Ravi K. Erukulla

Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion

The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.

Elastase activated liposomal delivery to nucleated cells

The specific activation of liposomes for delivery has been explored by enzyme mediated cleavage of a peptide substrate covalently conjugated to a fusogenic lipid. We have previously shown an elastase sensitive peptide conjugated to 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine [corrected] (DOPE) could be activated by enzymatic cleavage, triggering liposome-liposome lipid mixing and fusion with erythrocyte ghosts (Pak et al., Biochim. Biophys. Acta, 1372 (1998) 13-27). Further optimization of this system has been aimed at obtaining substrate cleavage at or below physiological elastase levels and to demonstrate triggered delivery to living cells. Therefore a new peptide-lipid, MeO-suc-AAPV-DOPE (N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE), has been developed that exhibits greater sensitivity and selectivity for elastase cleavage and subsequent conversion to DOPE. This peptide-lipid was used with DODAP (dioleoyl dimethylammonium propane, a pH dependent cationic lipid) in a 1:1 mol ratio with the expectation that endocytosis would lead to a liposome with an overall positive charge if enzymatic cleavage had occurred. Elastase treated liposomes displayed pH dependent enhancement of binding, lipid mixing, and delivery of 10000 MW dextrans, relative to untreated liposomes, when incubated with HL60 human leukemic cells. Heat denatured elastase did not activate DODAP/MeO-suc-AAPV-DOPE liposomes, indicating enzymatic activity of elastase is necessary. Liposomes bound to ECV304 endothelial cells at physiological pH could be activated by elastase to deliver an encapsulated fluorescent probe, calcein, into the cell cytoplasm. These results suggest enzyme substrate peptides linked to a fusogenic lipid may be used to elicit specific delivery from liposomes to cells.