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Cancer is the main problem of all developed and many developing countries of the world and the cause of death and disability of population. Today, the actual problem is receipt of reliable information about the boundaries of malignant neoplasms and the detection of pathology in the early stages.

International audience; Squamous cell carcinoma is the most common type of oral and oropharyngeal cancers. Incomplete surgical removal of the tumor is often the cause of local recurrence of the disease and appearance of metastases. We have developed the novel endoscope fluorescence video system for visualization of micrometer size objects in real-time mode. The novel system was tested on three-dimensional models of cancer cells. Chlorine e6 (Ce6) was used as a photosensitizer. The accumulation of Ce6 in cancer cells was assessed by its fluorescence excited at 635 nm. It was shown that the 500 µm multicellular tumor spheroids could be easily detected with a good resolution and sensitivity. We suppose that the new endoscope video system could be useful for developing the intraoperative fluorescence navigation method using Ce6-mediated techniques that is able to visualize the small cancer cell clusters.

Published in Journal of Physics: Conference Series, 1439:012032, IOPScience, 2020; International audience; Current paper presents the results of the usage of indocyanine green and pheophorbide nanoform on 2D and 3D models of FaDu cells culture. The 2D model or monolayer was used for investigation of nanoparticles distribution within individual cells and their organelles. The 3D model or multicellular tumor spheroids were used for estimation of cells' metabolic processes by the investigation of the nanophotosensitizers fluorescence distribution within spheroid's layers. It was shown that pheophorbide nanoparticles are accumulated in the external cell layers of spheroids, indocyanine green nanoparticles distribution demonstrates completely opposite status – in the central part of the spheroid.

Photodynamic therapy and fluorescence diagnosis of malignant tumors have been actively developed over the last 15 years by the scientists participating in the Moscow Government-funded “Fight against Cancer” program. New effective photosensitizers absorbing in the long-wave spectral region were revealed. Unique agents Alasens-lio, Alasens-gel, Hexasens-lio, Phthalosens-lio, Holosens-lio, and Holosensgel were produced and preclinically studied both in vitro and in vivo. New methods using low-intensity and pulse modes of irradiation and also combined methods of treatment by photodynamic therapy and well-known chemotherapy or laser hyperthermia were developed. Methodological recommendations for experimental studies of agents with photoinduced antitumor properties were published.

Novel hybrid molecule containing 2-mercaptoethylamine was synthesized starting from O-propyloxime-N-propoxy bacteriopurpurinimide (dipropoxy-BPI), which was readily oxidized in oxygen atmosphere yielding the corresponding disulfide analogue (disulfide-BPI). Spectral, photophysical, photodynamic, and biological properties of compound were properly evaluated. Compounds bearing disulfide moiety can directly interact with glutathione (GSH), thereby reducing its intracellular concentration. Indeed, mice sarcoma S37 cell line was treated in vitro with disulfide-BPI, yielding a CC50 value of 0.05 ± 0.005 μM. A relatively high level of singlet oxygen was detected. It was demonstrated (by fluorescence) that the PS was rapidly accumulated in a cancer nest (S37) at a relatively high level after 2 h upon intravenous administration. After 24 h, no traces of the molecule were detected in the tumor mass. Moreover, high photodynamic efficiency was demonstrated at doses of 150-300 J/cm2 against two different in vivo tumor models, achieving 100% regression of cancer growth.

The 13,15-N-(3'-hydroxypropylcycloimide) chlorin p6 (CIC), which absorbs at 711 nm, possesses considerable photoinduced cell-killing activity. It is 43-, 61- and 110-fold more active than chlorin p6, 3-formyl-3-devinyl chlorin p6 and Photogem, respectively, and has no cytotoxicity without irradiation as estimated on A549 human adenocarcinoma cells. To attain the highest intracellular penetration and activity the monomeric form of CIC should be stabilized. This stabilization in an aqueous environment can be achieved using 0.002-0.005% of Cremophor EL emulsion (polyoxyethylene derivative of hydrogenated castor oil). The intracellular accumulation of CIC occurs in cytoplasm in a monomeric form bound to cellular membranes. This form of the dye is characterized by a high quantum yield of singlet oxygen generation (0.66 +/- 0.02). Besides diffuse staining of intracellular membranous structures, CIC accumulates 3- to 4-fold more intensely in mitochondria and Golgi apparatus, thus indicating these organelles to be the initial targets of its photodynamic action. The incubation time providing 50% accumulation level of CIC in cells is 30 +/- 5 min. The time for 50% release of CIC from the cells is 60 +/- 10 min. A 10-fold decrease in CIC intracellular penetration at 22 degrees C proves that temperature-sensitive mechanisms of transport, rather than diffusion, are responsible for the dye uptake. The average cytoplasmic concentration of CIC was seven times the extracellular concentration in the 0.2-1.6 microM range, used for the photodynamic activity measurements. The concentration of CIC and the light dose that correspond to ca 50% level of phototoxicity induce predominantly an apoptotic-type of cell death, whereas the conditions providing 100% level of phototoxicity induced necrosis. The results obtained indicate that cycloimide derivatives of chlorin p6 may serve as a base for the development of an efficient near-IR photosensitizer.

Comparative study of 13,15-[N-(2-hydroxyethyl)]cycloimide chlorin p6 (2), 13,15-(N-acetoxy)cycloimide chlorin p6 (3), 13,15-(N-hydroxy)cycloimide chlorin p6 methyl ester (4) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (5) together with the previously investigated 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (1) was performed. The dependence of the key photodynamic properties of 1-5 on the introduced substituents was analyzed. The photoinduced cell-killing activity of 4 is 100- and 280-fold higher than that of chlorin p6 and Photogem, respectively, as estimated on A549 human lung adenocarcinoma cells. The activity is reduced eight times in the order 4 > 5 > 1 > 2 > 3. The intracellular accumulation of 1-5 occurs in cytoplasm in a monomeric form bound to the lipids of cellular membranes. This form of 1, 2, 3, 4 and 5 is characterized by the high quantum yield of singlet oxygen generation, which depends on the introduced substituents, 0.66, 0.59, 0.35, 0.51 and 0.73, respectively. The photostability is two-fold less for 1 and four-fold less for 2, 3 and 5 than for 4. The rates of cellular uptake and efflux of 1-5 vary widely, thus providing the way to optimize the pharmacological properties of the photosensitizer (PS) using the respective substituents. Modifying the substituents, 1-5 were targeted to different cellular organelles. The enhanced accumulation in the Golgi apparatus and mitochondria complemented with diffuse staining of intracellular membranous structures is a property of 1-4. Compound 5 accumulates selectively in the lipid droplets and stains weakly perinuclear structures. Temperature-sensitive mechanisms of transport are responsible for the 1-4 uptake. Diffusion can play a role in the internalization of 5 but not of 1-4. Endocytosis via caveolae, clathrin-dependent and adenosine triphosphate-dependent pathways are not noticeably involved in the 1-5 internalization. Independently from their intracellular localization 1, 4 and 5 are highly efficient near-IR PS, which induce predominantly an apoptotic type of cell death under conditions providing ca 50% level of phototoxicity and necrosis at the 100% level of phototoxicity.

Photosensitizers 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (HPC) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (MMC) absorb at 711 nm and possess high photoinduced cytotoxicity in vitro. Here we report, that photodynamic therapy with HPC and MMC provide considerable antitumor effect in mice bearing subcutaneous P338 lymphoma. The highest antitumor effect was achieved at a dose of 4 μmol/kg when 1.5 h delay between dye injection and light irradiation (drug–light interval) was used. According to the confocal spectral imaging studies of tissue sections this drug–light interval corresponds to a maximum of tumor accumulation of MMC and HPC (tumor to skin accumulation ratio is 8–10). Short (15 min) drug–light interval can be used for efficient vasculature-targeted photodynamic therapy with HPC at a dose of 1 μmol/kg, whereas MMC is ineffective at the short drug–light interval. Relationships between the features of tissue distribution and efficacy of photodynamic therapy at different drug–light intervals are discussed for HPC and MMC.

Metal-free sulfonated phthalocyanine with the average number of sulfonate groups per molecule 2.4 (H(2)PcS(2.4)) was recently proved to be an efficient photosensitizer for the photodynamic therapy. Fluorescence spectral imaging microscopy was applied here to study localization and relative concentration of H(2)PcS(2.4) with micron-scale resolution in subcutaneously transplanted murine tumors: Ehrlich mammary gland carcinoma (EC), Lewis lung carcinoma (LLC), P388 lymphoid leukemia (P388) and B16 melanoma (B16). The study of cryogenic tissue sections prepared 24 h after H(2)PcS(2.4) intravenous injection revealed that H(2)PcS(2.4) was present in all tissue structures in the monomeric photoactive state. The preferential accumulation of H(2)PcS(2.4) was documented in tumor cells and adjacent non-tumor tissues (skin structures, fatty tissue, connective tissue enriched in fibrous component and infiltrated with fibroblasts and macrophages) for all the studied tumor models. P388 and B16 were stained with H(2)PcS(2.4) less than adjacent skin structures, whereas EC and LLC accumulated H(2)PcS(2.4) alike or higher than particular skin structures. Staining of EC and LLC was similar and ca. 1.4 and 2 times higher than that of B16 and P388, respectively, thus revealing the differences in ability of particular tumor strains to H(2)PcS(2.4) accumulation. The H(2)PcS(2.4) concentration in remote healthy tissues (skin, muscles and connective tissue) was 2-3 times lower as compared with the analogous tissue structures from the tumor area, whereas subcutaneous fatty tissue staining did not depend on the tissue-to-tumor distance. The tissue distribution of H(2)PcS(2.4) predefines the combined action of two photodynamic damage mechanisms: eradication of tumor due to the direct tumor cell destruction and suppression of tumor growth due to the injury of growth supporting system.

Lipophilic derivatives of chlorin p6, 13,15-N-(carboxymethyl)cycloimide methyl ester (CIC1) and 13,15-N-(2-carboxyethyl)cycloimide methyl ester (CIC2), were shown to absorb light in 710 nm region and to be efficient IR photosensitizers. They exhibit similar phototoxicities on the cells of A549 human lung adenocarcinoma, which are 40- and 100-fold higher than those of chlorin p6 and the clinically used Photogem, respectively, and are not toxic in the absence of light irradiation. The confocal spectral imaging technique allowed us to demonstrate that the high phototoxicity of CIC1 and CIC2 is due to their ability to readily penetrate to cells and to be bound to the cell membranes and lipid-containing structures in the monomeric photoactive form. Under the irradiation, the membrane-bound CIC1 and CIC2 are characterized by high quantum yields of singlet oxygen generation (0.6 and 0.65, respectively) and the inability to produce hydroxyl radicals. A 1.5-microM content of CIC1 and CIC2 in the incubation medium provides for their average cytoplasmic concentrations of 21 and 16.5 microM, respectively. The incubation times to achieve 50% level of maximum accumulation for CIC1 and CIC2 in A549 cells are 30 +/- 6 and 24 +/- 12 min, and the times for 50% release of the dyes from the cells are 17 +/- 4 and 50 +/- 10 min, respectively. A diffuse distribution with the predominant accumulation in the membranes of the Golgi apparatus and mitochondria is characteristic of both CIC2 and CIC1, whereas, in addition, CIC1 is considerably accumulated in lipid droplets (cellular organelles responsible for the storage and metabolism of neutral lipids and steryl esters). Our results demonstrate that changes in the structure of the imide substituent could affect the intracellular localization and the rate of release of chlorin p6 cycloimide derivatives from cells while preserving their high photodynamic activity.

Upon interaction of hydrazine hydrate with bacteriopurpurin, the initially formed monohydrazide in an acidic medium readily reacts with the second carboxyl group to give a six-membered N-aminocycloimide of bacteriochlorin p6. The free amino group at the fused imide ring makes it easy to obtain N-alkyl and N-acyl derivatives. The compounds thus obtained exhibit high light-induced cytotoxicity on A549 human adenocarcinoma cells.

Four monocationic cycloimide derivatives of chlorin p(6) (CICD) were studied as photosensitizers and compared to a structurally similar neutral derivative. Cationic CICD are highly photostable (quantum yield of photobleaching is about 1 x 10(-5), generate singlet oxygen under irradiation (quantum yields are 0.3-0.45), can be involved in a photo-induced substrate-dependent generation of superoxide radicals, but do not produce OH . 17,18-delta-lacton 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) () and 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) methyl ester () possess high cancer cell killing photodynamic activity, but they provide no photoinduced bactericidal effect. Substitution of an ethyl group with a hydroxyethyl or acetyl group at position 3 of the macrocycle results in a decrease in extinction and intracellular accumulation that finally leads to the reduced photocytotoxicity. Cationic CICD are targeted to lysosomes, and their intracellular penetration occurs most probably via caveolae-dependent endocytosis. Photodynamic treatment with cationic CICD results in the cell death via necrosis at both sub-phototoxic (40-70% of dead cells) and phototoxic (90-100% of dead cells) regimes of cell treatment. Irradiation induces lysosome damage, leakage of CICD from lysosomes and development of protease activity in cytoplasm, whereas mitochondria are not affected with irradiation. A positive charge of cationic CICD modified drastically an internalization pathway, sites of intracellular localization and mechanisms of photoinduced cytotoxicity as compared to previously studied neutral and anionic CICD. Our experiments with different CICD show that varying charge and structure of substituents it is possible to modulate many cellular properties of CICD in order to find the best molecular template of the advanced near-IR photosensitizer for photodynamic therapy.

The effect of electron-accepting substituents in position 3 of the chlorine p6 macrocycle in neutral and carboxyl-containing negatively charged cycloimide derivatives of chlorin p6 (CIC) on the photochemical and biological properties of these photosensitizers was studied. A relationship between the structure and properties of CICs was analyzed on the basis of information on their photoinduced cytotoxicity, efficiency of the generation of reactive oxygen species, photostability, intracellular localization, quantitative parameters of accumulation in cells, and cellular pharmacokinetics. It was shown that these compounds can be used for the development of photosensitizers with intense light absorption at 740 nm, controlled intracellular localization, and a high photodynamic activity toward tumor cells.

Di- and tri-sulfonated derivatives of metal-free phthalocyanine and Photosens have been compared in this study. In vitro phototoxicity was studied on HEp2, A549, and Colo26 cell cultures. It has been shown that phototoxicity of di- and tri- sulfonated derivatives of metal-free phthalocyanine exceeds phototoxicity of Photosens 5- and 8-times on HEp2 cell line, 8- and 6-times on A549 cell line, and 22-times on Colo26 cell line, respectively. In vivo photo-induced antitumor activity of metal-free phthalocyanine and Photosens was evaluated on mice with transplanted P388 lymphocytic leukemia. Dependence of photodynamic therapy efficiency on photosensitizer dose and time interval between preparation administration and irradiation was studied. It has been shown that Photosens is the most efficient in 5 mgikg of animal body weight dose and at 24-hour interval between injection and irradiation (tumor growth inhibition (TGI) - 82.2-95.6%). Metal-free phthalocyanine is the most efficient in 0.5 mg/kg dose which is 10- times lower than Photosens dose and when irradiated 4 hours later the injection (TGI - 78.6-100%). Metal-free di- and tri-sulfonated phthalocyanines are promising photosensitizers for photodynamic therapy. Taking into account its physical and chemical properties metal-free tri-sulfonated phthalocyanine named "Phthalosens" has been chosen for further thorough study.

Reactive oxygen species generated by photosensitizers are efficacious remedy for tumor eradication. Eleven cycloimide derivatives of bacteriochlorin p (CIBCs) with different N-substituents at the fused imide ring and various substituents replacing the 3-acetyl group were evaluated as photosensitizers with special emphasis on structure–activity relationships. The studied CIBCs absorb light within a tissue transparency window (780–830 nm) and possess high photostability at prolonged light irradiation. The most active derivatives are 300-fold more phototoxic toward HeLa and A549 cells than the clinically used photosensitizer Photogem due to the substituents that improve intracellular accumulation (distribution ratio of 8–13) and provide efficient photoinduced singlet oxygen generation (quantum yields of 0.54–0.57). The substituents predefine selective CIBC targeting to lipid droplets, Golgi apparatus, and lysosomes or provide mixed lipid droplets and Golgi apparatus localization in cancer cells. Lipid droplets and Golgi apparatus are critically sensitive to photoinduced damage. The average lethal dose of CIBC-generated singlet oxygen per volume unit of cell was estimated to be 0.22 mM. Confocal fluorescence analysis of tissue sections of tumor-bearing mice revealed the features of tissue distribution of selected CIBCs and, in particular, their ability to accumulate in tumor nodules and surrounding connective tissues. Considering the short-range action of singlet oxygen, these properties of CIBCs are prerequisite to efficient antitumor photodynamic therapy. Abbreviations: AO, acridine orange; BODIPY ceramide, N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a, 4a-diaza-s-indacene-3-yl) phenoxy)acetyl)sphingosine; CIBC(s), cycloimide derivative(s) of bacteriochlorin p; CIC(s), cycloimide derivative(s) of chlorin p6; CrEL, polyoxyethylene derivative of hydrogenated castor oil, Cremophor EL; CSI, confocal spectral imaging; DR, distribution ratio, a ratio of average cytoplasmic concentration to the extracellular concentration of a photosensitizer; FCS, fetal calf serum; LSCM, laser scanning confocal microscopy; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide; NIR, near infrared; PDT, photodynamic therapy; ΦΔ, singlet oxygen quantum yield; RNO, 4-nitroso-N,N,-dimethylaniline; Rh123, rhodamine 123; ROS, reactive oxygen species

Molecular in vitro and in vivo properties of 3-devinyl-3-formylchlorin p6 (FCp6) were examined in order to characterize this derivative as a new prospective photosensitizer. The long-wavelength absorption maximum of FCp6 was 690-696 nm (depending on environment). FCp6 was found to bind readily to membranous structures and form complexes with some proteins. The dye was associated with the plasmalemma and distributed rather diffusely along the cytoplasm with ca a three-fold higher accumulation within mitochondria in A549 human adenocarcinoma cells. The spectral analysis revealed that the major part of FCp6 was bound to membranes within cells. The membrane-bound FCp6 was shown to generate singlet oxygen efficiently. The average cytoplasmic concentration of FCp6 in A549 cells achieved ca 80% of its extracellular concentration in complete medium. The dye was characterized by a very fast efflux (16-fold decrease in 2 h). The ex vivo analysis of FCp6 fluorescence in mice revealed that the maximal dye content in blood, tissues, organs and tumor was achieved in less than 1 h after injection, followed by a considerable (ca six-fold) decrease during the next 23 h and a long-term persistence at low level. A preferential accumulation of FCp6 in subcutaneously implanted Ehrlich carcinoma along with its higher retention level comparing to the surrounding skin and muscles were observed in mice treated with different dye doses. In vitro cytotoxic assays with A549 and Raji B-cell lymphoma cells as well as in vivo analyses using Ehrlich carcinoma in mice revealed the very low toxicity of FCp6 without light irradiation and the significant photodynamic activity of this compound.

Binary systems combining a transition metal complex and ascorbate have been proposed recently for catalytic therapy of malignant tumors. The killing effect on tumor cells is achieved by production of free radicals in the course of accelerated oxidation of ascorbate by dioxygen in the presence of transition metal complexes. Further progress in the development of binary catalytic systems (BCSs) requires a special method for their investigation in cells and tissues, because neither component of BCSs fluoresces. Here a resonance Raman confocal spectral imaging (RR CSI) technique was introduced as a unique approach to monitor quantitatively the transition metal complexes within living cells. Intracellular accumulation, localization, and retention of theraphthal (TP), a catalyst of the advanced TP/ascorbate BCS, were investigated in A549 cells with the RR CSI technique. The cellular analysis was complemented with the detailed study of molecular interactions of TP in solution and environmental factors affecting the RR spectrum of TP. TP does not penetrate into membranes, it binds very weakly to DNA and RNA, but it readily forms complexes with proteins. Binding with Ca 2+ cations and decreasing pH below 6 induce aggregation of TP. By analyzing RR spectra recorded from every point within a TP-treated cell, three states of the agent were discriminated, namely, monomeric TP in polar environment, TP bound to proteins, and aggregated TP. Their cytoplasmic and nuclear distributions were mapped at different stages of uptake and efflux. By introducing organelle-selective fluorescent probes into drug-treated cells and measuring intracellular localization of both the probe and the drug, compartmentation of TP was revealed. Cell growth suppression by the TP/ascorbate system was measured, and probable molecular and organelle targets of radical damage were characterized.

The CSI technique comprises the measurements of a 2D set of intracellular fluorescence spectra of an antitumor agent with a 3D confocal resolution and subsequent decomposition of these spectra into models describing different states and interactions of the drug [1, 2]. The models have to be taken from in vitro experiments. The results of the decomposition present themselves as maps of intracellular distribution of complexes and states of the agent. Each point of the cell can be characterized in terms of drug concentration after calibration procedure. This allows one to determine regions/compartments of predominant drug accumulation and to predict probable targets of its antitumor action.

Photodynamic therapy (PDT) is an innovative and attractive approach to the tumor treatment. It requires photosensitizer which is accumulated selectively in tumor tissue and upon absorption of light generates singlet oxygen and/or free radicals whose destructive action initiates mechanisms of tumor necrosis or apoptosis [1]. Photosense™, new very promising photosensitizer, is currently at the first stage of clinical trial. According to the synthesis procedure Photosense is a mixture of di-, tri- and tetra- sulfonated aluminum phthalocyanines (AlPcSn, n=2, 3, 4, respectively). To understand cellular pharmacology of Photosense in great depth its components were isolated and their subcellular localization and molecular interactions were studied with the CSI technique. Data obtained for AlPcSn were used after to characterize cellular uptake, localized binding and efflux of Photosense itself.

Teraftal (TF), a cobalt octacarboxyphthalocyanine, belongs to the new class of cytostatics — binary catalytic systems developed for the therapy of malignant tumors [1]. Its cytotoxic action occurs via free radical formation mechanism and requires the presence of its catalytic pair — ascorbic acid. TF absorbs strongly in the red region (λmax= 675 run, e675=1×105 M-1 cm-1) but does not fluoresce. Thus its detection inside cells is rather complicated. In order to reveal subcellular distribution and states of TF as well as features of agent uptake we employ here a resonance Raman (RR) confocal spectral imaging (CSI) technique [2, 3]. This method is based on recording RR spectra from within cell in confocal mode. Then experimental spectrum from every point of the cell is decomposed as a sum of reference spectra with appropriate coefficients. The reference spectra originate from in vitro model measurements and describe different states and/or complexes of the drug. Integral intensity of reference spectrum multiplied by decomposition coefficient for each point produces 2D map (spectral image) which reflects relative distribution of states and complexes of antitumor drug.

The comparative study of the photosensitizers with different chemical, spectral-luminescent and photophysical properties (phthalocyanines -- photosense, di- and trisulphonated zinc (II) phthalocyanines; chlorines -- chlorine p6, desvinyl-3- formyl-chlorine p6 and 3-desvinyl-3-acetyl-chlorine p6; xanthenes --rhodamine 123, rhodamine 6G-chloride, rhodamine 6G-acetate, rhodamine 6G-iodide; arylamines -- oxazinne perchlorate, oxazine-1 zinc salt, 9-diethylamino-5- ethylaminobenzophe-nothiazonium acetate, methylene blue) was performed. The cyto- and the phototoxicity of these compounds were studied on the cells of two human tumor cell lines (Raji B-cell lymphoma and A-549 lung adenocarcinoma) by MTT-test. It was shown that phthalocyanines and chlorines were not cytotoxic, whereas xanthenes and arylamines possessed dark toxicity. On the basis of the IC50 (the substance concentration, which induced 50% inhibition of cell proliferation in the cell culture) the rows of phototoxicity of the compounds of various classes were set out: in phthalocyanines: ZnPcS2 greater than Photosence greater than ZnPcS3; in chlorines: 3dV-3F-Chl p6 greater than 3 dV-3Ac Chl p6 greater than Chl p6; in xanthenes: R6G- Ac greater than R6G-Cl greater than R6G-I greater than R 123; in arylamines: Meth B greater than or equal to 9-DE-B- Ac greater than Ox1-1/2ZnCl42- very much greater than Ox1-ClO4-. Phototoxicity of the studied compounds depended on the nature of the substitutes and of the counter ions in photosensitizers molecules as well as on the concentration of the photosensitizer, on the light doses and on the regimes of irradiation. The fractionation of the light doses increased the efficiency of the phototoxic effect of the dyes on the tumor cells significantly. It was shown by the luminescent microscopy that the dynamics and the intensity of the accumulation of the rhodamines derivatives in lung adenocarcinoma cells depended on the nature of the counter ion in the photosensitizer molecule and correlated with their cyto- and pho-toxicity. Thus, photosense, di- and trisulphonated zinc phthalocyanines, 3- desvinyl-3-formyl-chlorine p6, rhodamine 6G-acetate and methylene blue turned out to be promising for their further study as photodynamic agents and efficient modifiers of chemoradiotherapy and of PDT.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Comparative study of 13,15-[N-(2-hydroxyethyl)]cycloimide chlorin p6 (2), 13,15-(N-acetoxy)cycloimide chlorin p6 (3), 13,15-(N-hydroxy)cycloimide chlorin p6 methyl ester (4) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (5) together with the previously investigated 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (1) was performed. The dependence of the key photodynamic properties of 1-5 on the introduced substituents was analyzed. The photoinduced cell-killing activity of 4 is 100- and 280-fold higher than that of chlorin p6 and Photogem, respectively, as estimated on A549 human lung adenocarcinoma cells. The activity is reduced eight times in the order 4 > 5 > 1 > 2 > 3. The intracellular accumulation of 1-5 occurs in cytoplasm in a monomeric form bound to the lipids of cellular membranes. This form of 1, 2, 3, 4 and 5 is characterized by the high quantum yield of singlet oxygen generation, which depends on the introduced substituents, 0.66, 0.59, 0.35, 0.51 and 0.73, respectively. The photostability is two-fold less for 1 and four-fold less for 2, 3 and 5 than for 4. The rates of cellular uptake and efflux of 1-5 vary widely, thus providing the way to optimize the pharmacological properties of the photosensitizer (PS) using the respective substituents. Modifying the substituents, 1-5 were targeted to different cellular organelles. The enhanced accumulation in the Golgi apparatus and mitochondria complemented with diffuse staining of intracellular membranous structures is a property of 1-4. Compound 5 accumulates selectively in the lipid droplets and stains weakly perinuclear structures. Temperature-sensitive mechanisms of transport are responsible for the 1-4 uptake. Diffusion can play a role in the internalization of 5 but not of 1-4. Endocytosis via caveolae, clathrin-dependent and adenosine triphosphate-dependent pathways are not noticeably involved in the 1-5 internalization. Independently from their intracellular localization 1, 4 and 5 are highly efficient near-IR PS, which induce predominantly an apoptotic type of cell death under conditions providing ca 50% level of phototoxicity and necrosis at the 100% level of phototoxicity.

It is generally assumed that a central metal is essential for the efficiency of phthalocyanines in photodynamic therapy (PDT) of cancer. Contrary to the set opinion, the results of the present study indicate that the metal-free sulfonated phthalocyanines (H2PcSn, where n is the number of sulfonate groups per molecule) possess a considerable photoactivity. The relative phototoxicities of H2PcS1.5, H2PcS2.4, H2PcS3.1 and H2PcS3.8 on HEp2 human epidermoid carcinoma cells were 3.3, 20, 3.3 and 1, respectively, thus demonstrating dependence of the activity on the sulfonation degree, known for metallo-PcSn. A significant delay in tumor growth and a decrease in tumor regrowth rate were observed in mice after PDT with H2PcS2.4. The antitumor effect declined in the order H2PcS2.4 > H2PcS3.1 > H2PcS1.5 and vanished for H2PcS3.8. We demonstrate here that the high photodynamic activity of H2PcS2.4 can be explained by its physicochemical properties in living cells and tissues. Thus, H2PcSn (n is about 2) can be considered as a new alternative in PDT of light-accessible neoplasms and further clinic-oriented studies are warranted.