The current study was conducted to present novel thermophysical data on tretinoin-loaded niosomes paired with a combination of span 60 and tween 80. Measurements were carried out to analyze the liquid mixture density and viscosity of the mentioned multilayered structures for the first time, with consideration given to the diverse molecular weights of surfactants and various stabilizers at different temperatures. Through the application of equations of state, this study has the ability to set the stage for thermodynamic modeling of solutions that involve niosomes, presenting a promising avenue for further research. So, tretinoin-loaded formulations were prepared by investigating the effects of different co-surfactants, including cholesterol or dodecanol, as well as the impact of surfactant molecular weight limited to 650.525–1090.175 g mol−1. This novel investigation was conducted to assess the superior stabilizing capabilities of dodecanol in comparison to cholesterol, with a specific emphasis on optimized vesicle size, highest incorporation efficiency, and lowest zeta potential. In particulars, the response surface methodology (RSM) was applied to optimize the operative factors and the number of experiments. The experimental evidence clearly indicates that the use of dodecanol in the manufacturing process significantly improves the stability of niosomes, while the inclusion of cholesterol leads to higher liquid mixture density and viscosity in the prepared niosomes.
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