Triggerable liposomal fusion by enzyme cleavage of a novel peptide–lipid conjugate

Abstract

A novel peptide-lipid sensitive to enzyme cleavage was designed to generate liposomes that could be triggered to fuse by enzymatic activation. Covalent linkage of dioleoyl phosphatidylethanolamine (DOPE) to an elastase substrate, N-acetyl-ala-ala-, resulted in a cleavable peptide-lipid (N-Ac-AA-DOPE) with no intrinsic fusogenic activity. Cleavage of N-Ac-AA-DOPE and concomitant conversion to the fusogenic lipid DOPE could be detected after treatment with human leukocyte elastase or proteinase K, two proteases with similar substrate specificities. A strategy to utilize this cleavage to trigger fusogenicity was tested by modeling the fusion of liposomes containing the expected product of complete cleavage. Based on these results liposomes were designed to contain N-Ac-AA-DOPE, DOTAP, and PE in the ratio of 15/15/70. These liposomes exhibited lipid mixing with acceptor liposomes after elastase or proteinase K protease treatment. Activation of fusion, as monitored by a lipid mixing assay, appeared to be dependent on protease activity, as (1) heat inactivated enzyme did not activate liposomal fusion, and (2) the time and concentration dependence of proteinase K mediated cleavage of N-Ac-AA-DOPE correlated with membrane mixing. Liposomes could also be formulated that exhibited lipid mixing and transfer of aqueous fluorescent probe with erythrocyte ghosts. These observations demonstrate fusogenic lipids conjugated to enzyme substrates serve as triggerable fusion systems that may be useful for gene and drug delivery.

Lipid Carriers for Gene Therapy

“…Enzymatic cleavage by phosphatase [47] and elastase [48] have been demonstrated to be potential triggers for endosome-specific cleavage of liposome linkers. The introduction of disulphide linkers leads to cleavage under the reducing environment within the endosome or cytoplasm.…”

Nanomaterials for Optical Imaging

“…To enhance the drug release kinetics, triggered release preparations can be designed by simply incorporating environmentally sensitive motifs in the membrane bilayers. Drug release can be triggered by either exogenous or endogenous stimuli such as pH [153,154] , enzymes [155,156] , temperature [157] , and light [158,159] . Upon activation, these liposomes may undergo structural changes, subsequent loss of membrane integrity, and release (leakage) of the encapsulated contents (Figure 9.10 ).…”

Protease-Activated Delivery and Imaging Systems

“…Protease-activated liposomes have been designed to either (a) enhance liposomal fusion with tumor or tumor microenvironment cells, facilitating targeted drug delivery ( Fig. 39.2) (Pak et al 1998(Pak et al , 1999Kondo et al 2004, Terada et al 2006, or (b) become substantially destabilized upon proteolysis, resulting in drug release extracellularly (Fig. 39.3) (Hu et al 1986, Sarkar et al 2005.…”

“…Human leukocyte elastase prefers uncharged amino acid side chains, especially short sequences of Ala or Val. Initially, the peptide-lipid acetyl-Ala-Ala-[1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine] (N-Ac-AA-DOPE) was constructed for creating elastase-targeted liposomes (Pak et al 1998). Human leukocyte elastase and proteinase K both hydrolyzed N-Ac-AA-DOPE when the peptide-lipid was incorporated into dioleoyl trimethylammonium propane (DOTAP)/phosphatidylethanolamine liposomes.…”

“…Human leukocyte elastase and proteinase K both hydrolyzed N-Ac-AA-DOPE when the peptide-lipid was incorporated into dioleoyl trimethylammonium propane (DOTAP)/phosphatidylethanolamine liposomes. Liposomal fusion with red blood cells was promoted by proteolysis (Pak et al 1998). Subsequently, the peptide-lipid N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE (MeO-suc-AAPV-DOPE) was applied for liposomal targeting, as it was more readily cleaved by human leukocyte elastase than N-Ac-AA-DOPE yet was less sensitive to proteinase K (Pak et al 1999).…”