JDDST-2010

23Potential applications of chitosan in oral mucosal delivery S. Şenel Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100-Ankara, Turkey *Correspondence: [email protected] The accessibility of the oral cavity makes application of drugs easy and acceptable to the patient, while permitting easy removal in the event of adverse reactions. Drugs penetrating the oral mucosa can be delivered systemically via direct entry into the systemic circulation thus avoiding the hepatic frst-pass effect and degradation in the gastrointestinal tract. Due to the low permeability of the oral mucosa, new strategies are needed to improve the delivery of drugs across the mucosa. In order to improve the availability of drugs across the oral mucosa, mucoadhesive systems are applied to prolong the retention time of the delivery system on the mucosa. Furthermore, availability can be improved by using permeation enhancers. Chitosan with its favorable properties such as bioadhesivity, biodegradability, biocompatibility and permeation enhancing activity, offers great advantages over other polymers that are used for oral mucosal delivery. A wide choice of chitosan delivery systems such as solutions, gels, sponges, flms, fbers, tablets and micro-/nanoparticles have been shown to be capable of delivering drugs into the oral cavity as well as across the oral mucosa. In this review, applications of chitosan for oral mucosal delivery will be reviewed and the possibilities and limitations discussed. Key words: Chitosan – Oral mucosa – Oral mucositis – Dental delivery. J. DRUG DEL. SCI. TECH., 20 (1) 23-32 2010 For drugs which are usually ineffective when administered via the oral route due to hydrophilic and enzymatic degradation in the gastrointestinal tract, delivery through various mucosal routes such as nasal, oral mucosal, rectal, vaginal, etc. has been widely applied as an alternative delivery route. Over the last decade, the delivery of drugs across the oral mucosa, especially buccal mucosa has gained more importance, and there has been a signifcant increase in the number of the buccal products [1] which are on the market. There are many advantages of oral mucosa which make it an attractive site for delivery. Firstly, it provides direct entry into the systemic circulation hence avoiding the hepatic frst-pass effect and degradation in the gastrointestinal tract. It is a readily accessible area, and allows self application. The delivery can be terminated at any desired time. It is not gender specifc, and patient compliance is reported to be very good. However, there are also some disadvantages associated with this route of drug delivery, mainly the low permeability and a smaller absorptive surface area in comparison to the absorptive surface area of the small intestines. Furthermore, salivation and resulting swallowing would effectively remove the drug from the preferred absorptive region. In order to overcome these limitations, new approaches are being explored dealing with permeation enhancers, mucoadhesion, etc. for better delivery which enables the delivery system to remain on the application site longer and improve availability by also enhancing the permeability of the oral mucosa. Due to its favorable properties such as bioadhesion and penetration-enhancing effect, besides its bioactive properties, in recent years, chitosan has become a very promising compound in the development of oral mucosal systems. After a short introduction to oral mucosa and delivery, this review will focus more on chitosan and its applications in oral mucosal delivery and treatment. For further information on oral mucosal delivery, the reader is asked to refer to the recently published review articles [1-4]. I. STRUCTURE AND PERMEABILITY PROPERTIES OF THE ORAL MUCOSA The anatomy and functions of the oral mucosa have been extensively reviewed in previous papers [5, 6], hence in this section the structure of the oral mucosa and its properties will be described briefy only to facilitate understanding how drugs, after applied to the oral mucosa, cross the mucosa to reach to the blood capillaries. The soft tissues lining the human oral cavity are covered with a stratifying squamous epithelium. In regions subject to mechanical forces associated with mastication (gingiva and hard palate), there is a keratinizing epithelium which is similar to that of the epidermis of the skin. The mucosa in these regions is known as masticatory mucosa. The mucosal lining of the foor of the mouth (sublingual) and buccal regions, which must be fexible so as to accommodate chewing or speech, is termed the lining mucosa and is covered with a nonkeratiniz- ing epithelium like the epithelium covering the esophagus or uterine cervix. Specialized mucosa is found on the top surface of tongue and is of lesser importance to drug permeation [5]. The masticatory mucosa represents approximately 25%, the specialized mucosa (dorsum of tongue) approximately 15%, and the lining mucosa approximately 60% of the total surface area of the oral lining [7]. Both the structure and the relative area of the different types of mucosa would infuence the penetration of substances across the oral lining. The basic drug transport mechanism for oral epithelium is the same as for other epithelia in the body. There are two major routes involved: transcellular (intracellular) route and paracellular (intercellular). The major pathway across the stratifed epithelium of large molecules is via the paracellular spaces, and there is a barrier to penetration as a result of modifcations to the intercellular substance in the superfcial layers [8]. In non-keratinized epithelium, as cells reach the upper third to quarter of the epithelium, membrane coating granules become evi- dent at the superfcial aspect of the cells and appear to fuse with the plasma membrane so as to extrude their contents into the intercellular space. This discharge forms a barrier to the permeability of various compounds. Furthermore, different compounds may penetrate the epithelium at different rates, depending on the chemical nature of the molecule and the type of tissue being traversed. Passive diffusion has been shown to be the primary mechanism of transport of the drugs across the buccal mucosa whereas carrier-mediated transport has been reported to have a small role [1]. II. ORAL MUCOSAL DELIVERY SYSTEMS In general, oral mucosal delivery systems are designed to provide either unidirectional or multidirectional drug release. For unidirectional release, they may be applied topically on the mucosa as a reservoir that can release material either into the oral cavity (Figure 1a) or across the mucosa (Figure 1b). In the frst case, there is no requirement for the 24 agents to penetrate the oral lining, and this may not even be desirable whereas in the latter case, the drug is desired to penetrate across the oral mucosa and reach the blood circulation. Agents can also be applied topically so as to treat local mucosal conditions that might range from mucositis and the common mouth ulcer to rarer lesions, such as the blistering or vesicular-bullous diseases (Fig. 1c). In this modality, the target of drug action is likely to be the deeper proliferative cells of the oral epithelium or the infammatory process in the underlying connective tissue. In either case, the compound will have to penetrate the superfcial barrier layers of the oral epithelium. For rapid oral mucosal delivery, the drug may be presented as lozenges, flms, sprays or compressed tablets having a fairly rapid in-mouth disintegration time (15 min or less). Where prolonged or reservoir action is required, the dosage form is usually bioadhesive and the drug is release-controlled for slow absorption through the oral mucosa or slow release into the oral cavity. 1. Bioadhesion The ability to maintain the delivery system at a particular site for an extended period of time is benefcial both for local disease treatment and also for systemic drug bioavailability. Bioadhesive polymers have been widely used to maintain an intimate and prolonged contact of the formulation with the oral mucosa [3, 9]. The term bioadhesion is defned as attachment of a synthetic or natural macromolecule to mucus and/or an epithelial surface [10]. When adhesion occurs in a biological setting it is often termed “bioadhesion”, furthermore if this adhesion occurs on mucosal membranes it is termed “mucoadhesion” [11]. In general the expressions “bioadhesion” and “mucoadhesion” are often used interchangeably. Nagai was among the frst to pioneer the bioadhesive drug delivery system in the early 1980s [12]. The frst product developed by his group contains a steroidal anti-infammatory agent, triamcinolone acetonide, and is still on the market for the treatment of aphthous stomatitis (AFTACH by Teijin Pharma Ltd, Japan) [13]. 2. Penetration enhancement Another approach to overcome the barrier properties of the buccal mucosa for drugs is the incorporation of chemical compounds into the formulations [14]. Substances that help to promote drug permeation through the buccal epithelium are referred to as penetration enhancers, permeation promoters, absorption enhancers [15]. Ideally chemicals used as penetration enhancers should be safe and non-toxic, non- irritant, pharmacologically and chemically inert and non allergenic. In addition, the tissue should revert to its normal integrity and barrier properties upon removal of the chemical. Penetration enhancers can be divided into many categories according to their structure, mechanism of action and the type of drugs whose permeation they enhance. Penetration enhancers used for buccal delivery, the description of which is precluded due to the brevity of the current article, have been extensively reviewed by several authors [16, 17]. Buccal penetration enhancement is reported to result from agents being able to (a) increase the partitioning of drugs into the buccal epithelium, (b) extract (and not disrupt) intercellular lipids, (c) interact with epithelial protein domains and/or (d) increase the retention of drugs at the buccal mucosal surface [16]. III. CHITOSAN Chitin, which is the most abundant polysaccharide in nature after cellulose, is the major component of exoskeletons of crustaceans and insects as well as of cell walls of some microorganisms, bacteria and fungi. Chitosan is obtained by alkaline deacetylation of chitin, by removal of acetyl groups from the molecular chain of chitin to give amino groups (-NH 2 ). It comprises copolymers of glucosamine and N-acetyl glucosamine. Chitosan differs from cellulose at the C-2 car- bon by having this amine group in place of a hydroxyl group. Hence, unlike cellulose, chitosan possesses positive ionic charges. The degree of acetylation represents the proportion of N-acetyl- D-glucosamine units with respect to the total number of units and can be used to differentiate between chitin and chitosan. Chitin with a degree of deacetylation of 65-70% or above is generally known as chitosan. The degree of deacetylation is an important property of chitosan which defnes its physicochemical and biological properties and hence determines its applications. While chitin is insoluble in most organic solvents, chitosan is readily soluble in dilute acidic solutions below pH 6.0. Its solubility and possession of free amino groups which are active sites for many chemical reactions makes chitosan more preferable than chitin. Chitosan exhibits a variety of physicochemical and biological properties resulting in numerous applications in felds ranging from waste and water treatment, agriculture, textiles, cosmetics, nutritional enhancement and food processing. Due to its biocompatibility, biodegradability and bioactivity it became a very attractive substance for diverse applications as a biomaterial in pharmaceutical and medical felds. As a non-toxic and non-allergenic bioadhesive polymer, chitosan has been extensively studied for mucosal delivery of drugs and vaccines as well as for its bioactive properties such as antimicrobial, anti-infammatory, hemostatic, tumor inhibition, antiviral, tissue regeneration, wound healing and immunogenic activities [18]. It has also been shown to enhance the permeation of the compounds across various mucosae such as buccal, nasal, intestinal and vaginal mucosa [19-22]. Like its composition, the molecular weight of chitosan varies with the raw material sources and the method of preparation. It is commercially available from a number of suppliers in various grades of purity, molecular weight, and degree of deacetylation. However, there is still a lack in standardization of chitosan for pharmaceutical and biomedical applications even though there is a monograph for chitosan chloride in the European Pharmacopeia (EP 2008) and a monograph for chitosan is being prepared for the United States Pharmacopoeia (USP). The major beneft of using chitosan within pharmaceutical applications is the possibility of adding various chemical groups, in particular to the C-2 position allowing for the formation of novel conjugates with additional functionalities. Using such modifcations, Figure 1 - Drug release from oral mucosal delivery systems: a) unidirectional-into the oral cavity; b) unidirectional-across the mucosa; and c) multidirectional-into the oral cavity and oral mucosa. Potential applications of chitosan in oral mucosal delivery S. Senel J. DRUG DEL. SCI. TECH., 20 (1) 23-32 2010 25 the properties of chitosan may be tailored to suit the requirements of specifc pharmaceutical-technological challenges. The positive surface charge of chitosan allows it also to interact with macromolecules like exogenous nucleic acids, negatively charged mucosal surfaces, or even the plasma membrane. It has been shown that chitosan and its degradation products are quickly eliminated via the renal route following intraperitoneal administration to mice, thus overcoming accumulation in the body [23]. In physiological conditions, chitosan is thought to be degraded by lysosymes or by chitinases. While chitin and chitin synthase do not exist in mammals, human chitinase family members have recently been described. Chitinases are ubiquitous chitin-fragmenting hydrolases. The frst human chitinase discovered was chitotriosidase that is specifcally expressed by phagocytes, and later the identifcation, purifcation, and subsequent cloning of a second mammalian chitinase was reported [24]. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and humans the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Bioadhesive and penetration-enhancing properties of chitosan Chitosan has been proposed as a bioadhesive polymer for use in oral mucosal drug delivery [11]. The bioadhesive property of chitosan is mediated by ionic interaction between the positively charged chitosan amino groups in chitosan and the negatively charged sialic acid residues in mucus [9, 25]. A wide variety of bioadhesives have been tested in the tissue culture model and the effect of mucin was also examined. Whilst many gels were found to adhere for 1-5 h, chitosan was shown to remain longer than a day [26]. Histologically, excellent tissue wetting properties were observed in the presence of chitosan. The absence of mucin, the control of gel hydration and swelling and wetting characteristics were identifed as key factors for prolonged adhesion. Many studies have been reported on the penetration-enhancing effect of chitosan across the buccal mucosa [2, 20, 27]. It has been shown that the interaction between various types of bioadhesive polymers and epithelial cells also has a direct infuence on permeability of the tissue [28]. In this way, the penetration of large and hydrophilic molecules across an epithelial barrier may be enhanced. Similarly, the enhancing effect of chitosan on buccal permeability can be explained by the bioadhesive nature of chitosan, which increases the retention of the drug at its application site. Furthermore, as chitosan has been shown to be capable of disrupting lipid micelles in the intestine [29], the permeabilizing effect may also be attributed to its interference with the lipid organization in the buccal epithelium, however, such a mechanism has not been proven. The bioadhesive and penetration-enhancing properties of chitosan and its derivatives have been investigated in numerous in vivo and in vitro models [30]. In vitro permeation studies are performed in diffusion cells using excised tissues such as porcine and bovine [1, 31]. Buccal cell cultures have also been used for in vitro permeation and bioadhesion studies [32-34]. A lectin-binding inhibition technique, involving an avidin-biotin complex and a colorimetric detection system was developed to investigate the binding of chitosan to buccal epithelial cells, without having to alter their physicochemical properties by the addition of marker entities [35]. From the wide range of polymer solutions screened, chitosan was found to give the greatest inhibition of lectin binding to the surface of buccal cells, while methylcellulose, gelatin, Carbopol 934P and polycarbophil also produced a substantial reduction. Another technique has been described by Kockisch et al. [36] to evaluate polymer adhesion to human buccal cells following exposure to aqueous polymer dispersion, both in vitro and in vivo. Adhering polymer was visualized by staining with 0.1% (w/v) of either Alcian blue (60 min) or Eosin (10 min) solution, uncomplexed dye being removed by 0.25 M sucrose washings. The extent of polymer adhesion was quantifed by measuring the relative staining intensity of control and polymer-treated cells by image analysis. Chitosan was found to adhere to human buccal cells and following in vivo administration as a mouthwash it was shown to stay on the human buccal mucosa for at least 1 h. Various derivatives such as trimethylated chitosan [37], mono-N- carboxymethyl chitosan [38], chitosan-EDTA conjugates [39], thiolated chitosan [40] have been developed to improve its mucoadhesive and/ or permeation enhancing properties as well as to enhance its solubility. It was shown that the mucoadhesive properties were also affected by the charge of the polymer [41]. The mucoadhesive and penetration enhancement properties of 5-methyl-pyrrolidinone chitosan, two low-molecular-weight chitosans and a partially reacetylated chitosan were studied ex vivo using porcine buccal mucosa. Acyclovir, as the model drug, was added to the polymer solutions at 5% (w/w) concentration. Methyl-pyrrolidinone chitosan was shown to exert the best mucoadhesive and penetration enhancement properties and the penetration of acyclovir was found to decrease by partial depolymerization of chitosan and disappear after partial reacetylation [22]. It has been shown that the mucoadhesion and penetration-enhancing properties of N-trimethyl chitosans (TMCs) depend on the degree of quaternization and molecular weight [42]. The mucoadhesive properties were found to increase with increasing degree of quaternization [42]. Similarly, the effect of partially substituted N,O-[N,N-diethylaminome- thyl (diethyldimethylene ammonium)n]methyl chitosans, containing different percentages of pendant quaternary ammonium groups, on the permeation of rhodamine 123 (Rh-123), (hydrophobic marker of the transcellular absorption route), and of fuorescein sodium (NaFlu), (polar marker of the paracellular route), was investigated across the excised porcine cheek epithelium. These chitosan derivatives, having varying degrees of substitution were shown to enhance the paracellular drug penetration across the buccal epithelium which was remarkably higher than that of trimethylated chitosan whereas the transcellular transport was substantially accelerated only by the most substituted derivative [43]. IV. APPLICATIONS IN DRUG DELIVERY Due to its bioadhesive property, permeation-enhancing property and biocompatibility, chitosan is an excellent candidate for delivering therapeutic compounds especially when prolonged release is desired. Chitosan and its derivatives in various forms such as gels, flms, sponges, tablets, micro-/nanoparticles have been used for the delivery of drugs into the oral cavity as well as across the oral mucosa. The ex vivo and in vivo studies published in the last decade on applications of chitosan are summarized in Table I. In the following sections, the application of chitosan as a delivery system will be reviewed focusing more on formulation technologies. 1. Applications in the treatment of oral mucositis In general, the use of bioadhesive gels reduces the frequency of application and the amount of drug administered and can also improve patient compliance and acceptance. Hence many studies have been reported on gel formulations of chitosan and derivatives for drug delivery. Chitosan in gel form has been studied by several groups in the treatment of oral mucositis, which is a particularly painful and debilitating consequence of cancer therapy and occurs as a result of damage to the oral mucosa by radiation or chemotherapy. For the mucositis patient, the occlusion and lubrication of the chitosan gel is expected to reduce the discomfort of the infammatory and ulcerative conditions. Associated with atrophy and ulceration of the oral mucosa is an increased risk of infection, particularly when there is immunosuppression. Therefore, chitosan is an excellent candidate for the treatment of oral mucositis, Potential applications of chitosan in oral mucosal delivery S. Senel J. DRUG DEL. SCI. TECH., 20 (1) 23-32 2010 26 Table I - Applications of chitosan and its derivatives in oral mucosal drug delivery and treatment. Dosage form Drug Chitosan type Other additives Activity Treatment Model Ref. Gel TGF-b Chitosan-H (DD: 80%, MW: 1400 kDa, Dainishiseika Col- our and Chem. MGF, Japan) - Penetration enhancer Buccal drug delivery Wound healing Ex vivo (porcine mucosa) 20 Gel Hydrocortisone Chitosan-H (DD: 80%, MW: 1400kDa) - Penetration enhancer Buccal drug delivery Antiinflamma- tory Ex vivo (porcine mucosa) 26 Tablet Nicotine Protasan CL212 (DD: 73%, MW: 272kDa) Carbopol 974P Magnesium hydroxide Buccal drug delivery Smoking ces- sation Human 30 Gel Nystatin Chitosan-H (DD: 80%, MW: 1400kDa) Protasan CL 213 (DD: 84%, MW: 272kDa, Pronova,Norway) - Antifungal Buccal and gingival drug delivery Oral mucositis Hamster Human 43 Film Ginsenoside Rb1 Chitosan glycerol (Wako Pure Chemical Industries, Osaka, Japan) Sodium alginate Buccal drug delivery Oral mucositis Hamster 44 Film patch AZMX (Astra- Zeneca) Chitosan (MW: 400 kDa) PVA, PEO, PVP sodium tauro- cholate Gingival drug delivery Model study Dogs 47 Tablet containing chitosan microspheres Chlorhexidine diacetate Chitosan (DD: 75-85%, Aldrich,USA) Mannitol Sodium alginate Saccharine Buccal drug delivery Antifungal Human 53 Blend film Ornidazole Carboxymethyl-chitosan (MW: 199.6 kDa, substituent degree of carboxymethyled: 0.93) PVA Buccal drug delivery Antimicrobial Rat Rabbit 59 Gel Denbufylline Palmitoyl glycol chitosan SGDC Carbopol 974 Buccal drug delivery Model study Rabbit 69 Disc Bromocriptine mesylate/ pluronic F-127 solid dispersion Chitosan Carbopol 974P Buccal drug delivery Pathologic hyperprolactinemia Human 73 Patch Miconazole Chitosan NaCMC, PVA, HEC, HPMC, PVP Buccal, gingival drug delivery Antifungal Human 78 Film Taurine Chitosan-H (DD: 80%, MW: 1400kDa) Collagenous membrane Wound healing Periodontal regeneration Beagle dogs 63 Gel Demineralized bone matrix Protasan UP CL213 (DD: 84%, MW 252 kDa) Collagenous membrane Tissue regeneration Periodontal regeneration Human 64 Scaffold Plasmid and virus encoding TGF-ß1 gene Chitosan (DD: 85%, Sigma, USA) Collagen Tissue regeneration Gene delivery Periodontal regeneration BALB/c 65 Sponge Platelet-derived growth factor- BB (PDGF-BB) - - Bone healing Periodontal regeneration Rat 79 DD: degree of deacetylation; HEC (hydroxyethyl cellulose); HPMC (hydroxypropylmethyl cellulose); MW: molecular weight; NaCMC: sodium carboxymethyl cellulose (NaCMC); polyethyleneoxide (PEO) PVA (polyvinyl alcohol); PVP (polyvinyl pyrrolidone); SGDC: sodium glycodeoxycholate;TGF-b: transforming growth factor offering not only delivery of therapeutic compounds but also exerting antimicrobial activity as well as the ability to stimulate cell proliferation and tissue organization. In a previous study [20], transforming growth factor-b (TGF-b), which was chosen as a candidate compound that might protect or promote the healing of the mucosal lining by having a direct effect on epithelial proliferation in patients with oral mucositis, was incorporated into chitosan gel [20]. To exert an effect after topical application, it Potential applications of chitosan in oral mucosal delivery S. Senel J. DRUG DEL. SCI. TECH., 20 (1) 23-32 2010 27 is essential that the compound reaches the proliferative (basal) com- partment of the epithelium. Our results showed a six- to seven-fold enhancement of permeability by chitosan for TGF-b to which the oral mucosa was reported to be rather impermeable. Tissue section- ing technique enabled the compound to be quantitated in different strata at different time points, which provided information as to the effectiveness of delivery of the bioactive peptide to the proliferating cell compartments. The quantity of the drug in the superfcial layers of the epithelium was found to increase in the presence of chitosan, and also more TGF-b reached the deeper layer. The hydrophilicity of the compound also seemed to have an effect, for there was relatively greater penetration of TGF-b into the deeper tissue layers as compared to our previous study using hydrocortisone, which is a water insoluble compound [27]. In another study, we prepared gel and flm formulations using chitosans at different molecular weights and in different solvents [44]. Nystatin, which is considered as a prophylactic agent for oral mucositis was incorporated into the formulations. The in vitro release of nystatin from the formulations was found to decrease with the increasing molecular weight of chitosan. The effect of the formulations was investigated in vivo in hamsters with chemotherapy (5-fuorouracil)- induced mucositis. Mucositis scores in groups treated with nystatin incorporated into gel and suspension formulations were signifcantly lower (p < 0.05) than those treated with the chitosan gel alone. Survival of animals in the treated groups was higher than that in the control group. The retention time and distribution of the gels in the oral cavity were investigated in healthy volunteers. A faster distribution of nystatin in the oral cavity was obtained using the nystatin suspension compared to the chitosan gels, but the nystatin saliva level also decreased rapidly. Drug concentration above the minimum inhibitory concentration (MIC) value for Candida albicans (0.14 μg/mL) was maintained for longer periods of time at the application site (90 min) in the oral cavity. Similarly, Watanabe et al. [45] studied the effect of ginsenoside Rb1 isolated from ginseng on 5-fuorouracil-induced oral mucositis in hamsters. Ginsenoside Rb1 was incorporated into chitosan-sodium alginate flm. The flms were attached to the oral mucosa, and then the healing process was examined by measuring the area of mucositis, myeloperoxidase (MPO) activity and microscopic aspects. G-Rb1- containing flms were shown to improve recovery which was found to be dose-dependent whereas flms without ginsenoside Rb1 were shown to have no effect in comparison to the control group. In a study in which the functional (mucoadhesion, viscosity) and bioactive (anti-infective, antioxidant and tissue repairing) properties of various chitosans were assessed, and compared to those of hyaluronic acid to fnd the best candidate for the treatment of oral mucositis, high molecular weight chitosan ascorbate was found to be the most effective among the other chitosans studied [46]. 2. Applications in dental medicine In dentistry, various applications of chitosan have been proposed in which the physical, mechanical, chemical properties of this material have been used. These studies were reviewed in a previous paper published by the author [47]. Recently, there has been increased interest in application of chitosan for its bioactive properties such as wound healing, antimicrobial and tissue regeneration, in addition to the drug delivery system in gel, flm, sponge, fber and particulate forms. In this section, after reporting on recent studies on chitosan-based systems for delivery in dental medicine, which are generally for local delivery, application of chitosan in tissue regeneration will be reviewed. 2.1. Local drug delivery Chitosan blends with hydrophilic polymers including polyviny- lalcohol (PVA), polyethyleneoxide (PEO) and polyvinylpyrrolidone (PVP), were investigated as candidates for oral gingival delivery systems [48]. The bioavailabilty conferred by the chitosan blend delivery systems, as concluded from dog studies, was shown to be comparable to that based on chitosan alone, especially for those blends involving high-molecular-weight hydrophilic polymers. The study also indicated that chitosan blends were superior in other properties compared to chitosan alone. These included improved comfort and reduced irritation, ease of processing, improved flm quality, improved fexibility, and enhanced dissolution. Bupivacaine-loaded chitosan beads were prepared, and the in vitro drug release was studied in different media [49]. Maximum release of bupivacaine was obtained between 100 and 120 h, depending on the presence of lysozyme in the release medium, the enzyme facilitating the release process. A constant release rate of the drug, between 11 and 15 mg/h, was observed for 30 h. In order to prolong bupivacaine release, the drug-loaded chitosan beads were coated with a poly (DL- lactide-co-glycolide) flm. The resulting device allowed the drug to be released in a sustained form; a constant release rate between 28.5 and 29.5 mg/h was obtained for 3 days, and the maximum release of bupivacaine took place at day 9. The developed system was suggested for use in the treatment of dental pain in the buccal cavity. Film dosage form was developed for sustained delivery of a synthetic favonoid derivative, iprifavone into the periodontal pocket [50]. For this purpose, monolayer composite systems made of iprifavone- loaded poly(d,l-lactide-co-glycolide) (PLGA) micromatrices in a chitosan flm form were obtained by emulsifcation/casting/evaporation technique. Multilayer flms, made of three layers of polymers (chitosan/ PLGA/chitosan), were also prepared and compared to monolayer flms for their “in vitro” characteristics. Signifcant differences in swelling, degradation and drug release were observed depending on flm structure and composition. The composite micromatricial flms were shown to be a suitable dosage form to prolong iprifavone release for 20 days. Topical delivery is the most widely accepted approach to prolong drug concentrations of an antimicrobial agent in the oral cavity. As most antifungals do not possess the inherent ability to bind to the oral mucosa, this is best achieved through improved formulations. Besides bioadhesive properties, chitosan has been shown to inhibit the adhesion of Candida albicans to human buccal cells and exert antifungal activity [51]. The antifungal agent, chlorhexidine gluconate (Chx), which has also been shown to reduce C. albicans adhesion to oral mucosal cells, was incorporated into chitosan gels (at 0.1 or 0.2% concentration) or flm, and in vitro drug release and the antifungal activity of the gels and flms in the presence and absence of Chx was studied [52]. Prolonged release was observed with flm formulations. The highest antifungal activity was obtained with 2% chitosan gel containing 0.1% Chx. The antimicrobial activity of similar chitosan formulations in gel and flm forms was investigated against a periodontal pathogen, Porphyromonas gingivalis [53]. The viscosity, bioadhesive properties and antimicrobial activity of chitosans at different molecular weights and deacetylation degrees were assessed in the absence or presence of Chx which was incorporated into the formulations at 0.1 and 0.2% concentrations. The fow property of the gels was found to be suitable for topical application on the oral mucosa and to syringe into the periodontal pocket. The bioadhesive properties of the formulations were shown ex vivo using freshly excised porcine buccal mucosa. Bioadhesion was found to be not affected by the incorporation of Chx. Chitosan is shown to have an antimicrobial activity against P. gingivalis, and this was found to be higher with high-molecular-weight chitosan. The combination of chitosan with Chx showed a higher activity when compared to that of Chx alone, which would provide Chx application at lower concentrations thus avoiding its undesired side effects. Chitosan-based flms and gels are suggested as promising delivery systems for local therapy of periodontal diseases with their bioadhesive property and antimicrobial activity. Buccal tablets based on Chx-containing chitosan microspheres were developed against buccal infections [54]. The antimicrobial Potential applications of chitosan in oral mucosal delivery S. Senel J. DRUG DEL. SCI. TECH., 20 (1) 23-32 2010 28 activity of the microparticles was investigated, and the incorporation of Chx into chitosan microparticles was shown to improve the antimicrobial activity, particularly the highest against C. albicans. Drug-free chitosan microparticles were shown to exert antimicrobial activity as well due to the polymer itself. Buccal tablets were prepared by direct compression of the microparticles with mannitol alone or with sodium alginate. Following in vivo administration, the tablets were shown to maintain prolonged release of the drug in the oral cavity. In the control group, the mouth was rinsed with a commercial mouthwash (Dentosan) containing 0.2% (w/v) Chx. For the chitosan treated group, the drug was detected even after 3 h, whereas in the control group, the salivary drug concentrations were very low, and no drug was detected after 2 h. Mucoadhesive microspheres for the controlled release of triclosan in oral care formulations, specifcally dental pastes were prepared using chitosan, Gantrez MS-955, Carbopol 974P, polycarbophil [55]. Triclosan was rapidly released into a sodium lauryl sulfate-containing buffer from all except for the chitosan microspheres. The loading effciency was found to be highest with the chitosan microspheres. For microspheres that were manufactured from Gantrez, Carbopol or polycarbophil, the release was found to be zero-order kinetics whereas with chitosan, the release profle was shown to ft the Baker and Lonsdale model. Chitosan microspheres were suggested as promising candidates for the sustained release of triclosan in the oral cavity. The antibacterial and plaque-reducing action of water-soluble chitosan has been shown when used as a mouth rinse reagent [56]. A chitosan-containing chewing gum formulation was developed, which could effectively suppress the growth of oral bacteria in saliva [57, 58]. Fifty healthy subjects, ranging in age from 19 to 32 years, were recruited and allowed chew the gum for 5 min and then rested for 5 min. Each subject chewed a total of eight pieces of gum, which was either supplemented with or without chitosan, for a total of 80 min. The amount of oral bacteria was found to signifcantly decrease in the chitosan group. In particular, the number of mutans streptococci was maintained at about a 20% level in comparison to that before gum chewing, even at 1 h after gum chewing. It was suggested that a supplementation of chitosan to gum would be an effective method for controlling the number of cariogenic bacteria in situations where it is diffcult to brush one’s teeth, such as when an individual is away from home all day or participating in outdoor training. Chitosan has been used for delivery into oral cavity of the essential oils, which exert antibacterial/antifungal activity. Encapsulation with chitosan was suggested to provide protection of the essential oils, avoiding oxidation reactions, and also a sustained delivery [59]. The flms composed of poly(vinyl alcohol) (PVA) and carboxymethyl-chitosan (CMCS) were prepared as local drug delivery system by blending/casting method, and loaded with ornidazole (OD), which is an effective compound in the treatment of susceptible protozoal infections and prophylaxis of anaerobic bacterial infections [60]. The blend flms were shown to exert pH-responsive swelling behavior, and moderate drug release action, and also exhibit a little antimicrobial activity against Escherichia coli and Streptococcus aureus strains. Those characteristics of CMCS/PVA blend flms were reported to be governed by the weight ratio of CMCS and PVA. Increasing the content of PVA in blend flm was shown to decrease swelling and decelerate the drug release whereas increasing the content of CMCS was shown to enhance the antimicrobial activity. The biocompatibility and bioactivity of the blend flm was also assessed using rabbit blood and Wistar rats. No hemolysis, no toxicity to rat periodontia and no cytotoxicity to the rat muscle were observed. After subcutaneously implanting the blend drug flms in Wistar rats, the systems maintained good retention at the application site and a high drug concentration over a long period of time (5 days) which was longer than the period of in vitro drug released (160 min). Results of the in vitro and in vivo studies showed that CMCS/PVA blend drug flm was an excellent candidate for local drug delivery system. It was suggested that the PVA/CMCS drug flm would be absorbed over a long period of time and that the wound would eventually be cicatrized by new forming tissues. 2.2. Periodontal regeneration Periodontal disease, which is characterized by infammation of periodontal tissues, eventually leads to degeneration of the periodon- tium. If periodontal disease is left untreated, tooth loss can occur. The main aim of periodontal therapy is to repair the damaged periodontal supporting tissues as a result of the infammatory disease process [61]. Recent studies have suggested that chitosan and its derivatives are promising candidates as a supporting material for tissue engineering applications owing to their porous structure, gel-forming properties, ease of chemical modifcation, high affnity to in vivo macromolecules [62]. The effect of chitosan on osteoblast and fbroblast cell attachment was studied in vitro [63]. Mouse MC3T3-E1 osteoblasts and 3T3 fbroblasts were grown in the presence of serum on acid soluble and water soluble chitosans. Cell attachment and immunofuorescent analyses were performed at various time points to analyze initial phenotypic profles. Our results suggested that chitosan supports the initial attachment and spreading of osteoblasts preferentially over fbroblasts, and that manipulation of the biopolymer can alter the level of attachment and spreading. An amino acid, taurine, which is considered to be benefcial for regulating the infammation process, was incorporated into the chitosan flm and the synergistic effects of taurine and chitosan in the experimental defects at the vestibular bone of maxillary canine teeth in dogs were investigated [64]. Cellular activity was observed both in the mitochondria of fbroblasts and macrophages. These ultrastructural alterations were thought to be the sign of the disturbed balance between the generated oxidants and antioxidant defense mechanisms. Taurine appeared to enhance the acceleration effect of chitosan on wound healing at early periods. This effect may be considered benefcial in tissue repair in destructive diseases like periodontitis. The effect of chitosan on periodontal regeneration was investigated in twenty chronic periodontitis patients [65]. Following initial therapy, the patients were treated either with chitosan gel (1% w/v); chitosan gel + demineralized bone matrix or chitosan gel+collagenous membrane. A fap was applied for control purposes. Clinical and radiographic measurements were recorded at baseline, day 90 and day 180 after surgery. Signifcant bone healing was observed when compared with baseline indicating that chitosan gel alone or its combination with demineralized bone matrix/collagenous membrane is promising for periodontal regeneration. The effects of many growth factors on periodontal tissue cells have been assessed for their involvement in periodontal tissue engineering using chitosan-based delivery systems [61]. Porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding human transforming growth factor-b1 (TGF-b1) [66], and were assessed in vitro by analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament cells (HPLCs) were seeded in this scaffold, and gene transfection was traced by green fuorescent protein (GFP). The expression of type I and type III collagen was detected with RTPCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that the pore diameter of the gene-combined scaffolds was smaller than that of pure chitosan/collagen scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to grow in the scaffold, demonstrating the potential of chitosan/collagen scaffold combined Ad-TGF-b1 as a good substrate candidate in periodontal tissue engineering. Strong and macroporous scaffolds were developed via absorbable fbers, biopolymer chitosan, and mannitol porogen [67]. MC3T3-E1 Potential applications of chitosan in oral mucosal delivery S. Senel J. DRUG DEL. SCI. TECH., 20 (1) 23-32 2010 29 osteoblast-like cells were cultured on the specimens and inside the calcium phosphate cement (CPC) composite paste carrier. The scaffold strength was found to be more than doubled via reinforcement. The cement injectability was increased from about 60% to nearly 100%. Cell attachment and proliferation on the new composite matched those of biocompatible controls. Cells were able to infltrate into the macro- pores and anchor to the bone mineral-like nano-apatite crystals. For growth factor delivery, CPC powder:liquid ratio and chitosan content was reported to provide the means to tailor the rate of protein release from CPC carrier. N-[1-hydroxy-3-(trimethylammonium)propyl]chitosan chloride (HTCC) was prepared, and the antibacterial activity of chitosan and HTCC against oral pathogens, their proliferation activity and effects on the ultrastructure of human periodontal ligament cells (HPDLCs) were investigated [68]. Their results indicated that four oral strains were susceptible to chitosan and HTCC with minimum inhibitory concentrations (MICs) ranging from 0.25 to 2.5 mg/mL. Chitosan at 2000, 1000, 100, and 50 μg/mL concentrations was shown to stimulate the proliferation of human periodontal ligament cells (HPDLCs). On the contrary, HTCC inhibited the proliferation at the same concentrations but accelerated the proliferation of HPDLCs at relatively low concentrations (10, 3, 1.5, 1, and 0.3 l g/mL). 3. Applications for systemic delivery There are numerous applications of chitosan in oral mucosal delivery for systemic delivery as well [69]. Various dosage forms have been developed for the delivery of drugs across oral mucosa to treat various diseases. A non-covalently cross-linked palmitoyl glycol chitosan (GCP) hydrogel was assessed as an erodible controlled-release system for the buccal delivery of hydrophilic macromolecules [70]. Samples of GCP with different degrees of hydrophobicity were synthesized and porous hydrogels were prepared by freeze-drying an aqueous dispersion of the polymer in the presence or absence of either a model macromolecule fuorescein isothiocyanatedextran (FITC-dextran, MW 4400) and/or amphiphilic derivatives Gelucire 50/13 or vitamin E d-a-tocopherol polyethylene glycol succinate. The hydration and erosion of the gels were governed by the hydrophobicity of the gel and the presence of the amphiphilic additives. GCP gels could be loaded with up to 27.5% (w/w) of FITC-dextran by freeze-drying a dispersion of GCP in a solution of FITC-dextran. The gels were bioadhesive but less than that of hydroxypropylmethylcellulose, Carbopol 974NF. Furthermore, these hydrogels were incorporated with a model hydrophobic drug, denbufylline and sodium glycodeoxycholate (GDC) was also added as a penetration enhancer [71]. The buccal absorption of denbufylline was investigated in the rabbit model. Carbopol 974NF (CP), denbufylline and GDC tablets were used as control. Denbufylline was found to reduce the porosity, erosion and hydration of the gels while GDC increased the hydration and erosion. All gels were mucoadhesive but less so than the control CP tablets. Denbufylline was detected in 0.5 h after dosing with the GCP formulation, and delivery was sustained for at least 5 h after dosing whereas with the CP tablets, drug delivery was not sustained, and drug was detected in 1 h after dosing. 5-methylpyrrolidinone chitosan (MPC) flms were prepared as carriers for the buccal delivery of protein drugs [72]. Myoglobin (MHb) was chosen as the model protein. The results obtained show that the modulation of MHb release was achieved only through chitosan cross-linking; the best results in release rate control were obtained by cross-linking performed at pH 6.5. Good bioadhesion properties were maintained even with high cross-linking degrees; the swelling index of MHb-loaded flms at different cross-linking degrees assessed at pH 7.4 and pH 6.4 were comparable to those of placebo flms. It was reported that protein release could be controlled without affecting bioadhesion by setting suitable tripolyphosphate cross-linking conditions for MPC flms. Films based on chitosan hydrochloride (HCS) and polyacrylic acid sodium salt (PAA) were prepared for the delivery of acyclovir [73]. A commercial cream containing acyclovir and an aqueous acyclovir suspension were used as references. The addition of PAA to HCS produced a decrease in flm hydration. The addition of PAA to HCS was found to decrease the drug release. All chitosan-based flms were shown to enhance the permeation of acyclovir across porcine cheek epithelium when compared to the suspension and the commercial cream. The penetration enhancement properties were affected by the mixing ratio of the two polymers. The clinical effectiveness of a chitosan-based bioadhesive unidirectional buccal bromocriptine mysylate/pluronic F127discs was investigated in hyperprolactinemic patients [74]. A total of 42 patients were randomly divided into two groups. Group A comprised 21 patients who used unidirectional buccoadhesive bromocriptine methylate discs once daily for 1 month. Group B included 21 patients who used vaginoadhesive bromocriptine methylate discs once daily for 1 month. Serum prolactin (PRL) levels were measured before and after therapy in all cases. A signifcant reduction in serum PRL levels was observed after 1 month of therapy in both groups showing no signifcant difference between the groups. Buccal adhesive discs were reported to have advantages over the vaginal disc such as being gender nonspecifc, avoiding manipulating the vagina, which could be inconvenient to some patients, being not dependent on cyclic estrogen levels, and may easily be used during menstruation. A new highly porous, fexible device was developed for buccal peptide administration by a very simple and mild casting/freeze-drying procedure, which consisted of a mucoadhesive chitosan layer containing insulin and an impermeable protective layer made of ethylcellulose [75]. This structure was expected to provide unidirectional drug release to the mucosa and avoid loss of drug due to washout with saliva. Insulin release was reported to be modulated by varying certain formulation variables (chitosan salt type and molecular weight, chitosan, solution pH, insulin dose). It was suggested that electrostatic interaction could occur between chitosan amino groups and the insulin carboxylic groups. The affnity of chitosan sponges to mucin surfaces was related to the swelling and solubility properties of the different salts of chitosan. Antisense oligonucleotide-loaded chitosan nanoparticles were prepared and the release of oligonucleotide from chitosan-tripolyphosphate (TPP)/oligonucleotide nanoparticles investigated [76]. The interaction between chitosan and oligonucleotide was confrmed by using capil- lary zone electrophoresis (CZE). The release of oligonucleotides from nanoparticles was found to be dependent on loading methods and pH conditions. Chitosan/oligomer-TPP nanoparticles were reported to show the lowest release percent of oligonucleotides with 41.3% at pH 7.0 among the loading methods. The released oligonucleotides from chitosan/ oligonucleotide nanoparticles were suffciently stable for 12 h in the 20% saliva solution. The sustained release of oligonucleotide from chitosan nanoparticles was suggested to be suitable for the local therapeutic application in periodontal diseases. Buccal bioadhesive tablet formulations of nicotine hydrogen tar- tarate (NHT) for nicotine replacement therapy (NRT) were developed using chitosan and carbomer at different ratios [31]. The release of NHT from the tablets increased with the increasing amount of chitosan while the bioadhesion decreased. In vivo studies were performed on healthy non-smoker volunteers in comparison to a commercially available transdermal patch. When compared to the transdermal patch, similar plasma nicotine levels were obtained with the developed buccal tablet but in a signifcantly shorter time (Cmax for buccal tablet: 3 h, and for Cmax for transdermal patch: 11.5 h). The developed buccal formulation was very promising for relieving acute craving and is suggested for use in combination with the transdermal patch for NRT. A matrix for buccal drug delivery composed of a different chitosan salts and poloxamer 407 was prepared by lyophilization of the blend and then compression into discs [77]. An experimental design (32) Potential applications of chitosan in oral mucosal delivery S. Senel J. DRUG DEL. SCI. TECH., 20 (1) 23-32 2010 30 was used to study the infuence of the type of chitosan salt and of the relative amount of poloxamer on drug release capacity, swelling, erosion, and mucoadhesiveness of matrices. It was shown that the matrix properties depended signifcantly on both the relative amount of poloxamer and chitosan salt type. The rank orders of chitosan salts for the four processes assessed were reported as follows: drug release: chitosan acetate > chitosan citrate > chitosan lactate; swelling: chitosan lactate > chitosan acetate = chitosan citrate; erosion: chitosan citrate > chitosan lactate > chitosan acetate; mucoadhesion: chitosan lactate > chitosan acetate = chitosan citrate. Mucoadhesion was particularly promoted when poloxamer 407 was present at about 30% (w/w). The matrix composed of chitosan lactate and poloxamer 407 was reported to show the best characteristics for buccal administration. * Chitosan is clearly an attractive bioadhesive polymer which has great potential for improving the delivery of drugs into the oral cavity as well as across the oral mucosa especially due to its biodegradability, biocompatibility and low toxicity. With its chemical versatility, it provides an excellent opportunity to tailor delivery systems with the desired properties. Furthermore, chitosan itself has bioactive properties, for this reason it offers benefts in treatment as well. On the other hand, standardization of chitosan and its derivatives remains the major issue to be solved to optimize the possibility of its commercialization. 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