DETERMINATION OF SPECIFIC SIZE OF SUPERCRITICAL CO2 FORMED SOLID LIPID MICROPARTICLES BY COMPUTER IMAGE ANALYSIS

Supercritical CO2 technologies allow the formation of solvent-free solid lipid particles, which are efficient carrier systems of bioactive lipophilic compounds. These particles commonly have a sponge-like morphology composed of merged-solid-units whose size is difficult to measure objectively. This research aimed at the application of computer image analysis in the determination of specific size of merged-solid-units in sponge-like supercritical CO2 formed solid lipid microparticles. For this, Brazil nut oil-loaded beeswax particles (1:1 w/w) formed by rapid expansion through a nozzle (203.2 µm) after one hour in contact with supercritical CO2 (300 bar and 60 °C) were used as a model. Scanning electron micrographs of the particles were captured at different magnifications (500, 1.0k and 2.0k) to assess its influence on the size measurements. The image analysis was performed in triplicate, for each magnification, using tools of FIJI software such as “make binary” for background elimination, “adjustable watershed” for division of merged-solid-units, and “analyze particles” for areas measurement. Finally, areas were converted to equivalent diameters and expressed as normalized size distributions for adjustment to Rosim-Ramler function and subsequent calculation of descriptors such as 10 percentile (D10), 50 percentile (D50), 90 percentile (D90) and mean diameter of De Brouckere (D[4,3]). The size distributions were successfully adjusted by Rosim-Ramler function (R2>99%) and their descriptors had a good repetitiveness (coefficient of variation<10%) in each magnification tested. The use of fewer magnifications increased the value of the size descriptors (r<-0.8, p<0.01), which could be understood as effect of binarization error. Thus, the highest magnification (2.0k) is recommended to measure specific size of merged-solid-units of D[4,3]= 6.9±0.6 µm (D10=2.7±0.1 µm, D50=5.1±0.3 µm, D90=7.7±0.7 µm, detected solid-units=129±9). Specific size was determined by image analysis in a simple and objective way, which could help to understand the formation mechanism of solid lipid particles in supercritical CO2.