Chemistry and Physics of Lipids 155 (2008) 7–15

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Chemistry and Physics of Lipids

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Which physical and structural factors of liposome carriers control theirdrug-loading efficiency?

Mariusz Kepczynskia, Kinga Nawalanya, Marta Kumorek a, Agnieszka Kobierskaa,Barbara Jachimskab, Maria Nowakowskaa,∗

a Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Polandb Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30-239 Kraków, Poland

a r t i c l e i n f o

Article history:Received 24 January 2008Received in revised form 29 April 2008Accepted 30 May 2008Available online 11 June 2008

Keywords:Liposomes

a b s t r a c t

The correlation between structural and physical properties of lipid membrane and its drug-loading effi-ciency were studied. The properties of bilayer were altered by incorporation of several lipidic modifiers:cholesterol, oleic acid, methyl oleate, and pegylated lipid. By using the molecular probe technique itwas demonstrated that the membrane properties, such as micropolarity, microviscosity and free volumewere considerably changed by incorporation of the modifiers. The partitioning of two different porphyrinsbetween the bulk aqueous phase and the modified liposomes was studied using the fluorescence methods,and liposome-binding constants were determined. It was found that cholesterol reduced the partitioningof both porphyrins into liposomal bilayer. On the contrary, the incorporation of methyl oleate and pegy-

Drug-loading efficiency

Free volumeFluidityPP

lated lipid causes a pronounced increase in the value of the binding constants of both porphyrins. It wasconcluded that the free volume rather than hydrophobicity of bilayer is a governing factor in the solute

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. Introduction

Liposomes are widely studied for their application in drugelivery systems, resulting in a number of clinical applications,specially in cancer chemotherapy and severe fungal infectionsGerasimov et al., 1999). The liposomal encapsulation of anticancerrugs significantly alters the biodistribution and pharmacokinet-

cs, resulting in some reduction in toxicities and in improvedargeting to desired tissues (Gregoriadis, 1988; Han et al., 2007).iposomes are spherical, closed structures composed of curvedipid bilayers. They are predominantly composed of amphiphatic

olecules such as phospholipids. Because phospholipids haven asymmetric structure with the hydrophobic fatty acid chainsnd hydrophilic headgroup (phosphate and choline), the physic-chemical and structural properties across the liposome bilayerre anisotropic. The structure of a fluid 1,2-dioleoyl-sn-glycero-3-hosphocholine (DOPC) bilayer was previously determined from

ombined X-ray and neutron diffraction measurements (Wienernd White, 1992). The ‘structure’ was defined as the time-averagedpatial distributions of the principal structural groups of the lipidcarbonyls, phosphates, etc.) projected onto an axis normal to

∗ Corresponding author. Tel.: +48 12 663 2250; fax: +48 12 634 0515.E-mail address: nowakows@chemia.uj.edu.pl (M. Nowakowska).

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009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.chemphyslip.2008.05.174

© 2008 Elsevier Ireland Ltd. All rights reserved.

he bilayer plane. Incorporation of lipids with covalently attachedoly(ethylene glycol) into liposomal bilayer membranes causesignificant stabilization of liposome suspensions, prevents theirggregation and inhibits protein and cellular interactions withiposomes thereby considerably prolonging their blood circulationime. These vesicles are known as pegylated or sterically stabilizediposomes (SSL) and are applied as effective drug delivery supportsWoodle, 1998; Piperoudi et al., 2006).

High drug-incorporation efficiency is preferable both in basicesearch and in clinical developments (Yamamoto et al., 2007). Onef the fundamental problems related to the loading efficiency of arug into liposomes is identification of the type and the role ofhemical and structural factors which control the partitioning drugolecules into membranes. Therefore, the study of the membrane

roperties as a function of their composition is of great interestot only from theoretical but also from practical point of view (e.g.pplications in drug delivery). It has been recognized that the pres-nce of even small amounts of lipidic additives can greatly affecthe properties of the liposomal membrane such as thickness, flu-dity, polarity, etc. and as a result change their biological activity.

or example, Sadzuka et al. (2006) have investigated Photofrin (PF)issolved in phosphate-buffered saline and incorporated into lipo-omes or poly(ethylene glycol)-modified liposomes. They foundhat the anti-tumor phototoxicity of PF in PEG-liposomes was sig-ificantly higher than that in solution or in neat liposomes.

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M. Kepczynski et al. / Chemistry

The aim of the present study was to determine how theartitioning of porphyrins (which are applied as model drugs)etween the bulk aqueous phase and the hydrophobic lipid mem-ranes is affected by the structural and physical properties of lipidembrane. Two porphyrin compounds of different structure and

roperties, i.e. hematoporphyrin (HP) and 5,10,15,20-tetrakis(4-ydroxyphenyl)porphyrin (mTHPP) were used in these studies. Theroperties of liposome membrane were altered by intercalation ofarious modifiers. The content of the modifiers introduced into theilayer varied. The following four compounds were used as addi-ives: cholesterol, oleic acid and its methyl ester, and pegylatedipid (lipid with covalently attached poly(ethylene glycol) chain).holesterol is one of the major components of biomembranes and

s present in most of liposome formulations applied as drug carri-rs. It plays a crucial role in membrane organization, dynamics andunction. Interestingly, at higher concentrations it is non-randomlyistributed forming domains or pools in biological and model mem-ranes (de Almeida et al., 2003). The pegylated lipid is an essential

ngredient of the SSL liposomes. The oleic acid and its methylster were used to determine the effect of the unsaturated fattycids and surface charge on the loading efficiency of liposomes. Inddition, the effect of the studied modifiers on the membrane prop-rties was tested by molecular probe technique using the followingompounds as molecular probes: pyrene (Kalyanasundaram andhomas, 1977), Nile Red (Mukherjee et al., 2007) and 1,6-diphenyl-,3,5-hexatriene (Chantres et al., 1996).

. Materials and methods

.1. Materials

l-�-Phosphatidylcholine (PC) type XIII-E from egg yolk (99%,olution of 100 mg/mL in ethanol) was obtained from Sigma Chem-cal Co. (St. Louis, MO). It was a mixture of lipids with the followingatty acid makeup: 33% C16:0 (palmitic), 13% C18:0 (stearic), 31%18:1 (oleic), and 15% C18:2 (linoleic) (other fatty acids beinginor contributors), which gives an average molecular weight

f approximately 768 g/mol. N-[Methoxy(polyethylene glycol)000]carbonyl-1,2-dipalmitoyl-sn-glycero-3-phospho-ethanola-ine, sodium salt (PEG-lipid) was obtained from Northern Lipids

nc. (Vancouver, British Columbia, Canada). Oleic acid (OA, ≥98%)nd diethyl ether (>99.8%) were obtained from Fluka ChemieBuchs, Switzerland). Methyl oleate (MOA, ≥99%), cholesterolChol, 99%), hematoporphyrin IX dihydrochloride (HP) and,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (mTHPP) wereeceived from Aldrich Chemical Co. (Milwaukee, WI). The probes:yrene (Pyr, Fluka, for fluorescence, ≥99.0%) and Nile Red (9-iethylamino-5H benzo[�]phenoxazine-5-one) (NR, Fluka, foruorescence, ≥98.0%) and 1,6-diphenyl-1,3,5-hexatriene (DPH,luka, for fluorescence, ≥97.5%) were used as received.

The dyes used in all experiments were dissolved in DMF to formstock solution of approximately 1 mM concentration. All exper-

ments were conducted in phosphate-buffered saline (PBS) at pH.5.

.2. Absorption and emission spectra

UV–vis absorption spectra at 25 ◦C of the samples wereeasured using a Hewlett-Packard 8452A diode-array spectropho-

ometer equipped with a HP 89090A Peltier temperature controlccessory.

Anisotropy experiments and steady-state fluorescence spectraf the samples were measured on an SLM-AMINCO 8100 spectroflu-rimeter at room temperature. Emission spectra were corrected for

3

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ysics of Lipids 155 (2008) 7–15

he wavelength dependence of the detector response by using annternal correction function provided by the manufacturer.

.3. Preparation of liposomes

Liposomes were composed of PC and additive at a defined con-ent. Chol, PEG-lipid, OA, or MOA were first dissolved in ethanol involumetric flask to form a stock solution. Appropriate volumes of

he stock solution were combined with 50 �L of the PC lipid solu-ion in a volumetric flask and gently dried under nitrogen. The dry

aterial was redissolved in diethyl ether which was then evapo-ated under nitrogen to form a thin film of lipid. 10 mM PBS bufferas added till a desired lipid concentration was attained (usu-

lly 2.5 mg/mL), and the sample was sonicated for 5 min at 20 ◦Cn a Bransonic ultrasonic bath. The resulting multilamellar vesi-le dispersion was subjected to five freeze-thaw cycles from liquiditrogen temperature to temperature of 60 ◦C, extruded 10 timeshrough 2 stacked membrane filters with 200-nm pores (Nucleo-ore Track-Etch Membrane Whatman filters) using the PPH Markeranual extruder, and then passed ten times through membrane

lters with 100 nm pores. Such procedure allows obtaining the unil-mellar vesicles of homogeneous size distribution. The z-averageiameter (dz) and the polydispersity index (PD) of the samplesere determined with a Malvern Nano ZS light-scattering appara-

us (Malvern Instruments Ltd., Worcestershire, UK). dz and PD of theiposomal vesicles were in the range of 104–109 nm and 0.025–0.08,espectively. There was no effect of the modifiers content on theesicle size. Vesicles were stored at 4 ◦C.

.4. Determination of liposome-binding constants

A spectroscopic titration technique was used to determinehe binding constants (Kb) of the porphyrin chromophores toipid vesicles. Details of this technique were described previouslyKepczynski et al., 2002; Roslaniec et al., 2000). To determine thencubation time required to reach equilibrium, the partitioninginetics of the porphyrin molecules into liposomes were studied.fter each addition of an aliquot of lipid, the system was equi-

ibrated and emission spectra of porphyrin chromophore wereecorded. Kb is given in units of (mg/mL)−1 throughout this study.

.5. Experiments with molecular probes

Stock solutions of DPH and NR in DMF were prepared. For label-ng, the liposome suspension (1 mg lipid/mL) was incubated inhe dark for 1 h with appropriate amount of the probe stock solu-ion and the final lipid-to-probe molar ratios were 1220 for DPHnd 1000 for NR. The fluorescence anisotropies of the samplesere determined using �exc = 350 nm and �em = 428 nm for DPH and

exc = 542 nm and �em = 618 nm for NR. The measurements wereepeated three times and the average value was calculated.

Stock solution of Pyr was prepared in methanol. Pyr was sol-bilized in liposomal bilayer by slow injection of the appropriateolume of the stock solution to liposomal suspension, followed bytirring during 2 h in the dark. The fluorescence spectra of Pyr wereecorded at the excitation wavelength of 333 nm.

. Results

.1. Characterization of the studied systems

The chemical structures of two investigated porphyrins, HPnd mTHPP, are shown in Fig. 1. The structure of HP consists ofhe tetrapyrrole ring with two propionic acid groups attached at

M. Kepczynski et al. / Chemistry and Physics of Lipids 155 (2008) 7–15 9

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Fig. 2. Fluorescence spectra of mTHPP (c = 1.1 × 10−7 M, �exc = 422 nm) in buffer (pH5.5, the dotted line) and in a suspension of liposomes containing 20 mol% of oleicacid at increasing concentration of lipid (in these representative spectra: 0, 0.006,0flt

wabt(

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Fig. 1. Chemical structures of the investigated porphyrins.

he same side of the ring in positions six and seven. Such struc-ure causes this compound to be amphiphilic. mTHPP has ringubstituents, 4-hydroxyphenyl groups, in the meso positions sym-etrically distributed around the ring. The partitioning of HP to

he lipid bilayer is strongly dependent on the pH value of envi-onment and reaches the maximum at pH 5 (Brault, 1990). In ourtudy we used PBS buffer at pH 5.5. It was a compromise valuef pH to avoid the fast degradation of HP occurring at pH ≤ 5, onne hand, and to ensure a high value of Kb (defined below), onhe other hand. At this pH, about 92.6% of the carboxylic groups ofleic acid (pKa = 4.4 (Drzymała, 1987)) are dissociated. The ioniza-ion constants for the two carboxylate groups of HP are 5.7 ± 0.1nd 6.9 ± 0.05 (Kepczynski and Ehrenberg, 2002). Therefore, at pH.5 about 60.5%, 38%, and 1.5% of the porphyrin chromophores areresent in solution as neutral, monoanionic and dianionic forms,espectively.

.2. Liposome-binding constants

The partitioning of HP and mTHPP between the lipid vesicles and

queous phase can be quantitatively described by so-called bindingonstant, which is defined as follows (Ehrenberg, 1992):

b = cL

cw[L](1)

flowmw

able 1xperimentally measured binding constants (Kb) of HP and mTHPP to modified liposomuorescence anisotropy of DPH and NR

odifier Concentration (mol%) Kb (mg/mL)−1 Pyr

HP mTHPP I3/I1

0 25.1 ± 2.1 105 ± 35 0.804

hol 10 22.1 ± 1.9 46 ± 11 0.83315 17.1 ± 1.4 35 ± 16 0.83820 18.1 ± 2.4 52 ± 31 0.85230 9.67 ± 1.30 36 ± 19 0.894

A 5 25.2 ± 2.9 73 ± 10 0.81310 31.4 ± 4.2 112 ± 26 0.83120 26.2 ± 3.4 111 ± 31 0.84330 46.9 ± 20.5 143 ± 42 0.835

OA 5 29.9 ± 2.7 273 ± 91 0.83210 34.9 ± 7.6 303 ± 78 0.83620 45.2 ± 5.1 351 ± 42 0.8430 46.4 ± 5.6 394 ± 82 0.847

EG-lipid 3 39.9 ± 8.8 237 ± 58 0.8435 38.3 ± 6.0 254 ± 20 0.8497 33.8 ± 6.2 270 ± 55 0.85

10 32.2 ± 4.0 269 ± 40 0.853

.012, 0.024, 0.036, 0.048, 0.072, 0.095, 0.117 and 0.172 mg/mL). The intensity of theuorescence at 662 nm is shown in the insert, together with the line, fitted accordingo Eq. (2).

here cL and cw are porphyrin concentrations in the lipid vesiclesnd aqueous phase, respectively; [L] is concentration of lipid. Theinding constant was examined as a function of modifier concentra-ion using the spectroscopic titration technique developed earlierKepczynski et al., 2002; Roslaniec et al., 2000).

The study of the porphyrins partitioning to liposomes was car-ied out in PBS buffer at pH 5.5 by recording the steady-statemission spectra of solutions made with various lipid concen-rations and a constant concentration of porphyrin. The lipidoncentration [L] was varied from 0 to 0.2 mg/mL. The assessmentf the binding constant was based on the pronounced spectralhanges that are observed upon transfer of a porphyrin moleculerom an aqueous phase to the lipid bilayer. Fig. 2 presents a typicalet of spectra obtained for mTHPP in the presence of lipid at variousoncentrations with 20 mol% content of OA. An amplification of the

uorescence intensity was observed with increasing concentrationf the lipid. Similar changes in the fluorescence spectra of mTHPPere observed for the system with different concentration of theodifiers. In the case of HP an amplification of fluorescence spectraas less pronounced, but it was accompanied by a red shift of the

es, the ratio of the fluorescence intensities of the peak 3 to peak 1 of Pyr and the

DPH anisotropy Microviscosity (Poise) NR anisotropy

IE/IM

0.819 0.1483 ± 0.0045 1.66 0.2077 ± 0.0080

0.732 0.1603 ± 0.0032 1.91 0.2102 ± 0.00940.563 0.1650 ± 0.0086 2.01 0.2081 ± 0.00930.674 0.1628 ± 0.0056 1.96 0.2062 ± 0.00960.707 0.1798 ± 0.0033 2.37 0.1949 ± 0.0073

1.354 0.1421 ± 0.0050 1.55 0.2004 ± 0.00781.404 0.1313 ± 0.0401 1.36 0.1984 ± 0.00571.382 0.1300 ± 0.0235 1.34 0.1904 ± 0.00731.312 0.1343 ± 0.0029 1.42 0.1844 ± 0.0074

1.322 0.1378 ± 0.0052 1.48 0.1948 ± 0.00721.347 0.1325 ± 0.0082 1.38 0.1812 ± 0.00661.451 0.1327 ± 0.0043 1.39 0.1791 ± 0.00791.344 0.1287 ± 0.0065 1.32 0.1739 ± 0.0069

1.023 0.1304 ± 0.016 1.35 0.2105 ± 0.00771.044 0.1325 ± 0.028 1.38 0.2100 ± 0.00811.147 0.1230 ± 0.016 1.24 0.2068 ± 0.00701.205 0.1348 ± 0.011 1.42 0.2057 ± 0.0074

10 M. Kepczynski et al. / Chemistry and Ph

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ig. 3. The effect of the modifier on the binding constant to liposomal vesicles ofa) hematoporphyrin IX and (b) 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin; (♦)hol, (�) PEG-lipid, (�) OA, (©) MOA.

uorescence band (the fluorescence maximum shifts from 618 to23 nm) upon entering the lipid domain.

The effective binding constant, Kb, was determined by fittinghe experimental data to the following formula (Kepczynski et al.,002):

= Finit + FcompKb[L]1 + Kb[L]

(2)

here Finit, F and Fcomp are the fluorescence intensity of the dyeeasured in the absence of the lipid, in the presence of the lipid at

oncentration [L] and the asymptotic value of the intensity at com-lete binding, respectively. To obtain the Kb, F versus [L] data werelotted and fitted to Eq. (2) by a nonlinear regression routine. The

nsert in Fig. 2 shows an example of fluorescence intensity changest 662 nm with [L] and the line fitted to the plot of the dependencef F at 662 nm versus lipid concentration. The calculated values ofb are collected in Table 1.

The Kb value received for mTHPP in the neat liposomess comparable with the values found for the other non-ionicetraarylporphyrins. For instance, Kb values for 5,10,15,20-etrakis-(m-hydroxyphenyl)chlorin and tetrabenzoporphyrin arequal to 136 ± 21 (mg/mL)−1 and 89 ± 25 (mg/mL)−1, respectivelyKepczynski et al., 2002).

The partitioning of porphyrins between aqueous phase and

ydrophobic lipid vesicles was examined as a function of the mod-

fier content in the membrane. The effect of the modifiers on Kbs depicted in Fig. 3a and b. The salient findings that are deducedrom the Fig. 3a and b as well as Table 1 are that: (1) introductionf the cholesterol into lipid bilayer causes a reduction of partition-

iotoe

ysics of Lipids 155 (2008) 7–15

ng of both porphyrins into liposomal membrane; the values of Kbor liposomes containing 30 mol% of Chol are about 2.6 and 2.9imes lower then those characteristic of the neat vesicles for HPnd mTHPP, respectively; (2) addition of the PEG-lipid to liposomeake-up results in an considerable rise of the binding constants

or both porphyrins; (3) the partitioning of both porphyrins intoiposomal membrane increases significantly with the higher con-ents of MOA. The increase of the Kb values with increasing MOAoncentration to 30 mol% is more pronounced for mTHPP (a 3.7-old factor) than for HP (1.8 times); (4) appearance of the negativeurface charge after incorporation of OA into bilayer has differentffect on each of porphyrin; in the case of HP the rise of the Kb values lower than that for MOA, but finally at 30 mol% the Kb reaches theame value as in the presence of the methyl derivative. In the casef mTHPP introduction of OA results in an initial reduction of theb value (at the concentration 5 mol% of OA) followed by a feeble

ncrease with increasing contents of OA.

.3. Fluorescence molecular probe studies

Fluorescent probes are very useful in membrane biology dueo their ability to monitor membrane organization and dynam-cs. Three conceptually different fluorescence probes, NR (annisotropy and a solvatochromic probe), DPH (an anisotropy probe)nd Pyr (a solvent polarity and an excimer-forming probe) haveeen used for fluorescence spectroscopic studies of the liposomalembrane properties.

.3.1. Nile Red anisotropy and fluorescenceNR is an uncharged positive solvatochromic probe, quite well-

oluble in organic solvents and lipids, but relatively insoluble inater. The fluorescence of NR is sensitive to the polarity of theedium (Mukherjee et al., 2007). The fluorescence spectra of NR

ncorporated into the neat and modified liposomes were recorded.he emission maximum of NR in liposomes in the absence ofholesterol was found to be 635 nm when excited at 542 nm. Thencorporation of Chol causes slight shift of the emission maximumoward lower wavelength (to 631 nm at 30 mol% Chol) and the fluo-escence intensity decrease. The fluorescence intensity dropped bypproximately 52% when Chol concentration increased to 30 mol%.ince the NR fluorescence lifetime markedly decreases with thencrease on the hydrogen-bonding capability of the medium (Csert al., 2002), the reduction in fluorescence intensity at higherhol concentration could be due to enhanced water penetra-ion in the membrane interfacial region induced by cholesterol.uch a phenomenon was previously reported by Subczynski et al.1994).

The effect of additives on the fluidity in the polar region of theiposomes was studied by the use of fluorescence anisotropy of NR.s was found previously, NR fluorescence anisotropy is wavelength-ependent and it decreases with an increasing value of wavelengthCoutinho et al., 2002). In our experiments the dye was excited at42 nm and the emission was monitored at 618 nm. Fig. 4a showshe average steady-state anisotropy of NR in liposomal vesicles as aunction of increasing concentration of the modifiers. It can be seenhat the NR anisotropy drops with increasing concentration of OAnd its methyl ester. This can indicate that both compounds reducehe microviscosity in the polar region of the egg lipid vesicles. Theeduction of anisotropy caused by the charged fatty acid is smallerompared to the ester emphasizing an effect of the charge in the

nterface region on the microviscosity of the lipid bilayer. In the casef PEG-lipid the value of the anisotropy is practically constant inhe range of the studied concentrations. Similarly, the introductionf Chol into the bilayer up to concentration of 15 mol% has littleffect on the local viscosity. At higher content of Chol, however, its

M. Kepczynski et al. / Chemistry and Physics of Lipids 155 (2008) 7–15 11

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Fig. 5. (a) The effect of the modifiers on the fluorescence intensity ratio of band3–1 (I3/I1) of pyrene (�exc = 333 nm, cPyr = 9.6 × 10−7 M) solubilized in lipid bilayer;(r(

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ig. 4. The effect of the modifiers on the fluorescence anisotropy of therobe incorporated into lipid bilayer: (a) Nile Red (�exc = 542 nm, �em = 618 nm;NR = 1.4 × 10−7 M); (b) DPH (�exc = 350 nm, �em = 428 nm; cNR = 9.6 × 10−8 M); (♦)hol, (�) PEG-lipid, (�) OA, (©) MOA.

dmixture decreases the microviscosity in the polar region of theiposomes.

.3.2. DPHDPH is the most widely applied rotational probe for estimating

icroviscosity and fluidity of liposomal membrane in the acyl sidehain region (Chantres et al., 1996). The liposomes with differentmount of the additives were labeled with DPH in the dark. Theodifiers incorporated into membrane have no effect on the shape

nd location of the emission band of the probe. The measurementf the fluorescence polarization was done and the steady-statenisotropy of DPH in liposomes as a function of increasing con-entration of modifiers is shown in Fig. 4b. It is seen that thenisotropy of DPH increased with increasing level of Chol in theiposomal membrane. In the presence of the other additives thePH anisotropy decreases while their concentration is rising.

Apparent microviscosities obtained for liposomes with increas-ng molar ratios of the modifiers are listed in Table 1. The values

ere calculated from the measured fluorescence anisotropies usinghe Perrin equation as described in literature (Pandey and Mishra,999).

.3.3. Pyrene

Pyr was used as a molecular probe to estimate alteration of

ocal micropolarity of the egg lecithin membranes caused by incor-oration of the modifiers. In the presence of micelles and otheracromolecular systems, Pyr is preferentially solubilized in the

nterior hydrophobic regions of these aggregates because of poor

sidmb

b) The effect of the modifiers on the excimer-to-monomer fluorescence intensityatio (IE/IM) of Pyr (�exc = 333 nm, cPyr = 4.8 × 10−5 M) incorporated into lipid bilayer;♦) Chol, (�) PEG-lipid, (�) OA, (©) MOA.

yrene’s solubility in water. It is known that the ratio of the inten-ities of the third and the first vibrational bands, I3/I1, in theuorescence emission spectrum of pyrene can serve as a mea-ure for the polarity of the microdomain’s interior. Namely, I3/I1s low in polar media and high in hydrophobic environmentsKalyanasundaram and Thomas, 1977). Thus, the polarity of theiposomal bilayer was monitored by recording fluorescence spec-ra of pyrene solubilized in the membrane. The plot of I3/I1 ratioersus the concentration of the modifiers is given in Fig. 5a. Asan be seen from this figure, the I3/I1 ratio raises almost linearlyith increasing concentration of Chol in the membrane. In the case

f PEG-lipid, MOA and OA after an initial increase the I3/I1 ratioeaches the plateau. Interestingly, with the exception of low con-entration, the effect of oleic acid and its methyl ester on the valuef I3/I1 is the same.

It is known that at the sufficiently high concentration Pyr is ableo form excimers. Excimers are formed when two Pyr molecules arelose enough to each other and can interact during the excited stateifetime of a Pyr molecule (Maier et al., 2002). The excimers havecharacteristic red-shifted fluorescence emission. The excimer-to-onomer fluorescence intensity ratio (IE/IM) reflects the extent of

yr excimer formation, which depends mainly on the monomerpatial distribution in the lipid bilayer. The excimer formation abil-

ty of Pyr has been widely used to monitor the changes in the lateraliffusion in membrane (Ioffe and Gorbenko, 2005). Diffusion ofolecules in a membrane is directly related to a free volume of

ilayer (the unoccupied volume enclosed in the bilayer). Therefore,

12 M. Kepczynski et al. / Chemistry and Physics of Lipids 155 (2008) 7–15

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ig. 6. A schematic representation of half of the membrane bilayer showing the lohown in red; carbon atoms, in black; hydrogen atoms, in grey; nitrogen atoms, ingure legend, the reader is referred to the web version of the article.)

yr fluorescence studies may prove to be useful for detecting thehanges in the phospholipid bilayer free volume.

As shown in Fig. 5b, intercalation of the modifiers into the lipidembranes resulted in the alternation of IE/IM ratio. These findings

uggest that free volume of the modified membranes varies withraction of the additives. The presence of OA and its methyl estern the lipid bilayer cause a pronounced increase of the IE/IM ratio.he explanation of that phenomenon may be that the unsaturatedatty acids in cis-conformation decrease acyl chain ordering of theipid in bilayer increasing in this way the membrane free volume.n the case of PEG-lipid the IE/IM ratio also increases.

As is seen in Fig. 5b, the introduction of Chol leads to reduc-ion of IE/IM value. As expected, Chol rigidifies the bilayers reducinghe free volume available for diffusion of Pyr molecules. Interest-ngly, the dependence of IE/IM versus Chol content exhibits a dip at5 mol% of Chol. The increase of IE/IM value at higher Chol concen-rations can be attributed to the increase of free volume resultedrom the mentioned earlier domain formation at concentrationigher than 20 mol% (de Almeida et al., 2003).

.4. Fluorescence quenching studies

Quenching of the fluorescence of porphyrins with iodide ionss one of the simplest ways to obtain information about the rel-tive vertical depth location of membrane-bound fluorophores inmembrane (Bronshtein et al., 2004). Fluorescence quenching is

ssociated with a collisional quenching constant, KSV, which isbtained from the Stern–Volmer equation,

F0

F= 1 + KSV[Q ] (3)

here F0 and F are fluorescence intensities in the absence and inhe presence of the quencher, respectively, KSV is the Stern–Volmeronstant, and [Q] is the concentration of the quencher. To deter-ine the KSV for two porphyrins embedded in the lipid membrane,

he dependence F0/F versus [Q] was plotted, and fitted to Eq. (3).inear Stern–Volmer plots were obtained up to a quencher con-entration of 0.15 mM. The following values of KSV were obtained:

.66 ± 0.09 M−1 and 5.75 ± 0.15 M−1 for mTHPP and HP, respec-ively. As can be seen, the fluorescence of both porphyrins isuenched by I−, but efficiency of quenching is different for eachompound. The value of KSV of mTHPP is lower than that for HP,hich suggests that this porphyrin is situated deeper in the bilayer.

l±mCt

tion of studied compounds in phosphatidylcholine membranes. Oxygen atoms arephosphorus atoms, in purple. (For interpretation of the references to color in this

. Discussion

In this paper we present the study on the effect of the modi-ying substances on the drug-loading efficiency of the liposomes,hich seems to be very important as far as the application of the

ipid vesicles as drug delivery vehicles is concerned. We studied theilayer membranes made of egg-yolk PC, which are a model of bio-

ogical membranes with mixed, saturated and unsaturated, alkylhains (see Section 2).

Incorporation of the molecular probes into the membrane is aommon way to obtain useful information about the membraneroperties. Knowledge of molecular locations within membranes

s important for understanding the obtained information with theentioned probes. There are two reliable methods to estimate

his parameter: (1) depth-sensitive fluorescence quenching anal-ses (FQA), (2) molecular dynamics simulations (MD simulations);osition, distribution, and alignment of the molecule in lipid mem-ranes can be studied by this technique. As the generally acceptedonvention, the location in the membrane is given as a distance infrom the hydrocarbon center.The location of DPH within DOPC bilayer was previously inves-

igated using FQA (Kaiser and London, 1998). The average distancef the center of fluorophore molecule from the center of the bilayeras estimated to be 7.8 and 6.7 Å in the absence and in the presencef 33 mol% Chol, respectively, which corresponds to the C10–C11toms of the hydrocarbon chains. The length of DPH was esti-ated to be about 14 Å between carbon atoms at each end using theOPAC module of Chem3D Ultra 8.0 (CambridgeSoft Corporation,

ambridge, USA). The localization of NR within DOPC liposomesas also previously investigated using FQA (Mukherjee et al., 2007).

he average distance of the center of fluorophore molecule from theenter of the bilayer was estimated to be 17.6, 16.6 and 15.8 Å in thebsence and in the presence of 20 and 40 mol% Chol, respectively.he length of NR chromophore was estimated to be about 8.5 Åetween carbon atoms at each end using the Chem3D (the wholeolecule is 12.9 Å long). Using MD simulations, Hoff et al. (2005)

ave shown that Pyr prefers a position inside the lipid membraneear the polar region. The long axis of pyrene (the length is 7 Å)

ies preferentially parallel to the bilayer normal within a range of30◦. The preferred position of Pyr in the membrane was also deter-ined with FQA (Herrenbauer, 2002) to be in the region of the

5–C6 atoms of the acyl chain. The cholesterol molecule containshree well-distinguished regions: a small polar hydroxyl group, a

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igid plate-like steroid ring, and an alkyl chain tail. When choles-erol intercalates into the membrane, its polar hydroxyl group isositioned near the middle of the glycerol backbone region of theC molecule (Worcetor and Franks, 1976). We assumed that theocalization of OA and its methyl ester is similar to the position ofhe unsaturated acyl chains of DOPC. The location of the doubleond of DOPC in liposomes was estimated to 7.88 Å (Wiener andhite, 1992).Fig. 6 shows schematically the location of the molecular probes

sed in this work in the half of the membrane bilayer. The chemicaltructures of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), asn example of lipid, cholesterol and oleic acid are also shown, andhey are all arranged according to the orientation and location inhe membrane. NR has been used as a rotational probe to estimatehe fluidity in the polar region of the bilayer. DPH has been useds a fluorescence probe to measure the fluidity of liposomal mem-rane in hydrophobic region. Pyr has been used as a hydrophobicity

ndicator of the hydrocarbon chain region near to the polar region.he above-mentioned localization of the probes can be shifted byhe introduction of modifiers. As was shown for Chol this shift isot drastic. One should keep in mind that the lipid bilayer is veryynamic structure and the fluctuation of the location due to thehermal motions occurs (Wiener and White, 1992).

Despite of a large number of papers concerning interactionetween porphyrins with lipid membranes present in literature,e have found only one study on depth-localization of the dye in

he bilayer. The localization of the series of protoporphyrins witharying lengths of alkyl carboxylate side groups was studied usinghe FQA method (Bronshtein et al., 2004). It was found that theaturation of the lipids and temperature have practically no effectn the depth-localization. The distance of the effective center ofhe protoporphyrin fluorophore from the midpoint of the bilayerhanges from ∼19 Å (the fluorophore is practically near the waternterface) to ∼7 Å upon elongation of alkyl carboxylate groups. Forhe same series of porphyrins Lavi et al. (2002) have reported thealues of KSV in the range of 4.74–0.65 M−1. Based on these data theollowing correlation can be found: the lower is the value of KSV, theeeper is the molecule located in the bilayer. Taking into accounthe KSV values found for HP and mTHPP, it is reasonable to assumehat HP is located at the bilayer interface with two carboxylic groupxposed to the water phase, whereas mTHPP can enter a bit deeperart of membrane, since this dye is less effectively quenched by

odide ions. This is schematically depicted in Fig. 6. Our assump-ion concerning the HP localization in liposomes is in line with therevious observation that at low concentration HP molecules pref-rentially interact with phospholipid headgroup-water interfacesRicchelli and Gobbo, 1995).

Incorporation of the modifiers into the lipid bilayers changesreatly the structural or physical properties of liposomal membraneuch as organization, free volume, thickness, stiffness (fluidity) andolarity (hydrophobicity) of bilayer. In our studies we have usedour substances as modifies of the liposomal membrane: (1) Chol,hich is present in most of formulations of liposomes applied asrug carriers, (2) PEG-lipid, which is an essential ingredient of theSL liposomes, (3) OA, an unsaturated fatty acid, and (4) its methylster, MOA, in order to study the effect of the cis-double bond andurface charge on the loading efficiency of liposomes.

Hydrophobicity of the phospholipid membrane is depth-ependent and is largely determined by the extent of waterenetration into the membrane (Griffith et al., 1974). As was shown

sing the pyrene probe the neat egg-yolk PC bilayer have hydropho-icity comparable to the level of methanol in the chain region nearo the polar region. A constant increase of the hydrophobicity inhis region of membrane with increasing concentration of Chol wasbserved (Fig. 5a). The hydrophobicity reaches that of 1-propanol

abati

ysics of Lipids 155 (2008) 7–15 13

fter introduction of 30 mol% Chol. Thus, water penetration into theydrocarbon phase in the membrane is suppressed by the presencef Chol. On the other hand, the augmented by Chol penetration ofater into the polar region of the membrane was shown using theR probe. The presence of Chol decreases hydrophobicity (increasesater penetration) in the polar headgroup region of the liposomes

Subczynski et al., 1994). The intercalation of the unsaturated fattycid, OA, or its ester decreases the micropolarity of the membrane.cis double bond in the alkyl chains increases hydrophobicity in

he membrane, but the effect is smaller compared to Chol. In thease of PEG-lipid the increase of the local hydrophobicity was alsobserved with the use of Pyr. Similar effect of the PEG-lipid on theolarity of membrane was observed previously by Yoshida et al.1999). As can be seen in Fig. 5a the incorporation of the PEG-lipidsnto liposomes causes a marked increase of the I3/I1 value. Afterddition of only 5 mol% of PEG-lipid the hydrophobicity reaches thealue that is characteristic of the PC bilayer modified with 20 mol%f Chol. Thus, the presence of lipid with covalently attached PEGhains at the liposomes interface effectively reduces the hydrationevel of the PC membranes up to the chain region near to the polaregion. Elimination of water from the lipid head group region of theiposomes induced by the intercalation of PEG-lipid was previouslyemonstrated by Tirosh et al. (1998).

The viscosity in the bilayer interior varies with the depth andemperature and can be related to changes in the free volume ofhe membrane. The effect of Chol on the microviscosity of bilayers rather complex and depth-dependent. It is well-known that Cholncreases the apparent microviscosities (reduces fluidity) of mem-ranes being in liquid phase. Our results have shown that theicroviscosity of egg PC membranes increase 1.4 times, when con-

entration of Chol increase to 30 mol%. Using MD simulation study itas recently shown that the Chol added to the membrane induces

he ordering of the PC acyl chains in liquid phase (Cournia et al.,007). The rigid sterol ring systems straighten the PC acyl chains by

nhibition of trans-gauche conformational transitions. Induced inhat way tightening of the lipids packing in the bilayers affects theiruidity, viscosity and lateral diffusion. As expected, introduction ofhol to the egg PC liposomes rigidifies the bilayers and reduces the

ree volume available for diffusion of Pyr molecules. We observedhis as a reduction of the IE/IM ratio (see Fig. 5b) up to Chol con-entration of 15 mol%. Using MD simulation study it was recentlyonfirmed that cholesterol modifies the free volume properties ofhe membrane in the hydrocarbon phase close to the polar regionJedlovszky and Mezei, 2003). In the region where the cholesterolH groups are located, at high cholesterol concentrations, there isore free volume available in cholesterol-rich than in cholesterol-

oor or cholesterol-free membranes. This can explain the increasef the IE/IM value (Fig. 5b) and the decrease of the NR anisotropyFig. 4a) at concentration of Chol higher than 20 mol%.

As could be expected, incorporation of OA and MOA into theilayer results in a reduction of the microviscosity (increase inhe fluidity) at all locations in the membrane. The decrease ofnisotropy was observed for both rotational probes, NR locatedhallowly in the bilayer (Fig. 4a) and DPH buried deeply in theydrocarbon region (Fig. 4b). This can be easily explained consid-ring the 30◦ bend of oleoyl chains at the C9 position, which causeshe steric nonconformability to the lipid molecules. Thus, the freeolume in the membrane significantly increases, as was observedsing pyrene probe (Fig. 5b). The effect of PEG-lipid on the microvis-osity of the membrane is depth-dependent as can be seen in Fig. 4a

nd b. As demonstrated with DPH probe the fluidity of the hydrocar-on region increases significantly after introduction of even smallmount of the modifier. On the other hand, the microviscosity athe interface between the chain and polar regions is almost notnfluenced by the presence of the PEG-lipid.

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Considering the above described localization of the modifiersnd effects on the bilayer one can consider which properties ofipid membranes are important in drug-loading efficiency. Free vol-me and the changes of the free volume caused by the modifiersre important factors governing the membrane-loading efficiency.he liposomal partitioning of both porphyrins increases remark-bly with addition of additives which improve the free volumeroperties of the membrane. In fact, the value of Kb increases after

ntercalation of MOA or PEG-lipid. As can be seen in Fig. 5b OAnd MOA caused a similar improvement of the free volume in theydrocarbon region of the bilayer. But, the introduction of OA isonnected with appearance of the negative charges in the polaregion of liposomes since oleic acid is almost completely ionizedn our experimental conditions. One could expect an effect of theseharges on the partitioning of porphyrin which is bearing nega-ive charges and almost no effect on porphyrin species which areot ionized in our experimental conditions. Indeed, in the casef HP, which is partially dissociated, the Kb values for liposomesodified with OA are lower than that with incorporated MOA (see

ig. 3a). Surprisingly, we observed that the additional charges athe head groups region inhibit the increase of Kb value of mTHPP.

e have attributed this phenomenon to an effect of the charges onhe kinetics of entrance of mTHPP molecules into liposomes. Theppearance of negative charges in liposomes can impede attain-ent of the equilibrium state of the porphyrin partitioning. We

ave observed that HP took just a few minutes to reach bindingquilibrium, whereas more hydrophobic mTHPP required muchonger periods of incubation to equilibrate with the lipid phase.hus, the effect of OA on kinetics would be more pronounced in thease of mTHPP. In our experiments the system with OA-modifiediposomes and mTHPP were incubated to the extent limited by thetability of liposomes in room temperature, but probably this times not sufficient to reach the equilibrium state of the partitioning.he changes of microviscosity in the hydrocarbon part of liposomesre directly connected with the changes of free volume. As cane seen in Figs. 4b and 5b, an increase of the local free volumentails the reduction of the local viscosity. Thus, the local viscosityffects the binding constants of mTHPP. The Kb values increase withncreasing concentration of modifiers, which reduce the microvis-osity in the chain region.

Contrary to the expectations, it seems that polarity of the chainegion is rather minor factor affecting loading efficiency. The intro-uction of Chol caused the reduction of the binding constants foroth porphyrin in spite of the fact the hydrophobicity of the hydro-arbon region increase influenced by the presence of Chol.

. Conclusions

We have applied a method of direct determination of the par-itioning affinity of the two porphyrins with liposomal bilayer

odified with cholesterol, oleic acid, methyl oleate and pegylatedipid. These modifiers are important because they are the com-

on additives or potential additives to the liposome formulation inrug delivery systems. Steady-state fluorescence spectroscopy haseen used to characterize the properties of liposomes from egg yolk

ipids, such as polarity, fluidity and free volume. The studies carriedut with the use of molecular probes allowed the formulation ofeneral rules regarding the effect of modifiers on the properties ofilayers. Based on our experimental results and literature data weave concluded that the free volume of the bilayer rather than its

ydrophobicity of the chain region is governing factor in solubiliza-ion/partitioning of porphyrins. Our results emphasize clearly thathe exact design of the membrane make-up is important for stabilerug incorporation into liposomes and can significantly (even sev-ral times) improve the drug accumulation inside liposomal bilayer.

R

ysics of Lipids 155 (2008) 7–15

eferences

rault, D., 1990. Physical chemistry of porphyrins and their interactions with mem-branes: the importance of pH. J. Photochem. Photobiol. B Biol. 6, 79–86.

ronshtein, I., Afri, M., Weitman, H., Frimer, A.A., Smith, K.M., Ehrenberg, B., 2004.Porphyrin depth in lipid bilayers as determined by iodide and parallax fluores-cence quenching methods and its effect on photosensitizing efficiency. Biophys.J. 87, 1155–1164.

hantres, J.R., Elorza, B., Elorza, M.A., Rodado, P., 1996. Deoxycholate alters theorder of acyl chains in freeze- thaw extrusion vesicles of l-�-dipalmitoylphosphatidylcholine: study of the 1,6-diphenyl-1,3,5-hexatriene steady-statefluorescence anisotropy. Int. J. Pharm. 138, 139–148.

ournia, Z., Ullmann, G.M., Smith, J.C., 2007. Differential effects of cholesterol,ergosterol and lanosterol on a dipalmitoyl phosphatidylcholine membrane: amolecular dynamics simulation study. J. Phys. Chem. B 111, 1786–1801.

outinho, P.J.G., Castanheira, E.M.S., Ceu Rei, M., Real Oliveira, M.E.C.D., 2002. NileRed and DCM fluorescence anisotropy studies in C12E7/DPPC mixed systems. J.Phys. Chem. B 106, 12841–12846.

ser, A., Nagy, K., Biczók, L., 2002. Fluorescence lifetime of Nile Red as a probe for thehydrogen bonding strength with its microenvironment. Chem. Phys. Lett. 360,473–478.

e Almeida, R.F.M., Fedorov, A., Prieto, M., 2003. Sphingomyelin/phospha-tidylcholine/cholesterol phase diagram: boundaries and composition of lipidrafts. Biophys. J. 85, 2406–2416.

rzymała, J., 1987. An estimation of the surface ionization constant of oleic acid inaqueous sodium chloride solution. Colloid Polym. Sci. 265, 613–618.

hrenberg, B., 1992. Assessment of the partitioning of probes to mem-branes by spectroscopic titration. J. Photochem. Photobiol. B Biol. 14, 383–386.

erasimov, O.V., Boomer, J.A., Qualls, M.M., Thompson, D.H., 1999. Cytosolic drugdelivery using pH- and light-sensitive liposomes. Adv. Drug Deliv. Rev. 38,317–338.

regoriadis, G., 1988. Liposomes as Drug Carriers. Recent Trend and Progress. JohnWiley & Sons, New York.

riffith, O.H., Dehlinger, P.J., Van, S.P., 1974. Shape of the hydrophobic barrier ofphospholipid bilayers (evidence for water penetration in biological membranes).J. Membr. Biol. 15, 159–192.

an, H.D., Lee, A., Hwang, T., Song, C.K., Seong, H., Hyun, J., Shin, B.C., 2007. Enhancedcirculation time and antitumor activity of doxorubicin by comblike polymer-incorporated liposomes. J. Control Release 120, 161–168.

errenbauer, M., 2002. Biosorption of Polycyclic Aromatic Hydrocarbons (PAH) toMicroorganisms and Liposomes. Shaker Verlag, Aachen, Germany.

off, B., Strandberg, E., Ulrich, A.S., Tieleman, D.P., Posten, C., 2005. 2H NMR studyand molecular dynamics simulation of the location, alignment, and mobility ofpyrene in POPC bilayers. Biophys. J. 88, 1818–1827.

offe, V., Gorbenko, G.P., 2005. Lysozyme effect on structural state of model mem-branes as revealed by pyrene excimerization studies. Biophys. Chem. 114,199–204.

edlovszky, P., Mezei, M., 2003. Effect of cholesterol on the properties of phospho-lipid membranes. 2. Free energy profile of small molecules. J. Phys. Chem. B 107,5322–5332.

aiser, R.D., London, E., 1998. Location of diphenylhexatriene (DPH) and its deriva-tives within membranes: comparison of different fluorescence quenchinganalyses of membrane depth. Biochemistry 37, 8180–8190.

alyanasundaram, K., Thomas, J.K., 1977. Environmental effects on vibronic bandintensities in pyrene monomer fluorescence and their application in studies ofmicellar systems. J. Am. Chem. Soc. 99, 2039–2044.

epczynski, M., Ehrenberg, B., 2002. Interaction of dicarboxylic metalloporphyrinswith liposomes. The effect of pH on membrane binding revisited. Photochem.Photobiol. 76, 486–492.

epczynski, M., Pandian, R.P., Smith, K.M., Ehrenberg, B., 2002. Do liposome-bindingconstants of porphyrins correlate with their measured and predicted partition-ing between octanol and water? Photochem. Photobiol. 76, 127–134.

avi, A., Weitman, H., Holmes, R.T., Smith, K.M., Ehrenberg, B., 2002. The depth ofporphyrin in a membrane and the membrane’s physical properties affect thephotosensitizing efficiency. Biophys. J. 82, 2101–2110.

aier, O., Oberle, V., Hoekstra, D., 2002. Fluorescent lipid probes: some propertiesand applications (a review). Chem. Phys. Lipids 116, 3–18.

ukherjee, S., Raghuraman, H., Chattopadhyay, A., 2007. Membrane localization anddynamics of Nile Red: effect of cholesterol. Biochim. Biophys. Acta 1768, 59–66.

andey, B.N., Mishra, K.P., 1999. Radiation induced oxidative damage modifica-tion by cholesterol in liposomal membrane. Radiat. Phys. Chem. 54, 481–489.

iperoudi, S., Fatouros, D., Ioannou, P.V., Frederik, P., Antimisiaris, S.G., 2006. Incor-poration of PEG-lipids in arsonoliposomes results in formation of highly stablearsenic-containing vesicles. Chem. Phys. Lipids 139, 96–106.

icchelli, F., Gobbo, S., 1995. Porphyrins as fluorescent probes for monitoring phasetransitions of lipid domains in biological membranes. Factors influencing the

microenvironment of haematoporphyrin and protoporphyrin in liposomes. J.Photochem. Photobiol. B Biol. 29, 65–70.

oslaniec, M., Weitman, H., Holmes, R.T., Smith, K.M., Ehrenberg, B., 2000. Liposomebinding constants and singlet oxygen quantum yields of hypericin, tetrahydroxyhelianthrone and their derivatives: studies in organic solutions and in liposomes.J. Photochem. Photobiol. B Biol. 57, 149–158.

and Ph

S

S

T

W

W

W

Y

M. Kepczynski et al. / Chemistry

adzuka, Y., Tokutomi, K., Iwasaki, F., Sugiyama, I., Hirano, T., Konno, H., Oku, N.,Sonobe, T., 2006. The phototoxicity of photofrin was enhanced by PEGylatedliposome in vitro. Cancer Lett. 241, 42–48.

ubczynski, W.K., Wisniewska, A., Yin, J.-J., Hyde, J.S., Kusumi, A., 1994. Hydrophobicbarriers of lipid bilayer membranes formed by reduction of water penetration

by alkyl chain unsaturation and cholesterol. Biochemistry 33, 7670–7681.

irosh, O., Barenholz, Y., Katzhendler, J., Priev, A., 1998. Hydration of polyethyleneglycol-grafted liposomes. Biophys. J. 74, 1371–1379.

iener, M.C., White, S.H., 1992. Structure of a fluid dioleoylphosphatidylcholinebilayer determined by joint refinement of X-ray and neutron diffraction data III.Complete structure. Biophys. J. 61, 434–447.

Y

ysics of Lipids 155 (2008) 7–15 15

oodle, M.C., 1998. Controlling liposome blood clearance by surface-grafted poly-mers. Adv. Drug Deliv. Rev. 32, 139–152.

orcetor, D.L., Franks, N.P., 1976. Structural analysis of hydrated egg lecithin andcholesterol bilayers II. Neutron diffraction. J. Mol. Biol. 100, 359–378.

amamoto, T., Yokoyama, M., Opanasopit, P., Hayama, A., Kawano, K., Maitani, Y.,

2007. What are determining factors for stable drug incorporation into poly-meric micelle carriers? Consideration on physical and chemical characters ofthe micelle inner core. J. Control Release 123, 11–18.

oshida, A., Hashizaki, K., Yamauchi, H., Sakai, H., Yokoyama, S., Abe, M., 1999. Effectof lipid with covalently attached poly(ethylene glycol) on the surface propertiesof liposomal bilayer membranes. Langmuir 15, 2333–2337.