The microparticles are micrometric systems, ranging from 1 μm to 1000 μm, which have been widely studied and employed in the medical and pharmaceutical areas, particularly when they are developed with biodegradable polymers, due to its safety and biocompatibility [1] - [5] . Their main advantages are drug protection, mucoadhesion, gastroresistance, and controlled drug release, reducing the dose and frequency of drug administration, obtaining the same therapeutic effect with reduced adverse local and systemic effects and toxicity [6] [7] . Polycaprolactone (PCL) is a biodegradable polymer widely used in the development of microparticles due to its high stability, and permeability, biocompatibility with various drugs, low toxicity and low degradation rate. Its melting temperature varies from 59˚C to 64˚C, and its glass transition temperature is about −60˚C [8] - [11] . Poly(vinyl alcohol) (PVAL) is an amphiphilic semicrystalline polymer, with good interfacial adsorption capacity, and for this reason PVAL has been used in the production of emulsions and microparticles, acting as an emulsifier, to increase the physical stability of microparticles and to encapsulate different drugs. Furthermore, it has low toxicity and it is biodegradable. Its glass transition temperature is around 75˚C and its melting temperature is around 150˚C [12] - [15] .

A good deal of recent research has been devoted to the development of nanostructured materials containing inorganic fillers dispersed in a polymer matrix. However, these new nanostructured materials must be tested for safety and effectiveness. Among nanoparticles, montmorillonite clay is safe for biomedical applications, since this clay is already used in pharmaceutical preparations [16] - [18] . Bentonite is plastic clay consisting mostly of montmorillonite, a natural clay of the smectite group. It is a type 2:1 lamellar silicate (2 silicon tetrahedrons:1 aluminum octahedron) having the general formula [Mx (Alx-4Mgx) Si8O20 (OH) 4], where M is a monovalent cation and x is the isomorphic substitution degree (0.5 to 1.3). Regarding its microstructure, the lamellae have diameters between approximately 100 - 200 nm and thickness of 1 nm. This clay is used as a functional excipient in tablets due to its ability to form gels at low concentrations by swelling in water, and it is also used as a binder and disintegrant [19] [20] . For these reasons, bentonite is often employed to produce nanostructured microparticles.

There are several studies in literature that describe the development of polymer/clay system, most of them are nanocomposites. Thus, it is relevant to comprise the clay effect in the PCL and PVAL matrix. The clay dispersion in PCL matrix generally decreases the crystallinity and the crystallite size, because of the dispersed silicate layers that represent a physical barrier and hinder PCL crystal growth. A small clay dispersion, less than 5 wt%, in the PCL matrix is able to reduce its water permeability, increase its stiffness and ductility and improve its thermal stability. Moreover, PCL can exhibit a “pseudo solid-like” behavior at silicate loading greater that 3 wt%, suggesting the maintenance of long-range order domains and a clay orientation in some directions [21] . The clay dispersion in the PVAL matrix can increase the mechanical, thermal and gas barrier properties when its content ranges from 3 wt% to 10 wt%. It was also noticed that up to 5 wt% clay loading, clay particles were highly dispersed in PVAL matrix without any agglomeration. However, some agglomerated structures were formed in the polymer matrix above a 7 wt% clay concentration [22] [23] .

Nevertheless, there are no reports of the development of nanosctructured microparticles containg PCL, PVAL and clay particles. However, Dong & Feng developed nanos- ctructured nanoparticles of Poly(D, L-lactide-co-glycolide)/montmorrilonitte (PLGA/ MMT) by emulsion/solvent evaporation method. It was observed that the MMT played the role of a co-emulsifier and the nanoparticles presented a mean size of around 310 nm [24] . Dyab et al. developed core/shell hybrid organic-inorganic polymer microsph- eres, using polystyrene and laponite nanoparticles. The formed emulsion showed excellent stability against droplet coalescence and against microparticles coagulation. Generally, the number of microparticles increased and their size decreased with the content of laponite particles, ranging from 1% to 4%, used in stabilizing and it was attributed to the formation of a rigid layer of the inorganic nanoparticles around the microparticles, increasing the stability [25] .

Several methods can be applied to produce polymeric microparticles, such as: 1) Oil/ water emulsion extraction/evaporation method, where a required amount of polymer is dissolved in an organic phase which is emulsified under stirring to form an emulsion and to evaporate the organic phase; 2) Spray dryer technique, where the organic solution with dispersed polymers is sprayed through a nozzle in a spray dryer under different experimental conditions; 3) Solution-enhanced dispersion method, the microparticles were prepared by spraying a solution of polymer in mixture of carbon dioxide and organic solvent into air was termed as rapid exposition of supercritical solutions. As an alternative, the organic polymer solution could be atomized into a vessel containing pressed carbon dioxide; and 4) Hot melt technique, where polymers with low melting point were fabricated into microspheres by hot melt technique [26] [27] .

In choosing among them, simplicity, reproducibility and yield should be considered. The emulsification/solvent evaporation technique is widely used in the preparation of microparticles and allows the incorporation of hydrophilic and hydrophobic drugs. However, one of the restrain factors of this technique is the system homogenization, which is typically carried out mildly, for 3 hours at 500 rpm, to form the microparticles by the solvent’s evaporation at the interface, causing the polymer to precipitate [4] [6] . In this context, this work initially used oil/water emulsion extraction/evaporation method, and then used a high speed mixing to emulsification, in order to achieve a narrow particle size distribution. The spray drying technique has been used after the emulsification process to promote solvent removal and form microparticles, in a shorter time. This method has many advantages, such as good reproducibility, control of particle size and less dependence on the solubility of the active ingredient in the polymer. However, this technique still has limitations, especially for hydrophobic polymers with low melting point, such as PCL [28] - [31] .

The main objective of this study was to produce nanostructured microparticles of PCL/PVAL containing a commercial sodium clay (NT-25®) as the nanoparticle, using the emulsification process followed by spray drying. The second objective was to characterize the systems by using nuclear magnetic resonance, scanning electron microscopy, infrared spectroscopy, differential scanning calorimetry, X-ray diffraction and dynamic laser light scattering.

Materials

The following polymers were used to produce the microparticles:

1) polycaprolactone, obtained from Sigma Aldrich, with melt index of 1.9 ± 0.3 g/10 min (ASTM D-1238), density of 1.14 g/cm³ and numerical molar mass (Mn) of 80,000 g/mol;

2) poly(vinyl alcohol), obtained from Vetec, with hydrolysis degree of 88.3% and numerical molar mass (Mn) of 4060 g/mol; and

3) sodium clay NT-25, obtained from Bentonit União Nordeste. This clay is a natural calcium bentonite, without the presence of organic modifier, with surface area of 139 m²·g⁻¹ and cation exchange capacity (CEC) of 0.8 meq/g. It has inter-layer spacing (d 001) equal to 1.51 nm [32] .

The solvent used was chloroform P. A., obtained from Tedia Brasil. The other reagents employed were of analytic grade and were used as received.

Methods

Preparation of microparticles

In the first step, in order to make a primary emulsion 5% w/v of PCL was dissolved in chloroform, under magnetic stirring with stir bar of 2.5 cm, at 500 rpm, for 24 hours, at 25˚C, until former a clear solution. In the second step, to be used as outer aqueous phase 5% w/v of PVAL was dissolved in distilled water and stirred with a magnetic stirrer of 2.5 cm, at 500 rpm, at 60˚C until completely dissolved. Then, when the temperature was around 25˚C, the aqueous phase was poured into the organic phase, under magnetic stirring; at 800 rpm, for 30 minutes, in a flask of 500 ml, to form an initial emulsion system. The systems were prepared in triplicate and separated into three fractions. The initial magnetic stirring is still a technique applied for the development of microparticles, in the research field.

The first fraction was submitted to spray drying in an LM 1.0 MSD (mini spray dryer) (LabMaq do Brasil, São Paulo, Brazil), under the following operating conditions: feed flow of 3.3 ml/min; air flow of 500 l/h; air pressure of 3 kgf/cm², inlet temperature of 110 ± 4˚C; outlet temperature of 95 ± 5˚C; atomizer nozzle diameter of 1.0 mm; and vacuum formation rate of 0.6 m³/min. This first sample was named PCL/PVAL.

The second fraction was homogenized using an Ultra-Turrax® (UT) high-power homogenizer for 2 minutes at 16,000 rpm, at 25˚C, forming a more stable emulsion. Then this emulsion was dried using the same spray dryer, with the conditions described above. This sample was named PCL/PVAL/UT.

For the third fraction, 3% w/w of sodium NT-25 clay was added to the organic phase, followed by 48 hours of stirring, at 25 ˚C. Then, the aqueous phase was dispersed in the organic phase containing the clay, and this mixture was homogenized in the UT for 2 minutes at 16,000 rpm, forming an emulsion. This emulsion was dried using the mini spray dryer under the conditions described above. This sample was named PCL/PVAL/ NT-25/UT. The clay was not sprayed in the system that are used the magnetic agitation, because the system did not form a homogenous emulsion with magnetic stirring.

The concentration of PCL, PVAL, clay and solvents used were chosen according to previous studies in the literature, since the main objective of this work was to verify whether the proposed method was effective in developing nanostructured particles. However, it is recommended to developed stable microparticles, by the emulsion method, using around 2 to 10 wt% of polymer in the organic phase. The variation in PVA concentration will affect the stability of emulsion and the size of the microspheres. More uniform sized and small microspheres are obtained on the concentration of PVA, varying from 2.5 wt % to 5 wt% [33] .

Clay is a stabilizing, supending, adsorvent and viscosity increasing agent and the amount recommended for NT-25 clay acts as a stabilizing agent, ranging from 0.5% to 5% [18] - [20] . Besides, the PCL nanocomposites developed with NT-25 clay, ranging from 1% to 5%, showed that 3 w/w % of NT-25 achieved the best dispersion in the polymer matrix [34] .

Characterization of the microparticles

Nuclear magnetic resonance

The ¹³C NMR analyses were performed at 300 MHz with a Varian Mercury VX 300 spectrometer. The procedures used to obtain the spectra and analytic parameters are described below: For ¹³C NMR analysis, the PCL, PVAL and the microparticles were prepared in the following solutions: 100 mg of PCL in 2 ml of deuterated tetrachloroethane (TCE); 100 mg of PVAL in 2 mL of deuterated water (D₂O); and approximately 100 mg of microparticles in 2 ml of deuterated dimethyl sulfoxide (DMSO). The samples were placed in a NMR tube (10 mm in diameter) and then in a 10 mm probe.

The parameters used in the ¹³C analysis were observation frequency of 75 MHz, analysis temperature of 90˚C, 8000 accumulations, pulse width applied (90˚) equal to 23.4 µs, interval between pulses of 1 second and spectral window of 18,000 Hz. The peak areas in the microparticles’ ¹³C spectra were integrated, allowing calculation of the proportion of each polymer in the developed system [35] [36] .

Infrared spectroscopy

The FTIR analyses were performed in attenuated total reflection mode (ATR), in order to identify the main functional groups, using a Thermo Scientific Nicolet™ iS™ 10 FTIR spectrometer, in a scanning range of 4000 - 675 cm⁻¹, collection time of 25 seconds, with 128 scans and normal resolution spectrum. The reference material (calibration) used in the ATR analysis was a geranium crystal and the samples were placed on both sides of the crystal [37] [38] .

X-ray diffraction

The X-ray diffraction evaluations of the crystalline structure and dispersion of the clay in the nanoparticles were carried out at room temperature with a Rigaku Miniflex X-ray diffractometer, with emission of CuKα radiation (λ = 1.5418 Å), 40 KV and 30 mA. The diffraction patterns were collected in a scanning range of 2˚ < 2θ < 30˚, for 3 seconds at steps of 0.05˚ [37] [38] .

Differential scanning calorimetry

The miscibility between the PCL and PVAL was evaluated by DSC using a TA Instruments Q1000 V9.8 Build 296 calorimeter, operating under N₂ flow of 50 mL/min, with a heating rate of 10˚C/min, in the range from ?70˚C to 150˚C for PCL, from 25˚C to 270˚C for PVAL and ?70˚C to 260˚C for the PCL/PVAL microparticles [37] .

Scanning electron microscopy

The surface texture and shape of the microparticles were observed by SEM with a Jeol JSM-5610 LV scanning microscope. The samples were previously sputtered with gold for 20 seconds (Denton Vacuum Desk II) and the photomicrographs were obtained using voltage of 20 kV and 4000 to 8000 x magnification [11] [12] .

Dynamic laser light scattering

The average diameter of the particles was determined after their dispersion in distilled water at a concentration of 1:1000. The diameter was measured in a Brookhaven multi-angle particle sizer, with detection angle of 90˚, in a quartz cell with 1 cm optical path, and the data were integrated using the MAS OPTION software [11] [12] .

Nuclear magnetic resonance is a spectroscopy that analyzes the nuclear spin movement and behavior in the presence of a strong external magnetic field. From this spectroscopy it is allowed to analyze different nuclei in a sample, employing a specific sequence of pulses. The NMR spectroscopy is widely used to study the chemical assignments and molecular dynamics of various types of materials detected through the intermolecular interaction, dispersion and the distribution of their components [37] - [39] . The solution ¹³C NMR spectrum allows identifying the different types of carbon in samples, facilitating study of the microstructure of microparticles [40] [41] .

The ¹³C NMR solution spectra of PCL, PVAL and microparticles produced are presented in Figure 1. Analyzing the ¹³C NMR solution spectrum of the PCL, carbonyl

group presents its chemical shift located at 174 ppm, the OCH₂ group (I) at 65.2 ppm, the CH₂ group (II) at 35.2 ppm, the CH₂ group (III) at 29.5 ppm, the CH₂ group (IV) at 26.7 ppm and the CH₂ group (V) at 25.7 ppm [42] . The spectrum of the PVAL showed two signals in the one referrer to the C-OH group located at 68.4 ppm and the other one due to the CH₂ group at 45.3 ppm [43] .

The ¹³C NMR solution spectrum of the microparticles presented peaks characteristic of both PCL and PVAL. The peaks with chemical shifts at 174 ppm, 35.2 ppm, 29.5 ppm, 26.7 ppm and 25.7 ppm correspond to the carbonyl, CH₂ group (II), CH₂ group (III), CH₂ group (IV) and CH₂ group (V) of the PCL, respectively. In turn, the peaks with chemical shifts located at 68.4 ppm and 45.3 ppm correspond to the C-OH and CH₂ PVAL groups, respectively. However, the peaks corresponding to the chemical shift of the functional groups of the PCL were less intense than the peaks of the PVAL, due to the lower ratio of PCL in the microparticles.

By integrating the areas under the peaks of the microparticles in the NMR ¹³C spectra, it was possible to confirm the proportion of each polymer in the system, as reported in Table 1. The proportion of PCL in the system was about 10%, with PVAL making up the other 90%. All the samples produced presented similar results. The proportion of each polymer in the systems can be correlated with its thermal properties and with the processing method used.

DSC Measurments

PCL has a glass transition temperature (Tg) of around −70 ˚C and melting temperature (Tm) of about 55˚C while PVAL has a Tg near 75˚C and Tm of about 150˚C, as can be seen from analyzing the DSC spectra in Figure 2. In the spray drying process, the incoming air temperature was 110˚C, set at this level to promote proper dehydration of the material. PVAL has a higher melting point than the air temperature used in the drying process, so it was not in the melted state during the drying process, causing it to have lower propensity to stick together or adhere to the surfaces of the drying chamber. In contrast, PCL’s melting point is lower than this air temperature, so it was in the melted state during the drying process, causing a greater tendency to stick and form aggregates. This phenomenon explains the higher proportion of PVAL than PCL in the microparticles, as can be seen from the ¹³C spectra obtained by NMR [12] [13] .

The DSC analysis Figure 2 was used to determine the miscibility of the polymer system developed. While it was not possible to observe the Tg of the PCL due to the limitations of the DSC instrument, the Tg of the pure PVAL was about 72˚C. The micro-