Journal of Controlled Release 100 (2004) 5 28 www.elsevier.

com/locate/jconrel

Review

Recent advances on chitosan-based micro- and nanoparticles in drug deliveryB

Sunil A. Agnihotri, Nadagouda N. Mallikarjuna, Tejraj M. Aminabhavi*
Drug Delivery Division, Center of Excellence in Polymer Science, Karnatak University, Dharwad 580 003, India Received 15 July 2004; accepted 12 August 2004

Abstract Considerable research efforts have been directed towards developing safe and efficient chitosan-based particulate drug delivery systems. The present review outlines the major new findings on the pharmaceutical applications of chitosan-based micro/nanoparticulate drug delivery systems published over the past decade. Methods of their preparation, drug loading, release characteristics, and applications are covered. Chemically modified chitosan or its derivatives used in drug delivery research are discussed critically to evaluate the usefulness of these systems in delivering the bioactive molecules. From a literature survey, it is realized that research activities on chitosan micro/nanoparticulate systems containing various drugs for different therapeutic applications have increased at the rapid rate. Hence, the present review is timely. D 2004 Elsevier B.V. All rights reserved.
Keywords: Microparticles; Nanoparticles; Chitosan; Chemically modified chitosan; Drug delivery

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . Methods of preparation of micro/nanoparticles 2.1. Emulsion cross-linking . . . . . . . . 2.2. Coacervation/precipitation . . . . . . . 2.3. Spray-drying . . . . . . . . . . . . . . 2.4. Emulsion-droplet coalescence method . 2.5. Ionic gelation . . . . . . . . . . . . . 2.6. Reverse micellar method . . . . . . . 2.7. Sieving method . . . . . . . . . . . . . . . . . . . of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 8 8 9 10 12 12 14 15

B This paper is CEPS Communication # 23. * Corresponding author. Tel.: +91 836 2779983; fax: +91 836 2771275. E-mail address: aminabhavi@yahoo.com (T.M. Aminabhavi).

0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.08.010

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

3. 4. 5.

Drug loading into micro/nanoparticles of chitosan Drug release and release kinetics . . . . . . . . . Pharmaceutical applications of chitosan particulate 5.1. Colon targeted drug delivery . . . . . . . . 5.2. Mucosal delivery . . . . . . . . . . . . . . 5.3. Cancer therapy . . . . . . . . . . . . . . . 5.4. Gene delivery . . . . . . . . . . . . . . . 5.5. Topical delivery . . . . . . . . . . . . . . 5.6. Ocular delivery . . . . . . . . . . . . . . . 5.7. Chitosan as a coating material . . . . . . . 6. Chemically modified chitosans . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

15 16 18 18 18 19 20 22 22 22 22 24 24 24

1. Introduction Chitosan (CS) is a polysaccharide, similar in structure to cellulose. Both are made by linear h(1Y4)-linked monosaccharides [see Fig. 1 (a)]. However, an important difference to cellulose is that CS is composed of 2-amino-2-deoxy-h-d-glucan combined with glycosidic linkages. The primary amine groups render special properties that make CS very useful in pharmaceutical applications. Compared to many other natural polymers, chitosan has a positive charge and is mucoadhesive [1]. Therefore, it is used extensively in drug delivery applications [2 6]. Chitosan is obtained from the deacetylation of chitin, a naturally occurring and abundantly available (in marine crustaceans) biocompatible polysaccharide. However, applications of chitin are limited compared to CS because chitin is structurally similar to cellulose, but chemically inert. Acetamide group of chitin can be converted into amino group to give CS, which is carried out by treating chitin with concentrated alkali solution. Chitin and CS represent long-chain polymers having molecular mass up to several million Daltons. Chitosan is relatively reactive and can be produced in various forms such as powder, paste, film, fiber, etc. [7,8]. Commercially available CS has an average molecular weight ranging between 3800 and 20,000 Daltons and is 66% to 95% deacetylated. Chitosan, being a cationic polysaccharide in neutral or basic pH conditions, contains free amino groups and hence, is insoluble in water. In acidic pH, amino groups can undergo protonation thus, making it soluble in water. Solubility of CS depends upon the distribution of

free amino and N-acetyl groups [9]. Usually 13% aqueous acetic acid solutions are used to solubilize CS. Chitosan is biocompatible with living tissues since it does not cause allergic reactions and rejection. It breaks down slowly to harmless products (amino sugars), which are completely absorbed by the human body [10]. Chitosan degrades under the action of ferments, it is nontoxic and easily removable from the organism without causing concurrent side reactions. It possesses antimicrobial property and absorbs toxic metals like mercury, cadmium, lead, etc. In addition, it has good adhesion, coagulation ability, and immunostimulating activity. If degree of deacetylation and molecular weight of CS can be controlled, then it would be a material of choice for developing micro/nanoparticles. Chitosan has many advantages, particularly for developing micro/nanoparticles. These include: its ability to control the release of active agents, it avoids the use of hazardous organic solvents while fabricating particles since it is soluble in aqueous acidic solution, it is a linear polyamine containing a number of free amine groups that are readily available for crosslinking, its cationic nature allows for ionic crosslinking with multivalent anions, it has mucoadhesive character, which increases residual time at the site of absorption, and so on. Chitin and CS have very low toxicity; LD50 of CS in laboratory mice is 16 g/kg body weight, which is close to sugar or salt. Chitosan is proven to be safe in rats up 10% in the diet [11]. Various sterilization methods such as ionizing radiation, heat, steam and chemical methods can be suitably adopted for sterilization of CS in clinical

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

Fig. 1. (a) Structure of chitosan [poly (h1 4-d-glucosamine)]. (b) Structure of cross-linked chitosan.

applications [12]. In view of the above-mentioned properties, CS is extensively used in developing drug delivery systems [7,8,1318]. Particularly, CS has

been used in the preparation of mucoadhesive formulations [1922], improving the dissolution rate of the poorly soluble drugs [14,23,24], drug targeting [25,26] and enhancement of peptide absorption [20,21,27]. Many reports are available on the preparation of CS microspheres [23,25,26,28,29]. Many methods used in the development of microparticulate polymeric drug delivery devices can also be used to prepare CS microspheres [3035]. Dodane and Vilivalam [3] reviewed new approaches on pharmaceutical applications of CS and discussed its mechanisms of action in various in vitro and in vivo models. Recent reviews [36,37] addressed the issues on biomedical, pharmaceutical and biological aspects of chitin, CS and their derivatives. Chitosan and its derivatives as a non-viral vector for gene delivery [38] and CS-based gastrointestinal delivery systems [39] have been discussed. The recent review by Sinha et al. [40] covers various methods of preparation and evaluation of CS microspheres, but no attempt has been made to discuss nanoparticulate CS systems. Different types of CSbased drug delivery systems are summarized in Table 1.

Table 1 Chitosan-based drug delivery systems prepared by different methods for various kinds of drugs Type of system Tablets Capsules Microspheres/Microparticles Method of preparation matrix coating capsule shell emulsion cross-linking Drug diclofenac sodium, pentoxyphylline, salicylic acid, theophylline propranolol HCl insulin, 5-amino salicylic acid theophylline, cisplatin, pentazocine, phenobarbitone, theophylline, insulin, 5-fluorouracil, diclofenac sodium, griseofulvin, aspirin, diphtheria toxoid, pamidronate, suberoylbisphosphonate, mitoxantrone, progesterone prednisolone, interleukin-2, propranolol-HCl cimetidine, famotidine, nizatidine, vitamin D-2, diclofenac sodium, ketoprofen, metoclopramide-HCl, bovine serum albumin, ampicillin, cetylpyridinium chloride, oxytetracycline, betamethasone felodipine clozapine gadopentetic acid DNA, doxorubicin insulin, ricin, bovine serum albumin, cyclosporin A doxorubicin adriamycin, nifedipine, bovine serum albumin, salbutamol sulfate, lidocaine HCl, riboflavin isosorbide dinitrate, chlorhexidine gluconate, trypsin, granulocyte-macrophage colony-stimulating factor, acyclovir, riboflavine, testosterone, progesterone, beta-oestradiol chlorpheniramine maleate, aspirin, theophylline, caffeine, lidocaine HCl, hydrocortisone acetate, 5-fluorouracil

coacervation/precipitation spray-drying

Nanoparticles

Beads Films

ionic gelation sieving method emulsion-droplet coalescence coacervation/precipitation ionic gelation reverse micellar method coacervation/precipitation solution casting

Gel

cross-linking

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

However, the micro/nanoparticulate drug delivery systems offer numerous advantages over the conventional dosage forms. These include improved efficacy, reduced toxicity and improved patient compliance [35,4143]. The present review addresses the recent trends in the area of micro/nanoparticulate CS-based drug delivery systems. Literature of the past decade has been covered and results are critically evaluated.

2. Methods of preparation of micro/nanoparticles of chitosan Different methods have been used to prepare CS particulate systems. Selection of any of the methods depends upon factors such as particle size requirement, thermal and chemical stability of the active agent, reproducibility of the release kinetic profiles, stability of the final product and residual toxicity associated with the final product. Different methods used in the preparation of CS micro/nanoparticles are discussed in this review. However, selection of any of these methods depends upon the nature of the active molecule as well as the type of the delivery device. Since we are concerned only with the micro/nanoparticulate systems of CS and its derivatives, we will restrict our discussions only on these aspects. 2.1. Emulsion cross-linking This method utilizes the reactive functional amine group of CS to cross-link with aldehyde groups of the cross-linking agent (see Fig. 1b). In this method, a water-in-oil (w/o) emulsion is prepared by emulsifying the CS aqueous solution in the oil phase. Aqueous droplets are stabilized using a suitable surfactant. The stable emulsion is cross-linked by using an appropriate cross-linking agent such as glutaraldehyde to harden the droplets. Microspheres are filtered and washed repeatedly with n-hexane followed by alcohol and then dried [44]. By this method, size of the particles can be controlled by controlling the size of aqueous droplets. However, the particle size of final product depends upon the extent of cross-linking agent used while hardening in addition to speed of stirring during the formation of emulsion. This method is schematically represented in Fig. 2. The emulsion cross-linking method has few drawbacks

Fig. 2. Schematic representation of preparation of chitosan particulate systems by emulsion cross-linking method.

since it involves tedious procedures as well as use of harsh cross-linking agents, which might possibly induce chemical reactions with the active agent. However, complete removal of the un-reacted crosslinking agent may be difficult in this process. Recently, [33] we have used the emulsion crosslinking method to prepare chitosan microspheres to encapsulate diclofenac sodium using three crosslinking agents viz, glutaraldehyde, sulfuric acid and heat treatment. Microspheres were spherical with smooth surfaces as shown in Fig. 3. The size of the microparticles ranged between 40 and 230 Am. Among the three cross-linking agents used, glutaraldehyde cross-linked microspheres showed the slowest release rates while a quick release of diclofenac sodium was observed by the heat cross-linked microspheres. In our continuing study on CS-based derivatives [34], we have also prepared the nifedipine-loaded microspheres of polyacrylamide-g-chitosan using three concentrations of glutaraldehyde as the cross-linking agent. Microspheres were spherical with the mean particle size of 450 Am. Glutaraldehyde extracted in toluene was used as a cross-linking agent by Al-Helw et al. [45] to prepare CS microspheres encapsulated with phenobarbitone. Uniform and spherical microspheres with loading efficiency up to 57.2% were produced. Loading efficiency was dependent upon the preparation conditions. Parameters affecting the preparation and performance of microspheres are molecular weight

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

Fig. 3. Scanning electron micrograph of chitosan microspheres produced by emulsion cross-linking method.

and concentration of CS as well as concentration of the stabilizing agent. Particle size of the microspheres varied in the range 274450 Am. Release rates of phenobarbitone from different formulations of microspheres showed high initial release (burst effect) of the drug and about 2030% of the drug was released in the first hour. Release was faster from the small size microspheres, i.e., almost 7595% of the drug was released within 3 h depending upon the molecular weight of CS. Denkbas et al. [46] used the mixture of mineral oil/petroleum ether in the ratio of 60/40 (v/v) as the external medium to prepare CS microspheres using glutaraldehyde as a cross-linking agent and Tween-80 as an emulsifier. Smaller microspheres with narrow distributions were produced when CS/solvent ratio and drug/CS ratio were lower. The 5-fluorouracil was loaded up to a concentration of 10.4 mg/g of CS. Thanoo et al. [29] prepared the CS microspheres by emulsion cross-linking of CS solution in paraffin oil as an external medium with glutaraldehyde using dioctyl sulfosuccinate as the stabilizing agent. Addition of stabilizing agent during particle formation produced microspheres with spherical geometry and smooth surfaces. Encapsulation efficiencies up to 80% were achieved for theophylline, aspirin or griseofulvin. These microspheres were used to study the drug release rates, which were influenced by cross-link density, particle size and initial drug loading. Sankar et al. [47] prepared the CS-based pentazocine micro-

spheres for intranasal delivery. Formulation parameters such as drug loading, polymer concentration, stirring speed during cross-linking and oil phase were altered to develop microspheres having good in vivo performance. In vivo studies indicated a significantly improved bioavailability of pentazocine. Application of in vitro data to various kinetic models indicated that these systems followed the diffusion controlled release kinetics. Jameela et al. [48] prepared smooth, highly spherical, cross-linked CS microspheres in the size range of 45300 Am for the controlled release (CR) of progesterone. An aqueous acetic acid dispersion of CS containing progesterone was emulsified in the dispersion medium consisting of liquid paraffin and petroleum ether stabilized by using sorbitan sesquioleate; droplets were hardened by glutaraldehyde crosslinking. Extent of cross-linking showed a significant influence on drug release characteristics. Highly cross-linked microspheres released only about 35% of steroid in 40 days compared to 70% release from the lightly cross-linked microspheres. Evaluation of in vivo bioavailability by intramuscular injection in rabbits showed that a plasma concentration of 1 to 2 ng/mL was maintained up to 5 months without showing any high burst release effect. These data suggest the usefulness of cross-linked CS microspheres as potential carriers for long-term delivery of steroids. Bugamelli et al. [49] developed insulin-loaded microparticles of CS by the interfacial cross-linking in the presence of ascorbyl palmitate. Disposition of ascorbyl palmitate at the water oil interface allowed the formation of covalent bond with the amino groups of CS when its oxidation to dehydroascorbyl palmitate took place during the formation of microparticles. This method produced microparticles with high loading efficiency and released the drug at a constant rate up to 80 h. 2.2. Coacervation/precipitation This method utilizes the physicochemical property of CS since it is insoluble in alkaline pH medium, but precipitates/coacervates when it comes in contact with alkaline solution. Particles are produced by blowing CS solution into an alkali solution like sodium hydroxide, NaOH-methanol or ethanediamine using a compressed air nozzle to form coacervate droplets

10

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

[50]. Separation and purification of particles was done by filtration/centrifugation followed by successive washing with hot and cold water. The method is schematically represented in Fig. 4. Varying compressed air pressure or spray-nozzle diameter controlled the size of the particles and then using a crosslinking agent to harden particles can control the drug release. In another technique [51], sodium sulfate solution was added dropwise to an aqueous acidic solution of CS containing a surfactant under stirring and ultrasonication for 30 min. Microspheres were purified by centrifugation and re-suspended in demineralized water. Particles were cross-linked with glutaraldehyde. Particles produced by this method have better acid stability than observed by other methods. Chitosan microspheres loaded with recombinant human interleukin-2 (rIL-2) have been prepared by dropping of rIL-2 with sodium sulfate solution in acidic CS solution [52]. When protein and sodium sulfate solutions were added to CS solution and during the precipitation of CS, the protein was incorporated into microspheres. This method is devoid of cross-linking agent. The rIL-2 was released from microspheres in a sustained manner for up to 3 months. Efficacy of the systems developed was studied by using two model cells viz., HeLa and Lstrain cell lines. Microspheres were taken up by the cells and rIL-2 was released from the microspheres.

ChitosanDNA nanoparticles have been prepared using the complex coacervation technique [53]. Important parameters such as concentrations of DNA, CS, sodium sulfate, temperature, pH of the buffer and molecular weights of CS and DNA have been investigated. At the amino to phosphate group ratio between 3 and 8 and CS concentration of 100 Ag/ mL, the particle size was optimized to 100250 nm with a narrow distribution. Surface charge of these particles was slightly positive with a zeta potential of 112 to 118 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosanDNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation. 2.3. Spray-drying Spray-drying is a well-known technique to produce powders, granules or agglomerates from the mixture of drug and excipient solutions as well as suspensions. The method is based on drying of atomized droplets in a stream of hot air. In this method, CS is first dissolved in aqueous acetic acid solution, drug is then dissolved or dispersed in the solution and then, a suitable cross-linking agent is added. This solution or dispersion is then atomized in a stream of hot air. Atomization leads to the formation of small droplets, from which solvent evaporates instantaneously leading to the formation of free flowing particles [54] as depicted in Fig. 5. Various process parameters are to be controlled to get the desired size of particles. Particle size depends upon the size of nozzle, spray flow rate, atomization pressure, inlet air temperature and extent of crosslinking. He et al. [54] prepared both un-cross-linked and cross-linked CS microparticles by spray-drying method for the delivery of cimetidine, famotidine and nizatidine. Microspheres were spherical with a smooth and distorted morphology. Particle size of the un-cross-linked microspheres varied between 4 and 5 Am, while cross-linked microspheres ranged from 2 to 10 Am; they were all positively charged. Particle size and zeta potential were influenced by the extent of cross-linking. A decrease in extent of cross-linking increased both the particle size and the zeta potential. Particle size was increased when the spray flow rate was increased using the large size nozzle. Micro-

Fig. 4. Schematic representation of preparation of chitosan particulate systems by coacervation/precipitation method.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

11

Fig. 5. Schematic representation of preparation of chitosan particulate systems by spray drying method.

spheres with smaller particle size were produced at greater airflow rates. However, particle size was less affected by the inlet air temperature between 140 and 180 8C. Conti et al. [55] produced microparticles by exposing the spray-dried particles to vapors containing cross-linking agents. Cetylpyridinium chloride, an anti-infective agent, was incorporated into CS microspheres produced by spray-drying technique. Extent of cross-linking was controlled by the time of exposure to cross-linking agent. Ganza-Gonzalez et al. [56] have demonstrated that spray-drying technique is fast, simple and reliable to obtain microspheres. Microspheres were prepared by spray drying of aqueous CS dispersions containing metoclopramide hydrochloride using different amounts of formaldehyde as a cross-linker. Microspheres released the drug for more than 8 h, independent of the pH of the medium. In another study [57], vitamin D2 (VD2), also called as ergocalciferol, was efficiently encapsulated into CS microspheres prepared by spray-drying method. The microencapsulated product was coated with ethyl cellulose. The sustained release property of VD2 microspheres was used for the treatment of prostatic disease [58]. Spray-drying method was also used to prepare ampicillin-loaded methylpyrrolidone CS microspheres [59] by taking different drug-to-polymer weight ratios. Spray-dried microparticles were almost

spherical in shape with smooth surfaces and narrowsize distributions. Lorenzo-Lamosa et al. [60] prepared the microencapsulated CS microspheres for colonic delivery of sodium diclofenac. Sodium diclofenac was entrapped into CS microcores by spray-drying and then, microencapsulated into EudragitR L-100 and EudragitR S100 using an oil-in-oil solvent evaporation method. By spray-drying, CS microspheres of 1.82.9 Am sizes were prepared and efficiently microencapsulated into EudragitR microspheres ranging in size between 152 and 223 Am to form the multireservoir system. Number of variables such as type and concentration of chitosan, the core/coat ratio and the type of enteric polymer have been investigated to optimize the microsphere properties. Huang et al. [61] prepared CS microspheres by the spray-drying method using type-A gelatin and ethylene oxide propylene oxide block copolymer as modifiers. Surface morphology and surface charges of the prepared microspheres were investigated using SEM and microelectrophoresis. Shape, size and surface morphology of the microspheres were significantly influenced by the concentration of gelatin. Betamethasone disodium phosphate-loaded microspheres demonstrated a good drug stability (less 1% hydrolysis product), high entrapment efficiency (95%) and positive surface charge (37.5 mV). In vitro drug release from the

12

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

microspheres was related to gelatin content. Microspheres containing gelatin/CS ratio of 0.40.6 (w/w) showed a prolonged release up to 12 h. 2.4. Emulsion-droplet coalescence method The novel emulsion-droplet coalescence method was developed by Tokumitsu et al. [62], which utilizes the principles of both emulsion cross-linking and precipitation. However, in this method, instead of cross-linking the stable droplets, precipitation is induced by allowing coalescence of CS droplets with NaOH droplets. First, a stable emulsion containing aqueous solution of CS along with drug is produced in liquid paraffin oil and then, another stable emulsion containing CS aqueous solution of NaOH is produced in the same manner. When both emulsions are mixed under high-speed stirring, droplets of each emulsion would collide at random and coalesce, thereby precipitating CS droplets to give small size particles. The method is schematically shown in Fig. 6. Gadopentetic acid-loaded CS nanoparticles have been

prepared by this method for gadolinium neutroncapture therapy. Particle size depends upon the type of CS, i.e., as the % deacetylation degree of CS decreased, particle size increased, but drug content decreased. Particles produced using 100% deacetylated CS had the mean particle size of 452 nm with 45% drug loading. Nanoparticles were obtained within the emulsion-droplet. Size of the nanoparticle did not reflect the droplet size. Since gadopentetic acid is a bivalent anionic compound, it interacts electrostatically with the amino groups of CS, which would not have occurred if a cross-linking agent is used that blocks the free amino groups of CS. Thus, it was possible to achieve higher gadopentetic acid loading by using the emulsion-droplet coalescence method compared to the simple emulsion crosslinking method. 2.5. Ionic gelation The use of complexation between oppositely charged macromolecules to prepare CS microspheres has attracted much attention because the process is very simple and mild [63,64]. In addition, reversible physical cross-linking by electrostatic interaction, instead of chemical cross-linking, has been applied to avoid the possible toxicity of reagents and other undesirable effects. Tripolyphosphate (TPP) is a polyanion, which can interact with the cationic CS by electrostatic forces [65,66]. After Bodmeier et al. [67] reported the preparation of TPPCS complex by dropping CS droplets into a TPP solution, many researchers have explored its potential pharmaceutical usage [6873]. In the ionic gelation method, CS is dissolved in aqueous acidic solution to obtain the cation of CS. This solution is then added dropwise under constant stirring to polyanionic TPP solution. Due to the complexation between oppositely charged species, CS undergoes ionic gelation and precipitates to form spherical particles. The method is schematically represented in Fig. 7. However, TPP/CS microparticles formed have poor mechanical strength thus, limiting their usage in drug delivery. Insulin-loaded CS nanoparticles have been prepared by mixing insulin with TPP solution and then adding this to CS solution under constant stirring [74]. Two types of CS in the form of hydrochloride salt (SeacureR 210 Cl and ProtasanR 110 Cl), varying in

Fig. 6. Schematic representation of preparation of chitosan particulate systems by emulsion-droplet coalescence method.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

13

Fig. 7. Schematic representation of preparation of chitosan particulate systems by ionic gelation method.

their molecular weight and degree of deacetylation, were utilized for nanoparticle preparation. For both types of CS, TPP concentration was adjusted to get a CS/TPP ratio of 6:1. Chitosan nanoparticles thus obtained were in the size range of 300400 nm with a positive surface charge ranging from +54 to +25 mV. Using this method, insulin loading was modulated reaching the values up to 55%. Efficiency of the method was dependent upon the deacetylation of CS, since it involves the gelation of protonated amino groups of CS. There are many ongoing investigations, which demonstrate the improved oral bioavailability of peptide and protein formulations. Bioadhesive polysaccharide CS nanoparticles would seem to further enhance their intestinal absorption. Pan et al. [75] prepared the insulin-loaded CS nanoparticles by ionotropic gelation of CS with TPP anions. Particle size distribution and zeta potential were determined by photon correlation spectroscopy. The ability of CS nanoparticles to enhance the intestinal absorption of insulin and the relative pharmacological bioavailability of insulin was investigated by monitoring the plasma glucose level of alloxan-induced diabetic rats after the oral administration of various doses of insulin-loaded CS nanoparticles. The positively charged, stable CS nanoparticles showed particle size in the range of 250 400 nm. Insulin association was up to 80%. The in vitro release experiments indicated initial burst effect, which is pH-sensitive. The CS nanoparticles enhanced the intestinal absorption of insulin to a greater extent than

the aqueous solution of CS in vivo. After administration of 21 I.U./kg insulin in the CS nanoparticles, hypoglycemia was prolonged over 15 h. The average pharmacological bioavailability relative to s.c. injection of insulin solution was up to 14.9%. Xu and Du [76] have studied different formulations of CS nanoparticles produced by the ionic gelation of TPP and CS. TEM indicated their diameter ranging between 20 and 200 nm with spherical shape. FTIR confirmed tripolyphosphoric groups of TPP linked with ammonium groups of CS in the nanoparticles. Factors that affect the delivery of bovine serum albumin (BSA) as a model protein have been studied. These include molecular weight and deacetylation degree of CS, concentrations of CS and BSA, as well as the presence of polyethylene glycol (PEG) in the encapsulation medium. Increasing molecular weight of CS from 10 to 210 kDa, BSA encapsulation efficiency was enhanced nearly twice. The total release of BSA in phosphate buffered saline pH 7.4 in 8 days was reduced from 73.9% to 17.6%. Increasing deacetylation degree from 75.5% to 92% promoted the encapsulation efficiency with a decrease in release rate. Encapsulation efficiency decreased greatly by increasing the initial concentration of BSA and CS. Higher loading capacity of BSA enhanced the BSA release from nanoparticles. However, adding PEG hindered the BSA encapsulation and increased the release rate. Ko et al. [77] prepared CS microparticles with TPP by the ionic cross-linking method. Particle sizes of

14

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

TPP-CS microparticles varied from 500 to 710 Am with drug encapsulation efficiencies more than 90%. Morphologies of TPP-CS microparticles have been examined by SEM. As the pH of TPP solution decreased and molecular weight of CS increased, microparticles acquired better spherical shape having smooth surface. Release of felodipine as a model drug was affected by the preparation method. Chitosan microparticles prepared at lower pH or higher concentration of TPP solution resulted in a slower release of felodipine. With a decreasing molecular weight and concentration of CS solution, the drug release increased. The release of drug from TPP-CS microparticles decreased when the cross-linking time was increased. 2.6. Reverse micellar method Reverse micelles are thermodynamically stable liquid mixtures of water, oil and surfactant. Macroscopically, they are homogeneous and isotropic, structured on a microscopic scale into aqueous and oil microdomains separated by surfactant-rich films. One of the most important aspects of reverse micelle hosted systems is their dynamic behavior. Nanoparticles prepared by conventional emulsion polymerization methods are not only large (N200 nm), but also have a broad size range. Preparation of ultrafine polymeric nanoparticles with narrow size distribution could be achieved by using reverse micellar medium [78]. Aqueous core of the reverse micellar droplets can be used as a nanoreactor to prepare such particles. Since the size of the reverse micellar droplets usually lies between 1 and 10 nm

[79], and these droplets are highly monodispersed, preparation of drug-loaded nanoparticles in reverse micelles will produce extremely fine particles with a narrow size distribution. Since micellar droplets are in Brownian motion, they undergo continuous coalescence followed by re-separation on a timescale that varies between millisecond and microsecond [80]. The size, polydispersity and thermodynamic stability of these droplets are maintained in the system by a rapid dynamic equilibrium. In this method, the surfactant is dissolved in a organic solvent to prepare reverse micelles. To this, aqueous solutions of CS and drug are added with constant vortexing to avoid any turbidity. The aqueous phase is regulated in such a way as to keep the entire mixture in an optically transparent microemulsion phase. Additional amount of water may be added to obtain nanoparticles of larger size. To this transparent solution, a cross-linking agent is added with constant stirring, and cross-linking is achieved by stirring overnight. The maximum amount of drug that can be dissolved in reverse micelles varies from drug to drug and has to be determined by gradually increasing the amount of drug until the clear microemulsion is transformed into a translucent solution. The organic solvent is then evaporated to obtain the transparent dry mass. The material is dispersed in water and then adding a suitable salt precipitates the surfactant out. The mixture is then subjected to centrifugation. The supernatant solution is decanted, which contains the drug-loaded nanoparticles. The aqueous dispersion is immediately dialyzed through dialysis membrane for about 1 h and the liquid is lyophilized to dry powder. The method is schematically represented in Fig. 8.

Fig. 8. Schematic representation of preparation of chitosan particulate systems by reverse micellar method.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

15

Mitra et al. [81] have encapsulated doxorubicin dextran conjugate in CS nanoparticles prepared by reverse micellar method. The surfactant sodium bis(ethyl hexyl) sulfosuccinate (AOT), was dissolved in n-hexane. To 40 mL of AOT solution (0.03 M), 100 AL of 0.1% CS solution in acetic acid, 200 AL doxorubicindextran conjugate (6.6 mg/mL), 10 AL liquor ammonia and 10 AL of 0.01% glutaraldehyde solution were added with continuous stirring at room temperature. This procedure produced CS nanoparticles encapsulating doxorubicindextran conjugate. Solvent was removed by rotary evaporator and the dry mass was resuspended in 5 mL of pH 7.4 TrisCl buffer by sonication. To this, 1 mL of 30% CaCl2 solution was added dropwise to precipitate the surfactant as calcium salt of diethylhexyl sulfosuccinate. The precipitate was pelleted by centrifugation at 5,000 rpm for 30 min at 4 8C. The pellet was discarded and the supernatant containing nanoparticles was centrifuged at 60,000 rpm for 2 h to pellet the nanoparticles. The pellet was dispersed in 5 mL of pH 7.4 TrisHCl buffer. 2.7. Sieving method Recently, Agnihotri and Aminabhavi [82] have developed a simple, yet novel method to produce CS microparticles. In this method, microparticles were prepared by cross-linking CS to obtain a non-sticky glassy hydrogel followed by passing through a sieve as shown in Fig. 9. A suitable quantity of CS was dissolved in 4% acetic acid solution to form a thick

jelly mass that was cross-linked by adding glutaraldehyde. The non-sticky cross-linked mass was passed through a sieve with a suitable mesh size to get microparticles. The microparticles were washed with 0.1 N NaOH solution to remove the un-reacted excess glutaraldehyde and dried overnight in an oven at 40 8C. Clozapine was incorporated into CS before crosslinking with an entrapment efficiency up to 98.9%. This method is devoid of tedious procedures, and can be scaled up easily. Microparticles were irregular in shape, with the average particle sizes in the range 543698 Am. The in vitro release was extended up to 12 h, while the in vivo studies indicated a slow release of clozapine.

3. Drug loading into micro/nanoparticles of chitosan Drug loading in micro/nanoparticulate systems can be done by two methods, i.e., during the preparation of particles (incorporation) and after the formation of particles (incubation). In these systems, drug is physically embedded into the matrix or adsorbed onto the surface. Various methods of loading have been developed to improve the efficiency of loading, which largely depends upon the method of preparation as well as physicochemical properties of the drug. Maximum drug loading can be achieved by incorporating the drug during the formation of particles, but it may get affected by the process parameters such as method of preparation, presence of additives, etc.

Fig. 9. Schematic representation of preparation of chitosan particulate systems by sieving method.

16

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

Both water-soluble and water-insoluble drugs can be loaded into CS-based particulate systems. Watersoluble drugs are mixed with CS solution to form a homogeneous mixture, and then, particles can be produced by any of the methods discussed before. For instance, cisplatin was loaded [83] during the formation of particles with encapsulation efficiency as high as 99%. The initial concentration of cisplatin and volume of glutaraldehyde had no effect on the encapsulation efficiency. Drug encapsulation increased as the concentration of CS increased. Water-insoluble drugs and drugs that can precipitate in acidic pH solutions can be loaded after the formation of particles by soaking the preformed particles with the saturated solution of drug. Diclofenac sodium, which precipitates in acidic pH conditions, has been loaded by the soaking method [33]. In this method, loading depends upon the swelling of particles in water. Percentage loading of drug decreased with increasing cross-linking due to decreased swelling. Water-insoluble drugs can also be loaded using the multiple emulsion technique. In this method, drug is dissolved in a suitable solvent and then emulsified in CS solution to form an oil-in-water (o/w) type emulsion. Sometimes, drug can be dispersed into CS solution by using a surfactant to get the suspension. Thus, prepared o/w emulsion or suspension can be further emulsified into liquid paraffin to get the oil-water-oil (o/w/o) multiple emulsion. The resulting droplets can be hardened by using a suitable cross-linking agent. In a study by Jameela et al. [84], bovine serum albumin (BSA) and diphtheria toxoid were loaded into preformed glutaraldehyde cross-linked CS microspheres by passive absorption from aqueous solutions. This method is an alternative to loading biological macromolecules that are sensitive to organic solvents, pH, temperature, ultrasound, etc. In vitro release of BSA showed a high burst effect. Coating of particles with paraffin or polylactic acid modulated the drug release. Diphtheria toxoid loaded CS microspheres showed constant antibody titres for 5 months. Hejazi and Amiji [85] have prepared CS microspheres by ionic cross-linking and precipitation with sodium sulfate. Two different methods were used for drug loading. In method I, tetracycline was

mixed with CS solution before simultaneous cross linking and precipitation. In method II, drug was incubated with the pre-formed microspheres for 48 h. Cumulative amount of tetracycline that was released from CS microspheres and stability of drug was examined in different pH media at 37 8C. Microspheres with a spherical shape having an average diameter of 2 3 Am were formed. When drug was added to CS solution before cross-linking and precipitation, only 8% (w/w) was optimally incorporated in the final microsphere formulation. When drug was incubated with the pre-formed microspheres, a maximum of 69% (w/w) could be loaded. About 30% of tetracycline either in solution or when released from the microspheres was found to degrade at pH 1.2 in 12 h. Preliminary results of this study suggested that CS microspheres can be used to incorporate antibiotic drugs, which may be effective when administered locally in the stomach against H. pylori .

4. Drug release and release kinetics Drug release from CS-based particulate systems depends upon the extent of cross linking, morphology, size and density of the particulate system, physicochemical properties of the drug as well as the presence of adjuvants. In vitro release also depends upon pH, polarity and presence of enzymes in the dissolution media. The release of drug from CS particulate systems involves three different mechanisms: (a) release from the surface of particles, (b) diffusion through the swollen rubbery matrix and (c) release due to polymer erosion. These mechanisms are shown schematically in Fig. 10. In majority of cases, drug release follows more than one type of mechanism. In case of release from the surface, adsorbed drug instantaneously dissolves when it comes in contact with the release medium. Drug entrapped in the surface layer of particles also follows this mechanism. This type of drug release leads to burst effect. He et al. [54] observed that cemetidine-loaded CS microspheres have shown burst effect in the early stages of dissolution. Most of the drug was released within few minutes when particles were prepared by spray drying technique. Increasing the cross-linking den-

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

17

Fig. 10. Mechanism of drug release from particulate systems.

sity can prevent the burst release. This effect can also be avoided by washing microparticles with a proper solvent, but it may lead to low encapsulation efficiency. Drug release by diffusion involves three steps. First, water penetrates into particulate system, which causes swelling of the matrix; secondly, the conversion of glassy polymer into rubbery matrix takes place, while the third step is the diffusion of drug from the swollen rubbery matrix. Hence, the release is slow initially and later, it becomes fast. This type of release is more prominent in case of hydrogels. Al-Helw et al. [45] observed a high initial release of the drug in all the prepared formulations. Nearly, 20 30% of the incorporated drug was released in the first hour. Release was dependent on the molecular weight of CS and particle size of the microspheres. The release rate from microspheres prepared from high molecular weight CS was slow compared to those prepared from medium and low molecular weight CS. This could be attributed to both lower solubility of high molecular weight CS and higher viscosity of the gel layer formed around the drug particles upon contact with the dissolution medium. The release within the first 3 h was fast (75 95%) from microspheres

within the size range of 250 500 Am, but for particles in the size range of 500 1,000 Am, drug release was 56 90% in 5 h. This is attributed to large surface area available for dissolution with a small particle size, thus favoring rapid release of the drug compared to larger microspheres. Kweon and Kang [86] prepared the CS-g poly(vinyl alcohol) matrix to study the release of prednisolone under various conditions. Relationship between the amount of drug release and square root of time was linear indicating the diffusion-controlled release. Drug release was controlled by the extent of PVA grafting, heat treatment or cross-link density, but it was less affected by the pH when compared to plain chitosan. Ganza-Gonzalez et al. [56] analyzed the drug release data using Higuchi equation [87]. Higuchi equation was used to describe the release of a solute from a flat surface, but not from a sphere [88], but the good fit obtained suggests that the release rate depends upon the rate of diffusion through the cross-linked matrix. Authors have also fitted the release data to equations developed by Guy et al. [89] to describe the diffusion from a sphere. The most commonly used equation for diffusioncontrolled matrix system is an empirical equation used by Ritger and Peppas [90], in which the early time

18

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

release data can be fitted to obtain the diffusion parameters, Mt kt n Ml 1

Here, M t /M l is the fractional drug release at time t , k is a constant characteristic of the drug-polymer interaction and n is an empirical parameter characterizing the release mechanism. Based on the diffusional exponent [91], drug transport is classified as Fickian (n =0.5), Case II transport (n =1), non-Fickian or anomalous (0.5bn b1) and super Case II (n N1). Drug release from the CS microspheres cross linked with glutaraldehyde, sulfuric acid and heat have shown [33] different n values varying from 0.47 to 0.61. The n values increase with increasing loading of diclofenac sodium in different cross-linked formulations. Recently, Agnihotri and Aminabhavi [82] analyzed the dynamic swelling data of CS microparticles using Eq. (1) to predict drug release from the water uptake data of the microparticles cross-linked with (5.0, 7.5 and 10.0)10 4 mL of glutaraldehyde/mg of CS. It was observed that as the cross-linking increases, swelling of CS microparticles decreases. Values of n obtained in the range of 0.160 to 0.249 indicating that the release mechanism deviates from the Fickian trend. The values of n are b0.5 due to the irregular shaped particles and these decrease systematically with increasing cross-linking. In the swelling controlled release systems, drug is dispersed within a glassy polymer. Upon contact with biological fluid, the polymer swells, but no drug diffusion occurs through the polymer phase. As the penetrant enters the glassy polymer, glass transition temperature of the polymer is lowered due to relaxation of the polymer chains. Drug could diffuse out of the swollen rubbery polymer. This type of system is characterized by two moving boundaries: the front separating the swollen rubbery portion and the glassy region, which moves with a front velocity and the polymer fluid interface. The rate of drug release is controlled by the velocity and position of the front dividing the glassy and rubbery portions of the polymer. Jameela et al. [48] have obtained a good correlation fit for the cumulative drug released vs. square root of time, demonstrating that the release from the microsphere matrix is diffusion-controlled and obeys

Higuchi equation [87]. It was demonstrated that the rate of release depends upon the size of microspheres. Release from smaller size microspheres was faster than those from the large size microspheres due to smaller diffusional path length for the drug and the larger surface area of contact of smaller particles with the dissolution medium. Orienti et al. [92] studied the correlation between matrix erosion and release kinetics of indomethacin-loaded CS microspheres. Release kinetics was correlated with the concentration of CS in the microsphere and pH of the release medium. At high concentrations of CS and at pH 7.4, deviations from Fickian to zero order kinetics have been observed. Variations induced by these parameters on drug diffusion and solubility in the matrix undergoing erosion have been analyzed.

5. Pharmaceutical applications of chitosan particulate systems Chitosan-based particulate systems are attracting pharmaceutical and biomedical applications as potential drug delivery devices. Some important applications are discussed below. 5.1. Colon targeted drug delivery Chitosan is a promising polymer for colon drug delivery since it can be biodegraded by the colonic bacterial flora [93,94] and it has mucoadhesive character [1]. The pH-sensitive multicore microparticulate system containing CS microcores entrapped into enteric acrylic microspheres was reported [60]. Sodium diclofenac was efficiently entrapped within these CS microcores and then microencapsulated into Eudragit L-100 and Eudragit S-100 to form a multireservoir system. In vitro release study revealed no release of the drug in gastric pH for 3 h and after the lag-time, a continuous release for 8 12 h was observed in the basic pH. 5.2. Mucosal delivery Nowadays, mucosal surfaces such as nasal, peroral and pulmonary are receiving a great deal of attention as alternative routes of systemic administration. Chitosan has mucoadhesive properties and therefore,

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

19

it seems particularly useful to formulate the bioadhesive dosage forms for mucosal administration (ocular, nasal, buccal, gastro-enteric and vaginal-uterine therapy) [95]. Nasal mucosa has high permeability and easy access of drug to the absorption site. The particulate delivery to peroral mucosa is easily taken up by the Peyers patches of the gut associated lymphoid tissue. Chitosan has been found to enhance the drug absorption through mucosae without damaging the biological system. Here, the mechanism of action of CS was suggested to be a combination of bioadhesion and a transient widening of the tight junctions between epithelial cells [27]. Genta et al. [95] studied the influence of glutaraldehyde on drug release and mucoadhesive properties of CS microspheres. A new in vitro technique was developed based on electron microscopy to study the effect of polymer cross-link density on the mucoadhesive properties of CS microspheres modulating the rate of theophylline release. The ability of insulinloaded CS nanoparticles to enhance the nasal absorption of insulin was investigated in a conscious rabbit model. Chitosan nanoparticles enhanced the nasal absorption of insulin to a greater extent than the aqueous solution of CS [74]. van der Lubben et al. [96] incorporated the model protein ovalbumin into CS microparticles and the uptake of ovalbumin associated with CS microparticles in murine Peyers patches was demonstrated using confocal laser scanning microscopy. In a further study, van der Lubben et al. [97] investigated the ability of CS microparticles to enhance both systemic and local immune responses against diphtheria toxoid (DT) vaccine after the oral and nasal administration in mice. Systemic and local IgG and IgA immune responses against DT associated to CS microparticles were strongly enhanced after the oral delivery in mice. Even though oral vaccination has numerous advantages over the parenteral injection, degradation of the vaccine in the gut and low uptake in the lymphoid tissue of the gastrointestinal tract still complicate the development of oral vaccines. In this direction, van der Lubben et al. [98] prepared the CS microparticles and characterized them for size, zeta potential, morphology- and ovalbumin-loading as well as release characteristics. The in vivo uptake of CS microparticles by murine Peyers patches was studied by using confocal laser scanning microscopy

(CLSM). Chitosan microparticles were prepared using a precipitation/coacervation method. The size of CS microparticles was 4.30.7 Am and were positively charged (201 mV). Since only microparticles smaller than 10 Am can be taken up by M-cells of Peyers patches, these microparticles were used as vaccination systems. The CLSM studies showed that the model antigen ovalbumin was entrapped within the CS microparticles. Field emission scanning electron microscopy demonstrated the porous structure of CS microparticles, thus facilitating the entrapment of ovalbumin. Ovalbumin loading in CS microparticles was about 40%. Release studies have shown the low release of ovalbumin within 4 h, but most of ovalbumin (about 90%) remained entrapped in the microparticles. Since CS microparticles are biodegradable, the entrapped ovalbumin was released after intracellular digestion in Peyers patches. Initial in vivo studies demonstrated that fluorescently labeled CS microparticles can be taken up by the epithelium of the murine Peyers patches. Since the uptake by Peyers patches is an essential step in oral vaccination, these results have shown that the porous CS microparticles developed are most promising vaccine delivery systems. 5.3. Cancer therapy Gadopentetic acid-loaded CS nanoparticles have been prepared for gadolinium neutron-capture therapy [62]. Their releasing properties and ability for longterm retention of gadopentetic acid in the tumor indicated that these nanoparticles are useful as intratumoral injectable devices for gadolinium neutroncapture therapy. The accumulation of gadolinium loaded as gadopentetic acid (Gd-DTPA) in CS nanoparticles designed for gadolinium neutron-capture therapy (Gd-NCT) for cancer have been evaluated in vitro in cultured cells [99]. Using L929 fibroblast cells, Gd accumulation for 12 h at 37 8C was investigated at Gd concentrations lower than 40 ppm. The accumulation leveled above 20 ppm and reached 18.02.7 (meanS.D.) Ag Gd/106 cells at 40 ppm. Furthermore, the corresponding accumulations in B16F10 melanoma cells and SCC-VII squamous cell carcinoma, which were used in the previous GdNCT trials in vivo were 27.12.9 and 59.89.8 Ag Gd/ 106 cells, respectively. This explains the superior

20

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

growth-suppression in the in vivo trials using SCCVII cells. The accumulation of nanoparticles in these cells was 100 200 times higher in comparison to dimeglumine gadopentetate aqueous solution (Magnevistw), a magnetic resonance imaging contrast agent. The endocytic uptake of nanoparticles was suggested from TEM. These findings indicated that nanoparticles had a high affinity to cells, thus contributing to the long retention of Gd in tumor tissue leading to significant suppression of tumor growth in in vivo studies. Tokumitsu et al. [100] demonstrated the potential usefulness of Gd-NCT using gadolinium-loaded nanoparticles. The potential of gadolinium neutron-capture therapy (Gd-NCT) for cancer was evaluated using CS nanoparticles as a novel gadolinium device. The nanoparticles incorporated with 1200 mg of natural gadolinium were administered intratumorally twice in mice-bearing subcutaneous B16F10 melanoma. The thermal neutron irradiation was performed for the tumor site, with the fluence of 6.321012 neutrons/ cm2, 8 h after the second gadolinium administration. After irradiation, the tumor growth in the nanoparticle-administered group was significantly suppressed compared to that in the gadopentetate solution-administered group, despite radioresistance of melanoma and the smaller Gd dose than that administered in past Gd-NCT trials. Jameela et al. [101] have prepared glutaraldehyde cross-linked CS microspheres containing mitoxantrone. The antitumor activity was evaluated against Ehrlich ascites carcinoma in mice by intraperitoneal injections. The tumor inhibitory effect was followed by monitoring the survival time and change in the body weight of the animal for 60 days. Mean survival time of animals which received free mitoxantrone was 2.1 days and this was increased to 50 days when mitoxantrone was given via microspheres. In another study [102], the in vitro release of mitoxantrone was controlled for 4 weeks in phosphate buffer at 27 8C. Mitra et al. [81] have encapsulated doxorubicin dextran conjugate into long circulating CS nanoparticles. In an attempt to minimize cardiotoxicity of doxorubicin, a conjugate with dextran was prepared and encapsulated in CS nanoparticles. Size of the nanoparticle was 10010 nm, which favors enhanced permeability and retention effect. Antitumor effect of these doxorubicin dextran-loaded nanoparticles was

evaluated in J774A.1 macrophage tumor cells implanted in Balb/c mice. The in vivo efficacy of these nanoparticles was determined by tumor regression and increased survival time compared to doxorubicin dextran conjugate and the free drug. These results suggest that the system not only reduced the side effects, but also improved its therapeutic efficacy in the treatment of solid tumors. Janes et al. [103] evaluated the potential of CS nanoparticles as carriers for doxorubicin (DOX). The challenge was to entrap a cationic, hydrophilic molecule into nanoparticles formed by ionic gelation of the positively charged CS. To achieve this objective, the authors have masked the positive charge of DOX by complexing it with dextran sulfate. This modification doubled the DOX encapsulation efficiency relative to controls and enabled real loadings up to 4.0 wt.% of DOX. Authors also investigated the possibility of forming a complex between CS and DOX prior to the formation of particles. Despite low complexation efficiency, no dissociation of the complex was observed upon the formation of nanoparticles. Fluorimetric analysis of the in vitro drug released showed the initial release phase, the intensity of which was dependent upon the association mode, followed by a very slow release. Evaluation of the activity of DOX-loaded nanoparticles in cell cultures indicated that those containing dextran sulfate were able to maintain cytostatic activity relative to free DOX, while DOX complexed with CS before the nanoparticle formation showed a slightly decreased activity. Additionally, confocal studies showed that DOX was not released in the cell culture medium, but entered the cells while being associated to nanoparticles. These studies have shown the feasibility of CS nanoparticles to entrap DOX and to deliver it to the cells in its active form. 5.4. Gene delivery Gene therapy is a challenging task in the treatment of genetic disorders. In case of gene delivery, the plasmid DNA has to be introduced into the target cells, which should get transcribed and the genetic information should ultimately be translated into the corresponding protein. To achieve this goal, number of hurdles are to be overcome by the gene delivery system. Transfection is affected by: (a) targeting the

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

21

delivery system to target cell, (b) transport through the cell membrane, (c) uptake and degradation in the endolysosomes and (d) intracellular trafficking of plasmid DNA to the nucleus. Chitosan could interact ionically with the negatively charged DNA and forms polyelectrolyte complexes. In these complexes, DNA becomes better protected against nuclease degradation leading to better transfection efficiency. DNA CS nanoparticles have been prepared [53] to examine the influence of several parameters on their preparation. The transfection efficiency of CSDNA nanoparticles was cell-type dependent. Typically, it was 3 to 4 orders of magnitude, in relative light units, higher than the background level in HEK293 cells, and 2 to 10 times lower than that DNA complexes. achieved by LipofectAMINEA The presence of 10% fetal bovine serum did not interfere with their transfection ability. The study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of 4-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve the gene expression level in HeLa cells by 130-fold. Conjugation of PEG on nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of PEGylated nanoparticles in mice following i.v. administration was slower than the unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed during the first hour. Self-aggregates were prepared [104] by hydrophobic modification of CS with deoxycholic acid in aqueous media. Self-aggregates have a small size (mean diameter of 160 nm) with an unimodal size distribution. Self-aggregates can form charge complexes when mixed with plasmid DNA. The usefulness of self-aggregates/DNA complex for transfer of genes into mammalian cells in vitro has been suggested. Several transfection studies using chemically modified CS have been reported. Trimethyl CS oligomers were examined for their potency as DNA carriers [105]. Chitosan and lactosylated CS carriers were investigated for their transfection efficiencies in vitro [106]. Recently, galactosylated CS-g dextran DNA complexes have been prepared [107]. Galactose

groups were chemically bound to CS for liver specificity and dextran was grafted to increase the stability of the complex in water. It was shown that this system could efficiently transfect liver cells. Chew et al. [108] studied the i.m. immunization with full-length Der p 1 cDNA induced significant humoral response to the left domain (approximately corresponding to amino acids 1 116), but not to the right domain (approximately corresponding to amino acids 117 222) of Der p 1 allergen. Authors explored the use of CS DNA nanoparticles for oral immunization to induce the immune responses specific to both left and right domains of Der p 1. DNA constructs pDer p 1 (1 222) and pDer p 1 (114 222), which were complexed with CS and delivered orally followed by an i.m. injection of pDer p 1 (1 222) after 13 weeks. Such an approach has successfully primed Th1-skewed immune responses against both domains of Der p 1. It was suggested that such a strategy could be further optimized for more efficacious gene vaccination for full-length Der p 1. Numerous studies have been reported on prophylactic and therapeutic use of genetic vaccines for combating a variety of infectious diseases in animal models. Recent human clinical studies with the gene gun have validated the concept of direct targeting of dendritic cells (Langerhans cells) in the viable epidermis of the skin. However, it is unclear whether the gene gun technology or other needle-free devices will become commercially viable. Cui and Mumper [109] investigated the topical application of CSbased nanoparticles containing plasmid DNA (pDNA) as a potential approach to genetic immunization. Two types of nanoparticles were investigated: (i) pDNA-condensed CS nanoparticles and (ii) pDNA-coated on pre-formed cationic CS/carboxymethylcellulose (CMC) nanoparticles. These studies have shown that both CS and a CS oligomer can complex CMC to form stable cationic nanoparticles for subsequent pDNA coating. Selected pDNAcoated nanoparticles (with pDNA up to 400 mg/ mL) were stable to challenge with the serum. Several different CS-based nanoparticles containing pDNA resulted in both detectable and quantifiable levels of luciferase expression in mouse skin 24 h after topical application and significant antigenspecific IgG titer to expressed h-galactosidase at 28 days.

22

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

Borchard [110] has recently published a review on the efficient non-viral gene delivery using cationic polymers as DNA-condensing agents. The gene delivery is dependent on several factors such as complex size, complex stability, toxicity, immunogenicity, protection against DNase degradation, intracellular trafficking and processing of the DNA. The review also examined the advances made in the application of CS and CS derivatives to non-viral gene delivery. It gives an overview of the transfection studies performed by using CS as a transfection agent. 5.5. Topical delivery Due to good bioadhesive property and ability to sustain the release of the active constituents, CS has been used in topical delivery systems. Bioadhesive CS microspheres for topical sustained release of cetyl pyridinium chloride have been evaluated [55]. Improved microbiological activity was shown by these microparticulate systems. Conti et al. [111] prepared microparticles composed of CS and designed as powders for topical wound-healing properties. Blank and ampicillin-loaded microspheres were prepared by spray-drying technique. In vivo evaluation in albino rats showed that both drug-loaded and blank microspheres have shown good wound healing properties. 5.6. Ocular delivery De Campos et al. [112] investigated the potential of CS nanoparticles as a new vehicle to improve the delivery of drugs to ocular mucosa. Cyclosporin A (CyA) was chosen as a model drug. A modified ionic gelation technique was used to produce CyA-loaded CS nanoparticles. These nanoparticles with a mean size of 293 nm, a zeta potential of +37 mV, high CyA association efficiency and loading of 73% and 9%, respectively were obtained. The in vitro release studies, performed under sink conditions, revealed the fast release during the first hour followed by a more gradual drug release during the 24-h period. The in vivo experiments showed that after topical instillation of CyA-loaded CS nanoparticles to rabbits, therapeutic concentrations were achieved in the external ocular tissues (i.e., cornea and conjunctiva) within 48 h while maintaining negligible or undetect-

able CyA levels in the inner ocular structures (i.e., iris/ ciliary body and aqueous humour), blood and plasma. These levels were significantly higher than those obtained following the instillation of CS solution containing CyA and an aqueous CyA suspension. The study indicated that CS nanoparticles could be used as a vehicle to enhance the therapeutic index of the clinically challenging drugs with potential application at the extraocular level. 5.7. Chitosan as a coating material Chitosan has good film forming properties and hence, it is used as a coating material in drug delivery applications. Chitosan-coated microparticles have many advantages such as improvement of drug payloads, bioadhesive property and prolonged drug release properties over the uncoated particles. Chitosan-coated microspheres composed of poly(lactic acid) poly(caprolactone) blends have been prepared [113]. These microspheres showed good potential for the targeted delivery of antiproliferative agents to treat restenosis. Shu and Zhu [73] have prepared the alginate beads coated with CS by three different methods. The release of brilliant blue was not only affected by CS density on the particle surface, but also on the preparation method and other factors. Chiou et al. [114] have used different molecular weight chitosans for coating the microspheres. The initial burst release was observed in the first hour with 50% release of lidocaine. But, 19.2% release occurred at 25th hour for the un-coated particles and 14.6% at the 90th hour for the CS-coated microspheres.

6. Chemically modified chitosans Various chemical modifications of CS have been studied to alter its properties. N-Trimethyl chitosan chloride (TMC), a quaternized CS derivative, has been proven to effectively increase the permeation of hydrophilic macromolecular drugs across- the mucosal epithelia by opening the tight junctions [115]. The study investigated the intestinal absorption of octreotide when it is co-administered with a polycationic absorption enhancer, TMC. Chitosan succinate and CS phthalate were synthesized and assessed as potential matrices for colon-specific orally

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

23

administered drug delivery applications. The prepared matrices resisted the dissolution under acidic conditions. On the other hand, improved drug release profiles were observed in basic conditions. These results suggested the suitability of the prepared matrices in colon specific and orally administered drug delivery applications [116]. In order to overcome the low solubility of CS in neutral pH, which is the major drawback to use this type of polymer as a transfection agent, N-trimethylated and N-triethylated oligosaccharides have been synthesized [105]. Lee et al. [104] synthesized the hydrophobically modified CS containing 5.1 deoxycholic acid groups per 100 anhydroglucose units by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction as shown in Fig. 11. Since deoxycholic acid can form self-assemblies in aqueous media, it was found that the modified CS also formed the selfaggregates. The self-aggregates were characterized by

fluorescence spectroscopy and dynamic light scattering method. A charge complex was produced between the cationically charged self-aggregates and the negatively charged plasmid DNA. The feasibility of self-aggregates as an in vitro delivery vehicle was investigated for the transfection of genetic material in mammalian cells. Microcrystalline CS has been investigated as a gel forming excipient [117]. Matrix granules of CS of differing physicochemical properties loaded with either ibuprofen or paracetamol as model drugs have been prepared. Varying the amount or molecular weight of the microcrystalline CS and to a lesser extent by the degree of deacetylation controlled release rate. Giunchedi et al. [59] prepared and characterized a new derivative of CS: methyl pyrrolidone CS. It randomly carries pyrrolidinone groups covalently attached to the polysaccharide backbone. This CS derivative combines the biocom-

Fig. 11. A scheme of the coupling mechanism between chitosan and deoxycholic acid using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) through amide linkage formation [taken from Ref. 104].

24

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528

patibility of CS [118] and hydrophilic characteristics of the pyrrolidinone moiety [119], being particularly susceptible to the hydrolytic action of lysozyme [120]. The microparticles were characterized by S.E.M., particle size analyzer, DSC and in vitro ampicillin release. Drug release characteristics depend upon the nature of CS used. Chen et al. [121] studied the modification of CS by coupling with linoleic acid (LA) through 1-ethyl-3-(3dimethylaminopropyl) carbodiimide-mediated reaction to increase its amphipathicity for improved emulsification. The micelle formation of linoleic acid-modified CS in 0.1 M acetic acid solution was enhanced by O/W emulsification with methylene chloride, an oil phase. Fluorescence spectra indicated that without emulsification, the self-aggregation of LA-CS occurred at the concentration of 1.0 g/L or above, and with emulsification, self-aggregation was greatly enhanced followed by a stable micelle formation at 2.0 g/L. Addition of 1 M NaCl solution promoted the self-aggregation of LA-CS particles both with and without emulsification. The nanosize micelles of LA-CS were formed ranging in size between 200 and 600 nm. The LA-CS nanoparticles were used to encapsulate the lipid soluble model compound, retinal acetate, with 50% efficiency. Chitosan was chemically modified [122] by graft copolymerization of poly(ethylene glycol) diacrylate macromonomer onto CS backbone. Microspheres based on chitosan and polymer grafted chitosan were prepared by a polymer dispersion technique. A comparative study in relation to structural deviation among CS and modified CS microspheres was evaluated. These chemically modified CS microparticles were hydrophilic in nature and formed aggregates. Chitosan derivative with galactose groups was synthesized by introducing galactose group into the amine group of CS [123]. The results indicated that although acyl reaction on the part of amino groups of CS took place, the degree of galactosylated substitution was 20%. Crystallinity, solubility, stability and other physical properties were different from CS. Microspheres of CS and galactosylated CS were prepared by the physical precipitation and coacervation techniques, respectively. Microspheres of CS and galactosylated CS were spherical in nature with an average diameter of 0.54 and 1.05 Am and an average zeta potential of +17 and +15 mV, respectively. It was

suggested that galactosylated CS microspheres could be used for passive and active hepatic targeting.

7. Conclusions Chitosan has the desired properties for safe use as a pharmaceutical excipient. This has prompted accelerated research activities worldwide on chitosan micro and nanoparticles as drug delivery vehicles. These systems have great utility in controlled release and targeting studies of almost all class of bioactive molecules as discussed in this review. Recently, chitosan is also extensively explored in gene delivery. However, studies toward optimization of process parameters and scale up from the laboratory to pilot plant and then, to production level are yet to be undertaken. Majority of studies carried out so far are only in in vitro conditions. More in vivo studies need to be carried out. Chemical modifications of chitosan are important to get the desired physicochemical properties such as solubility, hydrophilicity, etc. The published literature indicates that in the near future, chitosan-based particulate systems will have more commercial status in the market than in the past.

Acknowledgements Authors thank the University Grants Commission (UGC), New Delhi, India for a major grant (F1-41/ 2001/CPP-II) sanctioned to Karnatak University to establish Center of Excellence in Polymer Science.

References
[1] P.C. Berscht, B. Nies, A. Liebendorfer, J. Kreuter, Incorporation of basic fibroblast growth factor into methylpyrrolidinone chitosan fleeces and determination of the in vitro release characteristics, Biomaterials 15 (1994) 593 600. [2] L. Illum, Chitosan and its use as a pharmaceutical excipient, Pharm. Res. 15 (1998) 1326 1331. [3] V. Dodane, V.D. Vilivalam, Pharmaceutical applications of chitosan, Pharm. Sci. Technol. Today 1 (1998) 246 253. [4] O. Felt, P. Buri, R. Gurny, Chitosan: a unique polysaccharide for drug delivery, Drug Dev. Ind. Pharm. 24 (1998) 979 993. [5] K.D. Yao, T. Peng, Y.J. Yin, M.X. Xu, Microcapsules/ microspheres related to chitosan, J. Macromol. Sci., Rev. Macromol. Chem. Phys. C35 (1995) 155 180.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528 [6] H.S. Kas, Chitosan: properties, preparations and application to microparticulate systems, J. Microencapsulation 14 (1997) 689 711. [7] R.A.A. Muzzarelli, C. Jeuniauk, G.W. Gooday, Chitin in Nature and Technology, Plenum, New York, 1986. [8] G. Sjak-Braek, T. Anthonsen, P. Sandford, Chitin and Chitosan, Elsevier, New York, 1992. [9] T. Sannan, K. Kurita, Y. Iwakura, Studies on chitin, 2. Effect of deacetylation on solubility, Makromol. Chem. 177 (1976) 3589 3600. [10] S. Nicol, Life after death for empty shells, New Sci. 129 (1991) 46 48. [11] K. Arai, T. Kinumaki, T. Fujita, Toxicity of chitosan, Bull. Tokai Reg. Fish Lab. 43 (1968) 89 94. [12] T. Chandy, C.P. Sharma, Chitosan as a biomaterial, Biomater. Artif. Org. 18 (1990) 1 24. [13] W.M. Hou, S. Miyazaki, M. Takada, T. Komai, Pharmaceutical application of biomedical polymers. Part XVI. Sustained release of indomethacin from chitosan, Chem. Pharm. Bull. 33 (1985) 3986 3992. [14] S. Miyazaki, K. Ishii, T. Nadai, Pharmaceutical application of biomedical polymers. Part IV. The use of chitin and chitosan as drug carriers, Chem. Pharm. Bull. 29 (1981) 3067 3069. [15] T. Handa, A. Kasai, H. Takenaka, S.Y. Lin, Y. Ando, Novel method for the preparation of controlled-release theophylline granules coated with a polyelectrolyte complex of sodium polyphosphate-chitosan, J. Pharm. Sci. 74 (1985) 264 268. [16] S. Miyazaki, H. Yamaguchi, C. Yokouchi, M. Takada, W.-M. Hou, Sustained-release and intragastric-floating granules of indomethacin using chitosan in rabbits, Chem. Pharm. Bull. 36 (1988) 4033 4038. [17] Y. Sawayanagi, N. Nambu, T. Nagai, Use of chitosan for sustained-release preparations of water-soluble drugs, Chem. Pharm. Bull. 30 (1982) 4213 4215. [18] S. Shiraishi, T. Imai, M. Otagiri, Controlled release of indomethacin by chitosan polyelectrolyte complex: optimization and in vivo/in vitro evaluation, J. Control. Release 25 (1993) 217 225. [19] C.M. Lehr, J.A. Bouwstra, E.H. Schacht, H.E. Junginger, In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers, Int. J. Pharm. 78 (1992) 43 48. [20] H.L. Luehen, C.M. Lehr, C.O. Rentel, A.B.J. Noach, A.G. Boer, J.C. Verhoef, H.E. Junginger, Bioadhesive polymers for the peroral delivery of peptide drugs, J. Control. Release 29 (1994) 329 338. [21] L. Illum, N.F. Farraj, S.S. Davis, Chitosan as a novel nasal delivery system for peptide drugs, Pharm. Res. 11 (1994) 1186 1189. [22] T. Imai, S. Shiraishi, H. Saito, M. Otagiri, Interaction of indomethacin with low molecular weight chitosan, and improvements of some pharmaceutical properties of indomethacin by low molecular weight chitosans, Int. J. Pharm. 67 (1991) 11 20. [23] I. Genta, F. Pavanetto, B. Conti, P. Giunchedi, U. Conte, Spray-drying for the preparation of chitosan microspheres, Proc. Int. Symp. Control. Release Bioact. Mater. 21 (1994) 616 617.

25

[24] Y. Sawayanagi, N. Nambu, T. Nagai, Enhancement of dissolution properties of prednisolone from ground mixtures with chitin or chitosan, Chem. Pharm. Bull. 31 (1983) 2507 2509. [25] J.M. Gallo, E.E. Hassan, Receptor-mediated magnetic carriers: basis for targeting, Pharm. Res. 5 (1988) 300 304. [26] E.E. Hassan, R.C. Parish, J.M. Gallo, Optimized formulation of magnetic chitosan microspheres containing the anticancer agent, oxantrazole, Pharm. Res. 9 (1992) 390 397. [27] P. Artursson, T. Lindmark, S.S. Davis, L. lllum, Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (CACO-2), Pharm. Res. 11 (1994) 1358 1361. [28] J. Akbuga, G. Durmaz, Preparation and evaluation of crosslinked chitosan microspheres containing furosemide, Int. J. Pharm. 111 (1994) 217 222. [29] B.C. Thanoo, M.C. Sunny, A. Jayakrishnan, Cross-linked chitosan microspheres: preparation and evaluation as a matrix for the controlled release of pharmaceuticals, J. Pharm. Pharmacol. 44 (1992) 283 286. [30] K.S. Soppimath, A.R. Kulkarni, T.M. Aminabhavi, Controlled release of antihypertensive drug from the interpenetrating network poly(vinyl alcohol) guar gum hydrogel microspheres, J. Biomater. Sci., Polym. Ed. 11 (2000) 27 43. [31] K.S. Soppimath, A.R. Kulkarni, T.M. Aminabhavi, Water transport and drug release study of cross-linked guar gum grafted polyacrylamide hydrogel microspheres for the controlled release application, Eur. J. Pharm. Biopharm. 53 (2002) 87 98. [32] A.R. Kulkarni, K.S. Soppimath, T.M. Aminabhavi, Controlled release of cefadroxil using sodium alginate interpenetrating network with gelatin/egg albumin, Eur. J. Pharm. Biopharm. 51 (2001) 127 133. [33] S.G. Kumbar, A.R. Kulkarni, T.M. Aminabhavi, Crosslinked chitosan microspheres for encapsulation of diclofenac sodium: effect of cross-linking agent, J. Microencapsulation 19 (2002) 173 180. [34] S.G. Kumbar, T.M. Aminabhavi, Synthesis and characterization of modified chitosan microspheres: effect of the grafting ratio on the controlled release of nifedipine through microspheres, J. Appl. Polym. Sci. 89 (2003) 2940 2949. [35] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E. Rudzinski, Biodegradable polymeric nanoparticles as drug delivery devices, J. Control. Release 70 (2001) 1 20. [36] D.K. Singh, A.R. Ray, Biomedical applications of chitin, chitosan and their derivatives, J. Macromol. Sci., Rev. Macromol. Chem. Phys. C40 (2000) 69 83. [37] A.K. Singla, M. Chawla, Chitosan: some pharmaceutical and biological aspects an update, J. Pharm. Pharmacol. 53 (2001) 1047 1067. [38] W.G. Liu, K.D. Yao, Chitosan and its derivatives a promising non-viral vector for gene transfection, J. Control. Release 83 (2002) 1 11. [39] R. Hejazi, M. Amiji, Chitosan-based gastrointestinal delivery systems, J. Control. Release 89 (2003) 151 165. [40] V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K. Bansal, S. Dhawan, Chitosan microspheres as

26

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528 a potential carrier for drugs, Int. J. Pharm. 274 (2004) 1 33. J. Kreuter, Nanoparticles, in: J. Kreuter (Ed.), Colloidal Drug Delivery Systems, Marcel Dekker, New York, 1994, pp. 219 342. L. Brannon Peppas, Recent advances on the use of biodegradable microparticles and nanoparticles in the controlled drug delivery, Int. J. Pharm. 116 (1995) 1 9. P. Couvreur, L. Grislain, V. Lenaerts, F. Brasseur, P. Guiot, in: P. Guiot, P. Couvreur (Eds.), Polymeric Nanoparticles and Microspheres, CRC Press, Boca Raton, FL, 1986. J. Akbuga, G. Durmaz, Preparation and evaluation of crosslinked chitosan microspheres containing furosemide, Int. J. Pharm. 11 (1994) 217 222. A.A. Al-Helw, A.A. Al-Angary, G.M. Mahrous, M.M. AlDardari, Preparation and evaluation of sustained release cross-linked chitosan microspheres containing phenobarbitone, J. Microencapsulation 15 (1998) 373 382. E.B. Denkbas, M. Seyyal, E. Piskin, 5-Fluorouracil loaded chitosan microspheres for chemoembolization, J. Microencapsulation 16 (1998) 741 749. C. Sankar, M. Rani, A.K. Srivastava, B. Mishra, Chitosan based pentazocine microspheres for intranasal systemic delivery: development and biopharmaceutical evaluation, Pharmazie 56 (2001) 223 226. S.R. Jameela, T.V. Kumary, A.V. Lal, A. Jayakrishnan, Progesterone-loaded chitosan microspheres: a long acting biodegradable controlled delivery system, J. Control. Release 52 (1998) 17 24. F. Bugamelli, M.A. Raggi, I. Orienti, V. Zecchi, Controlled insulin release from chitosan microparticles, Arch. Pharm. 331 (1998) 133 138. K. Nishimura, S. Nishimura, H. Seo, N. Nishi, S. Tokura, I. Azuma, Macrophage activation with multiporous beads prepared from partially deacetylated chitin, J. Biomed. Mater. Res. 20 (1986) 1359 1372. A. Berthod, J. Kreuter, Chitosan microspheres improved acid stability and change in physicochemical properties by cross-linking, Proc. Int. Symp. Control. Release Bioact. Mater. 23 (1996) 369 370. S. Ozbas-Turan, J. Akbuga, C. Aral, Controlled release of interleukin-2 from chitosan microspheres, J. Pharm. Sci. 91 (2002) 124 125. H.Q. Mao, K. Roy, V.L. Troung-Le, K.A. Janes, K.Y. Lim, Y. Wang, J.T. August, K.W. Leong, Chitosan DNA nanoparticles as gene delivery carriers: synthesis, characterization and transfection efficiency, J. Control. Release 70 (2001) 399 421. P. He, S.S. Davis, L. Illum, Chitosan microspheres prepared by spray drying, Int. J. Pharm. 187 (1999) 53 65. B. Conti, T. Modena, I. Genta, P. Perugini, C. Decarro, F. Pavanetto, Microencapsulation of cetylpyridinium chloride with a bioadhesive polymer, Proc. Int. Symp. Control. Release Bioact. Mater. 25 (1998) 822 823. A. Ganza-Gonzalez, S. Anguiano-Igea, F.J. Otero-Espinar, J.B. Mendez, Chitosan and chondroitin microspheres for oral-administration controlled release of metoclopramide, Eur. J. Pharm. Biopharm. 48 (1999) 149 155. X.Y. Shi, T.W. Tan, Preparation of chitosan/ethylcellulose complex microcapsule and its application in controlled release of vitamin D-2, Biomaterials 23 (2002) 4469 4473. C.W. Bishop, J.C. Knutson, C.R. Valliere, Method of treating prostatic disease using delayed and/or sustained release vitamin D formulation, US Pat. 5 795 882, 1998. P. Giunchedi, I. Genta, B. Conti, R.A.A. Muzzarelli, U. Conte, Preparation and characterization of ampicillin loaded methylpyrrolidinone chitosan and chitosan microspheres, Biomaterials 19 (1998) 157 161. M.L. Lorenzo-Lamosa, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Design of microencapsulated chitosan microspheres for colonic drug delivery, J. Control. Release 52 (1998) 109 118. Y.C. Huang, M.K. Yeh, C.H. Chiang, Formulation factors in preparing BTM-chitosan microspheres by spray drying method, Int. J. Pharm. 242 (2002) 239 242. H. Tokumitsu, H. Ichikawa, Y. Fukumori, Chitosan gadopentetic acid complex nanoparticles for gadolinium neutron capture therapy of cancer: preparation by novel emulsiondroplet coalescence technique and characterization, Pharm. Res. 16 (1999) 1830 1835. A. Polk, B. Amsden, K.D. Yao, T. Peng, M.F.A. Goosen, Controlled release of albumin from chitosan-alginate microcapsules, J. Pharm. Sci. 83 (1994) 178 185. L.S. Liu, S.Q. Liu, S.Y. Ng, M. Froix, T. Ohno, J. Heller, Controlled release of interleukin-2 for tumor immunotherapy using alginate:chitosan porous microspheres, J. Control. Release 43 (1997) 65 74. Y. Kawashima, T. Handa, H. Takenaka, S.Y. Lin, Y. Ando, Novel method for the preparation of controlled-release theophylline granules coated with a polyelectrolyte complex of sodium polyphosphate-chitosan, J. Pharm. Sci. 74 (1985) 264 268. Y. Kawashima, T. Handa, A. Kasai, H. Takenaka, S.Y. Lin, The effect of thickness and hardness of the coating film on the drug release of theophylline granules, Chem. Pharm. Bull. 33 (1985) 2469 2474. R. Bodmeier, K.H. Oh, Y. Pramar, Preparation and evaluation of drug-containing chitosan beads, Drug Dev. Ind. Pharm. 15 (1989) 1475 1494. S. Shirashi, T. Imai, M. Otagiri, Controlled release of indomethacin by chitosan polyelectrolyte complex: optimization and in vivo: in vitro evaluation, J. Control. Release 25 (1993) 217 225. A.D. Sezer, J. Akbuga, Controlled release of piroxicam from chitosan beads, Int. J. Pharm. 121 (1995) 113 116. Z. Aydin, J. Akbuga, Chitosan beads for the delivery of salmon calcitonin: preparation and characteristics, Int. J. Pharm. 131 (1996) 101 103. P. Calvo, C. Remunan-Lopez, J.L. Vila-Jata, M.J. Alonso, Chitosan and chitosan: ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for protein and vaccines, Pharm. Res. 14 (1997) 1431 1436.

[41]

[57]

[42]

[58]

[43]

[59]

[44]

[60]

[45]

[61]

[46]

[62]

[47]

[63]

[48]

[64]

[49]

[65]

[50]

[51]

[66]

[52]

[67]

[53]

[68]

[69] [70]

[54] [55]

[71]

[56]

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528 [72] P. Calvo, C. Remunan-Lopez, J.L. Vila-Jata, M.J. Alonso, Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers, J. Appl. Polym. Sci. 63 (1997) 125 132. [73] X.Z. Shu, K.J. Zhu, A novel approach to prepare tripolyphosphate/chitosan complex beads for controlled release drug delivery, Int. J. Pharm. 201 (2000) 51 58. [74] R. Fernandez-Urrusuno, P. Cavlo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of insulin using chitosan nanoparticles, Pharm. Res. 16 (1999) 1576 1581. [75] Y. Pan, Y. Li, H. Zhao, J. Zheng, H. Xu, G. Wei, J. Hao, F. Cui, Chitosan nanoparticles improve the intestinal absorption of insulin in vivo, Int. J. Pharm. 249 (2002) 139 147. [76] Y. Xu, Y. Du, Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles, Int. J. Pharm. 250 (2003) 215 226. [77] J.A. Ko, H.J. Park, S.J. Hwang, J.B. Park, J.S. Lee, Preparation and characterization of chitosan microparticles intended for controlled drug delivery, Int. J. Pharm. 249 (2002) 165 174. [78] Y.S. Leong, F. Candau, Inverse microemulsion polymerization, J. Phys. Chem. 86 (1982) 2269 2271. [79] A. Maitra, Determination of size parameters of water Aerosol OT oil reverse micelles from their nuclear magnetic resonance data, J. Phys. Chem. 88 (1984) 5122 5125. [80] P.L. Luisi, M. Giomini, M.P. Pileni, B.H. Robinson, Reverse micelles as hosts for proteins and small molecules, Biochim. Biophys. Acta 947 (1988) 209 246. [81] S. Mitra, U. Gaur, P.C. Ghosh, A.N. Maitra, Tumor targeted delivery of encapsulated dextran doxorubicin conjugate using chitosan nanoparticles as carrier, J. Control. Release 74 (2001) 317 323. [82] S.A. Agnihotri, T.M. Aminabhavi, Controlled release of clozapine through chitosan microparticles prepared by a novel method, J. Control. Release 96 (2004) 245 259. [83] J. Akbuga, N. Bergisadi, Effect of formulation variables on cis-platin loaded chitosan microsphere properties, J. Microencapsulation 16 (1999) 697 703. [84] S.R. Jameela, A. Misra, A. Jayakrishnan, Cross-linked chitosan microspheres as carriers for prolonged delivery of macromolecular drugs, J. Biomater. Sci., Polym. Ed. 6 (1994) 621 632. [85] R. Hejazi, M. Amiji, Stomach-specific anti-H. pylori therapy. I: preparation and characterization of tetracyline-loaded chitosan microspheres, Int. J. Pharm. 235 (2002) 87 94. [86] D.K. Kweon, D.W. Kang, Drug-release behavior of chitosang-poly(vinyl alcohol) copolymer matrix, J. Appl. Polym. Sci. 74 (1999) 458 464. [87] T. Higuchi, Mechanism of sustained action medication, J. Pharm. Sci. 52 (1963) 1145 1149. [88] C. Washington, Drug release from microdisperse systems: a critical review, Int. J. Pharm. 58 (1990) 1 12. [89] R.H. Guy, J. Hadgraft, I.W. Kellaway, M.J. Taylor, Calculations of drug release rates from spherical particles, Int. J. Pharm. 11 (1982) 199 207.

27

[90] P.L. Ritger, N.A. Peppas, A simple equation for description of solute release. II. Fickian and anomalous release from swellable devices, J. Control. Release 5 (1987) 37 42. [91] N.A. Peppas, R.W. Korsmeyer, in: N.A. Peppas (Ed.), Hydrogels in Medicine and Pharmacy, vol. 3, CRC Press, Boca Raton, FL, 1987, p. 103. [92] I. Orienti, K. Aiedeh, E. Gianasi, V. Bertasi, V. Zecchi, Indomethacin loaded chitosan microspheres. Correlation between the erosion process and release kinetics, J. Microencapsulation 13 (1996) 463 472. [93] D. Pantaleone, M. Yalpani, M. Scollar, Advances in chitin and chitosan, in: C.J. Brine, P.A. Sandford, J.P. Zizakis, Proceed. 5th Int. Conf. Chitin and Chitosan, Princeton, New York, 1991. [94] H. Zhang, I.A. Alsarra, S.H. Neau, An in vitro evaluation of a chitosan-containing multiparticulate system for macromolecule delivery to the colon, Int. J. Pharm. 239 (2002) 197 205. [95] I. Genta, M. Costantini, A. Asti, B. Conti, L. Montanari, Influence of glutaraldehyde on drug release and mucoadhesive properties of chitosan microspheres, Carbohydr. Polym. 36 (1998) 81 88. [96] I.M. van der Lubben, F.A.J. Konings, G. Borchard, J.C. Verhoef, H.E. Junginger, In vivo uptake of chitosan microparticles by murine Peyers patches: visualization studies using confocal laser scanning microscopy and immunohistochemistry, J. Drug Target. 9 (2001) 39 47. [97] I.M. van der Lubben, G. Kersten, M.M. Fretz, C. Beuvery, J.C. Verhoef, H.E. Junginger, Chitosan microparticles for mucosal vaccination against diphtheria: oral and nasal efficacy studies in mice, Vaccine 21 (2003) 1400 1408. [98] I.M. van der Lubben, J.C. Verhoef, A.C. van Aelst, G. Borchard, H.E. Junginger, Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine Peyers patches, Biomaterials 22 (2001) 687 694. [99] F. Shikata, H. Tokumitsu, H. Ichikawa, Y. Fukumori, In vitro cellular accumulation of gadolinium incorporated into chitosan nanoparticles designed for neutron-capture therapy of cancer, Eur. J. Pharm. Biopharm. 53 (2002) 57 63. [100] H. Tokumitsu, J. Hiratsuka, Y. Sakurai, T. Kobayashi, H. Ichikawa, Y. Fukumori, Gadolinium neutron-capture therapy using novel gadopentetic acid-chitosan complex nanoparticles: in vivo growth suppression of experimental melanoma solid tumor, Cancer Lett. 150 (2000) 177 182. [101] S.R. Jameela, P.G. Latha, A. Subramoniam, A. Jayakrishnan, Antitumor activity of mitoxantrone-loaded chitosan microspheres against Ehrlich ascites carcinoma, J. Pharm. Pharmacol. 48 (1996) 685 688. [102] S.R. Jameela, A. Jayakrishnan, Glutaraldehyde cross-linked chitosan microspheres as a long-acting biodegradable drugdelivery vehicle-studies on the in-vitro release of mitoxantrone and in-vivo degradation of microspheres in rat muscle, Biomaterials 16 (1995) 769 775. [103] K.A. Janes, M.P. Fresneau, A. Marazuela, A. Fabra, M.J. Alonso, Chitosan nanoparticles as delivery systems for doxorubicin, J. Control. Release 73 (2001) 255 267.

28

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528 [114] S.H. Chiou, W.T. Wu, Y.Y. Huang, T.W. Chung, Effects of the characteristics of chitosan on controlling drug release of chitosan coated PLLA microspheres, J. Microencapsulation 18 (2001) 613 625. [115] M. Thanou, J.C. Verhoef, P. Marbach, H.E. Junginger, Intestinal absorption of octreotide: N-trimethyl chitosan chloride (TMC) ameliorates the permeability and absorption properties of the somatostatin analogue in vitro and in vivo, J. Pharm. Sci. 89 (2000) 951 957. [116] K. Aiedeh, M.O. Taha, Synthesis of chitosan succinate and chitosan phthalate and their evaluation as suggested matrices in orally administered, colon-specific drug delivery systems, Archiv.der Pharmazie 332 (1999) 103 107. [117] M. Sakkinen, U. Seppala, P. Heinanen, M. Marvola, In vitro evaluation of microcrystalline chitosan (MCCh) as gelforming excipient in matrix granules, Eur. J. Pharm. Biopharm. 54 (2002) 33 40. [118] R.A.A. Muzzarelli, V. Baldassarre, F. Conti, G. Gazzanelli, V. Vasi, P. Ferrara, G. Biagini, The biological activity of chitosan: ultrastructural study, Biomaterials 8 (1988) 247 252. [119] R.A.A. Muzzarelli, Depolymerization of methylpyrrolidinone chitosan by lysozyme, Carbohydr. Polym. 19 (1992) 29 34. [120] R.A.A. Muzzarelli, In vivo biochemical significance of chitin-based medical items, in: S. Dumitriu, M. Szycher (Eds.), Polymeric Materials for Biomedical Applications, Marcel Dekker, New York, 1992, pp. 179 198. [121] X.G. Chen, C.M. Lee, H.J. Park, O/W emulsification for the self-aggregation and nanoparticle formation of linoleic acids modified chitosan in the aqueous system, J. Agric. Food Chem. 51 (2003) 3135 3139. [122] K.L. Shanta, D.R.K. Harding, Synthesis and characterization of chemically modified chitosan microspheres, Carbohydr. Polym. 48 (2002) 247 253. [123] C. Zhang, Q. Ping, Y. Ding, Y. Cheng, J. Shen, Synthesis, characterization and microsphere formation of galactosylated chitosan, J. Appl. Polym. Sci. 91 (2004) 659 665.

[104] K.Y. Lee, I.C. Kwon, Y.H. Kim, W.H. Jo, S.Y. Jeong, Preparation of chitosan self-aggregates as a gene delivery system, J. Control. Release 51 (1998) 213 220. [105] B.I. Florea, M. Thanou, M. Geldof, C. Meaney, H.E. Junginger, G. Borchard, Modified chitosan oligosaccharides as transfection agents for gene therapy of cystic fibrosis, Proc. Int. Symp. Control. Release Bioact. Mater. 27 (2000) 846 847. [106] P. Erbacher, S. Zou, T. Beuinger, A.M. Steffan, J.S. Remy, Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability, Pharm. Res. 15 (1998) 1332 1339. [107] Y.K. Park, Y.H. Park, B.A. Shin, E.S. Choi, Y.R. Park, T. Akaike, C.S. Cho, Galactosylated chitosan graft-dextran as hepatocyte-targeting DNA carrier, J. Control. Release 69 (2000) 97 108. [108] J.L. Chew, C.B. Wolfowicz, H.Q. Mao, K.W. Leong, K.Y. Chua, Chitosan nanoparticles containing plasmid DNA encoding house dust mite allergen, Der p 1 for oral vaccination in mice, Vaccine 21 (2003) 2720 2729. [109] Z. Cui, R.J. Mumper, Chitosan-based nanoparticles for topical genetic immunization, J. Control. Release 75 (2001) 409 419. [110] G. Borchard, Chitosans for gene delivery, Adv. Drug Deliv. Rev. 52 (2001) 145 150. [111] B. Conti, P. Giunchedi, I. Genta, U. Conte, The preparation and in vivo evaluation of the wound-healing properties of chitosan microspheres, STP Pharma. Sci. 10 (2000) 101 104. [112] A.M. De Campos, A. Sanchez, M.J. Alonso, Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A, Int. J. Pharm. 224 (2001) 159 168. [113] T. Chandy, R.F. Wilson, G.H.R. Rao, G.S. Das, Changes in cisplatin delivery due to surface-coated poly(lactic acid)poly(q-caprolactone) microspheres, J. Biomater. Appl. 16 (2002) 275 291.