Irina V. Balalaeva

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

Recombinant targeted toxin based on HER2-specific DARPin possesses a strong selective cytotoxic effect in vitro and a potent antitumor activity in vivo.

DARPins fused with other proteins are promising non-immunoglobulin scaffolds for specific binding to target cells. In this study HER2-specific DARPin (DARPin_9-29) was used as a tumor-targeting moiety for the delivery of a cytotoxic agent - the fragment of Pseudomonas aeruginosa exotoxin A. It was determined that DARPin-PE40 possesses a considerable cytotoxic activity and induces apoptosis in HER2-positive cells. Cytotoxic effect of DARPin-PE40 strongly correlates with the HER2 expression level. The effect of intravenous administration of DARPin-PE40 was tested in the xenograft model of breast cancer. It was shown that treatment of animals with DARPin-PE40 caused strong and prolonged suppression of xenograft tumor growth.

Evaluation of oral mucosa collagen condition with cross-polarization optical coherence tomography

The goal of the research was analysis of the effect of collagen condition in formation of cross-polarized CP OCT images. We used of the CP OCT technique for studying collagen condition on an example of oral mucosa. Special histologic picrosirius red (PSR) staining of cheek mucosa specimens was used with subsequent assessing of the result of collagen staining in polarized light. High correlation (r = 0.692, p = 0.0001) between OCT signal standard deviation (SD) in cross-polarized images and brightness of PSR stained collagen fibers in cheek mucosa specimens was demonstrated in patients with inflammatory intestine and oral mucosa diseases. We have found that the OCT signal SD in cross-polarized images reflects two boundary conditions of collagen disorganization, namely, loss of fiber properties at active inflammation which attenuates the signal and fibrosis that occurs due to synthesis of a new remodeled collagen which amplifies the OCT signal.

A novel far-red fluorescent xenograft model of ovarian carcinoma for preclinical evaluation of HER2-targeted immunotoxins

We have created a novel fluorescent model of a human ovarian carcinoma xenograft overexpressing receptor HER2, a promising molecular target of solid tumors. The model is based on a newly generated SKOV-kat cell line stably expressing far-red fluorescent protein Katushka. Katushka is most suitable for the in vivo imaging due to an optimal combination of high brightness and emission in the “window of tissue transparency”. The relevance of the fluorescent model for the in vivo monitoring of tumor growth and response to treatment was demonstrated using a newly created HER2-targeted recombinant immunotoxin based on the 4D5scFv antibody and a fragment of the Pseudomonas exotoxin A.

HER2-specific recombinant immunotoxin 4D5scFv-PE40 passes through retrograde trafficking route and forces cells to enter apoptosis

Immunotoxin 4D5scFv-PE40 is a recombinant protein that comprises 4D5scFv antibody as a targeting module and fragment of Pseudomonas exotoxin A as an effector (toxic) one. The immunotoxin has shown pronounced antitumor effect on cancer cells overexpressing HER2 receptor in vitro and on HER2-positive experimental tumors in vivo. We clarified the mechanism of 4D5scFv-PE40 activity that is of particular importance in the case of targeted therapeutic agent aimed at personalizing treatment of disease in relation to molecular genetic characteristics of each patient. After specific binding to HER2 on the cell surface and clathrin-mediated endocytosis the immunotoxin passes through retrograde trafficking route. During this route the immunotoxin molecule is supposed to undergo enzymatic processing that ends in separation of C-terminal and N-terminal fragments of the immunotoxin. Finally, C-terminal functionally active fragment of 4D5scFv-PE40 arrests protein synthesis in cytoplasm followed by cell death via apoptosis.

Parameters of electrical signals and photosynthetic responses induced by them in pea seedlings depend on the nature of stimulus

Local damage induces generation and propagation of variation potentials (VPs) that affect physiological processes in plants. The aims of the work presented here were to investigate parameters of VP induced by burning, heating and mechanical injury in pea seedlings, and to undertake a theoretical analysis of the mechanisms underlying the differences in VP parameters and a study of the photosynthetic responses caused by VPs induced by the damaging factors. The velocity of propagation of burn-induced VP decreased with distance from the damaged area whereas the velocities of heating- and injury-induced VPs were constant. The amplitudes of burn- and heating-induced VPs did not depend on distance whereas the amplitude of VP induced by mechanical injury decreased. VP propagation has been simulated on the basis of wound substance spread. The simulation revealed two possible ways of wound substance propagation: turbulent diffusion from the damaged area and secondary active production in intact cells. The photosynthetic response (decrease in the quantum yield of PSII and raising the level of non-photochemical fluorescence quenching (NPQ)) developed in case of VP entering the intact leaf under heating and burn but was not registered after mechanical injury. An increase in NPQ level was biphasic under burn in comparison with a single-phase one under heating, and the NPQ amplitude was slightly higher under burn. We suggest that differences in photosynthetic responses may be determined by the parameters of VPs induced by stimuli of different nature.

Study of the tissue distribution of potential boron neutron-capture therapy agents based on conjugates of chlorin e6 aminoamide derivatives with boron nanoparticles

Boron neutron-capture therapy of cancer is based on the ability of the 10B isotope to capture thermal neutrons; it is one of the most promising techniques in radiation therapy. The high content and selective accumulation of 10B in the tumor tissue are the most important prerequisites for its efficacy. The purpose of this study was to determine the biodistribution of cobalt bis(dicarbollide) conjugates with chlorin e6 amino amide derivatives. Experiments were carried out in Balb/c mice with transplanted CT-26 murine colon carcinoma. Boron-containing conjugates were injected into the tail vein at a dose of 10 mg/kg. The conjugate accumulation in tumor tissue and organs was studied by laser scanning microscopy. Excitation was performed at the wavelength of 514 nm; the signals were recorded in the range of 560–710 nm in increments of 10 nm. To evaluate the amount of the boron conjugate, we calculated the intensity of the fluorescence signal of the samples under investigation. At 3 h after administration of the agent, a high level of fluorescence was observed in the liver, spleen, and lung. The tumor/muscle accumulation ratio was approximately 3. The study demonstrated that boron derivatives of chlorin e6 are promising agents for boron neutron-capture therapy.

Imaging of human ovarian cancer SKOV-3 cells by quantum dot bioconjugates.

41 Development of molecular approaches to treat ment of oncological diseases determines the necessity of development ultrasensitive and highly precise methods for rapid visualization of specific markers of tumor cells. The main role of such cellular molecular markers, which are determined immediately in tumor tissue, is that, by characterizing the molecular profile of each certain tumor, they may help to determine the immune status of the tumor and select individual ther apy course. Creation of fluorescent constructs able to specifically bind to such markers is a promising approach to early molecular diagnostic of cancer. Semiconductor nanocrystals (quantum dots) are a new class of fluorophores widely used in various bio logical studies. In comparison to organic dyes and flu orescent proteins, which are conventionally used in fluorescence diagnostics, quantum dots have a num ber of advantages, such as a high quantum yield, inten sity, high molecular extinction coefficient, broad absorption spectrum, high resistance to photobleach ing, and exceptional resistance to photodegradation and chemical degradation. Generalized results of studies performed in recent years showed that quan tum dots linked to various guiding agents specific for tumor markers are promising tools for fluorescence diagnostics and determination of immune status of tumors and visualization of peripheral metastases (for review, see [1]). Targeted delivery of quantum dots is successfully ensured using full length monoclonal antibodies linked to nanocrystals directly through various cross linking agents or using a three stage labeling system based on the biotin–streptavidin affinity pair (primary antibodies, biotinylated secondary antibodies, and conjugates of quantum dots with streptavidin). How ever, in some cases, the use of constructs based on the antigen binding fragments of antibodies is more promising. In contrast to full length antibodies, these small fragments contain no constant domain; this has no effect on their guiding properties but reduces the possibility of occurrence of side effects caused by interaction of constant domains with immune cell receptors and complement proteins. An example of such constructs are single chain Fv fragments (scFv antibodies)—recombinant polypep tides in which variable domains of the light and heavy immunoglobulin chains are linked through a flexible linker. Such small (25–30 kDa) antibodies rapidly reach target cells, effectively recognize the antigen, and are easily withdrawn from the body [2]. In this study, we described a construct based on flu orescent CdSe/CdS nanocrystals and genetic engi neering recombinant miniantibodies, intended for detecting cells overexpressing the HER2/neu onco marker. The HER2/neu protein belonging to the fam ily of epidermal growth factors is an important marker in diagnosing cancer diseases. Its overexpression on the surface of ovarian, cervical, bladder, colonic, stomach, esophageal, and breast tumor cell correlates with unfavorable prognosis of disease development and increased resistance to chemotherapy. For this reason, early detected of cells overexpressing HER2/neu is of great clinical importance [3].

Synthesis and biological evaluation of new water-soluble photoactive chlorin conjugate for targeted delivery

A new water-soluble conjugate, consisting of a chlorin-based photosensitizing part, and a 4-arylaminoquinazoline moiety with high potential affinity to an epidermal growth factor receptors (EGFR) and vascular endothelial growth factor receptors (VEGFR), suitable for photodynamic therapy (PDT), was synthesized starting from methylpheophorbide-a in seven steps. An increased accumulation of this compound in A431 cells with high level of EGFR expression, in comparison with CHO and HeLa cells with low EGFR expression was observed. The prepared conjugate exhibits dark and photoinduced cytotoxicity at micromolar concentrations with IC50dark/IC50light ratio of 11-18. In tumor-bearing mice, the conjugate preferentially accumulates in the tumor tissue.

New promising porphyrazine-based agents for optical theranostics of cancer

New porphyrazine bases containing peripheral benzyloxyphenyl groups have been synthesized by the template method. The procedure includes condensation of aromatic aldehydes with malononitrile, transformation of arylmethylidenemalononitriles to arylethenetricarbonitriles, template assembly of porphyrazine macrocycle on bis(indenyl)ytterbium(II) complex, and removal of the central metal ion. Luminescence properties of the synthesized porphyrazines and their dependence on the viscosity of the medium were studied, and the light and dark toxicities of the porphyrazines have been estimated. The obtained results suggest the possibility of using these porphyrazines as optical theranostic agents of new generation.