Liposome-cell Fusion Research Articles

Enhanced Liposomal Drug Delivery Via Membrane Fusion Triggered by Dimeric Coiled-Coil Peptides.

An ideal nanomedicine system improves the therapeutic efficacy of drugs. However, most nanomedicines enter cells via endosomal/lysosomal pathways and only a small fraction of the cargo enters the cytosol inducing therapeutic effects. To circumvent this inefficiency, alternative approaches are desired. Inspired by fusion machinery found in nature, synthetic lipidated peptide pair E4/K4 is used to induce membrane fusion previously. Peptide K4 interacts specifically with E4, and it has a lipid membrane affinity and resulting in membrane remodeling. To design efficient fusogens with multiple interactions, dimeric K4 variants are synthesized to improve fusion with E4-modified liposomes and cells. The secondary structure and self-assembly of dimers are studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The structures and membrane interactions of PK4 are supported by molecular dynamics simulations. Upon addition of E4, PK4 induced the strongest coiled-coil interaction resulting in a higher liposomal delivery compared to linear dimers and monomer. Using a wide spectrum of endocytosis inhibitors, membrane fusion is found to be the main cellular uptake pathway. Doxorubicin delivery results in efficient cellular uptake and concomitant antitumor efficacy. These findings aid the development of efficient delivery systems of drugs into cells using liposome-cell fusion strategies.

Light-Triggered Cancer Cell Specific Targeting and Liposomal Drug Delivery in a Zebrafish Xenograft Model.

Cell-specific drug delivery remains a major unmet challenge for cancer nanomedicines. Here, light-triggered, cell-specific delivery of liposome-encapsulated doxorubicin to xenograft human cancer cells in live zebrafish embryos is demonstrated. This method relies on light-triggered dePEGylation of liposome surfaces to reveal underlying targeting functionality. To demonstrate general applicability of this method, light-triggered, MDA-MB-231 breast cancer cell specific targeting in vivo (embryonic zebrafish) is shown using both clinically relevant, folate-liposomes, as well as an experimental liposome-cell fusion system. In the case of liposome-cell fusion, the delivery of liposomal doxorubicin direct to the cytosol of target cancer cells results in enhanced cytotoxicity, compared to doxorubicin delivery via either folate-liposomes or free doxorubicin, as well as a significant reduction in xenograft cancer cell burden within the embryonic fish.

Peptide-Mediated Liposome Fusion: The Effect of Anchor Positioning.

A minimal model system for membrane fusion, comprising two complementary peptides dubbed “E” and “K” joined to a cholesterol anchor via a polyethyleneglycol spacer, has previously been developed in our group. This system promotes the fusion of large unilamellar vesicles and facilitates liposome-cell fusion both in vitro and in vivo. Whilst several aspects of the system have previously been investigated to provide an insight as to how fusion is facilitated, anchor positioning has not yet been considered. In this study, the effects of placing the anchor at either the N-terminus or in the center of the peptide are investigated using a combination of circular dichroism spectroscopy, dynamic light scattering, and fluorescence assays. It was discovered that anchoring the “K” peptide in the center of the sequence had no effect on its structure, its ability to interact with membranes, or its ability to promote fusion, whereas anchoring the ‘E’ peptide in the middle of the sequence dramatically decreases fusion efficiency. We postulate that anchoring the ‘E’ peptide in the middle of the sequence disrupts its ability to form homodimers with peptides on the same membrane, leading to aggregation and content leakage.

Carotenoid based bio-compatible labels for third harmonic generation microscopy

The use of carotenoids as biologically friendly labels for third harmonic generation (THG) microscopy is demonstrated. Carotenoid containing liposomes are used to label cell structures via liposome cell fusion. The THG microscopy labels, called harmonophores, were characterized by measuring the third-order nonlinear susceptibility (χ((3))) of carotenoids: violaxanthin, neoxanthin, lutein, β-carotene, zeaxanthin, canthaxanthin and astaxanthin. The THG ratio method was used, which is based on measuring the THG intensity from two interfaces using a nonlinear optical microscope. The second hyperpolarizability values of carotenoids were extracted from χ((3)) measurements taking into account the refractive index at fundamental and third harmonic wavelengths. The length dependence of the second hyperpolarizability of conjugated polyenes from 9 to 13 double bonds with varying oxygen functional groups was investigated. It appears that the presence of epoxides can have a higher influence than an additional conjugated double bond. Furthermore, labelling of both Drosophila Schneider 2 cells and Drosophila melanogaster larvae myocytes with β-carotene was achieved. This study demonstrates that THG enhancement by carotenoids can be used for nontoxic in vivo labelling of subcellular structures for third harmonic generation microscopy.

Fusion of cationic liposomes with mammalian cells occurs after endocytosis

The interaction of cationic liposomes prepared using either dioleoyltrimethylammonium propane (DOTAP) or 3β-( N-( N′ , N′-dimethylaminoethane)carbamoyl)cholesterol (DC-CHOL) with model membranes and with cultured mammalian cells was examined using an assay developed for monitoring virus-cell fusion (Stegmann et al. (1993) Biochemistry 32, 11330–11337). Lipid mixing between cationic liposomes and liposomes composed of DOPE/dioleoylphosphatidylglycerol (DOPG) or dioleoylphosphatidylcholine (DOPC)/DOPG was insensitive to pH in the range of pH 4.5–7.0 and was not affected by sodium chloride concentration in the range of 0–150 mM. Lipid mixing was dependent on dioleoylphosphatidylethanolamine (DOPE), since cationic liposomes prepared using dioleoylphosphatidylcholine (DOPC) were incapable of lipid mixing with DOPC/DOPG liposomes. The interaction of cationic liposomes with Hep G-2 and CHO D − cells was also studied. For both cell types, liposome-cell lipid mixing was rapid at 37° C, beginning within minutes and continuing for up to 1 hour after uptake. The extent of lipid mixing was decreased at 15° C, especially at later (≥ 20 min) time points. This suggests that at least part of the observed lipid mixing occurred after reaching cellular lysosomes. No lipid mixing was seen at 4° C. Monensin inhibited lipid mixing between cationic liposomes and the cells, despite having no effect on liposome uptake. Inhibition of endocytic uptake of liposomes, either by incubation in hypertonic media or by depletion of cellular ATP with sodium azide and 2-deoxyglucose abolished liposome-cell fusion in both cell types. These data demonstrate that binding to the cell surface is insufficient for cationic liposome-cell fusion and that uptake into the endocytic pathway is required for fusion to occur.

Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections.

The fusion of liposomes with the plasma membrane of influenza virus-infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processing by freeze substitution and low temperature embedding. Before fusion, radioactivity was solely detected on the apical cell surface, indicating the absence of redistribution artifacts and demonstrating the reliability of lipid autoradiography on both a light and electron microscopical level. After induction of fusion by a low pH treatment, the basolateral plasma membrane domain became progressively labeled, indicative of rapid lateral diffusion of [3H]dipalmitoylphosphatidylcholine in the plasma membrane. Analysis of individual fusion events by freeze fracture after rapid freezing confirmed the rapid diffusion of the liposomal lipids into the plasma membrane, as intramembrane particle-free lipid patches were never observed. After the induction of liposome-cell fusion, well-defined intramembrane particles were present on the otherwise smooth liposomal fracture faces and on the fracture faces of the plasma membrane. Morphological evidence thus was obtained in favor of a local point fusion mechanism with an intramembrane particle as a specific structural fusion intermediate.

Antibodies introduced into living cells with liposomes localize specifically and inhibit specific intracellular processes

We have developed a system for efficiently packaging antibodies and other macromolecules into lipsomes and then delivering the encapsulated molecules into living cells through liposome-cell fusion. Fusion is very efficient, and all cells can be demonstrated to contain liposome-delivered antibodies by staining by staining with a fluorescent second antibody. Using lupus antibodies directed against small nuclear ribonucleoprotein components of the cell, we were able to demonstrate strong nuclear localization, while control antibodies showed a general diffuse distribution throughout the cell. Lupus antibodies directed against ribosomes, on the other hand, strongly localized in the nucleolus and the cytoplasm with very little nucleoplasmic localization. Antitubulin antibodies predominantly localized in the cytoplasm. These results show that antibodies can survive liposome packaging and can retain their ability to recognize and bind to their specific antigens in the living cell. It also indicates that the nuclear envelope does not present a barrier to the liposome-introduced antibodies in Drosophila tissue culture cells. To determine if the antibodies were capable of interfering with cellular processes in vivo, we measured the effects of liposome-introduced antiribosome antibodies on translation and antitubulin antibodies on mitosis. In both cases, there was a significant inhibition suggesting that the antibodies can be used to interfere with specific functions at specific times in vivo.

Liposome-cell interactions: In vitro discrimination of uptake mechanism and in vivo targeting strategies to mononuclear phagocytes

The interactions of liposomes with cells have been extensively studied to determine their potential use as vehicles for the delivery of drugs in vivo. Since intravenously administered liposomes are, for the most part, cleared by cells of the reticuloendothelial system (RES), considerable effort has been made to take advantage of this phenomenon rather than view it as an obstacle. Indeed, cells of the RES, in particular macrophages, have been shown to play a vital role in homeostasis and in host defence mechanisms against infection and neoplasia. In this article, we present an overview of liposome-cell interactions, with particular emphasis on the techniques used to monitor the interaction of liposomes with macrophages. Specifically, we discuss methodologies which can be used to differentiate between liposome-cell fusion, adsorption and endocytosis in vitro. In addition, we outline the various strategies that have been employed for both actively and passively targeting liposomes to macrophages in vivo. We also review the rationale and various techniques for designing liposomes for enhanced macrophage uptake, which, in certain cases, results in the selective release of liposome-entrapped compounds in situ.

Effects of liposome-adipocyte interaction on calcium binding and insulin action.

The interaction of phosphatidylserine (PS)-containing liposomes with isolated rat adipocytes under conditions of liposome-cell fusion or endocytosis was accompanied by an increase in adipocyte membrane calcium binding in both the basal and insulin-stimulated state. In vesicle-treated adipocytes, insulin-stimulated 2-deoxyglucose uptake was inhibited and not as dependent on external calcium concentration as in control cells. These data suggest that the inhibition of insulin-stimulated 2-deoxyglucose uptake in vesicle treated adipocytes may result from the binding of calcium to the exogenously introduced phospholipid at the expense of membrane constituents normally involved in insulin-stimulated calcium binding. This is supported by the observations that 1) the calcium ionophore A23187 increased intracellular calcium levels, yet had no effect on insulin-stimulated hexose uptake; and 2) treatment of intact adipocytes with phospholipase A2 after the cells had been incubated with vesicles resulted in a decrease in membrane associated Ca++ and exogenous phosphatidylserine, which is consistent with a direct role of membrane phosphatidylserine in calcium binding.

The incorporation of the calcium-activated photoprotein into isolated rat fat-cells by liposome-cell fusion [proceedings

Although Ca2+ ions appear t o be involved in the action of certain hormones (Hales era/., 1977; Rasmussen & Goodman, 1977), there have been few direct demonstrations of an effect of a physiological stimulus on the concentration of intracellular CaZt. Ca2+-activated photoproteins have proved to be useful tools for monitoring changes in the concentration of cytoplasmic Ca2+ (Ashley et a/., 1976). The aim of the present studies was t o incorporate the Ca2+-activated photoprotein obelin into isolated rat fatcells in order t o measure changes in the concentration of intracellular ionized Caz+. Micro-injection of photoproteins can only easily be achieved with giant cells. Recently it has been reported that material entrapped within liposomes can be released into the cytoplasm of isolated cells (Weinstein et al., 1977). Liposomes bearing different net surface charges, which are relatively impermeable to Caz+ ions, can be prepared containing obelin and 1251-labelled carbonic anhydrase (Dormer et al., 1977, 1978). The initial experiments of the present study were carried out t o investigate conditions required for association of liposomes containing obelin with fat-cells. The liposomes were composed of phosphatidylcholine with no added cholesterol or other phospholipids and bearing no net surface charge. Isolated rat fat-cells were incubated for 30min with liposomes containing obelin and '251-labelled carbonic anhydrase in a Krebs-Ringer medium containing Ca2+ ( 1 . 3 m ~ ) . The cells were then washed up to six times to remove free liposomes. The total obelin associated with the cells was determined by measurement of the luminescence in the presence of Ca2+ ( 2 5 m ~ ) and Triton X-100 (lo%, v/v). Under theseconditions, less thanO.O3%oftheobeIinentrappedwithin the liposomes was found associated with the cells, although approx. 1 % of the L251-labelled carbonic anhydrase inside the liposomes was taken up by the cells. In contrast, incubation of cells with liposomes in the absence of extracellular Caz+ and in the presence of EGTA (0.3-1 mM) resulted in approx.0.3 % of the obelin and L251-labelled carbonic anhydrase entrapped in the liposomes being associated with the cells. Incubation of liposomes with cells was performed at 2 5 T , since at 37°C the activity of obelin appeared t o be unstable. To rule out the possibility that liposomes might be associating with triacylglycerol from broken fat-cells, liposomes containing obelin were incubated with homogenized cells, and the degree of obelin association was compared with the uptake of liposomes by intact cells. The association of liposome-entrapped obelin with triacylglycerol from homogenized cells was approximately one-third of that observed with intact cells (Table 1). It was concluded that most of the obelin associated with cells was due to the presence of intact cell membranes. To investigate the intracellular location of cell-associated obelin, fractionation studies were carried out. After liposome incubation the cells were homogenized and centrifuged at lOOOg, lOOOOg and 1OOOOOg. The pellets and final supernatant were assayed for obelin and a number of enzyme activities (acid phosphatase, 5'-nucleotidase, cytochrome oxidase and lactate dehydrogenase). Although some obelin was found in all fractions, the lOOOOg pellet contained more than 50% of the acid phosphatase and obelin activities. Approx. 20% of the obelin was in the lOOOOOg supernatant. However, since there was a significant correlation between acid phosphatase and obelin in all fractions (linear regression coefficient r = 0.9), it was possible that some of the supernatant obelin might have resulted from its release from lysosomes. To investigate whether conditions expected t o increase the concentration of cytoplasmic Ca2+ resulted in the utilization of the cell-associated obelin, the cells were incubated for lOmin in a medium containing Ca2+ ( I mM) or in the absence of extracellular Caz+ with dinitrophenol(1OOp~) or the ionophore A23187 ( 9 . 5 , ~ ~ ) . Incubation of cells

Introduction of enzymes, by means of liposomes, into non-phagocytic human cells in vitro

Liposomes containing entrapped horsedish peroxidase were incubated with three human cell lines in vitro. Although these cells did not ingest latex particles, and took up less than 1 minut of free peroxidase/5 · 10 6 from the medium, significant amounts (41–164 munits/5 · 10 6) of peroxidase became cell-associated by 30 min if the enzyme was presented in negatively charged liposomes (phosphatidylchloline/dicetyl phosphate/cholesterol, 70 : 20 : 10 molar ratio). Uptake of liposome-entrapped peroxidase by lymphocytes or fibroblasts was enhanced 2–5-fold if one molar percent of lysophosphatidylcholine was incorporated as a “fusogen”, and was not appreciately diminished by cytochalasin B, an inhibitor of phagocytosis. Lysophosphatidylcholine containing liposomes did not release trapped peroxidase into the medium during incubation, and studies employing the metallochromic dye, arsenazo III demonstrated lack of access of external Ca 2+ to the internal, enzyme-laden, aqueous compartments; liposome-liposome fusion was also excluded by similar means. Ultrastructural cytochemstry demonstrated peroxidase within liposomes in the free cytosol of cultured cells 15–90 min after apparent liposome-cell fusion. Data provide evidence that multilamellar liposomes can be as vectors for the introjection of missing enzymes into non-phagocytic human cells.