A Novel, Potential Microflora-Activated Carrier for a Colon-Specific

May 6, 2009 - A novel polyelectrolyte complex (PEC) formed by sodium cellulose sulfate (NaCS) and chitosan was prepared as a potential material for a ...
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MATERIALS AND INTERFACES A Novel, Potential Microflora-Activated Carrier for a Colon-Specific Drug Delivery System and Its Characteristics Ming-Jun Wang, Yu-Liang Xie, Qiao-Dong Zheng, and Shan-Jing Yao* Department of Chemical and Biochemical Engineering, Zhejiang UniVersity, Hangzhou 310027, P. R. China

A novel polyelectrolyte complex (PEC) formed by sodium cellulose sulfate (NaCS) and chitosan was prepared as a potential material for a colon-specific drug delivery system. The characteristics of NaCS-chitosan film were measured by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), in vitro degradation, and in vitro drug release experiments. SEM data indicated that the NaCS-chitosan film had relatively homogeneous and smooth morphology at the initial state and deformed after being immersed in simulated colonic fluid (SCF). FTIR data indicated that the NH3+ of the chitosan had reacted with the SO4- of the NaCS. In vitro degradation behavior revealed that NaCS-chitosan could be degraded by colon microflora and be hydrolyzed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). It is found in the experiment of in vitro drug release that the capsules formed by the NaCS-chitosan complex could release about 80% of the drug loaded in the SCF during 4 h. All these results indicated that the NaCS-chitosan complex shows excellent behavior for colon specificity and could be a potential material for a colon-specific drug delivery system. 1. Introduction In order to achieve a successful colon-specific drug delivery system, carriers and relevant approaches are needed to protect the loaded drug from degradation, release, and/or absorption in the upper portion of the gastrointestinal (GI) tract and then ensure abrupt or controlled release in the proximal colon.1 Delivery of orally administered drugs specifically to the colon has important significance in the field of pharmacotherapy. First, the colon diseases, such as irritable bowel syndrome, Crohn’s disease, and ulcerative colitis, can be treated effectively when the antiinflammatory agents are applied directly to the affected area. Second, the colon was found to be a promising site for systemic absorption of peptides and proteins because of the less hydrolytic environment in comparison with the stomach and small intestine as well as the existence of specific transporters.2 Third, the drugs, which are destroyed by stomach acid and metabolized by pancreatic enzymes, are minimally affected in the colon.3,4 Further, colonic delivery of drugs may also be useful when intentionally delayed drug absorption is required such as in asthma, gastric ulcer, or arthritis.5,6 Likewise, a colonspecific drug delivery system would not only increase the bioavailability of the drug at the target site and reduce the dose to be administered but would also reduce the side effects.7 Some materials have been explored to be used in the colonspecific drug delivery design, but most of them have some distinct disadvantages such as toxicity, poor colon specificity, or complicated preparation; thus, few of them have been applied commercially.7 There are four kinds of approaches for colonspecificity: (i) pH-dependent, (ii) time-dependent, (iii) bacterially triggered, (iv) pressure-controlled. However, many colon-specific drug delivery systems, such as time- and/or pH-dependent delivery systems, were found to be not very reliable in terms of “site-specific release”, because * To whom correspondence should be addressed. Telephone: 86571-87951982. Fax: 86-571-87951015. E-mail: [email protected].

many factors influence the drug transit time and pH in the GI tract such as age, sex, diet, intestinal motility, disease state, etc.8-10 Another colon-specific drug delivery system, the microflora-activated system, has been developed to permit drug release in the colon through covalent bond cleavage by enzymes in the colon. Moreover, some colon-specific degradable materials, such as aromatic azo-polymers and poly- or disaccharides containing insoluble polymers, were investigated in the bacterially triggered colon-specific drug delivery system.10-12 Although a number of these synthetic azo-polymers have been evaluated for colon-specific drug delivery, these new chemical entities require a detailed toxicological test before being used as drug carrier materials. In this sense, use of naturally occurring polysaccharides is attracting alot of attention for drug targeting to the colon.7 The polysaccharides are inexpensive and available in a variety of structures with varied properties.13 They are highly stable, safe, nontoxic, biodegradable, and can be easily modified chemically and biochemically.7 The rationale for the development of a colon delivery system based on polysaccharides is the presence of a high level polysaccharidases of microbial origin in the human colon such as β-D-glucosidase, β-Dgalactosidase, amylase, pectinase, xylanase, β-D-xylosidase, dextranase, etc.4,14 A large number of polysaccharides have already been tested for their potential as colon-specific drug delivery systems, such as chitosan, pectin and its salts, chondroitin sulfate, cyclodextrin, dextrans, guar gum, inulin, amylose, and locust bean gum.4 Chitosan, in particular, is considered as a suitable candidate.15 Chitosan is a polycationic polysaccharide derived from natural chitin by alkaline deacetylation. Chemically, it is a poly-β(1-4)-D-glucosamine. Chitosan is nontoxic and biocompatible and can be digested by colonic bacteria.16 The degradation products of chitosan are nontoxic, nonimmunogenic, and noncarcinogenic. An experiment of chitosan intake by healthy volunteers was found to produce a significant decrease of the

10.1021/ie801295y CCC: $40.75  2009 American Chemical Society Published on Web 05/06/2009

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Table 1. sample code

DS of NaCS

apparent viscosity of chitosan

mixing ratio (NaCS:Chitosan)

concentration of chitosan (w/v), %

concentration of NaCS (w/v), %

ratio of SO4-/NH3+

D1 D2 D3

0.2 0.4 0.6

300 300 300

1:1 1:1 1:1

0.5 0.5 0.5

4 4 4

1.7 3.1 4.2

fecal phenols, p-cresol and indole, in analogy with other polysaccharides.17 Chitosan also inhibited the putrefactive activity of the intestinal microbiota, thus reducing the risk of disease states.18 In addition, it has the ability to be conjugated with a variety of substrates via its amine. These properties make chitosan a good candidate for the development of colon-specific drug delivery systems. However, chitosan can be dissolved in acidic solutions and thus rapidly dissolves in the gastric cavity and fails to protect the loaded drug during passage through the stomach and small intestine. To overcome this problem, chitosan has to be modified by chemical or other methods. A lot of publications reported the colon-specificity of chitosan.16,19-23 Because of two of the most important properties, cationic nature and high charge density in acidic solution, chitosan reacts with some water-soluble polyionic species and can form polyelectrolyte complexes (PEC) that are insoluble under neutral conditions. This newly formed material can pass through the stomach. A few polyelectrolyte complexes (PEC) have been applied as colon-specific carriers, such as a chitosan-pectin, chitosan-alginate, etc.20,24-27 Sodium cellulose sulfate (NaCS) is a polyanion, derived from cellulose by a heterogeneous sulfating process.28 It has favorable biological properties, such as nontoxicity, biocompatibility, biodegration,hydrosolubility,andgoodfilm-formingbehavior.29-31 In recent years, a noval microcapsule system formed by NaCS and polydimethyldiallyl-ammonium chloride (PDMDAAC) has been used to immobilize microorganisms, enzymes, and animal/ plant cells, etc.32-36 In this work, we report a novel PEC material formed with chitosan and NaCS as a potential carrier material for a colonspecific drug delivery system and further investigate its properties. We report the morphology and the formation of the NaCS-chitosan complex structure by SEM and FTIR spectroscopy and evaluate the characteristics of NaCS-chitosan degradation in vitro. In addition, a colon-specific drug delivery capsule is prepared, and the properties for in vitro drug release are evaluated. 2. Materials and Methods 2.1. Materials. Chitosan with an 85% degree of deacetylation and Mw of 1.03 × 103 kDa was supplied by Jinan Haidebei Co., Ltd. (China). The viscosity of a 1% w/v solution in acetic acid (1% v/v) is 300 mPa. 5-Aminosalicylic acid (5-ASA) was purchased from Shanghai Rida Chemical Reagent Co., Ltd. (China). NaCS (degree of substitution (DS) ) 0.2-0.6) was prepared by heterogeneous reaction as described previously in our laboratory.26,27 Sprague-Dawley (SD) rats (male, 240-260 g) were supplied by Zhejiang Academy of Medical Sciences (China). The gelatin used to produce the capsules was bovine hide type B gelatin (bloom 270.0 g; protein 87.8%; ash 0.20%; moisture content 12.0%) and was purchased from Sinopharm Chemical Reagent Co., China. Carrageenan (κ-carrageenan) was purchased from Fujun Carrageenan Co. Ltd., China. Its moisture content was 13% (w/w), and the material was used as received. Its molecular weight is 300 kDa. All other chemicals and reagents used were of analytical grade and were used without further purification.

2.2. Preparation of NaCS-Chitosan Film. NaCS-chitosan complex was produced from a pair of oppositely charged polysaccharides. A 4% (w/v) aqueous NaCS solution was prepared in deionized water. An aqueous chitosan solution was prepared by dissolving chitosan powder into the deionized water containing 1% (v/v) of acetic acid. Then two solutions were mixed together and stirred at 1500 rpm for 15 min at room temperature. The pH of the mixture was about 3.0-3.5. Thereafter a mixture of 50 mL was cast on a glass plate and thoroughly dried at 45 °C in vacuum. A yellow-white film was formed on the glass plate. The volume of film-forming solution per cm2 of the surface is about 0.76 mL. The thickness of the films is about 0.2 mm. Table 1 lists the composition of polyelectrolyte complex of NaCS-chitosan. Additionally, all samples are anion rich. 2.3. FTIR Spectroscopy. Fourier transform infrared spectrometer-attenuated total reflectance (FTIR-ATR) spectra for the NaCS, chitosan, and NaCS-chitosan films were obtained with a MAGNA-IR 560 spectrometer (Nicolet Instrument Corp., Wisconsin, USA). The spectra were scanned over the wavenumber range of 4000-400 cm-1 at ambient temperature. 2.4. In Vitro Experimental Conditions and Media. Preparation of pH 1.5 Solution. pH 1.5 solution was an aqueous solution of HCl that was adjusted to pH 1.5, and the final concentration of HCl is about 0.011 mol/L. Preparation of Simulated Gastric Fluid (SGF). Simulated gastric fluid (SGF) was an aqueous solution of 1% (w/v) pepsin that was adjusted to pH 1.5 using 0.1 M HCl. Preparation of Simulated Intestinal Fluid (SIF). Simulated intestinal fluid (SIF) was 0.05 M phosphate buffer, pH 7.5, with 1% (w/v) pancreatin present. Preparation of Simulated Colonic Fluid (SCF). Male Sprague-Dawley rats were anaesthetized, and the cecum was exteriorized for collection of the contents. The cecal contents were diluted with phosphate-buffered saline (PBS, pH 7.0) to 30% (w/v) dilution for release experiment. This step was conducted under N2 to maintain an anaerobic environment.37 2.5. In Vitro Degradation Behaviors and Morphology of the NaCS-Chitosan Complex. Degradation of the samples was monitored as the fractional weight loss. Initially at least three replicates (20 mm × 20 mm) of each dried NaCS-chitosan film were weighed (w0). The NaCS-chitosan films were immersed in the simulated gastric fluid for 1 h and then removed and immersed in the simulated intestinal fluid for 4 h. The NaCS-chitosan films were finally immersed in the simulated colonic fluid for 12 h. The samples were maintained at 37 °C and stirred at 100 rpm in a shaker. Then the NaCS-chitosan complex samples were removed, washed with distilled water five times, and dried in vacuum at 45 °C for 12 h. The masses of the samples were measured accurately and the weight loss expressed as a percentage of the original weight. The degradation percentage (Dt) at t (h) was expressed by eq 1: Dt )

w0 - wt × 100% w0

(1)

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Figure 1. The morphology and size of capsules based on NaCS-chitosan.

where wt was the weight of sample at time t, and w0 was the initial sample weight. 2.6. ScanningElectronMicroscopy(SEM).TheNaCS-chitosan complex samples at 0, 1, 5, 11, and 17 h were thoroughly dried and investigated by SEM. The NaCS-chitosan complex films were mounted on brass stubs using carbon paste. SEM photographs were taken by using a scanning electron microscope (Genenis 4000; Edax, New Jersey, USA) at required magnification at room temperature so as to obtain a topographical characterization of the films. 2.7. Preparation of Capsules with the NaCS-Chitosan Complex. First, a NaCS-chitosan complex mixture was prepared as described in Section 2.2. Second, the NaCS-chitosan complex mixture was placed in a water bath maintained at 80 °C. Then 8% w/v of gelatin and 2% w/v of carrageenan, with respect to the volume of NaCS-chitosan mixture, were added into the NaCS-chitosan mixture, while stirring continuously. Then capsules were prepared by dipping a stainless steel rod into the mixture prepared above, followed by subsequent drying in vacuum at 45 °C for 12 h. Then, the formed capsules were carefully denuded. Finally, the rim of each shell was clipped to form the capsule cap with a length of 13 mm and diameter of 6 mm. The size of the capsule was 0# whose length was 17 mm, inner diameter was 5.8 mm, and thickness was 0.2 mm. The capsules based on D1, D2, and D3 were named by C1, C2, and C3, respectively. The capsule was yellow and not transparent for the observance of NaCS-chitosan. The morphology of the capsule is given in Figure 1. 2.8. In Vitro Release Profile of the Capsule Based on NaCS-Chitosan. The release profile of the capsule based on NaCS-chitosan was investigated by using 5-ASA as a model drug. First, 20 mg of 5-ASA was manually filled into a hard capsule formed by NaCS-chitosan. The joint of the capsule body and cap was carefully sealed by pressing them so that it served as a lock mechanism. The capsules were immersed in the simulated gastric fluid for 1 h, removed, immersed in the simulated intestinal fluid for 4 h, and then removed and immersed in the simulated colonic fluid for 7 h. The rotation speed of the shaker was 100 rpm, and the temperature was maintained at 37 ( 0.5 °C. Capsules were tied to a paddle with a cotton thread in each dissolution vessel to prevent floating. At predetermined time intervals, 2 mL of medium from the vessel was sampled and replaced by an equal volume of fresh medium. The drug content was assayed by HPLC. The drug release percent was determined using eq 2 % released )

Rt × 100% L

(2)

where L and Rt represent the initial amount of drug loaded and the cumulative amount of drug released at time t.

2.9. HPLC Analysis. The HPLC consisted of a modular chromatographic system (model 1100 series pump, variable wavelength detector (VWD) and manual injection valve; Agilent, Waldbroon, Germany). The detector was set at 300 nm. Chromatography was performed on a reverse-phase column (Hypersil BDS2 C-18 5U column, 250 × 4.6 mm I.D.; Yilite Co., Dalian, China) at room temperature. The mobile phase was methanol-phosphate buffer, pH 6.8 (5:95, v/v), and the flow rate was 1 mL/min. 2.10. Statistical Analysis. All of the experiments were done in triplicate. Results were expressed as the mean ( SD, and differences between two means were considered significant, on the basis of the Student’s t test, at p < 0.05. 3. Results and Discussion The electrostatic attraction between the cationic amino groups of chitosan (the macro pKa value is about 6.5)38 and the anionic SO4- groups of NaCS is the main interaction leading to the formation of PEC. PEC reaction between NaCS and chitosan can be represented schematically as in Figure 2. The rationale of the formation of polyanion-polycation (polyelectrolyte) complexes was described previously.39,40 It is mainly driven by an electrostatic mechanism where charge neutralization and possible local overcompensation or bridging (such as hydrogen bonding, Coulomb forces, van der Waals forces, and transfer forces) mediated by a multivalent counterion induces attraction between topologically separated segments of the polyelectrolytes. 3.1. FTIR Analysis. To investigate the complex formation between NaCS and chitosan, FTIR studies were conducted. Figure 3 shows the FTIR spectra of NaCS, the NaCS-chitosan complex, and chitosan. The FTIR spectrum of chitosan shows a weak band of the C-H stretching at 2871 cm-1, the absorption band of the carbonyl (CdO) stretching of the secondary amide (amide I band) at 1645 cm-1, the bending vibration band of the N-H of nonacylated 2-aminoglucose primary amines at 1570 cm-1, and the bending vibrations of the N-H (amide II band) at 1557 cm-1.41 The peaks at 1411 and 1321 cm-1 belong to the N-H stretching of the amide and ether bonds and N-H stretching (amide III band), respectively. The bridge oxygen (C-O-C, cyclic ether) stretching bands at 1151, 1062, 1023, and 893 cm-1 are observed as well.40,42,43 NaCS shows the following distinct peaks: as a sulfate it shows strong absorption bands of SdO at 1218 cm-1 and C-O-S at 807 cm-1. The FTIR spectra of NaCS, NaCS-chitosan complex, and chitosan reveal that for the NaCS-chitosan complex, the N-H bending vibration of nonacylated 2-aminoglucose primary amines (band at 1570 cm-1), and the bending vibrations of the N-H (amide II band) at 1557 cm-1 became weak, possibly indicating that the NH3+ of the chitosan and the SO4- of NaCS could have formed PEC. 3.2. Degradation Behavior of NaCS-Chitosan Film in Vitro. The experiments of the degradation behavior of NaCSchitosan film were carried out by immersing the NaCS-chitosan films (samples D1, D2, and D3) in the SGF, SIF, SCF, and phosphate-buffered saline (PBS 7.0) in sequence. The chitosan samples were immersed in the SCF but not immersed in SGF and SIF. The degradation percentage of NaCS-chitosan films as a function of time is plotted in Figure 4. When the NaCS-chitosan films were in the SGF and SIF, no mass loss was observed. As can be seen in Figure 4, it was found that the NaCS-chitosan films maintained the intact structure and little mass loss at a time of 6 h. From the time of 7 h, the degradation rate increased. The NaCS-chitosan films

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Figure 2. Polyelectrolyte complex reaction between sodium cellulose sulfate (NaCS) and chitosan.

Figure 3. FTIR spectra of (i) sodium cellulose sulfate (NaCS), degree of substitution (DS) of NaCS ) 0.2; (ii) NaCS-chitosan complex sample D1; (iii) chitosan, viscosity ) 300 mPa · s.

deformed from original shape into irregular shape with a rough surface. Then the degradation rate of the NaCS-chitosan films

nearly remained slow after 15 h. The reason may be that the conformation of remnant PEC prevents further degradation

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Figure 4. Degradation behavior of NaCS-chitosan film in vitro. The NaCS-chitosan films (samples D1, D2, and D3) were immersed in the simulated gastric fluid (SGF), simulated intestinal fluid (SIF), simulated colonic fluid (SCF), and phosphate-buffered saline (PBS 7.0) in sequence. The degradation percentage of sample D1(9), sample D2 (b), sample D3 (2), sample D1 in PBS after 5 h (0), sample D2 in PBS after 5 h (O), sample D3 in PBS after 5 h (∆), chitosan control (]). Mean ( SD (n ) 3).

Figure 5. Schematic representation of the ionic interactions between NaCS and chitosan at (A) a lower degree of substitution (DS) of NaCS, (B) a higher degree of substitution (DS) of NaCS.

activity of the colonic enzymes. The mass loss of chitosan control was high than D1, D2, and D3 after 11 h, which convinced us that the PEC network could prevent degradation activity of the colonic enzymes. However, the mass loss of the control samples, which were immersed in PBS after 5 h, remained constant under the level of 4%, and no disintegration or deformation was observed. The mechanism of chitosan degradation is still ambiguous because there are too many kinds colonic bacterial enzymes in the colon. To date, only a few polysaccharide-degrading enzymes produced by colonic bacteria have been isolated and characterized. It is now known to us that the β-glucosidase in the colon can cleave the β-1,4-linkage in chitosan. Cellulase can also hydrolyze chitosan, but too little description of the characterization of the colonic enzymes was given. The degradation of NaCS-chitosan here could be attributed to coaction of a complex enzyme system.19 However, the effect of DS on the NaCS-chitosan degradation can be seen in Figure

4. For example, at a time of 17 h, the mass loss of NaCS-chitosan films was about 47%, 39%, and 30% for D1, D2, and D3, respectively. The mass loss of NaCS-chitosan films decreased with increasing DS of NaCS. The reason is that the low DS of NaCS results in less SO4- of the NaCS that can react with the NH3+ of the chitosan. The reason can be explained by the schematic presentation of the ionic interactions between chitosan and NaCS shown in Figure 5. Figure 5A shows that the lower DS of NaCS, the looser the PEC network. Therefore, the enzymatic chain cleavage occurs easily. In contrast to Figure 5A, the PEC network in Figure 5B is tighter, and enzymatic chain cleavage hardly occurs. In the experiment of in vitro degradation of the NaCS-chitosan film, when the NaCS-chitosan films were immersed in the SGF and SIF, no mass loss was observed, which reveals that the NaCS-chitosan film is insoluble in the SGF and SIF and cannot be degraded by pepsin and pancreatin. When the NaCS-chitosan samples were in the SCF for the first hour, little mass loss was

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Figure 6. A. Morphology of NaCS-chitosan films, at 0 h in the in vitro experiment, initial state. B. Morphology of NaCS-chitosan films, at 1 h in the in vitro experiment, taken out of the simulated gastric fluid (SGF). C. Morphology of NaCS-chitosan films, at 5 h in the in vitro experiment, taken out of the simulated intestinal fluid (SIF). D. Morphology of NaCS-chitosan films, at 11 h in the in vitro experiment, taken out of the simulated colonic fluid (SCF). E. Morphology of NaCS-chitosan films, at 17 h in the in vitro experiment, taken out of the simulated colonic fluid (SCF).

determined, and the degradation rate was slow. However, after 1 h, the degradation rate increased rapidly and more NaCS-chitosan film was degraded. There may be two reasons. First, the colon microflora needs a short time to produce enzymes which could degrade chitosan or NaCS. Second, enzymes which can degrade the NaCS-chitosan film would spend a short time to enter the network of the NaCS-chitosan complex before cleavage. However, little mass loss of the control samples in PBS was observed. These properties of NaCS-chitosan make it a good candidate for a colon-specific drug delivery system. 3.3. Morphology of NaCS-Chitosan Films in the in Vitro Experiment. In the experiment of in vitro degradation, the NaCS-chitosan sample D1 at 0 h, 1 h, 5 h, 11 h, and 17 h (Figure 4) was thoroughly dried and investigated by SEM. Figure 6A shows the SEM micrographs of the NaCS-chitosan

film at 0 h. It can be seen that the NaCS-chitosan film had relatively homogeneous and smooth morphology. Figure 6B shows the morphology of the NaCS-chitosan film which was taken out of the SGF at a time of 1 h. The NaCS-chitosan film shrank and wrinkled after being immersed in the SGF. Figure 6C shows the morphology of the NaCS-chitosan film which was taken out of the SIF at a time of 5 h. Similar to Figure 6B, the NaCS-chitosan film also shrank and wrinkled, after being immersed in the SIF. Figure 6D and Figure 6E show the morphology of the NaCS-chitosan film which was taken out of the SCF at time of 11 and 17 h, respectively. It could be seen that the surfaces of the NaCS-chitosan films became rough. The pores and umbos were clearly observed on the NaCS-chitosan film, and the amount of pores and umbos increased with increasing time in the SCF.

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Figure 7. In vitro drug release of 5-ASA from capsules based on NaCS-chitosan. The capsules were immersed in the simulated gastric fluid for 1 h, then removed and immersed in the simulated intestinal fluid for 4 h, and then removed and immersed in the simulated colonic fluid for 7 h. The percent of released drug of sample C1(9), sample C2(b), sample C3 (2), sample C1 in PBS after 5 h (0), sample C2 in PBS after 5 h (O), sample C3 in PBS after 5 h (∆), capsule without NaCS-chitosan (]). Mean ( SD (n ) 3).

3.4. In Vitro Drug Release. 5-ASA was chosen as a model drug to examine whether the capsule based on NaCS-chitosan may be degraded in the large intestine or not. The amount of 5-ASA released from the capsule based on NaCS-chitosan was determined in the presence of rat cecal contents. The releasetime profile of 5-ASA from capsules based on NaCS-chitosan is shown in Figure 7. In the SGF, no release of 5-ASA was found, and in the SIF, 5-ASA began to be released from the capsule at a low rate. During 5 h, about 10% 5-ASA was found in SIF. The reason is that water permeated into the capsule and dissolved the 5-ASA, and the drug began to be released from the capsule through the capsule shell. After the capsule immersion in the SCF, the release of 5-ASA was markedly increased in the presence of rat cecal contents. For the first hour in the SCF, 5-ASA in the capsule C1 was also released slowly, and then a burst effect was observed between 6 and 7 h. After 7 h, almost 92% 5-ASA was released from the capsule based on NaCS-chitosan. For the capsules C2 and C3, the burst effect was observed between 7 and 8 h and the maximum drug release of 5-ASA was 85% and 82% for capsules C2 and C3, which appeared at 9 and 11 h, respectively. In the control experiments, 5-ASA was released from the capsules based on NaCS-chitosan C1, C2, and C3 in phosphate-buffered saline (PBS, pH 7.0) at an almost constant rate and about 16-18% 5-ASA was released from the capsules at a time of 12 h. There was no significant difference in control experiments between capsules C1, C2, and C3. Compared to capsules C1, C2, and C3, the capsule without NaCS-chitosan released almost all the drug within the first hour in the SGF. From all of the above observations, it was found that the DS of NaCS had little effect on drug release in PBS but had significant effect on drug release in SCF. The higher the DS of NaCS, the later the appearance of the burst effect. The reason was that increasing the DS of NaCS resulted in decreased degradation in SCF and then resulted in the delay of drug release. Generally, drug can be permeated through polymeric shells (films or membranes) by two mechanisms: the pore mechanism

and the partition mechanism.44 In the pore mechanism, drugs pass through the shell by diffusion through pores within the shell at a rate controlled mainly by the pore size of the shell and the molecular volume of the drug. In the other mechanism, the partition mechanism, drugs permeate the shell by drug dissolution in the polymer structure followed by drug diffusion along and between the polymer segments that make up the film structure. In practice, drugs permeate probably by both mechanisms but one is more likely to predominate at different stages.45 While the capsules were in the SGF, no 5-ASA release was detected. The reason is that the capsule had a thick shell of about 0.2 mm and 5-ASA cannot permeate the shell of the capsule in 1 h. With increasing time, water permeated into the capsule and dissolved the 5-ASA, and the drug began to be released from the capsule through the capsule shell’s pores at a low rate. At this stage, the pore mechanism predominated. However, when the capsules were immersed in the SCF for the first hour (between 5 h and 6 h), the release rate of 5-ASA increased compared to the earlier time. At this stage, the enzyme produced by the colon microflora began to cleave the NaCS-chitosan, the capsule shell’s porosity would increase, and the drug permeation could be increased, too. In this stage, with the NaCS-chitosan complex degraded, both mechanisms worked, but it is not clear which mechanism predominated. A burst release of 5-ASA occurred during 6-8 h. During this stage, the partition mechanism predominated. The NaCSchitosan network was broken into segments by the colonic enzyme and the drug could permeate the capsule easily through the polymer segments. After 8 h, the percent of released drug decreased. The reason is that the 5-ASA was degraded or hydrolyzed by the colonic enzyme. Therefore, the curve after 8 h is the coordination between release and degradation. It could be seen that the earlier the burst release, the lower the percent of released drug. After 8 h, the broken capsule shells could be found in the SCF. In contrast, the capsule shells of control samples in PBS remained intact and 5-ASA release was at low rate in SCF.

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These results revealed that NaCS-chitosan film has a favorable colon-specificity and could be a potential candidate for a colonspecific drug delivery system. 4. Conclusions In the present work we have focused on the preparation of a novel colon-specific drug delivery material formed by the NaCS-chitosan complex and on the characterization of the NaCS-chitosan complex by SEM and FTIR. SEM data indicated that the NaCS-chitosan film has relatively homogeneous and smooth morphology at the initial state and degraded after being immersed in SCF. FTIR data indicated that the NH3+ of the chitosan has reacted with the SO4- of the NaCS. To investigate the degradation of NaCS-chitosan film and its application in colon-specific drug delivery, the degradation behavior and in vitro drug release profiles were studied. The results showed that NaCS-chitosan film could be degraded by colon microflora and be insoluble in the SGF and SIF. In the study of in vitro drug release, capsules formed by the NaCS-chitosan complex could almost release 80% of drug loaded in the SCF. The results reveal that the NaCS-chitosan complex has good behavior for colon specificity and could be a potential candidate for a colon-specific drug delivery system. Acknowledgment The authors thank the National Natural Science Foundation of China, the Hi-tech Research and Development Program of China (863 Program, 2006AA02Z210), and Scientific and Technologic Program of Zhejiang Province for their financial support. Literature Cited (1) Asghar, L. F.; Chandran, S. Multiparticulate formulation approach to colon specific drug delivery: current perspectives. J. Pharm. Pharm. Sci. 2006, 9 (3), 327–38. (2) Shen, H.; Smith, D. E.; Brosius, F. C., III. Developmental expression of PEPT1 and PEPT2 in rat small intestine, colon, and kidney. Pediatr. Res. 2001, 49 (6), 789–95. (3) Kinget, R.; Kalala, W.; Vervoort, L.; van den Mooter, G. Colonic drug targeting. J Drug Target. 1998, 6 (2), 129–49. (4) Minko, T. Drug targeting to the colon with lectins and neoglycoconjugates. AdV. Drug DeliVery ReV. 2004, 56 (4), 491–509. (5) Ishibashi, T.; Hatano, H.; Kobayashi, M.; Mizobe, M.; Yoshino, H. In vivo drug release behavior in dogs from a new colon-targeted delivery system. J. Controlled Release 1999, 57 (1), 45–53. (6) Wiwattanapatapee, R.; Lomlim, L.; Saramunee, K. Dendrimers conjugates for colonic delivery of 5-aminosalicylic acid. J. Controlled Release 2003, 88 (1), 1–9. (7) Sinha, V. R.; Kumria, R. Microbially triggered drug delivery to the colon. Eur. J. Pharm. Sci. 2003, 18 (1), 3–18. (8) Qi, M.; Wang, P.; Wu, D. A novel pH- and time-dependent system for colonic drug delivery. Drug DeV. Ind. Pharm. 2003, 29 (6), 661–7. (9) Takaya, T.; Ikeda, C.; Imagawa, N.; Niwa, K.; Takada, K. Development of a colon delivery capsule and the pharmacological activity of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in beagle dogs. J. Pharm. Pharmacol. 1995, 47 (6), 474–8. (10) Xu, C.; Zhang, J. S.; Mo, Y.; Tan, R. X. Calcium pectinate capsules for colon-specific drug delivery. Drug DeV. Ind. Pharm. 2005, 31 (2), 127– 34. (11) Van den Mooter, G.; Samyn, C.; Kinget, R. In vivo evaluation of a colon-specific drug delivery system: an absorption study of theophylline from capsules coated with azo polymers in rats. Pharm. Res. 1995, 12 (2), 244–7. (12) Rubinstein, A.; Nakar, D.; Sintov, A. Colonic drug delivery: enhanced release of indomethacin from cross-linked chondroitin matrix in rat cecal content. Pharm. Res. 1992, 9 (2), 276–8.

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ReceiVed for reView March 24, 2008 ReVised manuscript receiVed March 16, 2009 Accepted April 1, 2009 IE801295Y