Liposomes and influenza viruses as an in vitro model for membrane interactions II. Influence of vesicle size and preparation methods

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European Journal of Pharmaceutical Sciences 1 (1994) 333-341

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Liposomes and influenza viruses as an in vitro model for membrane interactions II. Influence of vesicle size and preparation methods Susanne Ott a'*, Peter Schurtenberger b, Heidi Wunderli-Allenspach a aDepartment of Pharmacy, Biopharraacy, Swiss Federal Institute of Technology, CH-8057 Zffrich, Switzerland bPolymer Institute, Swiss Federal Institute of Technology, CH-8092 Zfirich, Switzerland (Received 8 October 1993; accepted 28 March 1994)

Abstract

As previouslydemonstrated, membrane fusion and transfer of prodrug-type moleculesbetweenmembranes can be studied with the octadecylrhodamine B chloride (R18) marker. We use this assay with PR8 influenza viruses and small (SUV) or large (LUV) unilamellar liposomes containing the virus receptor GD1aas interacting partners in order to analyse the effectof membrane curvature on fusion and transfer. Computer analysis revealed no differencein the kinetics of fusion between PR8 with large or small liposomes respectively. Lipid transfer, however, is about 5 to 6 times faster from SUV than from LUV. We also tested the influence of the production method for liposomes, namely detergent dialysis, sonication and extrusion on membrane interactions. Fusion is not affected by either, whereas transfer is slower with liposomes produced by sonication. Key words: Fusion; Lipid transfer; Liposomes (SUV, LUV); Fluorescence marker R18

Abbreviations: GDla, disialoganglioside; HA, hemagglutinin; PC, phosphatidylcholine; R18, octadecylrhodamine B chloride; SUV, LUV, small and large unilamellar vesicles.

1. Introduction

In recent years, much interest has been focused on interactions between lipid bilayers. Such processes play an important role under physiological conditions (e.g. endocytosis, exocytosis, protein trafficking), in pathological situations (e.g. infection with enveloped viruses) and also for the prospective use of artificial membrane vesicles (liposomes) as drug carriers. Basically there are two mechanisms by which membrane-incorporated, lipophilic compounds can leave a liposome: (1) fusion between liposomes and target membranes, e.g. specific virus receptor-mediated fusion and (2) spontaneous transfer to other membranes or lipophilic sites. With *Corresponding author. Dept. Pharmacy ETH, Winterthurerstr. 190, CH-8057 Ziirich, Switzerland. Tel. (+41-1) 2576046; Fax (+41-1) 2625223. 0928-0987/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0928-0987(94)00009-0

hydrophilic drugs, leakage tends to be a problem. However, this can be dealt with by coupling such molecules to lipophilic side chains. These types of "prodrugs" can then be incorporated as lipophilic compounds into lipid bilayers. To develop efficient drug targeting systems with liposomes, several parameters which potentially influence membrane interactions have to be controlled. Of particular interest are the impact of vesicle size and of the production methods for liposomes (e.g. detergent dialysis resulting in residual detergent, sonication). For the study of membrane fusion processes, various in vitro assays have been developed in the past. They are based on quenching ph~omena of fluorescent dyes (for review see Diizgiines and~entz, 1988). In our group, we have adapted such an in v~tro fusion assay in order to study in parallel the kinetics of specific, receptor-mediated fusion and of non-specific transfer (Ott and Wunderli, 1994). The assay comprises influenza viruses and virus receptor-containing liposomes as interacting partners. Influenza virus fusion is mediated by the viral hemagglutinin (HA) protein, which is only active at low pH (Maeda et al., 1981; White et al., 1983). In a neutral milieu or after inactivation of HA, fusion does not

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S. Ottet al./European Journal of Pharmaceutical Sciences 1 (1994) 333 341

occur. Transfer, however, is found at low as well as at neutral pH (Wunderli and Ott, 1990). Thus, depending on the assay conditions, either fusion or lipid transfer will prevail. Quantitation of membrane interactions is achieved with the octadecylrhodamine B (R18) fluorescence marker (Hoekstra et al., 1984), which is quenched at high concentrations. From its molecular structure, R18 resembles a prodrug: the hydrophilic rhodamine residue is "lipophilised" by coupling to a fatty acid side chain (C18). Therefore, this compound can serve as a lipophilic model substance for transfer. This assay was used to systematically analyse the influence of liposome size and production methods on both fusion and lipid transfer. Fusion kinetics were found to be independent of the size of the donor liposomes, whereas lipid transfer is faster with small liposomes than with large ones as donor vesicles. In addition, we demonstrate that also unlabelled liposomes can serve as acceptor membranes for lipid transfer. In the latter case, transfer is again faster from small than from large liposomes. The size of the acceptor vesicle by contrast does not influence transfer kinetics. Furthermore, we show that trace amounts of detergent, as found in liposomes produced by detergent dialysis, influence neither fusion nor transfer. Sonication does not affect fusion; however, it seems to slow down lipid transfer.

2. Experimental procedures

Chemicals [3H]GD1a (1 Ci/mmol) was a generous gift from Dr. Tettamanti, Milano. All other chemicals are listed in the accompanying paper (Ott and Wunderli, 1994). Virus

Influenza PR8 virus [A/PR/8/34 (H1N1)] was grown in M D C K (Madin Darby canine kidney) cells. Details are given in Wunderli and Ott (1990). Viral HA-spikes were removed by bromelain treatment (Brand and Skehel, 1972).

Liposomes Liposomes were prepared by various methods with the following standard lipid composition: PC/PA/cholesterol/Gola = 0.625/0.075/0.231/0.069 (molar ratios). For labelled liposomes octadecylrhodamine B chloride (R18) was included at a molar ratio of 0.083. The total lipid concentration was 0.8 mg per ml. Trace amounts of radioactively labelled DPPC, cholesterol or GD~a were included in order to estimate lipid recoveries and test the equivalence of lipid composition between different liposome preparations (see Results). Particle numbers were calculated according to Huang and Mason (1978). If necessary, liposome concentrations were increased by the following method: Volumes up to 1 ml of liposomes

were filled into micro-collodion bags (Sartorius, G6ttingen, Germany), larger volumes into dialysis tubing (12000 MW cut-off, Sigma). The bags or tubing were inserted into polyethyleneglycol 6000 until the desired concentration was reached. Liposome stock solutions were filtered through 0.2 #m pore size sterile filters (Minisart ®, Sartorius) and stored at 4°C in the dark. Preparations were stable for up to 4 weeks (LUV) and 8 weeks (SUV) respectively, as judged by dynamic light scattering and by their behaviour in the dequenching assay. The present study was performed with liposomes not older than 2 to 4 weeks.

Detergent dialysis liposomes Lipids (5 mg, composition see above) were mixed with sodium cholate at a lipid to detergent ratio of 0.6 (M/M). The dried lipid film was dispersed in 6 ml PBS pH 7.4, and cholate was removed by dialysis for 18 h with a Liposomat ® (Dianorm, Munich, Germany). These liposomes have been previously characterised (Wunderli and Ott, 1990). They contain less than 1 molecule cholate per 270 lipid molecules. Lipid recovery was 8 0 ± 3% (n = 11).

Handshaken liposomes Liposomes were prepared by manual dispersion of a dry lipid film without detergent in PBS pH 7.4. After filtration through a 0.2 #m pore size filter (recovery 83%), liposomes were centrifuged in a Beckman L5-65 ultracentrifuge for 6 h (rotor SW 41 Ti: 41 000 rpm; 4°C) to sediment vesicles larger than 20 S, which corresponds to a particle diameter of > 30 nm (Schwendener, 1979). About 9 ± 2 % (n = 3) of the initial lipid could be recovered in the supernatant as SUV.

Freeze-thaw-filter liposomes In order to obtain large, homogeneous liposomes, we used a modification of the extrusion method described by Mayer et al. (1986). Liposomes were produced in PBS pH 7.4 by handshaking (see above). They were then subjected to 8 cycles of freezing (solid CO2/ethanol) and thawing (37°C water bath) before being filtered 8 times through a 0.2 #m pore size filter. Lipid recovery was 73 ± 5% (n = 9).

Sonicated liposomes "Freeze-thaw-filter" liposomes were sonicated with a Branson probe sonifier (Model B30), equipped with a titan micro tip. Liposomes (1 ml in a glass tube) were kept on ice to avoid heating. Pulsed sonication (20% duty cycle) at maximal energy output allowed for micro tips was applied for two cycles of 15 min each. Titan particles and remaining large liposomes were removed by centrifugation in a Beckman table-top ultracentrifuge (rotor Ti 100.1; 100000 rpm; 12 min; 4°C),

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under which condition all vesicles larger than 20 S are pelleted. The lipid recovery in the supernatant was 37 4- 6% (n = 7) of initial lipid.

Table 1 Characteristics of SUV and LUV Type

Preparation method

Size

Dynamic light scattering (DLS)

LUV I LUV 2

"Freeze-thaw-filter" liposomes Prepared from detergent dialysis liposomes (SUV3) by column chromatography High-speed supernatant from handshaken liposomes High-speed supernatant from "freeze-thawfilter" liposomes which had been sonicated prior to centrifugation Detergent dialysis liposomes, mixture of two populations Prepared from detergent dialysis liposomes (SUV3) by column chromatography High-speed supernatant from detergent dialysis liposomes (SUV3) which had been sonicated prior to centrifugation High-speed supernatant from LUV2 which had been sonicated prior to centrifugation

J~ 180 rtm ~ 110nm

The sizes of liposomes were determined by dynamic light scatting (DLS). For routine measurements, the automated Nicomp 370 Submicron Particle Sizer (Skan, Basel, Switzerland) was used. The various preparation methods were also evaluated with a more powerful light-scattering apparatus (Malvern 4700 PS/ MW) equipped with a Malvern autocorrelator (7032ES/136c) and an argon ion laser (Coherent Innova 200-10) as described by Schurtenberger and Hauser (1993). Data analysis was performed by means of a second-order cumulant fit and an inverse Laplace transform algorithm as described elsewhere (Schurtenberger and Hauser, 1993). Both instruments gave essentially the same results.

Gel chromatography Separation of liposomes by size was carried out by gel exclusion chromatography with Sepharose 2B (Pharmacia). Columns (5.3 cm2x 30 cm) were eluted with PBS pH 7.4 at a flow rate of 20 ml per hour. The void volume was determined with Blue Dextran 2000 (1 ml, 2% W/V, Pharmacia). Liposomes were applied in a volume of 1 ml (~ 650 #g lipid) and collected fractions (1.7 ml) were analysed for R18 fluorescence (see below) and radioactivity. Size calibration of the column was performed with bovine serum albumin (Stokes' radius ~ 3.5 nm) and latex beads (Balzers Union, Balzers, Lichtenstein) of 91 nm and 312 nm diameter. In the case of latex beads, 1% sodium dodecyl sulfate was included in the elution buffer.

Fluorescence measurements R18 fluorescence was measured as described by Ott and Wunderli (1994). For all liposomes used in this study quenching was between 92 and 96%. No significant difference was observed between quench curves of detergent dialysis liposomes and "freeze-thaw-filter" liposomes, a finding which excludes the possibility of a different quenching behaviour of R18 in SUV and LUV, respectively (data not shown). The fusion and lipid transfer assays were performed as described previously (Wunderli and Ott, 1990). Details on the incubation conditions are given in the figure legends. Dequenching (DQ) as a function of time is expressed as fraction of the dequenching obtained after complete disruption of liposomes in 1% Triton X-100.

Data analyses Data analysis is described by Ott and Wunderli (1994). Best fits (proFit 4.0, QuantumSoft, Switzerland) were always obtained with either a simple second-order

SUVI SUV 2

SUV 3 SUV4 SUV5

SUV 6

< 20 S < 20 S ~ 25 nm ~ 110 nm ~ 26 nm ~ 26 nm < 20 S

< 20 S

Various R18-1abelled SUV and LUV were prepared and characterised (for details see Experimental procedures).

reaction (1) or with a superposition of two secondorder reactions (2): DQ(t) - DQmax x t

(1)

t*+t ×t DQ(t) - DQmax1 )< t + DQmax2 ,

t~ + t

t2 + t

(2)

3. Results

Liposomes In order to test the influence of liposome size on the kinetics of membrane interactions, SUV and LUV of standard lipid composition were produced with various preparation methods (see Table 1 and Experimental procedures). Their size and homogeneity were checked by dynamic light scattering. Starting from handshaken liposomes, which represent a rather heterogeneous vesicle population, three types of homogeneous liposomes were obtained. (1) Upon high-speed centrifugation under conditions which pellet liposomes larger than 20 S (corresponding to ,,~ 30 nm diameter; Schwendener, 1979) about 9% of the original lipid was recovered in the supernatant as SUVl. (2) Repetitive freezing, thawing and filtration produced LUV1 with a diameter of 179 4- 19 nm. (3) After sonication of LUV~ and subsequent highspeed centrifugation, about 40% of the original lipid could be recovered in the supernatant as SUV2 with a diameter of 24.3 4- 1.4 nm as determined by DLS. In another approach, detergent dialysis of cholate/lipid micelles was performed to produce liposomes. They had a residual cholate content of < 1 molecule per 270 lipid molecules. DLS measurements revealed that two

336

S. Ott et al./European Journal of Pharmaceutical Sciences 1 (1994) 333 341 0.8

0.5 0.4

i o.~.~~

~ ~. ~.~

~

0.9 0.0 ~ 0

0.3

0.1

gO

1~0 1~0 time [mini

2~

~.~

0

50

100

150

2~

time [mini

Fig. 1. R18 dequenching kinetics upon interaction of PR8 influenza viruses with GDla-containing SUV and LUV. Rl8-1abelled liposomes were prewarmed to 37°C in PBS pH 5.3 (closed symbols) or pH 7.4 (open symbols) as indicated. The reaction was started by addition of the PR8 viruses. The incubation mixture (1 ml) always contained 160 ng liposomal lipid and 3 #g viral lipid. • [] LUV2 and • © SUV4. Fitted curves (see Experimental procedures) are shown for representative experiments. Curve parameters, DQmax and t*, are listed in Tables 2 and 3.

Fig. 2. R 18 dequenching kinetics upon interaction of HA-depleted PR8 influenza viruses with GDta-COntaining LUV. PR8 influenza viruses were treated with bromelain to remove the HA spikes (see Experimental procedures) and incubated with R18-1abelled, GD~a-COntaining LUV1. • A bromelain-treated PR8 viruses; • [] untreated control viruses. Open symbols represent incubations at pH 7.4, closed symbols incubations at pH 5.3. Fitted curves are shown for representative experiments.

size populations were actually present: mainly SUV with a diameter of 26.6±7.1 nm and a small amount (6.8 ± 2.9% liposomal lipid) of larger liposomes (diameter 108 ± 11.5 nm). Despite the small amount of large liposomes, detergent dialysis liposomes are designated as SUV 3. With size fractionation on a Sepharose 2B column, two peaks could indeed be resolved (data not shown). The bulk of lipid was found in the peak of smaller liposomes, hence called SUV4. Calibration of the column with Blue Dextran showed that the larger liposomes eluted with the void volume and thus could represent a very heterogeneous liposome mixture. This liposome population was designated as LUV2. Unfortunately the Sephacryl series, which comprises gels with a separation capacity in the size range of the produced liposomes, could not be used. We found, in agreement with Reynolds et al. (1983), substantial lipid adsorption (data not shown). This excludes the use for liposomes with more than one lipid component. By analysis of the pooled peak fractions with DLS, we could demonstrate that in both peaks a homogeneous liposome population was present with a diameter of 109 nm for LUV2 and 26 nm for SUV4. Two additional preparations of SUV were obtained by sonication of SUV3, hence called SUVs, or by sonication of LUV2, hence called SUV6. In both cases, homogeneous SUV populations were found in the supernatants after highspeed centrifugation of the sonicated samples. In summary, the following populations of liposomes were available for kinetic testing: six different SUV with diameters between 24 and 30 nm and two different preparations of LUV. It has to be noted that among the vesicles used in this study, LUV2 and SUV3, SUV4, SUVs, SUV6 possibly contained residual cholate (< 1 molecule per 270 lipid molecules) and SUV2, SUV5 and SUV6 had been exposed to high-energy sonication.

Fusion and lipid transfer kinetics The above-described R18-1abelled LUV and SUV were tested for their interaction with PR8 influenza viruses. Assays were performed either under fusion conditions, i.e. pH 5.3 at 37°C, or at neutral pH (37°C), where only lipid transfer occurs (Wunderli and Ott, 1990). A representative experiment with LUV2 and SUV4 is illustrated in Fig. 1. At both pH values, the dequenching curves differ significantly between LUV and SUV. Very similar pictures were obtained for dequenching curves with other SUV or LUV (see Fig. 2 for LUV1, other curves not shown). For quantitative comparison of the dequenching kinetics obtained with the different liposomes, curves were analysed with a non-linear fitting program (see Experimental procedures). With all SUV tested, dequenching data were best described with a hyperbolic function which corresponds to second-order kinetics. Fitted curve parameters i.e. the maximal dequenching, DQmax, and the time to reach half-maximal dequenching, t*, are listed in Table 2 for SUV. DQmax values between 0.64 and 0.77 were reached at both pH values. Under fusion conditions (pH 5.3, 37°C), a fast process with t* ~ 1-2 min occurred, whereas at neutral pH a slow dequenching with t* ~ 35-60 min was noted. Vesicles which had been exposed to high-energy sonication, i.e. SUV2, SUV 5 and SUV6, consistently showed a larger t* (,-~ 60 min) than preparations which had not been sonicated, i.e. SUV~, SUV3 and SUV4 (t* ~ 40 min). No difference was found between liposomes which possibly contain residual cholate, i.e. SUV3, SUV4, SUVs, SUV6, and those which do not, i.e. SUVI and SUV2. In the case of LUV (Table 3), non-linear fitting of the dequenching curves obtained at pH 5.3 revealed a superposition of two second-order processes: a fast one with t* in the range of 2-4 min and a second slower one with t*

337

S. Ott et al./European Journal of Pharmaceutical Sciences 1 (1994) 333-341 Table 2 PR8 influenza viruses incubated with various R18-1abelled, GDla-COntaining SUV pH 5.3

SUV~ (n = 2) SUV/(n=4) SUV3 (n=8) SUVa(n=3) SUV5 ( n = l ) SUV6 (n= 1)

Table 3 PR8 influenzaviruses incubated with various R18-1abelled,Greta-containing LUV pH 5.3

pH 7.4

pH 7.4

DQmax

t* (min)

DQmax

t* (min)

fast component

slow component

0.690 4- 0.005 0.6705:0.003 0.7135:0.003 0.7315:0.004 0.7664-0.003 0.636 5:0.005

1.05 5:0.08 1.075:0.02 1.21 5:0.02 1.565:0.04 1.11 5:0.02 2.42 5:0.08

0.684 4- 0.008 0.7695:0.006 0.7344-0.005 0.7585:0.013 0.753 5:0.003 0.657 5:0.006

36.4 5:1.6 62.05:1.4 35.55:0.6 45.05:2.4 58.1 4-0.8 56.2 5:1.7

DQmax 1 t~ (min)

DQmax2 t~ (min)

DQmax

t* (min)

0.192 1.6 5:0.004 +0.1 0.172 3.8 5:0.014 5 : 0 . 5

0.401 5:0.013 0.376 +0.012

0.517 5:0.009 0.642 5:0.005

234.7 5:7.1 243.5 4-3.6

Various R18-1abelled SUV were prepared (see Table 1 and Experimental procedures) and incubated at 37°C with PR8 influenza viruses at pH 7.4 or pH 5.3 as indicated. The incubation mixture (1 ml) contained 160ng liposomal lipid and 3#g viral lipid corresponding to a total particle concentration of about 6 × 10~° particles/ml. Mean data derived from n independent experiments were analysed with the proFit program. Curve parameters with error estimates from the fits are listed.

~ 100-180 min. About one-third of the maximal dequenching was reached in the fast, the rest in the slow reaction. At pH 7.4, by contrast, a single secondorder reaction satisfactorily describes the experimental data. The latter is in agreement with the situation for SUV (see above); however, t* for LUV is significantly increased (t* ,-~ 240 min), indicating a slower transfer process from LUV than from SUV. DQmax values were about the same for the reactions at pH 7.4 and pH 5.3. They tended to be lower for LUV (0.50 to 0.65) than for SUV (0.65 to 0.80). Residual cholate, possibly present in LUV2 but not in LUV1, did not seem to influence the dequenching kinetics significantly. For SUV we have demonstrated previously that the fast dequenching reaction at pH 5.3 can be abolished by removing H A with bromelain, and thus represents virus/liposome fusion (Wunderli and Ott, 1990). To test whether the dequenching measured with LUV and PR8 at pH 5.3 was related to specific fusion, LUVI were incubated with HA-depleted PR8 viruses (Fig. 2). Only the fast component disappeared after this treatment and could thus be identified as a HA-specific fusion reaction. The remaining dequenching curve at p H 5.3 was very similar to the curves obtained with PR8 viruses (bromelain-treated or untreated) at pH 7.4. Curve fitting yielded the following parameters for the interaction of LUV~ with bromelain-treated viruses: DQmax = 0.54 + 0.01 and t* = 235.2 q- 9.2 min at pH 7.4; DQmax -0.38+0.01 and t * = 166.54-7.6 rain at pH 5.3. A small but significant difference thus exists between transfer at pH 7.4 and pH 5.3 (for SUV see below). We wanted to exclude the possibility that the differences observed in interaction kinetics of PR8 viruses with small and large liposomes respectively, could arise through differences in the lipid composition between LUV and SUV. To test this hypothesis, SUV3 were

LUV~ (n=4) LUV 2 (n=3)

168.7 5:14.7 124.8 5:20.3

R18-1abelled LUV were prepared (see Table 1 and Experimental procedures) and incubated at 37°C with PR8 influenza viruses at pH 7.4 or pH 5.3 as indicated. The incubation mixture (1 ml) contained 160ng liposomal lipid and 3 #g viral lipid corresponding to a total particle

concentration of about 3.8 × 10~° particles/ml. Dequenching curves were fitted with the profit program (see Experimentalprocedures). labelled with R18 and an additional, radioactively labelled marker ([IaC]DPPC, [3H]cholesterol or [3H]GDla). After separation of the liposomes on a Sepharose 2B column, the distribution of the single markers between the two peaks, SUV4 and LUV2, was determined and the ratios for the two were compared (Table 4). In respect to DPPC, cholesterol and R18, a homogeneous distribution was found, whereas GD1a was about two-fold enriched in LUV2 as compared to SUV4. We have shown earlier (Wunderli and Ott, 1990), that the presence of the virus receptor GD1a is important for fusion, but does not affect lipid transfer. To study the influence of an increased amount of GD1a on membrane interaction kinetics, detergent dialysis liposomes were produced with twice the standard amount of GD1a. DLS showed that this did not change liposome size. The kinetic parameters for fusion and lipid exchange were: DQmax -- 0.71 + 0.02 and t* = 1.56 + 0.24 min for pH 5.3 incubations, and DQmax -- 0.81 q- 0.01 and t* = 35.9 + 1.9 min for pH 7.4 incubations. This means that for both reactions fitted parameters were not significantly different from the values obtained with standard SUV3.

Liposome-liposome interactions R18-1abelled SUV3 and R18-1abelled L U V 1 respectively were also incubated with unlabelled liposomes instead of viruses as acceptor membranes (Fig. 3). Upon incubation of labelled SUV3 with unlabelled SUV 3 in PBS pH 7.4, a maximal dequenching of 0.66+0.01 was observed, half-maximal dequenching was reached after 40.7 + 1.5 min ( n - - 4 ) . When LUV were used as acceptor vesicles instead of SUV, the same dequenching curve was obtained, i.e. DQmax = 0.66+0.01 and t * = 4 1 . 5 + 1 . 8 min ( n = 4 ) . On the other hand, transfer from labelled LUV to unlabelled membranes (SUV or LUV) was much slower. Fitted

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S. Ott et al./European Journal of Pharmaceutical Sciences 1 (1994) 333-341

Table 4 Distribution of various lipids between SUV4 and LUV2 prepared from standard dialysis liposomes Liposomes

LUV2 SUV4

[~4C]DPPC/R18 (n = 3)

[3H]cholesterol/R18 (n = 1)

[3H]GDla/R18 (n = 5)

0.93 + 0.20 1.01 ± 0.01

0.89 0.99

2.11 + 0.68 0.98 5:0.02

Standard dialysis liposomes (SUV3) labelled with the indicated markers were separated on a Sepharose 2B column and the fractions analysed for radioactivity and fluorescence. The ratios were calculated from the fractions in the respective peaks.

parameters for dequenching curves obtained with R18labelled LUV1 and unlabelled SUV 3 are: DQmax-0.36 q- 0.01 and t* = 240.5 ± 15.9 min (n = 2). Since no acid-induced fusion interferes upon incubation of liposomes with liposomes, the influence of pH on lipid transfer could also be tested in these experiments. Incubation of labelled and unlabelled standard liposomes (SUV3) at pH 5.3 yielded dequenching curves with the following parameters: DQmax = 0.53 ± 0.01 and t* = 18.70 ± 0.44 min (n = 2). Thus, transfer of R18 was about two times faster at acidic pH as compared to neutral p H (see also above, data with bromelain-treated virus). This pH-dependence was also found if LUV instead of SUV were used as donor vesicles (data not shown).

4. Discussion Data are presented here which show the influence of vesicle size and liposomal preparation methods on both fusion and lipid transfer. If R18-1abelled SUV and LUV are compared in the fusion assay, a striking difference is found (see Fig. 1). Kinetic analyses reveal that contrary to the situation with SUV, the dequenching curves obtained with LUV consist of two kinetic components: a fast one, which comprises only one-third of the maximal dequenching, and in addition a much slower process. The fast reaction can be abolished by pretreatment of PR8 viruses with bromelain which removes the H A fusion protein and thus is identified as a virus/LUV fusion process. The apparently reduced extent of the fusion reaction can easily be traced to the limited marker dilution which occurs if large R18-1abelled vesicles fuse with two to three times smaller viruses (in respect to diameter of the lipid bilayer). The resulting fusion product is still significantly quenched, but cannot undergo another round of fusion (Nir et al., 1986), since unbound fusion protein H A is inactivated at low pH (Yewdell et al., 1983). R18 dequenching in the fusion products continues as a consequence of marker transfer, which kinetically leads to a superimposed second process. Experimental evidence for this interpretation is

0.8

0.6

i

0.4-

0.2-

0.0 . 0

.

. 50

. 100

. 150

200

time [ m i n ]

Fig. 3. RI8 dequenching kinetics upon interaction of R18-1abelled liposomes with unlabelled liposomes. R18-1abelled liposomes (160 ng lipid) as donor membranes were incubated with unlabelled liposomes (6 #g lipid) as acceptors (PBS pH 7.4, 37°C). SUV 3 and LUV] as small and large liposomes respectively. © R18-SUV 3 with unlabelled SUV3; /~ RI8-SUV 3 with unlabelled LUV]; [] RI8-LUV~ with unlabelled SUV 3. Data points are mean values from 3 or 2 independent experiments. Lines correspond to fitted curves. For curve parameters see text.

also obtained with "freeze-thaw-filter" liposomes displaying lower initial quenching, i.e. containing less R18 in the membrane. In this case, one would expect less residual quenching in the fusion product, and thus a higher proportion of total possible dequenching would result from the fusion process. Indeed, over 50% of the maximal dequenching originated from the fast fusion reaction when LUV~ with only 55 instead of 96% initial quenching were investigated (data not shown). Times to reach half-maximal fusion tend to be slightly larger for incubations of PR8 viruses with LUV (t* ~ 1.5-4 min) than for those of PR8 viruses with SUV (t* ,,~ 1-2 min). In all experiments a fixed ratio of virus to liposome lipid of 12/1 is used. Thus, the amount of unlabelled virus membrane is always in excess of the labelled liposome membrane. However, taking into account the different sizes of the vesicles, the total amount of particles in the assay is about 1.6 times lower for incubations of viruses with LUV than with SUV. As we have demonstrated previously, the ratelimiting step in the fusion reaction is binding, a collision-mediated process. Therefore, t* does depend on the total particle concentration in the assay, i.e. a twofold decrease in particle concentration also yields a twofold decrease in t* for fusion of SUV with PR8 viruses (Ott and Wunderli, 1994). We therefore conclude that after correction for collision frequency there is no significant difference in the fusion rate between LUV or SUV with PR8 viruses. This means that despite the striking difference in the R18 dequenching curves for SUV or LUV, detailed kinetic analyses reveal no difference in the fusion kinetics of PR8 viruses with liposomes of higher or lower curvature. This result is in agreement with data by Van Meer et al. (1985) on influenza virus and by Sarkar and Blumenthal (1987/1988) on Sendai virus fusion. Comparison of the fusion kinetics of the

S. Ottet al./European Journal of Pharmaceutical Sciences 1 (1994) 333-341

six SUV preparations reveals that independently of the production method DQmax values lay between 0.65 and 0.77, and times to reach half-maximal dequenching are in the range of 1 to 2 min. This means that neither highenergy sonication nor residual cholate affect the fusion process. We could also demonstrate (unpublished results) that kinetic parameters for the fusion reaction do not change with liposomal preparations containing even higher concentrations of cholate (up to 100 times the amount of standard dialysis liposomes). R18 does spontaneously exchange from labelled liposomes to unlabelled virus membranes upon incubation at 37°C at neutral pH. This is true for dialysis liposomes (Wunderli and Ott, 1990) as well as for other SUV and LUV (this study). As we show here, transfer is about 5-6 times faster from small (diameter ~ 30 nm) than from large (diameter 110 or 180 nm) liposomes independently of the production method. Previously we have also shown that R18 transfer does occur, when labelled PR8 viruses are incubated with unlabelled liposomes (Wunderli et al., 1993), with a t* of about 80 to 120 min, a range expected for the intermediate size of PR8 viruses (SUV 20. These data confirm that the frequency of collision between the interacting vesicles does not control the lipid transfer reaction. They also show that the curvature of the donor and not that of the acceptor membrane determines the lipid exchange rate. This last conclusion is in agreement with results from Fugler et al. (1985), who did not find a difference in cholesterol transfer whether LUV or SUV were used as acceptor vesicles. In previous reports (Hoeckstra et al., 1984; Loyter et al., 1988), no R18 transfer from labelled viruses or from labelled LUV was detected. We explain this discrepancy with our results by the fact that the transfer reaction from such large structures only proceeds very slowly (over 100 times slower than specific fusion), and therefore does not significantly contribute to the dequenching during the short observation times ( < 10 min), which had been optimised to measure the fast

339

fusion process. Recently, Stegmann et al. (1993) also reported on R18 transfer from influenza and Semliki Forest viruses during fusion experiments followed over lh. The question was also asked whether the apparent difference in the dequenching curves between SUV and LUV might be due to differences in lipid composition. As we could show, DPPC and cholesterol as well as R18 were evenly distributed between the two peaks obtained with detergent dialysis liposomes. For GDla, however, twice the amount was found in large vesicles as compared to small ones. This is in agreement with Thomas and Poznansky (1989) who showed that in SUV, for sterical reasons, GD1a is limited to the outer membrane leaflet, whereas in LUV it distributes equally in both leaflets of the bilayer. Experiments with SUV6 on the one hand, and with SUV 3 containing twice the standard amount of GD1a on the other hand, indicate that a difference in GD1a content by a factor of 2 affects neither fusion nor lipid transfer. The latter confirms earlier data on R18 transfer from neuraminidase-treated liposomes or from GDla-free liposomes. No difference was then found to standard, GD~a-Containing detergent dialysis liposomes (Wunderli and Ott, 1990). Another aspect of the present study is the fact that R18 transfer does depend on the pH of the incubation medium. Upon comparison of t* for the reactions at pH 5.3 and pH 7.4 under "nonfusing" conditions (i.e. virus/liposome interaction with bromelain-treated viruses, or liposome/liposome interactions, or slow component in the LUV/virus reaction under fusion conditions), it becomes clear that R18 transfer at pH 5.3 proceeds 1.5-2 times faster than transfer from the same liposome tested at pH 7.4. We suppose that this pH-effect on R18 desorption may be caused by the negative charge in the liposomes. More experiments with differently charged liposomes are needed to further elucidate this point. The finding that transfer is faster from SUV than from LUV seems not to be limited to our model lipid R18. Thus, the same observation was made for the spontaneous cholesterol transfer, a reaction that seems to follow first-order kinetics (McLean and Phillips, 1984; Fugler et al., 1985; Yeagle and Young, 1986; Thomas and Poznansky, 1988). In addition, we found that radioactively labelled phospholipids and cholesterol are much more effectively transferred to acceptor vesicles from SUV than from LUV as donor vesicles (data not shown). Jones and Thompson (1990) measured spontaneous transfer of 1-palmitoyl-2-oleylphosphatidylcholine from charged to neutral liposomes at 45°C with LUV and SUV as donor vesicles. They also observed a much slower transfer when donor LUV were used instead of SUV, but attributed this difference entirely to a lower frequency of collision for larger particles. As we have discussed above, this interpretation does not hold for

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our data since lipid transfer is found to be independent of collision frequency in our experimental system (Ott and Wunderli, 1994). We explain the finding of a faster transfer from SUV than from LUV by the looser packing of PC molecules in highly curved SUV facilitating lipid desorption. High curvature seems to affect several membrane properties such as the thermal transition of lipids that has been found to be lower in SUV than in LUV (Schuh et al., 1982, and reference therein). Many other membrane characteristics depend on the membrane curvature, e.g. the penetration of adriamycin into the bilayer (Dupou et al., 1989) and the activity of phospholipase A2 (Wilschut et al., 1978). The two differing preparations of LUV used in this paper do not show a significant difference in the R18 transfer reaction, although their diameter is slightly different (110 nm and 180 nm). We believe that beyond a certain size the packing stress is relieved, and thus a further increase in the size is not crucial for transfer. Taken together, the production method for liposomes does not influence fusion or transfer kinetics in our model system, except that sonication seems to slightly reduce lipid transfer. However, the size of the liposome and thus the membrane curvature determines the transfer of lipids from liposomes to other membranes. For the preparation of liposomes with increasing circulation time in the blood, small liposomes are preferred over larger liposomes since the latter are more efficiently eliminated by the reticulo-endothelial system (Allen and Everest, 1983; Machy and Leserman, 1983; Hui and Sen, 1989; Mayer et al., 1989). On the other hand, the stability of liposomes in a biological environment such as plasma depends on the desorption of liposomal lipids, such as PC and cholesterol. Therefore, one should expect that small liposomes are less stable in plasma than larger liposomes (Scherphof and Morselt, 1984; Tilcock et al., 1990). This indicates that when preparing liposomes for slow-release formulations in plasma, size is an important factor that has to be controlled. The goal is to design liposomes that are large enough to be stable over the desired time range, but small enough to escape the reticulo-endothelial system. For this purpose, in vitro tests like the R18 dequenching assay may be valuable to quickly determine the lipid transfer from the liposome under study. Acknowledgements We thank Dr. Tettamanti (Milano) for his generous gift of tritiated ganglioside. Thanks are also due to Maja Giinthert for excellent technical assistance. References Allen, T.M. and Everest, J.M. (1983) Effect of liposome size and drug

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