BODIPY Dye Derivative for Irreversible Fluorescent Labeling of Eukaryotic Cells and Their Simultaneous Cytometric Analysis

Pakhomov A.A., Efremova A.V., Maksimova M.A., Kononevich Y.N., Ionov D.S., Dubinets N.O., Martynov V.I., Muzafarov A.M., Alfimov M.V.

Pakhomov A.A., Efremova A.V., Kononevich Y., Ionov D.S., Maksimova M.A., Volodin A.D., Korlyukov A.A., Dubinets N.O., Martynov V.I., Ivanov A.A., Muzafarov A.M.

AbstractFluorescent dyes which exhibit emission/excitation in the second near‐infrared (NIR‐II, 1000–1350 nm) region are currently attracting significant attention in bioimaging and diagnostics applications. Furthermore, dyes with high two‐photon absorption cross‐section (TPA), such as BODIPY derivatives, are of a particular interest due to deeper signal penetration into biological tissues, better image contrast, reduced phototoxicity and photobleaching. Herein we report the synthesis and properties of new monomeric and dimeric di‐styryl‐BODIPY dyes, which have absorption maxima near 625 nm and emission in the range of 600–800 nm (NIR‐I, 650–950 nm). For the first time, we used a femtosecond Cr:Forsterite laser with a wavelength of 1250 nm (NIR‐II) for the excitation of NIR‐I di‐styryl‐BODIPY dyes by TPA. A cooperative effect was observed for TPA for the dimeric di‐styryl‐BODIPY dyes. The results obtained may be of great interest due to their potential applications in bioimaging and photodynamic therapy.

Shipunova V.O., Komedchikova E.N., Kotelnikova P.A., Nikitin M.P., Deyev S.M.

Therapy for aggressive metastatic breast cancer remains a great challenge for modern biomedicine. Biocompatible polymer nanoparticles have been successfully used in clinic and are seen as a potential solution. Specifically, researchers are exploring the development of chemotherapeutic nanoagents targeting the membrane-associated receptors of cancer cells, such as HER2. However, there are no targeting nanomedications that have been approved for human cancer therapy. Novel strategies are being developed to alter the architecture of agents and optimize their systemic administration. Here, we describe a combination of these approaches, namely, the design of a targeted polymer nanocarrier and a method for its systemic delivery to the tumor site. Namely, PLGA nanocapsules loaded with a diagnostic dye, Nile Blue, and a chemotherapeutic compound, doxorubicin, are used for two-step targeted delivery using the concept of tumor pre-targeting through the barnase/barstar protein “bacterial superglue”. The first pre-targeting component consists of an anti-HER2 scaffold protein, DARPin9_29 fused with barstar, Bs-DARPin9_29, and the second component comprises chemotherapeutic PLGA nanocapsules conjugated to barnase, PLGA-Bn. The efficacy of this system was evaluated in vivo. To this aim, we developed an immunocompetent BALB/c mouse tumor model with a stable expression of human HER2 oncomarkers to test the potential of two-step delivery of oncotheranostic nano-PLGA. In vitro and ex vivo studies confirmed HER2 receptor stable expression in the tumor, making it a feasible tool for HER2-targeted drug evaluation. We demonstrated that two-step delivery was more effective than one-step delivery for both imaging and tumor therapy: two-step delivery had higher imaging capabilities than one-step and a tumor growth inhibition of 94.9% in comparison to 68.4% for the one-step strategy. The barnase*barstar protein pair has been proven to possess excellent biocompatibility, as evidenced by the successful completion of biosafety tests assessing immunogenicity and hemotoxicity. This renders the protein pair a highly versatile tool for pre-targeting tumors with various molecular profiles, thereby enabling the development of personalized medicine.

Swain S.M., Shastry M., Hamilton E.

The long-sought discovery of HER2 as an actionable and highly sensitive therapeutic target was a major breakthrough for the treatment of highly aggressive HER2-positive breast cancer, leading to approval of the first HER2-targeted drug — the monoclonal antibody trastuzumab — almost 25 years ago. Since then, progress has been swift and the impressive clinical activity across multiple trials with monoclonal antibodies, tyrosine kinase inhibitors and antibody–drug conjugates that target HER2 has spawned extensive efforts to develop newer platforms and more targeted therapies. This Review discusses the current standards of care for HER2-positive breast cancer, mechanisms of resistance to HER2-targeted therapy and new therapeutic approaches and agents, including strategies to harness the immune system. The discovery of the monoclonal antibody trastuzumab almost 25 years ago revolutionized treatment and drug development for HER2+ breast cancer. Here, Swain et al. review the current standard of care for HER2+ breast cancer, describe mechanisms of drug resistance and focus on next-generation platforms and therapies for the treatment of this disease.

Semenov A.N., Gvozdev D.A., Zlenko D.V., Protasova E.A., Khashimova A.R., Parshina E.Y., Baizhumanov A.A., Lotosh N.Y., Kim E.E., Kononevich Y.N., Pakhomov A.A., Selishcheva A.A., Sluchanko N.N., Shirshin E.A., Maksimov E.G.

Carotenoids are potent antioxidants with a wide range of biomedical applications. However, their delivery into human cells is challenging and relatively inefficient. While the use of natural water-soluble carotenoproteins capable to reversibly bind carotenoids and transfer them into membranes is promising, the quantitative estimation of the delivery remains unclear. In the present work, we studied echinenone (ECN) delivery by cyanobacterial carotenoprotein AnaCTDH (C-terminal domain homolog of the Orange Carotenoid Protein from Anabaena), into liposome membranes labelled with BODIPY fluorescent probe. We observed that addition of AnaCTDH-ECN to liposomes led to the significant changes in the fast-kinetic component of the fluorescence decay curve, pointing on the dipole-dipole interactions between the probe and ECN within the membrane. It may serve as an indirect evidence of ECN delivery into membrane. To study the delivery in detail, we carried out molecular dynamics modeling of the localization of ECN within the lipid bilayer and calculate its orientation factor. Next, we exploited FRET to assess concentration of ECN delivered by AnaCTDH. Finally, we used time-resolved fluorescence anisotropy to assess changes in microviscosity of liposomal membranes. Incorporation of liposomes with β-carotene increased membrane microviscosity while the effect of astaxanthin and its mono- and diester forms was less pronounced. At temperatures below 30 °C addition of AnaCTDH-ECN increased membrane microviscosity in a concentration-dependent manner, supporting the protein-mediated carotenoid delivery mechanism. Combining all data, we propose FRET-based analysis and assessment of membrane microviscosity as potent approaches to characterize the efficiency of carotenoids delivery into membranes.

Pakhomov A.A., Kim E.E., Kononevich Y.N., Ionov D.S., Maksimova M.A., Khalchenia V.B., Maksimov E.G., Anisimov A.A., Shchegolikhina O.I., Martynov V.I., Muzafarov A.M.

A series of multifluorophore compounds with two, four, six and eight BODIPY residues linked to a siloxane core via a flexible spacer was synthesized. An increasing number of BODIPY units caused an enhanced propensity to chromophore aggregation with characteristic photophysical effects evoked by the aggregation. The increase in the number of fluorophores additionally resulted in considerable increase in molar extinction coefficient . Meanwhile, a slight drop of the extinction coefficient as calculated per a single chromophore was detected. Multifluorophore conjugates showed a significant broadening of fluorescence emission bands in polar solvents and a substantial decrease in the fluorescence quantum yield due to the aggregation-caused quenching. Time resolved fluorescence measurements of multifluorophore compounds in different solvents showed that the fluorescence decays have a multiexponential character and strongly depend on the number of fluorophores in the conjugate, polarity/viscosity of the solvent and the detection wavelength. • A series of BODIPY-siloxane conjugates with 2,4,6, and 8 fluorophores in a single molecule was synthesized. • The dyes showed a pronounced solvatochromic behavior. • A moderate quenching of fluorescence. • Decrease of specific extinction coefficient calculated per a single chromophore. • Fluorescence lifetimes depend on the detection wavelength and solvent.

Martynov V.I., Pakhomov A.A.

Abstract Fluorescent compounds have become indispensable tools for imaging molecular activities in the living cell. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is currently one of the most popular fluorescent reporters due to its unique photophysical properties. This review provides a general survey and presents a summary of recent advances in the development of new BODIPY-based cellular biomarkers and biosensors. The review starts with the consideration of the properties of BODIPY derivatives required for their application as cellular reporters. Then review provides examples of the design of sensors for different biologically important molecules, ions, membrane potential, temperature and viscosity defining the live cell status. Special attention is payed to BODPY-based phototransformable reporters. The bibliography includes 339 references.

Manohar S.M., Shah P., Nair A.

Flow cytometry (FCM) is a sophisticated technique that works on the principle of light scattering and fluorescence emission by the specific fluorescent probe-labeled cells as they pass through a laser beam. It offers several unique advantages as it allows fast, relatively quantitative, multiparametric analysis of cell populations at the single cell level. In addition, it also enables physical sorting of the cells to separate the subpopulations based on different parameters. In this constantly evolving field, innovative technologies such as imaging FCM, mass cytometry and Raman FCM are being developed in order to address limitations of traditional FCM. This review explains the general principles, main applications and recent advances in the field of FCM.

Shipunova V.O., Komedchikova E.N., Kotelnikova P.A., Zelepukin I.V., Schulga A.A., Proshkina G.M., Shramova E.I., Kutscher H.L., Telegin G.B., Kabashin A.V., Prasad P.N., Deyev S.M.

When combined with immunotherapy, image-guided targeted delivery of chemotherapeutic agents is a promising direction for combination cancer theranostics, but this approach has so far produced only limited success due to a lack of molecular targets on the cell surface and low therapeutic index of conventional chemotherapy drugs. Here, we demonstrate a synergistic strategy of combination immuno/chemotherapy in conditions of dual regioselective targeting, implying vectoring of two distinct binding sites of a single oncomarker (here, HER2) with theranostic compounds having a different mechanism of action. We use: (i) PLGA nanoformulation, loaded with an imaging diagnostic fluorescent dye (Nile Red) and a chemotherapeutic drug (doxorubicin), and functionalized with affibody ZHER2:342 (8 kDa); (ii) bifunctional genetically engineered DARP-LoPE (42 kDa) immunotoxin comprising of a low-immunogenic modification of therapeutic Pseudomonas exotoxin A (LoPE) and a scaffold targeting protein, DARPin9.29 (14 kDa). According to the proposed strategy, the first chemotherapeutic nanoagent is targeted by the affibody to subdomain III and IV of HER2 with 60-fold specificity compared with nontargeted particles, while the second immunotoxin is effectively targeted by DARPin molecule to subdomain I of HER2. We demonstrate that this dual targeting strategy can enhance anticancer therapy of HER2-positive cells with a very strong synergy, which made possible 1000-fold decrease of effective drug concentration in vitro and a significant enhancement of HER2 cancer therapy compared to monotherapy in vivo. Moreover, this therapeutic combination prevented the appearance of secondary tumor nodes. Thus, the suggested synergistic strategy utilizing dual targeting of the same oncomarker could give rise to efficient methods for aggressive tumors treatment.

Kuo W., Lin J., Hsu H., Chen H., Yang A., Wu C.

Human epidermal growth factor receptor 2 (HER2) overexpression occurs in various types of cancers. Regarding the anti-HER2 targeted therapies showed superior treatment outcomes in several (pre)clinical studies, we used multimodality image to rapidly select novel HER2-targeting antibodies for further therapeutics development. The four anti-HER2 antibodies (H32 IgG, 75 IgG, 61 IgG, and trastuzumab) labeled with either In-111 or a DyLight680 fluorescent dye were applied to perform cellular uptake, endocytosis, optical/microSPECT/CT imaging and biodistribution studies. In vitro and in vivo relative effectiveness of these antibodies were also compared in an N87 gastric cancer xenograft model. The internalized radioactivity of [111In]61 IgG in N87 cells increased from 33% at 12 hr to 56% at 48 hr after incubation, while the majority of other antibodies stayed on the cell membranes. Among these antibodies, 61 IgG showed the highest accumulation in tumors with the tumor-to-muscle ratio (T/M) of 131 ± 61.4 and 19.13 ± 3.42 conducted by IVIS and microSPECT/CT, respectively. We demonstrated that multimodality imaging is a reliable approach for selecting potential antibodies and found that 61 IgG manifested significant tumor accumulation with elevated internalization rate thus could be a suitable candidate for further development of new HER2-targeted therapies.

TANG Q., ONITSUKA M., TABATA A., TOMOYASU T., NAGAMUNE H.

McKinnon K.M.

Flow cytometry is a technology that provides rapid multi-parametric analysis of single cells in solution. Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors such as photodiodes or photomultiplier tubes. These light signals are converted into electronic signals that are analyzed by a computer and written to a standardized format (.fcs) data file. Cell populations can be analyzed and/or purified based on their fluorescent or light scattering characteristics. A variety of fluorescent reagents are utilized in flow cytometry. These include fluorescently conjugated antibodies, nucleic acid binding dyes, viability dyes, ion indicator dyes, and fluorescent expression proteins. Flow cytometry is a powerful tool that has applications in immunology, molecular biology, bacteriology, virology, cancer biology, and infectious disease monitoring. It has seen dramatic advances over the last 30 years, allowing unprecedented detail in studies of the immune system and other areas of cell biology. © 2018 by John Wiley & Sons, Inc.

Pakhomov A.A., Deyev I.E., Ratnikova N.M., Chumakov S.P., Mironiuk V.B., Kononevich Y.N., Muzafarov A.M., Martynov V.I.

In nonpolar solvents, hydrophobic organic fluorophores often show bright fluorescence, whereas in polar media, they usually suffer from aggregation-caused quenching (ACQ). Here, we harnessed this solvatochromic behavior of a 1,3,5,7-tetramethyl-BODIPY derivative for cell staining and applied it to live-cell imaging and flow cytometry. As opposed to commercially available dyes, this BODIPY derivative showed excellent contrast immediately after staining and did not require any wash-off.

Pakhomov A.A., Mironiuk V.B., Kononevich Y.N., Korlyukov A.A., Volodin A.D., Pryakhina T.A., Martynov V.I., Muzafarov A.M.

A meso-decene-BODIPY dye was synthesized and conjugated to a polydimethylsiloxane. Fluorescence properties of the obtained polymer drastically changed in different solvents. The polymer showed an unusual for other siloxane polymers trend to structuration.

Marfin Y.S., Solomonov A.V., Timin A.S., Rumyantsev E.V.

The group of fluorophores on boron dipyrrin platform (4,4- difluoro-4-bora3a,4a-diaza-s-indacene, also known as BODIPY) has attracted much attention in the field of molecular sensorics, including sensing of biomolecules and bioprocesses. Structural diversity of existing BODIPY with ample opportunities of directed modification of compounds makes this class of fluorophores attractive for medical and biological purposes. The recent progress in the design and functionalization of BODIPY allows using them for modification of drug micro- and nanocarriers in order to improve their therapeutic effect in cancer treatment. At the same time, integration of BODIPY into drug carriers provides the possibility of in vitro and in vivo real time imaging of used drug carriers. The high fluorescent intensity and low toxicity of BODIPY granted for conjugation with different biomolecules.The present review focuses on the recent advances for application of individual BODIPY in medical diagnostics, antimicrobial activity, as well as establishing the role of BODIPY in labeling of biomolecules (e.g. proteins, hormones and DNA). Also the review highlights the potential of BODIPY in functionalization of drug micro- and nanocarriers in order to achieve better therapeutic efficiency compared with non-modified materials. The advantages derived from the use of BODIPY for preparation and modification of drug carriers are critically evaluated and potential for future challenges, especially concerning the design of innovative multi-functional BODIPY-based nanocarriers, is discussed in detail using representative examples from literature.Our objective was to show that BODIPY are powerful tools for bioimaging, labeling of biomolecules and construction of new multifunctional drug carriers.

Efimova A.S., Ustimova M.A., Frolova A.Y., Martynov V.I., Deyev S.M., Fedorov Y.V., Fedorova O.A., Pakhomov A.A.