Evaluation of affinity matured Affibody molecules for imaging of the immune checkpoint protein B7-H3.

B7-H3 (CD276), an immune checkpoint protein, is a promising molecular target for immune therapy of malignant tumours. Sufficient B7-H3 expression level is a precondition for successful therapy. Radionuclide molecular imaging is a powerful technique for visualization of expression levels of molecular targets in vivo. Use of small radiolabelled targeting proteins would enable high-contrast radionuclide imaging of molecular targets if adequate binding affinity and specificity of an imaging probe could be provided. Affibody molecules, small engineered affinity proteins based on a non-immunoglobulin scaffold, have demonstrated an appreciable potential in radionuclide imaging. Proof-of principle of radionuclide visualization of expression levels of B7-H3 in vivo was demonstrated using the [99mTc]Tc-AC12-GGGC Affibody molecule. We performed an affinity maturation of AC12, enabling selection of clones with higher affinity. Three most promising clones were expressed with a -GGGC (triglycine-cysteine) chelating sequence at the C-terminus and labelled with technetium-99m (99mTc). 99mTc-labelled conjugates bound to B7-H3-expressing cells specifically in vitro and in vivo. Biodistribution in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrated improved imaging properties of the new conjugates compared with the parental variant [99mTc]Tc-AC12-GGGC. [99mTc]Tc-SYNT-179 provided the strongest improvement of tumour-to-organ ratios. Thus, affinity maturation of B7-H3 Affibody molecules could improve biodistribution and targeting properties for imaging of B7-H3-expressing tumours.

Neuroprotective Effects of Tryptanthrin-6-Oxime in a Rat Model of Transient Focal Cerebral Ischemia.

The activation of c-Jun N-terminal kinase (JNK) plays an important role in stroke outcomes. Tryptanthrin-6-oxime (TRYP-Ox) is reported to have high affinity for JNK and anti-inflammatory activity and may be of interest as a promising neuroprotective agent. The aim of this study was to investigate the neuroprotective effects of TRYP-Ox in a rat model of transient focal cerebral ischemia (FCI), which involved intraluminal occlusion of the left middle cerebral artery (MCA) for 1 h. Animals in the experimental group were administered intraperitoneal injections of TRYP-Ox 30 min before reperfusion and 23 and 47 h after FCI. Neurological status was assessed 4, 24, and 48 h following FCI onset. Treatment with 5 and 10 mg/kg of TRYP-Ox decreased mean scores of neurological deficits by 35-49 and 46-67% at 24 and 48 h, respectively. At these doses, TRYP-Ox decreased the infarction size by 28-31% at 48 h after FCI. TRYP-Ox (10 mg/kg) reduced the content of interleukin (IL) 1ß and tumor necrosis factor (TNF) in the ischemic core area of the MCA region by 33% and 38%, respectively, and attenuated cerebral edema by 11% in the left hemisphere, which was affected by infarction, and by 6% in the right, contralateral hemisphere 24 h after FCI. TRYP-Ox reduced c-Jun phosphorylation in the MCA pool at 1 h after reperfusion. TRYP-Ox was predicted to have high blood-brain barrier permeability using various calculated descriptors and binary classification trees. Indeed, reactive oxidant production was significantly lower in the brain homogenates from rats treated with TRYP-Ox versus that in control animals. Our data suggest that the neuroprotective activity of TRYP-Ox may be due to the ability of this compound to inhibit JNK and exhibit anti-inflammatory and antioxidant activity. Thus, TRYP-Ox may be considered a promising neuroprotective agent that potentially could be used for the development of new treatment strategies in cerebral ischemia.

A Comparative Study of Cancer Cells Susceptibility to Silver Nanoparticles Produced by Electron Beam.

INTRODUCTION: Silver nanoparticles (AgNPs) have a wide range of bioactivity, which is highly dependent on particle size, shape, stabilizer, and production method. Here, we present the results of studies of AgNPs cytotoxic properties obtained by irradiation treatment of silver nitrate solution and various stabilizers by accelerating electron beam in a liquid medium. METHODS: The results of studies of morphological characteristics of silver nanoparticles were obtained by transmission electron microscopy, UV-vis spectroscopy, and dynamic light scattering measurements. MTT test, alamar blue test, flow cytometry, and fluorescence microscopy were used to study the anti-cancer properties. As biological objects for standard tests, adhesive and suspension cell cultures of normal and tumor origin, including prostate cancer, ovarian cancer, breast cancer, colon cancer, neuroblastoma, and leukemia, were studied. RESULTS: The results showed that the silver nanoparticles obtained by irradiation with polyvinylpyrrolidone and collagen hydrolysate are stable in solutions. Samples with different stabilizers were characterized by a wide average size distribution from 2 to 50 nm and low zeta potential from -7.3 to +12.4 mV. All AgNPs formulations showed a dose-dependent cytotoxic effect on tumor cells. It has been established that the particles obtained with the combination of polyvinylpyrrolidone/collagen hydrolysate have a relatively more pronounced cytotoxic effect in comparison to samples stabilized with only collagen or only polyvinylpyrrolidone. The minimum inhibitory concentrations for nanoparticles were less than 1 µg/mL for various types of tumor cells. It was found that neuroblastoma (SH-SY5Y) is the most susceptible, and ovarian cancer (SKOV-3) is the most resistant to the action of silver nanoparticles. The activity of the AgNPs formulation prepared with a mixture of PVP and PH studied in this work was higher that activity of other AgNPs formulations reported in the literature by about 50 times. CONCLUSIONS: The results indicate that the AgNPs formulations synthesized with an electron beam and stabilized with polyvinylpyrrolidone and protein hydrolysate deserve deep study for their further use in selective cancer treatment without harming healthy cells in the patient organism.

Sweetened Alkylated Verdazyls Effectively Kill Cancer Cells under Light Irradiation.

The development of novel photo-dynamic therapy (PDT) agents enabling to treat the oxygen-deficient tumors is the emerging tasks for the modern medicinal chemistry. Herein, we describe the design and preparation of water-soluble agents for PDT which generate active radical species upon light irradiation. Two conjugates of carbohydrates with 1,2,4,6-substituted-1,4-dihydro-1,2,4,5-tetrazin-3(2H)-ones (AlkVZs) demonstrated high oxygen-independent cytotoxicity on PC-3 and Jurkat cancer cells under light irradiation combining with low toxicity in the dark. The efficacy of prepared compounds was estimated using MTT and Alamar Blue tests as well as microscopic dead/live colored images and flow cytometry. The analysis of obtained results reveals the influence of sugar moiety on the activity of AlkVZs. We believe that obtained compounds have high potency as platform for design of new agents for photo-dynamic therapy.

Neuroprotective Effects of the Lithium Salt of a Novel JNK Inhibitor in an Animal Model of Cerebral Ischemia-Reperfusion.

The c-Jun N-terminal kinases (JNKs) regulate many physiological processes, including inflammatory responses, morphogenesis, cell proliferation, differentiation, survival, and cell death. Therefore, JNKs represent attractive targets for therapeutic intervention. In an effort to develop improved JNK inhibitors, we synthesized the lithium salt of 11H-indeno[1,2-b]quinoxaline-11-one oxime (IQ-1L) and evaluated its affinity for JNK and biological activity in vitro and in vivo. According to density functional theory (DFT) modeling, the Li+ ion stabilizes the six-membered ring with the 11H-indeno[1,2-b]quinoxaline-11-one (IQ-1) oximate better than Na+. Molecular docking showed that the Z isomer of the IQ-1 oximate should bind JNK1 and JNK3 better than (E)-IQ-1. Indeed, experimental analysis showed that IQ-1L exhibited higher JNK1-3 binding affinity in comparison with IQ-1S. IQ-1L also was a more effective inhibitor of lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) transcriptional activity in THP-1Blue monocytes and was a potent inhibitor of proinflammatory cytokine production by MonoMac-6 monocytic cells. In addition, IQ-1L inhibited LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. In a rat model of focal cerebral ischemia (FCI), intraperitoneal injections of 12 mg/kg IQ-1L led to significant neuroprotective effects, decreasing total neurological deficit scores by 28, 29, and 32% at 4, 24, and 48 h after FCI, respectively, and reducing infarct size by 52% at 48 h after FCI. The therapeutic efficacy of 12 mg/kg IQ-1L was comparable to that observed with 25 mg/kg of IQ-1S, indicating that complexation with Li+ improved efficacy of this compound. We conclude that IQ-1L is more effective than IQ-1S in treating cerebral ischemia injury and thus represents a promising anti-inflammatory compound.

Evaluation of an Affibody-Based Binder for Imaging of Immune Check-Point Molecule B7-H3.

Radionuclide molecular imaging could provide an accurate assessment of the expression of molecular targets in disseminated cancers enabling stratification of patients for specific therapies. B7-H3 (CD276) is a transmembrane protein belonging to the B7 superfamily. This protein is overexpressed in different types of human malignancies and such upregulation is generally associated with a poor clinical prognosis. In this study, targeting properties of an Affibody-based probe, AC12, containing a -GGGC amino acid sequence as a chelator (designated as AC12-GGGC) labelled with technetium-99m (99mTc) were evaluated for imaging of B7-H3-expressing tumours. AC12-GGGC was efficiently labelled with 99mTc. [99mTc]Tc-AC12-GGGC bound specifically to B7-H3 expressing cells in vitro with affinities in nanomolar range. In mice bearing B7-H3-expressing xenografts, [99mTc]Tc-AC12-GGGC showed tumour uptake of 2.1 ± 0.5 %ID/g at 2 h after injection. Its clearance from blood, normal organs and tissues was very rapid. This new targeting agent, [99mTc]Tc-AC12-GGGC, provided high tumour-to-blood ratio already at 2 h (8.2 ± 1.9), which increased to 11.0 ± 0.5 at 4 h after injection. Significantly (p < 0.05) higher tumour-to-liver and higher tumour-to-bone ratios at 2 h in comparison with 4 h after injection were observed. Thus, [99mTc]Tc-AC12-GGGC could be a promising candidate for further development.

Alkylverdazyls as a Source of Alkyl Radicals for Light-Triggered Cancer Cell Death.

Two alkylated verdazyl radicals (AlkVZs) were investigated as active compounds for photoinitiated controlled MCF-7 cell death. Observed results unambiguously showed that AlkVZ could be a potential structural moiety for the design of a novel family of photodynamic therapy agents. The main advantage of the proposed substances is an oxygen-independent generation of active radicals, which play a pivotal role in the treatment of oxygen-deficient tumors.

Composite Ferroelectric Membranes Based on Vinylidene Fluoride-Tetrafluoroethylene Copolymer and Polyvinylpyrrolidone for Wound Healing.

Wound healing is a complex process and an ongoing challenge for modern medicine. Herein, we present the results of study of structure and properties of ferroelectric composite polymer membranes for wound healing. Membranes were fabricated by electrospinning from a solution of vinylidene fluoride/tetrafluoroethylene copolymer (VDF-TeFE) and polyvinylpyrrolidone (PVP) in dimethylformamide (DMF). The effects of the PVP content on the viscosity and conductivity of the spinning solution, DMF concentration, chemical composition, crystal structure, and conformation of VDF-TeFE macromolecules in the fabricated materials were studied. It was found that as PVP amount increased, the viscosity and conductivity of the spinning solutions decreased, resulting in thinner fibers. Using FTIR and XRD methods, it was shown that if the PVP content was lower than 50 wt %, the VDF-TeFE copolymer adopted a flat zigzag conformation (TTT conformation) and crystalline phases with ferroelectric properties were formed. Gas chromatography results indicated that an increase in the PVP concentration led to a higher residual amount of DMF in the material, causing cytotoxic effects on 3T3L1 fibroblasts. In vivo studies demonstrated that compared to classical gauze dressings impregnated with a solution of an antibacterial agent, ferroelectric composite membranes with 15 wt % PVP provided better conditions for the healing of purulent wounds.