Chitosan Microparticles Production by Supercritical Fluid Processing

Phillips, G. O.; Takigami, S.; Takigami, M. Hydration characteristics of the gum exudate from Acacia Senegal. Food Hydrocolloids 1996, 10, 11. [Crossr...
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Chitosan Microparticles Production by Supercritical Fluid Processing Ernesto Reverchon and Alessandra Antonacci* Department of Chemical Engineering, UniVersity of Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy

In this work chitosan (CS) in 1% acetic acid aqueous solutions has been successfully micronized using supercritical assisted atomization (SAA), which is a new and very promising supercritical fluid process. Some process parameters such as precipitation temperature and solute concentration in the liquid solution have been explored to evaluate their influence on the morphology and size of precipitated particles. Noncoalescing spherical microparticles have been produced as confirmed by scanning electron microscopy analysis. Laser scattering particle size analysis has shown that these precipitates have a sharp distribution: 99% of particles produced show diameters ranging between 0.1 and 1.5 µm, when precipitated from solutions with chitosan concentrations ranging between 1 and 10 mg/mL. X-ray and differential scanning calorimetry analyses have also been used to investigate CS modifications induced by SAA processing. A decrease in crystallinity has been observed when higher precipitation temperatures have been used. SAA micronized CS microparticles are expected to be promising vehicles for drug delivery. 1. Introduction Chitosan (CS) is a nontoxic, biodegradable, and biocompatible polymer. It is a natural linear biopolyaminosaccharide derived by alkaline deacetylation of chitin, the second most abundant natural biopolymer found in nature after cellulose. CS is insoluble at neutral and alkaline pH values but forms salts with inorganic and organic acids such as glutamic acid, hydrochloric acid, lactic acid, and acetic acid. Chitosan salts are soluble in water, the solubility being dependent on the degree of acetylation and pH. Usually, 1-3% aqueous acetic acid solutions are used to solubilize CS. Chitosan has received increasing attention as a renewable polymeric material, since it can be used in many fields due to its chemical structure. Particularly, properties such as biodegradability, low toxicity, and good biocompatibility make CS particularly suitable for use in biomedical and pharmaceutical formulations.1-3 CS has shown excellent properties as excipient and has been used as a vehicle in compressed tablets, as a disintegrant, as a binder, and as a granulating agent in ground mixtures, as well as a co-grinding diluent for the enhancement of dissolution rate and bioavailability of water insoluble drugs.4-6 CS has also been widely investigated in the development of controlled release drug delivery systems. Particularly, it has mucoadhesive properties due to molecular attractive forces formed by electrostatic interaction between positively charged CS and negatively charged mucosal surfaces; therefore, it can promote drugs transmucosal absorption. It has been exploited for nasal and oral delivery of polar drugs, to include peptides and proteins, for vaccine delivery,7,8 as well as a drug carrier for sustained release preparations.9,10 Micro-/nanoparticulate drug delivery offers several advantages over the conventional dosage forms, such as higher efficacy, reduced drug blood level fluctuations, and improved patient compliance. The use of microsphere-based therapies allows careful tailoring release of the drug to the specific treatment site through the choice and formulation of various drugpolymer combinations. CS microspheres can be used to provide controlled release of many drugs and to improve the bioavailability of degradable substances such as protein or enhance the * To whom correspondence should be addressed. Tel.: +39089964025. Fax: +39089964057. E-mail: [email protected].

uptake of hydrophilic substances across the epithelial layers. CS microspheres are being investigated for parenteral and oral drug delivery and are the most widely studied drug delivery systems for the controlled release of drugs such as antibiotics, antihypertensive agents, anticancer agents, proteins, peptide drugs, and vaccines.11-13 Drug loading of microparticles can be obtained by using two methods: (i) the drug can be loaded during the preparation of particles, i.e., the direct production of composite particles formed by the polymer and the active compound; (ii) after the formation of particles, the drug can be physically embedded into the polymer matrix or adsorbed onto the surface. In any case, polymer particle formation is a crucial step for the preparation of drug-loaded polymer particles, since the dimension, the shape, and the structure of the polymeric matrix influence the way of administration to the patient and the drug release rate in the organism. Different methods have been used to prepare CS particulate systems. Some conventional processes for CS microparticle production are emulsion cross-linking, coacervation/precipitation, ionic gelation, reverse micelles formation, solvent evaporation, and spray-drying.10-13 These conventional processes show several drawbacks, such as the use of organic solvents and hardto-separate surfactants, that require many subsequent treatments to reduce solvent residue below the safety limits, high temperatures, and limited control of particle size and particle size distribution.14-16 To overcome the previous drawbacks, new techniques, capable of improving the properties of polymeric materials and the environmental compatibility of their manufacturing processes, have been proposed. For example, supercritical fluid (SCF) techniques have been investigated for polymer processing. SCF-based techniques are an improvement of conventional processes that take advantage of the unique properties of SCFs: tunability of viscosity and diffusivity between those of liquids and gases and the environmentally benign impact of SCFs such as carbon dioxide.17 SCF-assisted particle technology also offers advantages connected to the efficient extraction of solvents and impurities and the plasticization of polymers.18-21 Some papers and reviews have been published on polymer processing using these techniques.22,23

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CS processing using SCF technologies has been attempted by some authors. For example, CS porous structures have been obtained using supercritical carbon dioxide (SC-CO2)24 and CSindomethacine composites via SCF-assisted impregnation25 have been proposed. However, to date, CS microparticle production using SC-CO2-based processes has not been successful yet. Indeed, solubility of CS in SC-CO2 is nearly zero; therefore, processes based on the solvent effect of CO2 cannot be successfully applied. Moreover, since currently 1-3% aqueous acetic acid solutions are used to solubilize CS, processes based on the antisolvent effect of CO2 cannot be used, since SC-CO2 is not an effective solvent for aqueous solutions.26 Supercritical-based atomization techniques have also been proposed as efficient micronization processes. Among these, carbon dioxide-assisted nebulization with a bubble dryer (CANBD) is based on the mixing of a liquid solution and SC-CO2 in a near-zero volume tee connection and subsequent atomization through a capillary tube. It has been successfully tested for several materials.27-29 The supercritical assisted atomization (SAA) differs from the previously described process, since it is based on the solubilization of controlled quantities of SC-CO2 in the liquid solutions using a saturator that contains high surface packings and ensures long residence times. Therefore, a near-equilibrium solution is formed that is subsequently atomized through a nozzle.30,31 In this case, SC-CO2 plays both as cosolute, being miscible with the solution to be treated, and as pneumatic agent to atomize the solution in fine droplets. The solution formed in the saturator is then sent to a thin-wall injector and sprayed into the precipitator. A two-step atomization is obtained: the primary droplets produced at the outlet of the injector (pneumatic atomization) are further divided into secondary droplets by CO2 expansion from the inside of the primary ones (decompressiVe atomization). This technique can provide a good control over particle size and distribution; microparticles sizing between 0.1 and 10 µm can be easily produced.32-34 These characteristics, joined to the quality of a green and not aggressive process with respect to the substances treated, can be used to successfully micronize a wide range of compounds. SAA can operate not only with organic solutions but also when aqueous solutions are used. This characteristic suggests the possibility of CS microparticle production using this technique. Therefore, the aim of this work is to verify the processability of CS by SAA and the performance of this technique using a 1% (v/v) acetic acid aqueous solution as solvent. SAA experimentation on CS is characterized by a further original aspect: the innovative use of a multicomponent liquid mixture used as solvent in the SAA process. The effect of process parameters such as temperature in the precipitator and solute concentration in the liquid solution on CS particles morphology, size, and distribution will be studied to evaluate the possibility of particle size tailoring for specific applications. 2. Experimental Apparatus, Materials, and Methods 2.1. SAA Apparatus. The SAA laboratory apparatus consists of two high-pressure pumps (model 305, Gilson, Middleton, WI) delivering the liquid solution and liquid CO2 to a heated bath (Forlab model TR12, Carlo Erba, Milan, Italy) and, then, to the saturator. The saturator is a high-pressure vessel (internal volume, 25 cm3) loaded with stainless steel perforated saddles which ensure a large contact surface between liquid solution and CO2, thus enhancing the dissolution of the gaseous stream into the liquid solution. Typically, volumetric flow rates of the

Figure 1. Schematic representation of the SAA mechanism.

liquid solution of about 4-5 mL/min are used that produce long residence times (from 4 to 5 min) in the saturator, leading to near-equilibrium conditions. The solution obtained in the saturator is sprayed through a thin-wall 80-µm diameter injection nozzle into the precipitator. A controlled flow of N2 is taken from a cylinder, heated in an electric heat exchanger (model CBEN 24G6, Watlow, St. Louis, MO), and sent to the precipitator to facilitate liquid droplet evaporation. The precipitator is a stainless steel vessel (internal volume, 3 dm3) operating at atmospheric pressure. The saturator and the precipitator are electrically heated using thinband heaters. A stainless steel filter located at the bottom of the precipitator allows the powder collection and the gaseous stream flow out. SAA apparatus layout and further details on the experimental procedures were published elsewhere.30-35 A schematic representation of the postulated SAA process mechanism is reported in Figure 1. We produced about 3 g/run of micronized CS with a yield of about 95% (calculated as the ratio between the CS collected and the CS fed to the apparatus). About 5% of the injected CS was lost on the walls of the precipitator and inside the pores of the filter. 2.2. Materials. Low molecular weight chitosan with a degree of deacetylation of 84.0% was supplied by Sigma-Aldrich (Milan, Italy). A 1% (v/v) acetic acid aqueous solution was used as the solvent to solubilize CS. Water (HPLC grade) and acetic acid (purity, 99.8%) were supplied by Sigma-Aldrich. Carbon dioxide (CO2; purity, 99.9%) and nitrogen (N2; purity, 99.9%) were purchased from SON (Naples, Italy). 2.3. Powder Morphology by Scanning Electronic Microscopy. CS powders, sampled at different heights in the precipitation chamber, were observed by a scanning electron microscope (model LEO 420, Carl Zeiss SMT AG, Oberkochen, Germany). Powders were dispersed on a carbon tab previously stuck to an aluminum stub (Agar Scientific, Stansted, United Kingdom). Samples were coated with gold-palladium (layer thickness, 250 Å) using a sputter coater (model 108A, Agar Scientific). At least 20 scanning electron microscopy (SEM) images were taken for each run to verify the powder uniformity. 2.4. Particle Size Distribution. Particle size analysis was performed on CS microparticles using an image analysis software applied on SEM images and by laser scattering. Particle size distributions (PSDs) were measured from SEM images using the Sigma Scan Pro Software (release 5.0, Jandel Scientific, Erkrath, Germany). About 1000 particle diameters were considered in each PSD calculation. Histograms representing PSDs in terms of particle number were calculated using Microcal Origin Software (release 6.0, Microcal Software Inc.,

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Northampton, USA); then, they were converted into volumetric distributions and plotted in a cumulative form. Laser scattering (LS) particle size analysis was carried out using a Malvern Mastersizer S laser diffractometer (Malvern Instruments Ltd., Worcestershire, U.K.). The smallest particle diameter detectable with this instrument is 0.05 µm. CS microparticles were suspended in a 5% (w/v) ammonium thiocyanate 2-propanol solution and sonicated for 15 min before analysis. Particle size distributions obtained by the two techniques have been, then, compared. 2.5. Solid-State Characterization. Diffraction patterns of CS powders were obtained using an X-ray diffractometer (model D8 Discover, Bruker AXS, Inc., Madison, WI) with a Cu-sealed tube source. Samples were placed in the holder and flattened with a glass slide to ensure a good surface texture. The measuring conditions were as follows: Ni-filtered Cu KR radiation, λ ) 1.54 Å, 2θ angle ranging between 10 and 70° with a scan rate of 3 s/step and a step size of 0.2°. Thermograms of CS samples were obtained using a differential scanning calorimeter (DSC, model TC11, Mettler Toledo, Inc., Columbus, OH). Fusion temperature and enthalpy were calibrated with an indium standard (melting point, 156.6 °C). A 10 mg amount of CS samples were accurately weighed, crimped in an aluminum pan, and heated from 25 to 300 °C under a nitrogen purge at 10 °C min-1. X-ray and DSC analyses were performed in three replicates for each batch of material. 3. Results and Discussion 3.1. Selection of the Saturator Operating Parameters. The solubilization of SC-CO2 in the liquid solution inside the saturator is one of the key steps controlling the efficiency of the SAA process. The solubility of SC-CO2 in liquids depends on the chemical structure of the liquid and on temperature and pressure in the saturator, since it is related to high-pressure vapor liquid equilibria (VLEs) of the selected liquid-CO2 system. Moreover, it can be also influenced by the presence of solute dissolved in the liquid. Gas and liquid flow rates are selected to obtain residence times of some minutes (from 4 to 5 min) in the saturator to ensure efficient gas dissolution in the liquid solution. The mass flow ratio between CO2 and liquid solution is related to the equilibrium of the ternary system CO2/solvent/ solute in the saturator. Indeed, when pressure and temperature are set, the mass flow ratio between CO2 and the liquid solution fixes the operating point position with respect to the phase equilibria (VLEs) diagram of the ternary system CO2/solvent/ solute in the saturator. In general, the most efficient decompressive atomization (that enhances the efficiency of the SAA process) is obtained when the operating point falls into the onephase liquid region; i.e., all CO2 is dissolved into the starting liquid solution. To determine this condition, quantitative VLE data at high pressure on ternary systems should be known. Data on high-pressure solubilities for acetic acid-water mixtures containing SC-CO2 are available in the literature only in a limited range of temperature.26 Moreover, the possibility that the presence of solute can modify VLEs has to be taken into account; but, this information is not available in this case. Therefore, an empirical study of the effect of this parameter should be necessary. In the case of water, at relatively low pressures and temperatures, the solubility of CO2 in aqueous solutions is low; i.e., the operating point always falls into a twophase gas-liquid region. Thus, when aqueous solutions are used

Table 1. Effect of the Chitosan Concentration in the Liquid Solution and of the Temperature in the Precipitator on the Morphology of CS Particles Precipitated by SAA from 1% Acetic Acid Aqueous Solution chitosan concn (mg/mL)

temp in the precipitator (°C)

morphology

1 5

95 87 95 105 135 95 106 106 126

spherical microparticles solvent condensation spherical microparticles spherical microparticles spherical microparticles solvent condensation spherical microparticles solvent condensation spherical microparticles

10 15

as solvent, the effect of the mass flow ratio on the composition of the phases formed in the saturator is negligible; i.e., the decompressive atomization is weakly influenced by this process parameter. According to these considerations, we relied on our previous experience in this process when selecting the mass feed ratio. Indeed, in previous works we tested the influence of this parameter, concluding that a moderate excess of CO2 with respect to the expected saturation concentration should be used. A mass feed ratio (R) of 1.8 between CO2 and the liquid solution was used in all the experiments performed in this work, since it has been shown to be the most appropriate for SAA of aqueous solutions.30,32,35 According to these considerations, some tests were performed by setting the saturator operating conditions in a pressure range from 80 to 150 bar and in a temperature range between 70 and 90 °C. The best results in terms of the stability of the process and morphology of CS precipitated particles were observed operating at 105 bar and 85 °C. Therefore, these pressure and temperature conditions were used in all the SAA experiments proposed in this work. 3.2. Effect of Temperature in the Precipitator. Temperature optimization in the precipitator is required to assist droplet evaporation, minimizing the thermal stress on the treated compound. Preheated N2 at a flow rate of 0.8 (N m3)/h has been used, coupled to electrical heating of the precipitator walls, to set the precipitation vessel at the desired temperature. The effect of temperature on CS particle morphology was observed performing experiments at temperatures ranging between 87 and 135 °C. All these experiments were performed at a CS concentration in the liquid solution of 5 mg/mL; the results are summarized in Table 1. At precipitation temperatures between 90 and 135 °C, well-defined and noncoalescing spherical microparticles were obtained, as shown by SEM images reported in Figure 2. At temperatures lower than 90 °C substantially no powder was recovered on the precipitator walls and part of the liquid solvent was found at the bottom of the precipitator. The SEM image of the very small amount of powder collected in the precipitator revealed that collapsed and coalescing particles were obtained, as can be observed in Figure 2a. This result can be explained considering that these precipitation temperatures can induce a partial recondensation of the solvent on the precipitated particles, or even a nonefficient evaporation of the droplets; the liquid is then forced to pass through the stainless filter by the N2 stream and no solute is collected in the precipitator. Therefore, the subsequent experiments on CS were performed at 95 °C in the precipitator to avoid solvent condensation and to minimize the thermal stress on the precipitated microparticles. 3.3. Effect of Solute Concentration. Previous works performed on SAA showed that solute concentration in the liquid solution is a relevant parameter in the definition of particle size

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Figure 2. SEM images of chitosan microparticles precipitated by SAA from 1% acetic acid aqueous solution at different temperatures in the precipitator (R ) 1.8; Csol ) 5 mg/mL).

and particle size distribution of the precipitates.30,32-35 Indeed, an increase of solute concentration enhances viscosity and surface tension of the liquid solution, resulting in the formation of larger primary droplets and also influences the dimensions of the secondary droplets. Therefore, systematic experiments were performed operating at different CS concentrations (C) in the 1% acid acetic aqueous solution to explore the influence of this process parameter. CS concentrations between 1 and 15 mg/mL were explored. Performing the experiment at larger C, a marked increase of the boiling temperature of the solution was observed: condensation phenomena occurred in the precipitator. Therefore, higher precipitation temperatures were used as C was increased from 5 to 15 mg/mL, as summarized in Table 1. The powder recovered in the successful experiments was always formed by well-defined noncoalescing microparticles. Examples of the particles collected at different C are shown in SEM images reported in Figure 3. These images have been produced at the same enlargement (10 K); therefore, a qualitative evaluation of the increase in particle size with solute concentration is possible. Quantitative measurement of PSDs was performed by SEM image analysis and by laser scattering analysis (LS). PSDs in terms of the number of particles and the particle volume were calculated with use of the image analysis method and from LS data. PSDs obtained by image analysis showed that SAAprecipitated CS powders are formed by particles ranging between 0.1 and 11 µm, with 90% of the total volume of the powder occupied by particles smaller than 4.5 µm when precipitated at C ) 1 mg/ mL, smaller than 6.6 µm at C ) 5

Figure 3. SEM images of chitosan microparticles precipitated by SAA from 1% acetic acid aqueous solution at concentrations and precipitation temperatures of 1 mg/mL and 95 °C (a), 5 mg/mL and 95 °C (b), and 10 mg/mL and 106 °C (c), respectively.

Figure 4. PSDs in terms of the number of particle percentages of micronized chitosan at C ) 1 mg/mL and Tp ) 95 °C (0), C ) 5 mg/mL and Tp ) 95 °C (b), and C ) 10 mg/mL and Tp ) 106 °C (4).

mg/mL, and smaller than 8.5 µm at C ) 10 mg/mL, respectively. However, the SEM image technique does not allow one to verify if particles are really well-separated; therefore, LS analysis was performed. PSDs obtained by LS are reported in Figures 4 and 5 in terms of the number of particles and the particle volume, respectively, and they substantially confirm the results of image analysis. These figures show the effect of the solute concentration on the particle size: particle size increases and distribution broadens when more concentrated solutions are processed. When

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Figure 5. PSDs in terms of particle volume percentages of micronized chitosan at C ) 1 mg/mL and Tp ) 95 °C (0), C ) 5 mg/mL and Tp ) 95 °C (b), and C ) 10 mg/ mL and Tp ) 106 °C (4).

the particle number distributions are considered (Figure 4), the C effect is visible on particles smaller than 0.3 µm, whereas fairly good overlap of distributions occurs for larger particles; i.e., when less concentrated solutions are processed, the number of small particles increases, while few large particles are produced. In the case of volumetric curves (Figure 5), even a few large particles have a relevant impact on the overall distribution, because the particle volume increases as the cube of its diameter. For this reason, the C effect is more evident in volumetric distributions obtained by LS. 90% of the volume is occupied by particles with diameters smaller than 4.1 µm operating at C ) 1 mg/mL and by particles smaller than 8.1 µm at C ) 10 mg/mL, agreeing fairly well with data obtained by image analysis and confirming that no agglomeration of CS microparticles is present. In conclusion, the concentration of the solution is confirmed to be a parameter influencing CS particle size and distribution, although its effect is less marked than in previous SAA works.30,32-35 This is probably due to the relatively narrow range of concentrations examined; i.e., in processing more concentrated solutions, a more marked effect would be visible. However, as previously explained, an increase in concentration over 15 mg/mL causes a strong increase in viscosity and boiling temperature that interfere with process efficiency, due to difficulties in solution pumping and to the necessity to use higher temperatures in the precipitator. 3.4. Solid-State Characterization. Dissolution and drying procedures can influence CS solid state. In particular, a decrease in crystallinity can occur depending on the drying process; e.g., freeze-drying produces less crystalline CS than oven drying.36,37 Therefore, X-ray and DSC analyses were performed on untreated and SAA-processed CS to evaluate the effect of the SAA process on the solid state of this polymer. Diffraction patterns of untreated and SAA-processed CS precipitated at temperatures ranging between 103 and 135 °C are reported in Figure 6. X-ray analysis reveals that untreated CS has a semicrystalline structure. After SAA processing, CS powders show a lower crystallinity, and, particularly, the higher is the precipitation temperature, the larger is the amorphous content in CS microparticles. The solid state of SAA-processed CS depends on the precipitation temperature. A possible explanation can take into account a physicochemical modification of the polymer. Several authors observed that the type of crystalline structure, the degree of crystallinity, and the average crystallite size depends on the deacetylation degree of the material: the higher the deacetylation of the sample, the lower its crystallinity and the smaller the crystallite size.38-40 Indeed, dissolving CS in acid aqueous

Figure 6. X-ray diffraction patterns of untreated and SAA-processed CS at different temperatures in the precipitator (Tp).

Figure 7. DSC curves of untreated and SAA-processed CS at different temperatures in the precipitator (Tp).

solutions and vaporizing the solvent at high temperatures can cause a change in both the chemical structure and the supramolecular structure of the polymer; processes such as further deacetylation and cross-linking of the polymer can take place in the thermal treatment of CS salts.41-43 DSC thermograms on untreated and SAA-precipitated CS at different temperatures are reported in Figure 7. The endothermic peak related to the evaporation of water is expected to reflect physical and molecular changes of the polymer. Polysaccharides usually have a strong affinity for water, and in the solid state these macromolecules may have disordered structures that can be easily hydrated. The hydration properties of polysaccharides depend on their primary and supramolecular structures.44,45 As can be observed in Figure 7, the first thermal event registered in all the samples is a wide endothermic peak centered between 90 and 105 °C, due to water evaporation. Figure 7 shows some differences between SAA-precipitated samples and the untreated material in this peak area and position, indicating that these samples differ in water holding capacity and in the strength of water-polymer interaction. Micronized CS powders show larger peak areas and lower peak temperatures with respect to the raw material, i.e., higher water content and weaker water-polymer interactions, respectively. Moreover, the powder obtained at 135 °C shows a lower endothermic peak temperature and a larger area with respect to the powders precipitated at 103 and 105 °C, thus pointing out that SAA precipitation temperature affects hydration properties of CS microparticles. These results agree with the X-ray analysis, since the less ordered structure due to chemical modification may contribute significantly to the increase in the content of sorbed water. The bound water content of the samples can also depend on the structure of the polysaccharide chain, e.g., by the number of amine and carboxymethyl groups that are hydrophilic centers. The second thermal event is an exothermic peak occurring at temperatures higher than 250 °C: SAA-precipitated powders

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show the exothermic peak shifted to lower temperatures with respect to the untreated material. Also this behavior can be attributed to a decrease in thermal stability as a consequence of the decreased acetyl content and degree of polymerization. In conclusion, SAA-treated CS microparticles show a crystalline degree depending on the temperature set in the precipitator. This process parameter can be easily modulated and allows the production of CS microparticles characterized by different degrees of crystallinity, according to the selected application. This aspect can be particularly relevant, for example, in modifying drug release mechanisms in CS/drugs formulations. 4. Conclusions and Perspectives The SAA process is able to produce CS well-defined spherical microparticles with narrow PSDs. Particle size tailoring is possible by modulation of some process parameters, such as the solute concentration. The precipitation temperature influences the crystallinity of the precipitated microparticles. SAA-micronized CS microparticles are expected to be promising vehicles for drug delivery. Moreover, the positive results obtained by SAA micronization of pure chitosan introduce the possibility of direct CS-drug composite microparticles production using the SAA technique. Acknowledgment The authors thank MIUR (Italian Ministry of Scientific Research) for financial support and acknowledge Dr. R. Donatantonio for his help in performing part of the experiments proposed in this work. Literature Cited (1) Felt, O.; Buri, P.; Gurny, R. Chitosan: a unique polysaccharide for drug delivery. Drug. DeV. Ind. Pharm. 1998, 24, 979. (2) Di Martino, A.; Sittingerc, M.; Risbuda, M. V. Chitosan: A versatile biopolymer for orthopaedic tissue-engineering. Biomaterials 2005, 26, 5983. (3) Shia, Z.; Neoha, K. G.; Kanga, E. T.; Wang, W. Antibacterial and mechanical properties of bone cement impregnated with chitosan nanoparticles. Biomaterials 2006, 27, 2440. (4) Kristmundsdottir, T.; Ingvarsdottir, K.; Saemundsdottir, G. Chitosan matrix tablets: The influence of excipients on drug release. Drug. DeV. Ind. Pharm. 1995, 21, 1591. (5) Illum, L. Chitosan and its use as a pharmaceutical excipient. Pharm. Res. 1998, 15, 1326. (6) Shiraishi, S.; Arahira, M.; Imai, T.; Otagiri, M. Enhancement of dissolution rates of several drugs by low-molecular weight chitosan and alginate. Chem. Pharm. Bull. 1990, 38, 185. (7) Shimoda, J.; Onishi, H.; Machida, Y. Bioadhesive characteristics of chitosan microspheres to the mucosa of rat small intestine. Drug. DeV. Ind. Pharm. 2001, 27, 567. (8) Ahna, J. S.; Choib, H. K.; Chunb, M. K.; Ryuc, J. M., Jungc, J. H., Kimc, Y. U.; Cho, C. S. Release of triamcinolone acetonide from mucoadhesive polymer composed of chitosan and poly(acrylic acid) in vitro. Biomaterials 2002, 23, 1411. (9) Akbuga, J. Effect of the physicochemical properties of a drug on its release from chitosan malate matrix tablets. Int. J. Pharm. 1993, 100, 257. (10) Brannon-Peppas, L. Recent advances on the use of biodegradable microparticles and nanoparticles in the controlled drug delivery. Int. J. Pharm. 1995, 116, 1. (11) Chen, L.; Subirade, M. Chitosan/β-lactoglobulin core-shell nanoparticles as nutraceutical. Biomaterials 2005, 26, 6041. (12) Sinha, V. R.; Singla, A. K.; Wadhawan, S.; Kaushik, R.; Kumria, R.; Bansal, K.; Dhawan, S. Chitosan microspheres as a potential carrier for drugs. Int. J. Pharm. 2004, 274, 1. (13) Agnihotri, S. A.; Mallikarjuna, N. N.; Aminabhavi, T. M. Recent advances on chitosan-based micro- and nanoparticles in drug delivery. J. Controlled Release 2004, 100, 5. (14) Akbuga, J.; Durmaz, G. Preparation and evaluation of crosslinked chitosan microspheres containing furosemide. Int. J. Pharm. 1994, 111, 217.

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ReceiVed for reView February 23, 2006 ReVised manuscript receiVed June 6, 2006 Accepted June 10, 2006 IE060233K