Journal
of
Photoch;mistry Journal of Phorochemistry
ELSEVIER
Photodynamic
and Photobiology
B: Biology
42 ( 1998)
20-27
damage by liposome-bound porphycenes: comparison between in vitro and in vivo models ’ H. Toledano, R. Edrei, S. Kimel * Department
of Chemistry,
Received
Technion-Israel
Institute
of Technology,
24 June 1997; accepted 3 November
Haifa
32000.
Israel
1997
Abstract Photodynamic efficacy of four tetrakis( methoxyethyl)porphycene (TMPn) derivatives encapsulated in Iiposomes, was studied in vitro and in vivo. Fluorcsccnce and absorption measurements were used to determine aggregation in dipalmitoyl phosphatidylcholine (DPPC) liposomes; no spectral changes were found when dissolving in an organic solution or in an aqueous dispersion of DPPC liposomes. This indicates that the porphycenes were located in the lipophilic bilayer of the liposomes. Fluorescence quenching experiments with I- showed, specifically, that
porphycenes
locatrd
in the liposome
bilayer
at various
depths,
according
to the hydrophilicity
of the porphycene
side chains.
Dose-
response relations were established: increasing porphycene concentration or light doseenhancedthe damageproportionally.In cultured MDCK cells, photodynamic damage was in accordance with location: a porphycene ‘buried’ inside the bilayer did not cause damage to the cell culture. PDT efficacy was tested also in vivo by the damage to blood vessels of the chorioallantoic membrane (CAM) of the fertilized chick embryo. Unlike in the in vitro case, the porphycene ‘buried’ inside the bilayer did cause significant photodynamic damage in vivo. This difference suggests that in vitro photodynamic action follows contact-mediated sensitizer transfer to cell membranes from liposomes, which remain distinct from cells, whereas in vivo the photosensitizer is delivered to tissue via fusion of liposomes with endothelial cell membranes. 0 1998 Elsevier Science S.A. Kqvwordr:
Photodynamlc
therapy;
Porphycenes;
Liposomes;
Chorioallantoic
1. Introduction
Photodynamic therapy (PDT) is an experimentalmodality usedfor treating ncoplasms.It involves photochemical rcactions that require a photosensitizing drug, light at an appropriate wavelength, and oxygen. The primary damagecaused by short-lived reactive oxygen species,may be in the tumor cells (cellular mechanism) [ I] or in the blood vesselssupplying the malignant tissue (vascular mechanism) [2,3] leading to tumor regression and, ultimately, to necrosis. Photodynamic efficacy wastestedfor a seriesofporphycenes. These are structural isomers of porphine, based on a 16-memberedmacrocycle with two ethine bonds and two direct links betweenpyrroles, insteadof four methinebonds,
* Corresponding author. Fax: +972 4 8233735; chrl
[email protected] ’ Part of M.Sc. Thesis presented by H. Toledano to the Graduate Technion. 101 l-1344/98/$19.00 l-1344(97)001
PJJ SlOl
0
1998 Elsevier 10-3
Science
S.A. All rights
e-mail: School,
reserved
membrane;
Fluorescence
quenching
so that the porphycene macrocycle possessesrectangular symmetry rather than four-fold symmetry (such as in porphine) [ 41. Porphycenesare characterized by an absorption band in the red spectralregion with molar absorptioncoefficient ( l e63o 3 50 000 M- ‘cm- ’ ) about 1O-fold larger than that measured
for porphyrins.
Moreover,
some porphycenes
were found to generatesinglet molecular oxygen [ 5-71 and to possesssuperiortumor-localizing propertiesIS-101 which make them promising second-generationphotochemotherapeutic agents. We consider here four derivatives of tetrakis( methoxyethyl) porphycene (TMPn) with a specific substituentin the 9-position of the macrocycle, which imparts a varying degreeof hydrophilicity to the porphycene molecule (Fig. 1) ascorroborated by different valuesof the retention time, R,, in HPLC [ lo], (see Table 1) . Porphyceneswere encapsulatedin dipalmitoyl phosphatidylcholine (DPPC) liposomes[ 11,121.Theirphotodynamic efficiency was testedusing two models: (i) in vitro cultured epithelial MDCK cells, and (ii) in vivo blood vesselsof the
H. Toledano
et al. /Journal
of Photochemistry
and Photobiology
B: Biology
42 (1998)
2CL27
21
CpoTMPn
GlamTMPn H&O
H&O
‘1
f
OCH,
OH
H&O
H,CO
OCH,
OCH,
StoTMPn
NicamTMPn H&O
Fig. 1. Chemical
structure
of four 2,7,12,17
Table I Stern-Volmer quenching constants for porphycenes in DPPC liposomes HPLC retention time (R,) using a C 18 column 1 lo] Porphycene
K (Mu
GlamTMPn CpoTMPn NicamTMPn
‘)
tetrakis
and
R, (min)a
4.1 * 0.7 2.4 k 0.5
1.3 3.7
1.5+0.5 -0.2f0.3
StoTMPn
W
1.8 21.6
“values from Ref. [ lo].
chorioallantoic membrane (CAM) embryo.
2. Materials
of the fertilized chick
and methods
4
f
OCH,
(methoxyethyl)porphycene
(TMPn)
derivatives.
and the dried lipid film washydrated with wate:ror phosphate buffered saline (PBS), and sonicatedfor 15min (probe sonicator - VC 50 Vibra Cell). Liposomeswere passedthrough a 0.45 pm pore filter and were usedwithin 1 h. 2.3. Spectroscopicstudies Absorbanceand fluorescencespectraof porphyceneswere measured,respectively, with a diodearray spectrophotometer (HP 8452A) and a spectrofluorimeter (Perkin-Elmer LS50). Fluorescencequenching with I- ions in liposomesuspensionwascarried out by increasingthe concentration of KI (at the expenseof KCl) in a solution of KI + KCl, with constant ionic strength soas to avoid ‘salt effects’ [ 151. Na,S,O,, 0.1 mM, wasaddedto prevent formation of I, or 1, - ; suspensions were thus free of yellow coloration.
2.I. Photosensitizers 2.4. In vitro protocol The derivatives of 2,7,12,17-tetrakis(methoxyethy1) porphycene (TMPn) studied in the present investigation were synthesizedby Vogel et al. [ 13,141and usedwithout further purification. They differ in the type of substituentin the 9position of the macrocycle as follows: 9-glutaric acid amide TMPn (GlamTMPn) ; 9-nicotinic acid amide TMPn (NicamTMPn) ; 9-capronyloxy ( CpoTMPn); and9-stearoyloxy TMPn (StoTMPn) . Theseporphycenesare not soluble in water. Stock solutions of 1 mg ml-’ in dichloromethane were kept at 4 “C in the dark. Porphyceneconcentrationswere determinedfrom absorbancespectra. 2.2. Preparation of DPPC liposomes Small unilamellar vesicles(SUV) of DPPC wereprepared [ 151by co-dissolving 12.5 pg porphyceneper ml chloroform containing 10 mg ml -’ DPPC. The solvent was evaporated
2.4.1. Cell cultivation MDCK cells (epithelial cells from cocker :spanielkidney obtained from the Naval BiosciencesLaboratory, University of California, Berkeley) are characterized by fast proliferation; they form a uniform monolayer adherent to the plate surface. Cells were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 5% fetal calf serum (FCS), 4 mM I-glutamine, 100 Uml-’ penicillin, 0.1 mg ml - ’ streptomycin (all obtainedfrom Biological Industries,Beth Haemek, Israel) at 37°C in 5% CO,. For experiments, cells in passages14 to 20 were used.To set up microwell cultures, cells in the log growth phase were brought into suspensionwith trypsin/EDTA, washedwith calcium- and magnesium-free PBS and resuspendedin medium. Cells were counted, diluted with medium and seededinto 96-well tissueculture microplatesat a concentra-
tion of 5 X IO’ cells/well using a multichannel pipette. After a monolayer was formed, 10 mM HEPES buffer was added to maintain pH. Some microplates were exposed to anoxic conditions for 24 h, by inserting the microplate into a sealed tank which contained anoxic tablets (Anaerocult A, Merck, Germany) that bind oxygen and create anaerobic milieu. 2.4.2. Photodynamic treutment Cells were washed twice with PBS and then incubated for 4 h in FCS-free DMEM containing 1.25 kg ml ~ I liposomebound porphycene. A red-filtered halogen light source (delivering 8.7 mW cm-’ at A > 600 nm) was used to irradiate a tissue culture microplate through a frame, exposing 9 wells at a time, for 4, 8. 16 or 32 min. PDT efficacy was expressed as percentage cell death relative to ‘blank’, defined as number of cells in wells in the same microplate that were incubated with porphycene but not exposed to red light. After irradiation, cells were resupplied with complete medium and incubated for 24 h. All manipulations involving porphycenes were carried out under subdued light. 2.4.3. Methylene blue (MB) assay Cells in the wells of the microplate were counted 24 h after PDT using vital staining with MB [ 161. Briefly, the culture medium in each well was removed by gentle aspiration. The cell layer was fixed by adding 100 ~1 paraformaldehyde 4% (w/v) in PBS to each well for 20 min at room temperature. The fixative was shaken off and 100 p,l of filtered 17~ (w/v) MB in 0.01 M borate buffer (pH 8.5) was added to each well. After 30 min, excess dye was removed and the remaining unbound dye was washed off by serially dipping the microplate into each of four tanks containing 0.01 M borate buffer (pH 8.5), and shaking the buffer off before each immersion. To elute the dye, after the last rinse and shake. 100 ~1 of 1: I (v/v) ethanol (absolute, 995%) and 0.1 M HCI were added to each well. The plate was then gently shaken and directly afterwards the absorbance of MB at 620 nm. that is related to the number of living cells. was measured for each well by a microplate photometer (Elysa readerCERES UV900 HDI). 2.5. In vivo protocol 2.5. I. CAMprepamtion The CAM was used as an in-vivo assay for PDT [9]. Briefly, fertilized eggs were placed in a hatching incubator at 37°C and 60% humidity. On the third day of incubation, 2-3 ml albumin was withdrawn from each egg to create a false air sac. The following day, a 2 cm diameter hole was opened in the egg shell at the apex, exposing the CAM. The opening was covered with a blackened Petri dish and incubation was continued in a static incubator. On day 10 the CAM was complete and ready for experiments. 2.5.2. Porphyene uptake Liposome-bound porphycene at a concentration of 250 ng/ 20 p,l was administered topically in the area demarcated by
a 6 mm diameter teflon ring placed on the CAM. Fluorescence measurements during drug uptake into CAM tissue were performed using a bifurcated optic light guide, which transmits excitation light to the CAM surface and emission from CAMembedded porphycene to the detection system of the spectrofluorimeter. For each porphycene, the emission spectrum (A,,, = 375 nm; A, = 600-700 nm) was recorded at IO min intervals. Since initial fluorescence intensity F( t = 0) differed for individual CAMS, the evolution of the fluorescence intensity F(t) is presented as F(t) /F( t= 0). For each porphycene, the uptake dynamics in the CAM was determined by averaging data obtained from IO different CAMS.
Irradiation with HeNe laser light (633 nm, 14.3 mW) was started 30 min after porphycene administration at SO, 12.5or 250 ng/20 p,l, when fluorescence measurements indicated optimal uptake in the CAM. The laser was positioned such that the tissue-scattered beam filled the entire ring area, giving an incident irradiation of 50 mW cm-‘. Blood vessels in the irradiated area were inspected with a stereomicroscope 3 h post-irradiation in a double-blind fashion. Damage was classified as: 0, no damage; 1, slight damage - vasodilation/ constriction; 2. moderate damage - hemostasis. clotting, CAM denaturation; 3, severe damage - widespread occlusion, hemorrhage. For each light and drug dose the resulting damage in 12 CAMS was averaged.
3. Results and discussion 3. I. Absorption and,fluorescerw
properties
Absorption and fluorescence measurements showed no differences when porphycenes were dissolved in dichloromethane or in aqueous dispersion of DPPC liposomes, indicating similar microenvironment of the porphycenes (data not shown). The four porphycenes locate in the lipophilic bilayer of the liposomes, as is expected from their lipophilic nature. The only solvent effects were due to Rayleigh scattering by liposomes which caused increased background absorption at shorter wavelengths. Further support for the above was provided using Triton-X, a detergent that causes monomerization [ 171. Absorption and fluorescence spectra of porphycenes at the maximum concentration in liposomes ( 12.5 kg ml- ‘, which corresponds to 0.0 I9 mM for NicamTMPn, CpoTMPn or GlamTMPn. and 0.015 mM for StoTMPn), were compared with and without 1% T&on-X. Absorption spectra were similar, no shifts or new bands were observed. Background absorption in solutions containing Triton-X was reduced considerably as a result of liposome rupture that causes the solution to become more transparent. In the emission spectra, Triton-X increased the fluorescence yield by about a factor of two. This increase results from liposome rupture and from monomerization. Changes in opaqueness
H. T&dam
et ~1. /Journal
cf Photochrmist~
and
Photobiology
GlamTMPn
0.00
0.04
0.08
0.12
0.16
B: Biology
42 iIWH)
20-27
23
CpoTMPn
0.20
0.24
0.00
0.05
NicamTMPn
0.10
0.15
0.20
StoTMPn
2.0 I
0.00
0.04
0.08
0.12
0.16
0.20
0.240.00
0.04
0.08
plots
0.16
0.20
0.24
II-1 W)
[I-l CM) Fig. 2. Stern-Volmer
0.12
for lluoresccnce
quenching
of porphycenes
in lipohomes.
affected fluorescenceemission,particularly for short wavelengthsof excitation (A,,, z 375 nm) (data not shown). 3.2. Fluorescence
quenching
studies
Fluorescencequenching by iodide was usedto determine the influence of the substituentin the 9-position on porphycene location in the liposome membrane. Fluorescence quenching data were analyzed using the Stern-Volmer equation F”/F=l+K[Q]
(1)
where p and F denote, respectively, fluorescenceintensities in the absenceand presenceof the I- quencher; [Q] is the molar concentration of the quencher,and K is the quenching constant.Fig. 2 depicts Stem-Volmer plots for fluorescence quenching of the four porphycenesin DPPC liposomes. StoTMPn fluorescencewasnot quenchedby I-, meaning that StoTMPn is not accessibleto 1.. since it is buried deep insidethe liposomebilayer, commensuratewith its oneorderof-magnitude higher lipophilicity comparedto the three other
Fig. 3. Photodynamic of dead cells verws
damage irradiation
in cell time.
cultures
hy porphycenes:
percentage
24
H. Toledano
et al. /Journal
of Photochemistr?;
porphycenes. To ascertain that fluorescence can be quenched when StoTMPn is accessible to I-, we compared fluorescence of StoTMPn dissolved in a mixture of acetone and water ( I/ 1 v/v, without liposomes), and measured a decrease of 55% in the presence of 0.25 M iodide. Table 1 (column 2) summarizes the Stern-Volmer quenching constants in liposomes. Clearly, GlamTMPn, NicamTMPn and CpoTMPn were accessible to iodide ions, though to a different extent, roughly proportional to their hydrophilic nature, given in terms of R, values (Table 1, column 3).
Photobiology
B: Biology
Table 2 Porphycene damage
parameters.
42 (1998)
2@27
Normalized
values
of &,,,\ and photodynamic
Porphycene
Eah\ in cell cultures
Cell damage”
CAM
GlamTMPn CpoTMPn NicamTMPn StoTMPn
0.81 0.87 1 0.96
1.1 0.8 1 0.05
0.3 0.1’ 1 0.5
inju$
“After 32 min irradiation. “50 ngi20 ~1, after 8 min irradiation. ‘Corrected for dark toxicity.
3.3. Photodynamic damage to MDCK cells Fig. 3 presents the percentage of dead cells after PDT with the four porphycenes at a concentration of 1.25 kg ml - ‘, for different irradiation time. The three porphycenes that were accessible to iodide ions induced comparable photodynamic damage (Table 2, column 3) consistent with their similar hydrophilicity (Table 1, column 3). Lipophilic StoTMPn caused negligible damage; even when cultures were irradiated for 32 min, only 4.5% of the cells were dead, as compared to irradiation for 32 min without StoTMPn which caused 1.5% mortality. Cell cultures that were grown (before PDT) in normal conditions and in anoxic milieu gave similar results. Anoxic conditions, in some respects. mimic those inside a tumor in vivo. Inefficient vascular supply and the resultant reduction in tissue oxygen tension lead to a feedback response of neovascularization mediated by VEGF (vascular endothelial growth factor) protein, which is hypoxia inducible also in cultured cells [ 181. Therefore, we have chosen to present (Fig. 3) results only for MDCK cells grown under anoxic conditions. Differences in photodynamic efficacy of a porphycene (Fig. 3) may, perhaps, be a result of dissimilar light doses. The photon energy (Eabs) absorbed by a porphycene was determined as:
E,,,=jE(h)( 1-lO-A’“‘)dh
and
(2)
where E(A) and A( A) denote, respectively, the energy emitted by the red-filtered halogen source and the porphycene absorbance at A, in the range of overlap, 600 nm < A < 700 nm (as exemplified in Fig. 4 for StoTMPn). Table 2 (column 2) summarizes Eabsas determined by Eq. (2) for each porphycene. The energy absorbed by the porphycenes is similar so that considerations of absorbed photon energy cannot explain the inertness of StoTMPn in cell cultures, which therefore must be due to its location inside liposomes which prevents access to cells. 3.4. In vivo uptake and PDT ej$cacy of porphycenes in CAM Uptake curves for liposome-bound porphycenes are presented in Fig. 5; results are accurate to within 20%. A fast decrease of the fluorescence intensity F(t) over the first 30
oL)0.x -
2.0 StoTMPn
0.1 -
40 min. Since there is no significant difference in uptake kinetics between the four porphycenes, a uniform 30 min incubation time was chosen for sensitizer-CAM equilibration prior to irradiation. The uptake kinetics was similar to that previously determined for other porphycene derivatives [ 91. PDT efficacy, as assayed by injury to CAM blood vessels, is shown in Fig. 6. Each entry represents the average damage induced in 12 CAMS following photoactivation. The efficacy of the porphycenes, on a per weight basis. is depicted in Fig. 6 and ranked in Table 2, column 4. Dose-response relations were established: increasing the porphycene dose or the irradiation time enhanced the injury proportionally. CpoTMPn yielded poorly reproducible results and dark toxicity occurred even at 50 ng/20 ~1, which may be explained by the hydrolysis in vivo reported only for CpoTMPn [ lo]. NicamTMPn was most effective in damaging blood vessels, even at the lowest concentration and shortest irradiation time; in addition, it did not cause dark toxicity. Unlike in cell cultures, StoTMPn was effective in injuring blood vessels.
H. Toledano
et ul. /Journnl
ofPhotochemist~
and
Photobiology
B: Biolog!
1
42 (1998)
20-27
u
GlamTMPn
-A-
StoTMPn
0.9
0.8 G II E =; r. IL
0.7
0.6
0.4
I
i
10
0
20
30
Time g. 5. Uptake
kinetics
of liposome-encapsulated
Fig. 6. Vascular
porphycenes.
topically
40
after applied
50
application
60
as manifested
by relative
CpoTMPn
NicamTMPn
StoTMPn
induced
by porphycene
PDT in the CAM
80
(min)
on CAM.
GlamTMPn
injury
70
as a function
of drug dose
fluorescence
and irradiation
intensities,
time
F(f)
/F(
t=
0).
26
H. Toledano
et al. /Journal
ofPhotochemistp
4. Conclusions The similarity of spectroscopic data in dichloromethane and in liposomes allowed us to ascertain that the porphycenes located in the phospholipid bilayer of the liposome. Fluorescence quenching data provided more detailed information on intraliposomal localization of individual porphycenes. The proximity of the porphycenes to the membrane surface was in accordance with the hydrophilicity of the substituent in the 9-position (Table 1): GlamTMPn > CpoTMPn > NicamTMPn % StoTMPn. Photodynamic damage to MDCK cell cultures (grown in normal and in anoxic conditions), as assayed by vital staining with MB, could be correlated to porphycene location in liposome membranes. Trapped, not-integratedporphycenesclose to the Iiposome surface caused larger cell damage, whereas StoTMPn which was located inside the bilayer, did not cause any damage, even though the energy absorbed by all porphycenes was similar. The order of damage to cell cultures was (Table 2, column 3) : GlamTMPn > NicamTMPn > CpoTMPn > StoTMPn. The inverted order of NicamTMPn and CpoTMPn when considering damage to cell cultures, compared to the order of location in liposome membranes, might be due to different ‘%bS. In contrast to the in vitro case, porphycene location in liposome membranes was not relevant to CAM blood vessel injury; StoTMPn that was ‘buried’ inside the bilayer caused efficient PDT in vivo. The order of damage to blood vessels was (Table 2, column 4): NicamTMPn > StoTMPn > GlamTMPn > CpoTMPn. Examination of the above results leads to an important conclusion for sensitizers encapsulated in liposomes: the in vitro photodynamic mechanism is basically different from that in vivo. The proposed model assumes that when no serum was added, photodynamic damage occurred after contactmediated transfer of porphycenes from liposomes, which remain distinct, to cell membranes in culture. In contrast, in the CAM model the photosensitizer is delivered to blood vessels via fusion of liposomes and endothelial cell membranes. This model is supported by reports ] 19,201 of increased liposome permeability in vivo compared to in vitro where no blood compartments are present. Transfer of phospholipid from liposomes to lipoprotein serum in vivo appears to damage liposome integrity and to release liposomal content, even when embedded in the bilayer [ 19,20 J. Results obtained in this investigation provide some insight on the intraliposomal location of porphycenes in unilamellar vesicles of DPPC. This information may be important for optimizing delivery of liposome-bound drugs to cultured cells and to tissue. Acknowledgements This research was supported by the US-Israel Binational Science Foundation, grant 93-00154 and by Cytopharm Inc., Menlo Park, CA. We thank Professor David Gershon
and
Photobiolog):
B: Biology
42 (1998)
20-27
(Department of Biology, Technion) for his advice and for permission to use facilities at the Department of Biology. The porphycenes were kindly provided by Professor E. Vogel (University of KBln, Germany) References Ill
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[ I91 T.M. Allen A arudy of phosphollpld mteractions between high-density lipoproterns and small unilamellar vesicles. Biochlm. Biophys. Acta 640 ( 1981) 385-197. [201 G. Scherphof, F. Roerdink. M. Waite, J. Parks, Disintegration of phosphatidylcholine liposomes in p)asma as a result of interaction with high-den,q lipoproteiw Blochlm. Biophys. Acr.~ 542 (1978) 29h-.w7
