Stable High-Loading of All-trans Retinoic Acid in Nanoliposomes Achieved Through Optimized pH and Antioxidant Incorporation

Background: All-trans retinoic acid (ATRA) has been shown to have an immunomodulatory activity that could be explored to develop adjuvants for vaccination against intestinal or mucosal infections. However, progress has been limited by the poor chemical stability of ATRA and the high concentrations required. This study aimed to incorporate high-dose ATRA in a previously developed nanoliposome containing Toll-like receptor agonists (TLR) - (GLA) and 3M-052, optimising the excipient composition and the physicochemical stability of ATRA. Method: Different ratios of ATRA: lipid, as well as different pH values, were explored to enable the incorporation of up to 3 mg/mL ATRA in the liposomal formulation. A hydrophobic antioxidant, α- tocopherol, was evaluated for its capacity to increase the chemical stability of ATRA within the liposomes. The particle size was determined by dynamic light scattering, and ATRA content was determined by HPLC with a diode array detector. Stability was monitored under normal and stressed conditions. Results: Maximum ATRA incorporation and nanoliposome stability occurred at low ATRA: lipid ratio, elevated pH, and with the inclusion of α-tocopherol. This study showed that the formulation of ATRA depends on the ATRA: lipid ratio, the pH of the hydrating solvent, and the incorporation of α- tocopherol, which enhances the loading efficiency of ATRA and protects the formulation against oxidation and photodegradation. Conclusion: Nevertheless, whereas the optimised liposome formulation remained suitable for TLR7/8 agonist (3M-052) stability, a significant loss in the TLR4 agonist (GLA) content occurred following manufacture, likely due to the high pH condition.