Multilamellar protein-lipid hybrid nanovesicles for In vivo protein delivery

Abstract

Proteins are highly potent and specific with a low level of side effects compared to chemical drugs, hence taking up a very large proportion of the global pharmaceutical market. However, the short in vivo half-life and inefficient delivery imposes limitations on proteins being used in a wide range of applications. The stability of proteins has been improved by chemical modification and encapsulation into lipid vesicles or polymeric nanocapsules. Despite these efforts, proteins undergo insufficient delivery effects due to instability and low encapsulation efficiency according to harsh experimental conditions (high temperature, high pressure, use of organic solvents, etc.). In this study, a new strategy was established to self-assemble unilamellar lipid vesicles into multilamellar protein-lipid hybrid complexes by protein-mediated physical attraction, allowing proteins to maintain their stability and have high loading efficiency. The interaction between proteins and lipid membranes was identified to confirm the structure and formation mechanism of the complex, the structural, chemical, and biological stability of the protein in the complex, and its applicability. First, the protein-lipid complex was self-assembled with a combination of various proteins (anionic: epidermal growth factor (EGF), superoxide dismutase (SOD), ovalbumin (OVA), human serum albumin (HSA)

cationic: insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF)) and lipid (cationic: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)

anionic: 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG)) vesicles. The particle size, zeta potential, and structures at various proteins-lipids weight ratios were compared. Proteins and liposomes were simply mixed with mild conditions to prepare multilamellar protein-lipid vesicles (MPLVs). MPLVs generally exhibited increasing mean diameter and neutralizing the zeta potential by electrostatic attraction interaction between proteins and liposomes. Proteins generally had a loading efficiency of > 90 % in the complex and excellent long-term dispersion stability for 100 days. The optimal weight ratios for the complex between proteins and liposomes were different because of the different size and charge distribution of proteins. Second, the structure, assembly mechanism, and skin permeability of EGF-induced protein-lipid complexes were analyzed. EGF was structurally and chemically stable in EGF-DOTAP MPLVs, showing that activity was maintained for 100 days. The surface-side lipid molecules of cationic liposomes physically rearranged by EGF induced the engulfing-folding mechanism between liposomes, resulting in a multilamellar structure. EGF-DOTAP MPLVs increased the depth of penetration of EGF into the skin epidermal layer by about three than when only EGF was used. Third, an effective vaccine for antigen activity was developed by coating the surface of MPLVs prepared using OVA with an adjuvant. The particle surface charge was neutralized by coating the complex surface with negatively charged immune adjuvants, monophosphoryl lipid A (MPLA), for application in vivo. To confirm the vaccine effect of immune adjuvant coated-OVA-DOTAP MPLVs, antigen uptake, surface markers on cells, cytokines, and antibodies were analyzed using bone-marrow dendritic cells (BMDCs), spleen, and serum from mice. Through this study, the protein-lipid multilamellar structure with stabilization and high loading efficiency of proteins was designed in a very simple method using protein-mediated physical lipid membrane interfacial engineering without chemical bonding and harsh conditions. Researches in this Ph.D. thesis highlight that the self-assembled protein-lipid hybrid complex is a very stable and efficient carrier for protein in vivo delivery.