Determination of critical micelle temperature of Pluronic® in Pluronic/gel phase liposome mixtures using steady-state anisotropy

Stealth strategies have improved some properties of drug nanocarriers, including circulation time in vivo and kinetic stability, which led to the novel-generation of stealth liposomes. Among these, Pluronic®/lipid mixtures provide one of the simplest and lowest-cost stealth liposomes, although the formation of Pluronic micelles above its critical micelle temperature (CMT) concurrently lead to undesirable effects in a drug-carrier approach. Recently, we proposed a fluorometric method to determine the CMT of Pluronics in Pluronic/fluid phase liposome mixtures, but the method fails in the presence of gel phase liposomes (Calori et al., 2019). The present study aimed to develop a simple and highly sensitive fluorometric method based on steady-state anisotropy (r) of 1,6-diphenyl-1,3,5-hexatriene (DPH) to determine CMT of Pluronics in Pluronic/gel phase liposome mixtures. From our results it was showed that r_DPH is >0.2 in gel phase liposomes, but lower than that in Pluronic micelles. As a consequence, a drastic drop in r_DPH values occurs with temperature-dependent micelle formation in Pluronic/lipid mixtures, due to the partitioning of DPH, previously loaded in liposomes, in formed micelles. The 1/r_DPH values led to a clear and elegant deflection point assumed to be CMT, as confirmed by dynamic light scattering. By combining this novel method with response surface methodology, partial phase diagrams of the boundary between liposome and liposome plus Pluronic micelle can be obtained with few assays. This method may be also useful for the optimization of other stealth liposomes for application as drug delivery systems of prolonged circulation time in vivo.