Photodynamic Therapy: Tumor Targeting with Adenoviral Proteins

A brief summary of the mechanisms involved in photodynamic therapy (PDT) and the role of delivery vehicles for photosensitizer targeting is addressed. Phthalocyanines (Pc) have been coupled to adenovirus type 2 capsid proteins including the hexon, the penton base and the fiber to enhance their target selectivity. Adenovirus penton base proteins contain the arginine-glycine-aspartic acid peptidic sequence (RGD) motif known to bind with great affinity and high specificity to integrin receptors, expressed by several types of cancer. Tetrasulfonated aluminum phthalocyanine (AlPcS4) was covalently coupled to the various capsid proteins via one or two caproic acid spacer chains (A1 or A2) in 7:1 up to 66:1 molar ratios. The capacity of the bioconjugates for singlet oxygen production, as measured by an L-tryptophan oxidation assay, was strongly reduced, likely reflecting scavenging by the carrier. Cell adsorption and in vitro photocytotoxicity assays were carried out using the A549 and HEp2 human cell lines expressing integrin receptors, and one murine, the EMT-6 cell line, which lacks receptors for the RGD sequence. The AlPcS4A2-protein complexes induced greater cytotoxicity as compared to the analogous AlPcS4A1 preparations. The penton base-AlPcS4A2 derivative was the more phototoxic for all cell lines tested. Tumor response studies using Balb/c mice with EMT-6 tumor implants demonstrated that the free AlPcS4A2 induced complete tumor regression at a dose of 1 mumol/kg and 400 J/cm2, which is comparable to the activity of the known AlPcS2adj. A mixture of adenovirus type 2 soluble proteins covalently labeled with AlPcS4A2 required 0.5 mumol/kg to induce the same response with the same light dose, suggesting that the high affinity RGD/receptor complex is able to target Pc for PDT.     

Receptor-mediated targeting of phthalocyanines to macrophages via covalent coupling to native or maleylated bovine serum albumin

Targeted delivery of aluminum tetrasulfophthalocyanine (AlPcS4) to the scavenger receptor of macrophages, via coupling to maleylated bovine serum albumin (mal-BSA), was explored as a means to improve photodynamic efficacy. The AlPcS4 was covalently coupled to BSA (9:1 molar ratio) via one or two sulfonamide-hexanoic-amide spacer chains, followed by treatment with maleic anhydride to yield the mal-BSA-phthalocyanine conjugates. The latter were tested for singlet oxygen production, receptor-mediated cell uptake and phototoxicity toward J774 cells of macrophage origin and nonphagocytic EMT-6 cells. Cell uptake of 125I-mal-BSA showed specific binding for J774 cells but not for EMT-6 cells. Competition studies of the conjugates with 125I-mal-BSA showed that coupling of AlPcS4 to BSA resulted in recognition of the conjugate by the scavenger receptor, whereas coupling to mal-BSA further enhanced its binding affinity. This suggests that affinity for the scavenger receptor is related to the overall negative charge of the protein. Phototoxicity of the conjugates toward J774 cells paralleled their relative affinity, with mal-BSA-AlPcS4 coupled via two spacer chains showing the highest activity. The conjugates were less phototoxic toward the EMT-6 cell line. The activities in both cell lines of all conjugated AlPcS4 preparations were, however, lower than that of the free disulfonated AlPcS2. Possible implications for the in vivo use of protein-photosensitizer conjugates to target selectively various macrophage-associated disorders is discussed.     

Photodynamic properties of naphthosulfobenzoporphyrazines, novel asymmetric, amphiphilic phthalocyanine derivatives.

Metallo naphthosulfobenzoporphyrazines sulfonated to different degrees (M-NSBP) were prepared, and their potential as photosensitizers for the photodynamic therapy (PDT) of cancer was evaluated. M-NSBP can be viewed as hybrid molecules between sulfophthalocyanines and naphthalocyanines resulting in distinct differences in the absorption spectra between the mono-through tetrasulfonated derivatives. This feature greatly facilited their purification. Using V-79 Chinese hamster cells in vitro, the disulfonated derivatives were found slightly more photoactive than the hydrophilic trisulfonated derivatives while the monosulfonated derivative was inactive, in spite of a sixfold higher cell uptake. In the case of the di- and trisulfonated derivatives, differences in phototoxicity correlated well with their relative cell uptake. Substitution of Al for Zn had little effect on the extent of phototoxicity of the M-NSBP. In vitro PDT of the EMT-6 cells after in vivo dye administration, revealed a similar potency for direct cell killing between the di- and trisulfonated AlOH-NSBP, while the monosulfonated analog was inactive. PDT with the amphiphilic disulfonated AlOH-NSBP on the EMT-6 mammary tumor in BALB/c mice induced a significant tumor response, while the monosulfonated derivative was much less active.     

Photodynamic inactivation of retroviruses by phthalocyanines: the effects of sulphonation, metal ligand and fluoride.

The photodynamic inactivation of retroviruses was investigated using aluminium and zinc phthalocyanine (Pc) derivatives. The N2 retrovirus packaged in either of the two murine cell lines, Psi2 and PA317, was used as a model for enveloped viruses. AlPc derivatives were found to be more effective photodynamically for inactivation of the viruses than the corresponding ZnPc derivatives. Sulphonation of the Pc macrocycle reduced its photodynamic activity progressively for both AlPc and ZnPc. Fluoride at 5 mM during light exposure completely protected viruses against inactivation by AlPc. In the presence of F-, inactivation by the sulphonated derivatives AlPcS1 and AlPcS4 was reduced 2.5- and twofold respectively. In a biological membrane (erythrocyte ghosts), F- had no significant effect on AlPcS4-sensitized lipid peroxidation. Under similar conditions, cross-linking of spectrin monomers in ghosts is drastically inhibited (E. Ben-Hur and A. Orenstein, Int. J. Radiat. Biol., 60 (1991) 293-301). Since Pc derivatives do not inactivate non-enveloped viruses, it is hypothesized that inactivation occurs by photodynamic damage to envelope protein(s). Substitution of sulphonic acid residues reduces the binding of Pc derivatives to the envelope protein(s), thereby diminishing their photodynamic efficacy and the ability of F- to modify it.     

Photodynamic activity of substituted zinc trisulfophthalocyanines: role of plasma membrane damage

We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS3Cn) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS3C, by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS3C,. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS3C6-C9 is a major factor in their overall photodynamic activity.     

A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF-1 Tumors in C3H Mice

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to-tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine-bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF-1 tumors trans-planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear-swelling model. When C3H mice were exposed to 167 J/cm² of polychromatic visible light from a UVB-filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.     

PHTHALOCYANINE-INDUCED PHOTOHEMOLYSIS: STRUCTURE-ACTIVITY RELATIONSHIP AND THE EFFECT OF FLUORIDE

Abstract— Phthalocyanine (Pc) containing AI, Ga or Zn as central metal ligand and substituted with a varying number of sulfonic acid residues as well as additional benzene rings were synthesized and their photodynamic activity was assayed using photohemolysis of human erythrocytes as an endpoint. The Pc derivatives vaned > 300-fold in their photodynamic activity. Activity corrclated with binding of the dye to the cell, with the exception of some of the amphiphilic dyes where cell uptake was an order of magnitude higher than expected from the observed activity. Fluoride was shown to inhibit AIPcS_n-induccd photohemolysis. This effect occurred also with other AlPc and GaPc derivatives, but the concentration of F required to slow photohemolysis by a factor of two (K_i) varied between 4 μ M and 10 mM. Fluorescence spectral studies indicated complex formation between F⁻ and the dye, which was stronger for AlPc than GaPc derivatives. Ultrastructural studies using scanning electron microscopy showed that the photosensitized cells were converted to spherocytes and that F⁻ prevented this to a large extent.     

Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.     

THE EFFECT OF FLUORIDE ON BINDING and PHOTODYNAMIC ACTION OF PHTHALOCYANINES WITH PROTEINS

Fluoride inhibits chloroaluminum phthalocyanine tetrasulfonate (AlPcS)-induced photohemolysis when added to dye loaded cells prior to light exposure. The mechanism by which F- exerts this effect was studied by measuring the binding of phthalocyanine (Pc) to various proteins in the absence and presence of F-. Parallel measurements were made of the photodynamic action under these conditions. Fluoride reduced the binding to proteins of AlPcS and CoPcS. The binding of CuPcS, ZnPcS and H2PcS was not affected. When bound to bovine serum albumin and exposed to light, H2Pc, ZnPc and AlPcCl were bleached at a biphasic rate. Only the photobleaching of AlPcCl was affected by F-. The effect of F- was to inhibit the initial rapid phase without affecting the slower phase. In the presence of D2O only the second phase of photobleaching was enhanced, in the absence or presence of F-. No effect of F- was observed on tryptophan photooxidation or glyceraldehyde-3-phosphate dehydrogenase photoinactivation by AlPcS. Crosslinking of spectrin monomers photosensitized by AlPcS was inhibited by F- in parallel with the reduced binding of dye to the protein. It is concluded that F- exerts its effect by complexing with metal ligands of Pc. As a result, the dye may be released from the protein or the binding mode may be changed in such a way that effective photochemistry is prevented. Primary photophysical processes of Pc most probably are not affected by F-.     

The Relationship of Phthalocyanine 4 (Pc 4) Concentrations Measured Noninvasively to Outcome of Pc 4 Photodynamic Therapy in Mice

The ability to noninvasively measure photosensitizer concentration at target tissues will allow optimization of photodynamic therapy (PDT) and could improve outcome. In this study, we evaluated whether preirradiation tumor phthalocyanine 4 (Pc 4) concentrations, measured noninvasively by the optical pharmacokinetic system (OPS), correlated with tumor response to PDT. Mice bearing human breast cancer xenografts were treated with 2 mg kg⁻¹ Pc 4 iv only, laser irradiation (150 J cm⁻²) only, Pc 4 followed by fractionated irradiation or Pc 4 followed by continuous irradiation. Laser irradiation treatment was initiated when the tumor to skin ratio of Pc 4 concentration reached a maximum of 2.1 at 48 h after administration. Pc 4 concentrations in tumor, as well as in Intralipid in vitro , decreased monoexponentially with laser fluence. Pc 4-PDT resulted in significant tumor regression, and tumor response was similar in the groups receiving either fractionated or continuous irradiation treatment after Pc 4. Tumor growth delay following Pc 4-PDT correlated with OPS-measured tumor Pc 4 concentrations at 24 h prior to PDT ( R ² = 0.86). In excised tumors, OPS-measured Pc 4 concentrations were similar to the HPLC-measured concentrations. Thus, OPS measurements of photosensitizer concentrations can be used to assist in the scheduling of Pc 4-PDT.     

Direct near-infrared luminescence detection of singlet oxygen generated by photodynamic therapy in cells in vitro and tissues in vivo

Singlet oxygen (1O2) is believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). Measurement of 1O2 near-infrared (NIR) luminescence at 1270 nm in biological environments is confounded by the strongly reduced 1O2 lifetime and probably has never been achieved. We present evidence that this is now possible, using a new NIR-sensitive photomultiplier tube. Time-resolved 1O2 luminescence measurements were made in various solutions of aluminum tetrasulphonated phthalocyanine (AlS4Pc) and Photofrin. Measurements were also performed on suspensions of leukemia cells incubated with AlS4Pc, and a true intracellular component of the 1O2 signal was clearly identified. Time-resolved analysis showed a strongly reduced 1O2 lifetime and an increased photosensitizer triplet-state lifetime in the intracellular component. In vivo measurements were performed on normal skin and liver of Wistar rats sensitized with 50 mg/kg AlS4Pc. In each case, a small but statistically significant spectral peak was observed at 1270 nm. The 1O2 lifetime based on photon count rate measurements at 1270 nm was 0.03-0.18 micros, consistent with published upper limits. We believe that these are the first direct observations of PDT-generated intracellular and in vivo 102. The detector technology provides a new tool for PDT research and possibly clinical use.     

Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis.     

Preparation of a water-soluble fluorinated zinc phthalocyanine and its effect for photodynamic therapy

An amphiphilic fluorinated phthalocyanine, zinc tetracarboxyoctafluorophthalocyanine (ZnC4F8Pc) was synthesized and characterized. Its photodynamic efficiency for HeLa cells was compared with hydrophilic zinc octacarboxyphthalocyanine (ZnC8Pc) and hydrophobic zinc hexadecafluorophthalocyanine (ZnF16Pc). ZnC4F8Pc had a remarkable photodynamic effect among the phthalocyanines used. The effect is apparently caused by the fact that ZnC4F8Pc is mainly accumulated in the hydrophobic lipid membrane and is in the photoactive monomer form in HeLa cells.     

Evaluation of cytotoxic effect of photodynamic therapy in combination with electroporation in vitro

Under the influence of electric pulses cells undergo membrane electroporation (EP), which results in increased permeability of the membrane to exogenous compounds. EP is applied in oncology as a method to enhance delivery of anticancer drugs. For that reason it was essential to combine photodynamic tumor therapy (PDT)--the cancer treatment method based on the use of photosensitizers that localize selectively in malignant tumors and become cytotoxic when exposed to light, and EP, with the aim to enhance the delivery of photosensitizers into the tumor and therefore to increase the efficacy of PDT. Thus, the aim of study was to evaluate the cytotoxic effect of PDT in combination with EP. A Chinese hamster lung fibroblast cell line (DC-3F) was used. The cells were affected by photosensitizers chlorin e(6) (C e(6)) at the dose of 10 mug/ml and aluminium phthalocyanine tetrasulfonate (AlPcS4) at the dose of 50 microg/ml. Immediately after adding of photosensitizers the cells were electroporated with 8 electric pulses at 1200 V/cm intensity, 0.1 ms duration, 1 Hz frequency. Then, after 20 min of incubation the cells were irradiated using a light source--a visible light passing through a filter (KC 14, emitted light from 660 nm). The fluence rate at the level of the cells was 3 mW/m(2). Cytotoxic effect on cells viability was evaluated using MTT assay. Our in vitro data showed that the cytotoxicity of PDT in combination with EP increases fourfold on the average. Based on the results we suggest that EP could enhance the effect of PDT.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

Lysosomal signaling enhances mitochondria-mediated photodynamic therapy in A431 cancer cells: role of iron

In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT-induced cell killing by mitochondria-targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4-PDT-induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin-induced killing, cells were incubated with ammonium ferric citrate (30 μM) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin-mediated PDT and that lysosomal disruption augments PDT with Pc 4.     

Immunosuppressive Effects of Silicon Phthalocyanine Photodynamic Therapy

The purpose of this study was to determine if silicon phthalocyanine 4 (Pc 4), a second-generation photosensitizer being evaluated for the photodynamic therapy (PDT) of solid tumors, was immunosuppressive. Mice treated with Pc 4 PDT 3 days before dinitrofluorobenzene sensitization showed significant suppression of their cell-mediated immune response when compared to mice that were not exposed to PDT. The response was dose dependent, required both Pc 4 and light and occurred at a skin site remote from that exposed to the laser. The immunosuppression could not be reversed by in vivo pre-treatment of mice with antibodies to tumor necrosis factor-alpha or interleukin-10. These results provide evidence that induction of cell-mediated immunity is suppressed after Pc 4 PDT. Strategies that prevent PDT-mediated immunosuppression may therefore enhance the efficacy of this therapeutic modality.     

Role of the Immune System in Mediating the Antitumor Effect of Benzophenothiazine Photodynamic Therapy

The role of the host immune system in contributing to tumor regression following benzophenothiazine photodynamic therapy (PDT) was examined. Photodynamic therapy with 2-iodo-5-ethylamino-9-diethylaminobenzo[a]-phenothiazinium chloride (2I-EtNBS) eradicated EMT-6 mammary fibrosarcomas in 75-100% of treated mice. In contrast, PDT failed to inhibit tumor growth in T-cell-deficient nude mice. Furthermore, T-cell depletion studies with anti-CD8 antibody revealed that the CD8+ T-cell population was critical for an effective PDT response (tumor volume 14 days post-PDT: 262 mm3 vs 59 mm3 in controls; P < 0.01). Because anti-CD4 antibody inhibited tumor growth in the absence of PDT, the role of CD4+ T cells remains unclear. Depletion of natural killer (NK) cells in vivo with anti-asialo-GM1 antibody significantly reduced a suboptimal PDT effect relative to vehicle controls (tumor volume 13 days post-PDT: 513 mm3 vs 85 mm3, respectively; P < 0.001). However, splenic NK cells obtained from PDT-treated tumor-bearing mice were not cytotoxic in vitro against EMT-6 cells, suggesting that NK cells contribute to the PDT effect in vivo by an indirect mechanism. In addition, when mice with complete tumor regression following PDT were rechallenged 28 days later with 5 x 10(5) EMT-6 cells, tumor growth was significantly inhibited as compared to controls (tumor volume 40 days postrechallenge: 137 mm3 vs 833 mm3 in controls; P < 0.03; percent animals without tumor in five experiments: 67% vs 8% in controls). Collectively, these results demonstrate that CD8+ T cells are required to prevent tumor regrowth after 2I-EtNBS-PDT, NK cells contribute to this response and such PDT can elicit protective antitumor immunity.     

Spectroscopic properties and photodynamic effects of new lipophilic porphyrin derivatives: efficacy, localisation and cell death pathways

Photodynamic therapy (PDT) and photodynamic diagnostics (PDD) of cancer are based on the use of non-toxic dyes (photosensitisers) in combination with harmless visible light. This paper reports physicochemical properties, cell uptake, localisation as well as photodynamic efficiency of two novel lipophilic porphyrin derivatives, suitable for use as PDT sensitisers. Both compounds are characterised by high quantum yield of singlet oxygen generation which was measured by time-resolved phosphorescence. Photodynamic in vitro studies were conducted on three cancer cell lines. Results of cell survival tests showed negligible dark cytotoxicity but high phototoxicity. The results also indicate that cell death is dependent on energy dose and time following light exposure. Using confocal laser scanning microscopy both compounds were found to localise in the cytoplasm around the nucleus of the tumour cells. The mode of cell death was evaluated based on the morphological changes after differential staining. In summary, good photostability, high quantum yield of singlet oxygen and biological effectiveness indicate that the examined lipophilic porphyrin derivatives offer quite interesting prospects of photodynamic therapy application.