Reagents and solvents
The commercial zinc oxide (ZnO) sample (DXRD = 0.22 μm) was supplied by Brasóxidos Indústria Química Ltda. (Sertãozinho-Mauá, SP, Brazil). Chitosan (CH) (75%–85% DA), sodium alginate (SA), sodium tripolyphosphate (TPP) (85% PA) and zinc acetate dehydrate (99% PA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Acetic acid (99.7% PA), sodium hydroxide (99% PA), hydrochloric acid (37% PA), hydrated potassium phosphate (P.A.) and sodium chloride were purchased from Vetec Química Ltd. (Rio de Janeiro, RJ, Brazil).
Preparation of biopolymer solutions, zinc oxide dispersion and microcapsules
The CH solution at 2% (m/v) concentration was prepared by adding 2 g CH slowly to 100 mL of acetic acid 1% (v/v), under magnetic stirring. This solution was heated for 30 min and maintained for 12 h under stirring at room temperature. The SA aqueous solution at 2% (m/v) was prepared using 2 g of SA in 100 mL water with magnetic stirring for 12 h. The TPP solution at 3% (m/v) was prepared in 100 mL ultrapure Milli-Q water (18.2 MΩ·cm at 25 °C).
A zinc acetate dihydrate 0.2 M solution (13.2 g in 500 mL water) was prepared with Milli-Q water under magnetic stirring and at room temperature in a two-necked 1000 mL flask adapted to a heating mantle and a reflux condenser. The reaction mixture was refluxed for 30 min. After cooling, a 5 M NaOH solution was added dropwise, until achieving pH 11 [11], after which refluxing was continued for 3 h. The reaction product was decanted and the supernatant solution discarded. After successive washings with Milli-Q water, the product was oven-dried at 100 °C for 24 h, characterized and used for the preparation of the microcapsules.
Commercial ZnO (250 mg) was dispersed in water (Millipore, model Direct-Q3, São Paulo, SP, Brazil) at 4 g/100 mL composition, at 10.000 rpm with a Ultra-Turrax dispersing IKA T25 equipment (IKA®, São Paulo, Brazil) for 40 min, oven-dried at 100 °C for 24 h and characterized.
ZnO nanoparticles were added to 20 mL of acetic acid 1% solution in a 50 mL beaker, and manually dispersed until no more granules were observed. Subsequently, this dispersion was homogenized in a Turrax at 10,000 rpm for 30 min and 15 mL of the CH 2% solution was added, dropwise, with the aid of a Pasteur pipette, at a Turrax stirring speed of 5000 rpm. Then, 30 mL of the SA 2% solution was added, dropwise, still under stirring at 5000 rpm, resulting in ZnO nanoparticles and both CH and SA (ZnO/CA) and ZnO microparticles with CH, SA and TPP (ZnO/CAT), in which 15 mL of a TPP solution 3% in Milli-Q water were also added to the reaction mixture. The resulting mixtures were subjected to ultrasonic radiation for 15 min (40% amplitude, 1 s/1 s intervals, in ice baths) (750 W Sonics & Materials Inc., Newtown, CT, USA) and dried at 60 °C for further characterization.
Physicochemical and morphological characterizations
The XRD curve for the samples was obtained on a Miniflex diffractometer (Rigaku Corporation, Osaka, Japan) operating at a CuKα wavelength of 1.542 Å. The samples were exposed to the X-ray beam with the X-ray generator running at 30 kV and 15 mA. Scattered radiation was detected at ambient temperature in the angular region (2θ) of 2–80° at a rate of 3°/min and a step size of 0.05°. The diffractogram was smoothed (Savitsky-Golay, polynome = 2, points = 7) and the baseline was corrected. Scherrer’s equation (Eq. (1)) was used to estimate the average size of the ZnO crystallites.
$$ {\mathrm{D}}_{\mathrm{XRD}}=\mathrm{K}\lambda /\upbeta \cos \uptheta $$
(1)
where D is the average crystallite diameter in Å, K = 0.9 is the shape factor, λ = 1.5418 Å is the wavelength of the CuKα radiation and θ is the angle of Bragg diffraction. The β value was determined by Eq. (2)
$$ {\upbeta}^2={\left({\mathrm{B}}^2-{\mathrm{b}}^2\right)}^{1/2} $$
(2)
where B is the full width at half maximum (FWHM) of the (101) reflection of ZnO and b is the FWHM of the (101) reflection of the quartz standard, which was taken directly from the software provided with the equipment.
The ZnO, ZnO/CAT and ZnO/CA samples were characterized by FTIR spectroscopy on a Perkin Elmer spectrophotometer, Frontier model (Waltham, MA, USA) using KBr disks, with accumulation of 20 scans and 2 cm−1 resolution. The KBr used to prepare transparent disks was permanently maintained in an oven at 50 °C. Samples were thoroughly dried and carefully weighed (2 mg) before grinding with 100 mg of KBr.
Rheological measurements were performed at 25 °C using a controlled AR2000 stress rheometer (TA Instruments Inc., New Castle, DE, USA), fitted with a cone-and-plate geometry (2° cone angle, 40 mm diameter, 54 μm gap). The strain sweep was measured first as the evolution of the complex modulus at 6.28 rad s−1 for the determination of the linear viscoelastic region. Subsequently, the frequency sweep (mechanical spectra) was measured from 10−1 to 102 rad s−1 (at a strain value of 1%), within the viscoelastic region.
Thermogravimetric analysis (TGA) was performed under a controlled N2 atmosphere using a Q500 thermoanalyzer equipment from TA Instruments (New Castle, DE, USA). The measurements were performed at a heating rate of 10 °C/min from room temperature to 700 °C.
Zinc contents were determined according to the standard AOAC method (2005) using a Varian AA280 atomic absorption spectrometer (Les Ulis, France). Each sample was heated at 550 °C and the ash boiled with 10 mL of 20% HCl in a beaker and then filtered into a 100 mL standard flask. All samples were analyzed in triplicate.
Scanning electron microscopy (SEM) was used to visualize the samples with a FEI Quanta™ 400 (Hillsboro, OR, USA) scanning electron microscope, at the acceleration voltage of 20 kV. The samples were vacuum-coated with gold before measurements.
The particle size distribution (PSD) of the samples was determined by laser diffraction, using the Masterisizer 2000 Malvern equipment (Malvern Instruments, Malvern, UK). The samples were dispersed in water on a Hydro 2000SM apparatus until the laser obscuration index reached 10–12%. The PSD values for the samples were determined in triplicate and were expressed as equivalent volume diameters at 10% (d10%), 50% (d50%) and 90% (d90%) of the cumulative volume, as the average of the diameter values (D4, 3) and Span. The Span values indicated the particle polydispersity, and were calculated according to Eq. (3).
$$ \mathrm{Span}={\mathrm{d}}_{90\%}-{\mathrm{d}}_{10\%}/{\mathrm{d}}_{50\%} $$
(3)
Bioactivity of ZnO complexes
Two solutions were prepared: simulated gastric juice solution (SGS) with 2 g of NaCl in 7 mL of HCl, with pH adjusted to 2.5, and simulated enteric juice solution (SES) using 6.8 g of KH2PO4 in 250 mL distilled water, adding 77 mL of 0.2 N NaOH at pH 6.8 and distilled water to complete the volume of 1 L for each [12].
The assays were performed in triplicate, in 250 mL beaker, using 175 mL of SGS or SES with 10 mg of each composite. This was added to 25 mL of the same solution into previously hydrated dialysis membranes (MEMBRA-CEL® dialysis tubing, MWCO 7000, 34 mm, 14 × 100 CLR). The systems were kept at 39 °C under gently magnetic stirring and the solutions (SGS or SES) and 15 mL aliquots were collected in different time intervals (0, 15, 30 and 45 min) and the solutions were replenished with the same volume. Aliquots were stored and submitted to AAS analysis to determine the amount of Zn 2+ released each time in the simulated media.
The minimum bacteriostatic concentration (MBC) evaluation of commercial ZnO, ZnO nano, ZnO/CAT and ZnO/CA samples was performed by the well diffusion method and by liquid growth-inhibition assay against Escherichia coli DH5α (strain 00379 INCQS-Fiocruz) and Staphylococcus aureus ATCC 6538 (INCQS-Fiocruz), Gram negative and Gram positive bacteria, respectively. The same assay was performed to investigate the antimicrobial activity of CH used in the formulation of the samples. The strains were grown in LB medium pH 7.0 (Luria-Bertani BD™) in an orbital shaker at 200 rpm, at 37 °C, for 24 h. The cellular density was adjusted in a saline solution (0.8% of NaCl) where the turbidity equivalent to McFarland 0.5 standard (1.5 × 108 CFU/mL) was used as an inoculum in the presence of the composites at increasing concentrations, at 37 °C for 18 h at 200 rpm. Subsequently, cells were serially diluted in saline solution (0.8% of NaCl), plated on solid LB, and incubated at 37 °C for 18 h. Colony-forming units were counted. The MBC was taken as the concentration at which 100% growth inhibition was observed.