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Contact-Dependent, Immunecomplex-Mediated Lysis of Hapten-Sensitized Liposomes Bruce Babbitt,tJ Lisa Burtis,t Patrick Dentinger,? Panayiotis Constantinides,+*oLarry Hillis,' Barbara McGir1,ll and Leaf Huang'SllJ LipoGen, Inc., 10515 Research Drive, Knoxville, Tennessee 37932, and Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996-0840. Received October 26, 1992
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Large unilamellar liposomes (d 160 nm) composed of dioleoylphosphatidylethanolamine(DOPE) (80-90%), a negatively charged phospholipid stabilizer (10-20%), and a small amount (0.1-1%) of a haptenated lipid are unusually stable in divalent cation-free isotonic buffer at pH 7.4. The liposomes can be stored under this condition a t 4 "C for a t least 6 months without any detectable leakage of the entrapped fluorescent dye calcein. However, the liposomes undergo a rapid (1h) aggregation and lysis reaction in the presence of free bivalent anti-hapten antibody. The liposome destabilization was immunospecific in that it did not occur with the normal IgG or in the presence of excess free hapten. Liposome lysis was always accompanied by liposome aggregation. Aggregation and lysis of the liposomes was completed in 5 min if the incubation temperature was raised to 70-80 "C. Replacing DOPE with dioleoylphosphatidylcholine in the liposomes did not abolish the liposome aggregation, but no liposome lysis was observed even at 80 "C. Since liposome aggregation appeared to be a necessary (but not sufficient) prerequisite for liposome lysis, we have named this new class of liposome "contact-sensitive liposomes". The immunodiagnostic potential of the contact-sensitive liposome was demonstrated with liposomes containing theophylline-DOPE. The aggregation and lysis of the liposomes induced by a monoclonal anti-theophylline antibody could be inhibited by free theophylline at concentrations of therapeutic significance. The observation could be the basis of a homogeneous assay for theophylline.
INTRODUCTION The equilibrium state of an unsaturated phosphatidylethanolamine (PEY such as dioleoyl-PE (DOPE) at physiologicalpH and temperature is the reverse hexagonal (HII) phase (reviewed by Gruner et al., 1985). This is because the phosphorylethanolamine head group is relatively poor in attracting bound water (Cevc and Marsh, 1985; Sen and Hui, 1988) and the hydrophobic, cisunsaturated acyl chains are relatively bulky in size (Gruner et al., 1985). However, stable bilayer liposomes can be prepared by mixing DOPE with appropriate amounts of other amphiphiles which can increase the interfacial hydration. Amphiphiles, termed "liposome stabilizers", used for this purpose include fatty acids (Diizgiines et al., 1985; Collins et al., 19901, fatty acyl amino acid (Connor et al., 1984), gangliosides (Tsao and Huang, 1985; Pinnaduwage and Huang, 1988), acidic phospholipids (Tari and Huang, 1989; Nayar and Schroit, 1985; Wang and
* Corresponding author. +
LipoGen, Inc.
* Present address: Cellcor Therapeutics,Inc., 200 Wells Ave.,
Newton, MA 02159.
5 Present address: SmithKline Beecham Pharmaceuticals, Drug Delivery Department,P.O. Box 1539, King of Prussia, PA 19406-0939. 11 University of Tennessee. 1 Present address: Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Abbreviations used: biotin-PE, N-biotinylphosphatidylethanolamine; CE, hexadecyl cholestanyl ether; DMPG, dimyristoylphosphatidylglycerol; DOPA, dioleoylphosphatidic acid; DOPC, dioleoylphosphatidylcholine;DOPE, dioleoylphosphatidylethanolamine; DOPG, dioleoylphosphatidylglycerol;DOPS, dioleoylphosphatidylserine;LUVET, large unilamellar vesicles prepared by extrusion; MPB-PE, N-[4-@-maleimidophenyl)butyryl]phosphatidylethanolamine;PE, phosphatidylethanolamine.
1043-1802/93/2904-0199$04.00/0
Huang, 19841, acidic (Leventis et al., 1987; Collins et al., 1990) and basic (Felgner et al., 1987) double-chain amphiphiles, and amphipathic proteins (Taraschi et al., 1982; Pinnaduwage and Huang, 1989). Particularly interesting is the DOPE liposomes stabilized by amphiphiles which are capable of forming an immune complex. For example, liposomes comprising DOPE and N- [ (dinitropheny1)caproyll-PE rapidly lyse in the presence of antidinitrophenol antibody which has been immobilized on a solid support (Ho and Huang, 1985). DOPE liposomes stabilized with glycophorinA also lyse when the liposomes come into contact with a glass surface coated with antiglycophorin antibody (Ho and Huang, 1985). Similarly, liposomes containing DOPE and palmitoyl antibody against the gD glycoprotein of the herpes simplex virus also rapidly lyse in the presence of intact virus particles (Ho et al., 1986a,b,1987b,19881,cell membranes containing viralglycoproteins (Ho et al., 1986a,1987a),or latex beads covalently coated with purified gD (Ho et al., 1988). In all cases, multivalent binding between the liposome and the immobilized antigen or antibody is required for lysis. This is probably because the stabilizer molecules are laterally aggregated in the liposome membrane which triggers a bilayer-t0-H.n phase transition, leading to the destabilization of the liposomes (Ho and Huang, 1985;Ho et al., 1986a). This unusual type of liposome has been named "target-sensitive liposomes" (Ho et al., 1986a),and their potential uses in immunodiagnosis (Ho et al., 1986b, 1987b) and therapeutic drug delivery (Ho et al., 1987a) have been demonstrated. We now report a related but different observation that DOPE liposomes, stabilized by an amphiphile and additionally containing a haptenated lipid, aggregate and lyse in the presence of free anti-hapten antibody. There is no requirement for immobilizing the antibody on any solid support. We have shown that the liposome lysis is immunospecific and can be accelerated by incubation at 0 1993 American Chemical Society
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a high temperature such as 80 "C. Liposome aggregation is a prerequisite for lysis, because if the aggregation is inhibited by free hapten no liposome lysis occurs even at 80 "C.Furthermore, the inhibition of liposome aggregation and lysis by free hapten depends on the hapten concentration. This last observation is the basis of using this novel type liposomes as a reagent in the homogeneous immunoassays,such as the one demonstrated in this report for theophylline.
measured by adding a 5-pL liposome sample to 3 mL of PBS in a quartz cuvette and reading light-emission intensity on a Perkin-Elmer LS-5B luminescence spectrometer at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Total calcein fluorescence was monitored after the addition of 10 pL of 10% Triton X-100 to lyse the vesicles and release the entrapped dye. Percent fluorescence quenching of the entrapped calcein in the liposomes was calculated using the following equation:
EXPERIMENTAL PROCEDURES
Materials. DOPE, DOPS, DOPA, DOPC, DMPG, DOPG, MPB-PE, and biotin-PE were obtained from Avanti Polar Lipids, Inc. Theophylline-DOPE was prepared by the condensation of 8-(2-~arboxyethyl)theophylline and DOPE (Hashimotoet al., 1985). The theophylline derivative was prepared by the general method of Cook et al. (1976). The theophylline-PE gave the same physical data as previously reported (Haga et al., 1981). The goat anti-biotin antibody, calcein, and protein A-agarose were supplied by Sigma Chemical Co. The mouse monoclonal (IgG1) anti-theophylline antibody was purchased from Immunoresearch, Inc. Liquid scintillation cocktail, Scintiverse, was purchased from Fisher Scientific. Biogel A15m was obtained from Bio-Rad Laboratories. The 13H]CE was synthesized as previously described (Pool et al., 1982). The [3H]cholesteryl oleate was supplied by New England Nuclear. The goat anti-biotin antibody was dialyzed against PBS for approximately 48 h and then filtered through a 0.2-pm Gelman Sciences Acrodisc filter to a final concentration of approximately 2 mg/mL. Preparation of Liposomes. Lipid preparations were made of either DOPC or DOPE as the bulk phospholipid (80-90 mol %), a stabilizer (10-20 mol % of biotin-PE, MPB-PE, DOPS, etc.), and a haptenated lipid (0.1-1.0 mol % of biotin-PE or theophylline-DOPE). In some samples I3H1CEwas added to the lipid as a tracer. Lipid films were obtained by evaporation of the solvent from a chloroform-lipid mixture using a stream of Nz gas and then placing the lipids in a desiccator at reduced pressure for a minimum of 4 h. Lipids were hydrated a t room temperature for at least 30 min in 50 mM calcein in PBS at a lipid concentration of 10 mM. Calcein, 2',7'-bis[ [(carboxymethyl)aminolmethyllfluorescein is a selfquenching dye that, at concentrations greater than 5 mM, undergoes progressive collisional fluorescence quenching (Allen, 1983). It has been widely used for analysis of liposome stability and/or leakage since it is not necessary to separate the liposomes from released dye in order to quantitate lysis. The calcein solution was prepared a t 50 mM in distilled H2O and the pH adjusted to 7.4 with 10 N NaOH. The osmolarity of the calcein was adjusted with 1OX PBS to that of human serum, approximately 330 mosM. The lipid suspensions were formed by rigorous vortexing and sized by extrusion 10 times at 200 psi with a Lipex extruder through Nuclepore polycarbonate membranes (0.2-pm pore size). If small unilamellar liposomes were needed, the suspension was sonicated with a bathtype sonicator from Laboratory Supplies, Inc., for 7 min. Suspensions were left at room temperature for 1-2 h to allow for bilayer annealing before being fractionated by column chromatography. The size of the liposomes was determined by laser light scattering using the Coulter N4SD submicron particle analyzer. Free, unentrapped calcein was removed from liposomes by gel filtration utilizing a 10 mL Bio-Gel A15m column, preequilibrated, and eluted with PBS, pH 7.4. This gave a working lipid concentration of 7 mM. Initial calcein fluorescence was
Ft-Fi 7% quenching = -x 100
Ft where Fi is the total fluorescence of the liposomes without detergent and Ftis the total fluorescence after Triton had been added to release all of the entrapped calcein. Freshly prepared, unlysed liposomes usually showed about 80 % fluorescence quenching. Antibody-Mediated Liposome Lysis. Three to 10r L (21-70 nmol of lipid) of liposomes containing entrapped calcein were added to 200 pL of PBS, pH 7.4. Various concentrations of antibody were added, and incubation was carried out at various temperatures for different periods of time as specified in Table I or the figure captions. At the end of incubation the entire sample was diluted to 3 mL with PBS and the calcein fluorescence was measured as described above. The percent liposome lysis was calculated using the following equation:
F-Fi Ft-Fi
% lysis = -x 100
where Fi and F are the fluorescence of the liposomes before and after incubation, respectively. Ft is the total fluorescence after Triton had been added to release all of the entrapped calcein. Nonspecific Liposome Lysis by Osmotic Pressure. Five microliters (35 nmol) of liposomes with 50 mM entrapped calcein, containing either 85 mol % of DOPE and 15 mol % of DMPG as stabilizer and 1 mol % of biotin-PE or 80 mol % of DOPE and 20 mol % of DOPG as stabilizer and 1mol 5% of biotin-PE, were incubated in a final volume of 205 pL for 15 min at room temperature, 22 "C,at various aqueous dilutions of a stock PBS buffer, pH 7.4. The dilutions resulted in a range of osmotic strengths of the buffer from 45 mosM/kg of HzO to approximately 3360 mosmol/kg of H2O. Calcein fluorescence of the liposomes was measured as above. Nonspecific Lysis of Liposomes by Divalent Cations. Five microliters (35 nmol) of liposomes containing 50 mM entrapped calcein, composed of 90 mol 7% of DOPE and 10 mol % of MPB-PE as stabilizer and 1mol % of biotin-PE as the ligand, were mixed with various amounts of divalent cations (chloride salt) in a final volume of 205 p L of PBS, pH 7.4. The samples were incubated at room temperature, 22 "C, for 15 min before the liposome lysis was measured as described above, except that the diluted PBS contained 1 mM EDTA. Electron Microscopy. LUVET of DOPEMPB-PE: biotin-PE (9O:lO:l) (about 50 nmol of total lipid) were incubated for 1h a t 37 "Cwith 2.5 pg of anti-biotin antibody in a total volume of 15pL. The liposomes were negatively stained with 1%uranyl acetate and viewed in a Hitachi 600 electron microscope operated at 75 kV. RESULTS
We and others have previously shown that a number of amphiphiles, especially the charged ones, can stabilize the
Contact-Senslve Llposomes
Table I. Phospholipid Stabilizers for DOPE Liposomes minimum mole % minimum mole % required to form required to form stabilizer stable liposomesa stabilizer stable liposomesa 10 DMPG 15 biotin-PE 10 DOPA 20 MPB-PE DOPS 15 DOPG 20 15 DPPG
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a Stable liposomesare defied as those showingno loss of entrapped calcein after storage at 4 "C for 30 days.
small unilamellar vesicles of DOPE (Diizgiineset al., 1985; Connor et al., 1984;Tsao and Huang, 1985;Tari and Huang, 1989; Wang and Huang, 1984; Collins et al., 1990). Table I shows that various synthetic phospholipids can also stabilize DOPE liposomes of larger size, i.e. average diameter of 162 33 nm prepared by an extrusion method. These liposomes have been named LUVET (Hope et al., 1985). Also shown in the table are the minimum amounts of these phospholipid stabilizers which are required for the formation of stable liposomes. Stable liposomes are defined as those which have no loss of entrapped calcein after storage of the liposomes at 4 "C for at least 30 days. As can be seen from the table, two N-modified PES,i.e. biotin-PE and MPB-PE, had the highest stabilization activity; only 10 mol % was required to form stable liposomes. The stabilization activity of the phospholipid stabilizer does not seem to correlate with the charge content of the molecule, since the doubly negatively charged DOPA was not as active as other singly negatively charged phospholipids such as DOPS. Furthermore, disaturated PG (DPPG and DMPG) were more active than the unsaturated PG (DOPG). This is in agreement with our previous results using small unilamellar vesicles (Tari and Huang, 1989). The unsaturated chains tend to favor the HIIphase more than the saturated chains, due to the larger molecular volumes occupied by the unsaturated chains (Gruner et al., 1985). LUVET composed of DOPE and a phospholipid stabilizer could additionally contain 0.1-1 mol % of a haptenated lipid, such as biotin-PE or theophylline-DOPE, without affecting their size distribution, calcein-entrapment efficiency, and stability. These liposomes remained lysable by antibody (see below) even after a prolonged storage time (at least 6 months at 4 "C). However, the stability of the liposomes was dependent on the osmolarity of the storage medium. Data in Figure 1 show that the liposomes were most stable in the iso-osmotic medium. Both hypo- and hyperosmotic conditions promoted the leakage of the entrapped calcein even after a 15-min incubation a t room temperature. Large liposomes are known to be osmotically sensitive (Bangham et al., 1967). Our data agrees with this conclusion. The stability of the liposomes was also dependent on the divalent-cation content of the medium. Figure 2 shows the result of an experiment in which different amounts of three different divalent cations, i.e. Ca2+,Mg2+,and Mn2+, were added to the medium. It is clear that Mn2+was the most potent one to induce the liposome lysis; greater than 80% lysis was observed at concentrations as low as 1 mM. Ca2+had the second highest activity, requiring more than 3 mM to induce a significant liposome lysis. Mg2+was fairly ineffective, requiring much higher concentrations to achieve the same level of liposome lysis. The relative potency of divalent cations to induce the bilayer destabilization is probably related to their differential activities to dehydrate the bilayer, which leads to bilayer aggregation
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Figure 1. Stability of liposomes under osmotic stress. LUVET composed of D0PE:DMPG:biotin-PE (8515:l) (m) or DOPE: DOPGbiotin-PE (80201) (+) were incubated for 15 min at 22 "C in phosphate buffers of various osmolarity. Liposome lysis was measured as release of the entrapped calcein as described in Experimental Procedures. Approximately 400 mosM/kg of H2O was the iso-osmotic condition.
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Divalent Cation (mM) Figure 2. Stability of liposomes in solutionscontainingdivalent cations. LUVET composed of DOPEMPB-PE:biotin-PE (90: 101) were incubatedfor 15 min at 22 "Cin PBS buffer containing various amounts of Mg2+(a),Ca2+( O ) ,or MnZ+(A). Liposome lysis was measured as release of the entrapped calcein.
and fusion (Portis et al., 1979). We routinely use divalentcation-free PBS for the preparation and storage of the liposomes. LUVET composed of D0PE:MPB-PE (9:1) containing either 0.1 or 1%biotin-PE as a ligand were tested for the effect of antibody on the stability of liposome. Various amounts of a purified goat anti-biotin antibody were mixed with the liposomes and incubated at 37 "C for 1 h. As can be seen in Figure 3, there was a concentration-dependent lysis of the liposomes. The lysis was immunospecific, because it could be blocked by the addition of an excess of free hapten, Le. biotin. Liposome lysis also did not occur when normal goat IgG was used instead of the antibody (data not shown). The extent of liposome lysis depended on the surface density of the ligand; liposomes containing 1% biotin-PE could be lysed with lower concentrations of anti-biotin than those containing 0.1% biotin-PE. Liposomes containing no biotin-PE could not be lysed by the antibody (data not shown). We have noticed that liposome lysis mediated by the anti-biotin antibody was always associated with liposome aggregation as evidenced by the cloudiness or turbidity of the incubation mixture. Turbidity did not occur when excessbiotin was present in the incubation mixture, or when the normal goat IgG was used. The appearance of the incubation
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Antbody (MI Figure3. Antibody-mediatedliposome lysis. LUVET composed of D0PE:MPB-PE (90:10), additionally containing either 196 ( 0 )or 0.1 96 (A)biotin-PE, were incubated with goat anti-biotin antibody for 1 h a t 37 "C. In parallel experiments, 0.2 pmol of free biotin was added to the antibody before mixing with the liposomes (A,m). Liposome lysis was measured as release of the entrapped calcein.
mixture after incubation was of uniform cloudiness with a background of yellowish-green color (due to the leaked calcein). Figure 4 shows the morphology of the aggregated liposomes. The temperature dependenceof the antibody-mediated liposome lysiswas examined. LUVET composed of DOPE D0PS:biotin-PE (85:15:1) were mixed with 10 pg of antibiotin antibody and incubated for 5 min a t various temperatures ranging from 22 to 80 "C. The extent of liposome lysis increased with increasing incubation temperature (Figure 5). A t 80 "C, greater than 80% of the entrapped calcein was released after only a 5-min incubation with the antibody. Extensiveliposome aggregation was also observed. In the absence of the antibody, there was no liposome aggregation nor significant lysis even a t 80 "C. Also shown in Figure 5 is the effect of the phospholipidcomposition on the antibody-mediated lysis of the liposomes. LUVET composed of D0PC:DOPS: biotin-PE (85:15:1) did not lyse up to 80 "C incubation either in the presence or absence of the antibody, despite the fact that these liposomes could be aggregated by the antibody. Note the difference in the experimental conditions between the experiments described in Figures 3 and 5. The experiment described in Figure 3 was done with an incubation at 37 "C for 1h; whereas the experiment described in Figure 5 was done with only a 5-min incubation. Therefore, the antibody-mediated lysis was greatly accelerated at the elevated temperatures. Under the condition of shorter incubation at an elevated temperature, the effect of antibody concentration on the liposome lysis was examined over a wide range of antibody concentration. LUVET composed of D0PE:DOPS:biotinPE (85:15:1) were incubated at 70 "C for 5 min in the presence of anti-biotin antibody up to 200 pg in 210-pL incubation volume (Figure 6). With increasing concentration of the antibody, there was an increasing level of liposome aggregation and lysis which plateaued at an antibody concentration of 20-40 pg. At higher antibody concentrations, the extent of liposomee aggregation and lysis progressively decreased such that less than 20 % dye release was observed at the 200 pg antibody concentration. The overall doseresponse curve is bell-shaped. This result indicated that there was an optimal antibody concentration for the aggregation and lysis of the liposomes. We have examined in more detail the inhibition of the antibody-mediated lysis of liposomes by the free hapten.
Figure 4. Aggregation of liposomes by antibody. LUVET composed of D0PE:MPB-PE:biotin-PE (901O:l)were incubated for 1 h at 37 "C in PBS: (a) in the presence of 2.5 pg of goat anti-biotin antibody and (b) before addition of antibody, viewed with negative-stain electron microscopy. Magnification is 17 OOOX. Bar is 0.5 pm.
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Temperature ( O C ) Figure 5. Effect of incubation temperature on antibodymediated liposome lysis. LUVET composed of D0PE:DOPS: biotin-PE (85:15:1) (0,*)were mixed with (open symbols) or without (solid symbols) 10 pg of goat anti-biotin antibody and incubated for 5 min a t different temperatures. Liposome lysis was measured as release of the entrapped calcein.
In this experiment, LUVET composed of D0PE:DOPS: theophylline-DOPE = 85:15:1 were incubatedwith a mouse monoclonal anti-theophylline antibody at the optimal
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Figure 6. Effect of antibody concentration on the antibodsmidiated liposome lysis at a high-incubation temperature. LUVET comDosed of D0PE:DOPS:biotin-PE (85:15:1) were
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described here contain a phospholipid stabilizer which is itself not an antigen or hapten. An additional antigenic or haptenated lipid is added to the liposomes described here only at very low concentrations (19% or lower). The lysis of Ho and Huang's liposomes require the contact with a multivalent antibody, Le. antibodies immobilized on a solid support such as a glass coverslip; free, bivalent antibody in solution does not lyse the liposomes (Ho and Huang, 1985). In contrast, our liposomes readily lyse in the presence of free IgG antibodies (Figures 3 and 5-7). Thus, the liposomes described in this report represent a new class of liposomes which can be lysed by the formation of the immune complex. Since the liposome lysis requires the aggregation of liposomes, we have named them the "contact-sensitive liposomes". One of the outstanding features of the "contact-sensitiveliposome" is their stability in the absence of liposome aggregation. The liposomes can be stored at 4 OC in a divalent-cation-free, iso-osmotic buffer for prolonged periods of time (Table I, Figures 1 and 2). In the absence of the antibody-mediated liposome contact, either as aresult of the absence of antibody or the presence of excess free hapten, liposome lysis was not observed even at elevated incubation temperatures (Figures 3 and 5). Such an extraordinary stability can be attributed to the proper choice of the liposome stabilizer. Data in Table I indicate that a number of synthetic, negatively charged phospholipids exhibit strong liposomestabilization activities. Among them are biotin-PE, which is also a haptenated lipid, and MPB-PE, which is widely used as a sulfhydryl-reactive lipid for antibody conjugation to liposomes (Martin and Papahadjopoulous, 1982). In addition to the negatively charged phosphodiester bond, both lipids contain a relatively bulky, hydrophilic head group which presumably helps attract interfacial HzO for the stability of the bilayer. Other synthetic phospholipids are less potent in activity, but they are more available commercially and resemble the naturally occurring phospholipids. Free IgG antibody, polyclonal or monoclonal, mediates a liposome lysis which leads to the release of the entrapped fluorescent dye (Figures 3 and 5-7). That such lysis is immunospecific is supported by the facts that it can be blocked by the free hapten (Figures 3 and 7) and that it does not occur in the absence of the specific antibody (Figures 3 and 5). Furthermore, liposome lysis is closely correlated with liposome aggregation (Figure 4). When the aggregation is prevented by the saturation of the antibody to free hapten, no lysis of the liposomes occurs even with an 80 OC incubation (Figure 5). Evidently, the role of the IgG antibody is simply to cross-link the haptencontaining liposomes and to allow a close contact between the aggregated liposomes. This mechanism is supported by the bell-shaped antibody dose-response curve shown in Figure 6. It is well-known that immune complex formation exhibits an optimal antibody concentration as well as an optimal antigen concentration (Bellanti, 1985). Increasing amounts of antibody lead to increasing levels of liposome aggregation, until the antibody is present in excess, at which time only monovalent antibody binding to the hapten, instead of a bivalent cross-linkingof haptens between the neighboring liposomes, occurs. Antibodymediated liposome lysis is dependent on the hapten density of the liposomes; liposomes with a higher hapten density require lower antibody concentrations for aggregation and lysis and vice versa (Figure 3). This result is consistent with the mechanism of liposome cross-linking by the bivalent antibody. Higher hapten density of the liposomes would facilitate a greater chance of antibody-mediated
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Figure 7. Effect of free ligand on the antibody-mediated liposome lysis. Various amounts of free theophylline were incubated with a mouse monoclonal anti-theophylline antibody (0.5 pg) for 5 min a t 22 OC, followed by the addition of LUVET composed of DOPED0PS:theophylline-DOPE (8515:l). The mixtures were then incubated for 5 min at 70 "C before the liposome lysis was measured as release of the entrapped calcein.
concentration for 5 min at 70 "C. Approximately 80% of liposome lysis was measured. However, if the antitheophylline antibody had been preincubated with various amounts of free theophylline for 5 min at room temperature, there was an inhibition of the liposome lysis which was theophylline-concentration dependent (Figure 7). The liposomal theophylline-DOPE used in this experiment was 0.32 nmol. Fifty percent inhibition of the liposome lysis occurred at the free theophylline dose of approximately 0.7 nmol. Thus, it took a comparable amount of free hapten to inhibit the binding of the antibody to the liposomal hapten which presumably is a prerequisite of the liposome lysis. This result indicated that the binding affinity of the liposomal hapten to the antibody was similar to that of the free hapten. DISCUSSION
The principal difference between the liposomes described in this report and those described by Ho and Huang (1985) is in the lipid composition of the liposomes. Although DOPE is the common major phospholipid used in both liposome formulations, Ho and Huang's liposomes contain a t least 12% antigenic or haptenated lipid as a bilayer stabilizer (Ho and Huang, 1985)and the liposomes
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cross-linking of the liposomes and result in a higher level of liposome lysis. It is easily understood that liposome-liposome contact (via aggregation) is a prerequisite of the liposome lysis. Destabilization of DOPE bilayer involves a bilayer-to-HII phase transition which requires the close contact of the neighboring bilayers (Siegel, 1984, 1986). Such a requirement is most conveniently demonstrated by the secondorder kinetics of liposome lysis reaction (Pinnaduwage and Huang, 1988). DOPE lipids at the contact region spontaneously reorganize into the so-called “intermembrane intermediate” (IMI) structures which eventually leads to the extensive HII phase structure (Siegel, 1984). Although we have not yet studied the kinetic aspect of liposome lysis, our data are consistent with this mechanism. Furthermore, the data in Figure 5 indicate that liposomes mainly composed of DOPC, instead of DOPE, did not lyse despite the fact that extensive liposome aggregation had occurred. DOPC does not exhibit the capability of HII phase formation (Gruner et al., 1985). Aggregated DOPC bilayers do not undergo bilayer destabilization as the DOPE bilayers do. Thus, a HII phase forming lipid is a necessary ingredient of the “contact-sensitive liposomes”. The potential application of these liposomes in immunodiagnosis is demonstrated by the data in Figure 7.Lysis of liposomes containing a theophylline hapten can be progressively inhibited by the presence of free theophylline at the concentrations of therapeutic significance. Therefore, an inhibitory, competitive assay for the free theophylline can be theoretically established. The assay would not require any washing, centrifugation, or separation steps and would thus be a homogeneous assay. It would be significantly different from the other liposome homogeneous assays in which a lysis molecule such as complement (Haxby et al., 1969) or cytolysin (Litchfield et al., 1984) is required for liposome lysis. The fact that the “contactsensitive liposomes”are extraordinarily stable makes such an application very attractive. Furthermore, the incubation time is only 5 min, which greatly facilitates the speed of the assay. The immunodiagnostic aspects of the “contact-sensitiveliposomes”deserves further exploration. The haptenated lipid of the “contact-sensitive liposomes”can be substituted with lipid-antibody conjugates. Aggregation of the immunoliposomes in the presence of a multivalent antigen such as virus particles leads to rapid liposome lysis. Such a design of an immunoliposome has recently been reported (Pinnaduwage and Huang, 1992). ACKNOWLEDGMENT
We thank Carolyn Drake and Christine A. Hallahan for excellent help in word processing. LITERATURE CITED Allen, T. M. (1983) Calcein as a tool in liposome methodology. In Liposome Technology (G. Gregoriadis, Ed.) Vol. 3, p 177, CRC Press, Boca Raton. Bangham, A. D., de Gier, J., and Greville, G. D. (1967) Osmotic properties and water permeability of phospholipid liquid crystals. Chem. Phys. Lipids I , 225. Bellanti, J. A. (1985) Immunology 111, W. B. Saunders Co., Philadelphia. Cevc, G., and Marsh, D. (1985) Hydration of noncharged lipid bilayer membranes: theory and experiments with phosphatidylethanolamine. Biophys. J. 47, 21. Collins, D., Connor, J., Ting-Beall, H. P., and Huang, L. (1990a) Protons and divalent cations induce synergistic but mechanistically different destabilization of pH-sensitive liposomes composed of phosphatidylethanolamine and oleic acid. Chem. Phys. Lipids 55, 339.
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