3.5.2. Part 2: Optimization of Instrumental Parameters by Box–Behnken Design (BBD)

The second part of the optimization aimed to study the effect of different CPPs in the CQAs or dependent responses that were evaluated in the first part of the optimization related to the selection of the most suitable concentrations of formulation components. The rivastigmine-loaded NLC formulation was produced by ultrasound technique and HPH, and the tested instrumental parameters were the emulsification speed (rpm), amplitude of sonication, and number of HPH cycles.

The emulsification time and speed are important parameters in obtaining small nanoparticles with a narrow PDI [11,46]. Thus, the emulsification time was set to 5 min and the effect of increasing the rpm on CQAs was evaluated. The time and amplitude of sonication are also important parameters in producing small NLCs. Generally, as the time and amplitude of sonication increase, particle size decreases [11,51]. However, it has been reported that a high amplitude of sonication increases the lipid nanoparticle size due to the formation of aggregates [11,46]. Therefore, the effect of increasing the amplitude of sonication on CQAs was evaluated.

Regarding HPH, the pressure was kept constant at 1750 bar, which allows the reduction of the particle size due to the generated cavitation forces. However, to obtain small and uniform nanoparticles, several homogenization cycles should be performed [61,76–79]. Thus, the effect of increasing the number of HPH cycles on CQAs was evaluated. The emulsification speed, sonication amplitude, and number of HPH cycles were the independent variables that were studied at low (−1), medium (0), and high levels (+1). The DoE showing the different combinations of the tested CPPs is presented in Table 2. For these studies, 9 experimental runs were performed for each of the 6 selected formulations to evaluate the effect of each independent variable on the particle size, PDI, ZP, and EE.


**Table 2.** Design of experiment (DoE) using Box–Behnken design (BBD) to optimize rivastigmine-loaded nanostructured lipid carriers (NLC) formulations using different combinations of critical process parameters (CPPs).
