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Antibacterial photodynamic therapy (APDT) is a promising method of treatment of local infections, based on photochemical reaction of photosensitizer (PS) under irradiation with visible or near infra-red light of appropriate wavelength. A molecule of PS is activated by light energy and as a result singlet oxygen and other reactive oxygen species, which are toxic for microbes, are generated. Antibacterial action of new PS (synthetic polycationic bacteriochlorin derivatives with spectral maximum of absorbance at 760 nm) with improved features was investigated in vitro in this work. Tetra- and octacationic bacteriochlorins with lower molecular mass, as compared with ones previously proposed, turned out to be highly effective against Pseudomonas aeruginosa and Staphylococcus aureus in plankton and biofilm state: reduction of viable bacteria in biofilms reached 5log10. The dependence of the effect on PS concentration, the time of incubation with PS and light dose was ascertained. Light microscopy of biofilms after photodynamic treatment and Live/Dead staining confirmed the death of bacteria or damage of their membranes after exposure to PS and irradiation.

Microencapsulation and targeted delivery of cytotoxic and antibacterial agents of photodynamic therapy (PDT) improve the treatment outcomes for infectious diseases and cancer. In many cases, the loss of activity, poor encapsulation efficiency, and inadequate drug dosing hamper the success of this strategy. Therefore, the development of novel and reliable microencapsulated drug formulations granting high efficacy is of paramount importance. Here we report the in vitro delivery of a water-soluble cationic PDT drug, zinc phthalocyanine choline derivative (Cholosens), by biodegradable microcapsules assembled from dextran sulfate (DS) and poly-l-arginine (PArg). A photosensitizer was loaded in pre-formed [DS/PArg]4 hollow microcapsules with or without exposure to heat. Loading efficacy and drug release were quantitatively studied depending on the capsule concentration to emphasize the interactions between the DS/PArg multilayer network and Cholosens. The loading data were used to determine the dosage for heated and intact capsules to measure their PDT activity in vitro. The capsules were tested using human cervical adenocarcinoma (HeLa) and normal human dermal fibroblast (NHDF) cell lines, and two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Our results provide compelling evidence that encapsulated forms of Cholosens are efficient as PDT drugs for both eukaryotic cells and bacteria at specified capsule-to-cell ratios.

A comparative experimental study of the specific activity of drugs based on 5-aminolevulinic acid (5-ALA) and its hexyl ester (5-ALA HE) was carried out. Their ability to induce the synthesis of photoactive protoporphyrin IX in the healthy tissues of the rabbit bladder when instilling the drug solutions at various concentrations has been estimated. It was shown that 5-ALA HE results in the induction and accumulation of PPIX in the rabbit bladder epithelium at much lower concentrations than 5-ALA. Thus, a significant increase in the fluorescence intensity in comparison with the control was achieved by instillation of 5-ALA HE solution in the rabbit’ bladder at a concentration of only 0.0001% (fluorescence intensity 2.20±0.60 a.u.), and for 5-ALA – only when using a solution at a concentration of 0.3% (fluorescence intensity 2.60±1.02 a.u.).

Antibacterial photodynamic therapy is a promising method of treating local infected foci, especially surgical and burn wounds, trophic and diabetic ulcers. This work explores the photophysical and antibacterial properties of novel phthalocyanine- and synthetic-bacteriochlorin-based octacationic photosensitizers (PS). The results of the study confirm their low degree of aggregation at high concentrations, as well as high efficiency of photodynamic treatment of Gram-negative bacterial biofilms.

Cationic bacteriochlorin derivatives which molecules differing by lipophilicity and positive charge degree have been investigated in vitro as photosensitizers for photodynamic inactivation of biofilm bacteria.

We developed the original preparation Lymphotest on the basis of disulphoderivative of diaminotriphenylmet- anic dye, differing from Lymphazurin by positions if sulphogroups in benzene ring - N-[4-[[4-(diethylamino)phenyl] (2,5-disulfophenyl)methylene]-2,5-cyclohexadien-l-ylidene]-N-ethylethanaminium hydroxide, inner salt, sodium salt. The proposed compound practically does not differ by spectrum from Lymphazurin, but is much more accessible and highly lymphotropic. Lymphotest in the form of 1% aqueous solution is characterized by a high diagnostic efficiency when using direct color lymphography, low toxicity (imposed dose exceeded the anticipated maximum dose for a human in 50-1000 times) and has no locally-irritating effect when using subcutaneously.

Physico-chemical properties, biodistribution in animal tissues, and PDT efficacy of bacteriochlorin photosensitizers, namely cationic salts of synthetic meso-tetrakis(N-alkyl-3-pyridyl)bacteriochlorins were studied in НЕр2 cell line and in the LLC mouse model. The tested compounds showed high stability in the dark and high in vitro phototoxicity against НЕр2 cells (the half maximal inhibitory concentration LD50 in the range from 0.34 ± 0.03 to 1.5 ± 0.03 μm). Synthetic bacteriochlorins rapidly accumulate in mouse tumor tissue with tumor-to-normal tissue fluorescence contrast ratios of 2.3–3.3, possess high PDT activity against LLC cells: inhibition of tumor growth, TGI 85.8–100%, increase in life span, ILS 105.7–129.2%, response rate, RR 50–100%. The highest PDT efficacy was found for meso-tetrakis[1-(4′-bromobutyl)-3-pyridyl]bacteriochlorin tetrabromide (IC50 0.34 ± 0.03 μm in vitro, TGI and RR 100% in vivo).

Polycationic derivatives of synthetic bacteriochlorin with absorption in the near infrared range are promising for the creation of photosensitizers (PS) for antimicrobial photodynamic therapy. In the present work, the photophysical and antibacterial properties of PS based on tetracationic derivatives of synthetic bacteriochlorins: hydrophilic 3-Py4BC(EtBr)4Br4 in aqueous solution and amphiphilic 3-Py4BCHP4Br4 in the dispersion of Kolliphor ELP were studied. Analysis of absorption and fluorescence in a wide range of concentrations has demonstrated low aggregation of the PS over the entire range. A high efficiency of photodynamic inactivation of Gram-positive Staphylococcus aureus and Gramnegative Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii bacteria was observed.

A new method of synthesis of phthalocyanines with phosphonate moieties by reaction of chloromethyl substituted phthalocyanines with phosphorus trichloride in the presence of aluminum chloride followed by hydrolysis of the intermediately formed phosphonic acid chlorides under mild conditions is developed. A number of phosphonomethyl substituted phthalocyanines with different central metal atoms (aluminum, silicon, titanium, copper, zinc and gallium) and/ or with the presence of unreacted chloromethyl groups were obtained. Significant dependence of monomer – aggregate equilibrium and, as consequence, of photochemical and photophysical properties of the synthesized complexes on pH value was found. Quantum yields of singlet oxygen generation and photodegradation of complexes were determined. The photodestruction of the synthesized compounds, except of rather photostable copper phthalocyanine, proceeds with participation of self-sensitized singlet oxygen and is significantly accelerated by the presence of chloromethyl groups in the macrocycle.

Summary In 1992–2006 at P.A. Hertsen Moscow Oncology Research Institute photodynamic therapy (PDT) was performed in 48 esophageal cancer patients (total 48 lesions). For PDT we used Russian photosensitizers (Photogem, Photosens, Radachlorin, Alasens), Russian diode lasers (Crystall) and endoscopic equipment. As a result of PDT complete regression was in 77% of esophageal cancer lesions, partial regression was in 23%. The follow-up period was 3–11 years. Median of survival was in 4.59 years of esophageal cancer patient.

New oxidation-resistant hexaphenyl-substituted tetramethyltetraazachlorin, its nickel complex, and tetraphenyl-substituted octamethyltetraazabacteriochlorin and octamethyltetraazaisobacteriochlorin nickel complexes were synthesized for the first time by mixed condensation of diphenylm aleonitrile with tetramethylsuccinonitrile in the presence of lithium dimethylaminoethoxide or nickel chloride. The products were characterized by electronic absorption, 1H NMR, and mass spectra. Mixed condensation of diphenylsuccinonitrile with phthalonitrile in the presence of nickel chloride was found to give nickel complexes of phenylsubstituted benzo-fused tetraazaporphyrins rather than their hydrogenated derivatives.

The reaction with sodium cyanide of the μ-oxo-bridged complex of tetra-4-tert-butyl-substituted iron phthalocyanine (form ‘690’) and that of the product of its treatment with organic bases such as Py, Im, etc. (form ‘627’) result in the formation of the same ferrous bis-cyanide complex Na2[ PctFeII( CN )2] which can be readily oxidized to the analogous ferric complex Na [ PctFeIII( CN )2]. Form ‘690’ has been oxidized to the corresponding ferric μ-oxo complex (form ‘630’). Data for all μ-oxo-bridged complexes (chemical behavior; electronic, NMR, Mössbauer, X-ray photoelectron and ESR spectra; magnetic susceptibility) are discussed, and based on them, the following structures are proposed: ( HPctFeII)2O (form ‘690’), H2[( LPctFeII)2O ] (form ‘627’) and ( PctFeIII)2O (form ‘630’).

Nanoparticles of aluminum and zinc phthalocyanin and metal-free phthalocyanin (AlPc, ZnPc, and H2Pc), whose molecular forms are photosensitizers, can serve as effective “prophotosensitizers” in photodynamic therapy for malignant tumors. Transition (stimulation) of photo-inert nanoparticles into a photoactive photosensitizer is realized locally in the tumor node by its exposure to potent laser pulses. Systemic injection of AlPc, ZnPc, and H2Pc nanoparticles has not led to accumulation of their photoactive form in the skin, which can lead to the development of skin phototoxicity. Effective protocols of photodynamic therapy with ZnPc nanoparticles are determined. The use of these protocols in mice with S-37 sarcoma led to 92-70% tumor growth inhibition, 48% improvement of survival, and cure in 84% cases.

In photodynamic therapy (PDT), photosensitizers are required to arrive in high concentrations at selective targets like cancer cells avoiding toxicity in healthy tissue. In this work, we propose the application of porous calcium carbonate carriers in the form of polycrystalline vaterite for this task. We investigated the loading efficiency for the photosensitizer Photosens in vaterite micro- and nanocarriers. A possible release mechanism depending on the surrounding pH was studied, showing a fast degradation of the carriers in buffers below pH 7. These results hold out the prospect of a novel PDT drug delivery system. Variation of particle size or additional coatings allow custom-design of workload release curves. An intrinsic cancer-sensitivity can be expected from the pH-dependent release in the acidic microenvironment of cancer tissue.

Current work is devoted to investigation of tetra-3-phenylthio-tetra-5-t-butylphthalocyanine [(PhS)4(t-Bu)4PcH2], aluminium hydroxyde tetra-3-phenylthiophthalocyanine [(PhS)4PcAlOH] and zinc tetra-3-phenylthiophthalocyanine [(PhS)4PcZn] as potential photosensitizers of near-infrared range. Investigations were performed on F1 mice bearing Erlich tumor. Photosensitizers were administered intravenously in liposomal form at doses of 4-10 mg/kg. Dynamic and selectivity of sensitizers' accumulation in tumor were estimated in vivo from fluorescence and absorption spectra of sensitized tissue. Photosensitizers have shown high selectivity of accumulation in tumor comparing to normal tissue of mice. Maxima of selectivity for (PhS)4(t-Bu)4PcH2, (PhS)4PcZn and (PhS)4PcAlOH achieve the values up to 2.5:1, 5:1 and 8:1 respectively. All photosensitizers completely clear from the normal tissue in 7-8 days. For PDT investigations tumors were irradiated using 732 nm laser with power density of 100-500 mW/cm2 and light dose density up to 400 J/cm2. The photodynamic efficiency was estimated using the parameter of tumor growth inhibition (TGI). All photosensitizers had shown high photodynamic efficiency of relatively large tumors. PDT using (PhS)4PcAlOH and (PhS)4(t-Bu)4PcH2 caused pronounced TGI exceeding 80%. Using (PhS)4PcZn caused moderate TGI of 60%. Investigations have shown that liposomal forms of phenylthiosubstituted phthalocyanine derivatives may be used to develop new efficient photosensitizers for PDT.

The new aqueous gel compositions based on 5-aminolevulinic acid (ALA) for fluorescent diagnostics and photodynamic therapy of superficial diseases have been elaborated. Biodegradable polymer was used as gel-forming component and some additives — solubilizer, emulgator etc — were used to improve distribution uniformity and penetration ability. These new compositions represent colorless and transparent gels which are long-term stable at the storage temperature of ≤5°C. The effectiveness of aqueous gels as ALA delivery systems is demonstrated. After topical administration of the gel at the skin with Ehrlich tumor inoculated subcutaneously, ALA effectively induces the synthesis of protoporphyrine IX (PPIX) in the skin and in the tumor. Intensity of ALA-induced PPIX fluorescence grows with increasing ALA concentration in the gel and time of gel application. The deepness of ALA penetration in the tumor reaches 5-6 mm after 4 h gel application. A distribution of ALA-induced PPIX in mouse tumor tissue after 4 h gel application depends on ALA concentration in gel. Intensity of PPIX fluorescence in deep-located part of tumor was substantially higher with gel containing 20% ALA than with ones containing 10% or 5% ALA.

This work is devoted to investigation of possibility to use the liposomal form of aluminium hydroxide tetra-3-phenylthiophthalocyanine as photosensitizer of near-infrared range. Aluminium hydroxide tetra-3-phenylthiophthalocyanine has shown high selectivity of accumulation in tumor comparing to normal tissue of mice as well as high photodynamic efficiency on mice bearing Erlich tumor (ELD) and lympholeucosis P-388. This compozition can be used to develop new effective photosensitizer for photodynamic therapy and fluorescent diagnostics.

Optimized irradiation regimens for intraperitoneal photodynamic therapy with porphyrin and phthalocyanine photosensitizers are determined in in vitro and in vivo studies.The experimental study on НЕр2 cell line showed that reduce of power density for constant light dose increased significantly the efficacy of photodynamic therapy (the reduce of power density from 20-80 mW/cm2 to 10 mW/cm2 had the same results (90% cell death) for half as much concentration of the photosensitizer).The obtained results were confirmed in vivo in mice with grafted tumor S-37. For light dose of 90 J/cm2 and power density of 25 mW/cm2 none of animals in the experimental group had total resorption of the tumor. For the same light dose and decrease of power density to 12 mW/cm2 total tumor resorption was achieved in 34% of animals, 66% of animals died from phototoxic shock. For twofold decrease of light dose – to 45 J/cm2 with the same low-intensity power density (12 mW/cm2) we managed total tumor resorption in 100% of animals.In the following studies of optimized irradiation regimen for intrapleural photodynamic therapy the reaction of intact peritoneum of rats on photodynamic exposure was assessed and optimized parameters of laser irradiation, which did not cause necrosis and intense inflammatory reaction of peritoneum, were determined – light dose of 10 J/cm2 with power density of mW/cm2.Thus, the reasonability for use of low-intensity regimens of irradiation for intraperitoneal photodynamic therapy was confirmed experimentally with possibility of high efficacy of treatment without inflammatory reactions of peritoneum.