The First Selenoanhydride in the Series of Chlorophyll a Derivatives, Its Stability and Photoinduced Cytotoxicity.

In this work, we obtained the first selenium-containing chlorin with a chalcogen atom in exlocycle E. It was shown that the spectral properties were preserved in the target compound and the stability increased at two different pH values, in comparison with the starting purpurin-18. The derivatives have sufficiently high fluorescence and singlet oxygen quantum yields. The photoinduced cytotoxicity of sulfur- and selenium-anhydrides of chlorin p6 studied for the first time in vitro on the S37 cell line was found to be two times higher that of purpurin-18 and purpurinimide studied previously. Moreover, the dark cytotoxicity increased four-fold in comparison with the latter compounds. Apparently, the increase in the dark cytotoxicity is due to the interaction of the pigments studied with sulfur- and selenium-containing endogenous intracellular compounds. Intracellular distributions of thioanhydride and selenoanhydride chlorin p6 in S37 cells were shown in cytoplasm by diffusion distribution. The intracellular concentration of the sulfur derivative turned out to be higher and, as a consequence, its photoinduced cytotoxicity was higher as well.

Therapeutic properties of a vector carrying the HSV thymidine kinase and GM-CSF genes and delivered as a complex with a cationic copolymer.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system. METHODS: We studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models. RESULTS: We showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan. CONCLUSIONS: We demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier - a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.

Fluorescence imaging analysis of distribution of indocyanine green in molecular and nanoform in tumor model.

BACKGROUND: The efficient intraoperative identification of tumors requires the development of highly specific near-infrared (NIR) probes as contrast agents. One of the most effective dyes existing in clinic oncology is Indocyanine Green (ICG). However, ICG has a rapid excretion, thus ruling out its extended accumulation in pathological tissues therefore limiting its clinical applications. ICG colloid solution (ICG NPs) consists predominantly of J-aggregates and to a lesser extent of H-aggregates and monomers. In the present study we assessed the spectral properties of ICG nanoforms in preclinical models. METHODS: We used optical spectroscopy and video fluorescence navigation to monitor accumulation and distribution of ICG monomers and ICG NPs in various tissues in mice with xenografted laryngopharyngeal carcinoma after intravenous drugs injection. RESULTS: After i.v. injection, the molecular form of ICG was not retained in the tumor and its circulation cycle averaged 5 min. Alternatively, the nanoform of the drug had a different pharmacokinetics, reaching maximum accumulation 24 h after intravenous injection. Moreover, once in the circulation, we observed a progressive accumulation in the tumor of both ICG H-aggregates and ICG monomers, but not J-aggregates. CONCLUSION: Spectral characteristics of ICG NPs indicated the presence of several fractions, namely, J- and H-aggregates along with molecular forms. These fractions had different fluorescence spectra, allowing us to track the transformation of the drug in vivo conditions. After ICG NPs administration, J-aggregates induce accumulation of monomeric forms in the tumor, enabling extended intraoperative diagnostic, and as such further studies of J-aggregates for theranostic applications in oncological surgery are of great interest.

A New Glutathione-Cleavable Theranostic for Photodynamic Therapy Based on Bacteriochlorin e and Styrylnaphthalimide Derivatives.

Herein, we report a new conjugate BChl-S-S-NI based on the second-generation photosensitizer bacteriochlorin e6 (BChl) and a 4-styrylnaphthalimide fluorophore (NI), which is cleaved into individual functional fragments in the intracellular medium. The chromophores in the conjugate were cross-linked by click chemistry via a bis(azidoethyl)disulfide bridge which is reductively cleaved by the intracellular enzyme glutathione (GSH). A photophysical investigation of the conjugate in solution by using optical spectroscopy revealed that the energy transfer process is realized with high efficiency in the conjugated system, leading to the quenching of the emission of the fluorophore fragment. It was shown that the conjugate is cleaved by GSH in solution, which eliminates the possibility of energy transfer and restores the fluorescence of 4-styrylnaphthalimide. The photoinduced activity of the conjugate and its imaging properties were investigated on the mouse soft tissue sarcoma cell line S37. Phototoxicity studies in vitro show that the BChl-S-S-NI conjugate has insignificant dark cytotoxicity in the concentration range from 15 to 20,000 nM. At the same time, upon photoexcitation, it exhibits high photoinduced activity.

Photodiagnosis and photodynamic effects of bacteriochlorin-naphthalimide conjugates on tumor cells and mouse model.

Photo-induced cytotoxicity and antitumor activity of a series of dual function agents for photodynamic therapy (PDT) and fluorescent imaging based on bacteriochlorin photosensitizer conjugated with various naphthalimide fluorophores was studied in vitro using murine tumor cells of S37 sarcoma and in vivo on mice bearing murine S37 sarcoma. Upon irradiation at the absorption maximum of the photosensitizer, the activity of conjugates was as high as in the case of individual bacteriochlorin, while an additional excitation of the naphthalimide fragment led to an increase in the PDT efficacy due to resonance energy transfer from the fluorophore to photosensitizer. The fluorescence contrast and specific cytotoxic activity measurements indicate that the conjugate of bacteriochlorin with 3,4-dimethoxestyrene-substituted naphthalimide is the most promising agent for the application as theranostic in PDT.

Photophysical properties and in vitro and in vivo photoinduced antitumor activity of cationic salts of meso-tetrakis(N-alkyl-3-pyridyl)bacteriochlorins.

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±0.03µm). 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±0.03µm in vitro, TGI and RR 100% in vivo).