Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

One of the hallmarks of atherosclerosis is the appearance of lipid-loaded macrophages in the vessel wall. Continuous infiltration of monocytes and lymphocytes, together with a strong proliferation of smooth muscle cells, gradually leads to the narrowing of a vessel (Ross, 1986; Libby and Clinton, 1993). Current therapies for atherosclerosis, such as percutaneous angioplasty or bypass surgery, are limited by the recurrence or worsening of the atherosclerotic process.

Over the last few years, however, photodynamic therapy (PDT) has been designated as a promising new therapy for cardiovascular pathologies, such as atherosclerosis and restenosis (Nyamekye et al., 1996). PDT is based on the use of light-sensitive compounds, photosensitizers (PS), which can be activated by light of a specific wavelength that is matched to the absorption characteristic of the particular PS. Photoactivation in the presence of tissue oxygen leads to the formation of cytotoxic-reactive oxygen species such as singlet oxygen. Photoactivation of photosensitizers therefore may result in the damage of cellular components, eventually leading to cell death (Levy, 1994). So far, PDT has been clinically used for various forms of cancer, diseases of the skin, and the upper digestive tract (Schuitmaker et al., 1996). The rationale for using PDT in atherosclerosis is based on the observation that various PS, such as porphyrins and phthalocyanines, selectively accumulate in the atherosclerotic plaque as compared with the adjacent normal vessel wall (Eldar et al., 1990; Tang et al., 1993; Hsiang et al., 1993, 1994; Visona and Jori, 1993). Local intra-arterial application of light of the appropriate wavelength to the atherosclerotic vessel therefore may reduce the narrowing of the vessel.

The application of PDT in atherosclerosis and restenosis may be improved by the enhanced, specific delivery of the PS to the affected site. Selective targeting of the PS to the atherosclerotic plaque may reduce its possible side effects, such as skin hypersensitivity to sunlight. Candidate structures on the cell surface in the atherosclerotic plaque to which to target PS are the so-called scavenger receptors. Scavenger receptors efficiently mediate the uptake and processing of (oxidatively) modified low-density lipoprotein (OxLDL). Within the atherosclerotic plaque, high numbers of scavenger receptors are expressed on macrophages (Matsumoto et al., 1990), and the level of expression on macrophage-derived foam cells increases dramatically in the course of the disease (H. E. deVries, B. Buchner, T. J. C. van Berkel, and J. Kuiper, submitted for publication). The incorporation of PS into OxLDL therefore may lead to a selective uptake and accumulation of these complexes by macrophage-derived foam cells. The use of OxLDL as a specific carrier for PS may enhance the beneficial effects of PDT in cardiovascular diseases. In this study, we examined the in vitro application of complexes of OxLDL-aluminum phthalocyanine chloride (AlPc) to achieve selective accumulation of the PS AlPc in macrophages. This strategy of PDT for cardiovascular diseases may be very promising to treat initial phases and proliferating stages of atherosclerosis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. RAW 264.7 leukemia virus-transformed murine macrophages were obtained from American Type Culture Collection (Manassas, VA). Dulbecco's modified Eagle's medium (DMEM), bovine calf serum, and trypsin were obtained from Gibco BRL Life Technologies (Breda, The Netherlands). Multiwell clusters, tissue culture flasks (T75), and Transwell (polycarbonate; pore size, 3.0 µm; 12 mm) were purchased from Costar (Cambridge, MA). ¹²⁵I in NaOH was purchased from Amersham Life Science (Buckinghamshire, UK). All other chemicals were of analytical grade. Aluminum phthalocyanine chloride [(C₈H₄N₂)₄AlCl was obtained at Eastman Kodak company (Rochester NY].

Cell Culture. Murine RAW 264.7 macrophages were cultured in DMEM containing 25 mM HEPES, 3.7 g/liter NaHCO₃, pH 7.4), supplemented with 10% bovine calf serum, 2 mM L-glutamine, and 100 µg/ml penicillin-streptomycin in an incubator containing 5% CO₂ and 95% air in a humidified atmosphere. Passing of cells was performed by washing twice in PBS (136.9 mM NaCl, 0.3 mM Na₂HPO₄, pH 7.4) followed by 10 min of trypsinization with 0.25% trypsin and 0.1% EDTA.

Lipoprotein Isolation, Modification, and Labeling. LDL (1.024 < d < 1.055) was isolated from fresh human serum by density gradient ultracentrifugation according to Redgrave et al. (1975). LDL was acetylated with acetic anhydride as described by Basu et al. (1976). LDL was oxidatively modified by incubation of 200 µg/ml of LDL with 10 µM CuSO₄ at 37°C. After 20 h of incubation, the reaction was terminated by administration of EDTA to a final concentration of 1.01 mM. The negative charge of AcLDL and OxLDL was checked routinely by agarose gel electrophoresis using a 1% agarose solution in hippuric acid buffer (R_f = 0.52 and 0.53, respectively). For binding experiments, LDL was iodinated before oxidative modification of LDL. LDL was radiolabeled with ¹²⁵I by the ICl method of McFarlane (1958) as modified by Bilheimer et al. (1972). The specific activity of OxLDL ranged from 80 to 200 cpm/ng protein.

Preparation of Protein-Free Triglyceride-Rich Emulsions. To determine whether the uptake of the OxLDL-AlPc complexes is mediated via scavenger receptors, protein-free triglyceride-rich emulsions were used as a control. Emulsions were prepared according to the sonication and ultracentrifugation procedure of Redgrave and Maranhao (1985). Emulsions were prepared from egg yolk phosphatidylcholine, lysophosphatidylcholine, cholesterol oleate, and cholesterol as described (Rensen et al., 1997). Emulsions were fractionated into three size populations by consecutive ultracentrifugation steps in a Beckman SW 40 Ti rotor. After 22 min of centrifugation at 20,000 rpm at 20°C, an emulsion fraction containing large-sized particles was isolated (fraction 1). Similarly, fractions 2 and 3 were isolated upon subsequent centrifugation at 40,000 rpm for 22 min and 4 to 5 h, respectively. Particle size and homogeneity of the fractions were assayed by photon-correlation spectroscopy using a Malvern 4700 C system (Malvern Instruments, UK). Emulsion fraction 3 used in this study had a mean diameter of 47.7 ± 1.7 nm and was composed (% w/w) of 68 ± 2.4 triolein, 26.9 ± 2.2 phosphatidylcholine, 2.6 ± 0.3 cholesteryl oleate, and 2.8 ± 0.2 cholesterol (Rensen et al., 1997).

Receptor Binding and Competition of ¹²⁵I-OxLDL by RAW Macrophages in Culture. Receptor-binding experiments were performed as described before (H. E. de Vries, B. Buchner, T. J. C. van Berkel, and J. Kuiper, submitted for publication). Briefly, before the receptor-binding experiment, cell culture medium was displaced by DMEM containing 2% BSA for 2 h. Total binding of ¹²⁵I-OxLDL was measured after incubating cells for 2 h at 4°C with various amounts of ¹²⁵I-OxLDL in concentrations ranging from 0.5 µg/ml to 50 µg/ml. Nonspecific binding was determined after incubating the cells with various amounts of ¹²⁵I-OxLDL in the presence of a 10-fold excess of unlabeled OxLDL with a minimum of 100 µg/ml. After 2 h cells were washed and solubilized in 0.1 N NaOH. Solubilized cells were counted for radioactivity, and protein content was measured to determine the cell-associated radioactivity. Dissociation constant (K_d) and B_max were determined from the specific binding curve according to a single site-displacement model using a computerized nonlinear fitting program (minimizing the sum of the squares via the Simplex-iteration procedure of Graphpad Prism; Graphpad Software). Specificity of the binding of ¹²⁵I-OxLDL to macrophages was determined by competition studies. Cells were incubated with 5 µg/ml ¹²⁵I-OxLDL at 4°C for 2 h in the presence of various concentrations of unlabeled competitors: OxLDL, AcLDL, or polyinosinic acid (polyI). After 2 h cells were washed three times and solubilized in 0.1 N NaOH. Cell-associated radioactivity was determined as described above.

Incorporation of AlPc into OxLDL and Emulsions. AlPc was dissolved in 10 ml dimethylformamide at a final concentration of 5 and 50 mM. The solvent was removed by evaporation under reduced pressure. The residual dry films were incubated with 10 ml of OxLDL (0.2 µg/ml) for various periods at 37°C in a shaking water bath. After incorporation, OxLDL-AlPc complexes were purified by gel filtration using a Sepharose G-50 coarse in PBS (10 mM NaPO₄, 150 mM NaCl, pH 7.4). After purification, the concentration of AlPc in the OxLDL fraction was calculated by measurement of its absorption at 670 nm. Absorption measurement throughout the spectrum (450-750 nm) revealed that OxLDL-AlPc has a single peak of absorption at 670 nm in a DU 64 spectrophotometer (Beckman, Fullerton, CA).

Smart FPLC Analysis. The OxLDL-AlPc complexes were characterized by means of Smart FPLC analysis. An aliquot of 80 µl was injected into a Superose 6 Precision Column (PC) (Smart System, Pharmacia, Uppsala, Sweden) and eluted with PBS (pH 7.4). The eluent was monitored continuously for its absorption at 254 nm, and fractions were collected. Subsequently, fluorescence (excitation at 640 nm; emission at 680 nm) was measured in all fractions in an LS-50B luminescence spectrophotometer (Perkin-Elmer, Beaconsfield, UK). After careful characterization, OxLDL-AlPc complexes were used for cellular incubations.

Cellular Incubations. Macrophages were incubated for various periods with increasing concentrations of OxLDL-AlPc. After incubation, cells were illuminated for different periods with a 500 W Halogen lamp equipped with a cut-off filter ( > 600 nm) and a 1-cm water filter to avoid thermal effects. Light intensity was 42 mW/cm² as measured with a Gentec TMP-310 photometer (Quebec). After irradiation, cells were washed with fresh medium and were incubated for 24 h in an incubator. To detect cytotoxicity, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed. Cells were incubated with 0.5 mg/ml MTT in DMEM without serum for 3 h. Subsequently, cells were washed and 30% dimethyl sulfoxide (in PBS) was added. After 30 min, absorption at 550 nm was measured.

Plasma Distribution of OxLDL-AlPc Complexes. To study the distribution of AlPc over the various plasma (lipo-) proteins, OxLDL-AlPc complexes were incubated with freshly isolated murine serum for 1 h at 37°C (1:1 equimolar amounts). Subsequently, the mixture was fractionated by means of SMART FPLC analysis on a Superose 6 Precision Column. The fractions were collected and assayed for fluorescence (excitation at 640 nm; emission at 680 nm). Protein profiles were monitored continuously by UV (254 nm) measurement of the eluent.

Data Analysis. Cytotoxic concentrations of the OxLDL-AlPc complexes were measured, and concentration-response curves were generated. Half-maximal cytotoxic concentrations (EC₅₀) were calculated from the concentration-response curves fitted according to a single site-competition model using a computerized nonlinear fitting program (Graphpad Prism; Graphpad Software). Statistical analysis of the data was performed by a Fisher's exact test, followed by a one-way ANOVA and a Student's t test (P > .05).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Binding of OxLDL to RAW Macrophages. To determine the interaction of OxLDL with RAW macrophages, receptor-binding experiments were performed. The binding of radiolabeled OxLDL to RAW macrophages was determined, and a specific binding of ¹²⁵I-OxLDL with a K_d and a maximal binding of 11 ± 2 µg/ml ¹²⁵I-OxLDL and 702 ± 39 ng bound ¹²⁵I-OxLDL per mg cell protein was defined, respectively (Fig. 1). Specificity of the binding of ¹²⁵I-OxLDL to cultured RAW macrophages was determined by competition experiments. Macrophages were incubated with 5 µg/ml ¹²⁵I-OxLDL in the presence of various concentrations of unlabeled OxLDL, polyI, and AcLDL. Binding of ¹²⁵I-OxLDL to macrophages was competed for by unlabeled OxLDL for 88%, by AcLDL for 48%, and by polyI for 56%.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Binding of 125I-OxLDL to RAW macrophages. Cells were incubated for 2 h at 4°C with increasing concentrations of 125I-OxLDL in the absence () or presence () of an excess of unlabeled OxLDL. Specific binding () was determined as described in Materials and Methods. Data are expressed as ng bound 125I-OxLDL per mg of cell protein and represent the mean ± S.E.M. of three individual measurements.

Incorporation of Photosensitizer in OxLDL. Two residual dry films of AlPc (initial concentrations of 5 and 50 mM in dimethylformamide) were incubated with OxLDL for 30 min, 1 h, 3 h, 6 h, and overnight at 37°C. The concentration of AlPc in OxLDL increased time-dependently. After 6 h of incubation, maximal incorporation of AlPc in OxLDL was found. No difference was detected in the concentration of incorporated AlPc per mg (apo)protein of OxLDL after the incubation of the two dry films of the initial concentrations of AlPc (5 and 50 mM) with OxLDL. Therefore, incorporation was performed with the use of a residual dry film of 5 mM AlPc, which was incubated with OxLDL for 6 h at 37°C. The concentration of AlPc in OxLDL varied between 6 and 8 nmol of AlPc per mg (apo)protein of OxLDL throughout the various preparations.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Elution profile of OxLDL-AlPc complex by Smart FPLC. OxLDL-AlPc was injected into a Superose 6 Precision Column and eluted with PBS, and the eluent was monitored by UV (254 nm; ) measurement. Fractions were assayed for fluorescence (; excitation at 640 nm; emission at 680 nm).

Cellular Incubations. Macrophages were incubated for 24 h with increasing concentrations of OxLDL-AlPc, ranging from 0 to 50 µg/ml. After incubation, cells were irradiated with red light for 0, 5, 10, 20, 30, and 60 min (Fig. 3). After another 24 h, viability of the cells was measured and it was observed that cytotoxicity increased with the time of illumination. The effect at a concentration of OxLDL-AlPc complexes correlated with the duration of illumination (Table 1). After 5 min of illumination, OxLDL-AlPc complexes exerted a half-maximal cytotoxic effect at a concentration of about 45 µg/ml whereas after 60 min of illumination the cytotoxic EC₅₀ value was 5 µg/ml. Nonilluminated cells served as a control, and no cytotoxicity was found in these cells. These data indicate that photoactivation of cells, which are loaded with OxLDL-AlPc, induces cellular damage that is dependent on the time of illumination.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effect of illumination periods on the viability of RAW macrophages after incubation with OxLDL-AlPc. Cells were incubated with increasing concentrations of OxLDL-AlPc for 24 h. After incubation, cells were illuminated for 0 (), 5 (), 10 (), 20 (), 30 (), and 60 () min. Cell viability was measured by MTT test (Materials and Methods). Data are the mean of five individual experiments. S.D. values (not shown) were between 5 and 10% of the indicated values.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of incubation periods on the viability of RAW macrophages incubated with OxLDL-AlPc. Cells were incubated with various concentrations of OxLDL-AlPc for 6 h (, EC50 = 94.2 ± 1.6 µg OxLDL-AlPc/ml), 24 h (, EC50 = 25.7 ± 1.4 µg OxLDL-AlPc/ml), 48 h (, EC50 = 12.8 ± 1.4 µg OxLDL-AlPc/ml), and 72 h (, EC50 = 15.4 ± 1.2 µg OxLDL-AlPc/ml) and subsequently illuminated for 10 min, and cytotoxicity was measured. Nonilluminated cells incubated for 72 h () with OxLDL-AlPc served as a control. Data are the mean of five individual experiments. S.D. values (not shown) were between 5 and 10% of the indicated values.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Inhibitory effects of polyI on the OxLDL-AlPc-induced cytotoxicity of RAW macrophages. RAW macrophages were incubated for 24 h with various concentrations of OxLDL-AlPc complexes in the presence of 0 (), 50 (), 100 (), 150 (), and 200 () µg/ml polyI. Cells were illuminated for 40 min, and cytotoxicity was measured. Data are the mean of five individual experiments. S.D. values (not shown) were between 5 and 10% of the indicated values.

Plasma Distribution of OxLDL-AlPc Complexes. To mimic the in vivo behavior of these complexes, OxLDL-AlPc complexes were incubated with freshly isolated murine serum for 1 h at 37°C in equimolar amounts. An aliquot was used for Smart FPLC analysis, and the plasma distribution of the complexes was analyzed according to the fluorescence profile. Figure 6 shows a fluorescence profile of the murine serum incubated with OxLDL-AlPc. The plasma lipoproteins eluted at the following volumes: very low density lipoprotein at 0.92 ml, LDL at 1.22, high-density lipoprotein (HDL) at 1.55 ml, and the large albumin peak at 1.68 ml. SMART FPLC analysis of the serum incubated with OxLDL-AlPc showed that there was no redistribution of the AlPc-associated OxLDL to the plasma (lipo)proteins (Fig. 6). The fraction eluting at 1.22 ml contained 80 to 90% of the fluorescence, indicating that AlPc remained associated with the OxLDL particle after serum incubation.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Plasma distribution of OxLDL-AlPc in murine serum OxLDL-AlPc was incubated with freshly isolated murine serum (1:1, v/v) for 1 h at 37°C. Plasma distribution of AlPc was analyzed by SMART FPLC analysis, and the eluent was monitored for protein (UV = 254 nm, ). Samples were assayed for fluorescence (excitation, 640 nm; emission, 680 nm) to detect AlPc (). The elution volumes of the plasma proteins are as follows: very low density lipoprotein at 0.92 ml, (Ox)LDL at 1.22 ml, HDL at 1.55 ml, and albumin at 1.68 ml.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The remodeling of a vessel and reoccurrence of atherosclerosis after angioplasty limit the current clinical therapies for the treatment of cardiovascular diseases. Recently, the use of photodynamic therapy for the treatment of atherosclerosis and restenosis was suggested. Studies have reported that various photosensitizers, such as phthalocyanines, selectively accumulate in the atherosclerotic lesions compared with the adjacent normal vessel (Eldar et al., 1990; Nyamekye et al., 1995). The subsequent local irradiation of the lumen of the vessel with light of the appropriate wavelength may reduce the size of the atherosclerotic plaque. Moreover, the use of a specific delivery system to target compounds into the atherosclerotic plaque may lead to an enhanced accumulation of PS in atherosclerotic lesions (Visona and Jori, 1993; Allison et al., 1997).

In the present study, we suggest the use of OxLDL as a carrier for the photosensitizer AlPc. OxLDL itself is efficiently taken up and processed via so-called scavenger receptors expressed on the cell surface of macrophages. Four different scavenger receptors expressed on macrophages are able to interact with OxLDL. Two isoforms of the class A scavenger receptor with similar binding properties are characterized, scavenger receptor type A (SRAI/SRAII) (Goldstein et al., 1979; Brown et al., 1980; Kodama et al., 1990). These receptors are localized mainly on tissue macrophages and endothelial cells. CD36, a member of the class B scavenger receptors, is a membrane glycoprotein that can mediate the uptake of OxLDL but is not polyI-sensitive. Recently, the macrophage-protein macrosialin has been described to bind OxLDL (Endemann et al., 1993; Acton et al., 1994; De Rijke and van Berkel, 1994; Ottnad et al., 1995; Ramprasad et al., 1995, 1996; Alessio et al., 1996). In this study, we show that OxLDL can interact specifically with RAW macrophages via scavenger receptors. The binding of OXLDL to the RAW macrophages can be inhibited by polyI for 71 ± 5%, and this indicates that the polyI-insensitive binding to the RAW macrophages is mediated by CD36. In addition, the polymerase chain reaction analysis on mRNA extracted from RAW cells identified the presence of the four aforementioned scavenger receptors (our unpublished data).

The rationale for the use of OxLDL as a targeting device for scavenger receptors is the fact that these types of receptors are expressed on macrophages in the atherosclerotic plaque. For instance, SRAI/AII are localized on the monocyte-derived macrophages in the atherosclerotic plaque. High levels of the mRNA for and protein of the SRAI/AII are detected on macrophage-derived foam cells in atherosclerotic lesions by in situ hybridization and immunocytochemistry, respectively (Ylä-Herttuala et al., 1991; Matsumoto et al., 1990; Naito et al., 1992; Geng et al., 1995). In previous studies, we have shown that the expression level of receptors for OxLDL on macrophage foam cells and their capacity to process OxLDL increase during the atherosclerotic process (H. E. deVries, B. Buchner, T. J. C. van Berkel, and J. Kuiper, submitted for publication). Besides the high levels of expression, another attractive feature of using SRAI/AII as a target is that its expression is not down-regulated by the intracellular cholesterol content (Goldstein et al., 1979; Brown et al., 1980). In addition, CD36 is expressed in high amounts on monocyte-derived macrophages, is involved in the recognition and processing of OxLDL, and may be present on macrophage-derived foam cells (Endemann et al., 1993; Nicholson et al., 1995; Nozaki et al., 1995; H. E. deVries, B. Buchner, T. J. C. van Berkel, and J. Kuiper, submitted for publication). Therefore, these structures can be considered as major candidates for targeting strategies to the macrophage-rich atherosclerotic lesions.

Because OxLDL is a specific ligand for the aforementioned scavenger receptors, we used OxLDL as a drug-delivery system for the photosensitizer AlPc. OxLDL-AlPc complexes were prepared and characterized carefully by monitoring their electrophoretic mobility and their elution profile on Smart FPLC.

Photoactivation of cells loaded with OxLDL-AlPc complexes resulted in selective cell death that correlated with the time of illumination. The cytotoxic effect of OxLDL-AlPc on RAW cells is concentration- and incubation time-dependent. No cytotoxicity was observed in cells incubated for 72 h with increasing concentrations of OxLDL-AlPc that were not irradiated with red light. Therefore, it is concluded that cytotoxicity of the cells after illumination was the result of photoactivation of the AlPc. Photoactivation of AlPc may lead to the formation of reactive singlet oxygen, which interacts with cell membrane components and induces cell death. These results demonstrated that the AlPc, incorporated in OxLDL, is taken up intact and is still able to be photoactivated.

As a control for nonspecific uptake of AlPc in lipid-like particles in these cells, protein-free triglyceride-rich emulsions were loaded with AlPc. After incubation and illumination, cytotoxicity was 8- to 10-fold lower in comparison with cell death observed after incubation of the cells with OxLDL-AlPc complexes. Therefore, it was concluded that scavenger receptors on the RAW cells efficiently mediated the uptake of the OxLDL-AlPc complexes. These suggestions are confirmed by the observation that, in the presence of the polyI, a ligand for the scavenger receptor, the uptake of the complexes could be blocked in a concentration-dependent manner.

To mimic the in vivo behavior of the complexes, OxLDL-AlPc was incubated with freshly isolated serum. Serum profiles were analyzed by FPLC analysis and assayed for protein and fluorescence. No redistribution of the AlPc to the plasma lipoproteins was detected, and 80 to 90% of the AlPc remained associated with the OxLDL particle. These data suggest that OxLDL can be used as a stable carrier for photosensitizers and may function as a delivery system to scavenger receptors in atherosclerotic plaques. The local application of light of the appropriate wavelength may induce photoactivation of AlPc and subsequent cell death within the atherosclerotic plaque. The preparation of a stable delivery system as described here is in contrast with observations by Visona and Jori (1993). Systemic administration of unilamellar liposomes containing the phthalocyanine ZnPC led to the uptake of the photosensitizer in the atherosclerotic lesions but not in normal vessel. However, the ZnPC that was incorporated into unilamellar liposomes redistributed predominantly into the fraction of HDL (Visona and Jori, 1993). In addition, Allison et al. (1997) compared the delivery of the photosensitizer BPD incorporated in liposomes LDL and AcLDL to the atheromous regions of the vessel. AcLDL-BPD complexes accumulated in the atherosclerotic plaques of Watanabe Heritable Hyperlipidemic and balloon-injured New Zealand rabbits compared to LDL or liposomes loaded with BPD. The selective accumulation of AcLDL-BPD complexes in the atherosclerotic lesion may be mediated by the interaction of AcLDL with SRAI/AII. However, after systemic administration of the AcLDL-BPD complexes, a redistribution of the BPD to other lipoproteins was observed, indicating that AcLDL-BPD complexes are not stable in vivo (Allison et al., 1997). These observations show the novelty of our present approach: our data demonstrate that the photosensitizer AlPc remains associated with the OxLDL particle after incubation with serum, which can function as a stable delivery system. Therefore, the use of OxLDL as a stable carrier system for photosensitizers may lead to augmented accumulation of photosensitizer in the atherosclerotic plaque. This strategy may be an important contribution to the potential benefit of PDT in the treatment of cardiovascular diseases.