-induced fusion of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles

The fusogenic properties of sulfatide-containing 1,2-dioleoyl-3sn -phosphatidylethanolamine (DOPE) small unilamellar vesicles (SUVs) in the presence of CaCl 2 were studied by mixing membrane lipids based on an assay of fluorescence resonance energy transfer (FRET). Fusion of the vesicles was also confirmed by mixing aqueous contents with the Tb/dipicolinate (DPA) assay. The half-times of lipid mixing revealed that the fusion rate decreased with increasing molar concentration of sulfatide. This inhibitory effect was more obvious at sulfatide concentrations higher than 30 mol%, where hydration at the membrane surface reached its maximum and the fusion was no longer pH-sensitive in the range of pH 6.0 – 9.0. Similar inhibitory effect was also observed in Ca 2 1 -induced fusion of DOPE/ganglioside GM 1 vesicles but at a lower concentration of the glycosphingolipid (20 mol%). In contrast, increasing the concentration of phosphatidylserine (PS) in DOPE/PS SUVs resulted in an increase in the rate of Ca 2 1 -induced lipid mixing and the pH sensitivity of this system was not affected. These results are consistent with an increasing steric hindrance to membrane fusion at higher molar concentration and larger headgroup size of the glycosphingolipids. Interestingly, the pH sensitivity of the sulfatide-containing liposomes was retained when they were allowed to fuse with synaptosomes in the absence of Ca 2 1 by a mechanism involving protein mediation. —Wu, X., and Q-T. Li. Ca 2 1 induced fusion of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles. J. Lipid Res. 1999. 40: 1254–1262. Supplementary key words pH-sensitive liposomes • fluorescence resonance energy transfer • lipid mixing • headgroup steric hindrance • membrane fusion It is well documented that pH-sensitive liposomes made of phosphatidylethanolamine (PE), and one bilayer stabilizer possessing a titratable negatively charged headgroup, fuse spontaneously with other lipid bilayers at acidic pH (for review see ref. 1). Alternatively, fusion can also be induced by the presence of physiological stimuli such as Ca 2 1 (2–5). Because endocytosis is regarded as the principal pathway by which liposomes enter cells and the pH of the lumen of endocytic vesicles is mildly acidic (6), pHsensitive liposomes have been extensively studied as vehicles for cytoplasmic delivery of drugs, enzymes, and nucleic acids (7–9). It is believed that these liposomes would be destabilized in the endocytic lumen and fuse eventually with the endosome membranes, thereby releasing the entrapped contents into the cytoplasm before they are destroyed in lysosomes (10). In previous studies, we have shown that sulfatide helps to stabilize 1,2-dioleoyl-3sn -phosphatidylethanolamine (DOPE) bilayers, and the hydration and partial dehydration of its headgroup with changing pH play an essential role in determining the pH sensitivity of DOPE/sulfatide vesicles (11). Sulfatide is also known to be able to prolong liposome circulation half-lives (12), a clear indication that clearance by the reticuloendothelial system is reduced. Furthermore, this glycosphingolipid appears to be able to assist liposomes to pass through the blood–brain barrier, implying that the sulfatide-containing liposomes may be useful for treatment of various kinds of brain diseases (13–18). It is therefore interesting to examine the effect of sulfatide on Ca 2 1 -induced fusion of DOPE/sulfatide vesicles and see whether the fusion process is modulated by pH. The results obtained from such studies should aid in the design of sulfatide-containing liposomes for drug delivery and may also help to shed some light on the general features of liposome-cell membrane interactions. It is generally accepted that membrane fusion can be separated into two distinct stages that are coupled kinetically (19, 20). The first step is dehydration of the surfaces of apposed bilayers resulting mainly from charge neutralization. The next step is destabilization of the bilayer structure by formation of nonbilayer lipid intermediates, leading to mixing of membrane lipids and aqueous conAbbreviations: DOPE, 1,2-dioleoyl-3sn -phosphatidylethanolamine; SUVs, small unilamellar vesicles; FRET, fluorescence resonance energy transfer; DPA, dipicolinate; GM 1 , galactosylN -acetylgalactosaminyl ( N -acetylneuraminyl) galactosylglucosylceramide; PS, bovine brain phosphatidylserine; PE, phosphatidylethanolamine; DOPC, 1,2-dioleoyl-3sn phosphatidylcholine; PC, phosphatidylcholine; NBD-PE, N -(7-nitro-2, 1,3-benzoxadiazol-4-yl)phosphatidylethanolamine; Rh-PE, N -(lissamine rhodamine B sulfonyl)phosphatidylethanolamine; DNS-Lys, N « -dansyll Lys; TES, N -[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; H 11 , hexagonal phase. 1 To whom correspondence should be addressed. by gest, on S etem er 0, 2017 w w w .j.org D ow nladed fom