Experimental research on delayed responses in cochlear microphonics records in guinea pigs

The stability (with respect to encapsulated carboxyfluorescein release) of liposomes made from various fluorocarbon 1,2-or 1,3-di-O-alkylglycerophosphocholines (ether-connected) and their membrane permeability have been investigated in buffer and in human serum. Membranes and liposomes, whether formulated with fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon, 1,2- or 1,3-di-O-alkylglycerophospholipids, display lower permeability coefficients and are able to retain more efficiently encapsulated CF, even when incubated in human serum, than any of their conventional counterparts. These fluorinated liposomes are as stable as the first generation of liposomes formulated with their fluorocarbon ester-connected 1,2-di-O-acylglycerophosphocholine analogs. These results further confirm that a fluorinated intramembranar layer reduces the permeability of membranes (more significantly when they are in a fluid state), protects them from the destabilizing effects of serum components and increases even the stability of the fluorinated liposomes whose membranes are in the gel state when incubated in human serum. The impact of the modular structure of the fluorinated phospholipids (number of fluorocarbon chains, ether vs. ester bond, 1,2- vs. 1,3-isomer, etc...) and structure/permeability/ stability relationships are also presented.     

Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes

Synthetic coat protein complex I (COPI)-coated vesicles form spontaneously from large ( approximately 300 nm in diameter), chemically defined liposomes incubated with coatomer, Arf1p, and guanosine 5'-[gamma-thio]triphosphate. Coated vesicles are 40-70 nm in diameter, approximately the size of COPI vesicles formed from native membranes. The formation of COPI-coated buds and vesicles and the binding of Arf1p to donor liposomes depends on guanosine 5'-[gamma-thio]triphosphate. In contrast to the behavior of the COPII coat, coatomer binds to liposomes containing a variety of charged or neutral phospholipids. However, the formation of COPI buds and vesicles is stimulated by acidic phospholipids. In the absence of Arf1p, coatomer binds to liposomes containing dioleoylphosphatidic acid as a sole acidic phospholipid to form large coated surfaces without forming COPI-coated buds or vesicles. We conclude that Arf1p-GTP and coatomer comprise the minimum apparatus necessary to create a COPI-coated vesicle.     

Characterization of 5-fluorouracil loaded liposomes prepared by reverse-phase evaporation or freezing-thawing extrusion methods: study of drug release

Entrapment of the anti-tumoral drug 5-fluorouracil (5-FU) in unilamellar liposomes prepared by freeze-thawing extrusion technique (FATVET) and the reverse-phase evaporation method (REV) from natural (bovine brain) sphingomyelin (SM) and synthetic distearoylphosphatidylcholine (DSPC) phospholipids was studied. Reverse-phase evaporation vesicles obtained from DSPC sized through polycarbonate membranes of 0.2 micron pore size were found to entrap roughly double amounts of drug than did extruded liposomes (0.1 micron pore size); however, s-REV in these preparations were more heterogenous in vesicle size than FATVET. The rate of in vitro drug release from the liposomes was found to be dependent of the bilayer composition and the method used to prepare the vesicles. The permeability coefficient P obtained was approx. 10(-11) m/s. The results suggest that 5-FU release is kinetically controlled by an interfacial process seemingly dependent on the surface activity of the drug. Also, the physical state of the bilayer determines the retention capacity of the vesicles. Thus, liposomes consisting of distearoylphosphatidylcholine whose acyl chains were in a gel state at the working temperature (37 degrees C) retained 70% of encapsulated 5-FU after 1 h, whereas liposomes composed of natural bovine brain sphingomyelin retained only 15% over the same period.     

Protein-catalyzed exchange of phosphatidylcholine between sonicated liposomes and multilamellar vesicles

Phospholipid exchange protein from beef heart or beef liver does not catalyze the transfer of phosphatidylcholine from multilamellar vesicles of phosphatidylcholine. Certain combinations of phospholipids, however, do yield multilamellar vesicles that will exchange phosphatidylcholine with liposomes in the presence of exchange protein. Multilamellar vesicles of phosphatidylcholine:phosphatidylethanolamine:cardiolipin (70:25:5, mol%) can be used in place of mitochondria or erythrocyte ghosts as an improved acceptor particle in the study of liposome structure with phospholipid exchange proteins. These multilamellar vesicles act as a well-defined reservoir of unlabeled phosphatidylcholine with 7% exchangable phospholipid. When the distribution of phosphatidylcholine in liposomes is studied by the exchange protein technique, results can be influence by the choice of phospholipid acceptor particle. With mitochondria as acceptor particle, the percentage of phosphatidylcholine in the outer monolayer of a liposome appears to be 60%, whereas a value of 70% is obtained when multilamellar vesicles are the acceptor. The discrepancy can be explained by a heterogeneity in liposomes prepared by sonication. A size-dependent fusion or adsorption process occurs between liposomes and mitochondria; the very small liposomal vesicles, obtained by gel filtration, combine nearly quantitatively with the natural membrane. This phenomenon is not seen with multilamellar vesicles. Thus by using multilamellar vesicles one obtains a less biased estimate of phospholipid distribution between inner and outer layers of liposomes.     

A test of some predictions from the personality assessment system

Bovine brain gangliosides incorporated into phospholipid liposomes provide receptors for wheat germ agglutinin. Purified monosialogangliosides were mixed with egg phosphatidylcholine, and unilamellar liposomes were generated. Addition of wheat germ agglutinin induced the liposomes to fuse, and gel filtration analysis revealed that the lectin was incorporated into the fused liposomes. The fusion process was studied by following the changes in the 90 degrees light scattering. Increasing the proportion of the monosialoganglioside in the liposomes was found to increase both the extent of the lectin-induced liposome fusion and the rate of the reaction; below a threshold of approx. 5 mol%, the process was extremely slow. The increase in light scattering could be prevented by the addition of the hapten inhibitor, N-acetyl-D-glucosamine (1 mM). Addition of the inhibitor, subsequent to the lectin, caused a partial decrease in light scattering due to the dissociation of unfused vesicle aggregates. Electron microscopic examination revealed that the ganglioside-containing liposomes were vesicles, 244 +/- 25 A (S.D.) in diameter. Upon addition of wheat germ agglutinin, the vesicles appeared to fuse to form larger vesicles, corresponding to dimers and trimers of the initial vesicles. Inhibition studies with a variety of monosaccharides indicated that the sialic acid moieties present in the ganglioside acted as the lectin-receptor sites. This was confirmed by the observation that wheat germ agglutinin did not interact with phosphatidylcholine vesicles containing desialyated ganglioside.     

The use of chlorazepate dipotassium (Tranxene) in the states of restlessness and agitation]

Calcitonin-loading was studied in liposomes composed of phosphatidylcholine, cholesterol and stearylamine in relation to the vesicle preparation method. Liposomes entrapping calcitonin were prepared by extrusion, sonication or from mixed micelles through the elimination of cholate by gel filtration. To understand the mode of calcitonin encapsulation in the vesicles, riboflavin was entrapped within the vesicles and taken as a simple model for the encapsulation of molecules in the aqueous phase. Interactions of calcitonin with the liposomal membranes were evaluated by studying the fixation of radiolabelled calcitonin to the outer surface of empty liposomes, and by preparing calcitonin-loaded LDL-like nanoparticles composed of phosphatidylcholine and cholesteryloleate. Calcitonin entrapment in the vesicles depends largely on the vesicle preparation method. When vesicles are prepared by removal of cholate from mixed micelles, relatively little calcitonin entrapment in the liposomes is obtained. In this type of vesicle, calcitonin is exclusively embedded in the vesicle bilayer. When vesicles are prepared by extrusion or sonication, calcitonin is found both in the aqueous and lipidic phases of the vesicles. Optimal calcitonin encapsulation was obtained when the liposomes were prepared by sonication.     

Transmembrane pH-driven Na+ permeability of fluorinated phospholipid-based membranes

The permeability to the H+/Na+ exchange of fluorinated phospholipid-based membranes has been evaluated by measuring the dissipation rate of a liposomal transmembrane pH gradient in the presence of Na+. The fluorinated liposomes are made from fluorocarbon/hydrocarbon or fluorocarbon/fluorocarbon double-chain ether-connected glycerophosphocholines or amido-connected phosphocholines deriving from diaminopropanol or serine. The fluorocarbon/hydrocarbon mixed-chain phospholipids, as compared to the fluorocarbon/fluorocarbon ones, form membranes that are substantially more able to maintain a transmembrane pH gradient in the presence of NA+ and display a lower Na+ permeability. However, these membranes are more permeable to the H+/Na/ exchange than conventional DSPC (1,2-distearoylphosphatidylcholine) ones. Our results indicate a detrimental impact of the membrane fluorination degree on H+/Na+ permeability: the lower the fluorination degree of the membrane, the lower its H+/Na+ permeability. Concerning structure/permeability relationships, it appears that the replacement of the ester connecting bond in their fluorinated phosphatidylcholine analogues for an ether or amide one lowers the transmembrane H+/Na+ exchange.     

Highly fluorinated amphiphiles and colloidal systems, and their applications in the biomedical field. A contribution

Fluorocarbons and fluorocarbon moieties are uniquely characterized by very strong intramolecular bonds and very weak intermolecular interactions. This results in a combination of exceptional thermal, chemical and biological inertness, low surface tension, high fluidity, excellent spreading characteristics, low solubility in water, and high gas dissolving capacities, which are the basis for innovative applications in the biomedical field. Perfluoroalkyl chains are larger and more rigid than their hydrogenated counterparts. They are considerably more hydrophobic, and are lipophobic as well. A large variety of well-defined, modular fluorinated surfactants whose polar head groups consist of polyols, sugars, sugar phosphates, amino acids, amine oxides, phosphocholine, phosphatidylcholine, etc, has recently been synthesized. Fluorinated surfactants are significantly more surface active than their hydrocarbon counterparts, both in terms of effectiveness and of efficiency. Despite this, they are less hemolytic and less detergent. Fluorosurfactants appear unable to extract membrane proteins. Fluorinated chains confer to surfactants a powerful driving force for collecting and organizing at interfaces. As compared to non-fluorinated analogs, fluorosurfactants have also a much stronger capacity to self-aggregate into discrete molecular assemblies when dispersed in water and other solvents. Even very short, single-chain fluorinated amphiphiles can form highly stable, heat-sterilizable vesicles, without the need for supplementary associative interactions. Sturdy microtubules were obtained from non-chiral, non-hydrogen bonding single-chain fluorosurfactants. Fluorinated amphiphiles can be used to engineer a variety of colloidal systems and manipulate their morphology, structure and properties. Stable fluorinated films, membranes and vesicles can also be prepared from combinations of standard surfactants with fluorocarbon/hydrocarbon diblock molecules. In bilayer membranes made from fluorinated amphiphiles the fluorinated tails segregate to form an internal teflon-like hydrophobic and lipophobic film that increases the stability of the membrane and reduces its permeability. This fluorinated film can also influence the behavior of fluorinated vesicles in a biological milieu. For example, it can affect the in vivo recognition and fate of particles, or the enzymatic hydrolysis of phospholipid components. Major applications of fluorocarbons currently in advanced clinical trials include injectable emulsions for delivering oxygen to tissues at risk of hypoxia; a neat fluorocarbon for treatment of acute respiratory failure by liquid ventilation; and gaseous fluorocarbon-stabilized microbubbles for use as contrast agents for ultrasound imaging. Fluorosurfactants also allow the preparation of a range of stable direct and reverse emulsions, microemulsions, multiple emulsions, and gels, some of which may include fluorocarbon and hydrocarbon and aqueous phases simultaneously. Highly fluorinated systems have potential for the delivery of drugs, prodrugs, vaccines, genes, markers, contrast agents and other materials.     

Functional characterisation of tachykinin receptors mediating ion transport in porcine jejunum

Reconstructed human skin was prepared from human keratinoblasts. After 1 week of cultivation at the air-liquid interface a stratified layer developed, similar to native human epidermis. Liposomes with an average diameter of 50 nm, made of phosphatidylcholine (PC), phosphatidylserine (PS) and human stratum corneum lipids (hSCL) were applied on top of this culture system. The rate of penetration through the reconstructed human epidermis was 1.38, 0.55 and 0.013 ng lipidh-1cm-2 for PC, hSCL and PS liposomes, respectively. Electron microscopy and confocal laser scanning microscopy showed that PS and hSCL liposomes aggregated at the skin surface, while PC liposomes remained homogeneously dispersed. Fluorescence measurements demonstrated that vesicles, made of native human stratum corneum lipids rapidly mixed with PS liposomes, weakly with hSCL liposomes and did not mix with PC liposomes.     

Liposomes as immunoadjuvants and vaccine carriers: antigen entrapment

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microns in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 27% of the spores used are associated with generated giant liposomes of similar mean size.     

Comparison of polymerically stabilized PEG-grafted liposomes and physically adsorbed carboxymethylchitin and carboxymethyl/glycolchitin liposomes for biological applications

The stabilities of two types of polymerically stabilized liposomes consisting of PEG-grafted (DSPC:CHOL:DSPE-PEG1900, 5:4:1) and physically adsorbed carboxymethylchitin (CMC) and carboxymethyl/glycolchitin (CO) are compared. The polyelectrolyte is adsorbed on positive (DSPC:CHOL:DMTAP, 5:4:1) and neutral (DSPC:CHOL, 1:1) liposomes at different molecular weights (Mw). In PBS buffer (c(s) = 154 mM, pH = 7.4) the theoretical stability ratios (W) calculated using the classical DLVO Theory, indicate that the CMC-coated vesicles and the negative liposomes (DSPC:CHOL:DMPG, 5:4:1) are highly stable (W > 1) compared to the PEG-grafted (W = 0.9511) and CO-coated (W = 0.9550) liposomes. Meanwhile, experimentally determined values of W, prove that the PEG-grafted is the most stable suspension (W = 5.5). Computation of the theoretical values of W for liposome-red blood cell and liposome-macrophage indicates that the electrosterically stabilized suspensions and the negative liposomes are stable. Light scattering results show that the flocculation of liposomes in blood and plasma depends on polymer molecular weight, type of polyelectrolyte and surface charge of the uncoated liposome. Neutral liposomes coated with CMC of Mw = 1.01 x 10(5) and negative liposomes provide a more effective barrier to plasma macromolecular protein adsorption than the grafted PEG groups and are easy to resuspend in blood.     

Tacrolimus therapy for persistent or recurrent acute rejection after lung transplantation

Purified native F1 antigen from Yersinia pestis was used to assess controlled-release vaccine delivery systems in poly(lactide-co-glycolide) (PLG) microparticles and liposomes. Antigen encapsulated in PLG microparticles induced high serum titres when injected i.p. in mice: mucosal IgA was also detected. Mice immunized with F1 in Alhydrogel or PLGs were protected against subcutaneous challenge with Y. pestis. F1 antigen surface-labelled onto liposome vesicles stimulated high serum titres in Balb/c mice and also induced a mucosal response: F1-labelled liposomes protected mice against challenge with up to 1 x 10(5) organisms. These findings indicate that a significant immune response is induced by immunizing with F1 formulated in PLGs and liposomes and that protection was achieved after only one dose.     

Efficacy and safety of long-term ciprofloxacin in the management of severe bronchiectasis

We describe an 'in vitro' assay which allows rapid quantification of the binding of biotinated-vesicles to streptavidin immobilised on microtitre plates by estimating levels of a liposome encapsulated fluorescent molecule, rhodamine 123. It is shown that optimal vesicle binding to streptavidin occurs when a six carbon biotin spacer arm derivative of distearoylphosphatidylethanolamine (biotin-X-DSPE) is incorporated in liposomes. This alleviates steric hindrance arising due to the inclusion of small amounts of large bulky amphiphiles such as monosialoganglioside (GM1, 5 mol%) in vesicles. In contrast the ability of liposomes containing poly(ethylene glycol) derivatives of DSPE (PEG2000-DSPE, 5 mol%) to bind streptavidin was only marginally better when biotin-X-DSPE was substituted for biotin-DSPE in vesicles. It is further shown that amounts of biotinated-vesicles bound to streptavidin were minimally influenced by the fluidity of the liposome preparation when assayed at 4 degrees C. However, at elevated temperatures (37 degrees C) lipid estimates as determined by vesicle entrapped rhodamine 123 were low due to leakage of this marker from vesicles. This was shown by comparing amounts of biotinated-liposomes bound to streptavidin coated plates using the lipid marker [3H]cholesteryl hexadecyl ether to estimates determined by vesicle entrapped rhodamine 123. The 'in vitro' assay protocol described here is a general method applicable in the optimisation of other targeting protocols. In conclusion our work suggests that liposomes containing GM1 and the spacer arm derivative biotin-X-DSPE bind optimally to immobilised streptavidin which should aid in the use of biotinated-liposomes in 'in vivo' targeted delivery applications.     

Effect of serum components from different species on destabilizing hydrogenated phosphatidylcholine-based liposomes

It is accepted that some serum components play important roles in enhancing liposome permeability and in facilitating rapid liposome uptake by the mononuclear phagocytic system. In this study we systematically investigated the influence of serum components from different species on complement-mediated immune damage to hydrogenated phosphatidylcholine (HEPC)-based liposomes. Our results demonstrated that when liposomes were incubated with fresh serum from rats or bovines, there was obvious leakage of 5(6)-carboxyfluorescein (CF) from the liposome. However, when liposomes were incubated with fresh serum from humans, rabbits, guinea pigs, mice, and dogs, almost no pronounced leakage from the liposome was observed. These results indicate that the variability of damage to a liposome corresponds to the variability of the animal species from which the serum comes. In addition, leakage of CF from liposomes was completely inhibited by heating at 56 degrees C for 30 min or by treatment with EDTA. However, such leakage was not blocked by treatment with EGTA/Mg2+, suggesting that the mechanism of lysis of liposomes is due to complement activation via the alternative pathway rather than via the classical pathway. Studies on reconstitution and compatibility further confirm that some serum factors (complement activating factors, CAFs) induce the activation of the complement system, ultimately leading to the lysis of the liposomes. However, CAF from different animal species exhibited corresponding species differences. Meanwhile, under the condition of heating and dialysis experiments, it is obvious that the CAF is susceptible to heat and the dialysed serum sustains biological activity to destabilize liposome following dialysis against a buffer with Ca2+ and Mg2+, indicating that the CAF is not a type of low-molecular weight material but a serum protein.     

Formation of unilamellar liposomes from total polar lipid extracts of methanogens

Unilamellar liposomes were formed by controlled detergent dialysis of mixed micelles consisting of acetone-insoluble total polar lipids extracted from various methanogens and the detergent n-octyl-beta-D-glucopyranoside. The final liposome populations were studied by dynamic light scattering and electron microscopy. Unilamellar liposomes with mean diameters smaller than 100 nm were obtained with lipid extracts of Methanococcus voltae, Methanosarcina mazei, Methanosaeta concilii, and Methanococcus jannaschii (grown at 50 degrees C), whereas larger (greater than 100-nm) unilamellar liposomes were obtained with lipid extracts of M. jannaschii grown at 65 degrees C. These liposomes were shown to be closed intact vesicles capable of retaining entrapped [14C]sucrose for extended periods of time. With the exception of Methanospirillum hungatei liposomes, all size distributions of the different liposome populations were fairly homogeneous.     

The comparison of early fluid therapy in extensive flame burns between inhalation and noninhalation injuries

Dopamine hydrochloride bearing positively charged small liposomes was prepared by sonicating the multilamellar vesicles. These vesicles were characterized for their physical attributes (shape, size, charge, drug entrapment efficiency, and drug leakage). The drug release kinetics from the liposomes were also studied and found to be Fickian-type diffusion. In vivo performance of the drug-entrapped liposomes was assessed by periodic measurement of drug- (chlorpromazine) induced catatonia in Sprague-Dowley rats. These results were compared with the plain dopamine HCl and levodopa preparations as well with the marketed formulation of levodopa containing carbidopa (Syndopa). These studies revealed that the dopamine can be effectively delivered to the brain by incorporating it into liposomes, and its degradation in circulation can also be protected. The results of liposomal formulation were found to be superior compared to plain levodopa as well as Syndopa.     

Effect of membrane surface potential on the uptake and the inhibition of cationic compounds in rat intestinal brush-border membrane vesicles and liposomes

The effect of membrane surface potential on the uptake of tryptamine, an organic cation, by rat intestinal brush-border membrane vesicles was investigated. In the presence of an inside-negative K(+)-diffusion potential, the manner of initial uptake of tryptamine appeared to be pH-dependent and the uptake in the acidic medium was lower than that in the neutral medium. Changes in surface potential of brush-border membrane vesicles were monitored using 8-anilino-1-naphthalenesulfonic acid (ANS) and the results suggested that the membrane surface potential (negative charge on the membrane surface) decreased in the acidic medium. A good correlation was observed between the K(+)-diffusion potential-dependent uptake of tryptamine and membrane surface potential monitored by ANS at various pH levels. The uptake of tryptamine by liposomes (large unilamellar vesicles), which contained various amounts of dipalmitoylphosphatidylserine (DPPS), was also examined. The uptake of tryptamine decreased with a decrease of DPPS content in the liposomes, and was correlated with the membrane surface potential monitored by ANS. Moreover, the effect of organic cations on the uptake of tryptamine by intestinal brush-border membrane vesicles was examined. The uptake of tryptamine was inhibited by tetracaine and imipramine. The inhibitory effect of these cations was well correlated with changes in the membrane surface potential in the presence of tetracaine or imipramine. These results suggest that the K(+)-diffusion potential-dependent uptake of tryptamine by intestinal brush-border membrane vesicles is affected by membrane surface potential, and the inhibition of tryptamine uptake originates in changes in the membrane surface potential caused by the organic cations.     

X-ray structures of new dipeptide taste ligands

AIMS/BACKGROUND: The mechanism of interaction and the role played by the vesicle lipid composition for the selective association between liposomes and liver cells were studied, at the ultrastructural level, by investigating both in situ and in vitro the interaction between hepatocytes, Kupffer and endothelial liver cells with egg-phosphatidylcholine (eggPC) or eggPC/stearylamine (9:1; mol:mol) reverse-phase evaporation (REV) liposomes. METHODS: Liver cells from rats, isolated by enzymatic perfusion and purified by differential centrifugation, were incubated, in a rotating bath at 37 degrees C, with liposomes (2.5 mM final liposomal lipid concentration). Cell aliquots were withdrawn and processed for electron microscope observation at fixed time intervals. Parallel experiments were carried out by in situ liver perfusion with liposome suspensions. RESULTS AND CONCLUSIONS: Our first conclusions are: 1) lipidic composition affects the rate of liposomes uptake and internalization by hepatocytes; 2) liposome uptake by hepatocytes or Kupffer cells is likely an endocytic process; 3) endothelial cells internalize lipid vesicles as well; 4) liposome uptake was due to a phagocytic activity for all isolated liver cells, while in the in situ observation endothelial cells seem to use another mechanism (fusion); and 5) the rate of internalization is related to the viability of the treated cells. Experimental data seem to indicate that differential behaviour in the internalization of lipid vesicles exists among parenchymal, Kupffer and endothelial liver cells. These differences suggest that clearance of liposomes by these cells involves two mechanisms (i.e., endocytosis or fusion) with different rates of uptake and internalization that facilitate the design of carriers that can deliver drugs preferentially to a specific liver cell type.     

Lysosomal degradation on vesicular membrane surfaces. Enhanced glucosylceramide degradation by lysosomal anionic lipids and activators

According to a recent hypothesis (Sandhoff, K., and Kolter, T. (1996) Trends Cell Biol. 6, 98-103), glycolipids, which originate from the plasma membrane, are exposed to lysosomal degradation on the surface of intralysosomal vesicles. Taking the interaction of membrane-bound lipid substrates and lysosomal hydrolases as an experimental model, we studied the degradation of glucosylceramides with different acyl chain lengths by purified glucocerebrosidase in a detergent-free liposomal assay system. Our investigation focused on the stimulating effect induced by lysosomal components such as sphingolipid activator protein C (SAP-C or saposin C), anionic lysosomal lipids, bis(monoacylglycero)phosphate, and dolichol phosphate, as well as degradation products of lysosomal lipids, e.g. dolichols and free fatty acids. The size of the substrate-containing liposomal vesicles was varied in the study. Enzymatic hydrolysis of glucosylceramide carried by liposomes made of phosphatidylcholine and cholesterol was rather slow and only weakly accelerated by the addition of SAP-C. However, the incorporation of anionic lipids such as bis(monoacylglycero)phosphate, dolichol phosphate, and phosphatidylinositol into the substrate carrying liposomes stimulated glucosylceramide hydrolysis up to 30-fold. Dolichol was less effective. SAP-C activated glucosylceramide hydrolysis under a variety of experimental conditions and was especially effective for the increase of enzyme activity when anionic lipids were inserted into the liposomes. Glucosylceramides with short acyl chains were found to be degraded much faster than the natural substrates. Dilution experiments indicated that the added enzyme molecules associate at least partially with the membranes and act there. Surface plasmon resonance experiments demonstrated binding of SAP-C at concentrations up to 1 microM to liposomes. At higher concentrations (2.5 microM SAP-C), liposomal lipids were released from the liposome coated chip. A model for lysosomal glucosylceramide hydrolysis is discussed.     

Rat alveolar macrophages are susceptible to activation by free and liposome-encapsulated lymphokines

Alveolar macrophages (AM) obtained from F344 rats were rendered tumoricidal by incubation in vitro with cellfree culture supernatant fluids rich in macrophage-activating factor (MAF) activity harvested from mitogen-stimulated F344 rat lymphocytes. AM activated by this procedure destroyed syngeneic, allogeneic, and xenogeneic tumor cells but were not cytotoxic for nonneoplastic cells. MAF was encapsulated in multilamellar lipid vesicles (liposomes) and its ability to render AM tumoricidal was compared with that of free (unencapsulated) MAF. Liposome-encapsulated MAF rendered AM cytotoxic at concentrations up to 16,000 times lower than free MAF. These data demonstrate that AM can respond in vitro to lymphokines and that MAF encapsulated within liposomes is far more efficient in rendering AM tumoridical than free MAF.