[CANCER RESEARCH 43, 5826-5830, December 1983]

Photosensitization of Liposomal Membranes by HematoporphyrinDerivative1

Greesh C. Goyal, Aleksander Blum, and Leonard I. Grossweiner2

Biophysics Laboratory, Physics Department, Illinois Institute of Technology, Chicago, Illinois 60616

ABSTRACT

The putative tumor-localizing and -photosensitizing fraction of

hematoporphyrin derivative, the fastest migrating fraction ofhematoporphynn derivative separated by polyacrylamide gel filtration (HPD-A), photosensitized lipid peroxidation and mem

brane lysis in egg phosphatidylcholine liposomes. The rate ofmembrane damage was approximately 4-fold faster in oxygen

compared to anoxia, with evidence for the involvement of singletoxygen. The diffusion of HPD-A into small liposomes led to a

shift of the Soret band from 363 nm in buffer to 398 nmaccompanied by 4-fold enhancement of the fluorescence. Thepresence of human serum albumin retarded the diffusion of HPD-

A into small liposomes, which is attributed to partial complexingof the HPD-A. A different effect of serum albumin was the

protection of large liposomes from photosensitized lysis by incorporated HPD-A. This protection is attributed to scavenging

of singlet oxygen, as evidenced by oxidation of tryptophan in theprotein.

INTRODUCTION

The tumoricidal action in PRT3 is effected by exposing tumor

tissue to red radiation from 620 to 640 nm at fluence levels from25 to 200 J/sq cm, after i.v. injection of HPD. HPD is obtainedby alkaline hydrolysis and neutralization of a mixture of porphyrinacetates (7), which is prepared in turn from HP by the methodof Lipson ef al. (16). The latter is referred to as crude HPD in thispaper, and HPD is reserved for the hydrolyzed material. Thetumor-localizing and -photosensitizing component of HPD is

contained in the fastest moving fraction separated by polyacrylamide gel filtration (8). This material was the photosensitizerused in this work and is designated as HPD-A. The action

mechanism in PRT has been attributed to the in situ generationof singlet molecular oxygen (102), induced by optical excitation

of HPD and its attack on tumor tissue membranes (7). Theobjective of this work was to investigate the photosensitizationmechanisms of HPD-A using EPC liposomes as a membrane

model. It is a continuation of prior studies on liposome photosensitization by HP (10) and the components of HPD (9). The resultsprovide new evidence for the involvement of 'O2 in liposome

membrane damage photosensitized by HPD-A and information

1Supported by NIH Grant GM 20117 and Department of Energy Contract AC02-76EV02217. This is Publication DOE/EV/02217-48.

2To whom requests for reprints should be addressed, at Department of Physics,

Illinois Institute of Technology. HT Center. Chicago, III. 60616.3The abbreviations used are: PRT, photoradiation therapy; HPD, hematopor

phyrin derivative; HP, hematoporphyrin IX; HPD-A, fastest migrating fraction of

hematoporphyrin derivative separated by polyacrylamide gel filtration; EPC, eggphosphatidylcholine; HSA, human serum albumin; LMV, large multilamellar vesicles;SUV, small unilamellar vesicles; MDA, malonyldialdehyde; DABCO, 1.4-diazabicy-

clo|2 2.2|octane: OER, oxygen enhancement ratio; DHE, dihematoporphyrin ether(bis-1 -[3-(1 -hydroxyethyl)deuteroporphyrin-8-yl] ethyl ether).

Received June 1, 1983; accepted September 6, 1983.

about the role of the microenvironment and the mediating effectsof HSA.

MATERIALS AND METHODS

Chemicals. Crude HPD was obtained from Porphyrin Products, Provo,Utah, and prepared from HP dihydrochloride (7) with equivalent photochemical results. HPD was prepared by dissolving the crude material in0.1 M NaOH, stirring for 60 min in the dark, neutralizing with 1 M HCI,and adjusting to pH 7.0 or 7.4 with 10 mw phosphate buffer. The HPD-A fraction was obtained by filtration of HPD on Bio-Gel P-10 columns(Bio-Rad Laboratories, Richmond, Calif.), using the phosphate buffer aseluent. The gel bed was 27 cm x 10 mm, inside diameter, and 1.32-mlfractions were collected at a flow rate of 9.6 ml/hr. The HPD-A fraction

was identified with a Soret band at 363 ±1 (S.D.) nm, and the absorbanceratio, A^AMO, was between 1.4 and 1.9. The approximate yield of HPD-

A was 50% of the starting material for a column loading of 2 mg, but itwas lower with smaller loadings (9). HSA (Fraction V, <0.005% fattyacid) and the other biochemicals were obtained from Sigma ChemicalCo. St. Louis, Mo., and used as received. HP dihydrochloride wasobtained from Porphyrin Products and Sigma with no observable difference in the results.

Liposomes. The liposomes were prepared from egg yolk L-a-phos-phatidylcholine (Sigma type VII-E) received in chloroform solution. In atypical preparation, a 15-mg aliquot was evaporated to dryness with anitrogen gas stream in a 10-mm test tube, the dry lipid was dispersed in

2 ml of the buffer by vortexing for 3 min, and the liposomes were allowedto swell for 2 hr at 3°under nitrogen. These large liposomes (LMV) were

not sized but are known from prior work to have a broad size distributionwith a high fraction from 0.6 to 2 /im (24). In the present work, negativelycharged LMV were prepared with a modified composition of 78 partsEPC, 16 parts dicetylphosphate, and 6 parts cholesterol, and the HPD-

A was incorporated by swelling in 10 mw phosphate buffer, pH 7.4,followed by either dialysis through Spectrapor-1 membrane tubing for 20hr or by gel filtration through a column of Sephadex G-50 fine (Pharmacia

Fine Chemicals, Inc., Piscataway, N. J.) to remove the external sensitizer.The small liposomes (SUV) were prepared from 100% EPC LMV bysonicating for 2 hr under nitrogen at 30°with a Heat Systems-Ultrasonics

Model W-10 sonicator utilizing a 2.4-mm titanium alloy tip. The residual

large liposomes and metal were removed by centrifugation at 50,000 xg for 20 hr at 5°.The SUV size was estimated by filtration on a 30-cm

x 14-mm column of Sephacryl S-1000 (Pharmacia). Elution with pH 7.0

phosphate buffer gave a single band with the ratio of elution volume (V.)to void volume (V0)equal to 1.80 at 3.0 ml/hr. The supplier's information

and published results (20) with columns of similar dimensions and operating conditions indicate that the SUV ranged from 10 to 90 nm indiameter, with the mean near 40 nm. The HPD-A fraction (As«= 7/cm)

was mixed 1:1 with a suspension of the SUV (2 mg of EPC per ml) whichled to rapid uptake. Evidence of complete uptake of the HPD-A wasobtained by passing the dyed SUV through a Sephadex G-50 fine columnand comparing the light-scattering absorbance of the eluted liposomesat 335 with the HPD-A absorption at 398 nm. Both bands coincided at

V0, and there was no evidence of porphyrin absorption remaining in thecolumn. Although exact measurements of the HPD-A concentrations

were not feasible because of aggregation and the heterogeneous composition, it was desirable to estimate the approximate molarity in certain

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Liposome Photosensitization by HPD

experiments. This was done by taking the molar extinction coefficient ofHPD-A at 363 nm in buffer and in the LMV as 7 x 104 liters/mol cm (2)and in the SUV at 398 nm as 1 x 105 liters/mol cm, based on an assumedmolecular weight of 600." The liposomes were stable for at least 5 dayswhen stored at 3°under nitrogen.

Irradiations. The irradiation arrangement was the same as describedpreviously (10). The light source was a 200-watt mercuryixenon arc(Hanovia 901-B1) focused on the front face of a polystyrene cuvet (1 x

1x4 cm) containing 3 ml of the liposome suspension and located on awater-cooled holder with temperature control to within ±0.1°.The sam

ples were bubbled with oxygen or nitrogen at a controlled flow rate (6ml/min) for 1 hr prior to irradiation and were bubbled continuously duringirradiation. The incident radiation was passed through 1 cm of water anda Corning C.S. No. 0-52 filter. The incident fluence rate as estimatedwith an Eppley thermopile and sharp-cut glass filters was 200 milliwatts/

sq cm from 360 to 750 nm. The assays for membrane damage werelysis, as measured by the absorbance decrease at 750 nm; the formationof conjugated diene hydroperoxides, as measured by the absorbanceratio, A235:A215(14), and the production of MDA. The last was determinedwith the thiobarbituric acid test following the preferred procedure ofAsakawa and Matsushita (3). Aliquots of 0.1 ml were diluted 60-fold prior

to measurement of the 530 nm absorption with this procedure, whicheliminated any interference from the HPD-A absorption. Control experi

ments with unirradiated liposomes were done for each set of measurements. The SUV (2 to 4 mg EPC per ml) were irradiated in phosphatebuffer (pH 7.0) at 25.0°, following the procedures used in prior work (9,

10). The conditions were changed with LMV (0.8 mg EPC per ml) tophosphate buffer (pH 7.4) plus 0.9% NaCI solution at 39.0°to approxi

mate physiological conditions. Check experiments were done with theSUV at different temperatures to determine the effects on membranedamage. The effects of sodium azide and DABCO were determined byadding the appropriate concentrations to the external buffer after incorporation of the HPD-A. These agents had no effect on the absorption

spectra of the dyed liposomes. All experiments were repeated at leasttwice to minimize the probability of an atypical liposome preparation.

HSA Effects. In certain experiments, HSA was added to the externalbuffer of the LMV suspensions. Spectral changes were measured whenthe HPD was first added to the liposomes and when first added to theHSA at various times after mixing, as reported below. The columnchromatography of HSA plus HPD-A mixtures was carried out on Seph-arose 6B (Pharmacia) with a filtration range of 104 to 106. The gel bed

was 27 x 1.5 cm, and the eluent was phosphate buffer (pH 7.4) at 22ml/hr flow rate. For the photosensitization studies on HSA, the sampleswere irradiated in the same apparatus as were the liposomes, and thedamage was assayed by the loss of tryptophan fluorescence excited at295 nm and measured at 350 nm using a Farrand manual spectrofluo-

rometer.

RESULTS

Photosensitization of Membrane Damage in LMV. A typicalset of lysis curves for LMV incorporating 1.8% (w/w) HPD-A is

shown in Chart 1. The OER, a useful measure of the acceleratingeffect of oxygen, has been defined for liposome systems as feo(N2):f8o(O2), where fso is the irradiation time required for 20%lysis (9). The prior work on photosensitized lysis of LMV by 1.0%HP showed that the lysis curves are reproducible to ±20%withfresh liposome preparations and to ±10%with the same preparation (10). The results for 2 independent runs with HPD-A ledto an OER of 4.3 ±0.1. The quenching of 1O2 by azide and

DABCO in aqueous and micellar systems has been investigatedin considerable detail, e.g., the work of Lindig and Rodgers (15)and references therein. The protection against LMV lysis by 0.1

4 Evaporation of HPD-A in unbuffered distilled water led to 6.6 x 104 liters/mol

cm at 365 nm.

I.Of

y 0.6

2.0

O 20 40 60 80 100 IZO 140

IRRADIATIONTIME, MIN

Chart 1. Photosensitized lysis of large liposomes incorporating 1.8% (w/w)HPD-A based on decrease of light-scattering absorbance [phosphate buffer plus0.9% NaCI solution (pH 7.4) at 39°).O, oxygen; •,nitrogen; A, oxygen plus 0.1 Mazide; A, oxygen plus 1 M DABCO; , photobleaching of HPD-A at 365 nm.

M azide and 1 M DABCO provides strong evidence for 102

involvement, although the participation of other active oxygenintermediates is not completely ruled out by these results. Liposome lysis was accompanied by photobleaching of the incorporated HPD-A, as evidenced by the absorbance decrease at 365

nm (Chart 1). This effect was equally rapid with nitrogen saturation (data not shown) and apparently involves a type I interaction of the HPD-A with the lipid, although membrane lysis was

much slower under nitrogen. Measurements of MDA formationshowed that A530increased to 0.35/cm after 25 min of irradiation,after which there was little change for an additional 55 min ofirradiation. In comparison, the fractional extent of lysis was onlyabout 8% after 25 min, followed by a rapid change to 60% lysisafter 80 min (Chart 1). A similar relationship between the rate ofMDA production and lysis was reported for the photosensitization of EPC liposomes by toluidine blue (1), strongly suggestingthat lipid peroxidation is the precursor of membrane lysis.

Photosensitization of Membrane Damage in SUV. The smalllight scattering by the SUV made it possible to measure accuratevalues of A235:A215and A398in situ. The data in Chart 2 showthat the initial increase of A235:A215was 6-fold faster with oxygen

as compared to nitrogen bubbling. The addition of 0.01 M azidereduced the rate under oxygen by 4.3-fold, after correction forthe oxygen control, which is further indicative of 1O2involvement

(data not shown). However, the relatively low peroxidation rateunder nitrogen (Chart 2, Curve B) was faster than the nitrogencontrol (Chart 2, Curve D), suggesting a secondary, type Imechanism was operative. As in the case of the LMV, there wasrapid photobleaching of the HPD-A (Chart 2, dashed line), which

was equally rapid under oxygen and nitrogen. Table 1 summarizes the results obtained with a different SUV sample containing0.3% HPD-A and irradiated at different temperatures. There was

very little change in the peroxidation parameters A235:A2i5andA53o(MDA test) between 21°and 50°,although A398(HPD-A

absorption) decreased more rapidly at the higher temperatures.Spectral Properties of HPD-A in Liposomes. Chart 3 shows

that HPD and HPD-A have similar absorptions in buffer, withpossibly a smaller contribution from the 390 nm shoulder in HPD-A. When concentrated HPD-A was added to a suspension of the

SUV, there was a shift of the Soret band from 363 to 398 nmwithin 1 min, and the original 363 nm band was evident as a

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G. C. Gayal et al.

shoulder. Similar effects were observed when 80 MM HP wasadded to SUV, in which case the Soret band shifted from 375 to398 nm (data not shown). The incorporation of HPD-A in the

LMV led to a broadened Soret band, suggesting partial localization in the interior aqueous phases and partial localization in thebilayer (Chart 3). Chart 4 shows unconnected fluorescence spec

tra obtained by exciting at 535 nm, where the concentrationswere adjusted to give approximately the same absorbance atthis wavelength. These measurements do not resolve the 675nm emission of HPD-A and HP with the phototube used (2). Thefluorescence efficiency of HPD-A in buffer was about 9-fold lowerthan that of HP. However, there was a 4-fold increase in theHPD-A fluorescence induced by diffusion from buffer into the

SUV, after correction for the detector sensitivity with rhodamineB. The higher fluorescence intensity for HPD-A in LMV (Chart 4,

Curve C) compared to SUV (Chart 4, Curve D) is an artifact oflight scattering by the LMV, as evident from the region below600 nm. The actual fluorescence yields are approximately equal.

Effects of HSA. The rapid shift of the HPD-A Soret peak from

363 nm in buffer to 398 mm in the SUV was retarded when theHPD-A was mixed with excess HSA prior to adding the SUV

suspension. The data in Table 2 show the changes in the Soretband peak and a width parameter (A37o:A396)after one part ofHPD-A (Ases= 26/cm) was added first to one part of 1 mw HSA

in phosphate buffer (pH 7.4), plus 0.9% NaCI solution, and themixture was added to one part of the SUV suspension (7.5 mg

2O 40 60 80

IRRADIATION TIME, MIN

Chart 2. Photosensitized peroxidation of small liposomes incorporating 0.3%(w/w) HPD-A based on increase of Azjs'Azu (phosphate buffer, pH 7.0, at 25°).A,

oxygen; B, nitrogen; C, oxygen control; D, nitrogen control; , photobleachingof HPD-A at 398 nm. D, oxygen; •nitrogen, rei, relative.

Chart 3. Optical absorption spectra. A. 15 «¿MHPD in phosphate buffer, pH 7.4;8, 20 (jM HPD-A in phosphate buffer, pH 7.4; C, 1.9% (w/w) HPD-A in smallliposomes; D. 2.0% (w/w) HPD-A in large liposomes plus light scatter. The HPDand HPD-A concentrations are based on estimated extinction coefficients of 7 x104 liters/mol cm at 363 nm in buffer and 1.0 x 10s liters/mol cm at 398 nm in

small liposomes, and M, 600.

06

580 600 £20 640 660 680 700

A mm)

Chart 4. Fluorescence spectra (uncorrected) excited at 535 nm. A, 15 tM HP inphosphate buffer (pH 7.4); B. 25 »MHPD-A in phosphate buffer, pH 7.4; C, HPD-A in large liposomes plus light scatter; D, HPD-A in small liposomes. The absorbanceat 535 nm was approximately constant for all samples.

Table 1Photosensitization of small liposomes by HPD-A

Irradia-tiontimo

(min)"030609021°0.701.221.381.44ASMÉIS35°0.611.201.401.4450°0.521.241.391.4221°0.000.0850.150.22/W35»0.000.0950.1750.25550°0.000.1050.1750.24521°3.202.621.981.40/W35°3.232.591.851.1750°3.302.461.540.80

nru-A (U.ovo) incorporated in au" Formation of hydroperoxides.c Thiobarbituric acid test for MDA." HPD-A photobleaching.

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Liposome Photosensitization by HPD

Tabte2Effect of HSA on diffusion of HPD-A into small liposomes

Time"(hr)0.17

0.52.04.027.051.5X™»(nm)373

375380389395396/WA«.1.08

1.041.000.990.930.93

a Final concentrations, 330 ¿IMHSA plus 100 M" HPD-A + 2.5 mg EPC per mlSUV at 25°.

o 0.6

¡" 0.8

IRRADIATION TIME MlN

Chart 5. Photosensitized oxidation of 50 pu HSA by 2 »IMHPD-A based on lossof 350 nm fluorescence excited at 295 nm (phosphate buffer, pH 7.4, at 30°).O,oxygen; •,nitrogen; A, oxygen plus 1 mu azide; Q, oxygen control; •HPD-A

photobleachmg at 375 nm. rei. relative.

EPC per ml). The small red shift of the Soret band when HPD-A

was first mixed with HSA was reported previously and attributedto binding of HPD-A to HSA (9). This conclusion was furthertested by mixing dilute HPD-A (=100 MM) with 1 mw HSA in

phosphate buffer (pH 7.4) and passing the mixture through aSepharose 6B column, as described in "Materials and Methods."

The eluent was monitored by optical absorption, HPD-A fluores

cence at 628 nm (535 nm excitation), and HSA fluorescence at340 nm (295 nm excitation). The 3 elution bands were coincidentat Va/V0= 2, and there was no evidence of porphyrin in the later-eluting fractions. The HPD-A Soret band was the same as that

of original mixture and accounted for >95% of the startingmaterial. The results strongly suggest that the 10-fold excessHSA complexed the HPD-A, although they do not exclude thecoincidental possibility that free, aggregated HPD-A eluted at thesame time as did HSA. No spectral evidence of HPD-A extraction

from either LMV or SUV was observed when HSA was addedafter incorporation of the HPD-A. The prior work has shown thatthe LMV were protected against photosensitized lysis by HPD-A in the external buffer (Asea = 0.29) when 25 MM HSA was

present (9). Equivalent protective effects were obtained withLMV incorporating 1.4% HPD-A in the presence of 30 UM HSAin PBS at 39°(data not shown). Since HSA does not permeate

the liposome membrane or extract HPD-A, the protection isattributed to external scavenging of 1O;»generated within the

liposomes. This mechanism is supported by evidence for HSAphotosensitization by HPD-A (without liposomes) as assayed by

the loss of tryptophan fluorescence (Chart 5). The protection by1 m«azide and under nitrogen supports the involvement of 1O2.

The data show also that HPD-A was partially photobleached.

However, photobleaching was equally rapid with nitrogen saturation (data not shown) ruling out the involvement of 102 in the

photobleaching reaction. These findings on HSA photooxidationand HPD-A photobleaching closely parallel prior work with HP(22). The low azide concentration of 1 mw was used for the HSAstudies to eliminate possible quenching of the sensitizer tripletstate by the azide. The higher azide concentrations used withthe liposomes may have led to partial triplet state quenching,although this would depend on the permeability of the membranes to azide ion and the possibility for interacting within theliposomes. Other arguments in support of 102 generation by

HPD-A and the involvement in membrane damage are summarized under "Discussion."

DISCUSSION

The present results are relevant to several aspects of theaction mechanism in PRT: the properties of HPD-A; photosensitization of membrane damage by HPD-A; and the mediating

effect of serum albumin. Various authors have speculated on thestructure of HPD-A based on indirect evidence. Kessel and Chou

(13) concluded from analytical and biological properties of HPDthat the active component is moderately hydrophobic but notnecessarily the most hydrophobic or the most readily aggregatedconstituent. Berenbaum ef al. (4) proposed a covalent porphyrinor oligomer based on high-performance liquid chromatography

results. From a combination of chemical reactions and analyticalresults, Dougherty (6) tentatively identified the active componentas DHE. Evidence that DHE is strongly aggregated in aqueousmedia includes the strongly blue-shifted 363 nm Soret peak (9),

weak fluorescence (8), and the resistance of the Soret peak toa red shift after dilution in buffer (8). In contrast, dilution ofconcentrated HP in buffer led to a Soret shift from about 373 to390 nm (23) and enhanced fluorescence (2), which are indicativeof monomerization. The shift of the HPD-A Soret band peak from

363 nm in buffer to 398 nm in the SUV (Chart 3) strongly suggeststhat DHE monomerizes (i.e., to a covalent dimer) in the lipidbilayer. This conclusion is supported by results of Spikes (23)with other porphyrins. The incorporation of 400 MM HP in dipal-

mitoylphosphatidylcholine SUV led to a Soret shift from 374 in0.1 M phosphate buffer (pH 7.4) to 400 nm. The latter coincideswith the Soret band in chlorofomrmethanol (9:1) where HPaggregation is negligible. Furthermore, the nonaggregating porphyrin, HP dimethyl ester, has a 400 nm Soret band in theorganic solvent over a wide concentration range and in thedipalmitoylphosphatidylcholine liposomes, even after adding Triton X-100, which lysed the liposomes. In conjunction with the

results of Spikes (23) and this work, the fluorescence enhancement of HPD localized in tissue compared to the relativelynonfluorescent injected solution may be attributed to monomerization in the lipid regions, without the necessity of invoking morecomplicated types of intercellular conversion (12).

The results with SUV incorporating HPD-A demonstrate thatHPD-A photosensitizes lipid peroxidation and membrane dam

age (Table 1; Chart 2) for conditions when it is membrane boundand fluorescent (Chart 4). The LMV results (Chart 1) are lessdefinitive because the spectra indicate partial localization inaqueous regions (Chart 3). However, the prior work in whichHPD-A photosensitized LMV lysis when added to the external

buffer (9) shows that the aggregated state in an aqueous environment also is a photosensitizer. The OER for lysis was 2.7 inthe latter case, compared to 4.3 with incorporated HPD-A,indicative of a weaker type II contribution for aggregated HPD-A. Similarly, the prior work with HP showed that type II photo-

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sensitization dominates with monomeric and/or dimeric HP inthe external medium, or incorporated in thèliposomes, and typeI photosensitization dominates with aggregated HP in both cases(10). The possibility remains that HPD-A may be a weak anoxic

photosensitizer in an aqueous environment, although the presentresults do not establish this point. The weight of evidencestrongly supports the conclusion that 1O2is the dominant active

oxygen intermediate in the photosensitized damage to liposomalmembranes. The protection of HSA from photosensitized oxidation by 1 mw azide (Chart 5) indicates that HPD-A generates1O2,because it is highly unlikely that this low azide concentrationcan quench the HPD-A triplet state (10). The involvement of 1O2

in photooxidation of tryptophan in HSA (and bovine serum albumin) was demonstrated also for HP photosensitization, usingprotection by azide and DABCO as acceptors and the "D20test," which were strongly positive (22). The protection of the

liposomes from lysis photosensitized by HPD-A by HSA in thiswork, and the prior work with external HPD-A (9), are additionalevidence that 1O2 is the lytic intermediate. Moan ef al. (17)measured the yields of 1O2from the components of HPD sepa

rated by high-performance liquid chromatography, based on

tryptophan photooxidation in D2O and found that the componentequivalent to HPD-A (Component 7) has a relative quantum yield

of 0.2 compared to 2.4 with the component identified as HP(Component 2). However, Component 7 was a 14-fold more

efficient photosensitizer for inactivation of NHIK 3025 cells thanwas Component 2. These findings support the conclusion thatHPD-A is an efficient 1O2generator in a lipid environment and aweak 1O2 generator in aqueous media. Finally, it should be

emphasized that EPC liposome lysis and peroxidation mediatedby 1O2 have been documented for many other types of dye

photosensitizers, including toluidine blue (1), retinal (5), méthylène blue (19), and carboxyfluorescein (21), and therefore thegeneration of 1O2by HPD-A within or external to EPC liposomes

should lead to damaging effects.The retardation of HPD-A uptake by SUV induced by HSA

(Table 2) is indicative of binding. The coincident elution of HPD-A and 10-fold excess HSA on the Sepharose 6B column supportsthis conclusion, although a similar result would obtain if the HPD-

A were present in the free, aggregated state with 40 to 60 DHEunits. Moan and Sommer (18) concluded that serum binds theirComponent 7 from fluorescence measurements. Preliminary results of equilibrium dialysis measurements in this laboratory showthat the binding of HPD-A to HSA is cooperative and thatapproximately 15% of the HPD-A is free at concentrations and

conditions appropriate to PRT (to be reported elsewhere).Lipid peroxidation was evident in the present work at about

10 min of exposure at 200 milliwatts/sq cm, corresponding to120 J/sq cm (Chart 2). However, the absorbed dose for thewavelength range of 360 to 750 nm was higher than that requiredfor PRT or inactivation of cell cultures. For example, Hilf ef al.(11) obtained 50% inactivation of R3230AC cells (hamster mammary adenocarcinoma) at dose levels of from 1 to 10 J/sq cm,depending on the HPD concentration and incubation period,where the wavelength range was 300 to 750 nm. The discrepancy indicates that the cellular targets are more sensitive to 1O2

than are liposome membranes. The photobleaching of HPD-A

observed in this work required fluence levels comparable tomembrane peroxidation and probably is not an important factorin PRT.

In summary, the following results of this investigation are

relevant to the molecular aspects of HPD-A photosensitizationin PRT: (a) the diffusion of HPD-A from aqueous buffer into an

EPC bilayer is a fast, spontaneous process, leading to a shift ofthe Soret band from 363 to 398 nm and an approximately 4-foldincrease of the fluorescence efficiency; (b) lipophilic HPD-A photosensitizes lipid peroxidation and membrane lysis via ^-me

diated reactions; (c) the presence of HSA retards the diffusionof HPD-A into the SUV, which is attributed to partial binding ofthe HPD-A; (d) HPD-A sensitizes the photooxidation of trypto

phan in HSA in the absence of liposomes; and (e) photosensitization of liposomes and HSA is accompanied by photobleachingof the HPD-A at higher fluence levels than is used for PRT.

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5830 CANCER RESEARCH VOL. 43

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