Evaluation of the Immunosafety of Cucurbit[n]uril In Vivo.

Cucurbiturils are a family of macrocyclic oligomers capable of forming host-guest complexes with various molecules. Due to noncovalent binding to drug molecules and low toxicity, cucurbiturils has been extensively investigated as potential carriers for drug delivery. However, the immune system's interactions with different drug carriers, including cucurbiturils, are still under investigation. In this study, we focused on cucurbiturils' immunosafety and immunomodulation properties in vivo. We measured blood counts and lymphocyte subpopulations in blood, spleen, and bone marrow, and assessed the in vivo toxicity to spleen and bone marrow cells after intraperitoneal administration to BALB/c mice. When assessing the effect of cucurbit[6]uril on blood parameters after three intraperitoneal injections within a week in laboratory animals, a decrease in white blood cells was found in mice after injections of cucurbit[6]util, but the observed decrease in the number of white blood cells was within the normal range. At the same time, cucurbit[7]uril and cucurbit[8]uril did not affect the leukocyte counts of mice after three injections. Changes in the number of platelets, erythrocytes, and monocytes, as well as in several other indicators, such as hematocrit or erythrocyte volumetric dispersion, were not detected. We show that cucurbiturils do not have immunotoxicity in vivo, with the exception of a cytotoxic effect on spleen cells after сucurbit[7]uril administration at a high dosage. We also evaluated the effect of cucurbiturils on cellular and humoral immune responses. We founded that cucurbiturils in high concentrations affect the immune system in vivo, and the action of various cucurbiturils differs in different homologues, which is apparently associated with different interactions in the internal environment of the body.

Influence of Cucurbiturils on the Production of Reactive Oxygen Species by T- and B-Lymphocytes, Platelets and Red Blood Cells.

Reactive oxygen species (ROS) are highly reactive chemical molecules containing oxygen. ROS play an important role in signaling and cell homeostasis at low and moderate concentrations. ROS could be a cause of damage to proteins, nucleic acids, lipids, membranes and organelles at high concentrations. There are a lot of cells that can produce ROS to maintain functional activity. It is known that metal nanoparticles can increase production of ROS in cells. However, the effect of cucurbiturils on ROS production is still unknown. In our study, we evaluated production of ROS by the immune (T-, B-lymphocytes, NK-cells) and non-immune cells (red blood cells, platelets), as well as tumor cells line (1301, K562) after treatment with cucurbiturils in vitro. Assessment of reactive oxide species (ROS) were provided by using dihydrorhodamine 123 (DHR 123). Fluorescence intensity and percentage DHR123 were measured by flow cytometry. Platelets, erythrocytes and activated T-helpers were changed the level of ROS production in response to stimulation with cucurbiturils. It was found that the percentage of these ROS-producing cells was reduced by cucurbiturils. Thus, cucurbiturils may affect the production of ROS by cells, but further research is needed in this area.

Hybrid Mucin-Vaterite Microspheres for Delivery of Proteolytic Enzyme Chymotrypsin.

While the enteral delivery of proteolytic enzymes is widely established for combating many diseases as an alternative to antibiotic treatment, their local delivery only emerges as administration route enabling sustained release in a controlled manner on site. The latest requires the development of drug delivery systems suitable for encapsulation and preservation of enzymatic proteolytic activity. This study proposes hybrid microspheres made of mucin and biodegradable porous crystals of calcium carbonate (CC) as the carriers for chymotrypsin (CTR) delivery. CTR is impregnated into CC and hybrid CC/mucin (CCM) microspheres by means of sorption without any chemical modification. The loading of the CC with mucin enhances CTR retention on hybrid microspheres (adsorption capacity of ≈8.7 mg g-1  vs 4.7 mg g-1 ), recharging crystal surface due to the presence of mucin and diminishing the average pore diameter of the crystals from 25 to 8 nm. Mucin also retards recrystallization of vaterite into nonporous calcite improving stability of CCM microspheres upon storage. Proteolytic activity of CTR is preserved in both CC and CCM microspheres, being pH dependent. Temperature-induced inactivation of CTR significantly diminishes by CTR encapsulation into CC and CCM microspheres. Altogether, these findings indicate promises of hybrid mucin-vaterite microspheres for mucosal application of proteases.

Neutron and Synchrotron Imaging Studies of Preservation State of Metal of Cultural Heritage Objects.

This paper analyzes the results of studies carried out at the National Research Center "Kurchatov Institute", Moscow, using the methods of neutron and X-ray synchrotron tomography from the point of view of the preservation state of metal objects. Objects damaged by corrosion and exposure to fire were the focus of this study. To identify regions of metal preservation, the diffraction contrast on grains of metal, observed in tomographic projections, was used. The simultaneous use of neutron and synchrotron imaging is shown to be a powerful tool for identification of the constituents of an object.

The Effect of Cucurbit[7]uril on the Antitumor and Immunomodulating Properties of Oxaliplatin and Carboplatin.

Cucurbit[7]uril (CB[7]) is a molecular container that may form host-guest complexes with platinum(II) anticancer drugs and modulate their efficacy and safety. In this paper, we report our studies of the effect of CB[7]-oxaliplatin complex and the mixture of CB[7] and carboplatin (1:1) on viability and proliferation of a primary cell culture (peripheral blood mononuclear cells), two tumor cell lines (B16 and K562) and their activity in the animal model of melanoma. At the same time, we studied the impact of platinum (II) drugs with CB[7] on T cells and B cells in vitro. Although the stable CB[7]-carboplatin complex was not formed, the presence of cucurbit[7]uril affected the biological properties of carboplatin. In vivo, CB[7] increased the antitumor effect of carboplatin, but, at the same time, increased its acute toxicity. Compared to free oxaliplatin, its complex with CB[7] shows a greater cytotoxic effect on tumor cell lines B16 and K562, while in vivo, the effects of the free drug and encapsulated drug were comparable. However, in vivo studies also demonstrated that the encapsulation of oxaliplatin in CB[7] lowered the toxicity of the drug.

Assessment of the Biocompatibility of Cucurbiturils in Blood Cells.

Currently, cucurbiturils are being actively researched all over the world. Research is focused on the ways of improving the solubility and selectivity of cucurbiturils, increasing the stability of the complexes with other particles in various media and enhancing their ability to bind and release various substances. The most significant area of our research is the assessment of safety, studying the biological properties and synergistic effects of cucurbiturils during complexation with drugs. In this article, the hemocompatibility of erythrocytes and leukocytes with cucurbiturils was investigated. We demonstrated that cucurbiturils have no cytotoxic effect, even at high concentrations (1 mM) and do not affect the viability of PBMCs. However, cucurbiturils can increase the level of the early apoptosis of lymphocytes and cucurbit[7]uril enhances hemolysis in biologically relevant media. Despite this, cucurbiturils are fairly safe organic molecules in concentrations up to 0.3 mM. Thus, we believe that it will become possible to use polymer nanostructures as drug delivery systems in clinical practice, since cucurbiturils can be modified to improve pharmacological properties.

Evaluation of the Immunosafety of Cucurbit[n]uril on Peripheral Blood Mononuclear Cells In Vitro.

Cucurbiturils (CB[n]s) are nanoscale macrocyclic compounds capable of encapsulating a molecule or part of a molecule by forming host-guest complexes. Integration of drugs with CB[n] is used for the following purposes: controlling clearance; protection of the drug from biodegradation; targeted delivery to specific organs, tissues, or cells; reduction of toxicity; and improving solubility. One of the major problems encountered in the application of new drug delivery systems is lack of knowledge of their biological properties. CB[n], unlike many other often toxic nanoparticles, has extremely low toxicity, even at high doses. However, many aspects of the biological actions of these nanoscale cavitands remain unclear, including the immunotropic properties. In this study, we investigated the immunotoxicity and immunomodulation properties of CB[n]. It was found that CB[7] and CB[6] did not decrease the viability of mononuclear cells at all tested concentrations from 0.1-1 mM. Overall, the results indicated an immunomodulatory effect of different concentrations of CB[n]. In the case of a longer cultivation time, CB[n] had an immunostimulating effect, which was indicated by an enhancement of the proliferative activity of cells and increased expression of HLA-DR on lymphocytes.

The Key to Understanding the European Miocene Chalcides (Squamata, Scincidae) Comes from Asia: The Lizards of the East Siberian Tagay Locality (Baikal Lake) in Russia.

The early middle Miocene (MN 5) lizards from the East Siberian Tagay locality (Baikal Lake, Russia) in Asia are described here. The lizard fauna consists of two clades, Lacertidae and Scincidae. The skink material is allocated to Chalcides. While this taxon was previously reported from Europe, it has rarely been observed in the Neogene record with only jaw fragments and frontal bones described. Its taxonomy was therefore enigmatic. The Tagay material is almost identical to the European fossils of Chalcides from Austria and Hungary, but it also contains the parietal bone. While the material is also similar to the extant Ch. ocellatus, it exhibits several morphological differences. A new species is therefore erected-Chalcides augei sp. nov. These findings further support the connection of the Baikal Lake area with central Europe during the first half of the Miocene. The comparative anatomy of the frontals, parietals and lower jaws was evaluated by micro-CT in selected skink taxa. This comparison highlights several important differences, for example, paired frontals are present in Broadleysaurus (an outgroup taxon), in Acontias and all studied members of Scincidae herein. The character optimization in Mesquite supports fused frontals as being the condition at the basal node of the Ateuchosauridae + Sphenomorphidae + Eugongylidae + Lygosomidae + Egerniidae + Mabuyidae clade. While the parapineal foramen is restricted to the parietal in most taxa studied herein, it is absent (or vestigial) in Acontias and Feylinia. In contrast to all other skinks, this foramen is located on the frontal in Ateuchosaurus chinensis. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1901-1934, 2020. © 2019 American Association for Anatomy.

Mucin adsorption on vaterite CaCO3 microcrystals for the prediction of mucoadhesive properties.

Porous vaterite CaCO3 crystals are widely used as containers for drug loading and as sacrificial templates to assemble polymer-based nano- and micro-particles at mild conditions. Special attention is paid nowadays to mucosal delivery where the glycoprotein mucin plays a crucial role as a main component of a mucous. In this work mucoadhesive properties of vaterite crystals have been tested by investigation of mucin binding to the crystals as a function of (i) time, (ii) glycoprotein concentration, (iii) adsorption conditions and (iv) degree of mucin desialization. Mucin adsorption follows Bangham equation indicating that diffusion into crystal pores is the rate-limiting step. Mucin strongly binds to the crystals (ΔG = -35 ±â€¯4 kJ mol-1) via electrostatic and hydrophobic interactions forming a gel and thus giving the tremendous mucin mass content in the crystals of up to 16%. Despite strong intermolecular mucin-mucin interactions, pure mucin spheres formed after crystal dissolution are unstable. However, introduction of protamine, actively used for mucosal delivery, makes the spheres stable via additional electrostatic bonding. The results of this work indicate that the vaterite crystals are extremely promising carriers for mucosal drug delivery and for development of test-systems for the analysis of the mucoadhesion.

Chemical and biological properties of a supramolecular complex of tuftsin and cucurbit[7]uril.

Cucurbit[7]uril (CB7) is an uncharged and water-soluble macrocyclic host. CB7 binds to doubly protonated tuftsin, which is the tetrapeptide Thr-Lys-Pro-Arg, with moderate affinity (Ka=2.1×103M-1). In this study, the host-guest complexation was confirmed by fluorescence titration. This affinity would allow for easy release of the peptide under physiological conditions. According to density functional theory calculations, the structural binding motif involves hydrogen bonding. The most energetically stable form had the Arg side chain inside the CB7 cavity. The effects of the tuftsin-CB7 complex on the proliferation and cytokine activity of immune cells were studied. The complex had broader spectrum immunomodulation than free peptides, and caused statistically significant (p<0,05) changes in cytokine production (tumor necrosis factor-α, interleukin-2, interferon-γ, and interleukin-10) by mononuclear cells. By contrast, the free peptide only activated tumor necrosis factor-α production.

Supramolecular Adducts of Cucurbit[7]uril and Amino Acids in the Gas Phase.

The complexation of the macrocyclic cavitand cucurbit[7]uril (Q7) with a series of amino acids (AA) with different side chains (Asp, Asn, Gln, Ser, Ala, Val, and Ile) is investigated by ESI-MS techniques. The 1:1 [Q7 + AA + 2H](2+) adducts are observed as the base peak when equimolar Q7:AA solutions are electrosprayed, whereas the 1:2 [Q7 + 2AA + 2H](2+) dications are dominant when an excess of the amino acid is used. A combination of ion mobility mass spectrometry (IM-MS) and DFT calculations of the 1:1 [Q7 + AA + 2H](2+) (AA = Tyr, Val, and Ser) adducts is also reported and proven to be unsuccessful at discriminating between exclusion or inclusion-type conformations in the gas phase. Collision induced dissociation (CID) revealed that the preferred dissociation pathways of the 1:1 [Q7 + AA + 2H](2+) dications are strongly influenced by the identity of the amino acid side chain, whereas ion molecule reactions towards N-butylmethylamine displayed a common reactivity pattern comprising AA displacement. Special emphasis is given on the differences between the gas-phase behavior of the supramolecular adducts with amino acids (AA = Asp, Asn, Gln, Ser, Ala, Val, and Ile) and those featuring basic (Lys and Arg) and aromatic (Tyr and Phe) side chains.