Artificial Liposomes With Internal Docking Site for Protein Delivery

ABSTRACT In this study, we developed a liposomal system incorporating two distinct pH‐responsive polymers to enhance the efficiency of protein delivery. Two pH‐responsive polymers were synthesized by conjugating deoxycholic acid (DOCA) and pH‐sensitive moieties [2,3‐dimethylmaleic anhydride (DMA) or 3‐(diethylamino)propylamine (DEAP)] to hyaluronic acid (HA), resulting in HA conjugates with DOCA and DMA (HDOC‐DMA) and with DOCA and DEAP (HDOC‐DEAP). Liposomes were fabricated using the thin‐film hydration technique with hydrogenated soy phosphatidylcholine (HSPC), HDOC‐DEAP, and HDOC‐DMA. Lysozyme (Lyz) was encapsulated within the liposomes using the extrusion method, followed by the addition of hyaluronidase (HAase) to remove HA or HA conjugates from the outer surface of the liposomes. Meanwhile, the incorporation of HDOC‐DMA and HDOC‐DEAP within the liposomal hydrophilic core and hydrophobic shell facilitated electrostatic interactions between the negatively charged DMA and the positively charged Lyz. This interaction ensured the stable immobilization of Lyz within the liposomal core, leading to the formation of the final formulation, (Lyz/HDOC‐DMA/HDOC‐DEAP)@Lipo. Our experimental results demonstrated that (Lyz/HDOC‐DMA/HDOC‐DEAP)@Lipo significantly enhanced Lyz encapsulation efficiency within the liposomal interior. At pH 6.5, the hydrolysis of DMA (inducing electrostatic repulsion between aminated HA and Lyz) and the protonation of DEAP (destabilizing the liposomal structure) collectively facilitated accelerated Lyz release. This system effectively suppresses passive diffusion of the encapsulated protein at pH 7.4 while promoting targeted protein release in a mildly acidic environment, providing a highly efficient platform for protein.