Presence of anionic phospholipids rules the membrane localization of phenothiazine type multidrug resistance modulator.

Abstract

Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are mainly cationic amphiphiles. The molecular mechanism of their action can involve interactions with transporter proteins as well as with membrane lipids. The interactions between anionic phospholipids and MDR modulators can be crucial for their action. In present work we study interactions of 2-trifluoromethyl-10-(4-[methanesulfonylamid]buthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS). Using microcalorimetry, steady-state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug location in membrane depends on lipid type. The electrostatic attraction between drug and lipid headgroups presumably keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral ones. FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures. Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures resemble pure PC ones.

Full text

Page 1

Biophysical Chemistry 109 (2004) 399–412
0301-4622/04/$ - see front matter ? 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.bpc.2003.11.004
Presence of anionic phospholipids rules the membrane localization
of phenothiazine type multidrug resistance modulator
Olga Wesolowska *, Andrzej B. Hendrich , Noboru Motohashi , Masami Kawase ,
{
Piotr Dobryszycki , Andrzej Ozyhar , Krystyna Michalak
a,
abc
dda
˙
Department of Biophysics, Wroclaw Medical University, ul. Chalubinskiego 10, 50–368 Wroclaw, Poland
{
Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose-shi, Tokyo 204-8588, Japan
Faculty of Pharmaceutical Sciences Josai University, Keyakidai, Sakado, Saitama 350-0295, Japan
Division of Biochemistry, Institute of Organic Chemistry, Biochemistry & Biotechnology, Wroclaw University of Technology,
Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland
´
˙
a
{{
b
c
d
{
{
Received 25 July 2003; received in revised form 17 November 2003; accepted 20 November 2003
Abstract
Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are
mainly cationic amphiphiles. The molecular mechanism of their action can involve interactions with transporter
proteins as well as with membrane lipids. The interactions between anionic phospholipids and MDR modulators can
be crucial for their action. In present work we study interactions of 2-trifluoromethyl-10-(4-wmethanesulfonylam-
idxbuthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS). Using microcalorimetry, steady-
state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug
location in membrane depends on lipid type. The electrostatic attraction between drug and lipid headgroups presumably
keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral
ones. FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures.
Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures
resemble pure PC ones.
? 2003 Elsevier B.V. All rights reserved.
Keywords: Phenothiazine derivative; Fluorescence spectroscopy; Differential scanning calorimetry; Anionic phospholipids; Drug–
membrane interaction; Multidrug resistance modulation
Abbreviations: CPZ, chlorpromazine; DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol; DPH,
1,6-diphenyl-1,3,5-hexatriene; DSC, differential scanning calorimetry; DH, enthalpy change during phase transition; FPhMS, 2-
trifluoromethyl-10-(4-wmethanesulfonylamidxbuthyl)-phenothiazine; GP, Laurdan generalized polarization; MDR, multidrug resis-
tance; NPN, N-phenyl-1-naphtylamine; PC, egg yolk phosphatidylcholine; PS, bovine brain phosphatidylserine; T , lipid phase
transition temperature.
*Corresponding author. Tel.: q4871-7841402; fax: q4871-7840088.
E-mail address: olawes@biofiz.am.wroc.pl (O. Wesolowska).
{
m

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O. Wesolowska et al. / Biophysical Chemistry 109 (2004) 399–412
{
1. Introduction
Biological membranes constitute the outer bor-
der of the cell and control the fluxes of all
substances between the cell and its environment.
The selective removal of xenobiotics and other
toxic compounds from the cells is their basic line
of defense. The outward transport of substances
threatening the cell is a double-edged process,
however, as the ability of cancer cells to pump out
anticancer agents poses a major problem to a
successful chemotherapy. This active outward
efflux of the wide spectrum of cytotoxic com-
pounds contributes to the phenomenon of multi-
drug resistance (MDR). The drug transport is
performed by transmembrane proteins belonging
to ABC proteins superfamily (ATP-binding cas-
sette) such as P-glycoprotein (reviewed in w1x).
The substrate specificity of P-glycoprotein is
extremely wide. Apparently the vast majority of
cationic amphiphiles can be bound and translocat-
ed by this transporter (see w2x for review), includ-
ing some short chain lipid analogues w3,4x.
Although ABC transporters are believed to play
the main role in MDR there is more and more
evidence gathered pointing to the important role
of lipid phase of cell membranes in this process,
too. Cell membrane constitutes the environment
where MDR transporters recognize and bind their
substrates. Its biophysical properties can also influ-
ence P-glycoprotein activity (reviewed in w5x). On
the other hand, lipid phase of membranes consti-
tutes the medium for the diffusional, passive,
inward transport of the drugs.
Clinical importance of multidrug resistance that
appears during a course of chemotherapy in vast
number of malignancies has attracted a lot of
scientists’ attention to compounds able to reverse
MDR. The reduction of MDR transporters’ activity
results in increased concentration of cytostatics
inside the cancer cells and thereby restores drugs’
ability to kill them. Compounds called MDR
modulators share no common chemical structure.
The majority of them, however, are relatively
hydrophobic neutral or cationic molecules that
partition easily into lipid bilayers w6,7x. As major-
ity of P-glycoprotein substrates and MDR modu-
lators are cationic amphiphiles it seems much
probable that the presence of negatively charged
phospholipids in membranes could rule their inter-
actions with lipid bilayers. Recently, it has been
shown that MDR modulator verapamil binds
stronger to the membranes containing anionic lip-
ids and competes with anticancer drug doxorubicin
for membrane partitioning w8x. It also increases the
rate of passive import of doxorubicin across the
plasma membrane in this way reducing MDR.
Phenothiazines are clinically used as antipsy-
chotic drugs. They also constitute the group of
promising multidrug resistance modulatorsw9–11x.
Although their potency to reverse MDR has been
recognized for many years, little is known about
the molecular mechanism of anti-MDR action of
phenothiazine derivatives. Structural modifications
that increase compound’s hydrophobicity increase
also multidrug resistance potency of phenothia-
zines w12,13x. Together with extremely wide scope
of effects exerted by these compounds on mem-
branes it suggests that interaction with lipid phase
of cell membranes can be important for anti-MDR
activity of phenothiazines. In physiological pH
phenothiazine derivatives bear the positive charge
w14x. It is thus probable that many of phenothia-
zine-associated effects in biological membranes
are ruled by specific interactions of these cationic
compounds with anionic phospholipids. Phenothi-
azine derivatives are known to cause erythrocyte
hemolysis w15x and, in lower concentrations, to
induce stomatocytosis of red blood cells w16,17x.
According to bilayer couple hypothesis proposed
by Sheetz and Singer in 1974 w18x invaginations
of erythrocyte membrane are caused by com-
pounds that specifically interact with phosphati-
dylserine and intercalate into inner monolayer of
cell membrane. Chlorpromazine (CPZ) was also
reported to cause partial phospholipid scrambling
in erythrocyte membranes w19x.
There is also a lot of data from model membrane
experiments suggesting that phenothiazines inter-
act differently with charged phospholipids than
with neutral ones. We have shown previously by
microcalorimetry that trifluoperazine induces phase
separation in bilayers formed from zwitterionic
DMPC and DPPC but not anionic DMPG w20x.
Freeze-fracture electron
phosphatidic acid bilayers has shown that the
microscopyofCPZ:

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O. Wesolowska et al. / Biophysical Chemistry 109 (2004) 399–412
{
Fig. 1. Chemical structure of 2-trifluoromethyl-10-(4-wmetha-
nesulfonylamidxbuthyl)-phenothiazine (FPhMS).
addition of phenothiazine can result in hexagonal
H phase formation w21x. Specific interaction of
≤
phenothiazine derivatives with phosphatidylserine
was also observed. Pajeva et al. w22x have shown
by DSC that some of these compounds interact
with DPPC and DPPS in dissimilar way and that
new kind of phospholipid organization is formed
in charged bilayers in the presence of these drugs.
The recent microcalorimetry and
of Nerdal et al. w23x have demonstrated that CPZ
interacts weakly with DPPC models systems and
that strength of drug–lipid bilayer interaction rais-
es dramatically after introducing bovine brain PS
into membranes. The monolayer technique has
been employed to show that the surface area of
acidic phospholipids increases strongly after addi-
tion of chlorpromazine whereas the surface area of
neutral phospholipids remains constant in spite of
the presence of the drug w24x. Jutila et al. w25x
have observed CPZ-induced changes in lateral
organization of model membranes composed of
DPPC:brain PS mixtures.
2-Trifluoromethyl-10-(4-wmethanesulfonylam-
idxbuthyl)-phenothiazine (FPhMS) was designed
and synthesized with intention of its future use as
a multidrug resistance modulator. Its anti-MDR
activity was confirmed in drug-resistant cancer
cells in culture w11,26x. The interactions of this
compound with model membranes composed of
zwitterionic DPPC were studied in details using
DSC, EPR and fluorescence spectroscopy tech-
niques w27x. We have also shown previously that
FPhMS is able to induce phase separation in
phosphatidylethanolamine bilayers w28x.
The aim of the present work was to study the
interactions of phenothiazine-type MDR modulator
FPhMS with model membranes containing acidic
phospholipids. We have employed DSC technique
to compare the influence of this compound on
neutral DMPC and charged DMPG bilayers. Flu-
orescence spectroscopy was used to study the
binding of FPhMS to liposomes composed of
PC:PS mixtures. We have shown that phenothia-
zine derivative probably occupies slightly different
locations inside the lipid bilayer depending on the
membrane charge. Additionally, fluorescence spec-
troscopy revealed that the extent of effect induced
by phenothiazine derivative on membranes is not
C-NMR studies
13
proportional to the molar ratio of neutral to anionic
phospholipid in the bilayer.
2. Materials and methods
1,2-Dimyristoyl-sn-glycero-3-phosphatidylgly-
cerol (DMPG) and 1,2-dimyristoyl-sn-glycero-3-
phosphatidylcholine
(DMPC)
from Avanti Polar Lipids Inc. (Alabaster, AL,
USA). Bovine brain L-a-phosphatidylserine (PS)
and egg yolk L-a-phosphatidylcholine (PC) were
from Sigma (St. Louis, MO, USA). Lipids were
used without further purification. 1,6-Diphenyl-
1,3,5-hexatrien (DPH) and N-phenyl-1-naphtylam-
ine (NPN) were purchased from Sigma (St. Louis,
MO,USA).6-Lauroyl-2-(N,N-dimethylamino)
naphthalene (Laurdan) was from Molecular Probes
(Eugene, OR, USA).
2-Trifluoromethyl-10-(4-wmethanesulfonylam-
idxbuthyl)-phenothiazine (FPhMS) was synthe-
sized as described inw29,30x. Its chemical structure
is shown in Fig. 1. All other chemicals were of
analytical grade.
werepurchased
2.1. Microcalorimetry
Stock solution (3.5 mM) of phenothiazine deriv-
ative was prepared in chloroform:methanol (1:1,
vyv). For each sample 2 mg of appropriate lipid
was dissolved in FPhMS stock solution. The

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{
amount of FPhMS was chosen to obtain the desired
drug:lipid molar ratio in the sample.
The samples were dried under the stream of
nitrogen and the residual solvent was removed
under vacuum for at least 3 h. Samples were
hydrated by 20 ml of 20 mM Tris–HCl buffer
(150 mM NaCl, 0.5 mM EDTA, pHs7.4).
Hydrated mixtures were heated to temperature
;10 8C higher than main phase transition temper-
ature of a given lipid and vortexed until homoge-
neous dispersion was obtained. Samples were
sealed in aluminum pans and scanned at the rate
of 1.25 8Cymin. Calorimetric measurements were
performed using Rigaku calorimeter, which was
partially rebuilt in our laboratory. Samples were
scanned immediately after preparation. The tem-
perature at which maximal deviation of transition
peak from the base line was recorded was taken
as a phase transition temperature. The area under
the transition profiles was used to calculate the
molar enthalpychange
transition.
accompanyingphase
2.2. Steady-state fluorescence spectroscopy
To obtain PS, PC and PC:PS liposomes chloro-
form solutions of lipids were mixed in appropriate
amounts. The mixtures of following PC:PS molar
ratios were used: 5:1 (16.66 mol% of PS), 2:1
(33.33 mol% of PS), 1:1 (50 mol% of PS), 1:2
(66.66 mol% of PS) and 1:5 (83.33 mol% of PS).
Organic solvent was then evaporated under stream
of nitrogen and the samples were kept under
vacuum for at least 2 h. The dry lipids were
hydrated in 1y15 M Michaelis phosphate buffer
(pH 7.4) in case of DPH and NPN experiments or
in 20 mM Tris–HCl buffer (50 mM NaCl, 0.1
mM EDTA, pHs7.4) in case of Laurdan experi-
ments. Small unilamellar liposomes were obtained
by sonication.
DPH stock solution (1 mM) was prepared in
tetrahydrofuran. NPN (1 mM), Laurdan (1 mM)
and FPhMS (5 mM) were dissolved in dimethyl
sulfoxide. Liposomes (final phospholipid concen-
tration 200 mM) were incubated with fluorescent
probe (concentration 5 mM) in darkness for 30
min or 15 min (for DPH and NPN, respectively)
at room temperature. FPhMS was then added (at
concentration varying from 5–100 mM) and incu-
bation was continued under the same conditions
for the next 20 min (DPH) or 10 min (NPN). For
Laurdan experiments liposomes (200 mM) were
incubated with fluorescent probe (1 mM) for 30
min (darkness, room temperature).
Fluorescence measurements were performed
with LS 50B spectrofluorimeter (Perkin–Elmer
Ltd., Beaconsfield, UK) using emission and exci-
tation slits of 5 nm. All measurements were done
at 25 8C. Temperature was controlled by a water-
circulating bath and the content of the cuvette was
continuously mixed. DPH excitation and emission
wavelengths were 380 nm and 450 nm, respective-
ly. NPN fluorescence was excited at 350 nm and
emission spectra were recorded in the range of
360–580 nm. Excitation wavelength for Laurdan
was 320–400 nm and fluorescence emission spec-
tra were recorded in the range of 410–540 nm.
Data were collected and processed with FLDM
Perkin–Elmersoftware.
polarization was calculated according to the equa-
tion w31x:
Laurdangeneralized
I yI
B
I qI
B
R
GPs
(1)
R
where I
intensities at the blue and red edges of the emission
spectrum, respectively. The GP values were cal-
culated using emission intensities at 440 nm (I )
and 490 nm (I ).
R
DPH polarization degree (P) was calculated as:
and I
are the fluorescence emission
BR
B
I yGI
≤
H
PsI qGI
≤
H
where I and I
polarizer parallel (≤) and perpendicular (H) to the
direction of polarization of the excitation light and
G is the instrumental correction factor calculated
by FLDM software.
are the emission intensity with
≤
H
2.3. Time-resolved fluorescence spectroscopy
The samples for fluorescence lifetimes measure-
ments were prepared in the same way as for steady-

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O. Wesolowska et al. / Biophysical Chemistry 109 (2004) 399–412
{
Fig.
FPhMS:DMPC mixtures. Numbers on the figure represent
drugylipid molar ratios. Thermograms were normalised to
equal amount of lipid for each profile.
2. ThermogramsofDMPC
(upperprofile)
and
state fluorescence experiments. Concentration of
FPhMS varied from 5–50 mM. In higher drug
concentrations lifetime measurements were not
performed due to very low fluorescence intensities.
Measurements were performed with a SLM Amin-
co 48000S frequency–domain instrument using a
450 W xenon lamp as a light source and frequency
range of 1–250 MHz. Samples were excited at
350 and 380 nm for NPN and DPH, respectively.
The emitted light passed through a cut-off filter to
eliminate the light below 370 nm for NPN and
410 nm for DPH experiments. All measurements
were done at 25 8C unless stated otherwise. The
lifetimes were calculated by multiexponential anal-
ysis from phase shifts and demodulation parame-
ters usingthefluorimeter
fluorescence decay was not a single exponential
function the average lifetime NtM was calculated
according to the equation:
software. When
n
N M
t s
f t
i i
8
is1
where t is fluorescence lifetime and f is fractional
i
intensity contribution.
i
3. Results
3.1. Microcalorimetry
The thermograms of pure DMPC and its
mixtures with different amounts of newly synthe-
sized phenothiazine derivative are presented in
Fig. 2. DMPC is a zwitterionic lipid that thermo-
tropic polymorphism is well known. In pure lipid
(upper trace) we recorded the main phospholipid
phase transition at 23 8C as well as pretransition
centered at 13.8 8C. The addition of FPhMS to the
lipid caused disappearance of pretransition and
distinct symmetrical broadening of the gel–liquid
crystalline transition peaks. The influence of stud-
ied compound on DMPC bilayer structure was so
strong that main phospholipid phase transition
disappeared completely at drug:lipid molar ratio
0.12. Also the parameters characterizing gel–liquid
crystalline transition of DMPC: temperature (T )
and enthalpy change (DH) during transition were
strongly influenced by the presence of phenothia-
m
zine derivative. Both parameters monotonously
decreased with increasing FPhMS concentration
(Fig. 3a,b).
Thermotropic behavior of mixtures of phenothi-
azine derivative with anionic lipid DMPG was also
studied. For pure lipid two transitions were record-
ed: pretransition and main phospholipid phase
transition at 14 8C and 24 8C, respectively (Fig.
4). The pretransition was abolished even at the
smallest drug:lipid molar ratio studied. The addi-
tion of studied compound to DMPG model system
resulted in symmetric broadening of transition
peaks. This broadening was stronger than observed
in FPhMS:DMPC mixtures. Addition of increasing
concentrations of phenothiazine derivative to the
lipid caused the transition temperature shift to
lower values (Fig. 5a). The extent of T lowering
induced by the drug in DMPG was approximately
4 8C, the same as in phosphatidylcholine. The
effect of FPhMS on DH of anionic lipid was,
however, different than in case of DMPC (Fig.
5b). Up to drug:lipid molar ratio 0.04 transition
enthalpy remained constant. It dropped slightly
only at higherphenothiazine
concentrations.
m
derivative

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{
Fig. 3. Alterations of main phospholipid phase transition par-
ameters: temperature (a) and enthalpy (b) induced by FPhMS
in DMPC. Errors are given as standard deviation values of
eight measurements.
Fig.
FPhMS:DMPG mixtures. Numbers on the figure represent
drug:lipid molar ratios. Thermograms were normalized to equal
amount of lipid for each profile.
4.Thermograms ofDMPG
(upperprofile)
and
3.2. Fluorescence spectroscopy
The interactions of FPhMS with model mem-
branes composed of neutral phosphatidylcholine,
negatively charged phosphatidylserine and their
mixtures were studied by means of fluorescence
spectroscopy. Addition of studied compound to the
liposomes labeled with NPN resulted in strong
quenching of its fluorescence. Stern–Volmer plots
of quenching are presented in Fig. 6. In all types
of lipid compositions studied NPN quenching
increased with increasing of phenothiazine deriv-
ative concentration. The strongest quenching was
observed in liposomes composed from pure PS
and the weakest in pure PC. It is worth noticing
that for concentrations of phenothiazine derivative
higher than 50 mM the quenching of NPN fluores-
cence was slightly higher in PC:PS (1:1) mixtures
than in two other PC:PS systems studied.
In all lipid systems studied Stern–Volmer plots
of NPN quenching showed slight upward curvature
with increasing phenothiazine derivative concen-
tration. Only in case of pure PS significant curva-
ture was observed. To investigate further this
quenching phenomenon we measured NPN fluo-
rescence lifetimes in PS liposomes in presence of
FPhMS. NPN lifetime recorded in pure liposomes
was 2.35 ns. The lifetime dropped to 1.49 ns after
addition of 5 mM of the drug and remained
constant up to FPhMS concentration of 50 mM.
Apart from reducing NPN fluorescence intensity
FPhMS also caused the shift of fluorescence emis-
sion spectra maxima toward longer wavelengths
as compared to the spectra recorded in the absence
of this compound. Such a maximum red-shift was
recorded in all types of PC:PS mixtures with the
exception of PC:PS (1:2). The biggest red-shift
was observed in PS liposomes (5 nm), almost
twice smaller in equimolar PC:PS mixture (2.7
nm), even smaller in PC:PS (2:1) (2.4 nm) and
the smallest one in pure PC (1.7 nm).
The presence of phenothiazine derivative influ-
enced also the fluorescence of the probe DPH.
Addition of studied compound to all types of
liposomes resulted in concentration dependent

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{
Fig. 5. Alterations of main phospholipid phase transition par-
ameters: temperature (a) and enthalpy (b) induced by FPhMS
in DMPG. Errors are given as standard deviation values of
eight measurements.
Fig. 6. Stern–Volmer plots of NPN fluorescence quenching
caused by FPhMS in liposomes composed of PC:PS mixtures.
Symbols represent: m – PC, s – PC:PS (2:1), n – PC:PS
(1:1), j – PC:PS (1:2), d – PS.
Fig. 7. DPH polarization as a function of FPhMS concentration
in liposomes composed of PC:PS mixtures. Symbols represent:
m – PC, s – PC:PS (2:1), n – PC:PS (1:1), j – PC:PS
(1:2), d – PS.
DPH fluorescence polarization increase (Fig. 7).
Only in PS model membranes at FPhMS concen-
trations above 50 mM polarization stopped increas-
ing. The biggest drug induced changes in DPH
polarization values were recorded in pure PC. The
extent of polarization increase caused by FPhMS
decreased in order: PC)PC:PS (2:1))PC:PS
(1:2))PC:PS (1:1))PS.
Apart from changes in DPH polarization phe-
nothiazine derivative caused also fluorescence
quenching of this probe (data not shown). The
extent of quenching was similar for all lipids
studied. DPH fluorescence lifetimes were also
investigated (Fig. 8). DPH had the longest lifetime
in membranes composed of pure phosphatidylser-
ine. In pure lipid systems the fluorescence lifetime
decreased together with the decrease of the amount
of anionic lipid in the model membrane. The
addition of phenothiazine derivative to liposomes
caused DPH lifetime shortening. This change was
the most pronounced in PS liposomes and almost
negligible in PC liposomes. We decided also to
study the influence of FPhMS on DPH fluores-
cence in DPPC model membranes in different
phase states (Fig. 9). The addition of the drug
caused DPH lifetime shortening in both gel-like
and liquid–crystalline states, however the extent

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{
Fig. 8. DPH fluorescence lifetime as a function of FPhMS con-
centration and liposome composition: PC – stripped band,
PC:PS (2:1) – dotted band, PC:PS (1:1) – filled band, PC:PS
(1:2) – open band, PS – cross-hatched band.
Table 1
DPH polarization in liposomes composed of egg yolk PC,
bovine brain PS and PC:PS mixtures. Errors are given as stan-
dard deviation values of six measurements
Lipid compositionDPH fluorescence polarization
PC
PC:PS (2:1)
PC:PS (1:1)
PC:PS (1:2)
PS
0.099"0.005
0.102"0.003
0.113"0.004
0.107"0.004
0.119"0.004
Fig. 9. Influence of FPhMS on DPH fluorescence lifetime in
DPPC liposomes below (open bars; measured at 25 8C) and
above (filled bars; measured at 45 8C) transition temperature.
Fig. 10. Laurdan generalized polarization values in PC:PS lipo-
somes as a function of excitation wavelength. Symbols repre-
sent: m – PC, h – PC:PS (5:1), s – PC:PS (2:1), n – PC:PS
(1:1), j – PC:PS (1:2), e – PC:PS (1:5), d – PS.
of change due to increasing drug concentration
was greater in lipid bilayers below T .
The study on FPhMS-induced NPN fluorescence
quenching and DPH polarization changes revealed
that phenothiazine derivative incorporated in the
liposomes composed of PC:PS (1:1) mixture
behaved more similarly to liposomes of pure PS
than when incorporated in model membranes of
PC:PS (1:2). This suggested that the range of
model membrane perturbation induced by FPhMS
was not proportional to anionic PS content in the
system. Therefore we decided to characterize the
properties of PC:PS membranes in absence of any
m
drug more thoroughly. Table 1 presents DPH flu-
orescence polarization values obtained in lipo-
somes composed of PC and PS mixed in different
molar ratios. Again it could be noticed that DPH
polarization value recorded in PC:PS (1:1) mem-
branes was more similar to pure PS liposomes
than polarization value in PC:PS (1:2) mixture.
We have studied the properties of PC:PS model
membranes also employing Laurdan as a fluores-
cent probe. We observed an increase in the inten-
sity of the blue part of emission spectra with
increase of PS content in the model system (data
not shown). The Laurdan generalized polarization
as a function of excitation wavelength (l ) is
presented in Fig. 10. All the GP vs. l
negative slopes i.e. they presented the typical
features of the liquid–crystalline phase. The typi-
cal GP value recorded in this phase is approxi-
ex
plots had
ex

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O. Wesolowska et al. / Biophysical Chemistry 109 (2004) 399–412
{
Fig. 11. Laurdan generalized polarization values in liposomes
as a function of PS concentration. Excitation wavelength was
390 nm.
mately y0.2 if calculated for the same excitation
and emission conditions as in Ref. w32x. As pre-
sented in Fig. 11 at 25 8C Laurdan in membranes
composed of pure egg yolk PC possessed the most
‘liquid-crystalline’ GP values whereas in pure PS
liposomes the highest Laurdan GP values were
observed. It could be also noticed that the increase
in generalized polarization was proportional to the
anionic lipid content in the membranes. This
dependence was by no means linear, however.
Introducing even the smallest amount of PS to PC
membrane (16.66 mol%) caused the most dramatic
increase of GP value. Further raising phos-
phatidylserine content resulted in smaller changes
in Laurdan generalized polarization.
4. Discussion
The results of calorimetric experiments has
proved that FPhMS intercalates into membranes
of both neutral DMPC and charged DMPG chang-
ing the parameters that characterize lipid bilayers’
phase transitions. In both lipids phenothiazine
derivative caused T
lowering, gel–liquid crystal-
m
line transition peaks’ broadening and decrease of
transition enthalpy. Broadening of transition peaks
together with lowering of transition temperature
are typical changes induced by compounds that
localize in C –C methylene region of lipid bilayer
18
w33x. Such a localization of FPhMS molecule is
consistent with a model proposed by Nerdal et al.
w23xfor chlorpromazine. According to it the hydro-
phobic ring system of CPZ molecule penetrates
the acyl chain region and is oriented along the
chain direction. In the same time positively
charged side chain group of chlorpromazine is
positioned in the vicinity of the phospholipid
headgroups what allows their mutual electrostatic
interaction. Such a location of FPhMS molecule
insidethemembrane
between phospholipid molecules both in polar and
hydrophobic region of the bilayer that results in
changes of all transition parameters induced by
this drug.
Transition temperature of both lipids is affected
to similar extent by phenothiazine derivative stud-
ied. Enthalpy change during main phospholipid
phase transition is, however, much stronger affect-
ed by this compound in DMPC bilayers. Its influ-
ence is so strong that the transition is completely
abolished at drug:lipid molar ratio 0.12. Main
phase transition peaks appear on thermograms as
the result of cooperative melting of phospholipid
hydrocarbonchains. Peak’s
decreasing of its intensity are signs of perturbation
of interactions between phospholipid acyl chains.
At high drug:lipid molar ratios these interactions
in DMPCmodel membrane
destroyed. Stronger influence of FPhMS on DMPC
bilayers than on DMPG ones may seem a little
surprising as strong specific interactions of phe-
nothiazines with anionic
reported previously w23,24x. We propose that the
phenomenon that we observed could be explained
by slightly different location of FPhMS molecule
inside the bilayers composed of different lipids.
Cationic side chain group of phenothiazine could
interact strongly with negatively charged polar
headgroups of DMPG. This interaction would
anchor the drug molecule relatively close to
membrane surface and thereby it could prevent the
hydrophobic ring system from intercalating deeply
between the acyl chains of phospholipid. On the
other hand, in neutral DMPC interactions of
FPhMS with polar region of the membrane would
probably be weaker that could result in deeper
compound’s immersion
membrane. Such a localization of phenothiazine
derivative in zwitterionic lipid system would cause
affects theinteraction
broadeningand
are probably
phospholipids were
insidethe model

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O. Wesolowska et al. / Biophysical Chemistry 109 (2004) 399–412
{
a stronger perturbation of its transition enthalpy as
compared with charged lipid system and that is
what is observed.
The role of membrane charge in its interaction
with phenothiazine-type MDR modulator was also
studied by means of fluorescence spectroscopy.
NPN is a fluorescent probe that locates at polary
apolar interface of lipid bilayer. Quenching of its
fluorescence with concomitant red shift of its
emissionmaximumis
increased polarity in the fluorophore vicinity
caused by the presence of the studied compound
w34x. However, the upward curvature of Stern–
Volmer plots (especially significant in case of PS
liposomes) may suggest that more complex mech-
anism is responsible for NPN quenching caused
by FPhMS. Such effect observed in homogenous
solution could be explained by a mixture of
dynamic and static quenching, however in lipo-
somes also membrane effects must be taken under
consideration. NPNfluorescence
depends on phenothiazine derivative concentration,
whereas NPN fluorescence lifetime is lowered by
even the lowest drug concentration tested and
drug’s amount increase does not change the life-
time any further, as if the interaction became
saturated. In our opinion this point to some kind
of direct interaction, i.e. complex formation
between the drug and the probe inside the lipid
bilayer. The nature of this putative complex is
elusive; it could be formed by molecules in ground
or in excited states. The red-shift of NPN emission
maxima in the presence of FPhMS could point to
exciplex formation. In our opinion, however, the
observed shift is too small. If exciplex was formed
we would rather expect the appearance of new
band of fluorescence with distinctly different
maximum.
We have observed that FPhMS caused the
strongest NPN quenching in liposomes formed
from anionic PS. Conversely the influence of this
drug on fluorescence polarization of DPH was the
most potent in model membranes composed of
neutral PC. DPH is a hydrophobic probe locating
deeply in the membrane core. Its fluorescence
polarization changes can be due to the altered
molecularorderofhydrophobic
membrane andyor probe’s lifetime variations. As
usuallyattributed to
quenching
regionof
the
accompanied by quenching of DPH fluorescence
we decided to study the probe’s fluorescence
lifetimes, too. Our results show that DPH has the
longest fluorescence lifetime in lipid bilayers com-
posed of anionic phosphatidylserine and the short-
est one in neutral PC membranes. PS model
membranes are also characterized by the highest
DPH polarization degree and the highest Laurdan
GP values (see below). The addition of phenothi-
azine derivative to DPH labeled liposomes caused
the shortening of fluorescence lifetime clearly
visible in PS bilayers and almost negligible in
membranes with high PC content. Such a behavior
suggests that drug-induced DPH lifetime shorten-
ingis responsiblefor
increase in PS bilayers to much greater extent than
in case of PC membranes. In the latter case the
polarization degree increase is probably caused by
membrane ordering effects, i.e. rigidifying of the
model membrane. Therefore we can conclude that
at least PC model membranes and PC:PS mixtures
of high PC content (minimum 50%) are rigidified
by FPhMS.
As fluorescent probe’s lifetime variation can
affect the results of fluorescence polarization meas-
urements we decided to study the effect exerted
by FPhMS on DPH fluorescence parameters in
lipid systems in more detail. Previously, we have
observed the opposite FPhMS-induced changes of
DPH polarization depending on phase state of the
lipid bilayer w27x. DPH polarization was reduced
by phenothiazine derivative in membranes below
transition temperature and increased above T , i.e.
in liquid crystalline state. In the present study we
measured DPH fluorescence lifetimes in both
phase states of DPPC membranes in presence of
the studied compound. We have demonstrated that
DPH lifetime is shortened by FPhMS in both gel-
like and liquid–crystalline lipid bilayers.
The picture generated by fluorescence spectros-
copy is consistent with our hypothesis of slightly
different localization of FPhMS molecules in mem-
branes composed of neutral and charged lipids.
According to our model phenothiazine derivative
is localized closer to the bilayer surface in PS
liposomes. That is why it affects the fluorescence
of shallowly positioned probe NPN stronger in PS
FPhMS-inducedpolarization changes are
observed polarization
m

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O. Wesolowska et al. / Biophysical Chemistry 109 (2004) 399–412
{
than in PC model membranes. On the other hand,
fluorescence polarization of deeply inserted DPH
is affected by the studied compound to lesser
extent in PS than in PC liposomes that is consistent
with postulated deeper FPhMS insertion into PC
bilayers.
Proposed different localization of phenothiazine
derivative in model anionic and neutral membranes
allows also to eliminate the apparent discrepancy
between the results obtained by DPH fluorescence
polarization and lifetime measurements. This dis-
crepancy lies in the fact that FPhMS-induced
change in polarization is the most pronounced in
neutral PC systems whereas DPH lifetime is the
most affected by the drug in anionic PS mem-
branes. In PC liposomes molecules of fluorescent
label and drug are presumably located much closer
to each other than in PS bilayers. Thus in PC
systems direct interactions between the drug and
the probe molecules may lead to the observed
lifetime effects. Since all of FPhMS concentrations
used are much higher than the concentration of
DPH their possible interaction may be saturated
and that is why we observe no lifetime dependence
on drug concentration. The deeper localization of
phenothiazine derivative in PC model membrane
would also cause the pronounced DPH polarization
changes. On the other hand, in PS bilayers the
effect exerted by phenothiazine derivative on DPH
fluorescence lifetime is indirect and depicts the
influence of the drug on membrane properties. As
the lifetime shortening is often related to the
increase of water content inside the membrane
w35x we may assume that incorporation of FPhMS
molecule closer to the polaryapolar membrane
interface in PS liposomes may enhance water
penetration into the membrane. DPH fluorescence
polarization increase observed in PS liposomes is
not contradictory to the postulate of increased
water content in membrane as this polarization
measurements series can be significantly biased by
lifetime shortening. The above hypothesis seems
to be supported by our NPN quenching results,
which point to the increased polarity of probe
surroundings in PS reach PC:PS mixtures.
There is one problem that still needs elucidation,
however. We have observed that the changes of
both NPN and DPH fluorescence parameters
recorded in the presence of FPhMS are not pro-
portional to the content of anionic PS in liposomes.
Phenothiazine’s behavior in liposomes composed
of equimolar PC:PS mixture is more similar to its
behavior in pure PS membranes, whereas in PC:PS
(2:1) and PC:PS (1:2) mixtures it resembles more
the pure PC system. To address this problem we
decided to study the properties of PC:PS model
membranes in the absence of the drug. DPH
fluorescence polarization
shown that molecular ordering in PC:PS (1:1)
liposomes is more similar to pure PS than to pure
PC membranes. Conversely the experiments with
the use of Laurdan as a fluorescent probe have
demonstrated that changes in its fluorescence par-
ameters are proportional to the amount of PS in
the model system studied. Laurdan reports on
membrane region below the phospholipid ester
groups and its spectral properties are highly sen-
sitive to the polarity in the fluorophore vicinity
w32x. The increase of hydration in membranes (e.g.
caused by gel to liquid–crystalline phospholipid
phase transition) results in decrease of intensity of
Laurdan emission with concomitant red shift of its
maximum (of approx. 40 nm). Due to Laurdan
spectral properties its generalized polarization can
be used to assess the coexistence of gel-like and
fluid domains in membrane (for details see Ref.
w32x). Briefly, GP values are approximately 0.6 in
gel phase and approximately y0.2 in liquid–
crystalline phase if calculated for the same exci-
tation and emission conditions as in w32x. Also the
slope of GP vs. l
dependence is characteristic
ex
for a given phase. It is around zero in gel phase,
negative in liquid–crystalline phase and positive
when domains of both phases coexist in the sys-
tem. Our results have shown that increasing
amount of PS in liposomes results in the decrease
of the polarity in membrane region where Laurdan
molecules are localized. GP calculation has dem-
onstrated that there is no typical gel–fluid phase
separation in this system. Both lipids used: bovine
brain PS and egg yolk PC are in liquid-crystalline
phase under conditions of the experiment. It can
be noticed both from Laurdan GP and DPH polar-
ization studies that pure PC membranes are more
fluid than pure PS liposomes.
measurementshave

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O. Wesolowska et al. / Biophysical Chemistry 109 (2004) 399–412
{
Laurdan is able to detect phase coexistence in
membranes only when the two phases present
differ significantly in hydration. That is why, in
spite of the results of Laurdan experiments, we
suppose that higher similarity of equimolar PC:PS
mixture to pure PS membranes may be caused by
non-ideal mixing of these two lipids in the liquid-
crystalline phase. PC- or PS-enriched regions could
form in membrane as the result of different bio-
physical properties of the two phospholipids. How-
ever, such regions, if they existed, would be both
fluid and they would not differ in lipid hydration
enough to be detected by Laurdan fluorescence.
We have also to bear in mind that electrostatic
repulsion between negatively charged PS head-
groups could hinder PS molecules from aggregat-
ing. It was demonstrated previously by fluor-
escencespectroscopic
mixtures of PC with such anionic lipids as phos-
phatidylserine and phosphatidic acid but not phos-
phatidylglicerol phase separation occurs w36x. The
theoretical analysis of PC:PS mixing has led
Huang and Feigenson w37x to the conclusion that
the range of PS mole fraction where phase sepa-
ration could be observed depends only on the
strength of electrostatic repulsion between PS
headgroups. Assuming that the nonelectrostatic
excessmixingenergy
2.22=10 J the authors calculated that in room
temperature phase separation in PC:PS mixture
existed only when the amount of PS in the system
was between 25 and 65 mol%. Their expectations
have been confirmed by experimental results by
Hinderliter et al. w38x. Monitoring the binding of
Ca ions to PC:PS membranes and employing
X-ray diffraction technique they have shown that
phase separation occurs only in mixtures with 52–
62 mol% of phosphatidylserine. The region of
phase separation could be broadened up to 50–80
mol% of PS by increasing the ionic strength of
the solution (the addition of 800 mM of KCl) that
caused screening of electrostatic PS headgroups
repulsion. The experiments presented in our work
were performed in low ionic strength. It allows as
to presume that in our system phase separation
could occur only in PC:PS (1:1) mixture or
occurred in this mixture to the highest extent. In
the other PC:PS mixtures tested both kinds of
methods that influid
ofa PC:PSpair is
y21
2q
lipids are probably mixed more homogenously. In
equimolar PC:PS system the molecules of FPhMS
could preferentially bind to PS-enriched regions of
membrane or to PC:PS domain boundaries as
proposed by Jutila w25x for CPZ. In our opinion
such a behavior of phenothiazine derivative could
explain the greater similarity of its influence on
PC:PS
(1:1)
membranes
liposomes.
thanonpure PS
Acknowledgments
This work was supported by the State Commit-
tee for Scientific Research (KBN) Grant No. 6
P05A 01221. Olga Wesolowska is grateful to the
Foundation for Polish Science for the scholarship.
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