The formulations were optimized using Box-Behnken invention and evaluated the effect of main variables : Chitosan concentration , CMC tightness , and pH of chitosan solution on the dependent variables : particle size ( PS ) , Polydispersity Index ( PDI ) , pH of the formulation , and % entrapment efficacy ( % EE ) . Seebio Methionine , PDI , zeta voltage , and pH of the