Targosomes: Anti-HER2 PLGA nanocarriers for bioimaging, chemotherapy and local photothermal treatment of tumors and remote metastases.

Developing combined cancer therapy strategies is of utmost importance as it can enhance treatment efficacy, overcome drug resistance, and ultimately improve patient outcomes by targeting multiple pathways and mechanisms involved in cancer growth and progression. Specifically, the potential of developing a combination chemo&photothermal therapy using targeted polymer nanoparticles as nanocarriers offers a promising approach for synergistic cancer treatment by combining the benefits of both therapies, such as targeted drug delivery and localized hyperthermia. Here, we report the first targeted anti-HER2 PLGA nanocarriers, called targosomes, that simultaneously possess photothermal, chemotherapeutic and diagnostic properties using only molecular payloads. Biocompatible poly(lactic-co-glycolic acid), PLGA, nanoparticles were loaded with photosensitizer phthalocyanine, diagnostic dye Nile Blue, and chemotherapeutic drug irinotecan, which was chosen as a result of screening a panel of theragnostic nanoparticles. The targeted delivery to cell surface oncomarker HER2 was ensured by nanoparticle modification with the anti-HER2 monoclonal antibody, trastuzumab, using the one-pot synthesis method without chemical conjugation. The irradiation tests revealed prominent photothermal properties of nanoparticles, namely heating by 35 °C in 10 min. Nanoparticles exhibited a 7-fold increase in binding and nearly an 18-fold increase in cytotoxicity for HER2-overexpressing cells compared to cells lacking HER2 expression. This enhancement of cytotoxicity was further amplified by >20-fold under NIR light irradiation. In vivo studies proved the efficacy of nanoparticles for bioimaging of primary tumor and metastasis sites and demonstrated 93% tumor growth inhibition, making these nanoparticles excellent candidates for translation into theragnostic applications.

Selective targeting of α7 nicotinic acetylcholine receptor by synthetic peptide mimicking loop I of human SLURP-1 provides efficient and prolonged therapy of epidermoid carcinoma in vivo.

α7-Type nicotinic acetylcholine receptor (α7-nAChR) promotes the growth and metastasis of solid tumors. Secreted Ly6/uPAR-Related Protein 1 (SLURP-1) is a specific negative modulator of α7-nAChR produced by epithelial cells. Here, we investigated mechanisms of antiproliferative activity of recombinant SLURP-1 in epidermoid carcinoma A431 cells and activity of SLURP-1 and synthetic 21 a.a. peptide mimicking its loop I (Oncotag) in a xenograft mice model of epidermoid carcinoma. SLURP-1 inhibited the mitogenic pathways and transcription factors in A431 cells, and its antiproliferative activity depended on α7-nAChR. Intravenous treatment of mice with SLURP-1 or Oncotag for 10 days suppressed the tumor growth and metastasis and induced sustained changes in gene and microRNA expression in the tumors. Both SLURP-1 and Oncotag demonstrated no acute toxicity. Surprisingly, Oncotag led to a longer suppression of pro-oncogenic signaling and downregulated expression of pro-oncogenic miR-221 and upregulated expression of KLF4 protein responsible for control of cell differentiation. Affinity purification revealed SLURP-1 interactions with both α7-nAChR and EGFR and selective Oncotag interaction with α7-nAChR. Thus, the selective inhibition of α7-nAChRs by drugs based on Oncotag may be a promising strategy for cancer therapy.

Barnase-barstar Specific Interaction Regulates Car-T Cells Cytotoxic Activity toward Malignancy.

The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.

Artificial Scaffold Polypeptides As an Efficient Tool for the Targeted Delivery of Nanostructures In Vitro and In Vivo.

The use of traditional tools for the targeted delivery of nanostructures, such as antibodies, transferrin, lectins, or aptamers, often leads to an entire range of undesirable effects. The large size of antibodies often does not allow one to reach the required number of molecules on the surface of nanostructures during modification, and the constant domains of heavy chains, due to their effector functions, can induce phagocytosis. In the recent two decades, targeted polypeptide scaffold molecules of a non-immunoglobulin nature, antibody mimetics, have emerged as much more effective targeting tools. They are small in size (3-20 kDa), possess high affinity (from subnano- to femtomolar binding constants), low immunogenicity, and exceptional thermodynamic stability. These molecules can be effectively produced in bacterial cells, and, using genetic engineering manipulations, it is possible to create multispecific fusion proteins for the targeting of nanoparticles to cells with a given molecular portrait, which makes scaffold polypeptides an optimal tool for theranostics.

3D Models of Cellular Spheroids As a Universal Tool for Studying the Cytotoxic Properties of Anticancer Compounds In Vitro.

The aim of this work is to develop a 3D cell culture model based on cell spheroids for predicting the functional activity of various compounds in vivo. Agarose gel molds were made using 3D printing. The solidified agarose gel is a matrix consisting of nine low-adhesive U-shaped microwells of 2.3 × 3.3 mm for 3D cell spheroid formation and growth. This matrix is placed into a single well of a 12-well plate. The effectiveness of the cell culture method was demonstrated using human ovarian carcinoma SKOVip-kat cells stably expressing the red fluorescent protein Katushka in the cytoplasm and overexpressing the membrane-associated tumor marker HER2. The SKOVip-kat cell spheroids were visualized by fluorescence microscopy. The cell concentration required for the formation of same-shape and same-size spheroids with tight intercellular contacts was optimized. To verify the developed model, the cytotoxicity of the targeted immunotoxin anti-HER2 consisting of the anti-HER2 scaffold DARP 9_29 and a fragment of the Pseudomonas aeroginosa exotoxin, DARP-LoPE, was studied in 2D and 3D SKOVip-kat cell cultures. The existence of a difference in the cytotoxic properties of DARP-LoPE between the 2D and 3D cultures has been demonstrated: the IC50 value in the 3D culture is an order of magnitude higher than that in the monolayer culture. The present work describes a universal tool for 3D cultivation of mammalian cells based on reusable agarose gel molds that allows for reproducible formation of multicellular spheroids with tight contacts for molecular and cell biology studies.

"Green" Synthesis of Cytotoxic Silver Nanoparticles Based on Secondary Metabolites of Lavandula Angustifolia Mill.

In this study, we used "green" synthesis to prepare silver nanoparticles (NPs) from aqueous plant and callus extracts of the narrow-leaved lavender Lavandula angustifolia Mill. 35.4 ± 1.6 nm and 56.4 ± 2.4 nm nanoparticles, colloidally stable in phosphate-buffered saline, were synthesized using the plant extract and the callus extract, respectively. NPs were characterized by spectrophotometry, dynamic light scattering, and scanning electron microscopy. We studied the dynamics of the nanoparticle synthesis and evaluated the cytotoxic properties of the plant extract-based NPs. Modification of NPs with bovine serum albumin demonstrated that blockage of the nanoparticle surface completely suppressed NP cytotoxic activity in vitro. The synthesized NPs possess localized surface plasmon resonance properties and are of small sizes, and their surface can be modified with protein molecules, which makes them promising agents for cancer theranostics.

Nanoparticle-based drug delivery via RBC-hitchhiking for the inhibition of lung metastases growth.

Delivery of particle-based theranostic agents via their transportation on the surfaces of red blood cells, commonly referred to as RBC-hitchhiking, has historically been developed as a promising strategy for increasing the extremely poor blood circulation lifetime, primarily, of the large-sized sub-micron agents. Here, we show for the first time that RBC-hitchhiking can be extremely efficient for nanoparticle delivery and tumor treatment even in those cases when no circulation prolongation is observed. Specifically, we demonstrate that RBC-hitchhiking of certain small 100 nm particles, unlike that of the conventional sub-micron ones, can boost the delivery of non-targeted particles to lungs up to a record high value of 120-fold (and up to 40% of the injected dose). To achieve this remarkable result, we screened sub-200 nm nanoparticles of different sizes, polymer coatings and ζ-potentials and identified particles with the optimal RBC adsorption/desorption behavior. Furthermore, we demonstrated that such RBC-mediated rerouting of particles to lungs can be used to fight pulmonary metastases of aggressive melanoma B16-F1. Our findings could change the general paradigm of drug delivery for cancer treatment with RBC-hitchhiking. It is not the blood circulation lifetime that is the key factor for nanoparticle efficiency, but rather the complexation of nanoparticles with the RBC. The demonstrated technology could become a valuable tool for development of new strategies based on small nanoparticles for the treatment of aggressive and small-cell types of cancer as well as other lung diseases.

Synthesis of Magnetic Nanoparticles Stabilized by Magnetite-Binding Protein for Targeted Delivery to Cancer Cells.

A new method for obtaining biomodified magnetite nanoparticles for targeted delivery to cells was developed. The method is based on the use of the C-terminal fragment of the Mms6 protein, which is involved in the magnetite biomineralization during the synthesis of magnetosomes in magnetotactic bacteria Magnetospirillum magneticum AMB-1, and the barnase*barstar high-affinity protein pair. The Mms6 protein fragment is required for stabilizing magnetite, and the barnase*barstar pair mediates the interaction between nanoparticles and the component for modification. The efficiency of this method was confirmed in the synthesis of magnetite nanoparticles recognizing the HER2/neu tumor marker and in the selective labeling of HER2/neu with these nanoparticles on the surface of cancer cells.

Synthesis and Characterization of Hybrid Core-Shell Fe3O4/SiO2 Nanoparticles for Biomedical Applications.

The creation of markers that provide both visual and quantitative information is of considerable importance for the mapping of tissue macrophages and other cells. We synthesized magnetic and magneto-fluorescent nanomarkers for the labeling of cells which can be detected with high sensitivity by the magnetic particle quantification (MPQ) technique. For stabilization under physiological conditions, the markers were coated with a dense silica shell. In this case, the size and zeta-potential of nanoparticles were controlled by a modified Stober reaction. Also, we developed a novel facile two-step synthesis of carboxylic acid-functionalized magnetic SiO2 nanoparticles, with a carboxyl polymer shell forming on the nanoparticles before the initiation of the Stober reaction. We extensively characterized the nanomarkers by transmission electron microscopy, electron microdiffraction, and dynamic and electrophoretic light scattering. We also studied the nanoparticle cellular uptake by various eukaryotic cell lines.

MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions.

Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.

A comprehensive study of interactions between lectins and glycoproteins for the development of effective theranostic nanoagents.

A comprehensive study of the interactions between lectins and glycoproteins possessing different glycosylation profiles in the composition of nanoparticles was carried out in order to find specifically interacting protein pairs for the creation of novel classes of multifunctional nanoagets that based on protein-assisted selfassembly. We obtained information about specific interactions of certain lectins with selected glycoproteins as well as about the ability of certain monosaccharides to competitively inhibit binding of glycoproteins with lectins. These protein-mediated interactions may be involved in the formulation of self-assembled nanoparticles for therapy and diagnostics of various diseases.

Development of immunoassays using interferometric real-time registration of their kinetics.

A method for effective development of solid-phase immunoassays on a glass surface and for optimization of related protocols by highly sensitive quantitative monitoring of each assay step has been proposed and experimentally implemented. The method is based on the spectral correlation interferometry (SCI) that allows real-time measuring of the thickness of a biomolecular layer bound to the recognition molecular receptors on the sensor chip surface. The method is realized with compact 3-channel SCI-biosensors that employ as the sensor chips standard cover glass slips without deposition of any additional films. Different schemes for antibody immobilization on a glass surface have been experimentally compared and optimized toward a higher sorption capacity of the sensor chips. Comparative characterization of the kinetics of each immunoassay stage has been implemented with the optimized protocols: i) covalent immobilization of antibody on an epoxylated surface and ii) biotinylated antibody sorption on a biotinylated surface via a high-affinity biotin-streptavidin bond. We have shown that magnetic nanoparticles employed as labels with model detection of cardiac troponin I further amplify the SCI signal, resulting in 100-fold improvement of the detection limit. The developed protocols can also be used with the alternative immunoassay platforms, including the label methods based on registration of only the final assay result, which is the quantity of bound labels.