Lectin-bearing liposomes: differential binding to normal and to transformed mouse fibroblasts

The binding of covalent conjugates of concanavalin A (Con A) or wheat germ agglutinin (WGA) and liposomes (lectin-liposomes) to the surface of normal and transformed mouse fibroblasts was studied. Quantitation of the binding was performed by means of microfluorometry and radioactive lipid label counting using both sparse and dense cell cultures. It was found that 2.5-3 times more lectin-conjugated liposomes are bound to L or SV3T3 cells than to the mouse embryo fibroblasts and 3T3 cells in a broad concentration range. The binding of Con A- and WGA-liposomes was inhibited up to 70% in the presence of the corresponding carbohydrate inhibitors. A decreased binding of lectin-liposomes to cells was also observed when cells were pretreated with the free lectin. Trypsinization of the cells resulted in an increase in the Con A-liposomes binding to normal fibroblasts. When free fluorescent Con A or WGA was used in binding studies no profound differences in the binding of lectin to normal or transformed cells were detected. The relation of the lectin-liposome/cell to cell/cell interactions is discussed.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Ultrastructural localization of wheat germ agglutinin-binding sites on surfaces of chick embryo cells during early differentiation.

The objective of this work was to examine changes in a surface component of cells from the chick embryo during morphogenetic migrations of gastrulation. Two electron microscope techniques were used to localize cell-bound wheat germ agglutinin (WGA), a lectin which specifically binds N-acetyl glucosamine residues. One technique involved conjugation of peroxidase to WGA before reaction with the cells; the other technique used glucose oxidase to mark WGA which was already cell-bound. In both cases, binding was revealed using diaminobenzidine. Before formation of the primitive streak, all surfaces of the two-layered embryo bound WGA. After migration of cells through the streak, to form the three-layered embryo, not all cell surfaces bound WGA equally. Epiblast cells generally bound WGA lateral to the primitive streak but not during passage through the streak. Mesenchyme cells, after passage through the streak, bound WGA increasingly as they migrated away from the streak. A WGA-binding matrix was observed in the vicinity of the mesenchyme cells and on the dorsal surface of the endoblast. The ventral surface of the endoblast bound the lectin very poorly. In some instances, a peroxidase reaction product was consistently seen on certain surfaces which was not removable by addition of the simple hapten N-acetyl glucosamine. In these cases, the density of the deposit was lessened by use of diacetyl chitobiose as a hapten. This result, together with the reduction of reaction product following certain hyaluronidase treatments, suggests that WGA may be binding to hyaluronic acid as well as membrane glycoproteins.     

Characterization of metalloanticancer capacity of an agglutinin from wheat

Many anticancer drugs cannot recognize selectively tumor tissues, and cause destruction to normal ones. Although it is very toxic, cisplatin is still one of the most applied chemotherapeutics used for treatment of sarcomas, carcinomas, etc. It causes severe side effects as a result of the lack of selectivity of the drug to tumor tissue and acquired or intrinsic resistance occurs. Wheat germ agglutinin (WGA) is a lectin that specifically recognizes transformed cells: prostate cancer cells, pancreatic cells etc., and is uptaken into the tumor cells for which it appears to be a suitable target for anticancer agents. A fluorescence spectroscopy method was used to study the interaction of WGA with four metal-based anticancer drugs: cisplatin, Pt porphyrin and two gold porphyrins. The affinity constant (k(D)) for binding of cisplatin with WGA was k(D) = 6.67 ± 2.5 μM. The hyperbolic curve indicated the presence of a single cisplatin binding site. The affinity of Au and Pt porphyrin to WGA (k(D) = 0.08-0.49 μM) is almost two orders of magnitude higher than that for cisplatin. We found that Pt porphyrin could displace fluorescent dye ANS showing an increase in the fluorescence intensity with a concomitant blue shift of the emission maximum suggesting that the compounds accommodate the same binding site. Current research characterizes the metalloanticancer binding capacity of WGA. Our results indicate that four metal-based anticancer agents have high affinity for WGA. Since WGA recognizes transformed cells, the obtained data show that this protein might have putative usage as a drug delivery molecule in cancer.     

Fluorescence analysis of hormone binding activities of wheat germ agglutinin

Wheat germ agglutinin (WGA) from embryos of the monocotyledonous plant Triticum vulgaris (Graminaceae) is a carbohydrate binding protein characterized by high specificity to N-acetyl-d-glucosamine and N-acetyl-d-neuraminic acid. In this study we show that parallel to its carbohydrate binding activities, WGA binds with several orders of magnitude higher affinity adenine, adenine-related cytokinins: kinetin, zeatin and isopentenyl-adenine as well as abscisic and gibberellic acids (K(d) 0.43-0.65 microM). Its interactions with these ligands cause conformational rearrangements in the protein molecules and significant enhancement of the protein tryptophan fluorescence (up to 60%) allowing characterization of the protein-hormone complexes. Dimeric WGA molecules possess two different classes of binding sites for the fluorescent hydrophobic probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) as suggested by the sigmoid shape of the fluorescence titration curve and the value of the Hill coefficient (n(H) 1.6+/-0.3). The plant hormones displace part of the bound TNS probe and share the higher affinity TNS binding sites. These results characterize WGA as a hormone-binding protein.     

Differences in lectin binding in squamous metaplasia induced by benzo(a)pyrene and vitamin A deficiency in hamster tracheal explants

Summary Epidermoid metaplasia in the hamster trachea can be produced by treatment with benzo(a)pyrene (BP) or vitamin A deficiency. To elucidate distinguishing features of the two types of lesions, lectin binding to tissue sections of tracheal explants exhibiting metaplastic lesions was assessed. In squamous metaplasia induced by vitamin A deficiency, Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) showed faint (+) to moderate (++) binding in both basal and suprabasal cells; Concanavalin A (Con A) showed moderate binding (++) to suprabasal cells and no binding in basal cells. In the BP-induced lesions, PNA and WGA bound intensely (++++, +++, respectively) in basal cells and faintly (+) to moderately (++) in suprabasal cells. The staining seemed to be predominant at the periphery of the cells. Further, the intensity of PNA and WGA staining increased significantly after the neuraminidase treatment. DBA and Con A showed faint (+) to moderate (++) binding in the BP-induced metaplasia. The results show that in BP-induced metaplasia, cells in the basal region show preferential binding of PNA and WGA. This research was supported by grant RO1-HL32308 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Protein microarrays to study carbohydrate-recognition events

In order to expand areas in which protein microarrays can be used to solve important biological problems, we have investigated ways in which the technique can be employed for functional glycomics. Initially, our protein microarrays were used for the rapid identification of carbohydrate-binding proteins using trifunctional carbohydrate probes and fluorescent dye-labeled polysaccharides. Glycan probes were selectively bound to the corresponding lectins immobilized on the solid surface. In addition, these microarrays were also employed for profiling of carbohydrates on Jurkat T-cell surfaces. These cells adhered to ConA, RCA(120), SNA and WGA, indicating expression of alpha-Man, Gal, NeuNAcalpha2,6Gal and GlcNAc residues on their surfaces. Furthermore, we determined binding affinities between WGA and carbohydrates by measuring IC(50) values of GlcNAc that inhibited 50% of trivalent GlcNAc binding to WGA immobilized on the solid surface. All the experiments show that protein microarrays can be used to study carbohydrate-recognition events in the field of glycomics.     

Lectin binding to neural crest cells. Changes of the cell surface during differentiation in vitro

To examine possible changes in cell surface carbohydrates, fluorescent lectins were applied at various times during differentiation of neural crest cells in vitro. The pattern and intensity of binding of several lectins changed as the crest cells developed into melanocytes and adrenergic cells. Considerable amounts of concanavalin A (Con A) and wheat germ agglutinin (WGA) bound to all unpigmented cells throughout the culture period. Melanocytes, however, bound much less of these lectins. Soy bean agglutinin (SBA), unlike Con A and WGA, only bound later in development to unpigmented cells at about the time when catecholamines were detected histochemically. Binding of SBA could be induced in younger cultures by pretreating the cells with neuraminidase. Melanocytes, however, did not bind detectable amounts of SBA even if treated with neuraminidase. The SBA-binding sites were often concentrated on cytoplasmic extensions and on contact points between neighboring cells, even when receptor mobility was restricted by prefixation of the cells or adsorption of lectin at 0 degrees C. All three lectins bound to cell processes resembling nerve fibers in particularly high amounts.     

Distribution of lectin binding glycoprotein in osteoclasts

Summary Arachis hypogaea (PNA) lectin, specific for Gal-B-1, 3-GalNac disaccharide and Wheat germ (WGA) lectin, specific for (GlcNac) and terminal neuraminic acid were used to identify histiocytic giant cells, osteoclast like giant cells and osteoclasts. PNA lectin, without neuraminidase predigestion was not bound by the giant cells, while they showed a strong reaction with WGA lectin. Neuraminidase pretreatment decreased WGA lectin binding, which supports that neuraminic acid plays a role in the binding of WGA. On the other hand, neuraminidase digestion liberated large amounts of PNA binding sites in every type of giant cells examined, showing a strong, intracytoplasmic granular staining. This observation is indicative of presence of PNA binding sites masked by neuraminic acid. Instead of the intracytoplasmic PNA binding in some osteoclasts a well defined part of the cytomembrane was havily stained. We suppose that this PNA binding part of cytomembrane equals to the zone of resorption, characterized by the ruffled border of osteoclasts. Our findings indicate that a neuraminic acid substituted PNA binding glycoprotein is synthetized both in osteoclasts and histiocytic giant cells which may indicate a common origin of these cell types.     

A facile,sensitive and quantitative membrane‐binding assay for proteins

Soluble proteins that bind membranes function in numerous cellular pathways yet facile, sensitive and quantitative methods that complement and improve sensitivity of widely used liposomes‐based assays remain unavailable. Here, we describe the utility of a photoactivable fluorescent lipid as a generic reporter of protein‐membrane interactions. When incorporated into liposomes and exposed to ultraviolet (UV), proteins bound to liposomes become crosslinked with the fluorescent lipid and can be readily detected and quantitated by in‐gel fluorescence analysis. This modification obviates the requirement for high‐speed centrifugation spins common to most liposome‐binding assays. We refer to this assay as Proximity‐based Labeling of Membrane‐Associated Proteins (PLiMAP).     

Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: in vitro studies

Conjugates of three components, folic acid-poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-DSPE), derived from PEG with molecular masses of 2000 and 3350 Da were synthesized by a carbodiimide-mediated coupling of FA to H2N-PEG-DSPE. The conjugates were characterized by 1H NMR, MALDI-TOF, and HPLC analysis of enzymatic cleavage with carboxypeptidase G. As a prototype of a folate receptor (FR)-targeted system, the conjugates were formulated at 0.5 mol % phospholipid in hydrogenated phosphatidylcholine/cholesterol liposomes with or without additional methoxyPEG2000-DSPE. In vitro binding studies were performed with sublines of M109 (murine lung carcinoma) and KB (human epidermal carcinoma) cells each containing high and low densities of FR. FA-PEG-DSPE significantly enhanced liposome binding to tumor cells. The best binding was observed when FA-PEG liposomes contained no additional mPEG-lipid. In fact, our experiments showed that the presence of mPEG on liposomal surfaces significantly inhibited FA-PEG-liposome binding to FR. Increasing the molecular mass of the PEG tether from 2000 to 3350 Da improved the FR binding, particularly in the case of mPEG-coated liposomes. The FA-PEG liposomes bound to M109-HiFR cells very avidly as demonstrated by the inability of free FA (used in a 700-fold excess either at the beginning or at the end of the incubation) to prevent the cell binding. This is in contrast to the 5-10-fold lower cell binding activity of mPEG-FA compared to that of free FA, and likely to be related to the multivalent nature of the liposome-bound FA. Only 22% of FA-PEG3350 and 32% of FA-PEG3350/mPEG cell-associated liposomes could be removed by exposure to pH 3, conditions that dissociate FA-FR, suggesting that more than two-thirds of the bound liposomes were internalized during incubation for 24 h at 37 degrees C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.     

Unusual electrostatic effects on binding of C1q to anionic liposomes: role of anionic phospholipid domains and their line tension.

The binding of 125I-C1q to anionic liposomes was studied as a function of protein concentration, pH, ionic strength, and anionic lipid composition. The maximum amount of protein bound per micromole of lipid was very sensitive to electrostatic factors, increasing strongly with decreased pH and ionic strength or increased anionic lipid content. The apparent association constant was independent of these electrostatic factors, however, in marked contrast to studies on basic peptide binding to anionic lipid vesicles. Microscopic observations of large unilamellar liposomes containing fluorescently labeled C1q or phosphatidylglycerol demonstrated, under conditions causing strong electrostatic interactions, that C1q and anionic lipids colocalized into domains whose radii of curvature were higher than that of the surrounding lipid. These domains were observed to bud and pinch off into brightly fluorescent vesicles. We propose a model for all of these observations in which the line tension or edge energy at the boundary of the domain resists its increase in circumference as the domain grows by electrostatic effects on binding, eventually resulting in vesiculation. We propose that under favorable electrostatic conditions, as larger domains form the edge energy balances the increases in the electrostatic contribution to binding, resulting in a net binding energy independent of electrostatic factors.     

Incorporation of acylated wheat germ agglutinin into liposomes

Purified wheat germ agglutinin (WGA) was derivatized with palmitic acid at an average stoichiometry of one fatty acid per dinner. Palmitoyl WGA was readily incorporated into liposomes with a cholate-dialysis method. Liposome-bound WGA caused agglutination of red blood cells at a concentration eight-fold lower than that of the native lectin. Furthermore, enhanced binding of liposome-bound WGA to mouse spleen cells was also observed. Potential applications of the liposome-bound lectin are discussed.     

Effect of wheat germ agglutinin on the interleukin pathway of human T lymphocyte activation

Wheat germ agglutinin (WGA) inhibits proliferation of human peripheral blood mononuclear cells (PBMC) induced by mitogens and antigens. We investigated the mechanism by which WGA inhibits PHA-induced human lymphocyte proliferation with regard to the interleukin pathway. Our data revealed that although PBMC-proliferation was markedly suppressed by WGA, levels of IL 2 activity in WGA-inhibited cultures were not reduced, but instead were increased, suggesting failure to utilize IL 2. Furthermore, the addition of exogenous IL 2 failed to overcome the suppression. Consistent with these observations, culturing PBMC with PHA plus WGA markedly decreased the number of high-affinity IL 2 receptor per cell, as determined by binding of purified [3H]IL 2, relative to cultures containing PHA alone. WGA immobilized on support beads bound detergent-solubilized IL 2 receptors from PHA-activated T cells, but did not bind human IL 2. However, WGA did not competitively block the binding of [3H]IL 2 to PHA-induced lymphoblasts. These results suggest that WGA inhibits lymphocyte proliferation by binding to and decreasing the number of high-affinity IL 2 receptors displayed on T cells, without impairing IL 2 production.     

Specific binding of lectins with the nucleus of the sea urchin embryo and changes in the lectin affinity of the embryonic chromatin during the course of development

(1) Embryonic cells of sea urchins were made permeable by treating them with glycerol solution for the purpose of allowing penetration of macromolecules into the cell. With the use of such permeabilized cells, several kinds of fluorescent dye-labeled lectins were introduced into the cell, and it was found that some lectins showed notable affinity with the nucleus as compared with cytoplasmic structures. (2) Isolated chromatin was incubated with several kinds of fluorescent dye-labeled lectins in vitro, and the amount of lectins bound with the chromatin was measured by fluorometry. By means of this method, the lectin-binding capacity of chromatin was estimated and compared at various stages of development. It was found that lectins could be classified into three groups according to the mode of binding with the chromatin: (a) Extent of binding increased notably at the gastrula stage (Con A and RCA-120); (b) extent of binding showed a temporary decrease at the gastrula stage (TTA); and (c) very low level of binding was maintained throughout all stages, and no particular change was observed at any stage of development (WGA, SBA, and UEA-I). (3) These facts seem to suggest that lectin-binding components are contained in sea urchin chromatin, and that drastic changes occur in these components of chromatin at the stage of gastrulation. It was proposed that the lectin-binding components such as proteoglycans and glycoproteins may play regulatory roles in embryonic chromatin at early stages of development.     

Glycoconjugates of human sperm surface. A study with fluorescent lectin conjugates and lens culinaris agglutinin affinity chromatography

Distribution of glycocompounds in human spermatozoa was studied by using fluorescent lectin-conjugates. Con A bound predominantly to acrosomal and posterior head regions whereas RCA I bound to the acrosomal region of intact spermatozoa, stained in suspension. Other lectins used (LCA, WGA, SBA, PNA) stained the the entire sperm surface. In airdried sperm smears binding of both Con A and RCA I were identical with the staining pattern obtained with living cells whereas LCA, WGA, SBA and PNA now bound heavily into acrosomal region. As a similar staining pattern was obtained with permeabilized sperm cells, this staining is apparently due to binding to intracellular structures. The efficiency of Lens culinaris agglutinin affinity chromatography in purification of human sperm glycoproteins was tested after their external radiolabelling with the neuraminidase/galactose oxidase/sodium borohydride method. 22% of applicated radioactivity could be eluted from the column with the specific inhibitory saccharide, and most of the radiolabelled surface glycoproteins of the whole sperm lysate, were also present in the LCA affinity column eluate. LCA affinity chromatography seems thus be an effective method to enrich membrane glycoproteins of human spermatozoa.     

Distribution of lectin binding glycoprotein in osteoclasts

Arachis hypogaea (PNA) lectin, specific for Gal-B-1,3-GalNac disaccharide and Wheat germ (WGA) lectin, specific for (GlcNac) and terminal neuraminic acid were used to identify histiocytic giant cells, osteoclast like giant cells and osteoclasts. PNA lectin, without neuraminidase predigestion was not bound by the giant cells, while they showed a strong reaction with WGA lectin. Neuraminidase pretreatment decreased WGA lectin binding, which supports that neuraminic acid plays a role in the binding of WGA. On the other hand, neuraminidase digestion liberated large amounts of PNA binding sites in every type of giant cells examined, showing a strong, intracytoplasmic granular staining. This observation is indicative of presence of PNA binding sites masked by neuraminic acid. Instead of the intracytoplasmic PNA binding in some osteoclasts a well defined part of the cytomembrane was heavily stained. We suppose that this PNA binding part of cytomembrane equals to the zone of resorption, characterized by the ruffled border of osteoclasts. Our findings indicate that a neuraminic acid substituted PNA binding glycoprotein is synthetized both in osteoclasts and histiocytic giant cells which may indicate a common origin of these cell types.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density

Influenza virus gains access to the cytoplasm of its host cell by means of a fusion event between viral and host cell membrane. Fusion is mediated by the envelope glycoprotein hemagglutinin (HA) and is triggered by low pH. To learn how many hemagglutinin trimers are necessary to cause membrane fusion, we have used two NIH 3T3 fibroblast cell lines that express HA protein at different surface densities. On the basis of quantitations of the number of HA trimers per cell and the relative surface areas of the two cell lines, the HAb-2 cells have a 1.9-fold higher plasma membrane surface density than the GP4F cells. The membrane lateral diffusion coefficient and the mobile fraction for HA is the same for both cell lines. A Scatchard analysis of the binding of glycophorin-bearing liposomes to the cells showed 1700 binding sites for the GP4F cells and 3750 binding sites for the HAb-2 cells, with effectively the same liposome-cell binding constant, about 7 x 10(10) M-1. Binding was specific for glycophorin on the liposomes and HA expressed on the cells. A competition experiment employing toxin-containing and empty liposomes allowed us to quantitate the number of liposomes that fused per cell, which was a small constant fraction of the number of bound liposomes. For the HAb-2 cells, about 1 in every 70 bound liposomes fused and for the GP4F cells about 1 in every 300 bound liposomes fused. Hence, the HAb-2 cells showed 4.4 times more fusion per bound liposome, even though the surface density of HA was only 1.9 times greater. We conclude the following: (i) One HA trimer is not sufficient to induce fusion. (ii) The HA bound to glycophorin is not the HA that induces fusion. That is, even though each HA has a binding and a fusion function, those functions are not performed by the same HA trimer.     

Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.