Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles.

Hemagglutinin, the membrane fusion protein of influenza virus, is known to mediate a low-pH-dependent fusion reaction between the viral envelope and the limiting membrane of the endosomal cell compartment following cellular uptake of the virus particles by receptor-mediated endocytosis. Here we exploited this activity of hemagglutinin to achieve efficient gene delivery to cultured cells. Hemagglutinin was reconstituted in the presence of the monocationic lipid dioleoyldimethylammonium chloride (DODAC) to permit plasmid binding to the virosome surface. Virosomes with 30 mol% DODAC exhibited a distinct binding capacity for plasmid without causing aggregation. The virosome fusion activity was not affected by the cationic lipid DODAC as demonstrated by low-pH-dependent lipid mixing with erythrocyte ghosts. Efficient cell transfection of BHK-21 cells was observed with virosomes containing 30 mol% DODAC and plasmid encoding for beta-galactosidase (pCMV beta-gal) associated to their surface. The transfection activity observed was dependent on the functional activity of hemagglutinin. Contrary to DNA/cationic lipid complexes the transfection was not dependent on the cationic lipid to DNA charge ratio. Importantly, transfection of BHK-21 cells with pCMV beta-gal by DODAC-containing virosomes did not show any significant signs of cytotoxicity that is commonly observed with DNA/cationic lipid complexes. Together with the high levels of expression of the transgene this highlights the potential of DODAC-containing virosomes as a novel approach in nonviral gene transfer.

Comparison between intratracheal and intravenous administration of liposome-DNA complexes for cystic fibrosis lung gene therapy.

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-vector formulations are two strategies to obtain gene transfer to the lung, it is still uncertain, however, which of these two modes of delivery will be more effective in the treatment of cystic fibrosis and other lung diseases. In this study, we attempted to optimize formulations of the cationic liposome DODAC:DOPE (dioleoyldimethylammonium-chloride: dioleoylphosphatidylethanolamine) complexed to plasmids encoding chloramphenicol acetyltransferase for i.t. and i.v. injection into CD-2 mice and compared the two methods. Our results showed that both methods conferred reporter gene expression in the lung that was significantly higher relative to injection of plasmid DNA alone. Expression using either mode of administration was maximal 24 h after injection and declined to around 10% of day 1 levels 2 weeks after injection. For i.v. delivery of DODAC. DOPE-DNA complexes multilamellar vesicles were more effective than large unilamellar vesicles in all organs investigated. Recombinant DNA could be detected in the distal lung region following either route of administration. However, i.t. administration predominantly led to DNA deposition in epithelial cells lining the bronchioles, e.g. in clara cells, whereas i.v. administration resulted in DNA deposition in the alveolar region of the lung including type II alveolar epithelial cells.

Recent advances in liposome technologies and their applications for systemic gene delivery.

The recent clinical successes experienced by liposomal drug delivery systems stem from the ability to produce well-defined liposomes that can be composed of a wide variety of lipids, have high drug-trapping efficiencies and have a narrow size distribution, averaging less than 100 nm in diameter. Agents that prolong the circulation lifetime of liposomes, enhance the delivery of liposomal drugs to specific target cells, or enhance the ability of liposomes to deliver drugs intracellularly can be incorporated to further increase the therapeutic activity. The physical and chemical requirements for optimum liposome drug delivery systems will likely apply to lipid-based gene delivery systems. As a result, the development of liposomal delivery systems for systemic gene delivery should follow similar strategies.

Interactions of liposomes and lipid-based carrier systems with blood proteins: Relation to clearance behaviour in vivo.

Liposomes and lipid-based drug delivery systems have been used extensively over the last decade to improve the pharmacological and therapeutic activity of a wide variety of drugs. More recently, this class of carrier systems has been used for the delivery of relatively large DNA and RNA-based drugs, including plasmids, antisense oligonucleotides and ribozymes. Despite recent successes in prolonging the circulation times of liposomes, virtually all lipid compositions studied to date are removed from the plasma compartment within 24h after administration by the cells and tissues of the reticuloendothelial system (RES). Plasma proteins have long been thought to play a critical role in this process but only a few efforts were made to evaluate the relevant importance of plasma protein-liposome interactions in the clearance process. Strategies to increase the bioavailability of liposomal drugs have included altering lipid compositions and charge, increasing lipid doses, and incorporating surface coatings. All of these modifications can influence membrane-protein interactions. In this article, we will focus on our experiences with liposome-blood protein interactions and how alterations in the chemical and physical properties of the carrier system influence the interactions with blood proteins and circulation times.

Condensation of plasmid DNA with polylysine improves liposome-mediated gene transfer into established and primary muscle cells.

Cationic liposomes provide a means to introduce genes into cells both ex vivo and in vivo. In the past few years their use has been described in several tissues, e.g. lungs, liver, endothelium, brain. In this study we evaluated a commercially available poly-cationic liposome formulation in delivering a reporter gene into cultured myogenic cell lines from mouse and rat, and primary fetal human myoblasts. We also examined the effect of serum on liposome-mediated transfection and designed a new procedure to enhance transfection efficiency, based on the pre-condensation of plasmid DNA with polylysine. Polylysine pre-condensation was particularly effective when transfecting the cells in the presence of serum, a finding that could be significant for in vivo transfections.

Influence of dose on liposome clearance: critical role of blood proteins.

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.

Influence of cholesterol on the association of plasma proteins with liposomes.

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.

Recent advances in liposomal drug-delivery systems.

Liposomal drug-delivery systems have come of age in recent years, with several liposomal drugs currently in advanced clinical trials or already on the market. It is clear from numerous pre-clinical and clinical studies that drugs, such as antitumor drugs, packaged in liposomes exhibit reduced toxicities, while retaining, or gaining enhanced, efficacy. This results, in part, from altered pharmacokinetics, which lead to drug accumulation at disease sites, such as tumors, and reduced distribution to sensitive tissues. Fusogenic liposomal systems that are under development have the potential to deliver drugs intracellularly, and this is expected to markedly enhance therapeutic activity. Advances in liposome design are leading to new applications for the delivery of new biotechnology products, such as recombinant proteins, antisense oligonucleotides and cloned genes.

Beta 2 glycoprotein I is a major protein associated with very rapidly cleared liposomes in vivo, suggesting a significant role in the immune clearance of "non-self" particles.

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.

Liposome-complement interactions in rat serum: implications for liposome survival studies.

Serum complement opsonizes particles such as bacteria for clearance by the reticuloendothelial system. Complement has been reported to interact with liposomes and therefore may mediate the reticuloendothelial system clearance of liposomes. This study has used a rat serum model to define some of the characteristics of liposomes which modulate their ability to activate complement. Using functional hemolytic assays and C3/C3b crossed immunoelectrophoresis, we have demonstrated that liposomes activated rat complement in a dose-dependent manner with higher concentrations of liposomes activating higher levels of complement. The detection of complement activation required the inclusion of phospholipids bearing a net charge. Complement activation occurred via the classical pathway; no alternative pathway activation was detected. The presence of cholesterol contributed to complement activation in a dose-dependent manner. Phospholipid fatty acyl chain length did not influence complement activation while the introduction of unsaturated acyl chains markedly decreased levels of complement activation. Liposome size also influenced complement activation with 400 nm unilamellar vesicles more effectively activating complement than 50 nm vesicles for equivalent amounts of exposed lipid. These studies demonstrate that the composition of the liposome greatly affects the in vitro activation of rat serum complement and suggest that the biological half-life of liposomes in the circulation of rats may be altered by changing the liposome composition to reduce complement activation.

Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis.

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9.

Clusterin is a heterodimeric multifunctional protein expressed in a variety of tissues and cells. It forms high density lipid complexes in plasma and participates in the control of the lytic activity of the late complement complex (TCC, C5b-9). Together with vitronectin, clusterin binds to the nascent amphiphilic C5b-9 complex, rendering it water soluble and lytically inactive. To define the interactions that underlie the complement-inhibitory function of clusterin, we have examined the binding interactions between [125I]clusterin and the isolated components of the complex, C5b-6, C7, C8, and C9 and vitronectin. By using ligand blotting in the presence of Tween, specific binding of the labeled clusterin with C7, the beta-subunit of C8 and C9 was detected. Binding to C9 was competed by polymerized C9, but not by C8, C7, C6, and CD59, suggesting that the conformational change occurring during the hydrophilic-amphiphilic transition of C9 exposes the interaction site for clusterin. When thrombin-treated C9 was analyzed, clusterin was found to recognize the C9b fragment containing the hydrophobic membrane interaction segment. Both subunits of clusterin interact with C9 and are similarly potent in inhibiting C5b-9-mediated hemolysis and Zn+(+)-induced C9 polymerization. These results show that clusterin exerts its inhibitory effect by interacting with a structural motif common to C7, C8 alpha, and C9b.

Association of blood proteins with large unilamellar liposomes in vivo. Relation to circulation lifetimes.

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.

Separation of large unilamellar liposomes from blood components by a spin column procedure: towards identifying plasma proteins which mediate liposome clearance in vivo.

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.

The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes.

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.

Uptake of liposomes by cultured mouse bone marrow macrophages: influence of liposome composition and size.

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.

Large unilamellar liposomes with low uptake into the reticuloendothelial system.

Particulate drug carriers, including liposomes, are rapidly removed from blood by cells of the reticuloendothelial system (RES) with resulting adverse effects on this important host defense system. In order to overcome this and other major disadvantages of liposomes, we have altered liposome composition in an effort to achieve prolonged circulation half-lives. Gangliosides and sphingomyelin act synergistically to dramatically diminish the rate and extent of uptake of liposomes by macrophages in vivo. The significantly extended circulation times achieved by these modified large unilamellar liposomes overcome an important barrier to the targeting of particulate drug carriers to specific tissues in vivo.

The degradation of platelet-activating factor in the plasma of a patient with familial high density lipoprotein deficiency (Tangier disease).

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.