Photophysical studies of zinc phthalocyanine and ...cancer.cytoluminator.com/cancer-photodynamic... · in liposomes were inferred on the basis of their fluorescence and triplet state

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Braz J Med Biol Res 37(2) 2004

Studies of phthalocyanines incorporated into liposomesBrazilian Journal of Medical and Biological Research (2004) 37: 273-284ISSN 0100-879X

Photophysical studies of zincphthalocyanine and chloroaluminumphthalocyanine incorporated intoliposomes in the presence of additives

Departamento de Química, Faculdade de Filosofia,Ciências e Letras de Ribeirão Preto, Universidade de São Paulo,Ribeirão Preto, SP, Brasil

S.M.T. Nunes,F.S. Sguilla and

A.C. Tedesco

Abstract

The photophysical properties of zinc phthalocyanine (ZnPC) andchloroaluminum phthalocyanine (AlPHCl) incorporated into lipo-somes of dimyristoyl phosphatidylcholine in the presence and absenceof additives such as cholesterol or cardiolipin were studied by time-resolved fluorescence, laser flash photolysis and steady-state tech-niques. The absorbance of the drugs changed linearly with drugconcentration, at least up to 5.0 µM in homogeneous and heteroge-neous media, indicating that aggregation did not occur in these mediawithin this concentration range. The incorporation of the drugs intoliposomes increases the dimerization constant by one order of magni-tude (for ZnPC, 3.6 x 104 to 1.0 x 105 M-1 and for AlPHCl, 3.7 x 104

to 1.5 x 105 M-1), but this feature dose does not rule out the use of thiscarrier, since the incorporation of these hydrophobic drugs into lipo-somes permits their systemic administration. Probe location in bio-logical membranes and predominant positions of the phthalocyaninesin liposomes were inferred on the basis of their fluorescence andtriplet state properties. Both phthalocyanines are preferentially dis-tributed in the internal regions of the liposome bilayer. The additivesaffect the distribution of these drugs within the liposomes, a fact thatcontrols their delivery when both are used in a biological medium,retarding their release. The addition of the additives to the liposomesincreases the internalization of phthalocyanines. The interaction of thedrugs with a plasma protein, bovine serum albumin, was examinedquantitatively by the fluorescence technique. The results show thatwhen the drugs were incorporated into small unilamellar liposomes,the association with albumin was enhanced when compared withorganic media, a fact that should increase the selectivity of tumortargeting by these phthalocyanines (for ZnPC, 0.71 x 106 to 1.30 x 107

M-1 and for AlPHCl, 4.86 x 107 to 3.10 x 108 M-1).

CorrespondenceA.C. Tedesco

Departamento de Química

FFCLRP, USP

Av. Bandeirantes, 3900

14040-901 Ribeirão Preto, SP

Brasil

Fax: +55-16-633-8151

E-mail: [email protected]

Research supported by FAPESP.

Received January 9, 2003

Accepted September 9, 2003

Key words• Zinc phthalocyanine• Chloroaluminum

phthalocyanine• Bovine serum albumin• Liposomes• Cholesterol• Cardiolipin• Dimyristoyl

phosphatidylcholine

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Introduction

Photodynamic therapy (PDT) of canceris a noninvasive treatment of small and su-perficial tumors that is currently being usedin a number of countries (1). The therapy isbased on the systemic administration of atumor-localizing photosensitizer followed byillumination with light of appropriate wave-length. The resulting photodynamic reac-tions give rise to singlet oxygen (1O2) and toother active oxygen species that lead to tu-mor destruction (1). The efficiency of PDTdepends on the development of new drugsand the ability of these drugs to accumulateselectively in tumor tissues in comparisonwith normal tissues. Several new classes ofphotosensitizers for PDT have reached thestage of clinical trials during the past fewyears (2). Among the more promising sec-ond-generation photosensitizers are phthalo-cyanines (Figure 1). There has been consid-erable interest in phthalocyanines for use inPDT, mainly because of their high absorb-ance coefficient (at 650-680 nm), with opti-mal tissue penetration by light (3). Thephotophysical properties of phthalocyaninesare strongly dependent on the central metalion. Among the metal phthalocyanines, Zn(II) and Al (III) complexes (zinc phthalocya-nine, ZnPC, and chloroaluminum phthalo-cyanine, AlPHCl) present the most favor-able photophysical properties for applica-

tion in PDT (4), i.e., relatively long-livedexcited singlet states (ca. 3-8 ns) and long-lived triplet states that are produced in highquantum yields (5).

Unfortunately, these phthalocyanines(ZnPC and AlPHCl) are insoluble in water orbiologically compatible solvents. Thus, theymust be administered in vivo by means ofdelivery systems (6). The transport of por-phyrins in the bloodstream via liposomeshas been shown to provide a larger and moreselective accumulation of the drugs in neo-plastic tissues (7), a fact that is a basic re-quirement for PDT. Their association withserum proteins can also enhance the prefer-ential uptake of hydrophobic photosensitiz-ers by tumor tissues, since serum albumin isone of the key components in blood thatinfluences drug distribution. Since the in-trinsic fluorescence of proteins is usuallyquenched upon the binding of tetrapyrroliccompounds (8), this spectroscopic behaviorprovides a means to study the interactionbetween these drugs and bovine serum albu-min (BSA), permitting the determination ofthe binding constant, Kb, and of the bindingstoichiometry of the complex formed (9).

Nevertheless, phthalocyanines are proneto self-aggregation. The deviation from lin-ear Beer-Lambert behavior for these drugsin solution caused by dimer formation isnormally more pronounced in water than inorganic solvents. Dimers are reported to beinactive or much more inefficient than mono-mers as photosensitizers (10). Unfortunately,phthalocyanines have been reported to dis-play a strong tendency to form dimers inwater as a result of the large hydrophobicskeleton avoiding contact with the aqueousmedium (11), leading to the dimerizationprocess and affecting their photophysicaland photosensitizing properties and photo-dynamic action (12). Aggregation was foundto reduce the sensitizing ability of phthalo-cyanines in electron transfer reactions and inO2 (1∆g) photoproduction (13). Since incor-poration of photosensitizers into liposomes

R

RR

R

N N

N N

N N N

N

M

Figure 1. Typical phthalocyaninestructure where M is the centralmetal ion (Al, Zn, Co, Ga, Si) andR represents a multitude of pos-sible ring substituents includingSO3H, F, COOH, etc.

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Studies of phthalocyanines incorporated into liposomes

(used as a drug delivery system) results in ahigh local concentration, information aboutthe behavior of these drugs in these media isimportant for the understanding of their ac-tion in biological media.

In the present investigation we studiedthe aggregation parameters of ZnPC andAlPHCl in order to accurately determine thephotophysical properties of these drugs. Thepredominant positions of these phthalocya-nines in dimyristoyl phosphatidylcholine(DMPC) liposomes are analyzed, as well asthe triplet state lifetime and the binding ofthese exogenous drugs with BSA.

Material and Methods

Reagents

ZnPC, AlPHCl, methylviologen (MV2+),9,10-anthraquinone-2-sulfonate (AQS-, so-dium salt), and 9,10-anthraquinone-2,6-disulfonate (AQDS2-, disodium salt) werepurchased from Aldrich Chemical CompanyInc. (Milwaukee, WI, USA) and used with-out further purification. DMPC, cardiolipin,cholesterol, and phosphate-buffered saline(PBS) were obtained from Sigma (St. Louis,MO, USA). All other chemicals were com-mercially available reagents of at least ana-lytical grade.

All experiments were carried out with aphthalocyanine concentration of 5.0 µM.Stock solutions of ZnPC and AlPHCl (1.0mM) were routinely prepared in ethanol andDMSO, respectively, and stored in the darkat 4ºC (ZnPC was dissolved in 0.1% pyridinein ethanol, v/v). The concentrations of thephthalocyanines were estimated spectropho-tometrically using ε678 = 2.93 x 105 M-1 cm-1

for AlPHCl in DMSO and liposomes andε673 = 2.41 x 105 M-1 cm-1 for ZnPC inethanol and liposomes. Stock solutions ofMV2+ (0.3 M, ε = 20,500 M-1 cm-1 at 257 nm)and AQDS2- (17 mM, ε = 6760 M-1 cm-1 at328 nm) in water were stored in the dark at-15ºC. Stock solutions of AQS- (17 mM,

ε = 5450 M-1 cm-1 at 330 nm) in water werestored in the dark at room temperature. Thevalues of the extinction coefficients wereobtained from the literature (14).

Liposome preparation

Small unilamellar liposomes of 0.7 mMDMPC were prepared on the basis of theinjection method of Kremer et al. (15). Typi-cally, 0.380 ml of an ethanolic solution,which was 9.21 mM in DMPC and 66 µM inZnPC or AlPHCl, was injected with a sy-ringe into 5 ml PBS, pH 7.4. The injectionwas performed at 46ºC under magnetic stir-ring and at a rate of 1 µl/s. Mixed liposomeswere prepared by adding ethanolic solutionsof cholesterol (50% DMPC, w/w) or cardio-lipin (30% DMPC, w/w) to the ethanolicsolution.

Steady-state spectroscopic measurements

Absorbance spectra were recorded witha Hitachi U-3000 spectrophotometer and flu-orescence spectra were recorded with a Hi-tachi F-4500 spectrofluorometer. ZnPC andAlPHCl solutions were excited at 600 nmwavelength and their fluorescence emissionwas recorded in the 600-800 nm range. Allmeasurements were made at 25 ± 2ºC. Band-widths were fixed at 5 nm for excitation andemission.

Determination of the dimerizationequilibrium constants

In order to investigate the aggregationstate of a phthalocyanine solution and todetermine the equilibrium dimerization con-stant (KD), the absorbance spectra of a seriesof phthalocyanine solutions were monitoredwith increasing phthalocyanine concentra-tions. KD for ZnPC and AlPHCl were studiedin the following medium: a) organic solution(DMSO for AlPHCl and pyridine for ZnPC),and b) DMPC liposomal medium for both.

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S.M.T. Nunes et al.

The KD for the equilibrium 2 M ↔ D isdefined as

KD = [D]/[M]2 (Eq. 1)

where [M] and [D] are the molar concentra-tions of the monomer and the dimer, respec-tively. The total concentration of the drugbeing equal to

C = [M] + 2[D] (Eq. 2)

straightforward calculations lead to

(Eq. 3)

(Eq. 4)

The absorbance of a solution containingboth the monomer and the dimer is given by

Abs = (εM[M] + εD[D])l (Eq. 5)

where εM and εD are the extinction coeffi-cients of the monomer and the dimer, re-spectively, and l the optical path length.

Rearrangement of the above equationsleads to

(Eq. 6)

where C is the total concentration of thephotosensitizer (monomer and dimer), εM

and εD are the extinction coefficients of themonomer and dimer, respectively, and l isthe optical path length. KD was determinedby means of Equation 6 (16).

The plot of absorbance as a function oftotal drug concentration (C) according toEquation 6 was used to evaluate the pro-posed scheme. The values of KD, εM and εD

were computed by nonlinear regression basedon the Lavenberg-Marquardt algorithm (16)using the Igor software. An estimated valueof εM was obtained from measurements inorganic solutions within the concentrationrange in which the Beer-Lambert law holdsfor each drug. The value of εM, determinedindependently, was fixed in Equation 6 and

the absorbance data were fitted (allowed tovary) to obtain εD and KD by a nonlinear leastsquare procedure (16).

Fluorescence quenching studies

The quenching of the fluorescence emis-sion by ZnPC and AlPHCl incorporated intounilamellar liposomes of DMPC in the pres-ence and absence of additives (cholesterol orcardiolipin) was studied using MV2+, AQDS2-

and AQS- as quenchers. Phthalocyanine so-lutions (5.0 µM) were excited at 600 nm andtheir fluorescence emission was recorded inthe range of 640-750 nm. The fluorescencequenching data obtained were analyzed byStern-Volmer formalism (17): F0/F = 1 + KSV

[Q], which relates the decrease in fluorescenceintensity (F0/F) to quencher concentration [Q];KSV is the Stern-Volmer quenching constant.

Interaction of ZnPC and AlPHCl with bovineserum albumin in organic and liposomalmedium

The interaction of the phthalocyaninesZnPC and AlPHCl with BSA was studiedspectrofluorometrically at 25ºC by the“double logarithmic plot” (18). For eachdrug tested, two sets of experiments wereperformed. In the first, solutions of phthalo-cyanines at low concentration in PBS weretitrated with increasing BSA concentrationsin order to determine maximum quenching,F∞. The relative fluorescence intensities ofBSA saturated with phthalocyanines, F∞,were extrapolated from the experimental databy plotting 1/(F0 - F) against 1/[P], where F isthe measured fluorescence of a solution con-taining the protein and the phthalocyanine,F0 is the fluorescence of a solution of proteinalone, and [P] is the phthalocyanine concen-tration. In the second, a protein system heldat constant concentration (absorbance at anexcitation wavelength of no more than 0.05)was titrated with small aliquots of concen-trated phthalocyanine solutions. In both ex-

277



periments, the total dilution was kept below2%. The fluorescence of BSA was excited at280 nm and recorded between 300 and 450nm, with excitation and emission bandpassesof 5 nm. Fluorescence data were treatedaccording to the methods of Lehrer andFashman (19) and Chipman et al. (20), withlog [(F0 - F)/(F - F∞)] being plotted againstlog [P]. The slope of the plot obtained givesN, the number of binding sites, and the valueof log [P] at log [(F0 - F)/(F - F∞)] = 0 is equalto the logarithm of the dissociation constantKdiss. The reciprocal of Kdiss is the bindingconstant Kb.

Triplet state measurements

All measurements were made using ZnPCand AlPHCl (5.0 µM) in organic medium(pyridine and DMSO, respectively), and li-posomal medium in the presence and ab-sence of the additives cholesterol and car-diolipin. Triplet state was investigated usinga laser flash photolysis apparatus which al-lows the simultaneous capture of the tran-sient absorbance spectrum and of the tran-sient kinetics at a single wavelength. Thesystem uses an Nd-YAG laser as the excita-tion source (SURELITE I-10 of Continuum,Santa Barbara, CA, USA), operating at 355nm, ca. 50 mJ/pulse to give 10-ns pulses. Thedecay kinetics of the triplet state of ZnPC andAlPHCl was recorded at 480 and 490 nm,respectively - the triplet state absorbancemaximum. Ten laser shots were averaged foreach measurement. Decay profiles were fit-ted by an interactive nonlinear least-squaresroutine method using a data analysis soft-ware package from Edinburgh Instruments(Edinburgh, UK) on a personal computer.

Results and Discussion

Determination of the dimerizationequilibrium constants

Like most phthalocyanines, ZnPC and

AlPHCl are prone to aggregation. In aque-ous solution, even the soluble phthalocya-nines (tetrasulfonated) display a typicallyaggregated spectrum even at low concentra-tions. Previous studies with another solublephotosensitizer (bacteriochlorin a) in PBSrevealed that this hydrophilic photosensi-tizer is strongly aggregated in its dimericform with a KD estimated to be 106 M-1 (21).In the present study, it was not possible todetermine the KD of the drugs ZnPC andAlPHCl in aqueous solutions due to the lackof solubility in this medium. The solubiliza-tion of the drugs within DMPC liposomesinduced dye monomerization and permittedus to evaluate the KD in DMPC medium.

The trend of the phthalocyanines ZnPCand AlPHCl to form aggregates and the equi-librium KD were analyzed and evaluated us-ing absorbance spectroscopic analysis of theQ band at 673 nm. It was observed that, atleast up to the concentration of 5.0 µM, theincrease of absorbance with phthalocyanineconcentration was linear in organic and lipo-somal medium (Figure 2). This behaviorimplies that, at least within this concentra-tion range (0 to ca. 5.0 µM), these phthalo-cyanines are in the monomeric state, in agree-ment with the Beer-Lambert law. In contrast,the behavior observed in liposomal medium

Figure 2. Variation in the absorbance (673 nm) as a function of zinc phthalocya-nine (ZnPC) concentration in DMPC liposomes and in organic solution (pyridine)in the inset.

Abs

orba

nce

1.2

1.0

0.8

0.6

0.4

0.2

2

1

Abs

orba

nce

5 10

5 10 15 x 10-6

[ZnPC] M15 x 10-6

[ZnPC] M

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S.M.T. Nunes et al.

in the concentration range above 5.0 µM (5to 15 µM) indicated that at higher concentra-tions a progressive change in the systemoccurred, indicating a typical aggregationprocess of the drugs. An example of theprocedure for processing the data obtained isillustrated in Figure 2 for ZnPC. The pointsshow the experimental results and were ad-justed by nonlinear regression in the fullconcentration range studied (0 to 15.0 µM).

The KD of ZnPC and AlPHCl in organicand liposomal medium obtained by nonlin-ear regression in the full concentration rangestudied (0 to 15.0 µM) are shown in Table 1.

KD for photosensitizers such as porphy-rins and phthalocyanines range from 104 to107 M-1 and depend on the solvent and tem-perature (22). The results obtained confirmedthat KD strongly depend on the medium,since in liposome medium aggregation tendsto be higher than in organic medium. Inaddition, the results indicate that the com-plexed metal ions zinc and aluminum do notinterfere with the tendency to aggregate (samestability - same order of magnitude of KDvalues for both drugs, i.e., ZnPC and AlPHCl,in liposomal medium). We observed that theincorporation of the drugs into liposomesincreased the KD by one order of magnitude.These results are in agreement with the be-havior expected for phthalocyanines incor-porated into liposomes. However, the in-crease of one order of magnitude in the KD of

these phthalocyanines after their incorpora-tion into liposomes is not a negative factor.A KD with an order of magnitude of 105 is nota high value for these types of drugs (18).The advantage of the incorporation of thesehydrophobic drugs into drug delivery sys-tems such as liposomes is the increase intheir solubility, permitting their systemicadministration.

Fluorescence quenching studies

The locations of ZnPC and AlPHCl inDMPC liposomes mixed with cholesterol(50% DMPC, w/w) or cardiolipin (30%DMPC, w/w) were evaluated and comparedwith those obtained for the drugs incorpo-rated into DMPC liposomes in the absenceof these additives to assess how the distribu-tion of the phthalocyanines and the interac-tion mode of the quenchers with them areaffected by the physicochemical propertiesof the liposomes in the presence of the addi-tives.

The quenching of ZnPC and AlPHCl bythe quenchers was monitored by measuringthe decrease in the emission intensity withexcitation at 600 nm as a function of quencherconcentration. Stern-Volmer plots were con-structed from the relative integrated fluores-cence intensity (640-750 nm). When ZnPCand AlPHCl were incorporated into DMPCliposomes in the presence and absence ofadditives at 25ºC, MV2+, AQS-, and AQDS2-

yielded linear fluorescence quenching plots.Table 2 shows the quenching parametersobtained in these studies.

The quenchers used in the present inves-tigation did not penetrate the lipid bilayerand the quenching occurred by an electrontransfer process (14). According to Ford andTollin (14), in liposomal systems MV2+ isoriented co-planar to the liposomal surfacein order to maximize the contact between thepolar and nonpolar parts of the quencher andthe liposome. On the other hand, AQS- inter-acts perpendicularly to the bilayer surface,

Table 1. Dimerization constants of ZnPC andAlPHCl at 25ºC in organic solution and DMPCliposomes.

Medium KD (M-1)

ZnPC AlPHCl

Organic 3.6 x 104 3.7 x 104

DMPC 1.0 x 105 1.5 x 105

AlPHCl = chloroaluminum phthalocyanine; DMPC= dimyristoyl phosphatidylcholine; DMSO = di-methyl sulfoxide; KD = dimerization constant; or-ganic medium = pyridine for ZnPC and DMSO forAlPHCl; ZnPC = zinc phthalocyanine.

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with the sulfonate group probably orientedin an opposite direction to the liposome.Thus, in liposomal systems MV2+ interactsonly with the drugs located in the outer partof the lipid bilayer, while AQS- and AQDS2-

interact with the drugs in both the inner andouter regions of the lipid bilayer. However,neither of these quenchers is able to diffuseinto the lipid bilayer (quenching by an elec-tron transfer process). Thus, the KSV valuesobtained for the quenching by MV2+ belongto the drug population more exposed to theexternal medium, while KSV values obtainedfor anthraquinone-type quenchers representthe weighted average of the KSV’s of bothclasses of fluorophores (inner and outer).

The lower KSV values for MV2+ com-pared with those for the anthraquinonequenchers (Table 2) in DMPC liposomesindicate that the drugs are incorporated intothe bilayer structure, in the inner part of theliposomal structure, which lowers the acces-sibility of MV2+ to these drugs. The resultsshowed that AQS- and AQDS2- interactedwith ZnPC and AlPHCl in both the inner andouter lipid bilayers by an electron transferprocess, although the efficiency of fluores-cence quenching of the phthalocyanines byAQDS2- was lower compared with AQS-, assuggested by the drop of the KSV valuesobtained for AQDS2- (Table 2). In this case,the KSV values of AQDS2- are related to themolecules of ZnPC and AlPHCl mainly lo-cated in the outermost regions of the lipidbilayer, and the efficiency of quenching washigher for AlPHCl than for ZnPC.

Different locations of the phthalocyaninesin the lipid bilayer can be deduced from thechanges in these KSV values under the sameexperimental conditions. The AQS- quencherwas more efficient for both probes. How-ever, AQDS2- showed a higher quenchingefficiency for AlPHCl than for ZnPC. Theseresults indicate that AlPHCl was locatedcloser to the bilayer/water interface thanZnPC. The KSV values for these compoundsare in agreement with the values found for

other photosensitizers in other drug deliverysystems (23).

In the liposomes containing additives,the fluorescence quenching of the phthalo-cyanines by MV2+ was negligible. The effi-ciency of quenching by AQDS2- in liposo-mal medium with additives was also lowerthan the efficiency of quenching with AQS-

(Table 2). The presence of additives de-creased the KSV for both drugs studied.

Clusters of cholesterol appear in vesiclesat mol fractions above 0.67. Mol fractionshigher than 0.5 of cholesterol are requiredfor cluster formation with phosphatidylcho-line (24-26). When the cholesterol contentexceeds the saturation limit in phospholipidmembranes, crystalline cholesterol mono-hydrate forms (27,28). In general, for cho-line phospholipids the saturation limit is≈50% cholesterol, although a higher solubil-ity (65%) has been reported by Huang et al.(29) and a solubility higher than 60% wasobtained by Guo and Hamilton (27). In thisway, the cholesterol concentration used inthe experiments (50% of total lipid, w/w)does not exceed the saturation limit in phos-phatidylcholine membranes and does notform crystalline cholesterol monohydrate.

In a membrane bilayer, cholesterol in-serts normal to the plane of the bilayer, withits hydroxyl group in close vicinity to thephospholipid polar heads and its alkyl sidechain extending towards the bilayer center.

Table 2. Fluorescence quenching parameters of ZnPC and AlPHCl (5.0 µM) in DMPCliposomes in the presence and absence of additives at 25ºC.

KSV (M-1)

5 µM ZnPC 5 µM AlPHCl

MV2+ AQS- AQDS2- MV2+ AQS- AQDS2-

Without addition - 269.8 195 2.0 239.0 28350% cholesterol - 131.9 121 - 93.0 17230% cardiolipin - 45.1 59 - 38.1 56

AlPHCl = chloroaluminum phthalocyanine; AQDS2- = 9,10-anthraquinone-2,6-disulfonate; AQS- = 9,10-anthraquinone-2-sulfonate; KSV = Stern-Volmer quenchingconstant; MV2+ = methylviologen; ZnPC = zinc phthalocyanine.

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S.M.T. Nunes et al.

Cholesterol is intercalated in the membraneparallel to the phospholipid hydrocarbonchains, and the phospholipid carbons at po-sitions 2-10 have been estimated to lie inclose proximity to the sterol tetracyclic ringstructure (30,31). Although cholesterol canenhance lateral separation of lipids in bilay-ers consisting of a single lipid species, re-sulting in a higher water permeability inphosphatidylcholine bilayers and creating acavity that was probably occupied by watermolecules (32-34), the phthalocyanines didnot become more accessible to the quencherslocated in the external medium, as can beseen in Table 2 (lower KSV values in thepresence of cholesterol). Thus, the incorpo-ration of cholesterol into liposomes caused achange in the distribution of the moleculesof ZnPC and AlPHCl from the outer to theinner region of the lipid bilayer. This choles-terol-induced redistribution of ZnPC andAlPHCl may be correlated with a change inthe distribution of cholesterol itself (32-34).Above 30% DMPC (w/w), cholesterol pref-erentially dissolves in the inner lipid bilayer.When the liposomes contain equimolaramounts of phosphatidylcholine and choles-terol, the inner bilayer contains about 65%DMPC of cholesterol (w/w) and the externalregion of the bilayer contains about 40%DMPC (w/w) (32-34). If the drugs had beenlocated near the bilayer/water interface, theywould have become more accessible to thewater-soluble quenchers (outer location) inliposomes containing cholesterol than in li-posomes without it (14). Consequently, theseresults prove the inner location of the drugsin the lipid bilayer with cholesterol, and notavailable to the quenchers, since a drop inquenching efficiency was noted after theaddition of the additive.

Cardiolipin is found in the inner mem-brane of mitochondria (35). The results withcardiolipin demonstrate that the insertion ofthis lipid into the bilayer shows the samebehavior as observed for cholesterol. Thissuggests a preferential distribution of ZnPC

and AlPHCl in cardiolipin-rich domains ofthe inner part of the lipid bilayer and itsexclusion from DMPC-containing domains.

The results obtained here show that theadditives cholesterol and cardiolipin pro-mote a change of the drug molecules to-wards the inner part of the lipid bilayer,according to the lower KSV values obtainedafter the incorporation of the additives intothe liposomes (Table 2), but ZnPC continuesto be more internalized than AlPHCl (lowerKSV values obtained for ZnPC when com-pared with those obtained for AlPHCl for thequenching by AQDS2-) (Table 2). The dis-placement of the drug molecules to the innerpart of the lipid bilayer is probably related totheir tendency to prefer hydrophobic envi-ronments.

Interaction of ZnPC and AlPHCl with bovineserum albumin in organic and liposomalmedium

The interaction of serum albumin withphthalocyanines quenches the fluorescenceof this protein, providing a means to assessbinding quantitatively. The interaction be-tween ZnPC and AlPHCl with BSA can befollowed by fluorescence spectroscopy, asillustrated in Figure 3. ZnPC and AlPHClquench the fluorescence of BSA in propor-tion to the amount of drug added. The fluo-rescence intensity of BSA changes with theconcentration of phthalocyanine in a wayconsistent with a reversible formation of acomplex between BSA and the drugs.

The inset in Figure 4 provides a linearplot of F0/∆F versus 1/[P] for the binding ofZnPC-BSA, used for the determination ofthe fluorescence intensity of BSA saturatedwith the phthalocyanines (F∞) ([P] = phthalo-cyanine concentration). As described in theexperimental section, Kdiss was calculatedfrom the slope and interception of the plot oflog [(F0 - F) (F - F∞)] versus log [P](an example of such double-logarithmic plotobtained for ZnPC is given in Figure 4). In

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addition, the slope of the plot gives the num-ber of the sites of the protein able to bindZnPC and AlPHCl molecules.

The association constants obtained forthe phthalocyanines studied are presented inTable 3, together with the binding stoichi-ometry of the complex formed.

Previous studies have indicated thatphthalocyanines without SO3

- groups adja-cent to iso-indole rings exhibit a high affinitybinding site constant of 1-4 x 104 M-1 (36,37).Other studies (38) have reported mesoporphy-rin IX and magnesium mesoporphyrin parti-tioning between liposome and serum albuminwith Kb of 2.5 x 107 M-1 and 1.7 x 107 M-1.Our results agree with these data (Table 3).

The present results show that when phthal-ocyanines are incorporated into DMPC lipo-somes, their association with BSA is in-creased in comparison with organic medi-um. Calorimetric titration studies indicatedthe binding of empty liposomes to the albu-min species. The albumin molecules fromdifferent species adsorb strongly to phos-phatidylcholine liposomes due mainly to theaction of hydrophobic dehydration forcesand entropy gain (39). In this way, the lipo-somes themselves increase the Kb to BSA.The encapsulation of the drug in the lipo-somes increases the already high associationof the liposome/drug complex with BSA.The presence of a strong binding site andseveral weaker sites was observed for ZnPCand AlPHCl incorporated into liposomes.The present results indicate that the associa-tion of the drugs with lipid-based deliverysystems affects their interaction with serumalbumin. Liposomes of DMPC increase thebinding of ZnPC and AlPHCl to BSA, whichshould increase the selectivity of tumor tar-geting by these phthalocyanines, since BSAcan deliver the bound drugs to the vascularstroma of the tumors (7).

Triplet state measurements

The triplet state properties of ZnPC and

Figure 3. Effect of zinc phthalocyanine concentration on the fluorescence spec-trum of bovine serum albumin. From the top, in order of decreasing fluores-cence intensity with excitation at 290 nm, ZnPC concentration was 0.00, 0.13,0.38, 0.64, 0.89, and 1.14 µM.

Figure 4. Double log plot of the quenching of protein fluorescence by zincphthalocyanine in organic medium. Inset, Double reciprocal plot in organicmedium (F = measured fluorescence of a solution containing the protein and thephthalocyanine, F0 is the fluorescence of a solution of protein alone, F∞ is thefluorescence intensity of BSA saturated with ZnPC, and [PC] is the ZnPCconcentration).

AlPHCl are similar. The transient spectra ofZnPC and AlPHCl in liposomal medium (inthe presence and absence of additives) re-semble those obtained for a homogeneoussolution (organic medium). Figure 5 illus-trates the transient spectrum of ZnPC (5.0µM) incorporated into DMPC liposomes inthe presence and absence of cholesterol and

Fluo

resc

ence

int

ensi

ty

2500

2000

1500

1000

500

0300 350 400 450

Wavelength (nm)

Log

[(F0

- F)/(

F - F

∞)]

0.2

-1.00

1/(F

0 -

F)

2 4 61/[ZnPC] µM-1

-5.4

Log [ZnPC]

2

4

6x10-3

-5.6-5.8-6.0-6.2-6.4-6.6-6.8

-0.8

-0.6

-0.4

-0.2

0.0

0.4

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S.M.T. Nunes et al.

cardiolipin, and also serves to illustrate thetriplet-triplet absorbance centered at 480 nm,together with the Soret and Q band groundstate bleaches.

Triplet lifetimes (τT) were calculated fromkinetic analysis of the transient decays, car-ried out using the fitting program of theapparatus itself and are shown in Table 4.

The triplet state reacts with forms ofmolecular oxygen (O2) by an energy transferprocess leading to singlet oxygen that is thekey agent in cell damage in PDT. Transientabsorbance spectra for the drugs obtained bylaser flash photolysis showed the presenceof transient species, which decayed mono-exponentially with the characteristic life-times shown in Table 4. As a consequence ofthe incorporation, triplet lifetimes are in-creased in liposomes in comparison withhomogeneous solution. The increase in thetriplet lifetimes is attributed to the ability ofthe bulk aqueous phase to interact with thesensitizer at the binding site. Therefore, weconclude that the increase in the triplet statelifetimes of ZnPC and AlPHCl as a functionof the addition of cholesterol and cardiolipinto liposomes results from a progressive re-duction in the exposure of the sensitizer tothe bulk aqueous phase, confirming the re-sults obtained in the quenching studies. Un-der identical conditions, the maximum trip-let lifetimes of ZnPC are shorter than thosefor AlPHCl, demonstrating that AlPHCl ismore internalized than ZnPC.

The behavior observed in these studiesimplies that, at least up to ca. 5.0 µM, bothphthalocyanines studied (ZnPC and AlPHCl)are in the monomeric state in both media(liposomal and organic), in agreement withthe Beer-Lambert law. The increase of oneorder of magnitude in the dimerization con-stant of the phthalocyanines after their incor-poration into liposomes does not rule out theuse of this carrier, considering the increasedsolubility of the drugs after incorporationinto the drug delivery system, allowing theirsystemic administration, and the increased

Table 3. Equilibrium constants of ZnPC and AlPHCl binding to BSA.

Medium ZnPC AlPHCl

N Kb (M-1) N Kb (M-1)

Organic 1.05 0.71 x 106 1.37 1.30 x 107

DMPC liposomes 1.07 4.86 x 107 1.11 3.10 x 108

AlPHCl = chloroaluminum phthalocyanine; DMPC = dimyristoyl phospha-tidylcholine; Kb = binding constant; N = number of binding sites; organicmedium = pyridine for ZnPC and DMSO for AlPHCl; ZnPC = zinc phthalo-cyanine.

Table 4. Triplet lifetimes (τT) of ZnPC and AlPHCl in organic and liposomalmedium in the presence of O2.

Medium ZnPC AlPHCl

Organic medium 0.30 0.80DMPC 0.84 1.28DMPC with cholesterol 1.35 4.62DMPC with cardiolipin 1.28 2.50

AlPHCl = chloroaluminum phthalocyanine; DMPC = dimyristoyl phospha-tidylcholine; organic medium = pyridine for ZnPC and DMSO for AlPHCl;ZnPC = zinc phthalocyanine.

Abs

orba

nce

0.0

Abs

orba

nce

400 600 800Wavelength (nm)

Wavelength (nm)

0.5

1.0

600500400

0.1

0.0

-0.1

-0.2

-0.3

Figure 5. Transient absorption spectra of zinc phthalocyanine (ZnPC) in organic(pyridine) and liposomal medium, in the presence and absence of the additives- liposome with cholesterol, and liposome with cardiolipin - after excitation at670 nm (50 mJ per pulse), showing the absorption of the triplet state centeredat λ = 480 nm and the bleach of the ground state centered at around 350 and680 nm. Inset, Absorption spectra of ZnPC in organic medium.

Organic medium

Liposomal medium

Liposome with cholesterol

Liposome with cardiolipin

700

283



binding with BSA which should increase theselectivity of tumor targeting. As a conse-quence of the incorporation, triplet lifetimesare increased in liposomes compared withhomogeneous solution. Thus, the results ob-tained clearly indicate that the incorporationof these drugs into DMPC liposomes maxi-mizes their photodynamic action, allowingan effective activity of the photosensitizer-liposome complex against cancer.

The results of the present study also indi-cate that the distribution of ZnPC and AlPHClin unilamellar DMPC liposomes is heteroge-neous, with some predominant positions (Fig-ure 6). In the presence of additives there is ashift of the drug molecules towards the innerpart of the lipid bilayer. The results obtainedindicated that AlPHCl and ZnPC are prefer-entially distributed in the inner part of thephospholipid bilayer of DMPC liposomes inthe presence and absence of cholesterol (50%DMPC, w/w) and cardiolipin (30% DMPC,w/w). The increased internalization of thedrugs in liposomes containing additives is apositive aspect, since in this way the drugscan be protected while they circulate in thebloodstream after systemic injection.

Silicon (IV) phthalocyanine, the firstphthalocyanine approved by the FDA forclinical trials, has the highest potential ofthis class of drugs for PDT (40). However,

silicon (IV) phthalocyanine needs to be usedin a Cremophor emulsion, a fact that is anobstacle to the generalized use of this drug.Cremophor has been used clinically for thedelivery of hydrophobic drugs. The possibil-ity of the use of hydrophobic phthalocya-nines such as ZnPC and AlPHCl encapsu-lated in liposomes that act as a drug deliverysystem, maintaining all the basic photophysi-cal and photochemical properties of the sen-sitizers, is an important result, since the lipo-somal vehicle is more economically viablethan Cremophor. Thus, our results confirmthe hypothesis that the liposomal mediumrepresents an excellent vehicle for the ad-ministration of these drugs in biological sys-tems, making liposomal ZnPC and AlPHClpromising candidates for PDT from the pho-tophysical point of view.

Aqueousphase

Phospholipid Cholesterol

ZnPC AIPHCI Cardiolipin

Figure 6. Preferential locationsof the photosensitizers ZnPCand AlPHCl in mixed liposomesof DMPC.

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