Cholesterol- and Biosurfactant-Stabilized Ethosomes for Betanin: Formulation, Encapsulation and Controlled In Vitro Release

Betanin is a hydrophilic antioxidant with limited physicochemical stability, motivating the development of vesicular carriers to improve its retention and regulate release. This study employed a systematic, stepwise formulation-screening approach to develop and characterize cholesterol-containing ethosomes, including biosurfactant-modified formulations, for betanin encapsulation. The effects of preparation conditions, surfactant type and concentration, cholesterol content, and betanin loading on colloidal properties and storage stability were evaluated. Encapsulation efficiency, loading capacity, and in vitro release were also investigated. Formulations containing 2.5% (w/w) phospholipid, 30% (w/w) ethanol, 10% (w/w) ethylene glycol, 1% (w/w) cholesterol, and 0.625% (w/w) Tween 20 or Tween 20/Mirasoft® SL L60 mixture, prepared by 3 min homogenization, showed the most favorable performance. Betanin incorporation altered vesicle organization, while encapsulation and release strongly depended on surfactant composition and betanin loading. The highest encapsulation efficiency was 87.87 ± 3.22%, whereas the maximum loading capacity was 0.0271 ± 0.0026%. Release was incomplete and composition-dependent, ranging from 5.67 to 34.49% after 90 min at pH 5.8. The limited release indicated formulation-dependent betanin retention, which may favor controlled delivery but could also restrict active-compound availability. As skin permeation and biological safety were not evaluated, the relevance of these findings for dermal delivery remains to be established. Weibull analysis adequately described the nonlinear release profiles. These findings demonstrate that biosurfactant-modified ethosomes are tunable lipid nanocarriers with composition-dependent encapsulation and in vitro release properties.