Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12-16% that of brain microsomes. Three other lipid-synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X-100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5-4 x 10(-4) M), but the CDP-ethanolamine Km was lower for myelin (3-4 x 10(-5) M) than for microsomes (11 - 13 x 10(-5 M). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.

Modification of the surface of distearoyl phosphatidylcholine vesicles with synthetic glycolipids dramatically affects the rate of uptake of these vesicles by mouse peritoneal macrophage. The high rate of uptake of 6-aminomannose-modified vesicles is effectively inhibited by cytochalasin B and chloroquine but not by colchicine, indicating that the mechanism of vesicle uptake is phagocytosis. Other modified vesicles appear to have some effect on the rate of uptake of 6-aminomannose-modified vesicles suggesting that the various vesicle types compete for the same initial binding sites. Analysis of 6-aminomannose-modified vesicles by y-ray perturbed angular correlation spectroscopy shows that the rotational correlation time of the encapsulated "'In3' does not change when the vesicles associate with macrophage. This result is consistent with transmission electron microscopy, which indicates that the aminomannose-modified vesicles remain intact after phagocytosis as aggregates of fused and intact vesicles surrounded by a single bilayer membrane structure.There is a growing body of evidence that carbohydrate cell-sur, face determinants play a significant role in intercellular recognition processes. Examples of mammalian carbohydrate recognition systems include (i) the receptor for galactose-terminated glycoproteins found in hepatocytes (1, 2), (ii) the receptor for 6-phosphomannosyl-containing glycoproteins found in fibroblasts (3, 4), and (iii) the receptor for mannose-terminated glycoproteins found in macrophage and polymorphonuclear leukocytes of the reticuloendothelial system (5-7). The existence of these receptors suggests that it should be possible to control the tissue distribution and cellular uptake of phospholipid vesicles by attaching appropriate carbohydrate determinants to the vesicle surface (8)(9)(10)(11).The modification of the surface of distearoyl phosphatidylcholine vesicles with particular synthetic glycolipids has been shown to affect dramatically the tissue distribution and stability ofthese vesicles in mice (12,13). The use ofvesicles loaded with "'In enables the structural integrity of the vesicles to be determined in vivo by y-ray perturbed angular correlation (PAC) spectroscopy and the tissue distribution to be determined by standard gamma-counting techniques (14-16). After intravenous injection, vesicles modified with 6-aminomannose derivatives of cholesterol produced initial retention ofhigh levels of intact vesicles in the lung, followed by concentration of intact vesicles in the liver and spleen. These modified vesicles concentrated in the axillary spaces associated with aggregates of polymorphonuclear leukocytes and macrophage when administered subcutaneously.The unusual tissue distributions and long-lifetimes observed in vivo for aminomannose-modified vesicles must depend in a complex way on interactions of these vesicles with serum components and specific cell types. To establish a better basis for understanding the remarkably stereospecific mechanisms for recognition and trans...

Phospholipid vesicles have been extensively investigated because of their usefulness as models for biological membranes and their potential application as carriers for drug delivery. However, preparations of small sonicated vesicles tend to aggregate and fuse (on storage at room temperature and at 4°C), resulting in significant changes in turbidity, rate of uptake by macrophage, and proton NMR linewidths. By modification of the surface of phospholipid vesicles with charged groups such as 11-aminogalactose that extend significantly from the vesicle surface, it is possible to obtain preparations that are stable for >7 days.Phospholipid vesicles have been extensively investigated because of their usefulness as models for biological membranes and their potential application as carriers for drug delivery (1-4). We have recently found that modification of the surface of distearoyl phosphatidylcholine vesicles by specific synthetic glycolipid determinants can affect the rate of uptake of these vesicles by mouse peritoneal macrophage in vitro and the differential tissue distribution of these vesicles in vivo in mice (5-9). However, small sonicated vesicles are thermodynamically unstable, and the properties of these vesicles can change significantly in the temperature range at which phase transitions occur (10-12). In particular, small sonicated vesicles tend to aggregate and fuse below the phase transition temperature, resulting in an increase in vesicle size as a function oftime (13-16). As these changes will ultimately affect the practical usefulness of phospholipid vesicles for drug delivery, we report in this paper studies of the effect of surface modification on the aggregation and fusion of phospholipid vesicles and on the rate of uptake of these vesicles by mouse peritoneal macrophage. MATERIALS AND METHODSMaterials. L-a-Distearoyl phosphatidylcholine (Ste2PtdCho) from Calbiochem and cholesterol (Chol) from Sigma were used without further purification. Mannosyl, aminomannosyl, and aminogalactosylderivatives ofChol [6-(5-cholesten-3/3-yloxy)hexyl 1-thio-a-D-mannopyranoside (ManChol), 6-(5-cholesten-316 yloxy)hexyl 6-amino-6-deoxy-1-thio-a-D-mannopyranoside (NH2ManChol), and 6-(5-cholesten-3f3-yloxy)hexyl 6-amino-6-deoxy-l-thio-,f-D-galactopyranoside (NH2GalChol), respectively] were synthesized at Merck. [oleate-1-'4C]Cholesteryl oleate (specific activity, 51 Ci/mol; 1 Ci = 3.7 x 10'°becque-rels) was purchased from New England Nuclear.Newborn calf serum, medium-199, and penicillin/streptomycin were purchased from Microbiological Associates (Los Angeles, CA) and plastic Petri dishes (35 x 10 mm) were obtained from Falcon. D20 (99.8% D) was purchased from Aldrich.Preparation of Liposomes. Small unilamellar vesicles were prepared according to the method ofMauk and colleagues (5-9). Briefly, a lipid mixture was prepared by mixing Ste2PtdCho, Chol, NH2ManChol (or NH2GalChol), and A23, 2:0.5:0.5:0.004 (mol/mol) or as otherwise specified. The mixture was dried in vacuum overnight and then probe sonicated in phosph...

The stability and tissue distribution of lipid vesicles modified at the surface by the incorporation ofeither a galactosyl ceramide (GalCer)'or a galactosyl'cholesterol (GalChol) glycoconjugate have been studied in. mice by measuring the release of vesicle-entrapped "'1In. Although the tissue distributions of both vesicle types were similar, the GalCer-containing vesicles were markedly less stable than those. prepared with GalChol, whether administered orally or by intraperitoneal injection. Physical characterization of the vesicles in vitro suggests that the increased disruption rate for GalCer vesicles in vivo is related to. structural instabilities induced by the cerebroside, which can then result in either an increased rate of vesicle uptake by tissues or a greater susceptibility to lysis. These studies demonstrate the importance ofthe nonpolar anchoring groups in determining the fate of surface-modified vesicles in vivo.The targeting ofencapsulated agents, such as drugs or enzymes, to specific tissues is one of the goals in the development of liposomes as exogenous delivery systems. To achieve this result, a number of strategies have been attempted, including the addition ofcharged lipids to neutral vesicles (1), the covalent binding of antibodies to vesicle surfaces (2), and the use oflocalized hyperthermia to induce phase transitions in synthetic liposomes (3). The finding that mammalian hepatocyte surfaces contain a galactose-specific glycoprotein binding site (4) has led to studies on the usefulness ofcarbohydrate-modified vesicles in targeting encapsulated materials (5-7). Initial work has shown great promise (7), leading to further investigation ofvariables that can affect the system. Due to the amphiphilic nature of vesicle bilayers, it is convenient to incorporate polar carbohydrate molecules by attaching them to nonpolar lipid groups. In view' of the importance of lipid shape to vesicle structure (8), it is likely that the choice of lipid conjugate or anchoring group will, in addition to the type of carbohydrate used, affect the fate of modified vesicles in vivo. In the present communication; we report studies of carbohydrate-modified vesicles containing glycolipids differing in the structure oftheir nonpolar group. The results, comparing the effects of N-stearoyl-DL-dihydrogalactocerebroside (galactosyl ceramide, GalCer, II) with those of 6-(5-cholesten-3f-yloxy)hexyl-l-thio-(3-D-galactopyranoside (galactosyl cholesterol, GalChol, I) demonstrate the importance of the carbohydrate anchoring group in maintaining stable vesicles in vivo.

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