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Experimental Therapeutics |
Wellman Laboratories of Photomedicine, Massachusetts General Hospital [M. R. H., J. L. M., I. R., B. O., T. H.], Boston, Massachusetts 02114, and Departments of Dermatology [M. R. H., B. O., T. H.] and Molecular Endocrinology [E. V. M.], Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Conjugation of PS to macromolecular delivery vehicles may improve their performance in PDT by increasing specificity and/or uptake in tumors or other pathological lesions, favorably altering pharmacokinetics or biodistribution (e.g., less skin photosensitivity) or decreasing phototoxicity to normal tissue (8) . The physical properties of macromolecular-PS conjugates such as size, charge, hydrophobicity, and degree of aggregation can be easily altered, while keeping the same PS molecule joined to the conjugate. The conjugation of PEG to macromolecules that are administered i.v. (9) has been used to extend serum half-life and reduce the immunogenicity of injected proteins (10) . PEG has also found wide application in the preparation of sterically stabilized (Stealth) liposomes (11) , which show reduced uptake by macrophages, and has been shown to increase solubility and reduce aggregation (9) . pl may be used as the macromolecular backbone for attaching PSs such as ce6 to some of the epsilon amino groups, and, in addition, the remaining amino groups are available to attach PEG and subsequently modify the overall charge by acetylation or succinylation (12) .
i.p. macrophages may take up large amounts of PS when administered i.p. (13) and release inflammatory mediators upon illumination (14) . This may be responsible for the systemic toxicity observed after high-dose i.p. PDT (2) . We have previously shown that the acetylated pl-ce6 -ac conjugate has significantly higher photooxicity in vitro compared with the unmodified cationic conjugate and the anionic succinylated conjugates (15) . We hypothesized that attachment of PEG to these conjugates would reduce aggregation, reduce the uptake by macrophages, increase the selectivity for cancer cells, and possibly increase phototoxicity. To test this hypothesis, the uptake, phototoxicity, cellular localization, and oxygen consumption of pegylated and nonpegylated pl-ce6-ac were investigated in vitro with two cell lines, one a human ovarian cancer and the other a mouse macrophage cell line. A preliminary study was then carried out involving the i.p. injection of the conjugates in an established human ovarian xenograft model growing i.p. tumors in nude mice. The amounts of PS in the tumor and normal tissue and the degree of penetration of the PS into i.p. tissue were investigated.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthesis and Characterization.
pl-ce6 was synthesized as described previously (15)
from pl hydrobromide (150 mg; degree of polymerization, 46; average Mr, 5000; Sigma Chemical Co., St. Louis, MO) and 20 mg of preformed ce6 N-hydroxysuccinimide ester. After exhaustive dialysis to remove DMSO, 1 ml of 10% Na2CO3 solution was added to the resulting solution, which was then split into two equal parts. To one part was added 100 mg methoxypolyoxyethylene imidazolyl carbonyl (average Mr, 5000; Sigma Chemical Co.) and allowed to stand at 25°C in the dark for 12 h. This provided two solutions containing pl-ce6 and pl-ce6-PEG. To each of these was added an excess of acetic anhydride (1 ml). After standing in the dark for 12 h, both preparations were then exhaustively dialyzed as before. The absorbances at 400 nm measured in 0.1 M sodium hydroxide/1% SDS were similar as were the absorption spectra, and the extinction coefficients were assumed to be ce6 equivalent (150,000 M-1cm-1). Calibration curves were constructed for each conjugate so that the fluorescence measured in 0.1 M sodium hydroxide/1% SDS could be converted into mol ce6 equivalent.
Aqueous 2-phase partition experiments were performed as described previously (16) , with the conjugates present in mixtures of 4% (w/v) solution of PEG 8000 in PBS and a 5% (w/v) solution of dextran (average Mr, 500,000) at a final concentration of 10 µM ce6 equivalent.
The fraction aggregated was calculated by measuring fluorescence in the supernatant fractions after centrifugation and comparing it with samples that were agitated, based on the assumption that the aggregated material would be sedimented. Sufficient conjugate was added to two aliquots of 1.5 ml of RPMI 1640 (containing 10% FCS) to give a ce6 equivalent concentration of 20 µM. Seven hundred fifty µl of each aliquot were then serially diluted with 750 µl of complete medium 11 times to give two identical series of 2-fold serial dilutions from 20 µM to 10 nM. One set of dilutions was then centrifuged at 16,000 x g for 15 min at 4°C to sediment the aggregated portion of the conjugate while the other set was gently agitated to prevent any slow sedimentation. Three aliquots were taken from the supernatant of the centrifuged tubes and from the agitated tubes, and each aliquot was added to 1.4 ml of 0.1 M sodium hydroxide/1% SDS and the fluorescence measured from the peak height scanned between 580 and 720 nm, after excitation at 400 nm.
Oxygen Consumption in Solution.
A LICOX oxygen partial pressure monitor (Gesellschaft für medizinische Sondentechnik mbH, Kiel-Mielkendorf, Germany) was used to monitor the consumption of oxygen when the conjugates (2-µM solutions in RPMI 1640 containing 10% FCS) were illuminated by 75 mWcm-2 666 nm light in a plastic cuvette with a path length of 1 cm. After calibrating the Clarke electrode with pure N2 and pure O2, the conjugate solutions were oxygenated to 
500 mm pO2, and illumination commenced. The cuvette was stirred by a Hellma Cuvette-O-Stir (Model 333; Hellma USA, Plainview, NY). The signal from the electrode was captured by LICOX software on a personal computer via an RS232C interface. The pO2 showed an exponential decline, and fluorescence measurements before and after illumination gave a measure of photobleaching. The experiment was repeated three times by adding fresh aliquots of each conjugate and reoxygenating. The initial rate of oxygen consumption was determined by drawing a tangent to the exponential pO2 decay curve.
Uptake.
OVCAR-5 or J774 cells (105) in 1 ml RPMI 1640 with 10% FCS were seeded into each well of 24-well plates that were allowed to grow to 70–80% confluency. Fresh medium with 10% FCS containing conjugate (1 µM ce6 equivalent) was added, and after 4 h, the cells were washed three times with 1 ml of PBS and incubated with 1 ml of trypsin-EDTA for 10–30 min. The resulting cell suspension was then centrifuged, the trypsin supernatant was aspirated and retained, and the pellets dissolved in 1.5 ml of 0.1 M sodium hydroxide/1% SDS for at least 24 h to give a homogeneous solution. The fluorescence was then determined as described above and converted into mol ce6 equivalent. The protein content of the entire cell extract was then determined by a modified Lowry method (17)
using BSA dissolved in 0.1 M sodium hydroxide/1% SDS to construct calibration curves. The trypsin supernatant was also checked for the presence of fluorescence, which was <5% of that found in cell pellets. Results were expressed as mol of ce6 per mg cell protein.
Confocal Microscopy.
OVCAR 5 cells (2 x 105) were plated on a 20 x 20 mm microscope coverslip and incubated for at least 24 h. The conjugates were dissolved in complete medium, added to the cells and incubated for 3 h. For the final 20–30 min, 25 nM rhodamine 123 (R123; Eastman Kodak, Rochester, NY) or LysoTracker Green (Molecular Probes; Eugene, OR) were added. The coverslips were rinsed in PBS and mounted with PBS on a microscope slide using 0.02-mm-thick distance holders to prevent compression of the cells. A confocal laser microscope (Leica Mikroskopie und System GmBH, Wetzlar, Germany) consisting of a Leica TCS 4D scanner attached to a Leitz DM IRD inverted microscope was operated using the TCS-NT software package (Leica Lasertechnik, Heidelberg, Germany). 488 nm radiation from an argon laser was used for excitation. A 100x oil immersion objective were used to image at a 1024 x 1024 pixels resolution. Two channels collected fluorescence signals in either the green range (580-nm dichroic mirror plus 530-nm bandpass filter) or the red range (580-nm dichroic mirror plus 590-nm longpass filter). The green and red images (false color output) were superimposed for the figures.
Phototoxicity.
This was carried out essentially as previously described (15)
. Briefly, 2.5 x 104 cells in 0.1 ml of RPMI 1640 with 10% FCS were seeded in each well of 96-well plates and cultured for 24 h until 70% confluent. Conjugates were added in fresh medium at a final concentration of 1 µM ce6 equivalent for 4 h. Cells were washed twice and fresh medium was added. An argon-pumped dye laser was tuned to 666 nm and coupled into a 1-mm quartz fiber and objective lens to create a homogeneous spot which delivered light from below the wells with fluences ranging from 0 to 10 Jcm-2 at an irradiance of 50 mWcm-2. After illumination, cells were incubated with fresh medium for 24 h. Controls were: light alone, conjugate, and kept out of the incubator in the dark for the duration of the illumination, neither light nor conjugate. Survival fraction was quantified with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (18)
and read in an automatic microplate reader at 480 nm. Survival fraction was the mean formazan absorbance from PDT-treated cells divided by the mean absorbances from dark controls incubated with conjugate.
Nuclear Morphology after Illumination.
Cells were seeded in single-chamber tissue culture slides (Nunc Inc., Naperville, IL) and, at 60% confluency, were treated with conjugate (1 µM ce6 equivalent for 3 h) followed by 666 nm light. After 24 h, any floating cells were spun down onto the slide by cytospin centrifuge (StatSpin Technologies, Norwood, MA), and the slide fixed with 4% paraformaldehyde for 5 min, rinsed with PBS for 5 min, and soaked for 5 min in Hoescht 33258 solution (20 µg/ml; Calbiochem, San Diego, CA). Fluorescence images were obtained with a charge coupled device camera (Optronics Engineering, Goleta, CA) using excitation bandpass filter at 352 nm and emission longpass filter at 450 nm. Nuclear areas were measured using IP Spectrum Plus image analysis program (Signal Analytics Corp, Vienna, VA). Cells were counted as having abnormal nuclei when their areas were less than 66% of the mean area of control cells.
Oxygen Consumption in Cells Loaded with Conjugate.
To obtain sufficient ce6 in the cells to give a measurable consumption of oxygen, P100 plates with either J774 or OVCAR-5 cells were grown to near confluency (1.2–1.5 x 107 cells), and conjugates were added in the case of J774 at 2 µM and of OVCAR-5 at 4 µM ce6 equivalent concentrations in 10 ml of RPMI 1640 with 10% FCS, and incubated for 24 h. The cells were then washed with PBS and trypsinized to give a single-cell suspension, centrifuged to give a cell pellet, which was resuspended in 3 ml of PBS, and 2 ml of this was introduced into the stirred cuvette fitted with the LICOX electrode described above. Three 100-µl aliquots of the cell suspension were taken to determine the ce6 and cell protein concentrations as described. The cell suspension was illuminated with 75 mWcm-2 666 nm light, and the oxygen consumption trace was recorded as described above. After completion of illumination, three 100-µl aliquots of the cell suspension were taken to quantify photobleaching of the cellular fluorescence.
In Vivo Experiments.
All of the experiments were approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital. Mice were housed in laminar flow racks under specific pathogen-free conditions and were monitored daily for general health status. A xenograft model for human epithelial ovarian carcinoma that was developed in our laboratory (19)
was used in these experiments. In brief, female Swiss athymic nude mice (Cox Breeding Laboratories, Cambridge, MA), 6–8 weeks old, were given injections i.p. of 35 x 106 OVCAR-5 cells in 2 ml of PBS. Two weeks after tumor cell injection, they were placed on a synthetic diet (ICN Pharmaceuticals) lacking in plant ingredients and the associated chlorophyll that has been shown to give rise to significant levels of chlorin-like autofluorescence especially in the skin and organs of the digestive tract (20
, 21)
. Three weeks after tumor cell injection, either pl-ce6-ac or pl-ce6-ac-PEG was injected i.p. at a dose of 1 mg of ce6 equivalent per kg body weight, dissolved as a 1-mM solution in PBS. Three h after injection, blood (50 µl) was drawn from the retro-orbital sinus, the mice were sacrificed by carbon dioxide asphyxiation, and the tumor, normal liver, small intestine, kidney, bladder, spleen, skin, and muscle were harvested. Wet tissue samples (about 100 mg) were weighed immediately after resection and suspended in 2 ml of 1 M sodium hydroxide/0.2% SDS for 7 days when the tissue was completely dissolved. Serum was prepared from the blood sample and treated similarly. The ce6 content of the tissue extracts was determined by fluorescence spectrophotometry, and quantification against standard curves was prepared from each conjugate as previously described (22)
. Portions of peritoneal wall that did not contain gross tumor were snap-frozen in liquid nitrogen, and 10-µm sections were prepared with a cryotome for fluorescence microscopy. This was carried out using the Leica confocal scanning microscope as described above and images from the red channel (ce6 fluorescence) were overlaid on images from the green channel (tissue autofluorescence).
Statistical Methods.
Differences between two means were evaluated by the unpaired, two-sided Student’s t test assuming equal or unequal variation in the SDs as appropriate. SDs of the ratios of two means were obtained by calculating in quadrature (23)
. Ps of less than 0.05 were considered significant.
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RESULTS |
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. The substitution ratio (i.e., the number of ce6 attached to each pl chain) in this study was estimated by absorption spectroscopy to be 4 ce6 per chain of 46 lysine residues (8.7%). Two-phase partition in PEG 8000/dextran 500 mixtures has been reported (16)
to be a good indicator of covalent PEG modification (increased partitioning into the upper PEG phase). In the present case, the ce6 partitioned between the two liquid phases with none found at the interface, and the attachment of PEG, increased the partition coefficient 2-fold compared with the non-PEG conjugate (Table 1)
. The fraction of the nonpegylated conjugate that was aggregated increased in a concentration-dependent fashion. Pegylation significantly reduced the degree of aggregation, especially at the higher concentrations (Table 1)
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550 mm Hg. Each illumination lasted for 12 min and was repeated three times for each conjugate (see Fig. 2a
for a representative trace for pl-ce6-ac). From the trace, the initial rate of oxygen consumption in mm Hg per min could be determined by measuring the slope of a tangent drawn to the exponential curve and the total amount of oxygen consumed. Fluorescence was measured before and after illumination to compare the extent of photobleaching between the conjugates. The means of the values of the three initial rates of oxygen consumption are shown in Table 1
. The pegylated conjugate had a significantly lower initial rate of oxygen consumption than did the nonpegylated form. The percentage of fluorescence photobleached after 12-min illumination was also significantly decreased after pegylation, which implies that the reduction in oxygen consumption seen when the conjugate is pegylated is not simply attributable to easier photobleaching. By dividing the number of mol of O2 consumed by photo-oxidative processes (assuming water at 25°C with pO2 of 740 mm Hg is 1.27 mM O2 (24)
) by the number of mol of ce6 consumed by photobleaching, the average number of molecules of O2 used by each ce6 molecule before being destroyed could be calculated. Pegylation significantly reduced the number of O2 molecules consumed (by 75%).
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. The macrophage cell line takes up more ce6 from both PEG (three times the amount) and non-PEG (1.3 times the amount) conjugates. For both cell lines, pegylation reduced the uptake compared with the uptake from the non-PEG conjugate, but the reduction was less in the case of J774 cells (to 50% of the non-PEG values) than for OVCAR-5 (to 25% of the non-PEG values).
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. In OVCAR-5 cells the non-PEG conjugate (Fig. 3a)
is substantially in endocytic vessels that stain green with the lysosomal stain. There are some red aggregates visible in addition to the yellow areas. The mitochondrial stain (Fig. 3b)
shows relatively little overlap. After pegylation, the lysosomal overlap image (Fig. 3c)
shows extralysosomal red membrane staining in addition to lysosomal localization, whereas the mitochondrial overlap image (Fig. 3d)
shows considerable overlap with the green fluorescence. The images (Fig. 3, e and f)
from the J774 cells and the non-PEG conjugate are similar to those from the OVCAR-5 cells, with largely lysosomal localization. However, after pegylation, the images do not show the same changes seen with the OVCAR-5 cells. There is more, not less, aggregation (visible as very dense red fluorescence, apparently outside the cells; Fig. 3g
) and no increase in mitochondrial overlap (Fig. 3h)
. It should be noted that increases in the intensity of red fluorescence do not necessarily correlate with increased cellular uptake of the PS, as the intracellular aggregation state has been reported to dramatically affect the fluorescence intensity.
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. It can be seen that the nonpegylated acetylated conjugate is more phototoxic than the pl-ce6-ac-PEG is to macrophages, whereas the reverse is true for the cancer cells. Both phototoxicity curves show the expected logarithmic dependence of the survival fraction on the fluence delivered.
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. Pegylation of the acetylated conjugate dramatically increased the relative phototoxicity toward OVCAR-5 cells (9-fold), while decreasing it toward the macrophages (to one-fourth).
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Oxygen Consumption in Cells.
The consumption of oxygen by a cell suspension that had been loaded with conjugate and illuminated in a stirred cuvette could be determined by the LICOX pO2 monitor. A representative trace is shown in Fig. 2
. Each conjugate was repeated twice with each cell line, and the mean initial rate of O2 consumption for each illumination was divided by the mean number of picomoles of ce6 in the 2 ml of cell suspension. The extent of photobleaching was determined by taking aliquots of cell suspension before and after illumination and extracting the fluorescence. The total moles of oxygen consumed was divided by the number of moles of ce6 destroyed by photobleaching (calculated from the concentration of ce6 in the cuvette and the percentage of photobleaching). The results are shown in Table 2
. Pegylation reduces the initial rates of O2 consumption per mole of ce6 taken up in both cell lines and, in addition, reduces the number of molecules of oxygen consumed by each ce6 molecule in both cell lines. The numbers of molecules of O2 consumed per ce6 molecule when loaded into cells is significantly higher (up to six times) than the same number found when the conjugates were illuminated in solution (compare Table 1
).
In Vivo Experiments.
The in vivo tumor-targeting ability of the pegylated and nonpegylated conjugates were tested by injecting them i.p. in our nude mouse xenograft model of ovarian cancer (19)
. The amount of ce6 in various tissues was quantified by fluorescence extraction and spectrofluorimetry. Table 3
shows that the mean absolute amount of ce6 extracted from several i.p. tumor nodules was significantly higher for the pegylated conjugate compared with the nonpegylated (13.8 ± 1.4 versus 7.3 ± 2.5 nmol/g tissue; P < 0.01). In addition, the amount of ce6 in several normal organs was determined, and this allowed tumor:normal tissue ratios to be calculated. These ratios were significantly higher for pl-ce6-PEG-ac compared with pl-ce6-ac for all organs except the bladder, for which the difference in ratios was nonsignificant, and except for serum, for which the ratios were similar.
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, show confocal fluorescence micrographs of sections of peritoneal wall taken from mice at necropsy. It can be seen that plCe6-ac (Fig. 6A)
gives lower levels of red ce6 fluorescence compared with plCe6-PEG-ac (Fig. 6B)
, and, in addition, the fluorescence appears to penetrate farther beyond the basement membrane (arrows) in the case of the pegylated conjugate.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We hypothesized that covalent pegylation would have beneficial effects on the cellular uptake and/or phototoxicity of conjugates of pl with ce6. The hydrophilic nature of the PEG chains might be expected to reduce aggregation of the conjugates and increase solubility, as has been reported (26) . It is known that the photophysical properties of tetrapyrrole PS are strongly affected by their aggregation state (27) , and it was expected that pegylation would affect the photodynamic process and that this might be reflected in the oxygen consumption. As expected, pegylation of the pl-ce6-ac reduced the degree of aggregation in serum-containing medium, but surprisingly it reduced both the initial rate and total amount of oxygen consumption when illuminated in solution.
The consumption of oxygen by photochemical oxidation mechanisms when PSs are illuminated in the presence of an oxidizable substrate can proceed through a type II mechanism (transfer of energy from the PS triplet to ground state triplet O2 producing singlet O2) or a type I mechanism involving radicals and superoxide anion (probably produced from the PS radical cation; for a comprehensive review, see Ref. 1 ). It is generally thought that aggregated PS are less photoactive, i.e., they are less fluorescent, generate less singlet oxygen in solution, and produce less triplet-state PS as determined by laser flash photolysis (28) . It has been shown that hematoporphyrin in liposomes changes from a type II to a type I mechanism on raising the concentration, which leads to aggregation (29) . Another report (30) compared two preparations of hematoporphyrin derivative (Photofrin I and II) in the photo-oxidation of epidermal microsomes. It was found that Photofrin I, which contains less aggregates, followed a type II mechanism, whereas Photofrin II (with more aggregates) had a significant contribution from type I mechanism. It was reported that the addition of PEG to a solution of hematoporphyrin stimulated photooxidation of amino acids (including tyrosine, which proceeds via a type I mechanism; Ref. 31 ). This effect was attributed to alteration of the photophysical properties by a layer of "vicinal water" on the polymer molecules. ce6 has been reported (32) to be extremely easily photobleached (1 to 3 orders of magnitude higher rate constant) compared with other porphyrins and phthalocyanines when illuminated in FCS. Spikes (33) investigated photobleaching mechanisms of several porphyrins. He found that photobleaching was significantly greater when the illumination took place in the presence of photo-oxidizable substrates, especially serum albumin, and that the mechanism seemed to be via an oxygen-dependent type I process.
Macromolecular conjugates have been reported to be capable of being taken up into cells only via a process of endocytosis because of their size, which prohibits them from passing through the plasma membrane (34) . After endocytosis, the PS will mainly end up in endosomes and lysosomes, unless it is metabolized into small molecules and released from the lysosomes, or otherwise translocated along the endosomal pathway. Little has been published about the effect of pegylation on the cellular uptake, subcellular localization, and metabolism of macromolecules. One report (35) details the effect of pegylation on the receptor-mediated uptake and intracellular metabolism of asialofetuin. Uptake was reduced by as much as 75%, whereas the lysosomal degradation of internalized molecules was only 12% of the non-PEG asialofetuin. In concordance with this, results of the present study indicate that pegylation significantly reduces the cellular uptake in both cell lines. Opposite differences in the phototoxicity were observed between the cell lines; pegylation increases the killing in the ovarian cancer cell line, but decreases the killing in the macrophages. This effect is even more pronounced when the phototoxicity is corrected for the uptakes of ce6 by the different cells. Pegylation makes each molecule of ce6 up to nine times more phototoxic to OVCAR-5 cells and up to four times less phototoxic to macrophages. Pegylation reduced both the initial rate of oxygen consumption and the total number of oxygen molecules consumed after both cell types had been loaded with conjugate and illuminated. Only OVCAR-5 cells were observed to undergo apoptosis, and this was achieved at a significantly lower light dose with the pegylated conjugate.
How can the opposite effects of pegylation on phototoxicity in the two different cell lines be explained? We believe that a possible answer may be gained from considering the change in subcellular localization. In the OVCAR-5 cells, but not J774 cells, pegylation led to more membrane staining, including the mitochondrial membrane. It has now been well established by work from Kessel and Luo (36) that PSs that localize in mitochondrial membranes lead to a rapid induction of apoptosis after illumination characterized by cytochrome c release from mitochondria followed by activation of caspases (37) . It has been suggested that PSs are relatively less phototoxic when located in lysosomes (38) . There have been reports of the aggregation state of intracellular PSs affecting their localization with the more aggregated species tending toward lysosomal localization (39 , 40) . Macrophages have more and larger lysosomes than epithelial cells and, therefore, the relative phototoxicity of lysosomally located PS would be expected to be lower for macrophages. There is a recent report of the same PSs having different localizations (mitochondrial or lysosomal) in different cell lines (41) . The decreased consumption of oxygen during illumination of PS-loaded cells after pegylation, together with the increased relative phototoxicity, suggests that PS localized in lysosomes may produce large amounts of singlet oxygen that is relatively harmless, whereas membrane-localized PS may cause considerable damage after producing only a small amount of reactive oxygen species.
A second explanation may also be relevant. Macrophages have been shown to express high levels of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase (42) . These enzymes would be expected to significantly protect the cells from phototoxicity caused by a type I mechanism but not necessarily that mediated by a type II mechanism, and this could account for the lower level of macrophage phototoxicity after pegylation, if it could be shown that pegylation indeed shifted the mechanism from type II to type I. Antioxidant enzymes levels have been found to be significantly lower in most cancer cells (43) , which would help to account for the increased phototoxicity to OVCAR-5. The ability to increase the phototoxicity toward cancer cells while sparing macrophages may have significance in the i.p. PDT of ovarian cancer, for which functional peritoneal macrophages may be important to destroy partially damaged tumor cells (44) .
We carried out preliminary experiments to test the ability of the i.p. injection of the pegylated and nonpegylated conjugates to target OVCAR-5 cells growing as disseminated i.p. tumors in nude mice. Pegylation of pl-ce6-ac increased the absolute amount of PS taken up by tumors 3 h after i.p. injection. In addition, tumor:normal tissue ratios for normal organs ranged from 2- to 5-fold higher for the pegylated conjugate compared with the nonpegylated form. The higher amounts taken up into tumor may be caused by the reduction of aggregation observed after pegylation, and this may also account for the reduction in accumulation in other organs. The increased penetration observed with the pegylated conjugate may be important, because the degree of penetration into tumor after an i.p. injection is likely to define the therapeutic effectiveness of the treatment. Additional experiments are under way in our laboratory to explore the effect of pegylation on the biodistribution, pharmacokinetics, and tumoricidal properties of these conjugates. In conclusion, this study shows that pegylation of PS conjugates may be a way of still further increasing the selectivity of PDT for ovarian cancer by shifting the PS targeting away from macrophages and toward cancer cells, by increasing the phototoxicity in vitro, and by improving tumor uptake and penetration when administered i.p. in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant R01 AR40352 from NIH (to T. H.) and by Grant N00014-94-1-0927 from the Department of Defense Medical Free Electron Laser Program (M. R. H., B. O.) 
2 Present address: Department of Biomedical Engineering, Cleveland Clinic Foundation, Cleveland, OH 44195.
3 To whom requests for reprints should be addressed, at Department of Dermatology, Massachusetts General Hospital, 50 Blossom Street WEL224, Boston, MA 02114-2698. Phone: (617) 726-6996; Fax: (617) 726-8566; E-mail: thasan{at}partners.org
4 The abbreviations used are: PDT, photodynamic therapy; ce6, chlorine6; PEG, polyethylene glycol; pl, poly-l-lysine; pl-ce6-ac, acetylated pl-ce6 conjugate; PS, photosensitizer.
Received 4/24/01. Accepted 8/ 2/01.
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