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. The relationship between the structure and properties of CICs was analyzed on the basis of information on their photoinduced cytotoxicity, efficiency of generation of reactive oxygen species, photostability, intracellular localization, quantitative parameters of accumulation in cells, and cellular pharmakinetics. 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.

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.

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.

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-μM content of CIC1 and CIC2 in the incubation medium provides for their average cytoplasmic concentrations of 21 and 16.5 μM, 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 sterol 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.

Metal-free sulfonated phthalocyanine with the average number of sulfonate groups per molecule 2.4 (H2PcS2.4) 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 H2PcS2.4 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 H2PcS2.4 intravenous injection revealed that H2PcS2.4 was present in all tissue structures in the monomeric photoactive state. The preferential accumulation of H2PcS2.4 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 H2PcS2.4 less than adjacent skin structures, whereas EC and LLC accumulated H2PcS2.4 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 H2PcS2.4 accumulation. The H2PcS2.4 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 H2PcS2.4 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.

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°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 μM 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.

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.

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.

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLex-PAA-flu, Sia2-PAA-flu, GlcNAc2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLex-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLex-PAA-3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLex-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

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 confocal spectral imaging (CSI) technique is described, its basic principles are considered, and a brief review of its applications to the study of biologically active compounds (BAC) within living cells and in tissue slices is presented. This technique is based on measurements and analysis of fluorescence or resonance Raman spectra in each point of the specimen under microscope with a three-dimensional resolution of about cubic micrometer. This technique is applicable to the study of stained fluorescent and nonfluorescent compounds. Unlike the conventional approaches based on the optical microscopy, the CSI technique opens the opportunity for the identification of complexes and microenvironment of BAC in intact cells and thin tissue slices (slices or sections), as well as for the analysis of localization and distribution of compounds of interest and their complexes in cellular organelles and tissue structures. The use of CSI technique in combination with the conventional biochemical and cytological methods makes it possible to significantly expand the informativeness of investigation of modes of action of new BAC.

The confocal spectral imaging (CSI) technique is described, its basic principles are considered, and a brief review of its applications to the study of biologically active compounds (BAC) within living cells and in tissue slices is presented. This technique is based on measurements and analysis of fluorescence or resonance Raman spectra in each point of the specimen under microscope with a three-dimensional resolution of about cubic micrometer. This technique is applicable to the study of stained fluorescent and nonfluorescent compounds. Unlike the conventional approaches based on the optical microscopy, the CSI technique opens the opportunity for the identification of complexes and microenvironment of BAC in intact cells and thin tissue slices (slices or sections), as well as for the analysis of localization and distribution of compounds of interest and their complexes in cellular organelles and tissue structures. The use of CSI technique in combination with the conventional biochemical and cytological methods makes it possible to significantly expand the informativeness of investigation of modes of action of new BAC.

With the aim to perform spectroscopic studies and spectral images inside living cells, a microspectrofluorometer has been designed for two-dimensional spectral imaging in the visible and in the near-UV region. The main advantage of the device relies on its ability to scan the laser beam along one direction of the sample. This scanning is optically coupled with one direction of a bidimensional detector, allowing an instantaneous recording of a one-dimensional spectral image. The overall scanning of the sample is achieved by means of submicrometric displacements of the stage in the perpendicular direction. The main characteristics and performances of the microspectrofluorometer in terms of sensitivity (detection of a few molecules), spatial resolution (0.5 x 0.5 x 1 microm), and spectral resolution (1 nm) are presented. Finally, applications of this new apparatus concerning in situ localization and spectral characterization of two dyes are shown with Drosophila salivary glands (ethidium bromide) and T47D tumor cells (Hoechst 33342).

The incorporation of 10–6 M ethidium bromide (EB) was studied in viable Drosophila melanogaster salivary glands with a spatial resolution reaching a few µm3, using a confocal laser microspectrofluorometer designed for spectral analysis. Spectra were recorded with the 514 nm Argon laser line during excitation times of 1 second (20 µW on the preparation) at 5 min intervals for 30 or 60 min, either at points in determined cell sites or serially throughout the cells. The fluorescence intensity time-course indicated that the EB intake was not an all-or-none process, but rather a graded, sensitive indicator of the functional state of the cell. On the micrometer scale, the cytoplasm behaved as an homogeneous substrate with the fluorescence intensity depending on EB intake and intracellular diffusion. In the nucleus, however, localized enhancement of the emission intensity was observed. Spectral analysis allowed us to characterize the interactions. The mean values of λmax in the cytoplasm (600 nm), in the nucleus (601 nm) and outside the glands (602 nm) were less than for free EB in aqueous solution (630 nm); values of full width at half maximum were between 92 and 96 nm, which is much lower than the 120 nm observed for free EB. The recorded spectra were analyzed using a linear combination of two spectral models, namely free and DNA intercalated EB. In the nucleus, the free EB model spectra was found to represent up to 10% of the recorded spectra whereas it was near zero in the cytoplasm. The present data suggest that the intranuclear concentration of free EB (allowing for its lower fluorescence quantum yield) might be at least equal to that of the bound EB.

We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C-terminus of a cytotoxic T-cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class-I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion-peptide moiety bound to the plasma membrane of living cells. When the fusion peptide was linked to the C-terminus of the CTL epitope, the chimeric peptide (M2-F) adopted a marked beta-sheet conformation. In contrast, when the fusion peptide was linked to the N-terminus of the T-cell epitope (F-M2), the chimeric peptide adopted an alpha-helical conformation in the presence of trifluoroethanol. The immunogenicity of the two chimeric peptides for class-I restricted CTL was also significantly different, the one adopting the alpha-helical conformation being more immunogenic. Probably due to its obvious conversion to an alpha-helical conformation, the F-M2 peptide could have a higher propensity to insert into membranes, as shown by microspectrofluorometry, with a resultant better immunogenicity than the M2-F peptide.