| Abstract|| |
The aim of this study was to develop a controlled release system targeting antibiotic delivery to the stomach. The hydrogels were synthesized by using chitosan, poly (acrylic acid) and poly (vinyl pyrrolidone) polymers crosslinked with glutaraldehyde and N,N'-methylenebisacrylamide. Interpenetrating polymeric network (IPN) hydrogels were prepared by varying the concentration of crosslinking agent (glutaraldehyde). The amount of chitosan, poly (acrylic acid), poly (vinyl pyrrolidone) and N,N'-methylenebisacrylamide were kept constant in all formulations. The effect of glutaraldehyde concentration on the swelling and release characteristics were evaluated. Modalities used to assess the most optimal hydrogel formulation included high liquid chromatography, FTIR analysis, differential scanning calorimetry, swelling studies, in vitro drug release study, mucoadhesive study and scanning electron microscopy. The result showed that IPN hydrogels were greater in swelling, more mucoadhesive and released more drug at lower pH values. Thus, it is believed that the antibiotic concentration in the stomach might be sustained through this formulation.
Keywords: Controlled release, glutaraldehyde, IPN, stomach targeted
|How to cite this article:|
Gupta AK, Siddiqui AW, Datta MS, Ramchand D. Interpenetrating polymeric network hydrogel for stomach-specific drug delivery of clarithromycin: Preparation and evaluation. Asian J Pharm 2010;4:179-84
|How to cite this URL:|
Gupta AK, Siddiqui AW, Datta MS, Ramchand D. Interpenetrating polymeric network hydrogel for stomach-specific drug delivery of clarithromycin: Preparation and evaluation. Asian J Pharm [serial online] 2010 [cited 2013 May 25];4:179-84. Available from: http://www.asiapharmaceutics.info/text.asp?2010/4/4/179/76738
| Introduction|| |
Site-specific controlled release systems offer many distinctive advantages over classical method of drug delivery. These include localized delivery of the drug to a particular part of the body. Controlled release systems that have been developed so exhibit pH-dependent drug release.  Hydrogels are three dimensional, hydrophilic, polymer networks capable of imbibing large amounts of water or biological fluids. Interpenetrating polymeric networks (IPN) comprise two or more independent polymer networks that are formed in presence of one another. IPN properties such as porosity, elasticity, degree of swelling and responsive behavior to a stimulus can be tuned by the appropriate choice of network forming polymers and suitable crosslinking agent and its proportion. IPN hydrogels have emerged as useful materials for certain applications such as localized antibiotic delivery in the acidic environment of gastric fluid. 
Chitosan, a natural polysaccharide, exhibits favorable biological properties such as biocompatibility, biodegradability and nontoxicity. For several years chitosan has been largely evaluated as a potential vehicle for oral dosage forms. Chitosan, poly (acrylic acid) and poly (vinyl pyrrolidone) have been much investigated as a stimulus sensitive drug release system and glutaraldehyde is the most common crosslinking agent chosen for chitosan-based hydrogels. 
Helicobacter pylori lives deep within gastric mucus layer and prolonged local application of drug is needed sufficiently to diffuse bacteria. It has been demonstrated that H. pylori is one of the major causative micro-organisms for peptic ulcer disease. This bacterium release enzyme urease, which convert urea into ammonia and bicarbonate, which aids in neutralizing acidic medium and allow the bacteria to colonize in gastric mucosa. 
In the present study, an attempt has been made to formulate IPN hydrogels of clarithromycin using chitosan, poly (acrylic acid) and poly (vinyl pyrrolidone) polymers. Chitosan hydrogels provide pH-responsive release profile by swelling in gastric fluid (low in pH value) to release drug over a prolonged period of time.
| Materials and Methods|| |
Clarithromycin was gift sample from Ranbaxy Laboratories, Gurgaon, India. Chitosan (MW=3.5 Χ10 5 , >80% deacetylated) was gift sample from Central Institute of Fisheries Technology, Cochin. Poly (vinyl pyrrolidone) and glutaraldehyde were purchased from Central Drug House, New Delhi. Poly (acrylic acid) and N,N'-methylenebisacrylamide were purchased from Loba Chemie, Mumbai. All other chemicals were of analytical grade and double distilled water was used throughout the experiment.
Preparation of interpenetrating hydrogels
IPN hydrogel containing clarithromycin was prepared by chemical crosslinking process.  Chitosan gel was prepared in 1% acetic acid solution. A known amount of drug was added to chitosan solution and stirred for 15 min using mechanical stirrer. Poly (vinyl pyrrolidone), acrylic acid and crosslinkers were added to the solution of chitosan under continuous mixing. Chitosan, poly (vinyl pyrrolidone) and acrylic acid was used in 1:1:1 (w/w) ratio. The glutaraldehyde added in the IPNs was 0.25, 0.5, 1.0 and 2.0% w/v. Amount of N,N'-methylene-bis-acrylamide added was 1% to the weight of acrylic acid. Ammonium persulfate solution (25 ml) was lastly added. The mixture was placed in petridish and placed in room temperature to form gel. Gel was cut into equal dimensions (2Χ2mm) and washed with distilled water to remove any unreacted monomer or crosslinking agent. The washings were collected and preserved for analysis. They were then dried in air and vacuum, and stored for further use. [Figure 1] showed schematic preparation of hydrogel.
Determination of amount of drug entrapped
The amount of drug entrapped: in the IPN hydrogels was determined by an indirect method. After the gel preparation washings are collected, filtered with a 0.45 ΅m milipore filter and analysed by reversed-phase high performance liquid chromatography (RP-HPLC). The detection was done on RP-HPLC with the detection chosen at 210 nm. The difference between the amount of drug initially employed and the drug content in the washing is taken as an indication of the amount of drug entrapped.
% Drug entrapment = A2 / A1 Χ 100
A1 - Amount of drug initially loaded.
A2 - Amount of drug in washings.
Fourier transform infrared spectral analysis
The prepared IPN hydrogel pieces were subjected to fourier transform infrared (FTIR) analysis by KBr pellet method using FTIR spectrophotometer, [8201 PC (4000-400/cm), Shimadzu, Japan]. This was employed to ascertain the compatibility of drug with excipients.
Differential scanning calorimetry
Differential scanning calorimetry (DSC) was performed on pure sample of carithromycin and its formulation using STARe SW 9.00 apparatus. DSC thermogram of 4-5 mg sample was recorded at a heating rate of 5ºC/min. over a temperature range of 50-500ºC.
Surface morphology/scanning electron microscopy
The surface morphology of IPN hydrogel in unswollen state and kept in acidic pH (pH 2.0) was determined separately by using PHILIP 505 scanning electron microscope. Swollen hydrogel sample was dried in vaccum. Hydrogel samples were mounted on a scanning electron microscope holder (aluminium sample mount) with a double-sided adhesive tape and coated with gold palladium. The hydrogel samples were analyzed with PHILIP 505 scanning electron microscope at different magnifications and an acceleration voltage of 14.9 KV.
Swelling studies of IPNs were done as dynamic equilibrium swelling. In dynamic swelling experiment IPNs were placed in 0.01 N HCl (pH 2.0) at 37±1ºC. During swelling, gels were removed from the swelling bath at regular time intervals and their surface dried with filter paper and weighed; thereafter they were returned in to the same swelling bath.
The swelling ratio can be calculated as function of time and is calculated from the following relationship:
Where Ws represents the weight of the swollen state at a given time, Wd is the weight of sample at dry state, and We is the weight of the swollen state of a sample at equilibrium.
The mucoadhesive property of prepared IPN hydrogel was evaluated by in vitro mucoadhesive testing method known as washoff method as reported previously by Shantha and Harding  A rat stomach mucosa was tied on the glass slide using a thread. About 50 hydrogel pieces were spread on to wet rinsed tissue specimen and prepared slide was hung on to one of the grooves of a USP tablet disintegration apparatus. By operating the disintegrating test apparatus the tissue specimen was given a slow regular up and down movement in the test fluid at 37±1ºC. At every 1 hr-interval the equipment was stopped and the number of pieces still adhering to tissue was counted. Percent mucoadhesion was given by the following formula:
% Mucoadhesion = P1/P2 Χ 100
P1- no. of adhered hydrogel pieces
P2- no. of applied hydrogel pieces
In vitro drug release study
The release of clarithromycin from hydrogel was determined as described by Miyazaki et al.  using USP dissolution test apparatus with a paddle stirrer at 50 r/min. The dissolution medium used was 500 ml of 0.01 N HCl (pH 2.0), and temperature was maintained at 37±1 ºC. Hydrogel were transferred to the dissolution media. At specific time interval, a precisely measured sample of the dissolution medium was removed and replenished with prewarmed (37ºC) fresh medium to maintain sink condition. Samples were withdrawn at predetermined time intervals and neutralized with sodium hydroxide (0.05 M) solution prior to analysis by RP-HPLC using a mobile phase consisting of acetonitrile-aqueous 0.05 M phosphate buffer solution of pH 4.0 (40/60 v/v). The apparatus used for HPLC analysis was an Agilent 1100 quarternary pump, with a variable wavelength detector, thermostatted autosampler and column. A Hypersil ODS C 18 column (250Χ4.5 mm ID, 5 ΅m Thermo, UK) was fitted with a Phenomenex guard column packed with octadecyl C 18 . The column temperature was maintained at 40ºC and flow rate at 1 ml/min. The detector was set at 210 nm.
| Result and Discussion|| |
Chitosan, a natural polymer which exhibits pH sensitivity, was chosen to develop IPN hydrogels for oral-controlled drug delivery to the stomach region in combination with poly (vinyl pyrrolidone) and poly (acrylic acid). Acrylic acid is converted to poly (acrylic acid) by free radical polymerization using ammonium persulfate as a free radical polymerization initiator. Free radical polymerization is a process in which there is generation of free radical acts as a catalyst for the polymerization of monomers.
Drug entrapment efficiency
The entrapment efficiency of different IPN hydrogel formulation was calculated as percent total drug entrapped. The entrapment efficiency of clarithromycin in different formulation of IPN hydrogel was found to be 94.2±2.4%, 95±2.1%, 95.4±2.7% and 96.8±1.9% for FC1, FC2, FC3 and FC4 respectively. According to the method of preparation of IPN hydrogel the entrapment efficiency should be 100%, but the observations shows that entrapment efficiency is <100% in all formulations. This may be due to loss of drug during washing of hydrogel. [Table 1] The data indicate that entrapment efficiency depends on crosslinker ratio, at lower crosslinker ratio entrapment efficiency decreases because lower crosslinker ratio results into higher swelling during washing of hydrogel that will cause greater amount of drug loss during washing, and higher crosslinker ratio results in greater entrapment efficiency because of lesser swelling during washing that will results in lesser amount of drug loss during washing.
FTIR spectral analysis
The chitosan spectrum exhibits band at 3379.05 (OH stretching) and 3436.91 (-NH 2 stretching). The absorption band at 1118.64 (antisymmetric stretching of C-O-C bridge) and 2923.88 (-CH2 stretching). The band at 1652.88 due the chitosan spectrum was attributed to the formation of C=N, due to imine reaction between amino group of chitosan and aldehyde group in aldehyde [Figure 2].
|Figure 2: Fourier transform infrared spectra showing compatibility of drug with polymers in formulation FC2|
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From the DSC thermogram, it was observed that a sharp endothermic peak at 226 ºC was seen for clarithromycin (corresponds to its melting point) as well as for its formulation. Since the peaks are identical, it proves that there is no chemical interaction between the drug and the polymer employed for the preparation of IPN hydrogels [Figure 3].
|Figure 3: Comparative differential scanning calorimetric thermogram: DSC thermogram of clarithromycin (A) and IPN hydrogel formulation FC2 (B)|
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Swelling curves of IPN hydrogel with different amount of crosslinking agent shown that swelling increased with time and it will also show that swelling decreased with increasing crosslinking agent in IPN hydrogel. The degree of swelling of IPN hydrogels depend on its network structure, which is controlled by the concentration of crosslinking agent. Results indicate that IPN hydrogels exhibited greater swelling at lower crosslinker ratio [Figure 4]. This can be explained in terms of ionic interaction of amino group of chitosan in stomach pH condition. The protonation of amino group in chitosan ensures chain relaxation, thus leads to faster hydrogen bond dissociation and efficient solvent diffusion. 
|Figure 4: Percentage swelling of interpenetrating polymeric hydrogels prepared|
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An increase in amount of crosslinking agent leads to denser crosslinking bridges. Such structures can be characterized by lower and slower penetration of solvent through the chain structure of polymer, suggesting that the swelling ratio and release characteristics of the IPN hydrogel can be controlled by varying crosslinking agent concentration used during manufacturing process. Swelling of hydrogels may result in to mobility of crosslinking chains, facilitating solvent diffusion and rapid release of drug through polymer. Since glutaraldehyde is responsible for the formation of crosslinks, increasing the concentration of glutaraldehyde will increase the network density, resulting in reduction of chain mobility and formation of more stable and rigid hydrogel that show a slower tendency to swell. Sahin et al, reported that a decrease in the swelling ratio in terbutaline sulfate loaded albumin microsphere was observed with an increase in the glutaraldehyde concentration.  Raymond et al found out that the release of phenytoin sodium from gelatine microsphere can be delayed by the addition of glutaraldehyde to the microsphere formulation. 
In vitro drug release study
The release of clarithromycin from IPN hydrogel was studied in 0.01 N HCl (pH 2.0) at 37±1 ºC. The release of drug significantly increased with decreasing crosslinking agent concentration. The diffusion of drug molecule out of IPN hydrogels containing chitosan was enhanced because of swelling at lower pH. The extent of release increase as the hydrogel swelling increase at lower pH, which leads to ionization of amino groups. This selective release will ensure maximum availability of the drug in the stomach thereby maintaining bactericidal concentration of the antibiotic in the stomach. The formulation FC1 shows best releases profile and it will release about 88.54±4.1% drug respectively in 12 hr, so justifying itself as an optimized formulation in terms of drug release profile [Figure 5].
The mucoadhesive property of the hydrogels was evaluated by wash-off method. At the end of 5 hr, % mucoadhesion was found to 80±2.15%, 60±3.40%, 62±3.67%, 62±2.68% for FC1, FC2, FC3, FC4 formulations [Figure 6]. Formulation FC1 shows highest % mucoadhesion. The basis of mucoadhesion that a dosage form can stick to the mucosal surface. A salt bridge effect has been proposed for the interaction of the positively charged mucoadhesive hydrogel particles with the negatively charged mucous glycoprotein.  Chitosan possesses OH and NH 2 groups that can give rise to hydrogen bonding. These properties are considered essential for mucoadhesion. Further cationic polyelectrolyte nature of chitosan could provide a strong electrostatic interaction with mucosal surface.  The rank order of mucoadhesion for formulations was to be FC1 > FC2 > FC3 > FC4.
Surface morphology/scanning electron microscopy
Surface morphology of the IPN hydrogels was examined by scanning electron microscopy (SEM) by using PHILIP 505 scanning electron microscope [Figure 7]. It was observed that surface of the unswollen hydrogels (FC1) showed no surface pores [Figure 8]a. The IPN hydrogel pieces kept in 0.01N HCl (pH 2.0) showed sponge like porous surface [Figure 8]b.
|Figure 8: Scanning electron micrograph of unswollen (a) and swollen (b) IPN hydrogel|
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Thus, IPN hydrogels of clarithromycin could be prepared using chitosan, poly (vinyl pyrrolidone) and acrylic acid monomer, and glutaraldehyde and N, N'-methylene bis acrylamide as crosslinking agents. The prepared IPN hydrogel particles showed swelling in acidic pH. Formulation FC1 with low crosslinking agent showed better swelling and release of drug.
| Conclusions|| |
A Stomach retentive IPN hydrogel was prepared successfully by chemical crosslinking process. The results of in vitro studies showed that by appropriate modification of crosslinking agent concentration the extent of swelling and rate of drug release can be modulated. Mucoadhesive study showed that, "IPN hydrogel prepared" have good mucoadhesion property and retained in gastric environment of stomach for prolonged period of time. Thus IPN hydrogel prepared maintain antibiotic concentration in stomach for prolonged period of time, can be used as a drug delivery system for treatment of H. pylori infection and in management of peptic ulcer. Further modification of these interpenetrating hydrogels can lead to the successful application for the targeting a drug delivery system (or device) to the stomach with extended release.
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Anish Kumar Gupta
Department of Pharmacy, Lloyd Institute of Management and Technology, Greater Noida-201 306, U.P
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]