Effect of Photodynamic Pretreatment on the Susceptibility of Murine Tumor Cells To Macrophage Antitumor Mechanisms

Abstract— In vitro photodynamic treatment of YAC-1 murine T-lymphoma cells with the hematoporphyrin derivative Photosan 3 and red light resulted in dose-dependent phototoxicity. Photodynamic pretreatment, however, did not render these cells more susceptible to macrophage-me-diated tumor cytotoxicity or the cytotoxic effects of mac-rophage-derived antitumor mediators like tumor necrosis factor aL (TNF-aL) or interferon bT (IFN-bT). Independent of the degree of photosensitization used, the cytotoxicity values obtained with macrophages or the different mediators were shifted by the respective values for phototoxicity, suggesting these effects to be additive and thus not interdependent. These data show that while higher overall tumor cytotoxicity can be achieved by a combination of photodynamic treatment and macro-phage-mediated tumor destruction, this apparently is not a result of enhanced sensitivity of photodynamically treated tumor cells to macrophage antitumor mechanisms in general     

Activation of mouse macrophages by in vivo and in vitro treatment with a cyanine dye, lumin.

The cyanine photosensitizer, lumin, is a potent macrophage activating agent: 4 days after administration of small amounts of lumin to mice (20-40 ng mouse-1), peritoneal macrophages exhibited a greatly enhanced Fc-mediated ingestion activity; higher doses (more than 3000 ng mouse-1) did not have this effect. The in vitro photodynamic activation of macrophages in mouse peritoneal cells exposed to white fluorescent light (3 J m-2 s-1) was also studied in media containing various concentrations of lumin. A short light exposure (45 J m-2) with 10 ng lumin ml-1 produced a maximum ingestion activity of macrophages. Lumin has absorption peaks at 670 and 760 nm. Therefore we designed experiments in which peritoneal cells were exposed to a red fluorescent light (emission, 660 nm; 0.5 J m-2 s-1). In a medium containing 3 ng lumin ml-1 with 7.5 J m-2 of red light, a markedly enhanced ingestion activity of macrophages was observed. The photodynamic treatment of peritoneal macrophages alone did not stimulate phagocytic activity, but the photodynamic treatment of a mixture of non-adherent (B and T) cells and macrophages resulted in a greatly enhanced ingestion activity of macrophages. Thus non-adherent cells are required for the photodynamic activation of macrophages, implying that an activating factor is generated within the non-adherent cells and transmitted to the macrophages. This hypothesis was confirmed by the observation that co-cultivation of photodynamically treated non-adherent cells with untreated macrophages resulted in a greatly enhanced ingestion capacity.     

TUMORICIDAL CAPACITIES OF MACROPHAGES PHOTODYNAMICALLY ACTIVATED WITH HEMATOPORPHYRIN DERIVATIVE

Four days after administration to mice of small amounts (30-600 ng/mouse) of hematoporphyrin derivative (HPD), peritoneal macrophages exhibited a greatly enhanced Fc-receptor mediated phagocytic capacity as assayed by ingestion activity of IgG-coated sheep erythrocytes. Much higher doses (greater than 3000 ng/mouse) did not have this effect. The peritoneal macrophages activated by administration of HPD have tumoricidal capacity for IgG-coated retinoblastoma cells. We then studied in vitro photodynamic activation of macrophages by white and red fluorescent light irradiation of mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) in media containing very low concentrations of HPD. A short (5 s) white fluorescent light exposure (1Wm-2) of peritoneal cells in a medium containing 0.03 ng HPD/mL produced the maximal level of ingestion activity of macrophages. A 15 s red fluorescent light exposure (1Wm-2) of peritoneal cells in a medium containing 0.1 ng HPD/mL produced the maximal level of ingestion activity of macrophages. Thus, photodynamic activation of macrophages with white fluorescent light is more efficient than that with red fluorescent light. This can be explained by the fact that HPD has a large absorption peak at about 364 nm which extends into the visible range, and decreasingly smaller absorption bands at 500, 535, 570 and 630 nm. In vitro photodynamically activated macrophages showed efficient tumoricidal activity regardless of the type (white or red) of light used. These results suggest that a low level of HPD promotes therapeutic immunopotentiation.     

EFFECTIVENESS OF PHOTOFRIN II IN ACIWATION OF MACROPHAGES AND in vitro KILLING OF RETINOBLASTOMA CELLS

Abstract Administration of a small dose (300 ng/mouse) of photofrin II (PII) to mice, followed by 4 days of exposure to only ambient fluorescent light in animal quarters, induced Fc-receptor-mediated phagocytic and superoxide-generating capacities of peritoneal macrophages by five- and seven-fold, respectively. When these mice were kept in the dark for 4 days, no activation of macrophages was observed. These results suggest that macrophage activation is a consequence of photodynamic activation. Much higher doses (> 3000 ng/mouse) suppressed macrophage activity. However, 2 months after administration of 3000 ng PII/mouse, greatly enhanced phagocytic and superoxide-generating capacities of peritoneal macrophages were observed.
In vitro photodynamic activation of macrophages was analyzed after white or red fluorescent light exposure of mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) in media containing PII. A short (10 s) white fluorescent light treatment of peritoneal cells in a medium containing 0.03 ng PII/mL produced the maximal level of phagocytic activity of macrophages. Illumination with the same total fluence of red fluorescent light requires a threefold higher concentration of PII to achieve the same extent of enhanced phagocytic activity of macrophages. Thus, photodynamic activation of macrophages with PII by white fluorescent light was more efficient than by red fluorescent light. Similarly, photodynamic killing of retinoblastoma cells was more efficient with white than red fluorescent light. The concentration of hematoporphyrin (HP) or PII required for direct photodynamic killing of retinoblastoma cells was roughly four orders of magnitude greater than that required for activation of macrophages. These results suggest that effective photodynamic therapy may be achieved with milder treatments that stimulate macrophage activity, an important component of immunopotentiation.     

Elevation of intracellular cyclic adenosine 3',5'-monophosphate levels in macrophages activated with lipopolysaccharide for tumor cell killing.

The macrophage activation for tumor cytotoxicity with lipopolysaccharide (LPS) was remarkably inhibited by adding indomethacin (5 x 10(-6) M) or 10mM LiCl which is known to inhibit adenylate cyclase activity. The tumor cytotoxicity of macrophages inhibited with these agents was recovered by adding dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) (10(-4) or 10(-5) M) and furthermore tumor cell killing activity was augmented as compared with LPS-activated macrophage. Macrophages showed a 5 to 6 times increased intracellular cAMP concentration over the control within 30 min when incubated with LPS. However, the increased intracellular cAMP concentration was decreased by adding LiCl (10 mM). Thus, these findings indicate that there is an important relation between intracellular cAMP concentration and the mechanism of macrophage activation. One can then conclude that at least the initial enhancement of intracellular cAMP was important for tumor cell killing as a signal transmission in macrophage activated by LPS.     

5-Aminolevulinic acid-induced protoporphyrin-IX accumulation and associated phototoxicity in macrophages and oral cancer cell lines

Studies were carried out on 5-aminolevulinic acid (ALA)-induced protoporphyrin (PpIX) synthesis in mice peritoneal macrophages and two human oral squamous cell carcinoma (OSCC) cell lines NT8e and 4451. Cells were treated with 200 microg/ml ALA for 15 h and PpIX accumulation was monitored by spectrofluorometry and phototoxicity to red light (630+/-20 nm) was measured by MTT assay. PpIX accumulation was higher in macrophages as compared to OSCC cells under both normal serum concentration (10%) and conditions of serum depletion. The results on phototoxicity measurements correlated well with the levels of PpIX accumulation in both macrophages and cancer cells. While red light caused 20% phototoxicity in macrophages, no phototoxicity was seen in 4451 cells at 10% serum. Decrease in serum concentration to 5% and 1% led to higher phototoxicity corresponding to 40% and 70% in macrophages and 10% and 15% in 4451 cells. Similar results were obtained in NT8e cell line. Propidium iodide staining followed by fluorescence microscopic observations on photodynamically treated co-culture of murine or human macrophages and cancer cells showed selective damage to macrophages. These results suggest that in OSCC, macrophages would contribute more to tumor PpIX level than tumor cells themselves and PDT may lead to selective killing of macrophages at the site of treatment. Since macrophages are responsible for production and secretion of various tumor growth mediators, the effect of selective macrophage killing on the outcome of PDT would be significant.     

EFFECT OF PHOTODYNAMIC THERAPY ON ANTITUMOR IMMUNE DEFENSES: COMPARISON OF THE PHOTOSENSITIZERS HEMATOPORPHYRIN DERIVATIVE AND CHLORO-ALUMINUM SULFONATED PHTHALOCYANINE

The effects of the two photosensitizers chloroaluminum sulfonated phthalocyanine (ClAlSPc) and hematoporphyrin derivative (HpD) on the functional activities of macrophages and natural killer (NK) cells, two immunocyte populations implicated in the control of tumor development and spread, have been investigated. Murine peritoneal macrophages treated in vivo with ClAlSPc or HpD at 10 mg/kg body weight showed no impairment of Fc-mediated phagocytic capacity and only minor disturbances of in vitro tumoricidal/tumoristatic function. The NK cell activity of splenocytes obtained from photosensitizer-treated mice, assayed 24 or 48 h after i.v. injection of ClAlSPc or HpD at 10 mg/kg was unaffected compared to controls. However significant inhibition of NK activity was observed when splenocytes obtained from mice with or without subcutaneous Colo 26 tumors, treated with ClAlSPc plus laser therapy (675 nm) were used as effector cells. The results show that impairment of some anti-tumor activity can be observed in phthalocyanine treated or phthalocyanine + laser-treated animals but this relatively minor impairment may augur well for the use of systemic phthalocyanine administration in photodynamic therapy.     

Sub-lethal Photodynamic Damage to ARPE-19 Cells Transiently Inhibits Their Phagocytic Activity†

Efficient phagocytosis of photoreceptor outer segments (POS) membranes by retinal pigment epithelium (RPE) plays a key role in biological renewal of these highly peroxidizable structures. Here, we tested whether photodynamic treatment, mediated by merocyanine 540 (MC 540), rose Bengal or a zinc-substituted chlorophyllide inhibited phagocytic activity of ARPE-19 cells in vitro. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS isolated from cow retinas and nonspecific phagocytosis of fluorescent polystyrene beads were measured by flow cytometry. Photodynamic treatment, mediated by all three photosensitizers with sub-threshold doses, induced significant inhibition of the cell-specific phagocytosis. The nonspecific phagocytosis was inhibited by photodynamic treatment mediated only by MC 540. The inhibition of phagocytosis was a reversible phenomenon and after 24 h, the photodynamically treated cells exhibited phagocytic activity that was comparable with that of untreated cells. This study provides proof of principle that sub-threshold photodynamic treatment of ARPE-19 cells with appropriate photosensitizers is a convenient experimental approach for in vitro study of the effects of oxidative stress on specific phagocytic activity of RPE cells. We postulate that oxidative damage to key components of the cell phagocytic machinery may be responsible for severe impairment of its activity, which can lead to retinal degeneration.     

Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6

Effect of varying extracellular pH on mode of cell death induced by photodynamic action of chlorin p6 was investigated in human colon carcinoma (Colo-205) cells. At an extracellular pH of 7.4, compared to cells treated with chlorin p6 in dark, the photodynamically treated cells showed reduction in mitochondrial membrane potential, an increase in ADP/ATP ratio (1:2) and a large percentage of cells with chromatin condensation. In contrast, when photodynamic treatment and post irradiation incubation was carried out in acidic medium (pH 6.5), total loss of mitochondrial membrane potential, a marked increase in ADP/ATP ratio (1:33) and increased damage to plasma membrane were observed. Further, cells subjected to photodynamic treatment in a medium of pH 7.4 showed twofold increase in caspase-3 activity as compared to photodynamic treatment at pH 6.5. These results suggest that chlorin p6 mediated photodynamic action induces apoptotic cell death when extracellular pH is 7.4 whereas necrosis is more predominant under condition when extracellular pH is 6.5.     

PHOTODYNAMIC TREATMENT OF HERPES SIMPLEX VIRUS INFECTION IN VITRO

Abstract— The effects of photodynamic action on in vitro herpes simplex virus infections of CV-1 monkey kidney fibroblasts or human skin fibroblasts were determined using proflavine sulfate and white fluorescent lamps. Photodynamic treatment of confluent cell monolayers prior to virus infection inactivated cell capacity, i.e. the capacity of the treated cells to support subsequent virus growth as measured by plaque formation. The capacity of human cells was more sensitive to inactivation than the capacity of monkey cells when 6 μM proflavine was used. Treated cell monolayers recovered the capacity to support virus plaque formation when virus infection was delayed four days after the treatment. Experiments in which the photodynamically treated monolayers were infected with UV-irradiated virus demonstrated that this treatment induced Weigle reactivation in both types of cells. This reactivation occurred for virus infection just after treatment or 4 days later. A Luria-Latarjet-type experiment was also performed in which cultures infected with unirradiated virus were photodynamically treated at different times after the start of infection. The results showed that for the first several hours of the virus infection the infected cultures were more sensitive to inactivation by photodynamic treatment than cell capacity. By the end of the eclipse period the infected cultures were less sensitive to inactivation than cell capacity. Results from extracellular inactivation of virus grown in monkey cells at 6 μM proflavine indicated that at physiological pH the virus has a sensitivity to photodynamic inactivation similar to that for inactivation of cell capacity. The combined data indicated that photodynamic treatment of the cell before or after virus infection could prevent virus growth. Thus, photodynamic inactivation of infected and uninfected cells may be as important as inactivation of virus particles when considering possible mechanisms in clinical photodynamic therapy for herpes simplex.     

Synergistic Interaction of Photodynamic Treatment with the Sensitizer Aluminum Phthalocyanine and Hyperthermia on Loss of Clonogenicity of CHO Cells

Abstract— When CHO cells were exposed to hyperthermia and subsequently to photodynamic treatment, the combined effects were additive but in the reverse sequence the interaction was synergistic. The synergistic interaction comprised two quite different components: (1) photodynam-ically induced sensitization of cellular proteins and/or supramolecular structures for thermal inactivation and (2) a photodynamically induced inhibition of the cellular repair system for sublethal thermal damage. The first component of the synergistic interaction was reflected by a change of the Arrhenius parameters of thermal cell killing. A lowering of the activation energy of this process was responsible for the synergistic interactions, whereas a concomitant decrease of the frequency factor, opposing this effect, actually caused a much lower degree of synergism at higher temperatures. This component of the synergistic interaction did not respond to the insertion of an intermediate incubation period between the two treatments. The second component of the synergistic interaction, viz. the interference with the ability of cells to survive sublethal thermal damage, was reversible, as an intermediate incubation between photodynamic treatment and hyperthermia resulted in its repair. The photodynamically induced inhibition of the ability of cells to survive sublethal thermal damage was not related to ATP or glutathione depletion, inhibition of de novo protein synthesis or impairment of degradation of damaged protein molecules. Restoration of the repair system for sublethal damage depended on a metabolic process and required free intracellular Ca²⁺, suggesting that a cell signaling pathway may be involved. Thus, in a practical sense the magnitude of the synergistic interaction between photodynamic treatment and hyperthermia depends on the length of the interval between the two treatments and on the temperature and duration of the subsequent thermal treatment. This may have significant consequences for the development of clinical protocols.     

PHOTODYNAMIC ACTION OF CHEMICALLY RELATED FLAVONES

Abstract— Flavone, polyhydroxyflavones (apigenin, fisetin, kaempferol, luteolin, myricetin, quercetin, resokaempferol and robinetin), polymethoxyflavones and acetylated and benzylated flavones were tested for photodynamic activity using Tetrahymena pyriformis T as the test organism. Among these compounds, polymethoxyflavones showed the highest order of activity, followed by flavone and then flavone derivatives with OH and OCH₃ groups. Resokaempferol was the only active polyhydroxyflavone, the remainder being inactive such as the benzyl-derivative. The methoxyl group in the 5–position and an increase in number of methoxyl groups from one to three in the phenolic portion of the flavonoid tended to decrease photodynamic activity. Tetrahymena killed photodynamically by polymethoxyflavones were morphologically altered by blister-like blebs. Polymethoxyflavones showed the lowest cytotoxicity and the greatest photodynamic activity among those flavonoids tested. The majority of the favonoids in this series have absorption spectra in the 320–370 nm region.     

Toward Precise Antitumoral Photodynamic Therapy Using a Dual Receptor-Mediated Bioorthogonal Activation Approach

Targeted delivery and specific activation of photosensitizers can greatly improve the treatment outcome of photodynamic therapy. To this end, we report herein a novel dual receptor-mediated bioorthogonal activation approach to enhance the tumor specificity of the photodynamic action. It involves the targeted delivery of a biotinylated boron dipyrromethene (BODIPY)-based photosensitizer, which is quenched in the native form by the attached 1,2,4,5-tetrazine unit, and an epidermal growth factor receptor (EGFR)-targeting cyclic peptide conjugated with a bicycle[6.1.0]non-4-yne moiety. Only for cancer cells that overexpress both the biotin receptor and EGFR, the two components can be internalized preferentially where they undergo an inverse electron-demand Diels–Alder reaction, leading to restoration of the photodynamic activity of the BODIPY core. By using a range of cell lines with different expression levels of these two receptors, we have demonstrated that this stepwise “deliver-and-click” approach can confine the photodynamic action on a specific type of cancer cells.     

PHOTODYNAMIC EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE UPTAKE OF RHODAMINE 123 BY MITOCHONDRIA OF INTACT MURINE L929 FIBROBLASTS AND CHINESE HAMSTER OVARY Kl CELLS

Abstract— It was shown that the cationic fluorescence probe rhodamine 123 accumulates in mitochondria of murine L929 fibroblasts and Chinese hamster ovary Kl epithelial cells due to the driving force of both plasma membrane and mitochondrial membrane potentials. Photodynamic treatment of L929 cells with hematoporphyrin derivative resulted in an increased uptake of rhodamine 123 and a diminished uptake of 1,1,3,3,3',3'-hexamethylindocarbocyanine iodide. This indicates a considerably increased mitochondrial membrane potential, which most likely is the result of a direct or secondary inhibition of the ATP-synthetase, and a decreased plasma membrane potential. The oxygen consumption rate and the ATP level decreased due to photodynamic treatment. Post-incubation of L929 cells subsequent to photodynamic treatment revealed that the uptake of rhodamine 123. the ATP content and the oxygen consumption rate were restored. For all parameters similar results were obtained with CHO-K1 cells, with the exception that during post-incubation the intracellular ATP content remained at the level reached after illumination. These results indicate that photodynamically induced disturbance of mitochondrial functions and the ATP level are not crucial for the loss of clonogenicity of L929 cells. In CHO-K1 cells however, the continuously lowered ATP level may have detrimental consequences for cell survival. The photodynamic stimulation of the rhodamine 123 uptake may be a rather general phenomenon. Because rhodamine 123 exhibits a much higher toxicity towards carcinoma cells than towards other cells, a synergistic interaction between this drug and photodynamic therapy (PDT) may be anticipated, if PDT also stimulates mitochondrial rhodamine 123 accumulation in carcinoma in vivo.     

INTERACTION OF PHOTODYNAMICALLY INDUCED CELL KILLING and DARK CYTOTOXICITY OF RHODAMINE 123

Abstract— Loss of clonogenicity of Chinese hamster ovary (CHO) cells, murine L929 fibroblasts and human bladder carcinoma T24 cells caused by photodynamic treatment (PDT) with hematoporphyrin derivative (HPD) is synergistically enhanced by subsequent incubation with rhodamine 123 in the dark. For CHO and L929 cells this synergistic interaction can be explained by an increased uptake of rhodamine 123 as the result of the photodynamic treatment. With aluminum phthalocyanine (AIPc) as photosensitizer only additive effects were observed in the three cell lines. Incubation in the dark with rhodamine 123, followed by a photodynamic treatment with HPD, resulted in an antagonistic interaction with regard to loss of colony formation. With AIPc the combination of treatments resulted in an additive effect with L929 and T24 cells, whereas with CHO cells a slight antagonistic interaction was observed. An antagonistic effect was also observed in model experiments, treating histidine photodynamically with HPD and measuring oxygen consumption. A possible explanation of these results could be an interaction or complex formation of rhodamine 123 with HPD resulting in a diminished singlet oxygen production. With AIPc this does not take place.     

Role of complement anaphylatoxin C3a in photodynamic therapy-elicited engagement of host neutrophils and other immune cells

Tumor treatment by photodynamic therapy (PDT) provokes a host-protective inflammatory and acute-phase response and an immune reaction. Neutrophilia manifested in this context is driven by multiple mediators of neutrophil chemotaxis orchestrated by an activated complement system. Mouse FsaR fibrosarcoma was used in this study to further investigate neutrophilia induced by Photofrin-based PDT. The complement anaphylatoxin C3a was identified as a major chemoattractant in the advanced phase of PDT-induced neutrophilia, because injecting mice with antibodies blocking its receptor C3aR significantly inhibited the increase in neutrophil levels 8 h after PDT. At the same time point, an increased C3aR expression was detected in neutrophils, monocytes and B lymphocytes in the blood of host mice. Peritoneal macrophages and mast cells harvested from treatment-naive mice exhibited elevated C3aR expression after coincubation in vitro for 8 h with PDT-treated FsaR cells. Thus, C3a emerges as one of the key effector molecules engaged in PDT-induced host response.     

Type I macrophage activator photosensitizer against hypoxic tumors

Photodynamic immunotherapy has emerged as a promising strategy to treat cancer. However, the hypoxic nature of most solid tumors and notoriously immunosuppressive tumor microenvironment could greatly compromise the efficacy of photodynamic immunotherapy. To address this challenge, we rationally synthesized a type I photosensitizer of TPA-DCR nanoparticles (NPs) with aggregation-enhanced reactive oxygen species generation via an oxygen-independent pathway. We demonstrated that the free radicals produced by TPA-DCR NPs could reprogram M0 and M2 macrophages into an anti-tumor state, which is not restricted by the hypoxic conditions. The activated M1 macrophages could further induce the immunogenic cell death of cancer cells by secreting pro-inflammatory cytokines and phagocytosis. In addition, in vivo anti-tumor experiments revealed that the TPA-DCR NPs could further trigger tumor immune response by re-educating tumor-associated macrophages toward M1 phenotype and promoting T cell infiltration. Overall, this work demonstrates the design of type I organic photosensitizers and mechanistic investigation of their superior anti-tumor efficacy. The results will benefit the exploration of advanced strategies to regulate the tumor microenvironment for effective photodynamic immunotherapy against hypoxic tumors.

The photosensitizer-triggered macrophage-mediated photodynamic immunotherapy is reported. The TPA-DCR NPs induce the ICD of hypoxic tumor by generating type I ROS to polarize macrophage, then promote tumor infiltration of T cells.     

A Gene-Editable Palladium-Based Bioorthogonal Nanoplatform Facilitates Macrophage Phagocytosis for Tumor Therapy

Macrophage phagocytosis of tumor cells has emerged as an attractive strategy for tumor therapy. Nevertheless, immunosuppressive M2 macrophages in the tumor microenvironment and the high expression of anti-phagocytic signals from tumor cells impede therapeutic efficacy. To address these issues and improve the management of malignant tumors, in this study we developed a gene-editable palladium-based bioorthogonal nanoplatform, consisting of CRISPR/Cas9 gene editing system-linked Pd nanoclusters, and a hyaluronic acid surface layer (HBPdC). This HBPdC nanoplatform exhibited satisfactory tumor-targeting efficiency and triggered Fenton-like reactions in the tumor microenvironment to generate reactive oxygen species for chemodynamic therapy and macrophage M1 polarization, which directly eliminated tumor cells, and stimulated the antitumor response of macrophages. HBPdC could reprogram tumor cells through gene editing to reduce the expression of CD47 and adipocyte plasma membrane-associated protein, thereby promoting their recognition and phagocytosis by macrophages. Moreover, HBPdC induced the activation of sequestered prodrugs via bioorthogonal catalysis, enabling chemotherapy and thereby enhancing tumor cell death. Importantly, the Pd nanoclusters of HBPdC were sufficiently cleared through basic metabolic pathways, confirming their biocompatibility and biosafety. Therefore, by promoting macrophage phagocytosis, the HBPdC system developed herein represents a highly promising antitumor toolset for cancer therapy applications.     

SENSITIVITY OF ACTIVATED MURINE PERITONEAL MACROPHAGES TO PHOTODYNAMIC KILLING WITH BENZOPORPHYRIN DERIVATIVE

Abstract— This study compared the ability of highly purified resting and activated DBA/2 mouse peritoneal macrophages to survive treatment with the photosensitizer benzoporphyrin derivative (BPD, verteporfin) and light. Culture of macrophages with recombinant murine interferon-γ (rIFN-γ, 100 U/mL) for 72 h imparted a phenotypic and functional activation by dramatically increasing cell surface expression of major histocompatibility complex Class II (Ia) molecules and the formation of nitric oxide. The rIFN-γ-activated macrophages were significantly (P < 0.05) more sensitive (lethal dose to cause a 50% reduction in cell survival, LD₅₀= 14.4 ± 1.1 ng/mL) to photodynamic killing with BPD and light (10 J/cm²) than cells (LD₅₀= 18.2 ± 2.0 ng/mL) cultured in medium alone. In contrast, macrophages treated with different concentrations of bacterial lipopolysaccharide (LPS) were as resistant or more resistant to photodynamic killing than cells cultured in medium alone. No cytotoxic effect of BPD was detected in cultures containing the drug but protected from light. Comparable amounts of BPD were taken up in vitro by unactivated and rIFN-γ-activated macrophages, as detected by flow cytometric analysis. However, cells cultured with LPS (10 μg/mL) took up more BPD than macrophages cultured in medium alone or with rIFN-γ. The DBA/2 P815 mastocytoma cells took up greater amounts of the drug and were subsequently more vulnerable to treatment with BPD and light (LD₅₀= 6.9 ng/mL) than macrophages cultured under any condition. The explanation for the increased vulnerability of rIFN-γ-activated macrophages and the greater resistance of LPS-activated macrophages, relative to medium-cultured macrophages, to photodynamic killing with BPD is uncertain. However, the increased susceptibility of macrophages, activated with the immunomodulatory cytokine IFN-γ, to treatment with BPD and light might indicate how photodynamic therapy could interfere with the development of experimental autoimmune disease, conditions in which activated macrophages are known to be involved.