*2.4. Formulation of SLN Using Fractional Factorial Screening Design*

SLNs containing clarithromycin, lipid, surfactant, and cosurfactant were formulated by high-speed stirring and the ultra-sonication method described in the literature [26]. High speed mixing generally produces an emulsion, which on cooling forms SLNs. Based on the solubility study of drug in lipid, the amounts of lipid used for the screening design were 150 mg and 200 mg, and the concentrations of surfactants selected were 1 and 3%.

Preliminary studies were carried out using different surfactant ratios (surfactant to cosurfactant; 1:9, 3:7, 5:5, 7:3, and 9:1) and evaluated for percent entrapment efficiency and percent drug loading. The selection of the surfactant ratio was based on the target set for percent entrapment efficiency (>85%) and for percent drug loading (>30%).

Screening designs were used to screen the main significant effects from various potential factors that can affect the formulation. Fractional factorial design is one type of screening design. It was applied for screening of all the selected factors utilized for the development of a clarithromycin-loaded SLN formulation. Based on the number of factors, the total number of experimental runs was calculated as sixteen by the equation, 2*n*−<sup>1</sup> , where *n* = number of factors. Selected independent variables included for the screening method were process-related factors, such as homogenization speed and sonication time, in addition to formulation variables like amount of lipid, surfactant concentration, and surfactant ratio. The dependent variables investigated were particle size (R1), entrapment efficiency (R2), and drug loading (R3). The coded and actual values of independent variables for fractional factorial screening design are shown in Table 1.


**Table 1.** Coded and actual values of independent variables for fractional factorial design.

In brief, an accurately weighed quantity of stearic acid and clarithromycin were solubilized in ethanol by heating on a water bath at 65–70 ◦C, while surfactant and cosurfactant were dissolved in aqueous phase. SLNs were obtained by slowly adding the molten organic phase drop wise into the aqueous phase under continuous stirring using a homogenizer (Remi Motors, Mumbai, India) set at respective speeds for screening design batches, as shown in Table 2. The precipitated SLN-containing dispersions were subjected to further mixing in cold water using a probe sonicator (Brookfield Engineering Laboratories, MA, USA). The SLNs formed were washed with distilled water and filtered. Table 2 shows the coded and actual values for 16 batches of fractional factorial design.
