Biological Evaluation of Optimized Nanoparticles
Hesperetin was effectively encapsulated into poly (d,l-lactic-co-glycolic acid) nanoparticles by using experimental design methods. A seven-factor Plackett–Burman design was used in order to determine the major process parameters. A significant linear equation, which shows the effect of each process parameter on encapsulation efficiency was developed, and then the most effective factors were determined. Further investigation and optimization was carried out by applying the three-factor three-level Box–Behnken design. Significant second-order mathematical models were developed by regression analysis of the experimental data for both responses: encapsulation efficiency and nanoparticle size. The two step experimental design allowed the synthesis of the desired nanoparticle formulations with maximum encapsulation efficiency (80.5 ± 4.9%) and minimum particle size (260.2 ± 16.5 nm) at optimum process conditions: 0.5% polyvinyl alcohol (PVA) concentration, 5.13 water:organic phase ratio, and 3.59 ml min−1 flow rate of the emulsified solution into 0.1% PVA. Furthermore, the biological activity of these optimized nanoparticles were determined with antimicrobial activity and cytotoxicity studies; results were then compared to the free hesperetin. The cytotoxicity result revealed that hesperetin and hesperetin-loaded nanoparticles were biocompatible with normal cell line L929 fibroblast cells up to 184.83 and 190.88 μg ml−1 for 24 h, and up to 133.24 and 134.80 μg ml−1 for 48 h, respectively. In the antimicrobial study, the optimized nanoparticle showed inhibition activity (minimal inhibitory concentration (MIC) values were 125 μg ml−1 for Escherichia coli, and 200 μg ml−1 for Staphylococcus aureus), while the free hesperetin did not demonstrate activity in both strains (MIC value >200 μg ml−1). These in vitro results may provide useful information for the investigation of hesperetin-loaded nanoparticles in diagnostic and therapeutic applications.