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Orientational photorefractive gratings in nematic liquid crystals (NLCs) on a poly(N-vinyl-carbazole) film dispersed with 5,10,15,20-tetra-phenylporphyrinatozinc across poly(vinyl alcohol) film were observed. The maximum diffraction efficiency of 0.3-0.5% was measured.

Our recent in vivo studies have investigated the surface adsorption property of various circulating liposomes to blood proteins, and have related this property to liposome clearance behavior. In particular, we have investigated liposomes composed of different charged or neutral lipids, fatty acyl chain length and saturation, and cholesterol content. From these studies an apparent inverse relationship between the amount of blood protein that associates with large unilamellar vesicles and the circulation half-lives of the liposomes is observed, indicating that protein-mediated liposome clearance mechanisms are dominant. Furthermore, by comparing the protein profiles of rapidly cleared liposomes with liposomes exhibiting enhanced circulation times, key blood proteins have been identified and implicated in the clearance process.

The proteins associated with liposomes in the circulation of mice were analyzed in order to determine whether bound proteins significantly influence the fate of liposomes in vivo. Liposomes were administered intravenously via the dorsal tail vein of CD1 mice and were isolated from blood after 2 min in the absence of coagulation inhibitors using a rapid "spin column" procedure. Various negatively charged liposomes exhibiting markedly different clearance properties were studied; notably, these included liposomes containing 10 mol % ganglioside GM1 which has been previously shown to effectively limit liposomal uptake by the fixed macrophages of the reticuloendothelial system. The protein binding ability (PB; g of protein/mol of lipid) of the liposomes was quantitated and related to the circulation half-life (tau 1/2) of the liposomes. Liposomes having similar membrane surface charge imparted by different anionic phospholipids were found to exhibit markedly different protein binding potentials. Furthermore, PB values determined from the in vivo experiments were found to be inversely related to circulation half-lives. PB values in excess of 50 g of protein/mol of lipid were observed for rapidly cleared liposomes such as those containing cardiolipin or phosphatidic acid (tau 1/2 less than 2 min). PB values for ganglioside GM1-containing liposomes (tau 1/2 greater than 2 h) were significantly less (PB less than 15 g of total protein/mol of total lipid). PB values were also determined for liposomes recovered from in vitro incubations with isolated human serum; relative PB values obtained from these in vitro experiments were in agreement with relative PB values measured from in vivo experiments. PB values, therefore, could be a useful parameter for predicting the clearance behavior of liposomes in the circulation. Liposomes exhibiting increased PB values in vivo were shown by immunoblot analysis to bind more immune opsonins, leading to a higher probability of phagocytic uptake. Finally, based on results obtained using the in vitro system, it is suggested that the mechanism by which ganglioside GM1 prolongs the murine circulation half-life of liposomes is by reducing the total amount of blood protein bound to the liposomes in a relatively nonspecific manner.

In order to facilitate the isolation of liposomes from blood components, we have developed a simple and rapid procedure combining chromatographic and centrifugal methods. This 'spin column' procedure was used to isolate liposomes from incubation mixtures with human serum or from the blood of CD1 mice after intravenous administration of liposomes. An advantage of this procedure is that processing times are fast (typically minutes) such that the isolation procedure can be done in the absence of chelators or other coagulation inhibitors which may affect protein/liposome interactions. Furthermore, several samples can be analyzed together and small sample volumes can be processed. In addition, we show that this spin column procedure can be employed to isolate large unilamellar vesicles averaging 100 nm in diameter from lipoproteins and plasma proteins. The applicability of this spin column procedure in studying protein/liposome interactions is demonstrated by quantitating the amount of human complement component C3 bound per liposome using a C3 competitive ELISA assay after incubation with human serum. The proteins associated with the recovered liposomes were further analyzed by conventional SDS-polyacrylamide gel electrophoresis. We show that egg phosphatidylcholine/cholesterol (55:45, mol/mol) or egg phosphatidylcholine/cholesterol/dioleoylphosphatidylserine (35:45:20, mol/mol) liposomes isolated from the circulation of CD1 mice within minutes of administration have distinct, complex profiles of associated proteins. By isolating circulating large unilamellar liposomes using the spin column method and characterizing the proteins associated with their membranes, this protein fingerprinting approach will expedite identifying protein interactions which affect liposome stability and clearance in vivo.

It is well established that the circulation half-life of liposomes increases with increasing dose. This effect is commonly attributed to ‘saturation’ of the fixed and free macrophages of the reticuloendothelial system resulting in reduced clearance rates. However, it is also known that the clearance rate of liposomes is dependent on the amount of associated blood protein, leading to the possibility that dose-dependent increases in circulation lifetimes could be due to decreases in the amount of blood protein associated per liposome. In order to test this hypothesis, the protein binding and clearance properties of large unilamellar liposomes composed of distearoylphosphatidylcholine/cholesterol and egg phosphatidylcholine/dioleoylphosphatidic acid/cholesterol were examined in mice. Liposomes were injected over a dose range of 10 to 1000 mg lipid/kg body weight, and the circulation lifetime and liver and spleen accumulation monitored. As expected, longer circulation half-lives were observed at higher doses for both liposome compositions. However, it was also found that at higher liposome doses, significantly less protein was bound per liposome. The results indicate that there is a limited pool of blood proteins that is able to interact with liposomes of a given composition. At higher lipid doses these blood proteins are distributed over more liposomes resulting in lower protein binding values and longer circulation lifetimes.

The in vivo association of blood proteins with large unilamellar liposomes composed of saturated phosphatidylcholines was analyzed to determine the effect of membrane fluidity and hydrocarbon chain length on liposome-plasma protein interactions and liposome clearance. Liposomes composed of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diarachidoylphosphatidylcholine (DAPC) were administered via the lateral tail vein of CD-1 mice and were subsequently isolated from the blood at 2 min postinjection. The protein binding ability (PB, grams of protein bound per mole total lipid) of the liposomes was quantified and related to their circulation half-lives. Liposomes composed of long-chain saturated phospholipids that exist in the gel (frozen) state at 39 degrees C (DPPC,DSPC and DAPC) bound large quantities of blood proteins, in excess of 48 g of protein per mole total lipid, and were found to be rapidly cleared from the circulation. The incorporation of cholesterol into DSPC liposomes resulted in significantly decreased PB values and enhanced circulation lifetimes for this lipid system. This cholesterol effect plateaued at 30 mol % cholesterol, corresponding to the loss of the gel-liquid crystalline phase transition, and resulted in PB values of 23-28 grams of protein per mole of total lipid. The types of blood proteins binding to DSPC liposomes were not significantly altered by the inclusion of cholesterol. This is the first demonstration of rapid clearance of neutral large unilamellar liposomes having high levels of bound protein.

Liposomes recovered from the blood of liposome-treated CD1 mice were previously reported to have a complex protein profile associated with their membranes (Chonn, A., Semple, S.C., and Cullis, P.R. (1992) J. Biol. Chem. 267, 18759-18765). In this study, we have further characterized and identified the major proteins associated with very rapidly cleared large unilamellar vesicles. These liposomes contained phosphatidylcholine, cholesterol, and anionic phospholipids (phosphatidylserine, phosphatidic acid, or cardiolipin) that dramatically enhance the clearance rate of liposomes from the circulation. These anionic phospholipids are normally found exclusively in the interior of cells but become expressed when cells undergo apoptosis or programmed cell death, and thus, they are believed to be markers of cell senescence. Analysis of the proteins associated with these liposomes by SDS-polyacrylamide gel electrophoresis revealed that two of the major proteins associated with the liposome membranes are proteins with electrophoretic mobilities corresponding to M(r) of 66,000 and 50,000-55,000. The 66-kDa protein was identified to be serum albumin by immunoblot analysis. Using various biochemical and immunological methods, we have identified the 50-55-kDa protein as the murine equivalent of human beta 2-glycoprotein I. beta 2-glycoprotein I has a strong affinity for phosphatidylserine, phosphatidic acid, and cardiolipin inasmuch as the levels of beta 2-glycoprotein I associated with these anionic liposomes approach or even exceed those of serum albumin, which is present in serum at a concentration 200-fold greater than beta 2-glycoprotein I. Further, we demonstrate that the amount of beta 2-glycoprotein I associated with liposomes, as quantitated by an enzyme-linked immunosorbent assay, is correlated with their clearance rates; moreover, the circulation residency time of cardiolipin-containing liposomes is extended in mice pretreated with anti-beta 2-glycoprotein I antibodies. These findings strongly suggest that beta 2-glycoprotein I plays a primary role in mediating the clearance of liposomes and, by extension, senescent cells and foreign particles.

Alterations in the lipid composition of biological membranes can have dramatic effects on their ability to interact with soluble proteins. In a series of studies employing large unilamellar vesicles (LUVs) produced by an extrusion technique, we have characterized the influence of membrane components on protein-membrane interactions. Much of our understanding of how and why proteins interact with seemingly inert membrane surfaces stems mainly from studies involving one or two protein component systems. These studies, however, do not accurately reflect the interactions that occur in the complex biological milieu (reviewed by Horbett and Brash, 1987). There have been very few studies reported on the interactions of proteins with liposomal systems incubated with whole blood. There are two main reasons for this. First, the large majority of studies on the association of plasma proteins with liposomes in vitro have been performed employing multilamellar systems. Due to the variable lamellarity of liposomes of different lipid compositions, quantification of the amount of various proteins associated per liposome has not been possible. Second, convenient techniques have not been available for the isolation of liposomes, particularly LUVs, from blood components. We have recently described a rapid method for the isolation of well-defined large unilamellar liposomes from the blood of liposome-treated mice (Chonn et al., 1991). With such a procedure now available, we have started to biochemically and immunologically characterize the amount and type of proteins associated with liposomes exposed to the complex biological milieu.

A wide range of liposome compositions have previously been examined in vivo for their ability to affect the uptake of liposomes into cells of the reticuloendothelial (RE, mononuclear phagocyte) system (Allen, T.M. and Chonn, A. (1987) FEBS Lett. 223, 42-46; Allen et al. (1989) Biochim. Biophys. Acta 981, 27-35). In this study we have examined the ability of cultured murine bone marrow macrophages to endocytose liposomes of various compositions and have looked for correlations between the in vivo and the in vitro observations. Compounds which substantially decreased RE uptake of liposomes in vivo, such as monosialoganglioside (GM1) and a novel synthetic lipid derivative of polyethyleneglycol (PEG-PE), also greatly decreased liposome uptake by bone marrow macrophages in a concentration-dependent manner. Lipids which increase bilayer rigidity, such as sphingomyelin (SM) and cholesterol (CHOL), decreased both in vivo and in vitro uptake of liposomes. Likewise, positive correlations were observed between the in vivo behavior of liposomes containing phosphatidylserine (PS) or various gangliosides and the ability of these liposomes to be taken up by bone marrow macrophages. Total liposome uptake by macrophages increased with incubation time at 37 degrees C while very little liposome association with the macrophages was observed at 4 degrees C. Liposome uptake increased with liposome concentration and for liposomes composed of egg phosphatidylcholine (PC) uptake plateaued at 40 nmol lipid per mg cell protein. There was an inverse correlation between liposome size of extruded large unilamellar vesicles and their uptake by macrophages.

Platelet Activating Factor (PAF) (1-O-alkyl-2-acetyl sn-glycerol 3- phosphocholine) has been characterized by its ability to aggregate platelets at low concentrations and its profound hypotensive effects. There is evidence that the rate of catabolism of this compound in the plasma regulates its concentration. In humans, we and others have shown that a PAF acetylhydrolase is associated with low density lipoprotein (LDL). The LDL particle in the plasma of patients with Tangier disease is quite different from normal as its lipid core appears to be enriched with triacylglycerol. Thus, we have studied the potential of this abnormal lipoprotein to degrade PAF. The assay for PAF acetylhydrolase was based on the release of 3H from PAF that was labelled in the acetate moiety of the sn-2 position. Tangier disease plasma had approximately 3.3-fold higher PAF acetylhydrolase activity (208 +/- 9 nmol/min/mL) than controls (63 +/- 18 nmol/min/mL). This increase was brought about by an increase in the Vmax (400 +/- 40, Tangier disease; 54 +/- 5, controls) and Km for PAF (120 +/- 20 mumol/L, Tangier disease; 28 +/- 4 mumol/L, controls). The activity appears to be a specific acetylhydrolase rather than a phospholipase A2 as preincubation of the substrate with 0 to 100 mumol/L phosphatidylcholine did not affect the amount of [3H] acetate released. The role of PAF, and its degradation by LDL-bound PAF acetylhydrolase in the phenotypic expression of this patient with Tangier disease, is not known. However, this is the first patient so far described who has an increased ability to degrade PAF in the plasma.