INTERACTIONS BETWEEN LIPID VESICLES AND CELL MEMBRANES* R. C . MacDonald, V. M. Carr, R. I. MacDonald, P. P. Carballo, and J. A. Fisher Department of Biological Sciences Northwestern University Evanston, Illinois 60201
INTRODUCTION The importance of membrane fusion with respect to a variety of cellular functions has long been appreciated. Interest in the mechanism by which membranes fuse, however, has grown largely as the potential experimental and clinical uses of artificial and natural membrane-bound vesicles have begun to be realized. For example, an investigator's ability to introduce foreign substances into cells by fusing them with liposomes containing those substances would appear to resolve problems of genetic engineering and drug therapy.l The study of eukaryotic cell genetics has also been advanced by the virus- or liposome-induced fusion of genetically unlike cells.' The study of the mechanism of membrane fusion has proceeded along two main avenues. The investigation of the composition and changes thereof in normally fusing cells such as differentiating myoblasts and in eukaryotic cells pathologically induced to fuse by paramyxoviruses represents one approach to the problem.* Another approach involves the use of chemical agents to fuse cells. In the latter case, the presumption is that identification of compounds that cause fusion will enable the recognition of their common characteristics. Thus, it will become apparent which elements of the membranes of fusing cells are critical to the fusion process. Although that presumption now appears untenable, we will attempt to show that the approach it dictated has led to a better understanding of the complexities of membrane fusion. The induction of fusion by defined chemical compounds has been accomplished with hydrophobic compounds such as retinol, oleic acid, and lysolecithin or with liposomes composed of phospholipids and the hydrophobic comp o u n d ~ . ~ -There " is an obvious difference in the physical states of these two classes of reagents when they are dispersed in water, namely, that the latter present to the cell membrane a structure not greatly different from that membrane, whereas the former are present as micellar dispersions or microemulsions. Although it is not clear that cell fusion induced by these two classes of agents is necessarily different, the use of liposomes offers a clear advantage in that they need not be composed entirely of bilayer-forming lipids. Thus, one can incorporate agents such as long-chain cations into liposomes, which thus acquire a positive charge so as to cause agglutination of cells, the event that must obviously precede fusion. Perhaps more importantly, the liposome is a more tract-
* This investigation was supported by United States Public Health Service Grant No. A1 11428. 200 0077-8923/78/0308-0200$01.75/0@ 1978,New York Academy of Sciences
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able delivery system for some fusogens, allowing better control of the concentrations of those fusogenic and potentially lysogenic substances. By varying the proportions of lipid backbone and fusogen in the liposome, one can more easily correlate the amount of fusogen with its effect. This communication describes the use of liposomes composed of a phospholipid bilayer backbone, a positively charged component, and a third ingredient, either a “fusogen,” a compound with a known effect on biological membranes, or simply a compound which on the basis of its structure was expected to have some perturbing effect on membranes. A surprisingly wide variety of compounds have been found to endow phosphatidylcholine-stearylamine liposomes with the capacity to induce cell fusion. The fact that this group of effective “fusion factors” represents such a broad range of chemical structures has foiled attempts to identify a unique chemical entity in the cell membrane critical to its ability to fuse with another membrane. On the other hand, the existence of a common end result for a widely divergent series of compounds suggests that these compounds may be fusogenic by virtue of their effects on membrane permeability and/or cytoskeleton which can be exerted by a variety of mechanisms. COMPOUNDS
THAT
ENABLE PHOSPHATDYLCHOLINE-STEARYLAMINE
LIPOSOMESTO FUSEERYTHROCYTES We have previously shown that di-isostearoylphosphatidylcholine (PC) stearylamine (SA) liposomes cause agglutination of cells in suspension.’ Agglutination, expressed as the hemagglutination titer of the agglutinating agent, is absolutely dependent upon the presence of SA. According to a variety of criteria-including effects of pH and ionic strength variation and correlations between surface potential of both liposomes and cells-agglutination of erythrocytes could be ascribed entirely to electrostatic interactions of positively charged liposomes and negatively charged cell surfaces. PC-SA liposomes, although they agglutinate Ehrlich ascites cells, do not cause fusion of these cells. If lysophosphatidylcholine (LPC) is added to the liposome-generating mixture in addition to PC and SA, the resultant liposomes can induce cell fusion.s Up to the point at which cell lysis becomes substantial, PC-SA-LPC liposomes induce fusion in proportion to LPC and SA content. In the presence of high molecular weight dextran, these liposomes also induce fusion of chicken erythrocytes. Although electron microscopy revealed that PC-SA-LPC liposomes do fuse with cell membranes, we could not determine whether cell fusion resulted from simultaneous fusion of the cells with one liposome or whether incorporation of liposome constituents into cell membranes predisposed the latter to fuse with each other.8 Acquisition of liposome constituents, especially LPC, could have occurred by fusion of the liposome with the cell membrane or by release of constituents from a liposome attached to the cell surface and subsequent incorporation into the cell membrane. If the former were the prevalent mechanism, we would expect that cell fusion via simultaneous fusion with a liposome would predominate, whereas if liposome constituents were to enter cell membranes by diffusion upon contact, we would expect cell fusion to occur by direct interaction of cell membranes. To distinguish between these two possibilities, as well as to provide useful information on mechanisms of membrane fusion, one
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might determine conditions under which liposomes fuse with cell membranes. In theory this can be done by measuring the amount of marker free in the cell cytoplasm, the marker having been previously confined to the aqueous compartment of the liposomes. If marker appears to be transferred from liposomes to cells under conditions in which endocytosis of intact liposomes does not occur and in which leak of the marker from the liposome into the medium and subsequently into the cell is negligible, then liposome-cell membrane fusion can reasonably be assumed to have occurred. In recent months, attempts to demonstrate and quantitate fusion of human erythrocyte ghosts with PC-SA-LPC liposomes have been undertaken. The system on which we expended most effort was based on the appearance of a fluorescent compound within the ghost. We loaded liposomes with the enzyme aryl sulfatase and loaded ghosts with umbelliferone sulfate, a fluorogenic substrate of this enzyme. The availability of inhibitors of the sulfate transport pathway, e.g., stilbene derivatives or dipyrimadole, a membrane-impermeant inhibitor (human and guinea pig serum contain a potent nondialyzable inhibitor-perhaps heparin-of aryl sulfatase) , as well as the opportunity to assay loss of substrate from the ghosts by adding enzyme to the medium, provided the means of potentially distinguishing interaction of enzyme and substrate as a result of fusion from interaction as a result of conditions other than fusion of liposome with erythrocyte ghost. Measurement of the fluorescence of individual ghosts with a microspectrofluorometer would then permit quantitation of liposome-ghost fusion. A variety of technical problems were encountered in this study, and although we are still uncertain that all of these were satisfactorily solved, it appears that if PC-SA-LPC liposomes do fuse with erythrocyte ghosts, then it is less, perhaps much less, than one volume of liposome contents per ten volumes of ghost contents. Subsequent to this investigation we turned our attention from LPC to other substances which, when combined with PC and SA, might yield liposomes with an unmistakable capacity to fuse with cell membranes. We thus resorted to the “shotgun” approach, since LPC appeared insufficiently fusogenic. We decided to prepare liposomes containing PC, SA and one of a rather large selection of compounds that were either known to have specific effects upon membranes or that might, by a modest stretch of the imagination, be expected to have effects on either the structure o r function of cell membranes. A fourth component, a fluorescent lipid-soluble dye (dioctadecylcarbocyanine toluene sulfonate; D ) was also to be included and we would then simply monitor the interaction of these liposomes at various doses and under various conditions of incubation. Given fusion of a typical multilayered liposome with a cell, the outermost layer of the liposome should appear as a patch of fluorescence on the cell surface and a fluorescent (n-1) layered liposome would appear inside the cell. Presumably the (n-1) liposome, because of its positive charge would be quickly covered by hemoglobin and would therefore float freely in the cytoplasm. If the liposome were to induce cell fusion, this might be observed as it occurred. If nucleated erythrocytes were used, cell fusion would be obvious from the appearance of giant cells or polykaryons as well as the appearance of fluorescent liposomes within the cell. TABLE1 is a list of the compounds that were tested in liposomes as the fourth constituent in addition to PC, SA, and D. For all initial tests the ratio of PC:SA:D was held constant at 100:6:1, and the proportion of the potentially fusogenic compound varied over a range-in most cases from 450 to 1/40 the amount of PC. As may be seen from the TABLE,a rather large proportion of
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the compounds tested are listed as beii g cffective in causing human erythrocyte fusion. 1 to be accurate with respect to the capacity of the comWe believe TABLE pounds to endow otherwise inert liposomes with the ability to induce cell fusion under our test conditions. It should be noted, however, that in about half of the instances in which positive results are indicated, cell fusion was not observed directly but was inferred from the appearance, after an incubation period of a few hours to a day or more, of cells or ghosts with diameters significantly (by a factor of 1.5 or more) larger than those of untreated cells. Human erythrocytes were used because of their availability, so the appearance of polykaryons was precluded. The amount of PC present in the liposomes would form a bilayer with an area that exceeds that of the erythrocyte by a factor of about 5, so that abnormally large cells could be produced by incorporation of PC into
TABLE 1 SUBSTANCES TESTEDFOR AN ABEITYTO ENDOWPC-SA LIPOSOMES WITH THE CAPACITY TO INDUCE HUMANERYTHROCYTE FUSION Strong Positive Effect
Weak Effect
No Effect
Gramicidin N y statin Alpha-tocopherol Strychnine Glycerol mono-oleate Retinol Octane Hexane Oligomycin Vitamin K1 Tetraphenylalanine Polyleucine Leucylglyclphenylalanine PolyphenyIalanine Bacitracin Ouabain
Squalene Heptadecane Decane Dicyclohexyl Chloroform Cytochalasin B
Triton X-100 Brij 58 Lysolecithin Vinblastine
the cell membrane without cell-cell fusion. Although large-scale transfer of liposomal lipid to cell membranes could have ensued in individual instances, evidence to be presented indicates that cell growth occurred in all instances predominantly by the single mechanism of cell fusion. The inclusion of compounds in the ambiguous and negative categories in TABLE 1 should be regarded as provisional. Work done after completion of the summary revealed that the initial test conditions were very probably not optimal with respect to the pH and divalent ion concentration of the medium. In addition, it is possible that an increase in SA content would move a given liposome composition from a lower to a higher category in the table. For example, the supposedly nonionic detergents Brij 58 and Triton X-100evidently contained sufficient anionic impurity to neutralize a portion of the SA. Liposornes with a high content of these detergents were noticeably inferior aggluti-
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nating agents, and had the SA content of Brij or Triton containing liposomes been increased, they might have been more fusogenic. LPC, which is also in the negative or ineffective category by the present test, is effective under other conditiom6 Looking at the group of effective compounds in TABLE1, we are hardpressed to say what these substances have in common that would account for their effectiveness. Examination of the structures of these compounds, which vary from an alkane of molecular weight less than 100 daltons to polypeptides of molecular weight in excess of 40,000 daltons, would suggest that the fusogenic compounds have nothing in common. It may well be that the members of this group act by many different modes, but the morphologic changes undergone by erythrocytes subjected to liposomes containing these agents suggest that if such is the case, the net result of the action of all of these liposomes is the same. The morphologic change that is common to all cells treated with liposomes effective in initiating fusion is the assumption, prior to fusion, of a spherical shape by the target cell with a diameter (8.5 microns) equal to that of the major diameter of a normal discoid erythrocyte. Liposome treatment causes human erythrocytes initially to become echinocytes or spherocytes, both of which are refractile and have diameters smaller than the major diameter of the discoid cell. Cells that do not eventually fuse retain this morphology; those that eventually fuse swell by several microns to become weakly refractile, spherical cells which appear dark gray through phase-contrast optics. This swelling has also been noted by Lucy and coworkers to be a prelude to fusion induced by substances such as retinol in aqueous dispersion.’ The common characteristic of liposomes containing the effective compounds listed in TABLE 1, then, seems to be their ability to initiate swelling. Since swelling is evidently an osmotic phenomenon, “effective” liposomes share the ability to perturb the volume-regulating capacity of the cell and perhaps to impair the normal function of the cell’s cytoskeleton. Additional arguments for this proposition are given below. Before concluding this section, we should point out that the second event we had hoped to see-namely, fusion of a liposome with an erythrocyte-was never observed. Liposomes were easily visible due to their content of the fluorescent dye, but they remained adherent to the surface of the cells for times that exceeded the experimenter’s patience. The possibility cannot be excluded, of course, that liposomes too small to see did fuse with the cell membranes. The membranes of swollen cells and fused cells were invariably uniformly fluorescent, suggesting that, by some means, these membranes had acquired at least the dye from liposomes. The bulk of the fluorescence of such cells, however, was confined to a small patch on the surface where liposomes might have congregated. Such regions on human erythrocytes are apparently analogous to the region of the surface of swollen and fused chicken erythrocytes that borders the nucleus (see below). OF ERYTHROCYTE FUSION INDUCED BY QUANTITATION GRAMICIDINCONTAINING LIPOSOMES
Gramicidin ( G ) was the first compound found to endow PC-SA liposomes with the ability to induce cell fusion. It remains one of the most effective agents. For these reasons the bulk of our investigations have been carried out with PC-SA-G liposomes.
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The majority of the investigations reported here deal with human erythrocytes. In order to quantitate the effects of the interactions of liposomes with these cells, we expressed the observed increases in size as an “apparent fusion index” (AFI) . The AFI was calculated by the following method. After a given experimental treatment, the diameters of a sample of the cell population were measured under the phase microscope. A histogram of cell diameters of 50 or more cells was prepared and the area represented by each class of diameters was calculated. These areas were then added and the sum divided by the area of erythrocytes swollen in extremely hypotonic medium. (Areas obtained were 145 p* for human erythrocytes and 385 p 2 for chicken erythrocytes.) The result is the AFI. Its meaning is the same as the fusion index defined by Kohn? if it is assumed that liposome constituents do not become incorporated into the cell membrane, which would result in an increase in area independent of cell fusion. The question of whether liposome lipids do contribute significantly to the increase in cell diameters observed is important relative to the actual frequency of cell fusion induced by liposomes. Some estimates were made with nucleated (chicken) erythrocytes in order to compare fusion indices obtained from nuclear and cell counts with the AFI obtained by the area measurement procedure. Although the comparison is not as straightforward as expected for reasons outlined below, it is clear that there is not wholesale transfer of liposome constituents to erythrocyte membranes. 1 presents data on the number of nuclei contained The upper part of FIGURE within cells of various diameters (given in microscope-eyepiece reticle units) that are observed after treatment of a chicken erythrocyte suspension with PC-SA-G-D liposomes. Each dot on the graph represents one cell of the diameter and the number of nuclei as given by the coordinates of the dot. Before lysing, chicken erythrocytes swell to diameters of 7 units. Using this diameter for a single cell and assuming the area of fused cells to be the sum of the areas of their constituent cell membranes (fused cells are spherical; hence water must flow into the cells during fusion to make up the volume deficit), we drew the dotted line of the graph as the expected relationship between nuclear number and cell diameter. It is seen that the data correspond reasonably well with predicted values. Liposome treatment causes extensive crenation; crenated cells account for most of the diameters under 7 units. 1 is a frequency distribution for cells Whereas the upper part of FIGURE containing 100 nuclei, the bottom part of the figure is an appearance distribution for several thousand cells. A dot on the lower part signifies that among the cells measured, at least one with the diameter and nuclear number coordinates of the dot was seen. These data reveal that when a large number of cells are surveyed, some cells are found with numbers of nuclei far out of correspondence with cell diameter. The explanation of this phenomenon requires a consideration of the stages of cell fusion, which we will discuss briefly. As noted, the obvious result of liposome treatment subsequent to agglutination is crenation. Whereas human erythrocytes swell symmetrically from the crenated state to become spherical, crenated chicken erythrocytes swell asymmetrically by first blowing a “balloon,” i.e., at some point on the surface of the cell, usually on the long side near the nucleus, a bleb will appear and grow until it resembles a basketball pressed against a football (the nucleus being the football). If cell fusion occurs at this stage, it is invariably the basketball portion that participates. Fusion may also occur at a later stage, at which time the
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