Anastasiya V. Ryabova

Near-infrared fluorescent proteins

Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

Genetically encoded immunophotosensitizer 4D5scFv-miniSOG is a highly selective agent for targeted photokilling of tumor cells in vitro

Tumor-targeted delivery of cytotoxins presents considerable advantages over their passive transport. Chemical conjugation of cytotoxic module to antibody is limited due to insufficient reproducibility of synthesis, and recombinant immunotoxins are aimed to overcome this disadvantage. We obtained genetically encoded immunophotosensitizer 4D5scFv-miniSOG and evaluated its photocytotoxic effect in vitro. A single-chain variable fragment (scFv) of humanized 4D5 antibody was used as a targeting vehicle for selective recognition of the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) overexpressed in many human carcinomas. As a phototoxic module we used a recently described photoactivated fluorescent flavoprotein miniSOG. We found that recombinant protein 4D5scFv-miniSOG exerts a highly specific photo-induced cytotoxic effect on HER2/neu-positive human breast adenocarcinoma SK-BR-3 cells (IC50= 160 nM). We demonstrated that the 4D5scFv-miniSOG specifically binds to HER2-positive cells and internalizes via receptor-mediated endocytosis. Co-treatment of breast cancer cells with 4D5scFv-miniSOG and Taxol or junction opener protein JO-1 produced remarkable additive effects.

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.

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.

Application of aluminum phthalocyanine nanoparticles for fluorescent diagnostics in dentistry and skin autotransplantology

This paper deals with the possibility of application of aluminum phthalocyanine (AlPc) nanoparticles in clinical practice. AlPc fluoresces in the molecular form but in the form of nanoparticles it does not. Separation of molecules from an AlPc nanoparticle and therefore the appearance of fluorescence occurs under the effect of a number of biochemo-physical factors. Owing to this feature the application of AlPc nanoparticles followed by the measurement of fluorescence spectra is proposed as a diagnostics method. It was shown that after AlPc nanoparticle application on a tooth surface the fluorescence intensity in the enamel microdamage area is 2-3 times higher than that in the normal enamel area. The appearance of fluorescence after application of AlPc nanoparticles on skin autografts testifies to the presence of inflammation.

Fluorescence investigation of the detachment of aluminum phthalocyanine molecules from aluminum phthalocyanine nanoparticles in monocytes/macrophages and skin cells and their localization in monocytes/macrophages

BACKGROUND Nanoparticles made from aluminum phthalocyanine (AlPc) are non-fluorescent in the nanoparticle form. Once AlPc molecules become detached from the particle, fluorescence occurs. Preliminary work showed the benefit of using aluminum phthalocyanine nanoparticles (nAlPc) for the rating of the rejection risk of skin autografts in mice by measuring fluorescence intensities of detached AlPc. Skin autografts showing a high fluorescence intensity were finally rejected suggesting an inflammatory process. In contrast, autografts with normal autofluorescence were accepted. This work was focused on the mechanism of this finding. The aim is detecting inflammatory processes and the potential use of nAlPc for PDT as a new treatment modality. METHODS The effect of the lipopolysaccharide-stimulated monocyte/macrophage murine cell line J774A.1 on the monomerization of internalized nAlPc was tested. Further, we investigated the influence of J774A.1 cells and the normal skin cell lines L-929 or HaCaT on the dissolution of nAlPc by laser scanning microscopy and flow cytometry. Localization of AlPc molecules after uptake and dissolution of nanoparticles by the cells was surveyed. RESULTS In co-culture models composed of J774A.1 and HaCaT/L-929 cells, the AlPc fluorescence intensity in J774A.1 cells is 1.38/1.89 fold higher, respectively. According to localization measurements in J774A.1 cells it can be assumed that nAlPc is taken up via endocytosis and remains in endosomes and/or lysosomes dissolving there. Detached molecules of AlPc cause rapture of the endosomal and/or lysosomal membrane after irradiation to become quite uniformly distributed in the cytoplasm. CONCLUSIONS Evidence for monocytes/macrophages being the origin of the measured AlPc fluorescence in rejected skin autografts was confirmed.

Synthesis, Characterization, and Selective Delivery of DARPin–Gold Nanoparticle Conjugates to Cancer Cells

We demonstrate that the designed ankyrin repeat protein (DARPin)_9-29, which specifically targets human epidermal growth factor receptor 2 (HER 2), binds tightly to gold nanoparticles (GNPs). Binding of the protein strongly increases the colloidal stability of the particles. The results of experimental analysis and molecular dynamics simulations show that approximately 35 DARPin_9-29 molecules are bound to the surface of a 5 nm GNP and that the binding does not involve the receptor-binding domain of the protein. The confocal fluorescent microscopy studies show that the DARPin-coated GNP conjugate specifically interacts with the surface of human cancer cells overexpressing epidermal growth factor receptor 2 (HER2) and enters the cells by endocytosis. The high stability under physiological conditions and high affinity to the receptors overexpressed by cancer cells make conjugates of plasmonic gold nanostructures with DARPin molecules promising candidates for cancer therapy.

Laser heating of the Y 1-x Dy x PO 4 nanocrystals

We propose a novel material prepared by microwave-hydrothermal treatment, the tetragonal xenotime-type yttrium orthophosphate YPO4 nanocrystals doped by different concentrations of Dy3+. It may be suitable for laser-induced local heating of cancer tumors for hyperthermia. We heated a powder consisted of the nanoparticles by focused quasi-CW laser irradiation at different wavelengths in the near IR spectral range fitting the transparency window of biological tissues. The local temperature on the surface of the powder in the place of irradiation increases linearly with increasing laser power and increasing the Dy3+ concentration. At the same time the efficiency of local heating Φ = ΔT / (P f) (ΔT is a local temperature increase, f is an oscillator strength of absorption transition, and P is the quantity of laser power) is proportional to the energy of the initially excited electronic level. The proposed method allows for high rates of heating and cooling. The laser power used for heating was rather low, tens of milliwatts that together with short heating time to required temperature may result in extremely low doses of laser irradiation for heating.

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.

Selective staining and eradication of cancer cells by protein-carrying DARPin-functionalized liposomes

The proteoliposomes containing large quantities of highly fluorescent protein, mCherry or a fragment of Pseudomonas exotoxin A, PE40 and functionalized with the designed ankyrin repeat protein (DARPin), which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub‐nanomolar concentrations human breast adenocarcinoma cells, overexpressing HER2, respectively. ABSTRACT Since their discovery, liposomes have been widely employed in biomedical research. These nano‐size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand‐targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80–90nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme‐containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9–29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub‐nanomolar concentrations HER2‐positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor‐positive cells demonstrated here makes the DARPin‐functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy.