Synthesis and Preparation of Chitosan/Clay Microspheres: Effect of Process Parameters and Clay Type

This work aimed to prepare chitosan/clay microspheres, by the precipitation method, for use in drug carrier systems. The influence of the process parameters, particularly two airflows of the drag system (2.5 and 10 L·min−1) on the microspheres physical dimensions and properties, such as microstructure, degree of swelling and porosity were evaluated. The samples were characterized by optical microscopy (OM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). Water absorption and porosity tests were also performed. The results showed that the process parameters affected the size of the microspheres. The diameter, volume and surface area of the chitosan/clay microspheres decreased when they were prepared with the higher airflow of the drag system. The microspheres presented a porous microstructure, being the pore size, percentage of porosity and degree of swelling affected not only by the process parameters but also by the type of clay. Hybrids (chitosan/clay) with intercalated morphology were obtained and the hybrid prepared with montmorillonite clay at higher airflows of the drag system presented the greatest interlayer spacing and a more disordered morphology. Thus, it is certain that the chitosan/clay nanocomposite microspheres prepared with montmorillonite (CL clay) at higher airflows of the drag system can have good drug-controlled release properties.


Introduction
Chitosan is a biopolymer, derived by the deacetylation of chitin, which consists of D-glucosamine and N-acetyl-D-glucosamine units and is frequently used as excipient in pharmaceutical dosage forms [1][2][3][4]. Chitosan has been widely used since it exhibits a number of unique properties such as biocompatibility, biodegradability and non-toxicity [5]. It is a weak base with pKa in the range of 6.2-7.0, being insoluble in water at neutral and alkaline pH. However, chitosan is soluble at acidic pH, since the amino groups are protonated. Importantly, positively charged chitosan can interact with polyanions and form crosslinked matrices [6][7][8][9]. Furthermore, positively charged chitosan can However, the preparation of chitosan/clay microspheres using this inexpensive apparatus, has not been studied yet. Therefore, the objective of this work was to prepare chitosan/clay microspheres, by the precipitation method, using an inexpensive apparatus, aiming at the use in drug carrier systems. The influence of the process parameter (airflow in the drag system) and the clay type on the dimensions and physical properties of the microspheres, such as microstructure, degree of swelling and porosity, was evaluated.

Materials
Medium molecular weight chitosan from shrimp shells, Mv = 114 kDa determined by viscometry [39] and degree of deacetylation DD = 92 % determined by infrared spectroscopy method [40] was supplied by Polymar-Fortaleza/CE-Brazil (Fortaleza, Brazil). Sodium containing natural montmorillonite take-off with the trade name of Cloisite Na + (CL) was provided by Southern Clay Products Inc. (Gonzales, TX, USA) and Argel bentonite clay (AG) was supplied by Bentonit União Nordeste-São Paulo/SP-Brazil (São Paulo, Brazil). The CL clay presents a cation exchange capacity (CEC) of 92.6 meq/100 g, given by the supplier and the AG clay of 92 meq/100 g, as determined by our research group in an earlier study [41]. These clays were used without any further purification. Glacial acetic acid and sodium hydroxide were provided by Vetec Química-Brazil (Duque de Caxias, Brazil) and sodium acetate was supplied by Nuclear-Brazil (Angra dos Reis, Brazil). All aqueous solutions were prepared using distilled water and all reagents and solvents were analytical grade and used as received.

Preparation of Chitosan/Clay Microspheres
Chitosan/clay microspheres were prepared by the precipitation method, employing experimental equipment developed in our laboratory [38] (Figure 1). Briefly, chitosan solution (4% w/v) was prepared using an aqueous solution of acetic acid (5% v/v) containing 4% of sodium acetate under magnetic stirring at room temperature (23 ± 2 • C) for 2 h. After this time, the pH of the solution was adjusted to 4.9, with the addition of a 1 M NaOH solution and then a clay suspension, previously prepared, was slowly added to chitosan solution in appropriate amount to reach a final clay concentration of 10 wt.% (based on chitosan weight). The chitosan/clay mixtures were maintained under mechanical stirring at 600 rpm and 50 ± 2 • C for 4 h and 30 min. Afterwards, the chitosan/cay mixtures were added dropwise, through a drip system constructed from polymeric pipe and a 0.45 mm diameter nozzle, into a gently stirred coagulation liquid (aqueous solution of NaOH, 8% v/v). The effects of manufacturing parameters on the characteristics of the resulting microspheres were studied by setting the injection flow rate at 0.150 mL·min −1 and the airflow rate of the drag system at 2.5 and 10.0 L·min −1 . The formed microspheres suspension was filtered and washed with distilled water until neutrality and then dried in an oven at 50 • C for 24 h. Moreover, from time to time, the reactions of same sets of parameters were duplicated and the reproducibility was found to be excellent. Chitosan microspheres were manufactured by the same method and used for comparison purposes. studied by setting the injection flow rate at 0.150 mL⋅min −1 and the airflow rate of the drag system at 2.5 and 10.0 L⋅min −1 . The formed microspheres suspension was filtered and washed with distilled water until neutrality and then dried in an oven at 50 °C for 24 h. Moreover, from time to time, the reactions of same sets of parameters were duplicated and the reproducibility was found to be excellent. Chitosan microspheres were manufactured by the same method and used for comparison purposes.

Determination of the Dimensions of Chitosan/Clay Microspheres
Optical microscopy (OM) of the chitosan and chitosan/clay microspheres was conducted in a microscope model Q734ZT series 059 from DP Scientific Instruments (Cambridge, MA, USA). The micrographs obtained were used to determine the diameter and volume of the prepared microspheres. To this end, a small amount of dried microspheres (about 10 microspheres) was placed on a clean glass slide. The slide containing the microspheres was allocated to the base of the microscope and the images obtained were analyzed using the software Pixcavator 6.0.

Microspheres Morphology
The surface topography of the microspheres was examined under a scanning electron microscope (Shimadzu SSY-550, Tokyo, Japan). A small amount of dry microspheres, at least 10, was placed on aluminum stubs and made electrically conductive by coating with a thin layer of gold. A scanning electron photomicrograph was taken at the acceleration voltage of 30 kV, chamber pressure of 0.6 mm Hg. The images obtained were analyzed using the software Gwyddion 2.50.
X-ray diffraction (XRD) analysis of the chitosan and chitosan/clay microspheres, prepared with 10% by weight of clay, were conducted in Shimadzu XRD-7000 apparatus (Shimadzu, Tokyo, Japan), using copper Kα radiation (1.5418 Å), in a range of 2θ between 2 and 70 degrees, voltage of 40 kV, current 30 mA and speed of 1 • /min. The basal spacing (d001) value of the pristine clay was determined by Bragg's law, according to Equation (1) [42]. With this technique, it is possible to confirm the intercalation of the chitosan in the clay galleries by the expansion of basal spacing this one and to investigate if a microcomposite or nanocomposite was produced.
where θ is the measured diffraction angle in degree.

Water Absorption
Water absorption was determined according to Depan, et al. [43]. Briefly, the chitosan and dry chitosan/clay microspheres of known mass were immersed in distilled water at 25 • C for 1 day (24 h). After this time, the microspheres were removed, quickly placed on absorbent paper, to remove surface water and then weighed. The water absorption percentage (A) of these samples was calculated using the Equation (2): where m and m 0 are the masses of the wet and dry samples, respectively.

Optical Microscopy
The images of chitosan and chitosan/clay microspheres, obtained by OM, prepared with an injection flow rate of 0.150 mL·min −1 and airflows of 2.5 and 10 L·min −1 are present in Figure 2. These images were analyzed using the software Pixcavator 6.0 and the diameter and volume values were determined. The results obtained are in Table 1.  It is evidenced that the airflow rate of the drag system (2.5 and 10 L·min −1 ) affected the dimensions of the prepared microspheres. The diameter and volume of all microspheres (CS, CS/10%CL and CS/10%AG) decreased when they were manufactured with the higher airflow of the drag system (Table 1). According to Barbosa,et al. [38], the air rate flowing parallel to the needle is the main responsible for the drop drag, preventing their growth. Thus, the higher the airflow rate the larger the drag force, preventing the growth of droplet, resulting in a microsphere with smaller diameter.
The incorporation of the CL and AG clays within chitosan also affected the size of the microspheres. This may be explained on the basis that an increase in viscosity of the chitosan solution with the addition of clays prevents the growth of droplet, resulting in a microsphere with smaller size. The lower dimensions were exhibited by microspheres formed from CS/10%CL as compared to those from CS/10%AG, in the same injection condition. The reason for this is believed to be a consequence of the higher viscosity of the chitosan solution prepared with CL clay due the higher electrostatic interaction between CS and CL, corroborate with WAXD data. The viscosity of the chitosan solution and chitosan/clay suspensions was determined at pH 4.9 with a Brookfield-02 rheometer (Braseq Brasileira de Equipamentos LtdaAv, Jarinu, Brazil) operating at 50 rpm and 20 °C  It is evidenced that the airflow rate of the drag system (2.5 and 10 L·min −1 ) affected the dimensions of the prepared microspheres. The diameter and volume of all microspheres (CS, CS/10%CL and CS/10%AG) decreased when they were manufactured with the higher airflow of the drag system (Table 1). According to Barbosa,et al. [38], the air rate flowing parallel to the needle is the main responsible for the drop drag, preventing their growth. Thus, the higher the airflow rate the larger the drag force, preventing the growth of droplet, resulting in a microsphere with smaller diameter.
The incorporation of the CL and AG clays within chitosan also affected the size of the microspheres. This may be explained on the basis that an increase in viscosity of the chitosan solution with the addition of clays prevents the growth of droplet, resulting in a microsphere with smaller size. The lower dimensions were exhibited by microspheres formed from CS/10%CL as compared to those from CS/10%AG, in the same injection condition. The reason for this is believed to be a consequence of the higher viscosity of the chitosan solution prepared with CL clay due the higher electrostatic interaction between CS and CL, corroborate with WAXD data. The viscosity of the chitosan solution and chitosan/clay suspensions was determined at pH 4.9 with a Brookfield-02 rheometer (Braseq Brasileira de Equipamentos LtdaAv, Jarinu, Brazil) operating at 50 rpm and 20 • C for 10 min. The viscosity measurements were carried out every 30 s; thus, 20 viscosity readings were done for each sample. The viscosity data were evaluated using analysis of variance (ANOVA) and the significance of the model was verified with the F test. In the significant models, the averages were compared with Tukey's test, with a significance level of 95% (p < 0.05) using Sisvar 5.6. According to statistical data, the viscosity of CS solution, CS/10%AG suspension and CS/10%CL suspension were, respectively, 264, 316 and 344 cP. Thus, it is possible that the entrapment efficiency of drugs in CS/10%CL microspheres is greater since an increase in viscosity of the chitosan solution may prevents drug crystals from leaving the droplet. In addition, the increase in viscosity of the chitosan solution, which influences the diffusion of the drug as well as erosion of the microspheres, may result in slowest sustained release [31]. Thus, this sample (CS/10%CL-10), being small in size, have large surface to volume ratios and can be used as a potential carrier for prolonged delivery of drugs, macromolecules and targeted drug delivery. Figure 3 shows the surface morphology of the chitosan and chitosan/clay microspheres, prepared with 2.5 and 10 L·min −1 airflows, at the 50× and 2000× magnifications. All microspheres presented a drop shape with small deformations at the break point; nevertheless, the chitosan/clay microspheres are more elongated, especially that prepared with AG clay (CS/10%AG). In addition, under higher magnification, differences are observed in the morphologies of the microspheres. Chitosan microspheres showed distributed nodular domains (in the form of crystals) along the microspheres, probably due to the presence of agglomerates of polymer chains resulting from the interaction in aqueous medium as reported by Orrego and Valencia [44] in a similar study. Chitosan/clay microspheres presents surfaces free of nodular domains, with a rather rough smooth surface. The dimensions of microspheres decreased when they were manufactured with the higher airflow of the drag system corroborated with OM data. The incorporation of the CL and AG clays within chitosan also affected the size of the microspheres. Compared to chitosan microspheres, the size of chitosan/CL microspheres decreased but the one of chitosan/AG slightly increased. CS/10%CL microspheres is greater since an increase in viscosity of the chitosan solution may prevents drug crystals from leaving the droplet. In addition, the increase in viscosity of the chitosan solution, which influences the diffusion of the drug as well as erosion of the microspheres, may result in slowest sustained release [31]. Thus, this sample (CS/10%CL-10), being small in size, have large surface to volume ratios and can be used as a potential carrier for prolonged delivery of drugs, macromolecules and targeted drug delivery. Figure 3 shows the surface morphology of the chitosan and chitosan/clay microspheres, prepared with 2.5 and 10 L·min −1 airflows, at the 50× and 2000× magnifications. All microspheres presented a drop shape with small deformations at the break point; nevertheless, the chitosan/clay microspheres are more elongated, especially that prepared with AG clay (CS/10%AG). In addition, under higher magnification, differences are observed in the morphologies of the microspheres. Chitosan microspheres showed distributed nodular domains (in the form of crystals) along the microspheres, probably due to the presence of agglomerates of polymer chains resulting from the interaction in aqueous medium as reported by Orrego and Valencia [44] in a similar study. Chitosan/clay microspheres presents surfaces free of nodular domains, with a rather rough smooth surface. The dimensions of microspheres decreased when they were manufactured with the higher airflow of the drag system corroborated with OM data. The incorporation of the CL and AG clays within chitosan also affected the size of the microspheres. Compared to chitosan microspheres, the size of chitosan/CL microspheres decreased but the one of chitosan/AG slightly increased. For all microspheres, voids or porous are observed and by using the Gwyddion 2.50 software, it was possible to calculate the average pore size of the microspheres; the data are in Table 2. The airflow rate of the drag system (2.5 and 10 L·min −1 ) affected the average pore size of the chitosan and chitosan/CL microspheres. The higher airflow rate resulted in the higher mean pore size. On the other For all microspheres, voids or porous are observed and by using the Gwyddion 2.50 software, it was possible to calculate the average pore size of the microspheres; the data are in Table 2. The airflow rate of the drag system (2.5 and 10 L·min −1 ) affected the average pore size of the chitosan and chitosan/CL microspheres. The higher airflow rate resulted in the higher mean pore size. On the other hand, this property not change for chitosan/AG microspheres. The incorporation of the CL and AG clays within chitosan also affected the average pore size of the microspheres. In the same injection condition, chitosan/clay microspheres exhibited the smallest pore size, especially the ones prepared with AG clay. The topography images, with which it was possible to calculate the data shown in Table 2, using the Gwyddion 2.50 software, of the chitosan and chitosan/clay microspheres ( Figure 4) present several peaks that indicate rough and porous morphology of the samples. The darkest and largest peaks refer to the highest roughness. According to these images it is evidenced that the microspheres prepared with CL clay using the higher airflow rate (CS/10%CL-10) presented a more homogeneous morphology relative to pore distribution and surface roughness. Thus, this composition may be more effective for use as controlled drug delivery system. As described by Van de Belt, et al. [45], the total amounts of drug released increase linearly with bulk porosity, pointing to a diffusion mechanism, indicating that the kinetics of drug release are initially controlled to some extent by surface phenomena, while the sustained release over a time span of several days depends on the penetration depth as determined by the bulk porosity of the material. Therefore, by adjusting surface roughness and bulk porosity of microspheres, one can control both the initial as well as the sustained release of the drugs.
The water absorption data of the chitosan and chitosan/clay microspheres prepared at the airflow velocities of the 2.5 and 10 L·min −1 drag system is shown in Figure 5. The analyses were performed in triplicate and the results of this physical property was calculated as the average of three independent experiments and subjected to statistical analysis. The results were evaluated using analysis of variance (ANOVA) with Tukey's test (p < 0.05) using Sisvar 5.6 and the statistical data are in Table 3. The water absorption data of the chitosan and chitosan/clay microspheres prepared at the airflow velocities of the 2.5 and 10 L·min −1 drag system is shown in Figures 5. The analyses were performed in triplicate and the results of this physical property was calculated as the average of three independent experiments and subjected to statistical analysis. The results were evaluated using analysis of variance (ANOVA) with Tukey's test (p < 0.05) using Sisvar 5.6 and the statistical data are in Table 3.    It was found that the airflow velocities and clay type influenced on water absorption of the microspheres prepared ( Figure 5, Table 3). According to statistical data (Table 3), the chitosan microspheres manufactured with CL clay at higher airflow velocities of drag system (CS/10%CL-10) exhibited the higher water absorption (p < 0.05) ( Table 3). In the same injection condition (10 L·min −1 ), the water absorption of the microspheres decreased in the order CS/10%CL-10 > CS/10%AG-10 > CS-10 (Table 3). This can be due the hydrophilic nature of the clays. Our results are in agreement with result of Lavorgna, et al. [46] that showed chitosan/clay nanocomposite had more water sorption compared to neat chitosan film. Thus, it can be concluded that CS/10%CL-10 microspheres are more indicated to sustained release seeing that this kind of release requires the penetration of dissolution fluids into the interconnecting pores and cracks, which is dictated by the wettability of the polymer matrices and by the number and sizes of the pores in the polymer matrix [45]. Figure 6 shows the wide-angle XRD patterns (WAXD) of chitosan (CS), montmorillonite clay (CL), bentonite clay (AG) and chitosan/clay hybrids (CS/10%CL-10 and CS/10%AG-10). The X-ray diffraction pattern of chitosan (CS) showed a broad band of low intensity between 8-12 • , typical of semi-crystalline material, corroborating with the data obtained by Baskar and Kumar [47] and Luo, et al. [48]. As described by Wan, et al. [49], this peak (2θ = 10 • ) corresponds to the crystalline form I of the chitosan structure, this form is orthorhombic and has a unit cell with a = 7.76 Å, b = 10.91 Å and c = 10.30 Å, which is related to the diffraction plane (100). Chitosan also shows a peak in the 20 • . These peaks (10 • and 20 • ) are typical fingerprints of chitosan related to hydrated and anhydrous crystals, respectively [50]. Montmorillonite and bentonite clays (CL and AG), presented reflection peaks (001) with approximately 2θ at 5.90° and 6.02°, corresponding to a basal interplanar distance (d001) of 1.49 nm and 1.47 nm, respectively, characteristic of montmorillonite sodium [51]. After the incorporation of CL and AG within CS, the (001) peak of CL and AG moves to lower angles from 2θ = 5.90° to 2θ = 2.81° and from 2θ = 6.02° to 2θ = 5.47°, corresponding to a d001 value of 3.14 nm and 1.61 nm for Montmorillonite and bentonite clays (CL and AG), presented reflection peaks (001) with approximately 2θ at 5.90 • and 6.02 • , corresponding to a basal interplanar distance (d 001 ) of 1.49 nm and 1.47 nm, respectively, characteristic of montmorillonite sodium [51]. After the incorporation of CL and AG within CS, the (001) peak of CL and AG moves to lower angles from 2θ = 5.90 • to 2θ = 2.81 • and from 2θ = 6.02 • to 2θ = 5.47 • , corresponding to a d 001 value of 3.14 nm and 1.61 nm for CS/10%CL-10 and CS/10%AG-10, respectively. The increase in the interlayer distance for these nanocomposites was calculated from its corresponding d 001 . Considering the thickness of the montmorillonite layer of 0.96 nm [42], an increase of 2.18 and 0.65 nm in the interlayer spacing for the CS/10%CL-10 and CS/10%AG-10, respectively, was found, indicating the intercalation of bilayers of CS into the interlayers of CL and AG clays and the formation of an intercalated nanostructure. The higher value presented by CS/10%CL-10, suggests that the interlayers of almost all of CL clay were intercalated with bilayers of CS as described by Tan, et al. [52]. The intercalation of bilayer chitosan into the montmorillonite was also obtained by Darder, et al. [53], for chitosan to clay ratios of 5:1 and 10:1. This interaction was possibly favored by the electrostatic interaction of the second layer (−NH + 3 ) with the acetate ions of the chitosan solution, which allowed the access to the sites for anion exchange [26].
Due to the fact that clays have different compositions, the clay type also influenced the morphology of the hybrids. The hybrids containing CL clay (CS/10%CL-10) presented the greatest interlayer spacing and a more disordered morphology when compared to hybrids containing AG clay. As a result, it can be assumed that CL clay interacted more strongly with chitosan than AG clay. Since it is generally accepted that electrostatic attractive forces could improve the controlled-drug release properties [54][55][56] and a longer path is required for drug molecules to diffuse out of the more disordered morphology microspheres, it is possible that the CS/10%CL-10 microspheres, may exhibit the subsequent slowest sustained release [57,58].

Conclusions
Microspheres of chitosan and chitosan/clay were obtained by the precipitation method. The process parameters and the type of clay affected their dimensions (diameter, volume and surface area). Regarding the water absorption and porosity, the clay type had great influence, being montmorillonite (CL clay) responsible for the highest values. In general, the microspheres presented a porous microstructure, being the pore size, percentage of porosity and degree of swelling affected not only by the process parameters but also by the clay type. Chitosan/clay hybrids with intercalated morphology were obtained and the CS/10%CL-10 hybrid presented the greatest interlayer spacing and a more disordered morphology. Thus, it is certain that the chitosan/clay hybrid microspheres prepared with montmorillonite (CL clay) at the airflow velocities of 10 L·min −1 drag system can have good drug-controlled release properties. Furthermore, CL clay can provide mucoadhesive and toxin adsorption capabilities, which further suggest strongly that chitosan/CL clay nanocomposite microspheres are quite suitable to be applied in drug-delivery systems in comparison with pure chitosan microspheres. In summary, chitosan-clay nanocomposite is a versatile polymer nanocomposite for biomedical applications, including tissue engineering and controlled drug delivery.