Irina G. Meerovich

Fluorescence diffuse tomography for detection of red fluorescent protein expressed tumors in small animals.

A fluorescence diffuse tomography (FDT) setup for monitoring tumor growth in small animals has been created. In this setup an animal is scanned in the transilluminative configuration by a single source and detector pair. To remove stray light in the detection system, we used a combination of interferometric and absorption filters. To reduce the scanning time, an experimental animal was scanned using the following algorithm: (1) large-step scanning to obtain a general view of the animal (source and detector move synchronously); (2) selection of the fluorescing region; and (3) small-step scanning of the selected region and different relative shifts between the source and detector to obtain sufficient information for 3D reconstruction. We created a reconstruction algorithm based on the Holder norm to estimate the fluorophore distribution. This algorithm converges to the solution with a minimum number of fluorescing zones. The use of tumor cell lines transfected with fluorescent proteins allowed us to conduct intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Ibr, Mel-Kor, and human embryonic kidney HEK293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. The emission of red fluorescent proteins (RFPs) in the long-wave optical range permits detection of deep-seated tumors. In vivo experiments were conducted immediately after subcutaneous injection of fluorescing cells into small animals.

Lifetime imaging of FRET between red fluorescent proteins

Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.

Study of action of cyclophosphamide and extract of mycelium of Pleurotus ostreatus in vivo on mice, bearing melanoma B16-F0-GFP

In this work we studied in vivo the combined action of cyclophosphamide and the extract of mycelium of Pleurotus ostreatus on mice bearing melanoma B16-F0, expressing green fluorescent protein (GFP). This model allows to recognize small-size tumors and metastases, unrecognizable by other methods. It was found that combined administration of cyclophosphamide (300 mg/kg) and the extract of mycelium of Pleurotus ostreatus (100 mg/kg), administered for 10 days after cyclophosphamide injection, as well administration of cyclophosphamide alone, cause inhibition of tumor growth about 97%. It was shown that administration of the extract of mycelium of Pleurotus ostreatus alone leads to inhibition of tumor growth of 61%. It was found that in case of combined administration of cyclophosphamide and the extract of mycelium of Pleurotus ostreatus, leucopenia was less expressed than in case of administration of cyclophosphamide alone.

Three-dimensional in vivo imaging of tumors expressing red fluorescent proteins.

3D imaging of genetically-engineered fluorescent tumors enables quantitative monitoring of tumor growth/regression, metastatic processes, including during anticancer therapy in real-time.Fluorescent tumor models for 3D imaging require stable expression of genetically encoded fluorescent proteins and maintenance of the properties of tumor cell line including growth rate, morphology, and immunophenotype.In this chapter, the protocol for 3D imaging of tumors expressing red fluorescent protein are described in detail.

Aluminium hydroxide tetra-3-phenylthiophthalocyanine as new photosensitizer for photodynamic therapy and fluorescent diagnostics

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.

In vivo evaluation of accumulation of sensitizers for oncological diagnostics and therapy using the method of diffuse reflectance spectroscopy

The article is devoted to in vivo investigations of absorption in biological tissue sensitized by preparations used for diagnostics and therapy of tumors. Efficiency of method of difhse reflectance spectroscopy for in vivo evaluation of accumulation of sensitizers is demonstrated.

Fluorescent and photodynamic properties of infrared photosensitizer bacteriochlorophyllide-serine

The study is devoted to investigation of fluorescent and photodynamic properties of near-infrared photosensitizer bacteriochlorophyllide-serine. The peculiarities of its fluorescence under different laser excitation conditions, dynamics of concentration and selectivity of accumulation were investigated. Photodynamic therapy of mice and investigation of tissue deoxygenation during tumor irradiation were performed. It was shown that bacteriochlorophyllide-serine is an effective photosensitizer for PDT and fluorescent diagnostics of tumors.

High-throughput screening system for the study of phototoxicity of photosensitizers in vitro

The technique and the device for studying of phototoxic properties of photosensitizers in vitro on cell monolayers in 96-well microplate was developed. It allows to irradiate independently each well of microtiter plate, and study simultaneously all points of dependence of phototoxic effect on the light dose for certain conditions of investigation. Also it allows to study and compare at the same conditions the dependence of phototoxic effect on the light dose for different concentrations of photosensitizer or for several photosensitizers. Developed device includes powerful source of light based arc xenon lamp with elliptical reflector, special filters and multi-fiber bundle. To determine the degree of the cell viability under the photodynamic treatment proliferative test with fluoresceinediacetate was used. Using this device and technique phototoxic action of a number of different derivatives of aluminium phthalocyanines was studied. The results of this screening have a good correlation with the results obtained in vivo on mice.

Phototoxic properties of dibiotinylated aluminum sulphophthalocyanine in vitro and in vivo

The investigations of photodynamic activity of the dibiotinylated aluminium sulphophthalocyanine in vitro and in vivo were performed. The results obtained showed that in vitro dibiotinylated aluminium sulphophthalocyanine provides an effective damage of small cell lung carcinoma OAT-75. In vivo dibiotinylated aluminium sulphophthalocyanine induces a total damage of Erlich carcinoma with expressed vascular damage even in a concentration 0.5 mg/kg of body weight.

Fluorescent and photodynamic properties of near-infrared photosensitizer bacteriochlorophyllide-serine

Study is devoted to investigation of fluorescent and photodynamic properties of near-infrared photosensitizer bacteriochloriphyllide-serine. The peculiarities of its fluorescence excited by different lasers, dynamics and selectivity of accumulation were investigated. Photodynamic therapy of mice and investigation of tissue de-oxygenation during tumor irradiation were performed. It was shown that bacteriochloriphyllide-serine is an effective photosensitizer for PDT and fluorescent diagnostics of tumors.