Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy

Chemistry and Physics of Lipids 104 (2000) 35 – 47 www.elsevier.com/locate/chemphyslip

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy Aladdin Pramanik, Per Thyberg, Rudolf Rigler * Department of Medical Biochemistry and Biophysics, Di6ision of Medical Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden Received 15 April 1999; received in revised form 24 June 1999; accepted 17 July 1999

Abstract Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phospholipid vesicle membrane; Rhodamine; Fluorescence correlation spectroscopy; Lipid-peptide molecular interaction; Melittin; Channel formation; Magainin; Vesicle membrane breakage

1. Introduction Phospholipid vesicles, artificial membrane systems, have been studied intensively as a tool to determine the properties of biological membranes. With vesicles it is easier to manipulate the various * Corresponding author. Tel.: +46-8-728-6800; fax: +468-32-6505. E-mail address: [email protected] (R. Rigler)

parameters of the membrane system and thus, to investigate membrane-bound enzymes and membrane transport systems without possible interfering reactions that occur in the cell membrane. The most important use of vesicles is in drug therapy as carriers of drugs. Vesicles, prepared with drugs entrapped inside, are being used as carriers for these drugs to target organs (Ranade, 1989) and thus, the effect of drugs lasts for a longer period (Perez-Soler, 1989).

0009-3084/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 9 9 ) 0 0 1 1 3 - 9

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In the extrusion technique, a straightforward and rapid protocol for production of large unilamellar vesicles of homogeneous size (Mayer et al., 1986), the total absence of potentially toxic agents (organic solvents or detergents) is obviously beneficial when vesicles are used as carriers of drugs (Mayer et al., 1985; Ranade, 1989). Since the interior of the vesicle is an aqueous environment, with the extrusion technique it is possible to prepare vesicles with different substances/dye molecules entrapped inside. Thus, the external and internal environment of phospholipid vesicles can be manipulated and studies can be conducted on a variety of properties of these synthetic membranes, including their ability to incorporate or release molecules and their interaction with various substances (peptides, proteins, nucleic acids). In earlier studies interactions between proteins and vesicles have been performed using different biochemical procedures (Eytan et al., 1976; Ingelman-Sundberg and Glaumann, 1980; Popot et al., 1981; Le´vy et al., 1992; Schu¨rholz et al., 1992; Tranum-Jensen et al., 1994). In this paper using fluorescence correlation spectroscopy (FCS) we will demonstrate molecular interactions of the peptides melittin, (Habermann, 1972; Bernheimer and Rudy, 1986; Dempsey, 1990; Berne´che et al., 1998) and magainin (Zasloff, 1987) with vesicles. FCS is a powerful biophysical tool for examining molecular interactions of biological important substances with very high specificity (Rigler, 1995). In FCS, the fluorescence of single dye-labelled molecules excited by a sharply focused laser beam is observed. From the autocorrelation function of the fluorescence intensity fluctuations, which are due to varying numbers of molecules in the laser volume element of observation, the average number of molecules can be calculated. Furthermore, from the characteristic correlation times dynamic processes can be analyzed (Magde et al., 1972; Ehrenberg and Rigler, 1974; Elson and Magde, 1974; Rigler et al., 1993; Eigen and Rigler, 1994). Thus, FCS is used for measuring the diffusion velocities of fluorescence particles/ molecules in solutions (Rigler et al., 1993; Rigler, 1995; Tjernberg et al., 1999) and at the cell surfaces (Rigler, 1995; Pramanik et al., 1999; Rigler et al., 1999). With FCS one can analyze a mixture

of several components with different molecular weights (M1, M2, M3…) and correspondingly with different diffusion times (tD1, tD2, tD3…). The beauty of this technique is that there is no need for separating unbound component from bound one (Rigler, 1995). Moreover, measurement can be done in a very tiny volume (1–5 ml) in a nanomolar range within very short time (5 s or even less). The single molecule sensitivity of FCS (Rigler and Mets, 1993; Mets and Rigler, 1994; Rigler, 1995) at subnanomolar range and below opens up a new era in molecular interactions.

2. Materials and methods

2.1. Chemicals Rhodamine (tetramethylrhodamine-5-(and-6)isothiocyanate) was purchased from Molecular Probes Europe BV (Leiden, The Netherlands). Melittin and magainin were purchased from Bachem Feinchemikalien (Bubendorf BL, Switzerland). Stock solutions of melittin and magainin were made as 1 mg/ml in water and stored at − 20°C into small portions. The lipid POPC (1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) was obtained from Avanti Polar Lipids (Alabaster, AL). HEPES (N-(2-hydroxy-ethyl)piperazine-N%-2-ethanesulfonic acid) was bought from Bioprobe, (Chemie Brunschwig AG, Basel, Switzerland). Both NaCl and Na2EDTA·2H2O were supplied by Merck (Darmstadt, Germany). All other chemicals are of standard grade.

2.2. Vesicle preparation Phosphatidylcholine (PCh) has been used for vesicle preparation. As is known, due to amphipathicity PCh, with a hydrophilic head and a hydrophobic tail, interact with each other in an aqueous system. At certain concentration and condition, PCh interacts to form a bimolecular leaf structure with two layers of lipid in which the polar head groups are at the interface between the aqueous medium and the lipid, and the hydrophobic tails interact to form an environment that excludes water. Since this lipid bilayers of PCh are

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extremely stable structures and water is essentially excluded from their interior, we disrupted these bilayers with the extrusion technique so that they can self-seal. Thus, a lipid bilayer of PCh closed in on itself, forming a spherical vesicle separating the external space from an internal compartment (Fig. 1).

2.2.1. Lipid films For all vesicle preparation we made lipid films as follows. Ten mg of POPC was dried by rotary evaporation and then placed overnight under an oil pump vacuum. The films were stored at − 20°C under argon in order to prevent oxidation processes. 2.2.2. Preparation of 6esicles from unlabelled phospholipids together with added free rhodamine entrapped inside (REV) In the studies, unilamellar vesicles of 100 nm diameter were prepared from lipid films using the extrusion technique (Hope et al., 1985; Mayer et al., 1986). Prior to extrusion, with vortex mixing a dried lipid film was dispersed in 2 ml buffer solution (10 mM HEPES (pH 7.4), 107 mM NaCl, 1 mM Na2EDTA·2H2O) (Rex, 1996) containing 1 nM rhodamine. Then a freeze – thaw

Fig. 1. Schematic presentation of cross sections of vesicles. (A) Vesicles are made from phospholipids together with added rhodamine where rhodamine is entrapped inside (RLV). (B) Vesicles are made from rhodamine-labelled phospholipids (RLV). In RLV rhodamine is covalently bound to the head groups of phospholipids. Thus, rhodamine is not entrapped inside of RLV, i.e. the inside of RLV is free of dye molecules.

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cycle of lipid mixture was done five times. The freeze–thaw cycle was obtained by freezing the lipid mixture in liquid nitrogen/dried ice and thawing at 40°C. After the freeze–thaw cycle the lipid suspension was extruded ten times by the Avanti mini extruder (Avanti Polar Lipids) through one polycarbonate membrane of the 100 nm pore size (Nucleopore Corporation, Pleasanton, CA). The REV entrapped free rhodamine inside (Fig. 1A), which can be released out of REV if pores/channels are formed into vesicles, or they are broken. MicroSpin™ S-200 HR Columns (Pharmacia Biotech, Uppsala, Sweden) were used to remove non-entrapped rhodamine after vesicle preparation. REV were stored at 4°C under argon and they are stable at least for a month.

2.2.3. Preparation of 6esicles from rhodamine-labelled phospholipids (RLV) With the similar procedure as in REV preparation, we also made vesicles from rhodaminelabelled phospholipids (rhodamine-1-palmitoyl-2oleoyl - sn - glycero - 3 - phosphatidylcholine (rhodamine-labelled POPC)). In RLV preparation the content of rhodamine-labelled lipids is 5% and unlabelled phospholipids were added to rhodamine-labelled phospholipids in order to compensate the phospholipid concentration. We therefore believe that the property of the lipid surface in the RLV vesicles is represented by the non-labelled phospholipids. However, it cannot be excluded that dye tag may affect local properties of phospholipids. In contrast to REV, RLV did not entrap free rhodamine inside since rhodamine is covalently bound to the head groups of phospholipids (Fig. 1B). Therefore, the formation of channels into RLV cannot result in rhodamine release in solution and only breakage of RLV can make an appearance of free rhodamine in solution. RLV were stored at 4°C under argon and they are stable at least for a month. 2.3. Fluorescence correlation spectroscopy (FCS) 2.3.1. FCS instrumentation FCS was performed with confocal illumination of a volume element of 0.23 fl in an instrument as described previously (Rigler et al., 1992, 1993). As

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focusing optics a Zeiss Neofluar 63× NA 1.2 was used in an epi-illumination setup. For separating exciting from emitted radiation a dichroic filter (Omega 540 DRL PO2) and a bandpass filter (Omega 565 DR 50) were used. Rhodamine and rhodamine-coupled vesicles were excited with the 514.5 nm line of an Argon laser. The fluorescence intensity fluctuations were detected by an avalanche photo diode (EG & SPCM 200) and were correlated with a digital correlator (ALV 5000, ALV, Langen, Germany).

2.3.2. FCS data e6aluation The observed fluorescence intensity fluctuations dI(t) when correlated with fluorescence intensity fluctuations at time t + t yield the normalized intensity autocorrelation function G(t): G(t)=1+

dI(t) dI(t + t) I2

(1)

where the brackets describe the time average and ŽI the mean fluorescence intensity (Ehrenberg and Rigler, 1974; Elson and Magde, 1974). The intensity autocorrelation function G(t) for Brownian motion of molecules/particles in a 3D Gaussian volume element is described (Rigler et al., 1993) as follows:

G(t)= 1 +

1 N

or

: ;: ; 1 4Dt 1+ 2 v

: ;:

1 4Dt 1+ 2 z

1 N

1



G(t)=1+

+y

< : ;:   ; : ;:   ; = 1 (1−y) N

1+

1+

t

tD F

1 v 1+ z

t

tD V

1 v 1+ z

2

1/2

t

tD F

1/2

2

t

(3)

tD V

where N is the number of molecules, tD v = v 2/ 4Dv is the diffusion time for vesicles; tD F = v 2/ 4DF is the diffusion time for free rhodamine; y is the fraction of vesicles diffusing with tD v; (1− y) is the fraction of free rhodamine diffusing with tD F; v (0.5 mm) is the radius of the volume element of the laser beam and z (2 mm) is its length. In order to assess the presence of discrete free rhodamine molecules and vesicles we analyzed G(t) representing the distribution of diffusion times P(tDi ) according to a model of multiple components:

G(r)= 1+

;

1

1

1/2

1/2 1 (2) v 2t t 1+ 1+ tD z tD where N is the number of fluorescence molecules, tD the diffusion time (tD =v 2/4D, D the diffusion coefficient), v (0.5 mm) the radius and z (2 mm) the length of the volume element of the laser beam. The above-mentioned intensity autocorrelation function G(t) (Eqs. (1) and (2)) is valid when the particle size is smaller than the volume element. With a volume element of a diameter of 1 mm and a vesicle size of 100 nm this condition is fulfilled.

G(t)= 1+

To calculate the average number of molecules per volume element, and diffusion coefficients of free rhodamine (DF) and vesicles (DV) the intensity autocorrelation function G(t) is analyzed as follows:

1 m % N i=1

: ;: Pi

1+

t tDi



1 v 1+ z

2

t tDi

;

1/2

(4)

where Pi represents the distribution of diffusion time tDi. For parametrization and fitting of the autocorrelation function G(t) non-linear least squares minimization according to the Marquardt algorithm (Marquardt, 1963) was used. For evaluation of the distribution of diffusion times P(tDi ) the CONTIN algorithm (Provencher, 1982a,b) was applied.

2.3.3. FCS experiments Free rhodamine was used to calibrate the instrument and as a reference to estimate diffusion times of the vesicles, and to justify whether the

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vesicles are intact or broken. The diffusion times of free rhodamine and vesicles were determined separately. The stock solutions of the peptides melittin and magainin were diluted in the same buffer (10 mM HEPES pH 7.4), 107 mM NaCl, 1 mM Na2EDTA·2H2O) as used in vesicle preparation in order to maintain osmolarity when vesicles were incubated with the peptides. Melittin and magainin were diluted in the buffer to concentrations ranging from 1 to 100 mM. The effects of melittin or magainin on vesicles were investigated by incubating the vesicles together with the peptides for 1 h or more at room temperature under continuous shaking. The concentration ratio of peptide:phospholipid ranged from 1:1 to 1:40. At least the ratio of 1:20 (peptide:phospholipid) was necessary to obtain pronounced effects of peptides on vesicles. Hanging droplets (20 ml) of the samples were analyzed for 30 s up to 10 min in the FCS instrument, at several time points of incubation, ranging from minutes to hours. All experiments were performed at 20°C.

3. Results and discussion

3.1. Autocorrelation functions of 6esicles In FCS measurements the fluorescence intensity fluctuations are recorded from only those molecules that diffuse through the confocal laser volume element. The time required for the passage of fluorescent molecules through the volume element is determined by the diffusion coefficient, related to the size and shape of molecules. From the autocorrelation functions of fluorescence intensity fluctuations, the average number (N) and the diffusion time (tD) of molecules crossed through the confocal volume are analyzed (Eq. (3)). Intensity autocorrelation functions G(t) of free rhodamine and vesicles are shown in Fig. 2. Diffusion times (tD) of free rhodamine in solution (Fig. 2A), of REV vesicles with rhodamine entrapped inside as well as of RLV vesicles with rhodamine covalently bound to the head groups of phospholipids and not entrapped inside (Fig. 2B – C) are found to

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be average 0.061 9 0.002 and 9 9 1 ms, respectively. Similar diffusion times are obtained when autocorrelation functions (Fig. 2A–C) are analyzed with the CONTIN algorithm (Fig. 2D–F, see Eq. (4)). There are indications that RLV vesicles prepared under similar condition as REV ones appeared to be slight larger ( 5% in tD or hydrodynamic radius) (Fig. 2D–F). It can only be speculated that the hydrodynamic radius of RLV vesicles might be affected by the structure of rhodamine-labelled phospholipids, i.e. rhodamine-labelled POPC. Due to difference in diffusion times between free rhodamine and vesicles it is possible to quantify the fractions of free rhodamine and vesicles. It is noteworthy that there is no need of separation step for this quantification. Both the preparations of vesicles are found to be well pure since only a non-significant amount of free rhodamine is found in REV (Fig. 2E) and rhodamine-labelled phospholipid in RLV (Fig. 2F).

3.2. Melittin releases rhodamine from REV Intensity autocorrelation functions G(t) and distribution of diffusion times P(tDi ) of the effects of melittin on REV are exhibited in Fig. 3. In control tD of REV is 9 ms (Fig. 3A, C) whereas after exposure of melittin to REV tD is found to be 0.07 ms (Fig. 3B, D). The tD = 0.07 ms corresponds to free rhodamine (see Fig. 2A, D), indicating that rhodamine is not inside of the vesicles. In addition, the number of molecules has been increased after addition of melittin to vesicles, pointing out that the increase in the number of molecules can be contributed only by rhodamine molecules released. These results are consistent with the earlier studies on the melittin-induced leakage of vesicle membranes (Benachir and Lafleur, 1995; Rex and Schwarz, 1998). The results suggest that melittin either breaks vesicles or makes channels into them (see the model I, Fig. 7A). In analogy of our data FCS can be used to study vesicles, entrapped drugs inside. Thus, FCS can be of great pharmaceutical significance if it will be applied as a rapid protocol to test vesicles as drug carriers.

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Fig. 2. (A) The intensity autocorrelation function G(t) of free rhodamine in solution, tD =0.063 ms, N =2.4. (B) G(t) of rhodamine entrapped vesicles (REV), tD = 7.5 ms, N =0.8. (C) G(t) of rhodamine-labelled phospholipid vesicles (RLV), tD =9.1 ms, N =0.6. D – F are the CONTIN distributions of diffusion times P(tDi ) from A, B and C respectively.

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Fig. 3. Effects of the peptide melittin on rhodamine entrapped vesicles (REV). (A) The intensity autocorrelation function G(t) of REV in the absence of melittin (control), tD = 9 ms, N= 0.4. (B) G(t) of REV in the presence of melittin, tD =0.07 ms, N= 1.8. C and D are the CONTIN distributions of diffusion times P(tDi ) from A and B, respectively. Vesicles were incubated with melittin (5 mM) in a ratio of 1:20 (peptide:phospholipid) for 1 h at room temperature. Incubation mixture was shaken continuously on a shaker. Control vesicles were also shaken in a similar way.

3.3. Melittin makes channels into 6esicles Fig. 4 demonstrates G(t) and P(tDi ) of the melittin effects on RLV. tD of RLV in control (Fig. 4A, C) or in the presence of melittin (Fig. 4B, D) is found to be similar ( 7.5 ms). The tD =7.5 ms does not correspond to free rhodamine since it is much longer compared to that of free rhodamine (see Fig. 2A, D), showing that there is no free rhodamine in solution, i.e. outside of the vesicles. As compared to REV, the number of molecules is not changed when

RLV is incubated with melittin. Since RLV does not entrap free rhodamine, broken pieces of RLV are the only possibility for the appearance of rhodamine in solution, which in turn can increase the number of molecules. Thus, the absence of rhodamine molecules in solution and no change in the number of molecules verify that these vesicles are not broken into rhodamine-labelled phospholipid fragments, confirming the suggestion that melittin makes channels into vesicles (see the model I, Fig. 7A– B).

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The results are consistent with the earlier data that melittin interacts both with planar lipid bilayers and vesicles by forming pores into planar and vesicle membranes. In these reports the mechanisms of the melittin-induced pore formation are addressed with reference to structure, location, orientation, and conformation of melittin in lipid membranes, melittin concentration, membrane potential, and the lipid composition and structure of membranes by various approaches (Tosteson and Tosteson, 1981; Hanke et al., 1983; Vogel and Jahnig, 1986; Vogel, 1987; Brauner et al., 1987; Kuchinka and Seelig, 1989; Beschiaschvili and

Seelig, 1990; Pawlak et al., 1991; Stankowski et al., 1991; Otoda et al., 1992; Belmonte et al., 1994; Pawlak et al., 1994; Smith et al., 1994; Rex, 1996; Matsuzaki et al., 1997; Berne´che et al., 1998; Biggin and Sansom, 1999). Melittin is an amphiphilic peptide and adopts an a-helical conformation when interacts with lipid membranes. Melittin induces pores in membranes in the form of tetramers. The hydrophilic sides of four helices spans bilayers facing each other in order to form a hydrophilic pore through the membrane (Vogel and Jahnig, 1986; Vogel, 1987; Smith et al., 1994; Pawlak et al., 1994; Biggin and Sansom, 1999).

Fig. 4. Effects of the peptide melittin on rhodamine-labelled phospholipid vesicles (RLV). (A) The intensity autocorrelation function G(t) of RLV in the absence of melittin (control), tD = 7.4 ms, N= 0.3. (B) G(t) of RLV in the presence of melittin, tD =7.5 ms, N = 0.25. C and D are the CONTIN distributions of diffusion times P(tDi ) from A and B, respectively. Experimental conditions were the same as described in Fig. 3.

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Fig. 5. Effects of the peptide magainin on rhodamine entrapped vesicles (REV). (A) The intensity autocorrelation function G(t) of REV in the absence of magainin (control), tD = 8.5 ms, N =0.5. (B) G(t) of REV in the presence of magainin, tD =0.07 ms, N = 1.9. C and D are the CONTIN distributions of diffusion times P(tDi ) from A and B, respectively. Vesicles were incubated with magainin (50 mM) in a ratio of 1:20 (peptide:phospholipid) for 1 h at room temperature. Incubation mixture was shaken continuously on a shaker. Control vesicles were also shaken in a similar way.

The structural features of the lipid composition (e.g. POPC or POPG, POPC-POPG mixture) of bilayers play a key role when melittin interacts with membranes. It has been shown by NMR measurement that melittin exerts a pronounced effect on a conformational change of the phosphocholine headgroup of a POPC bilayer (Seelig and Seelig, 1980; Kuchinka and Seelig, 1989; Beschiaschvili and Seelig, 1990). It is noteworthy to mention that in a very sensitive and suitable way FCS has confirmed the earlier studies of

the melittin effects on lipid membranes performed by other important techniques (circular dichroism (CD), nuclear magnetic resonance (NMR), electron spin resonance (ESR), Raman spectroscopy, fluorescence energy transfer, fluorescence quenching…).

3.4. Magainin releases rhodamine from REV Both G(t) (Fig. 5A–B) and P(tDi ) (Fig. 5C– D) show that addition of magainin to REV has

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A. Pramanik et al. / Chemistry and Physics of Lipids 104 (2000) 35–47

decreased the tD from 8.5 (Fig. 5A, C) to 0.07 ms (Fig. 5B, D). As the tD =0.07 ms is much faster compared to 8.5 ms and corresponds to free rhodamine (see Fig. 2A, D), indicating that rhodamine is free in solution, i.e. is not inside of the vesicles. Furthermore, after incubation of REV with magainin as with melittin, the number of molecules has been increased. The data suggest that magainin either makes channels into vesicles, which may allow rhodamine molecules to be released, or breaks vesicles (see the model II, Fig. 7C).

3.5. Magainin breaks 6esicles tD of RLV in control is 11 ms (Fig. 6A, C) whereas after exposure of magainin to RLV (Fig. 6B, D) is found to be 0.07 ms (average tD = 0.0749 0.002 ms). However, the small peak observed in Fig. 6C represents the non-significant amount of rhodamine-labelled phospholipids not appeared in vesicle forms. POPC was labelled with rhodamine. On the basis of molecular weights of POPC (760), rhodamine (443) and magainin (2410) theoretical tD for Rhodamine-

Fig. 6. Effects of the peptide magainin on rhodamine-labelled phospholipid vesicles (RLV). (A) The intensity autocorrelation function G(t) of RLV in the absence of magainin (control), tD =11 ms, N =0.3. (B) G(t) of RLV in the presence of magainin, tD = 10 ms, N= 2. C and D are the CONTIN distributions of diffusion times P(tDi ) from A and B, respectively. Experimental conditions were the same as described in Fig. 5.

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Fig. 7. Model I: melittin makes channels into phospholipid vesicle membranes. (A) Rhodamine-entrapped vesicles (REV) plus melittin. (B) Rhodamine-labelled phospholipid vesicles (RLV) plus melittin. Model II: magainin breaks phospholipid vesicle membranes. (C) REV plus magainin. (D) RLV plus magainin.

POPC is calculated to be about 0.080 ms and that for Rhodamine– Magainin to be about 0.112 ms. Thus, the average tD =0.074 9 0.002 ms found in the presence of magainin corresponds to monomeric rhodamine-labelled phospholipid, i.e. rhodamine-labelled POPC. It obviously does not correspond to RLV since it is much faster compared to tD of RLV (see Fig. 2C, F or Fig. 6A, C), showing that there are rhodamine-labelled phospholipids in solution. In contrast to melittin, incubation of RLV with magainin has resulted in the increase of the number of molecules. As the inside of RLV does not contain free rhodamine molecules, broken pieces of RLV are the only

source to cause rhodamine-labelled phospholipid release in solution and thereby to increase the number of molecules. Thus, the appearance of rhodamine-labelled phospholipid molecules in solution and the increase in the number of molecules provide evidence that these vesicles are broken into rhodamine-labelled phospholipid fragments, confirming the suggestion that magainin breaks vesicles (see the model II, Fig. 7C– D). This finding is consistent with the fact that at higher concentrations (40–100 mM) magainin causes cell membrane lysis (Ludtke et al., 1994a). Magainin at the concentration of 50 mM breaks

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vesicle membranes by inserting into phospholipid membranes as discussed earlier (Ludtke et al., 1994a) or it seems that magainin affects the vesicles by solubilizing phospholipid membranes. However, magainin at lower concentrations (below 10 mM) was found not to break vesicles (data not shown). This is also in line with the earlier report that at low concentrations magainin is only able to cause the membrane thinning but not to break it (Ludtke et al., 1994b). Studies of vesicle breakage by FCS can also be linked to the use of vesicles as drug carriers to target organs. By knowing whether drugs break vesicles or not, FCS may be useful for selection of drugs when they are suggested to be administered in vesicles.

4. Conclusions The results demonstrate that melittin and magainin interact with phospholipid vesicles membranes in different ways. In molecular interactions with vesicles melittin forms channels into vesicle membranes whereas magainin breaks vesicle membranes (Fig. 7). This study suggests that FCS is a sensitive and a rapid technique to study molecular interactions of biologically important substances with vesicles, e.g. it can be used as a rapid protocol to test vesicles as drug carriers.

Acknowledgements This work was supported by the Swedish Natural Science Research Council as well as the Swedish Technical Science Research Council. We gratefully acknowledge the excellent technical assistance of Ms Ebba Hagman and Ms Brith Larsson.

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