*2.5. Statistical Analysis*

Statistical di fferences were determined using analysis of variance (ANOVA), followed by Tukey's test for comparisons between groups. The significance level was taken as 95% (*p* < 0.05). Factorial design data were analyzed using STATISTICA 7.0.

#### **3. Results and Discussion**

The lipid composition and concentration used in the preparation of NLC were chosen based on work reported by Gokce et al. [19] in which the optimal liquid lipid concentration (Miglyol 812 ®) was 15% of the whole lipid phase (Compritol ® 888 ATO and Miglyol 812 ®) [19]. Poloxamer ® 188 and Tween ® 80 were used as stabilizers of the formulations. Compritol ® 888 ATO is a solid lipid composed of glycerol tribehenate (28–32%), glycerol dibehenate (52–54%), and glycerol monobehenate (12–18%). The main fatty acid is behenic acid (C22) (>85%), but other fatty acids (C16–C20) are also present [38]. Miglyol 812 ® is a medium chain triglyceride composed mainly of caprylic (C8:0; 50–80%) and capric (C10:0; 20–50%) fatty acids with a minor level of caproic (C6:0; ≤2%), lauric (C12:0; ≤3%), and myristic (C14:0; ≤1%) fatty acids [39].

Mohammadi et al. [39] and Kovasevic et al. [40] demonstrated that Miglyol 812 ® can be used in the range of 10–60% of total lipid without a ffecting the mean particle size and the distribution of NLC.

The choice of stabilizers is also a very important step in the preparation of NLC formulations because they control the particle size and the stability, preventing their aggregation during storage [39,41]. Currently, the non-ionic surfactants Poloxamer 188 ® and Tween ® 80 are the most used for the preparation of these formulations [42]. According to Tamjidi et al. [42], the steric repulsion is the major colloidal interaction among NLC stabilized with non-ionic surfactants, yielding good stability to the variations of concentration and pH of electrolytes, and to the freeze–thaw stages. Moreover, non-ionic surfactants have lower toxicity and irritation potential than ionic ones [43].

However, NLC prepared with non-ionic surfactants may undergo a weak flocculation, as well as requiring large amounts of surfactants to cover the particle surface compared to those stabilized by electrostatic repulsion [44].

In Poloxamer ® 188, the hydrophobic polypropylene oxide chains are adsorbed onto the particle surface as the "anchor chain", while the hydrophilic polyethylene oxide chains are pulled out from the surface to the aqueous medium, thereby creating a stabilizer layer [45]. In addition, Poloxamer ® 188 exhibits low toxicity, can control release and targeted delivery applications, and is stable at high temperatures [46]. Tween ® 80 is a polyethoxylated sorbitan and oleic acid derivative that has high surface activity and low toxicity [39]. The coating with Tween ® 80 improves the stability of the lipid present in NLC by hydration in the surface layer [47,48].

In this work, we used a combination of these surfactants because they produce a layer at the interface, generating high coverage as well as adequate viscosity to improve the stability and synergism in the particle size reduction [39]. Aiming to obtain particles in the nanometer range, NLC was produced by an association of high shear homogenization (HSH) [19] and ultrasound method (US) [20]. HSH produced particles in the micrometer range (pre-emulsion) and the ultrasound method reduced the microparticles to the nanometer range.

The e ffects of the formulation variables (independent variables)-shear intensity and homogenization time on the response parameters (dependent variables)-mean particle size (PS) and polydispersity index (PDI), were evaluated using full factorial design 22 with triplicate of the

central point. For the factorial design study, a total of seven experiments were required. Zeta potential, encapsulation efficiency (EE), production yield (Y), and instability index were also measured.

Table 3 shows the influence of shear intensity and homogenization time on NLC production (RSV-loaded NLC and NLC without RSV = placebo).

The combination of HSH and US methods produced placebos (NLC) with sizes ranging between 100 nm and 260 nm, and RSV-loaded NLC (NLC-RSV) with sizes ranging between 125 nm and 190 nm.

Particle size of less than 200 nm was attributed to the efficiency of the emulsion step. Gokce et al. [49] observed that the Compritol® 888 ATO tends to return to solid form during mixing because this lipid is a mixture of mono-, di-, and triglycerides. It is known that the longer the fatty acid triglyceride, the higher the temperature needed to convert it from the solid state to liquid (melt) state. However, the presence of Miglyol® 812 helps to distribute the heat energy more homogeneously due to the high concentration of unsaturated fatty acids reducing the melting point of the system. This results in a more efficient emulsification, which in turn has an effect on the size of the particles formed. After cooling, the pre-emulsion shows smaller particles, which may result in even smaller nanoparticles [50,51]. Thus, the stability is related to the lipid composition, since NLC presents a disordered lipid matrix conferred by the presence of liquid lipid and to polysorbate surfactant (Tween® 80) used in its preparation [52]. All NLC formulations showed a PDI of above 0.2 and negative ZP around −12 mV.

The PDI has an important effect on the physical stability and uniformity (distribution) of NLC. The values should be as low as possible to ensure the long-term stability. PDI values of 0.1–0.25 show a narrow size distribution, while PDI values greater than 0.5 indicate a very broad distribution [53]. The PDI values obtained from placebo and RSV-loaded NLC above 0.2 indicated a non-monodisperse distribution with the presence of aggregated suggesting lower long-term stability. This type of distribution is usual in NLC produced using the HSH and US method, where it is very difficult to achieve a unimodal distribution of sizes [20].

ZP is also an indirect measurement of the long-term physical stability of NLC. It relates to the trend of particles to aggregate. According to Lakshimi and Kumar (2010), in electrostatically stabilized NLC, a good stability is achieved in ZP above ±30 mV, whereas in a combination of electrostatic and steric stabilization, a minimum of ZP of ±20 mV is desirable [53,54]. In addition, ZP of ±0–5 mV produces a maximum flocculation [32,55,56]. As shown in Table 3, all NLC had a negative ZP around −12 mV, indicating moderate stability regardless of RSV incorporation, suggesting that RSV did not significantly alter the ZP of the formulations (*p* > 0.05).

Besides the ZP, the long-term stability was also assessed by the instability index. The instability index is a dimensionless number between 0 (more stable) and 1 (more unstable), calculated based on the clarification at a given separation time, divided by the maximum clarification. For that, we used the LUMiSizer® equipment, which allows the measurement of the transmitted light intensity during centrifugation, as a function of time and position, over the entire sample length [57,58].

In spite of the ZP values, with the exception of the NLC-RSV2 and NLC-RSV4 formulations, the dispersion analysis indicated a good simulated physical stability of the NLC containing RSV, expressed as instability index (<0.05). This observation suggests that these particles will remain stable and have a good dispersion quality in long-term storage.

The results of encapsulation efficiency showed that a large amount of RSV (EE > 92%) was incorporated in all RSV-loaded NLC formulations, suggesting its preferential partition into lipid matrix of the nanoparticles [15]. Gokce et al. [14] also obtained the EE of 91% using the same formulation. In addition, the production yield of both placebo and RSV-loaded NLC was found to be satisfactory, with an average above 60%.

Figure 1 shows the micrographs obtained by TEM of NLC and NLC-RSV. TEM analysis confirmed the colloidal sizes of particles. NLC was almost spherical and uniform in shape with smooth surfaces, while NLC-RSV showed more amorphous shapes. No crystallization of RSV was observed on the surface of NLC-RSV. Thus, our study suggests that the lipid matrix used solidified upon cooling, but it remained in the amorphous state, helping with the accommodation of RSV in a lipid matrix [38].


*Antioxidants* **2019**, *8*, 272

**Table 3.** Influence of shear intensity and homogenization time on the production of NLC.

**Figure 1.** Micrographs obtained by TEM of (**a**) NLC4 and (**b**) NLC-RSV4. Scale bar = 500 nm.

Figure 2 shows the Pareto chart of the standardized effects and Figure 3 shows the surface response charts of experimental design for the production of placebos. As shown in Figure 2a,b, the PS and their PDI were not significantly influenced by tested parameters; neither was the interaction between variables.

**Figure 2.** Pareto charts of the standardized effects for the placebo obtained for (**a**) particle size (Z-average) and (**b**) polydispersity index (PDI).

**Figure 3.** Surface response charts of experimental design of the placebo obtained for (**a**) particle size (Z-average) and (**b**) polydispersity index (PDI).

For the mean particle size, the p-value obtained by shear intensity was −1.53564, homogenization time was −0.57553, and the interaction was 1.220829, while for the PDI, the *p*-value obtained by shear intensity was −1.04762, homogenization time was 1.079277, and the interaction was 1.456501. These parameters and their interaction were reported not to be statistically significant. However, the response surface charts of experimental design (Figure 3a,b), show that increasing the shear intensity decreases the average size and the PDI. Moreover, in Figure 3, we observed that the average PS is slightly affected by the homogenization time, while PDI is not affected.

Comparing NLC1 with NLC2 and NLC3 with NLC4, we observed two trends where the PS goes down in NLC1/NLC2and where PS goes up in NLC3/NLC4by increasing the homogenization time.

Thus, although neither variable is statistically significant when the placebos are subjected to a lower homogenization time and shear intensity, they tend to be larger, i.e., approximately 263 nm, and the PDI is >0.40. The placebo produced with shear intensity of 19,000 rpm and homogenization time of 6 min showed a smaller PS, around 105 nm.

The influence of each independent variable and their interactions on RSV-loaded NLC were also evaluated by Pareto charts (Figure 4) and surface response (Figure 5). As shown in Figure 4a,b, the PS and their PDI were not significantly influenced by tested parameters; neither was the interaction between variables. For the mean particle size, the p-value obtained by shear intensity was −1.50165, homogenization time was 0.3316068, and the interaction was −0.84409, while for the PDI, the *p*-value obtained by shear intensity was −2.85191, homogenization time was −127996, and the interaction was 1.081873. These parameters and their interaction were reported not to be statistically significant. However, the response surface charts of experimental design (Figure 5a,b), shows that increasing the shear intensity decreases the average size and the PDI. Moreover, in Figure 5, we observed that both particle size and PDI are slightly affected by the homogenization time. Thus, although neither variable is statistically significant when the RSV-loaded NLC are subjected to a smaller homogenization time and intensity shear, the PDI is >0.54. We observed that smaller particles are obtained by increasing shear intensity. However, comparing NLC-RSV1 with NLC-RSV2 and NLC-RSV3 with NLC-RSV4, we observed two trends where the PS goes up in NLC-RSV1/NLC-RSV2 and where PS goes down in NLC-RSV3/NLC-RSV4 by increasing the homogenization time.

**Figure 4.** Pareto charts of the standardized effects for RSV-loaded NLC obtained for (**a**) particle size (Z-average) and (**b**) polydispersity index (PDI).

**Figure 5.** Surface response charts of experimental design of RSV-loaded NLC obtained for (**a**) particle size (Z-average) and (**b**) polydispersity index (PDI).

The RSV-loaded NLC produced at the central point with the shear intensity of 19,000 rpm and homogenization time of 6 min showed a smaller PS, around 135 nm.

As the experimental results of PS and PDI of NLC-RSV4 were similar to the results obtained for NLC-RSV5, NLC-RSV6, and NLC-RSV7, we also used the instability index to select as optimal parameter.

Thus, based on the results of the experimental design and instability index, it was concluded that the shear rate of 19,000 rpm and the shear time of 6 min are the optimal parameters for RSV-loaded NLC production.
