Elastase activated liposomal delivery to nucleated cells

The specific activation of liposomes for delivery has been explored by enzy me mediated cleavage of a peptide substrate covalently conjugated to a fuso genic lipid. We have previously shown an elastase sensitive peptide conjuga ted to 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) could be a ctivated by enzymatic cleavage, triggering liposome-liposome lipid mixing a nd fusion with erythrocyte ghosts (Pak et al., Biochim. Biophys. Acta, 1372 (1998) 13-27). Further optimization of this system has been aimed at obtai ning substrate cleavage at or below physiological elastase levels and to de monstrate triggered delivery to living cells. Therefore a new peptide-lipid , MeO-suc-AAPV-DOPE (N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE), has been dev eloped that exhibits greater sensitivity and selectivity for elastase cleav age and subsequent conversion to DOPE. This peptide-lipid was used with DOD AP (dioleoyl dimethylammonium propane, a pH dependent cationic lipid) in a 1:1 mol ratio with the expectation that endocytosis would lead to a liposom e with an overall positive charge if enzymatic cleavage had occurred. Elast ase treated liposomes displayed pH dependent enhancement of binding, lipid mixing, and delivery of 10 000 MW dextrans, relative to untreated liposomes , when incubated with HL60 human leukemic cells. Heat denatured elastase di d not activate DODAP/MeO-suc-AAPV-DOPE liposomes, indicating enzymatic acti vity of elastase is necessary. Liposomes bound to ECV304 endothelial cells at physiological pH could be activated by elastase to deliver an encapsulat ed fluorescent probe, calcein, into the cell cytoplasm. These results sugge st enzyme substrate peptides linked to a fusogenic lipid may be used to eli cit specific delivery from liposomes to cells. (C) 1999 Elsevier Science B. V. All rights reserved.