**2. Employed methods**

challenges associate to this area are immense. They include control over the distribution in size and dispersion of the nanosize constituents, tailoring and understanding the role of interfaces between structurally or chemically dissimilar phases on bulk properties. Large scale and

**Figure 3.** (a) Rubber tree plantation, *Hevea brasiliensis* species, (b) latex collection process using the bleeding method;

An special class of composites and nanocomposites is the one formed by polymer and ceramic materials. In general, choosing a polymer as a matrix or continuous phase is interesting, since many of them have appreciable mechanical and thermal properties. Other properties are also regarded, e.g. hydrophobic/hydrophilic balance, chemical stability and bio-compatibility. The nanometric component is usually inorganic, and called dispersed phase. It can provide high mechanical and thermal stability and novel properties and functionalities that depend on component chemical nature, structure, size and crystallinity [17]. The dispersed phase provides or improves the redox properties, electronic, magnetic, density, refractive index, and others. In most cases, the main features of each of the components present in the nanocomposite is preserved or even improved and, in addition, one can obtain new properties resulting from the synergy of both components. Typical examples of polymer/ceramic nanocomposites of technological interest are formed by ceramic nanoparticles such as barium strontium titanate phase in a matrix with low dielectric loss [18] or nickel-zinc ferrite (Ni0.5Zn0.5Fe2O4 or NZF),

controlled processing of many nanomaterials has yet to be achieved.

detail: storage vessel, and (c) dry natural rubber, "Brazilian pale crepe" type.

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dispersed in a polymeric matrix such as vulcanized natural rubber (NR) [19].

When mechanical properties of composites and nanocomposites are investigated, it is seen that the main contribution comes from the polymeric matrix. However, an appropriate nanoparticle engineering and dispersion process can act amplifying, reducing or creating new features in mechanical properties of nanocomposites. Interface and interaction between nanoparticles/matrix exert a significant influence on the mechanical properties, mainly due to the reorganization of chemical bonds and physical attractions of electrostatic nature. Therefore, properties of nanoparticles such as shape, size, surface activity, crystallinity and network microstrain become relevant. Depending on nanocomposites composition, external factors such as temperature, application of electric and magnetic fields can alter and modulate their properties, expanding application options for these materials. Thus, nanocomposites can be used in intelligent membranes, new catalysts and sensors, new generations of photovoltaic and fuel cells, intelligent micro-electronics systems, micro-optical and photonic components,

In the next topics, the preparation methods used in nanoparticles synthesis will be explored. The nanoparticles mentioned are magnetic (nickel-zinc ferrite, with stoichiometry Ni0.5Zn0.5Fe2O4 (NZF)), ferroelectric (strontium potassium niobate, stoichiometry KSr2Nb5O15 (KSN)), besides magnetic and ferroelectric nanocomposites based on vulcanized natural rubber. Characterization techniques used are also covered, as well as biological testing of cell viability and against leishmaniasis.

#### **2.1. Preparation of ceramic nanoparticles**

Preparation of ceramic phases KSr2Nb5O15 (KSN) and Ni0.5Zn0.5Fe2O4 (NZF) was performed using Modified Polyol Method [22, 23]. The main advantages of this method are high chemical homogeneity, the possibility of obtaining single phase powders and the large material portion produced in a single synthesis process (10 to 100g). Chemical formula and purity of starting reagents employed in oxides synthesis are listed in Table 1.


**Table 1.** Chemical formula and purity of the materials used in the preparation of ferroelectric and paramagnetic nanoparticles (respectively, KSr2Nb5O15 and Ni0.5Zn0.5Fe2O4).

**• Description:** in a 2 L beaker, under stirring and heating, the dissolution in nitric acid of all precursor oxides was performed in proper proportion to the desired oxide stoichiometry. 50 g of niobate oxide and ferrite were prepared for each synthesis and stoichiometric calculations were based on this mass value. Upon dissolution of all starting materials, 100 ml of ethylene glycol were added. In a chapel, the temperature was raised to 180 °C using a magnetic stirrer. With the gradual increase of temperature occurred the emanation of a yellowish-brown coloured gas, due to decomposition of NO3 groups, similar to the process developed in synthesis via Pechini method [24]. After this initial process, the material generated in the beaker was placed in a chamber-type oven.


Figure 4 presents a flowchart of the steps for preparing and calcining the niobate and ferrite by modified Polyol method until to characterization stage.

**Figure 4.** Flowchart of ferroelectric KSr2Nb5O15 and paramagnetic Ni0.5Zn0.5Fe2O4 ceramic phases preparation by Modi‐ fied Polyol Method. In blue, starting reactants from phase KSr2Nb5O15 and in red, starting reactants from phase Ni0.5Zn0.5Fe2O4.

#### **2.2. Nanocomposite magnetic and ferroelectric preparation**

calculations were based on this mass value. Upon dissolution of all starting materials, 100 ml of ethylene glycol were added. In a chapel, the temperature was raised to 180 °C using a magnetic stirrer. With the gradual increase of temperature occurred the emanation of a yellowish-brown coloured gas, due to decomposition of NO3 groups, similar to the process developed in synthesis via Pechini method [24]. After this initial process, the material

**• Pre-calcining:** precursors pre-calcination was carried out in two stages, under an O2 atmosphere with a flow of 500 ml/min for the niobate phase and under a N2 atmosphere with a flow of 300 ml/min for the ferrite phase. In the first step, the temperature was increased from room temperature at a rate of 10 °C/min to 150 °C, which was held constant for 2 hours for elimination of low molecular mass molecules such as water vapor and some organic groups. In the following, keeping the same heating rate, temperature was raised to 300 °C and maintained for 1 h, in order to remove part of non-stoichiometric elements of the phase. During pre-calcination significant elimination of organic material fraction occurs, thus obtaining a black precursor powder for KSN and reddish-brown powder for NZF.

**• Calcination:** Both precursors were calcined with a final temperature of 450 °C. For niobate phase, a ten-hour threshold (600 m) was performed at 300 °C for disposal of organic wastes, and a two-hour threshold (120 m) in the final calcination temperature. A heating rate of 5 °C/min and nitrogen flow of 150 mL/min were used during heating, for avoiding sample oxidation in second phase formation. For ferrite, a three-hour threshold (180 minutes) was performed at final calcination temperature, in order to provide sufficient time for occurring of diffusional mass processes. A heating rate of 5 °C/min and air flow equal to 7 L/min were used during heating. For both phases, the cooling process was performed at a natural rate.

Figure 4 presents a flowchart of the steps for preparing and calcining the niobate and ferrite

**Figure 4.** Flowchart of ferroelectric KSr2Nb5O15 and paramagnetic Ni0.5Zn0.5Fe2O4 ceramic phases preparation by Modi‐ fied Polyol Method. In blue, starting reactants from phase KSr2Nb5O15 and in red, starting reactants from phase

generated in the beaker was placed in a chamber-type oven.

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by modified Polyol method until to characterization stage.

Ni0.5Zn0.5Fe2O4.

Magnetic and ferroelectric nanocomposites were obtained from mechanical blending of dry natural rubber, various concentrations of ceramic nanoparticles and vulcanization system. Chemical formula and purity of the starting reactants employed in preparation of vulcanized natural rubber nanocomposites are listed in Table 2.


**Table 2.** Names, chemical formula, and purity of materials used in preparation of functional nanocomposites based on vulcanized natural rubber.

Nanocomposites preparation was initiated with dry mechanical mixing of the activation system in a open chamber mixer for 20 minutes. The activation system consists of 4 phr of zinc oxide and 3 phr of stearic acid with various concentrations of nanoparticles and 100 phr of dry natural rubber. At this stage the samples are called "activated samples". These samples were stored at a temperature of 25 °C and without light exposure for 24 hours.

After the storage step, vulcanization (2 phr of sulfur) and acceleration (1 phr of 2-mercapto‐ benzothiazole) agents were added to the activated samples by using the same mixing route. At this stage the samples are termed "accelerated samples". Accelerated samples were then thermo-conformated in thicknesses equal to 200 µm, 2 mm and 6 mm in a press with a heating system at 150 °C for 8 min and 30 s, and closing uniaxial pressure equal to 2.5 MPa. Vulcani‐ zation temperature and pressure used are indicated for natural rubber [25], and the vulcani‐ zation time parameter can be determined through rheometry test [26, 27].

Two sets of vulcanized natural rubber nanocomposites were prepared. The first set (NR/KSN) with KSr2Nb5O15 ferroelectric nanoparticles, and the second set (NR/NZF) with Ni0.5Zn0.5Fe2O4 nanoparticles, both at various concentrations (1, 2, 3, 4, 5, 10, 20 and 50 phr). Figure 5 presents NR, NR/NZF and NR/KSN films and membranes with 5 phr of nanoparticles. Images of other samples with different concentrations and temperatures are visually similar and were not included in this section.

#### **2.3. Main characterizations of nanoparticles and nanocomposites**

**• XDR:** characterization by X-ray diffraction of KSN and NZF phases was performed on a Xray diffractometer with Cu-Kα radiation (λ = 1.54060 Ǻ), angular range of 5° ≤ 2θ ≤ 80°, and variation rate (or step) of 0.02°. Diffraction data were refined using the software FullProf

**Figure 5.** Thin films with a thickness of 200 µm (a, b and c) and membranes with a thickness of 2 mm (d, e and f) of NR, NR/NZF, NR/KSN and NR/KSN/NZF respectively, with 5 phr of nanoparticles.

[28]. KSN, with a bronze tungsten tetragonal structure was indexed to JCPDS-34-0108 and NZF, with a inverse spinel structure was indexed to JCPDS-08-0234 [29].


#### **2.4. Cell viability or toxicity assays**

Assays of cell viability or the nanoparticles toxicity, vulcanized natural rubber and nanocom‐ posites compared to mammalian cells were performed using "violet crystal method", as described by J. Moraes et al [30]. Mammalian cell lineage used in these experiments was of Vero cells ATCC CCL-81, originating from "American Type Culture Collection" (Manassas, VA, USA), a cell line from African green monkey *Cercopithecus aethiops (L.)* kidney. In the experiments, Vero cells were grown in culture plates of 96 wells containing nanoparticles at concentrations between 15.6 and 1000 µg/mL or nanocomposites based on vulcanized natural rubber at concentrations between 250 and 4000 µg/mL in DMEM (Dulbecco's Modified Eagle Medium) environment, supplemented with 10% serum at 37 °C in an CO2 atmosphere of 5%. Anova and Kruskal-Wallis tests were used to compare multiple normal or non-normal samples, respectively. Student´s t-tests and Mann-Whitney test were used to compare two normal or non-normal samples, respectively. The BioEstat 5.0 software package [ACHO QUE ERA LEGAL COLOCAR UMA NOTA DE RODAPÉ FALANDO SOBRE ONDE OBTER O PROGRAMA] (Belém, Brazil, 2007) was used for performing the statistical tests and for graphical representations.

After 24 and 48 hours, supernatants were removed and adhered cells were fixed and stained with crystal violet 0.2% in methanol 20%v. It is noted that tests were carried out with concen‐ trations of 150 mg/ml, concentrations significantly higher than those reported in the literature and no significant changes were observed as compared to essays up to 4000 µg/mL. Toxicity was evaluated from the absorbance of control wells containing cells in DMEM environment. Throughout the incubation period, cultures were monitored daily in inverted optical micro‐ scope. All assays were performed in triplicate and the obtained average standard deviation was less than 2%.

#### **2.4. Leishmaniasis assays**

[28]. KSN, with a bronze tungsten tetragonal structure was indexed to JCPDS-34-0108 and

**Figure 5.** Thin films with a thickness of 200 µm (a, b and c) and membranes with a thickness of 2 mm (d, e and f) of

**• TEM:** images of transmission electron microscopy of KSN and NZF nanoparticles at a temperature of 25 °C were obtained from the supernatant fraction of the dispersion, nanoparticles and methanol, deposited on a polymer film. A field-emission (FEG) micro‐ scope with tungsten filament was used; accelerating voltage between 40 and 100 kV, CCD

**• SEM:** scanning electron microscopy images of vulcanized natural rubber and nanocompo‐ sites NR/KSN and NR/NZF were performed using a microscope with field emission (FEG) and energy dispersive analysis of x-ray analysis (EDX). Images were obtained on the sample

**• AFM:** atomic force microscopy AFM/STM was used in contact mode. AFM were performed to characterize nanoparticles morphology, vulcanized natural rubber and functional nanocomposites. The public domain software Gwyddion was used to generate the threedimensional projection of the sample surface from the height mode AFM images (height).

Assays of cell viability or the nanoparticles toxicity, vulcanized natural rubber and nanocom‐ posites compared to mammalian cells were performed using "violet crystal method", as described by J. Moraes et al [30]. Mammalian cell lineage used in these experiments was of Vero cells ATCC CCL-81, originating from "American Type Culture Collection" (Manassas, VA, USA), a cell line from African green monkey *Cercopithecus aethiops (L.)* kidney. In the

NZF, with a inverse spinel structure was indexed to JCPDS-08-0234 [29].

NR, NR/NZF, NR/KSN and NR/KSN/NZF respectively, with 5 phr of nanoparticles.

422 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

chamber.

and cryogenically fractured surfaces.

**2.4. Cell viability or toxicity assays**

*In vitro* population growth kinetics: In a BHI (brain heart infusion) environment supplemented with 10%v fetal bovine serum (FBS), 2% v human urine, 100 µg/ml potassium G penicillin and 100 µg/mL of streptomycin sulfate, a sample with rectangular dimensions 10x10x2mm of vulcanized natural rubber or nanocomposites with an inoculum of five hundred thousand parasites in the promastigote form of *Leishmania braziliensis* species, ARQ-1 strains isolated from clinical cases of Santa Cruz do Rio Pardo city, São Paulo state, in 1997.

From that instant, every three hours for a week, cell counts on the supernatant portion of the colony were performed using a Neubauer chamber. With data count a curve of parasite colony development was sketched. For comparison, control colonies, i.e. without the introduction of samples or natural rubber nanocomposites were also investigated. Throughout the tests, the temperature was maintained between 27 and 32 ºC, and hydrogen potential (pH) between 6.0 and 6.9. All assays were performed in triplicate and the average standard deviation obtained was less than 1%.
