Barnase*Barstar-guided two-step targeting approach for drug delivery to tumor cells in vivo.

For precise ligation of a targeting and cytotoxic moiety, the use of Barnase-Barstar pair as a molecular glue is proposed for the first time. Targeting was mediated through the use of a scaffold protein DARPin_9-29 specific for the human epidermal receptor 2 (HER2) antigen that is highly expressed on some types of cancer and Barnase*Barstar native bacterial proteins interacted with each other with Kd 10-14 M. The approach proposed consists of prelabeling a target tumor with hybrid protein DARPin-Barnase prior to administration of cytotoxic component-loaded liposomes that have Barstar covalently attached to their surface. Based on in vivo bioimaging we have proven that DARPin-based Barnase*Barstar-mediated pretargeting possesses precise tumor-targeting capability as well as antitumor activity leading to apparent tumor-growth inhibition of primary tumors and distant metastases in experimental animals. The results obtained indicate that the new system combining DARPin and Barnase*Barstar can be useful both for the drug development and for monitoring the response to treatment in vivo in preclinical studies.

[Spectrophotometric assessment of photodynamic teeth bleaching]. / Otsenka effektivnosti fotodinamicheskogo otbelivaniya zubov.

THE AIM: Clinical analysis of the effectiveness of photodynamic tooth whitening according to the spectrophotometric method. MATERIAL AND METHODS: Photodynamic tooth whitening was performed in 35 patients aged 20-45 years, on the teeth of the frontal group. To record the optical characteristics of enamel, the spectrophotomerism method was used. RESULTS: The study reliably shows that the average change in color saturation «C¼ ranged from 0.5 to 2 units. The optical characteristic of luminosity "L" before and after photodynamic exposure remained within the limits of healthy enamel, and the optical characteristic of whiteness on the scale of whitened teeth was 2 units. CONCLUSION: The method of photodynamic therapy can be used in dental practice to conduct a teeth whitening procedure.

Theoretical and experimental modeling of interstitial laser hyperthermia with surface cooling device using Nd3+-doped nanoparticles.

To improve methods of laser hyperthermia for the treatment of bulk malignant neoplasms, an urgent task is the development of techniques and devices that automatically control heating at a given tissue depth and ensure its uniformity. The article proposes the concept of a system for performing hyperthermia with real-time spectroscopic temperature control and surface cooling, which allows to record spectra of diffusely scattered radiation and fluorescent signal from various depths of biological tissues by the means of the variation of the angle and distance between the fiber source of laser radiation and the receiving fiber. Theoretical and experimental modeling of the spatial distribution of diffusely scattered radiation and temperature inside the tissue with a fiber optic device providing surface cooling of the irradiated tissue, and recording spectral information from a given depth in real time, is presented. Simulation of radiation propagation in biological tissues, depending on the distance between the source and the receiver and the angle of their tilt, was carried out using the Monte Carlo method. Modeling of the temperature distribution inside the tissues was carried out by means of a numerical solution of the heat conduction equation. Experimental modeling was carried out on phantoms of biological tissues simulating their scattering properties as well as accumulation of the investigated nanoparticles doped with Nd3+ ions. It was shown that inorganic nanoparticles doped with rare-earth Nd3+ ions can be used as temperature labels for feedback to the therapeutic laser. According to the results of the theoretical simulation, optimal configurations of the relative arrangement of the fibers were chosen, as well as the optimum surface cooling temperatures for the given power densities. The heating of the phantom of the neoplasm containing the investigated nanoparticles doped with Nd3+ ions by laser radiation with an 805-nm wavelength and power density of 1 W/cm2 up to 42 °C at a depth of 1 cm while maintaining the surface temperature within the limits of the norm was demonstrated.

[Analysis of photosensitizer gel penetration through the teeth structure]. / Analiz glubiny proniknoveniia gelia fotosensibilizatora v strukturu tkanei zubov.

As a result of the studies carried out on 20 extracted frontal teeth it was reliably shown that after 10 minutes the concentration of the photosensitizer in enamel at full depth reached values sufficient for performing photodynamic teeth whitening. At the same time, the penetration of the photosensitizer into the deep layers of dentin was not detected which allows to carry out the photodynamic bleaching procedure only within the enamel and enamel-dentine boundary, thus not affecting the pulp of the tooth.

Phototoxicity of flavoprotein miniSOG induced by bioluminescence resonance energy transfer in genetically encoded system NanoLuc-miniSOG is comparable with its LED-excited phototoxicity.

Photodynamic therapy (PDT) is a clinical, minimally invasive method for destroying cancer cells in the presence of a photosensitizer, oxygen, and a light source. The main obstacle for the PDT treatment of deep tumors is a strong reduction of the excitation light intensity as a result of its refraction, reflection, and absorption by biological tissues. Internal light sources based on bioluminescence resonance energy transfer can be a solution of this problem. Here we show that luciferase NanoLuc being expressed as a fusion protein with phototoxic flavoprotein miniSOG in cancer cells in the presence of furimazine (highly specific NanoLuc substrate) induces a photodynamic effect of miniSOG comparable with its LED-excited (Light Emitting Diode) phototoxicity. Luminescence systems based on furimazine and hybrid protein NanoLuc-miniSOG targeted to mitochondria or cellular membranes possess the similar energy transfer efficiencies and similar BRET-induced cytotoxic effects on cancer cells, though the mechanisms of BRET-induced cell death are different. As the main components of the proposed system for BRET-mediated PDT are genetically encoded (luciferase and phototoxic protein), this system can potentially be delivered to any site in the organism and thus may be considered as a promising approach for simultaneous delivery of light source and photosensitizer in deep-lying tumors and metastasis anywhere in the body.

[Bifunctional Toxin DARP-LoPE Based on the HER2-Specific Innovative Module of a Non-Immunoglobulin Scaffold as a Promising Agent for Theranostics].

We have generated and characterized HER2-specific targeted toxin based on the low-immunogenic variant of Pseudomonas exotoxin A (LoPE), in which most of the human immunodominant B-cell epitopes have been inactivated. Nonimmunoglobulin DARPin-based HER2-specific protein was used as a targeting module for toxin delivery to the cellular target. Using confocal microscopy, it has been found that both domains in this hybrid toxin retained their functionality, i.e., the specific interaction with HER2 receptor, as well as the internalization and effective transport to ER typical of the wild-type Pseudomonas exotoxin A. The HER2-dependent cytotoxic effect correlated with receptor expression level at the cell surface, as shown in vitro using cell lines with different levels of HER2 expression. Due to the very high selective cytotoxicity against HER2-positive human tumor cells, as well as expected low immunogenicity, we believe that this new targeted toxin may be promising for future in vivo studies as a therapeutic agent for HER2-positive tumors.

Cytotoxicity of targeted HER2-specific phototoxins based on flavoprotein miniSOG is determined by the rate of their internalization.

The concept of targeted therapy implies the development of bifunctional agents complementing the therapeutic module with a targeting one. A promising target for the delivery of imaging and/or toxic modules is the HER2 (ErbB2) receptor. Earlier, we have functionally characterized the targeted photosensitizers 4D5scFv-miniSOG and DARPin-miniSOG, causing the death of HER2-overexpressing cells when irradiated with blue light. However, the cytotoxicity of targeted toxins 4D5scFv-miniSOG and DARPin-miniSOG (both having functionally active targeted and cytotoxic modules in recombinant proteins) against human breast adenocarcinoma cells differs 5 times. The study of the dynamics of internalization of 4D5scFv-miniSOG and DARPin-miniSOG proteins in the complex with HER2 in this work showed that the rate of internalization contributes most significantly to the toxicity of these photosensitizers, because it determines the duration of the presence of the phototoxin in the lipid bilayer of the cell membrane, where its damaging effect is maximum.

Gonarthritis photodynamic therapy with chlorin e6 derivatives.

BACKGROUND: The new methods of osteoarthritis treatment are in constant demand due to the complexity of the early diagnosis and therapy. Specific features of Сhlorin e6 derivative (Ce6) accumulation in knee joint tissues and the efficiency of photodynamic therapy (PDT) of gonarthritis were studied. METHODS: The experimental research was conducted on the model of posttraumatic gonarthritis on rabbits. The analysis of dynamics of change of Ce6 concentration in tissues of a knee joint was carried out by the method of fluorescent diagnostics. The intra-joint PDT was carried out using 662nm laser with energy density of 120-150J/cm(2) and a sapphire diffuser. An analysis of slices was conducted to confirm the anti-inflammatory effect through apoptosis. RESULTS: The method of fluorescent spectroscopy revealed that the highest amount of Ce6 was accumulated in the synovial membrane of a damaged knee joint 2.5h after its intravenous introduction. On 14th day after gonarthritis modeling but before PDT the synovial membrane showed signs of synovitis. On 21st day after PDT the synovial membrane possessed noticeable villous structure, and no cells of inflammatory nature were observed. CONCLUSION: Fluorescent diagnostics in knee joint tissues can be used in clinical practice of gonarthritis before, during and after PDT for monitoring the Ce6 accumulation and for treatment control. Optimal radiation energy density was determined to be 150J/cm(2). In the studied time intervals (5-25min) no dependency of PDT effect on irradiation time at the same energy density was observed. The analysis of results of clinical and morphological research shows that PDT is a low-invasive method of gonarthritis treatment with a high degree of efficiency and selectivity.

Effect of laser optoperforation of the zona pellucida on mouse embryo development in vitro.

The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida.

A new anticancer toxin based on HER2/neu-specific DARPin and photoactive flavoprotein miniSOG.

Cytotoxic effects of a new targeted phototoxin DARPin-miniSOG and mechanism of its action were investigated in vitro. It was determined that DARPin-miniSOG causes light-induced death of HER2/neu-positive cancer cells (IC50 0.8 µM). Treatment of the cells with DARPin-miniSOG in the presence of ascorbic acid eliminated the light-induced cytotoxic action of the protein. This observation suggests the involvement of oxidative stress in the mechanism of the phototoxin action. DNA fragmentation analysis, caspase-3 activity assay and PI-staining of HER2/neu-positive cancer cells treated with DARPin-miniSOG indicated that phototoxin induces necrotic cell death under blue light illumination. Co-localization analysis showed that DARPin-miniSOG accumulates mostly in endosomes and lysosomes.

[CRITERIA OF DIFFERENCE EVALUATION FOR VARIOUS TYPES OF HEMORRHAGIC FEVER WITH RENAL SYNDROME FOCI. HFRS FOCI IN VARIOUS BIOTOPES OF TYPICAL BARRENS].

AIM: Examine features of natural, natural-anthropourgic and anthropourgic foci of hemorrhagic fever with renal syndrome (HFRS) in various, mostly forest, biotopes of typical barrens due to differences of non-specific HFRS prophylaxis in foci of various types. MATERIALS AND METHODS: Epizootological and epidemiologic data from 1998 to 2012 were analyzed, gathered in HFRS foci of all types in Saratov area of Saratov Region (typical barrens). 14,606 trap-nights were worked off and 2669 small mammals were procured. The most significant population-ecologic and ecologic-epizootological methods and criteria were used for comparative analysis of differences for 3 types of foci. RESULTS: Based on analysis of multi-year data seasonal differences for HFRS foci of various types were shown by 10 population-ecologic and ecologic-epizootologic criteria. CONCLUSION: The results obtained allow to state that modern means and methods of non-specific prophylaxis of HFRS and other zoonoses in foci of various types different significantly. This allows the most rational use of material and financial resources.

[Criteria of difference evaluation for-various types of hemorrhagic fever with renal syndrome foci. HFRS foci in various biotopes of northern barrens].

AIM: Determine qualitative and quantitative criteria of evaluation of features for natural, natural-anthropourgic and anthropourgic foci of hantaviruses (using hemorrhagic fever with renal syndrome--HFRS foci as an example), that have formed in various areas of Saratov Region. MATERIALS AND METHODS: Epizootological and epidemiologic data from 1998 to 2012 were analyzed. During this period in all the types of foci of Atkarsk area (Northern barrens) 13,004 trap-nights were worked off, 2577 small mammals were procured. Genetic, population-ecological and ecological-epizootological methods and criteria were used for comparative analysis of 3 types of loci: PCR diagnostics and sequencing of pathogenic for humans hantaviruses, species cadaster and features of season dynamics of species composition of hantavirus carriers, domination indexes, parameters of quantity and infection of carriers by hantaviruses; distribution of the latter by biotopes, degree and character of population contacts with natural-foci complexes, epizootic potential of foci. RESULTS: Based on multi-year data analysis features of 3 types of HFRS foci were shown in various, mostly forest, biotopes of Northern and a part of typical barrens. A stable domination of Pumala genotype hantavirus was noted; a weakly expressed circulation in parasite systems with Apodemus genus carriers, first of all Dobrava genotype Apodemus agrarius, was established as well. CONCLUSION: A certain specter of most universal criteria;that are closely interconnected, are necessary during typing of HFRS foci and a number of other zoonozes.

Fluorescence quenching in the lichen Peltigera aphthosa due to desiccation.

Photoprotective mechanisms were studied on the tripartite lichen Peltigera aphthosa that exhibits external cephalodia. Using the methods of steady-state and time-resolved fluorescence microscopy, we studied the dynamics of the rehydration process in different parts of the lichen thalli. It was found that apical, medial and basal parts of the thallus are not only morphologically different, but also show completely different chlorophyll induction curves and other spectral characteristics. In dry state, significant contribution to the fluorescence spectrum of lichen gives a green fluorescence of hyphae forming the upper crust, which is rapidly and almost completely quenched during the rehydration process. Probably this is one of the protective mechanisms that reduce the amount of light reaching the PS II reaction centers in the dry state. In the process of rehydration, we observed an increase in the intensity of the chlorophyll fluorescence of the photobiont at 680 nm, with significant changes of the fluorescence lifetimes and the amplitude ratios of fast and slow components of fluorescence decay kinetics. While in dry state, chlorophyll fluorescence is strongly quenched (opposite to the fluorescence of the hyphae), and the fluorescence time constants recover to the typical decay times of active photosynthetic organisms during rehydration. The quantitative behavior of these changes differs largely between the apical, medial and basal parts of the thallus, probably due to the complex interactions of the fungus, algae and cyanobacteria.

Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu.

Here we propose a simple and reliable approach for detection of the tumor marker HER2/neu using the targeting fluorescent hybrid protein DARPin-mCherry. As a targeting module, we used DARPin9-29, which is a member of a novel class of non-immunoglobulin targeting proteins that can highly selectively recognize the extracellular domain of the epidermal growth factor receptor HER2/neu. The red fluorescent protein mCherry was used as the detecting module. The hybrid protein DARPin-mCherry was prepared with high yield in a bacterial expression system and purified in one step by affinity chromatography. The purified protein is not prone to aggregation. The specificity of DARPin-mCherry binding with the HER2/neu tumor marker was demonstrated using confocal microscopy, flow cytofluorimetry, and surface plasmon resonance. The dissociation constant of the DARPin-mCherry protein complex with the HER2/neu receptor determined by surface plasmon resonance was calculated to be 4.5 nM. These characteristics of the hybrid protein DARPin-mCherry suggest it as a promising agent for immunofluorescent assay and an attractive alternative to antibodies and their fragments labeled with fluorescent dyes that are now used for this purpose.