Liposomes and influenza viruses as an in vitro model for membrane interactions I. Kinetics of membrane fusion and lipid transfer

EUROPEAN

ELSEVIER

European Journal of Pharmaceutical Sciences 1 (1994) 323-332

JOUUNAL

OF

PHARMACEUTICAL SCIECES

Liposomes and influenza viruses as an in vitro model for membrane interactions I. Kinetics of membrane fusion and lipid transfer Susanne Ott*, Heidi Wunderli-Allenspach Department of Pharmacy, Biopharmacy, Swiss Federal Institute of Technology, CH-8057 Zfirich, Switzerland (Received 8 October 1993; accepted 28 March 1994)

Abstract

To study membrane interactions, we use a previously described model system with intact PR8 influenza viruses and small liposomes containing the virus receptor Gola. The assay is based on the fluorescent membrane marker octadecylrhodamine B chloride, R18, incorporated in liposomes. With this assay two processes can be studied in parallel: hemagglutinin-dependent virus/liposome fusion and spontaneous marker transfer. We present a detailed kinetic analysis of fusion and transfer which takes into account the contribution of dequenching and quenching in donor and acceptor vesicles, respectively. Both fusion and transfer follow secondorder kinetics; they differ, however, in the interacting species. Fusion is collision-mediated, i.e. it depends on the total particle concentration. For R18 transfer, the rate is independent of the particle concentration, but determined by the R18 surface density, i.e. the R18/lipid ratio, which is responsible for the initial quenching of the labelled species. Key words: Fusion; Lipid transfer; Kinetics; Fluorescence marker R18

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

1. Introduction

The concept of using liposomes as drug carriers has gained much interest in recent years. Lipophilic molecules are directly incorporated into lipid bilayers, whereas hydrophilic ones appear in the inner, aqueous space of vesicles. To increase the loading capacity of the latter and to reduce leakage of hydrophilic substances, various molecules with modified physico-chemical characteristics (prodrugs) have been constructed in the past. A classical approach is the coupling of a hydrophilic molecule with a lipophilic side chain which can then be anchored in a lipid bilayer. Examples are cyto*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 S S D I 0928-0987(94)00008-N

sine arabinoside, 5-fluoro-2'-deoxyuridine and others (Tagushi, 1980; Schwendener et al., 1985; Rubas et al., 1986). Such prodrugs leave the lipid bilayers upon interactions between liposomes and other membranes, and/or by passive partitioning between liposomes and the surroundings. In vivo liposomes can undergo various membrane interactions: binding to cell surfaces non-specifically or specifically via receptors, endocytosis and, under certain conditions, fusion either at the cell surface or in prelysosomes (see e.g. Weinstein and Leserman, 1984). For quantitative investigations on membrane interactions, fluorescent membrane probes proved useful (reviewed in Dtizgiines and Bentz, 1988). Special interest has been put on octadecylrhodamine B chloride (R18), a membrane marker which can be integrated into pre-existing biological or artificial membranes (Hoekstra et al., 1984). At high concentrations R18 fluorescence is quenched. Dequenching, which occurs through marker dilution upon interaction of the labelled with unlabelled membranes, can be monitored in a spectrofluorometer. The original R18 assay, designed for fusion processes, has been widely applied (Struck et al., 1981; Nir et al., 1986; Stegrnann et al., 1986; Morris et al., 1989). We have

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S. Ott, H. Wunderli-Allenspach/European Journal of Pharmaceutical Sciences 1 (1994) 323 332

shown that it can also be used to study lipid transfer (Wunderli and Ott, 1990). Recently Stegmann et al. (1993) also reported on R18 dequenching due to a transfer reaction. From its molecular structure R 18 represents a type of the above-mentioned prodrugs: the hydrophilic rhodamine residue is lipophilised by coupling to a fatty acid side chain (C18), which serves as an anchor and permits incorporation into liposomal membranes. Thus R18, besides being a membrane marker, can also be used as a model for a liposomally incorporated prodrug interacting with membranes. In an in vitro assay PR8 influenza viruses were incubated with R18-1abelled, virus receptor (GDla)containing liposomes as fusion partners (Wunderli and Ott, 1990). At pH 5.3, hemagglutinin-specific fusion and non-specific transfer occur simultaneously. At neutral pH the influenza fusion protein is not active, and only non-specific prodrug, i.e. R18, transfer occurs. This assay can thus be used to study under the same experimental condition two processes by which a prodrug can leave the liposome, fusion and transfer. In this first paper of a series we present a detailed kinetic analysis of the two processes which takes into consideration the contribution of dequenching and quenching of R18 fluorescence in the donor and acceptor vesicles respectively. Both follow second-order kinetics, but the interacting species are different: the rate-limiting step in the specific fusion reaction is the collision frequency of the interacting partners, i.e. fusion depends on the total particle concentration in the assay. For the transfer process, however, the collision frequency is not rate-limiting. In this case, it is the initial surface density, i.e. the R18/lipid ratio, of the donor that determines the rate of the transfer reaction. The rate-limiting factor is therefore the initial concentration of the transferred model molecule R18. Dequenching assays are useful for fusion as well as for transfer studies provided that the conditions are optimised with respect to the interacting species. In a second paper (Ott et al., 1994) we analyse the influence of liposomal factors such as size and production method on fusion and transfer kinetics.

2. Experimental procedures Chemicals

Egg yolk phosphatidylcholine (PC) and phosphatidic acid (PA), both grade 1, were purchased from Lipid Products (Nutfield, UK). Ganglioside GDla was from Bachem (Bubendorf, Switzerland), cholesterol from Sigma (Buchs, Switzerland) and octadecylrhodamine B chloride (R18) from Molecular Probes (Junction City, OR, USA). [14C]dipalmitoylphosphatidylcholine (55.7 mCi/mmol) was obtained from Amersham and [3H]cholesterol (30 Ci/mmol) from NEN. Phosphate buffer (10 mM

Na2HPO4 and KH2PO4 each) containing 130 mM NaC1 (PBS) at pH 7.4 or pH 5.3 as indicated, was degassed and filtered (0.2 #m pore size) before use. Virus

Influenza PR8 virus [A/PR/8/34 (H1N1)] was grown in M D C K (Madin Darby canine kidney) cells. Details on virus growth and partial purification have been published elsewhere (Wunderli and Ott, 1990). The protein content was measured by the method of Bradford (1976) with the Bio-Rad microassay (Bio-Rad Laboratories, Munich, Germany). The amount of viral lipid was calculated with the assumption that influenza viruses consist of 20% (w/w) lipid and 70% (w/w) protein (Barrett and Inglis, 1985), and the number of viruses was estimated on the basis of the viral particle weight (4 × 10 -16 g per particle) (Klenk, 1991). Liposomes

Detergent dialysis liposomes were prepared with sodium cholate (lipid to detergent ratio 0.6 M/M) as described (Wunderli and Ott, 1990) with a diameter of 27 -4- 5 nm (n -- 6) as measured by dynamic light scattering (Nicomp 370, submicron particle sizer). Large unilamellar liposomes (180 + 20 nm, n = 8) were prepared with a repetitive freeze-thaw-filtration method (see Ott et al., 1994) similar to the extrusion method. The number of liposome particles was calculated according to Huang and Mason (1978). The following lipid composition was used: egg yolk phosphatidylcholine, phosphatidic acid, cholesterol and ganglioside GD1a at molar ratios of 0.625/0.075/0.231/0.069. In standard liposomes octadecylrhodamine B chloride, R 18, was included at a molar ratio of 0.083 leading to an initial quenching (Qinit) of about 95%. The amount of R18 in the labelled particles was expressed as surface density r, i.e. the number of R18 molecules per nm 2 lipid surface (inner and outer). A Qinit of 96% corresponds to r = 0.122. For experiments in which the influence of the R18 surface density r was investigated, the molar ratio of R18/lipid was lowered as indicated. To establish quench curves, R18 labelling was performed by exogenous addition of the marker to preformed liposomes. They were only used after equilibration at room temperature for at least 24 h. No significant difference was then found between the quench curve after exogenous or endogenous labelling. In the case of viruses, only exogenous labelling can be performed. Quench curves were established after equilibration for 24 h in analogy to exogenously labelled liposomes. Fluorescence measurements

Fluorescence measurements were carried out with a LS-SB spectrophotofluorometer (Perkin Elmer) at excitation and emission wavelengths of 545 and 585 nm respectively (slit width 10 nm). For kinetic experiments

S. Ott, H. Wunderli-Allenspach/EuropeanJournalof PharmaceuticalSciences 1 (1994) 323-332

325

R18-1abelled liposomes were pipetted into a thermostated cuvette at 37°C in PBS pH 5.3 or 7.4 as indicated, and the dequenching reaction was started by the addition of PR8 viruses. Standard concentrations were 0.16 #g liposomal and 3 #g viral lipid per ml unless otherwise stated, i.e. unlabelled membranes were always present in excess. Details on the assay conditions were published elsewhere (Wunderli and Ott, 1990). Fluorescence was recorded as a function of time. All measurements were related to a standard, which was obtained by complete disruption of liposomes in 1% Triton X-100. As R18 fluorescence varies with the milieu in which the probe is diluted, the "Triton standard" only represents a relative value; however, it allows direct comparison of parameters of reactions performed with varying amounts of the R18 marker. Total dequenching DQtot was calculated as follows:

2k

FpBS -- BGRpas DQtot - FTriton -- BGRTriton

Data analyses for quench curves Quench curves of liposomes and PR8 viruses, i.e. quench values as a function of the respective R18 surface densities r, were analysed by a Stern-Volmer plot (Arbeloa, 1981).

(1)

where F~,Bs, FTriton correspond to fluorescence measured in PBS or Triton, and BGRpa s, BGRTriton to background PBS or Triton. The initial dequenching of the liposomes DQinit ( - - 1 - Qinit) was subtracted from DQtot(t ) to obtain dequenching due to membrane interactions: DQ(t) = DQtot(t ) - DQinit

(2)

Data analyses for dequenching kinetics Dequenching data were evaluated on Macintosh Ilci with the proFit4.0 non-linear curve-fitting program (QuantumSoft, Zfirich, Switzerland). This program allows several user-defined functions to be sequentially fitted to the data points with the Levenberg-Marquard algorithm as described (Press et al., 1986). The choice of the best fit was based on chi square values and the respective residuals. In our case, dequenching curves were analysed with formulas describing first-order or secondorder reactions or superposition thereof as follows. First-order reaction: DQ = DQmax(1

--

e -'kt)

which corresponds to v ~- lk (DQmax - DQ)

(3) the

integrated

form

of

lk first-order rate constant (time -l) DQmax maximal dequenching reached Second-order reaction: DQ = DQmax x t

/(1

)

~k × DQmax + 1

= OQmax × t/(t* + t) which corresponds to v = 2k(DQmax - DQ) 2

(4) the

integrated

form

of

t* ci

second-order rate constant (concentration x time) -1. It equals (t* ~
For direct comparison of reactions in which the concentration of the interacting species, e.g. particle concentration (sum of viruses and liposomes) or R18 surface density, is varied, we use t~, the value normalised to the parameter t* obtained with standard incubation conditions (see Results). Best fits for DQ(t) were always obtained with the second-order-derived formula (4).

1/DQ= l+al r al

a2

xr+a2xr

2

(5)

R18 molecules per nm 2 lipid surface (inner plus outer) reflects the contribution of R18 monomers (r) to the quenching. It comprises 2 components: t °, unquenched fluorescence lifetime; km, rate constant for the quench process reflects the contribution of R18 monomer/ monomer interactions (r 2) to the quenching. It comprises 3 components: t°, unquenched fluorescence lifetime; Kd, dimerisation constant of R18; kqd , rate constant for dequenching by nonfluorescent dimers

3. Results

Kinetics of dequenching for fusion between R18-labelled S U V and PR8 viruses The kinetics of R18 dequenching upon incubation of Gola-containing, R18-1abelled SUV with PR8 influenza viruses at pH 5.3 (37°C) under standard conditions (see Experimental procedures) are illustrated in Fig. 1. As indicated, data analysis with a non-linear fit program (see Experimental procedures) revealed that fitting with a second-order function was clearly superior to fitting with a first-order function (see Figure legend). To study the influence of the total particle concentration on the R18 dequenching kinetics, incubations were performed as described above except that the total particle

326

S. Ott, H. Wunderli-Allenspach/European Journal of Pharmaceutical Sciences 1 (1994) 323 332 0.8

~__~.7 . . ~

0.6

c 4)

0,4

~

Table 2 Effect of collision frequency and RI8 surface density on the R18 dequenching kinetics at pH 7.4

~.-~ m"

Total conc. (particles/ml)

Qint (%)

r (R18/nm 2)

DQmax

O" ~)

~:

0.2

--.0.0

s e c o n d order fit

A

first order fit

12 x 6 × 2 x 1x

i

20 4'0 t i m e [rain]

6'0

Fig. 1. Dequenching kinetics upon incubation of PR8 viruses with R 18labelled GDta-containing SUV at p H 5.3, 37°C under standard conditions. The solid line represents the computer fit (proFit4.0 program, see Experimental procedures) with the second-order-derived formula (4). The dashed line shows the fit obtained using first-order kinetics, formula (3).

concentration was varied between 1 × 101° and 1.2 × 1011 per ml (standard conditions: 6 × 101° per ml). The ratio of unlabelled PR8 viruses to labelled liposomes was kept constant (~ 2). For quantitative comparison the fitted second-order curve parameters, i.e. the maximal dequenching reached, and t*, the time to reach half-maximal dequenching, are listed in Table 1A. An increase in t* is observed with decreasing particle concentrations, e.g. with 1.2 × 1011 particles per ml t*

Table 1 Effect of collision frequency and R18 surface density on the R18 dequenching kinetics at p H 5.3 Total conc. (particles/ml)

A 12 × 6× 2× 1×

Qint (%)

r (R18/nm z)

Fit parameters DQma×

t* (min)

101° 10 I° 101° 101°

96 96 96 96

0.122 0.122 0.122 0.122

0.74 4- 0.01 0.694-0.01 0.684-0.01 0.644-0.01

0.59 4- 0.03 1.124-0.04 2.744-0.13 5.464-0.30

10 ~° 10 l° 10 ~° 10 ~° 101°

96 93 87 77 54

0.122 0.092 0.064 0.056 0.0315

0.82 ± 0.01 0.80 4- 0.01 0.81±0.01 0.66±0.01 0.654-0.01

1.02 4- 0.03 1.25 4- 0.03 1.644-0.05 1.02±0.02 1.29±0.10

t~ (min)

1.2 1.1 0.9 0.9

B 6 × 6× 6× 6× 6×

Fit parameters

R18-labelled SUV were incubated with PR8 viruses at pH 5.3 and the dequenching measured. Data were analysed with proFit (see Experimental procedures). The fit parameters DQmax and t* with their fit errors are listed. A: The total particle concentration (SUV plus viruses) was varied while Qinit of the liposomes was kept constant. To illustrate the dependence of the reaction at pH 5.3 on the collision frequency, t* values were normalised to the total particle concentration of the standard assay (6 × 10 t° particles/ml) by t~ = t* × [total conc.]/ 6 × 101°. B: Liposomes with decreasing initial surface densities of R18 were used under standard assay conditions at p H 5.3.

101° 101° 101° 10 ~°

96 96 96 96

0.122 0.122 0.122 0.122

0.944-0.02 0.97 ± 0.01 0.77 4- 0.02 0.70 4- 0.02

101° 10 I° 101° 101° 101°

96 93 87 77 54

0.122 0.092 0.064 0.056 0.0315

0.95±0.02 0.664-0.01 0.82 4- 0.02 0.91 ± 0 . 0 2 0.754-0.02

t~ (min)

t* (min)

36.4 33.7 37.9 37.0

=~ 1.7 ± 1.1 ± 1.6 ± 2.0

B 6× 6× 6× 6× 6×

36.35:1.15 31.45:2.0 66.2 ± 4.8 88.8±3.1 133.2±6.7

36 28 37 43 35

Experimental conditions were as described in the legend in Table 1, except that the experiments were carried out at pH 7.4. A: Variation of the total particle concentration while keeping the Qinit of SUV constant. B: Variation of initial R18 surface density under standard assay conditions, i.e. with constant particle concentration. Curves were fitted as described (see Experimental procedures). The fit parameters DQmax and t* with their fit errors are listed. To illustrate the dependence of the reaction at pH 7.4 from the initial R18 quenching of the respective preparations, t* were normalised to a constant RI8 surface density of 0.122 molecules R18 per n m 2 t~ = t* × r/0.122.

equals 0.6 min, whereas with 1 × 101° particles per ml it takes 5.5 min to reach half-maximal dequenching. DQmax is between 0.64 and 0.74 independently of the particle concentration. In general, an increase in the particle concentration leads to an increase in the frequency of collision between interacting particles. Therefore, t* values can be normalised to a constant relative collision frequency (see legend to Table 1). Normalised values, t~, of 0.9-1.2 min are obtained over the whole concentration range tested. These experiments clearly indicate that fusion kinetics at pH 5.3 are of second order in relation to the total particle concentration. We have also investigated the influence of the initial R18 surface density (r) in labelled liposomes on the dequenching reaction at pH 5.3. Liposomes with varying concentrations of R18 were produced. Their initial quenching ranged from 96% (0.122 molecules R18 per nm 2) to 54% (0.0315 molecules R18 per nm2). These liposomes were incubated with PR8 viruses under standard assay conditions (0.16 #g liposomal and 3 #g viral lipid per ml, 37°C), and the increase in R18 fluorescence was monitored. No significant difference in the dequenching kinetics, i.e. DQmax and t*, was observed for liposomes of varying initial quench values (see Table 1B). The mean t* value was calculated as 1.2:1:0.3 min, which is in the range of the previously determined value (see above). This shows that the marker concentration in SUV does not influence the fusion reaction.

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S. Ott, H. Wunderli-Allenspach/European Journal of Pharmaceutical Sciences 1 (1994) 323-332

Dequenching kinetics upon R18 transfer between labelled SUV and PR8 viruses To characterise the kinetics of the R18 transfer reaction, analogous experiments to those described above were performed at neutral pH where no fusion with influenza viruses occurs. Incubations were carried out at pH 7.4 under standard conditions with variation of either the total particle concentration or the initial R18 surface density. Analysis of all experimental data obtained with incubation at pH 7.4 revealed that best fits were again obtained assuming second-order kinetics. Fitted curve parameters are listed in Table 2. DQmax values are always between 0.70 and 0.97, i.e. in the same range as found for the fusion reaction. Under standard conditions (see Experimental procedures), t* is about 30-fold increased as compared to the t* value of the fusion reaction at pH 5.3. Variation of the total particle concentration in the assay revealed that this parameter does not influence the transfer kinetics (Table 2A), i.e. t* values stay constant (34 to 38 min) and DQmax values are between 0.70 and 0.94. To study the influence of pH on the transfer reaction, we tested the effect of particle concentration and thus collision frequency on the dequenching reaction at acidic pH in the absence of fusion. For this purpose virus particles devoid of HA were used. The latter were obtained by digestion with bromelain which is known to cleave the HA protein resulting in the loss of the specific fusion activity of the virus (Brand and Skehel, 1972). Under these conditions, the reaction was somewhat faster (t* ,,~ 20 min), but again it was found to be independent of the particle concentration (data not shown). Thus, R18 transfer both at pH 7.4 and pH 5.3 shows second-order kinetics, although the collision frequency of the viruses and liposomes is not rate-limiting for the reaction. A different picture was obtained if the influence of the initial R18 surface density on the transfer reaction was tested (same initial quench values as tested in fusion experiments). In this case again no systematic variation was found for the DQmax values; however, t* ranged from 30 to 130 min (Table 2B). With decreasing R18 surface density, t* increased, i.e. transfer was slowed down. Normalisation to the surface density of standard liposomes, 0.122 R18 molecules per nm 2, yielded t~ values between 28 and 43 min. From these results we conclude that R18 molecules represent the interacting species determining the second-order kinetics of transfer (see below). General aspects of dequenching kinetics data analysis Analysis of dequenching kinetics is complicated by the fact that several processes may overlap. In the case of fusion, marker dilution after collision occurs within one, i.e. the fused, particle through diffusion along the concentration gradient. The rate-limiting step is the collision frequency of labelled and unlabelled vesicles (see above).

80

40

0a 20

0

,

,

,

,

1

2

3

0

0.08

0.16

r [R10 molecules/nm 2]

Fig. 2. Stern-Volmer plot of quench curve for R18-labelled SUV and PR8 viruses respectively. PR8 viruses (A) or liposomes with standard lipid composition (B) were exogenously labelled with decreasing amounts of R18. Quenching of each preparation was determined and DQ -l = (1 - Q)-~ plotted against the respective R18 surface densities r, i.e. the number of R18 molecules per nm 2 of lipid surface. A polynomial fit (n = 2) was used for both quench curves (proFit4.0 program). The intercept (r = 0) equals 1 for liposomes and viruses. Fit parameters for a I and a2 are listed in the Results section.

Both the fusion itself and the subsequent distribution of the marker molecule on the fusion product are obviously faster processes and do not show up in the dequenching kinetics (see Supplement). In transfer reactions, the situation is more complex. The marker surface density of the labelled donor vesicles decreases, whereas on the unlabelled acceptor it increases simultaneously. For R18 this means that dequenching in the donor is accompanied by a gradual increase of quenching in the acceptor particles. The contribution of each of the two overlapping processes to the total dequenching can be calculated with help of the quench curves of the interacting particles. In Fig. 2, the quench curves of SUV and PR8 viruses respectively are illustrated with a SternVolmer plot (see Experimental procedures). For SUV, quenching and dequenching respectively are functions of the square of the molar marker surface density: (DQ -1) = 1 + a2 x r 2. The same result was found for LUV (data not shown). For both SUV and LUV the a2 constant equals 1154 + 116 (n :-7) (Wunderli et al., 1993). In the case of R18-1abelled PR8 viruses about 20 times more of the marker was needed to reach the same quenching. In this case monomers and dimers were involved in the quench process: (DQ -1) = 1 + a~ x r + a2xr 2, with a~ -~ 9 and a2 --- 3 (see legend Fig. 2). Calculations for the contribution of donor and acceptor vesicles to the total dequenching during transfer (pH 7.4) were performed using the respective quench curves for liposomes and PR8 viruses (see Supplement, formula (S10)). This combined quench curve was used to attribute to each experimentally determined DQtot(t ) the corresponding amount of transferred R18 expressed as surface density r. A linear relationship was found between DQtot , measured up to 120 min, and r. This is illustrated in Fig. 3 where the transferred R18 is

328

S. Ott, H. Wunderli-Allenspach/European Journal of Pharmaceutical Sciences 1 (1994) 323 332

1.0

~

Table 3 Interaction of PR8 viruses and R18-1abelled LUV at pH 5.3 Total conc. pH 5.3 fast component pH 5.3 slow component (particles/ml) DQmax t* t~ DQmax t*(min) (min) (min)

0.5

8.1× 10 ~° 0.7× 10 ~° 0.0 0.0

015

1.0

r / rlnit Fig. 3. DQtot as a function of the transferred fraction of R18, r/rinit. For experimentally derived DQtot (0-120 min) the corresponding r values were calculated with formula (S 11) (see Supplement) and displayed as r/ rinit for various rinit: • 0.122 (96% Qinit), • 0.056 (75% Qinit); • 0.0315 (54% Qinit). In addition, curves were extrapolated for DQtot at t > 120 min using the fit parameters listed in Table 2: [] 0.122 (96% Qinit), C) 0.056 (75% Qinit);/~ 0.0315 (54% Qinit). The y-intercept of the respective curves is identical to the corresponding initial quenching of the liposome preparations.

expressed as fraction r/rinit t o allow direct comparison between various Qinit. For extended times (> 120 min) linearity is not preserved in all cases as can be seen if curves are extrapolated t o r/rinit > 0.9. With Qinit > 90°/o the linear relationship is maintained up to a transferred fraction of about 0.8, whereas with Qinit < 800/0 deviations from linearity occur at lower fractions of transferred R18, e.g. already around 0.3 for Qinit = 52o/o. The time-dependent transfer of R18, r(t), can finally be expressed by replacement of the experimental DQtot data points with the corresponding r values (see Supplement). With this approach r(t) was calculated for various Qinit for times up to 120 min and the resulting data sets fitted with the profit4.0 program (see Experimental procedures). In all cases best fits resulted with a hyperbolic, i.e. second-order-derived function (see Experimental procedures), t~ values of the r(t) curves obtained with Qinit between 52 and 96% were 33-43 min. This is in the same range as found for DQ(t) curves (see Table 2B), which means that transfer kinetics of R18 molecules are not distorted by the overlapping dequenching/quenching in the donor and acceptor vesicles respectively. The t~ value of 40 min therefore reflects the actual time to reach half-maximal transfer of the marker. The corresponding rate constant for transfer equals 2k ,,~ 0.2 [(R18 molecules per nm2) -~ × min-l].

0.18±0.01 1.06±0.13 0.10±0.03 9.27±0.04

1.4 1.1

0.39±0.02 112.3±15.7 0.40±0.02 118.7±24.9

Incubations of LUV (Qinit 92%) with PR8 viruses were carried out at pH 5.3 with two different particle concentrations under the same assay conditions as used for SUV. The lipid concentration corresponding to 8.1 × 10 ~° total particles/ml for the assay with LUV is 340ng liposomal lipid and 6.4 mg viral lipid. Dequenching curves from two independent experiments were analysed as described. Best fits were obtained by superposition of two second-order reactions, t* of the fast component was normalised to the standard particle concentration of SUV (6 × 10 ~°, see above) as described in the legend to Table 1.

viruses diameter 55-70 nm (only lipid bilayer), LUV diameter 180 nm and SUV diameter 27.5 nm. Maximal possible marker dilution upon interaction of PR8 viruses with R18-1abelled SUV is 5-fold, whereas for R18labelled LUV with viruses it is only 1.25-fold. Therefore, dequenching will be incomplete after fusion and will proceed subsequently through marker transfer to unlabelled viruses present in excess. Thus, dequenching at pH 5.3 results from overlapping fusion and transfer. The resulting dequenching curves can be analysed with the p r o f i t program. Best fits were obtained assuming superposition of two second-order reactions: a fast component related to fusion and a slower component related to transfer. Studies were performed in this system to test the influence of the total particle concentration. In Table 3, we show that the fusion reaction between LUV and PR8, i.e. the fast component, is affected. The t* value increases with decreasing particle concentration. Normalisation to the standard particle concentration (see figure legend) yields the same result as for SUV. At the concentrations tested (0.7 and 8.1 × 10 l° particles per ml), t~ is 1.4 or 1.1 min. For lipid transfer (slow component) t* does not show a substantial difference for the two particle concentrations tested (t* 110-120 min). Thus the results obtained above with small liposomes are confirmed by using LUV. Transfer from LUV is, however, slower as compared to the one determined above from SUV.

Interaction of R18-labelled L U V with PR8 viruses

4. Discussion

To exclude the possibility that the analysed dequenching kinetics were only peculiar to the interaction between SUV and PR8 viruses, R18-1abelled LUV were tested in the same assay. The interaction of PR8 viruses with R18labelled LUV yields more complex dequenching kinetics than the one with SUV. This is due to the size of the respective interacting particles (see Ott et al., 1994):

A schematic diagram of the membrane interactions between R18-containing liposomes and PR8 viruses is shown in Fig. 4. Best fits for the experimental, timedependent dequenching at pH 5.3 as well as at pH 7.4 were obtained assuming second-order kinetics. Rate constants in both cases thus depend on the concentration of

S. Ott, H. Wunderli-Allenspach/European Journal of Pharmaceutical Sciences 1 (1994) 323-332

(L)R18 -t- (V) ~ '

(/)RI~ (V)

~x ? ~~ ~'1"~

(L)R1a + IV)R18

~

(LRlsVm~

Fig. 4. Schematic representation of membrane interactions between R18-1abelled liposomes and PR8 viruses. Fusion and lipid transfer as measured with the R18 marker are schematically represented in this diagram. 2kb second-order binding constant, rate-limiting for the fusion reaction; lk F first-order fusion constant; 2kTr second-order transfer (i.e. dequenching) constant, rate-limiting for R18 transfer.

the interacting species. In the case of fusion this was found to be the total particle concentration in the assay. With linearly increasing particle concentrations a linear decrease was found for t* at pH 5.3, which shows that the rate-limiting step in the fusion process is particle collision. At very high particle concentrations a change should occur to a pseudo-first-order reaction. Due to experimental limitations (light scattering of particles, detection range of the fluorescence spectrophotometer) this could not be substantiated in our assay. However, as previously demonstrated (Wunderli and Ott, 1990), dequenching occurs with a very fast first-order process ( h / 2 ~ 0.1 s), ifPR8 viruses and R18-1abelled SUV were prebound in the cold before exposure to fusion conditions (pH 5.3, 37°C). For the dequenching observed at pH 7.4 data analysis also shows second-order kinetics. This reaction can, however, by kinetic criteria be clearly distinguished from fusion: the rate-limiting step is not the collision frequency but rather the local R18 concentration, i.e. the surface density r. The higher the local surface density of the model molecule R18, the faster the transfer reaction. As we demonstrate (Supplement), the same result is obtained if we take into account the simultaneous dequenching in the acceptor and quenching in the donor. Lipid transfer so far has usually been measured after separation of the donor from the acceptor vesicles. As demonstrated here, the principle of fluorescence dequenching, which is largely applied for fusion and leakage studies, can also be used for kinetic studies of lipid transfer. Rate constants calculated here for fusion and lipid transfer cannot be compared directly, since they differ in the respective interacting species. Yet we can discuss them comparatively using the t* values. Under standard c o n d i t i o n s , (Qinit = 96%, particle concentration 6× 101°) per ml, the time to reach 50% of the respective reactions is 1 min for fusion and 40 min for transfer. Thus, although fusion is a very fast process, the rate-limiting step (binding) in this assay is only about 10-100 × faster as compared to the non-specific transfer. In vivo a targeted liposome should ideally reach its site of action

329

before specific binding and fusion occur. Since transfer does not depend on binding, a considerable part of the drug can be lost non-specifically. From the data presented here it seems that lower membrane concentration helps to reduce transfer. Transfer processes, as described here for R18, are well documented for other lipids like phosphatidylcholine derivatives or cholesterol (Roseman and Thompson, 1980; Nichols and Pagano, 1982; Fugler et al., 1985). In agreement with our results, they have usually been described to be independent of the concentration of the interacting vesicles. In contrast, however, to our results they were reported to follow first-order kinetics (Gardam et al., 1989) and only at very high vesicle concentrations was an additional, particle concentration-dependent process observed by Jones and Thompson (1989). The question remains, whether transfer of R18 occurs by passive partitioning between the lipophilic (membrane) and the hydrophilic phase as has been claimed for other transfer reactions (Nichols and Pagano, 1982; Fugler et al., 1985; Gardam et al., 1989), or whether collision of donor and acceptor vesicles is a prerequisite although not rate-limiting. At the moment, we cannot decide between the two possibilities. From experiments with bromelain-treated viruses as acceptors, we know that specific binding is not required for transfer, since the same transfer rate was observed with HA-depleted as with intact viruses and also with intact viruses incubated with Gola-free liposomes (Wunderli and Ott, 1990). Moreover, even unlabelled liposomes can equally well be used as acceptor vesicles (data not shown). In all cases, the initial R18 surface density remains ratelimiting. The advantage of the R 18 fluorescent probe over other fusion markers lies in the fact that it easily partitions into pre-existing membranes and thus can be used to label biological membranes directly. This is certainly the reason why it has been widely used in recent years (Struck et al., 1981; Nia" et al., 1986; Stegmann et al., 1986; Morris et al., 1989). On the other hand we have shown that R18 also tends to partition out of labelled membranes. Our kinetic analyses show that R18 is still a very useful fusion marker, provided that the necessary controls are performed. It even offers the advantage that both processes, fusion and transfer, can be studied simultaneously as they can be distinguished due to the kinetic differences. From its molecular structure R18 represents a type of a lipophilised prodrug. Thus R18, besides being a membrane marker, can also serve as a model to test the membrane interactions of lipophilised prodrugs incorporated into various liposomes. In the following paper (Ott et al., 1994) we use this model to analyse the effect of liposomal size and production method on fusion and transfer kinetics. In summary, the presented dequenching assay proved helpful to characterise and compare the kinetics of

s. ott, H. Wunderli-Allenspach/European Journal of Pharmaceutical Sciences 1 (1994) 323-332

330

t r a n s f e r a n d fusion, t w o m e c h a n i s m s by w h i c h a lipophilic d r u g c a n l e a v e l i p o s o m e s . B o t h r e a c t i o n s f o l l o w s e c o n d - o r d e r kinetics. T h e y c a n , h o w e v e r , be disting u i s h e d since f u s i o n d e p e n d s o n t h e t o t a l p a r t i c l e c o n c e n t r a t i o n in t h e a s s a y w h e r e a s t r a n s f e r is a f u n c t i o n o f t h e R I 8 s u r f a c e density. T h i s m e a n s t h a t , d e p e n d i n g o n e x p e r i m e n t a l c o n d i t i o n s in r e g a r d to p a r t i c l e c o n c e n t r a t i o n a n d initial q u e n c h i n g o f the l a b e l l e d species, o n e o r t h e o t h e r r e a c t i o n is f a v o u r e d .

Acknowledgement We thank assistance.

Maja

Gfinthert

for

excellent technical

References Arbeloa, I.L. (1981) Dimeric and trimeric states of the fluorescein dianion: part 2. Effects on fluorescence characteristics. J. Chem. Soc. Faraday Trans. 77, 1735-1742. Barrett, Th. and Inglis, S.C. (1985) Chapter 6: Growth, purification and titration of influenza viruses. In: Mahy, B.W.J. (ed.) Virology: a Practical Approach. IRL Press, Oxford, pp. 119-150. Bradford, M.M. (1976) A rapid and sensitive method for the quantiration of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Brand, C.M. and Skehel, J.J. (1972) Crystalline antigen from the influenza virus envelope. Nature New Biol. (London) 238, 145-147. Diizgiines, N. and Bentz, J. (1988) Fluorescence assays for membrane fusion. In: Loew, L.M. (ed.) Spectroscopic Membrane Probes, Vol. 1. CRC Press Inc., Boca Raton, FL, pp. 117-159. Fugler, L., Clejan, S. and Bittman, R. (1985) Movement of cholesterol between vesicles prepared with different phospholipids or sizes. J. Biol. Chem. 260, 4098-4102. Gardam, M.A., Itovitch, J.J. and Salves, J.R. (1989) Partitioning of exchangeable fluorescent phospholipids and sphingolipids between different lipid bilayer environments. Biochemistry 28, 884-893. Hoekstra, D., De Boer, T., Klappe, K. and Wilschut, J. (1984) Fluorescence method for measuring the kinetics of fusion between biological membranes. Biochemistry 23, 5675-5681. Huang, C. and Mason, J.T. (1978) Geometric packing constraints in egg phosphatidylcholine vesicles. Proc. Natl. Acad. Sci. USA 75, 308-310. Jones, J.D. and Thompson, T.E. (1989) Spontaneous phosphatidylcholine transfer by collision between vesicles at high lipid concentration. Biochemistry 28, 129-134. Klenk, H.-D. (1991) Family - - Orthomyxoviridae: genus - - influenza virus A and B: type species - - influenza virus: A/PR/8/34 (H1N1). In: Francki, R I.B., Fauquet, C.M., Knudson, D.L. and Brown, F. (eds.) Classification and Nomenclature of Viruses. 5th Report of the International Committee on Taxonomy of Viruses. Springer Verlag, Vienna, pp. 263-272. Morris, S.J., Sarkar, D.P., White, J.M. and Blumenthal, R. (1989) Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. J. Biol. Chem. 264, 3972-3978. Nichols, J.W. and Pagano, R.E. (1982) Use of resonance energy transfer to study the kinetics of amphiphile transfer between vesicles. Biochemistry 21, 1720-1726. Nir, S., Stegmann, T. and Wilschut, J. (1986) Fusion of influenza virus

with cardiolipin liposomes at low pH: mass action analysis of kinetics and extent. Biochemistry 25, 257 266. Ott, S., Schurtenberger, P. and Wunderli-Allenspach, H. (1994) Liposomes and influenza viruses as an in vitro model for membrane interactions. II. Influence of vesicle size and preparation methods. Eur. J. Pharm. Sci. 1,333 341. Press, W.H., Flannary, B.P., Teukolsky, S.A. and Vetterling, W.T. (1986) Numerical Recipes - - the Art of Scientific Computing. University Press, Cambridge. Roseman, M.A. and Thompson, T.E. (1980) Mechanism of the spontaneous transfer of phospholipids between bilayers. Biochemistry 19, 439-444. Rubas, W., Supersaxo, A., Weder, H.G., Hartmann, H.R., Hengartner, H., Schott, H. and Schwendener, R. (1986) Treatment of murine L1210 lymphoid leukemia and melanoma B16 with lipophilic cytosine prodrugs incorporated into unilamellar liposomes. Int. J. Cancer 37, 149 154. Schwendener, R.A., Supersaxo, A., Rubas, W., Weder, H.G., Hartmann, H.R., Schott, H., Ziegler, A. and Hengartner, H. (1985) 5rO-palmitoyl- and 3',5'-O-dipalmitoyl-5-fluoro-2rdeoxyuridine - novel lipophilic analogues of 5-fluoro-2'deoxyuridine: synthesis, incorporation into liposomes and preliminary biological results. Biochem. Biophys. Res. Comm. 126, 660-666. Stegmann, T., Hoekstra, D., Scherphof, G. and Wilschut, J. (1986) Fusion activity of influenza virus - - a comparison between biological and artificial target membrane vesicles. J. Biol. Chem. 261, 10966-10969. Stegmann, T., Schoen, P., Bron, R., Wey, J., Bartoldus, I., Ortiz, A., Nieva, J.-L. and Wilschut, J. (1993) Evaluation of viral membrane fusion assays comparison of the octadecylrhodamine dequenching assay with the pyrene excimer assay. Biochemistry 32, 1133011337. Struck, D.K., Hoekstra, D. and Pagano, R.E. (1981) Use of resonance energy transfer to monitor membrane fusion. Biochemistry 20, 4093-4099. Tagushi, T. (1980) Review of a new antimetabolic agent 1-hexylcarbamoyl-5-fluorouracil (HCFU). Recent Results in Cancer Research, Vol. 70. Springer-Verlag, Heidelberg, pp. 125-130. Weinstein, J.N. and Leserman, L.D. (1984) Liposomes as drug carriers in cancer chemotherapy. Pharmacol. Therapeut. 24, 207 233. Wunderli-Allenspach, H. and Ott, S. (1990) Kinetics of fusion and lipid transfer between virus receptor containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay. Biochemistry 29, 1990-1997. Wunderli-Allenspach, H., Giinthert, M. and Ott, S. (1993) Inactivation of PR8 influenza virus through the octadecylrhodamine B chloride membrane marker. Biochemistry 32, 900-907.

Supplement Detailed kinetic analysis of dequenching in the interacting particles T h e f o l l o w i n g c a l c u l a t i o n s a r e b a s e d o n f o r m u l a s (1) to (5) i n t r o d u c e d in E x p e r i m e n t a l m e t h o d s .

1. Fusion 1.1. Definitions r rinit

surface density of RI8 initial R 1 8 s u r f a c e d e n s i t y in l i p o s o m e

S. Ott, H. Wunderli-Allenspach/European Journal of Pharmaceutical Sciences 1 (1994) 323-332 rF

Ltot

L DQtot OQlip DQF

R18 surface density on the fusion product after diffusion of the marker total amount of liposomes in assay amount of fused liposomes dequenching of R18 in the assay (see formula (1)) dequenching of R18 in the unfused liposome dequenching of R18 in the fusion product

DQ(t) = (L/Ltot)(DQF - DQinit) = (L/Ltot) × C

L/Ltot = (l/C) × DQ(t)

2. Transfer

Unfused liposomes.

2.1. Definitions

DQlip = DQinit = 1/(1 + 1154 × r~nit)

(S1)

Fusion products. Experiments are performed with excess PR8 virus (viruses/liposomes=2/1). Under these conditions one virus fuses with one liposome (Nir et al., 1986). The R18 surface density, r, on the fusion product changes with time from rinit to a final r F which depends on the respective diameters of the fusion partners, i.e. of the liposomes and viruses. According to the Stern-Volmer equation: DQF(r) = 1/(1 + 1154 x r 2)

($2)

amount of R18 in liposome, i.e. in donor amount of R18 transferred to virus = Rlip q- Rvir, total amount of R18 in assay Rtot initial R18 surface density in liposome rinit r difference between rinit and residual surface density on donor surface density of R18 on virus after transfer of rvir Rvi r f r o m liposome rinit -- r surface density of R18 on liposome after transfer of Rvir to virus DQtot dequenching of R18 in the assay (see formula (1)) dequenching of R18 in the liposome (donor) DQlip DQvir dequenching of R18 in the virus (acceptor) Rlip Rvir

2.2. Quenching in donor and acceptor particles

As diffusion of R18 on the fused particles is very fast, particle collision being the rate-limiting step (see Results), fusion products have a constant R18 surface density rv and hence a constant DQF:

Donor.

DQF = 1/(1 + 1154 x ~v)

DQlip(r ) = 1/(1 + 1154 x (rinit -- r) 2)

($3)

1.3. Total dequenching in the fusion assay

After transfer of Rvir molecules R18 to the acceptor (virus), the R18 surface density in the donor (liposome) is reduced to (rinit -- r). According to Stern-Volmer: ($7)

Acceptor.

At time t, R18 will be distributed on L fused particles and (Ltot - L ) unfused liposomes. DQtot can thus be calculated from (S1) and ($3): DQtot = [1 - (L/Ltot) ] x DQinit + (L/Ltot) x DQF

($6)

This indicates that with the experimentally determined DQ(t) a second-order function also results for L(t) as expected when taking into account the collisionmediated character of the fusion reaction.

1.2. Quenching in liposomes and fusion products

Quenching in the unfused liposomes can be calculated with the Stern-Volmer equation (see (5)). During the fusion reaction the R18 surface density in the unfused liposomes (rirat) does not change, thus DQlip is constant:

331

($4)

After transfer of Rvi r molecules R18, the surface density in the virus is r v i r . DQvir(r ) can be calculated as follows: DQvir(r) = 1/(1 + 9 x rvir + 3 x rv2ir)

(S8)

1.4. Time dependence of the variable L

2.3. Total dequenching in the transfer assay

Combination of the total dequenching as determined experimentally (formula (2)) and the total dequenching expressed as a function of the R18 surface density r (formula ($4)) yields the time-dependent change of L:

For the total dequenching, DQtot , in the transfer assay the following aspects have to be considered. The number of donor and acceptor particles is constant throughout the experiment. At time t, Rvir will be transferred to the virus particles resulting in a R18 surface density of rvir. Under standard experimental conditions rvir equals (r/20) as a 20-fold excess of unlabelled acceptor

DQinit -F DQ(t) = [1 - (L/Ltot) ] × DQinit -q- (Z/Ztot) × DQF

($5)

S. Ott, H. Wunderli-Allenspach/European Journal of Pharmaceutical Sciences 1 (1994) 323 332

332

•

,

0.08

rinit

]

r - -

rinit

~ 0.04 n-

40

80

120

l i m e Ira|n]

membranes is present. Calculations for DQtot can thus be performed with Eqs. (1) and (5): FpBs(tot) = FpBs(donors) + Fpas(acceptors ) D Q t o t x R t o t = Rlip × D Q l i p q- Rvir × D Q v i r

(Rlip/Rtot)

× DQli p +

D Q t o t = [(Rto t -

Rvir)/Rtot]

D Q t o t = [(rinit -

r)/rinit]

D Q t o t = [1 -

(r/rinit)]

(Rvir/Rtot)

x D Q l i p q-

× DQlip +

x D Q l i p q-

× DQvir

(Rvir/Rtot)

(r/rinit)

(r/rinit)

× DQvir

x DQvir

x DQvir

(S9)

For the total dequenching in the assay ($7) and ($8) can be substituted in (S9): DQtot(r )

=

(1

---- r) rinit

r rinit

--X

1

×

1 + 1154

r

(~)+3x

r

(~)

2

(S10)

2.4. Time dependence of the variable r

Fig. S1. Transferred R18 as a function of time for various Qinit. For liposomes of various initial quenching the transferred RI8 marker, expressed as surface density r, was calculated from the experimentally determined DQtot(t ) at pH 7.4 and plotted against the corresponding times (see Supplement): ~ r = 0.122 (96% Qinit), • r = 0.056 (75% Qinit); • r = 0.0315 (54% Qinit). Calculated data were fitted (solid lines) with the following second-order-derived function: r = rmax× t/(t* + t). The following curve parameters were obtained: t* = 36.2± 1.5 min and rmax = 0.110 ± 0.002 for Qinit 96%; t* = 96.9 + 4.1 rain and rmax = 0.049 d: 0.001 for Qinit 75%; t* = 146.9 ± 16.9 min and rmax = 0.029 ± 0~002 for Qinit 54%.

DQtot =

l+9x

× (rinit - r) 2 q-

This means that during transfer DQtot depends on the transferred amount of R18 and the corresponding quenching in the donor and the acceptor respectively.

0.00

0

×

1 + 1154 1

x (rinit -

1 1 + 9 X rvir q- 3 X flvir

q-

r) 2

Combination of the experimentally determined dequenching as a function of time t (formula (2)) and the total dequenching expressed as a function of the R18 surface density r (formula (S10)) yields the timedependent change of r, i.e. r(t): DQini t + D 0 ( t ) = (1 _ _ _ r ) rinit

×

1 + 1154 x 1

- + - r- - × rinit 1 + 9 ×

1 (rinit - r) 2

+ 3×

( 01 (Sll)

The expression for the change of R 18 surface density r(t) is complex; however, for each measured DQ(t) the corresponding r can be calculated. For this purpose the spline function (proFit4.0) was used which allows free approximation of any complex curve without choosing a defined function. The resulting r(t) were analysed again with proFit4.0. In all cases best fits were obtained with a second-order-derived formula:

r(t) = (rmax × t)/(t* + t)

(S12)

Thus, a second-order function also results for the timedependent change in R18 surface density during transfer of the marker. Fig. S1 illustrates the calculated r(t) curves for Qinit = 96%, 75% and 54% together with the corresponding optimal fits. Fitted parameters for various Qinit a r e listed in the figure legend.