of released drug at pH 7.4 just by changing the pectin coating. This in vitro study suggests that PCT@CHT/LDH systems can be proposed as promising DDS systems in view to procure the delivery of drugs at the intestinal tract in a controlled manner. These systems will profit from the stability of pectin at acid pH to avoid the drug release in the stomach, as well as from the resistance of chitosan at basic pH to procure a slow liberation of 5ASA in the intestinal tract. In addition, the mucoadhesive properties of chitosan could be especially relevant for the colon-targeted delivery of the encapsulated drug.

4. Conclusions Considering the major interest in the development of oral administration DDS for controlled delivery of specific drugs within the intestinal tract, this work introduces a new approach consisting in the preparation of bio-hybrid chitosan beads, in which the drug is intercalated in a LDH host, coated with pectin that have been successfully tested in in vitro release experiments. These ternary systems were designed in order to combine advantages from the three components: (i) the external pectin coating protects the DDS in low pH ambient, as that found in the stomach; (ii) the chitosan matrix in which the drug is incorporated presents mucoadhesive properties of interest for specific adsorption in the intestinal tract; and (iii) the immobilization of the drug in the LDH host solid offers the possibility to control the kinetics of the drug release. These systems were tested using 5ASA as model drug, due to its interest in the treatment of the ulcerative colitis and Crohn’s disease. In vitro assays simulating changes of pH and time of residence that occur during the in vivo passage of the drug through the gastrointestinal tract proved the efficiency of the PCT@CHT/LDH-5ASA systems compared to those based on only one of the biopolymers or on the direct immobilization of the drug without the LDH host. Moreover, the kinetics of the drug release can be also tuned by changing the thickness of the pectin coating. These findings may result of special relevance in view to prepare adequate formulations for the application of this drug and others of interest

in the treatment of intestinal diseases in its specific sites of action. In addition, it could be envisaged the possibility of including more specific functionalities in chitosan in order to make even more specific the location of the release. Other advantages of these DDS are the availability, biocompatibility and the low price of the biopolymers, which make them as a very promising approach for treatment of different colonic diseases. Acknowledgments This work was supported by the CICYT (Spain; MAT2012-31759 project). A.C.S.A. acknowledges the CSIC (Spain) for her JAE-PreDoc fellowship. L.N.M.R. acknowledges the CAPES (Brazil) for her fellowship. The authors also thank Mr. A. Valera and Mr. C. Sebastián for the FE-SEM studies. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2013.12.035. References Aisawa, S., Kudo, H., Hoshi, T., 2004. Intercalation behavior of amino acids into Zn–Al-layered double hydroxide by calcination–rehydration reaction. J. Solid State Chem. 177, 3987–3994. Alcântara, A.C.S., Aranda, P., Darder, M., Ruiz-Hitzky, E., 2010. Bionanocomposite based alginate/zein layered double hydroxide materials as drug delivery systems. J. Mater. Chem. 20, 9495–9504. Alves, N.M., Mano, J.F., 2008. Chitosan derivatives obtained by chemicalmodifications for biomedical and environmental applications. Int. J. Biol. Macromol. 43, 401–414. Ambrogi, V., Perioli, L., Ricci, M., Pulcini, L., Nocchetti, M., Giovagnoli, S., Rossi, C., 2008. Eudragit® and hydrotalcite-like anionic clay composite system for diclofenac colonic delivery. Microporous Mesoporous Mater. 115, 405–415. Aranda, P., Alcântara, A.C.S., Ribeiro, L.N.M., Darder, M., Ruiz-Hitzky, E., 2012. Bionanocomposites as drug delivery systems. In: Choi, H., Choy, J.H., Lee, U., Varadan, V.K. (Eds.), Proc SPIE on Nanosystems in Engineering and Medicine, 8548, #85486D. Ashford, M., Fell, J.T., Attwood, D., Sharma, H., Woodhead, P.J., 1993. An in vitro investigation into the suitability of pH-dependent polymers for colonic targeting. Int. J. Pharm. 95, 193–199. Boateng, J.S., Matthews, K.H., Stevens, H.N.E., Eccleston, G.M., 2008. Wound healing dressings and drug delivery systems: a review. J. Pharm. Sci. 97, 2892–2923. Cavani, F., Trifiro, F., Vaccari, A., 1991. Hydrotalcite-type anionic clays: preparation, properties and applications. Catal. Today 11, 173–301. Choy, J.H., Kwak, S.Y., Jeong, Y.J., Park, J.S., 2000. Inorganic layered double hydroxides as nonviral vectors. Angew. Chem. (Int. Ed.) 39, 4041–4045. Choy, J.H., Choi, S.J., Oh, J.M., Park, T., 2007. Clay minerals and layered double hydroxides for novel biological applications. Appl. Clay Sci. 36, 122–132. Coimbra, P., Ferreira, P., Sousa, H.C., Batista, P., Rodrigues, M.A., Correia, I.J., Gil, M.H., 2011. Preparation and chemical and iological characterization of a pectin/chitosan polyelectrolyte complex scaffold for possible bone tissue reparation. Int. J. Biol. Macromol. 48, 112–118. Constantino, V.R.L., Pinnavaia, T.J., 1995. Basic properties of Mg2+1 − x Al3 + x layered double hydroxides intercalated by carbonate, hydroxide, chloride and sulfate anions. Inorg. Chem. 4, 883–892. Costantino, U., Ambrogi, V., Nocchetti, M., Perioli, L., 2008. Hydrotalcite-like compounds. Versatile layered hosts of molecular anions with biological activity. Microporous Mesoporous Mater. 107, 149–160. Costantino, U., Nochetti, M., Sisani, M., Vivani, R., 2009. Recent progress in the synthesis and application of organically modified hydrotalcites. Z. Kristallogr. 224, 273–281. Costantino, U., Nocchetti, M., Tammaro, L., Vittoria, V., 2012. Modified hydrotalcitelike compounds as active fillers of biodegradable polymers for drug release and food packaging applications. Recent Pat. Nanotech. 6, 218–230. Crepaldi, E.L., Valim, J.B., 1998. Hidróxidos duplos lamelares: síntese, estrutura, propriedades e aplicac¸ões. Quím. Nova 21, 300–311. Darder, M., Lopez-Blanco, M., Aranda, P., Leroux, F., Ruiz-Hitzky, E., 2005. Bionanocomposites based on layered double hydroxides. Chem. Mater. 17, 1969–1977. Darder, M., Aranda, P., Ruiz-Hitzky, E., 2007. Bionanocomposites: a new concept of ecological, bioinspired, and functional hybrid materials. Adv. Mater. 19, 1309–1319. de Souza, J.R.R., de Carvalho, J.I.X., Trevisan, M.T.S., Paula, R.C.M., Ricardo, N.M.P.S., Feitosa, J.P.A., 2009. Chitosan-coated pectin beads: characterization and in vitro release of mangiferin. Food Hydrocolloid. 23, 2278–2286.

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