Melatonin was obtained from Acros Organics (Geel, Belgium). Poly(methacrylic acid-co-methyl methacrylate) (Eudragit S100®) was supplied by Almapal (São Paulo, Brazil). The caprylic/capric triglyceride mixture was obtained from Brasquim (Porto Alegre, Brazil). Sorbitan monooleate (Span 80®), polysorbate 80 and triethanolamine were acquired from Delaware (Porto Alegre, Brazil). Carbopol 940® was obtained from BF Goodrich (Charlotte, NC, USA). Lactose and maltodextrin were purchase from Henrifarma (São Paulo, Brazil) and Roquette (Lestrem, France), respectively. All other chemicals and solvents were of pharmaceutical grade and used as received.
Preparation and characterization of nanocapsule suspensions
Melatonin-loaded polymeric nanocapsules (NC) were prepared (n = 3) by interfacial deposition of the preformed polymer according to the method described by Fessi and co-workers . The organic phase consisted of melatonin (0.0125 g), caprylic/capric triglyceride mixture (0.8 mL), Eudragit S100® (0.25 g), and Span 80® (0.1915 g) in acetone. This lipophilic solution was poured into a hydrophilic phase containing polysorbate 80 (0.1915 g). Acetone was removed, and the suspensions were concentrated by evaporation under reduced pressure to obtain a final volume of 25 mL (0.5 mg mL−1 of melatonin) .
The mean size and polydispersity of the nanocapsules were determined at 25 ± 2°C by photon correlation spectroscopy (Zetasizer Nano ZEN3600, Malvern, UK). Suspensions were diluted 500-fold in MilliQ® water (Millipore Co., Billerica, MA, USA). Zeta potential was measured by an eletrophoretic technique, using the same equipment. For these measurements, the suspensions were diluted (1:500) with 10 mM NaCl aqueous solution. The pH measurements were carried out directly in the samples using a Micronal B474 potentiometer (Micronal, São Paulo, Brazil).
The melatonin content of the nanocapsules was determined after their dissolution in acetonitrile and assayed by high-performance liquid chromatography (HPLC) . The system consisted of an SPD-10A Shimadzu detector, LC-10 AD Shimadzu pump, SIL-10A Shimadzu injector (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan), and Lichrospher® RP-18 column provided by Merck (Darmstadt, Germany). The mobile phase consisted of acetonitrile/water (55:45, v/v) at a flow rate of 0.7 mL min−1. Melatonin was detected at 229 nm. The encapsulation efficiency (percentage) was calculated by difference between the total content and free melatonin concentration in the nanocapsule suspension. The free melatonin concentration was determined using the ultrafiltration-centrifugation technique (Microcon 10,000 KDa, Millipore) .
Preparation and characterization of spray-dried polymeric nanocapsules
Spray-dried melatonin-loaded nanocapsules were prepared (n = 3) using an MSD® 1.0 spray dryer (Labmaq, São Paulo, Brazil). Lactose and maltodextrin at 10% (w/v) were evaluated individually as drying adjuvants. The drying adjuvant was added to the nanocapsule suspension under magnetic stirring. The stirring was maintaining for 10 min before the formulation was fed into the spray dryer. A two-fluid nozzle with a cap orifice diameter of 0.7 mm and a co-current flow was used. The inlet temperature in the drying chamber was maintained at 150 ± 10°C, and the feeding rate was set at 0.3 L/h . Powders prepared with lactose and maltodextrin were named as D-NC-L and D-NC-M, respectively.
The process yield (percentage) was calculated as the ratio between the total weight of powder recovered in the sample collector and the total dry mass of the components used. The residual moisture content (percentage) of each spray-dried product was measured by Karl Fischer titration in dry methanol (Titro Matric 1 S, Crison Instruments, Barcelona, Spain). Measurements were performed in triplicate. In order to determine the melatonin content in the spray-dried powders, samples were dispersed in methanol and kept under magnetic stirring for 10 min. After centrifugation, melatonin was assayed by HPLC according to the method described above.
The size of the spray-dried powder particles was measured by laser diffractometry (Mastersizer 2000®, Malvern, UK). Measure of the width of the distribution of particle size (SPAN) values were used, and they were calculated by (d0.9
, and d0.1
are the particle diameters determined respectively at the 90th, 50th, and 10th percentile of undersized particles. The deagglomeration profile of the dried particles was evaluated as a function of time after their dispersion in water (Hydro SM small volume sample dispersion unit, Malvern, UK). Aliquots were analyzed every 5 min for 60 min. The aliquots acquired from 0 and 60 min were also centrifuged, filtered (0.45 μm, Millipore), and analyzed by photon correlation spectroscopy (Zetasizer Nano Series ZEN3600, Malvern, UK). Furthermore, nanoparticle tracking analysis (NanoSight LM10 and NTA 2.0 Analytical Software, NanoSight, Wiltshire, UK) was also used as a tool to estimate the particle size distribution and to gain visual information. This experiment was carried out using 0.5 mL of the diluted samples (1:5,000 in milliQ water v
) introduced into the chamber by means of a syringe. The chamber was placed on the optical microscope, and the particles were illuminated by a laser diode (635-nm wavelength). The video images of the Brownian motion of the individual particles were obtained real time via CCD camera and analyzed using the NTA 2.0 Analytical Software (NanoSight, UK). In the nanoparticle tracking analysis (NTA) method, the particle sizing system is based on the scattering of light, where all visible particles in the sample and each separate light scattering center are seen as an individual particle during filming. Each video clip was captured over 120 s. The automatic detection threshold was enabled, and the maximum particle jump was set at 10 in. the NTA software. The size values obtained by NTA were used to evaluate the redispersion efficiency (RE) of the spray-dried systems. The RE was calculated using Equation 1
, where values close to one indicate a narrow redispersion of the powders
where dRP is the mean particle size of the redispersed spray-dried powder and dS is the mean particle size of the original nanocapsule suspension.
Morphological analysis of spray-dried powders was carried out by scanning electron microscopy (JEOL scanning microscope JSM-5800, Tokyo, Japan). Samples were analyzed after they had been gold sputtered (JEOL Jee 4B SVG IN, Tokyo, Japan), and the analysis was performed at the Electron Microscopy Center of the Federal University of Rio Grande de Sul State (Centro de Microscopia Eletrônica - UFRGS).
Preparation and characterization of hydrophilic gels
Hydrogels were prepared using 0.5% of Carbopol 940®, 0.2% of diazolinidyl urea, and triethanolamine. Four different formulations were prepared: (a) a hydrogel prepared by adding Carbopol 940® directly to the nanocapsule suspension (G-NC), (b) a hydrogel prepared by adding the spray-dried nanocapsules (prepared with lactose) to a preformed hydrogel (G-NC-L), (c) a hydrogel prepared by adding the spray-dried nanocapsules (prepared with maltodextrin) to a preformed hydrogel (G-NC-M), and (d) a hydrogel prepared by adding directly the melatonin dispersed in water containing polysorbate 80 at 0.77% to preformed hydrogels (G-M). All formulations were prepared at a final concentration of 0.5 mg g−1 of melatonin. Hydrogels containing redispersed spray-dried nanocapsules (G-NC-L and G-NC-M) were prepared by adding 1.57 g of powder in 10 g of each hydrogel previously prepared, reaching a final concentration of 0.5 mg g−1 of melatonin. The pH values of the gels were determined using a potentiometer (Micronal B474, São Paulo, Brazil) through the direct immersion of the electrode in semisolids (n = 3). The physical stability of the hydrogels was evaluated by multiple light scattering (Turbiscan Lab, Formulaction, L'Union, France). The samples were poured into glass cells without any dilution and analyzed using scan mode every 6 min for 20 h at room temperature.
In vitro drug release profiles
In vitro drug release profiles (n = 3) from all formulations (nanocapsule suspensions, dried nanocapsules, and hydrogels) were studied under sink conditions using a cellulose dialysis bag (MWCO = 12,000 to 14,000 Da, Sigma-Aldrich Corporation, St. Louis, MO, USA). Nanocapsule suspensions (10 mL) and the hydrogels (10 g) were transferred directly to the dialysis bags, which were placed in a beaker containing 150 mL of 5% polysorbate 80 aqueous solution (at 37°C) with slow magnetic stirring. For the spray-dried nanocapsules, the powders were previously redispersed in 10 mL of water. The initial concentration of melatonin in the dialysis bag was 0.5 mg mL−1 for all samples. Aliquots of 2 mL were withdrawn periodically and replaced with the same volume of fresh medium. The concentration of melatonin released at each time was determined by HPLC using a validated methodology previously described . Drug release profiles were analyzed by model-dependent methods (mono-exponential and bi-exponential models) using the software MicroMath Scientist® (St. Louis, MO, USA). The best model to describe the release profile was selected based on the highest model selection criterion (MSC) and the highest correlation coefficient (r), as well as the best curve fitting.
In vitro skin permeation studies
The in vitro permeation experiments were performed using Franz type diffusion cells and pig abdomen skin at 37°C. Pig skin was obtained from a local slaughterhouse. The skin was cleaned to remove the hair and adipose tissue and kept at −20°C until use. The thickness of the skin piece (1.5 to 2 mm) was measured using a micrometer (Dial Thickness Gage, 2046 S, Mitutoyo Corporation, Kanagawa, Japan). The dermal side of the porcine flank skin was exposed to the receptor fluid (5.0% (v/v) polysorbate 80 aqueous solution), and the stratum corneum was exposed to the air (non-occlusive conditions). The effective permeation area was 16 cm2, and the volume of the receiver chamber was around 50 mL.
The hydrogels were applied and weighed in the donor compartment (50 mg/cm2). The flux of melatonin from the nanocapsules through the skin was calculated by determining the drug concentration in the receptor medium at predetermined times (2, 4, 6, 8, 12, and 24 h) by liquid chromatography (as previously described). Results represent the mean of six independent replicates (n = 6). The cumulative amounts of melatonin per diffusion area were calculated for each time point and plotted versus the sampling time points. Flux corresponds to the slope calculated by the linear regression of these data points.
All analyses were carried out in triplicate. Results are expressed as the mean ± standard deviation. Parameters of the mathematical modeling (k, A, α, B, and β) and the data of in vitro melatonin permeation were statistically evaluated using one-way analysis of variance at a significance level of 5% (StatGraphics Plus 5.1, STATPOINT TECHNOLOGIES, INC., Warrenton, VA, USA).