Thiacalixarene Carboxylic Acid Derivatives as Inhibitors of Lysozyme Fibrillation.

Amyloid fibroproliferation leads to organ damage and is associated with a number of neurodegenerative diseases affecting populations worldwide. There are several ways to protect against fibril formation, including inhibition. A variety of organic compounds based on molecular recognition of amino acids within the protein have been proposed for the design of such inhibitors. However, the role of macrocyclic compounds, i.e., thiacalix[4]arenes, in inhibiting fibrillation is still almost unknown. In the present work, the use of water-soluble thiacalix[4]arene derivatives for the inhibition of hen egg-white lysozyme (HEWL) amyloid fibrillation is proposed for the first time. The binding of HEWL by the synthesized thiacalix[4]arenes (logKa = 5.05-5.13, 1:1 stoichiometry) leads to the formation of stable supramolecular systems capable of stabilizing the protein structure and protecting against fibrillation by 29-45%. The macrocycle conformation has little effect on protein binding strength, and the native HEWL secondary structure does not change via interaction. The synthesized compounds are non-toxic to the A549 cell line in the range of 0.5-250 µg/mL. The results obtained may be useful for further investigation of the anti-amyloidogenic role of thiacalix[4]arenes, and also open up future prospects for the creation of new ways to prevent neurodegenerative diseases.

Antibacterial Activity of Various Morphologies of Films Based on Guanidine Derivatives of Pillar[5]arene: Influence of the Nature of One Substitute on Self-assembly.

The progress of the pillar[5]arene chemistry allowed us to set out a new concept on application of the supramolecular assemblies to create antimicrobial films with variable surface morphologies and biological activities. Antibacterial films were derived from the substituted pillar[5]arenes containing nine pharmacophoric guanidine fragments and one thioalkyl substituent. Changing the only thioalkyl fragment in the macrocycle structure made it possible to control the biological activity of the resulting antibacterial coating. Pretreatment of the surface with aqueous solution of the amphiphilic pillar[5]arenes reduced the biofilm thickness by 56 ± 10% of Gram-positive Staphylococcus aureus in the case of the pillar[5]arene containing a thiooctyl fragment and by 52 ± 7% for the biofilm of Gram-negative Klebsiella pneumoniae in the case of pillar[5]arene containing a thiooctadecyl fragment. Meanwhile, the cytotoxicity of the synthesized macrocycles was examined at a concentration of 50 µg/mL, which was significantly lower than that of bis-guanidine-based antimicrobial preparations.

Photo- and X-ray Induced Cytotoxicity of CeF3-YF3-TbF3 Nanoparticle-Polyvinylpyrrolidone-"Radachlorin" Composites for Combined Photodynamic Therapy.

The Ce0.5Y0.35Tb0.15F3 nanoparticles with a CeF3 hexagonal structure were synthesized using the co-precipitation technique. The average nanoparticle diameter was 14 ± 1 nm. The luminescence decay curves of the Ce0.5Y0.35Tb0.15F3 nanoparticles (λem = 541 nm, 5D4-7F5 transition of Tb3+) conjugated with Radachlorin using polyvinylpyrrolidone coating as well as without Radachlorin were detected. Efficient nonradiative energy transfer from Tb3+ to the Radachlorin was demonstrated. The maximum energy transfer coefficients for the nanoparticles conjugated with Radachlorin via polyvinylpyrrolidone and without the coating were 82% and 55%, respectively. The average distance between the nanoparticle surface and Radachlorin was R0 = 4.5 nm. The best results for X-ray-induced cytotoxicity were observed for the NP-PVP-Rch sample at the lowest Rch concentration. In particular, after X-ray irradiation, the survival of A549 human lung carcinoma cells decreased by ~12%.

Single-Stage Microfluidic Synthesis Route for BaGdF5:Tb3+-Based Nanocomposite Materials: Synthesis, Characterization and Biodistribution.

Rare-earth-doped nanoscaled BaGdF5 is known as an efficient contrasting agent for X-ray micro-CT and NMR as well as a promising candidate for X-ray photodynamic therapy, thereby opening an opportunity for theragnostic applications. Conventional synthesis of Ln-doped BaGdF5 consider a long-lasting batch procedure, while a conjugation with photosensitizer usually implies a separate stage requiring active mixing. To the best of our knowledge, in this work, we for the first time obtain BaGdF5:Tb3+ nanophosphors in a microfluidic route at temperatures as low as 100 °C while decreasing the time of thermal treatment down to 6 min. The proposed synthesis route allows for the obtaining of single-phase and monodisperse BaGd1-xF5:Tbx3+ nanoparticles with an averaged particle size of ca. 7-9 nm and hydrodynamic radius around 22 nm, as estimated from TEM and DLS, respectively. In addition, X-ray-excited optical luminescence has been recorded in situ for the series of nanophosphors synthesis with varied flow rates of Tb3+ and Gd3+ stock solutions, thereby anticipating a possible application of microfluidics for screening a wide range of possible co-dopants and reaction conditions and its effect on the optical properties of the synthesized materials. Moreover, we demonstrated that BaGd1-xF5:Tbx3+@RoseBengal conjugates might be obtained in a single-stage route by implementing an additional mixer at the synthesis outcome, namely, by mixing the resulting reaction mixture containing nanoparticles with an equivalent flow of photosensitizer aqueous solution. In vitro cytotoxicity test declares moderate toxicity effect on different cell lines, while the results of flow cytometry indirectly confirm cellular uptake. Finally, we report long-term biodistribution monitoring of the synthesized nanocomposites assessed by X-ray micro-CT in the in vivo experiments on balb/c mice, which depicts an unusual character of agents' accumulation.

The Synthesis and Antibacterial Properties of Pillar[5]arene with Streptocide Fragments.

The growing problem of bacterial resistance to antimicrobials actualizes the development of new approaches to solve this challenge. Supramolecular chemistry tools can overcome the limited bacterial resistance and side effects of classical sulfonamides that hinder their use in therapy. Here, we synthesized a number of pillar[5]arenes functionalized with different substituents, determined their ability to self-association using DLS, and characterized antimicrobial properties against S. typhimurium, K. pneumoniae, P. aeruginosa, S. epidermidis, S. aureus via a resazurin test. Biofilm prevention concentration was calculated for an agent with established antimicrobial activity by the crystal-violet staining method. We evaluated the mutagenicity of the macrocycle using the Ames test and its ability to affect the viability of A549 and LEK cells in the MTT-test. It was shown that macrocycle functionalized with sulfonamide residues exhibited antimicrobial activity an order higher than pure streptocide and also revealed the ability to prevent biofilm formation of S. aureus and P. aeruginosa. The compound did not show mutagenic activity and exhibited low toxicity to eukaryotic cells. The obtained results allow considering modification of the macrocyclic platforms with classic antimicrobials as an opportunity to give them a "second life" and return to practice with improved properties.

The Effect of Ficin Immobilized on Carboxymethyl Chitosan on Biofilms of Oral Pathogens.

In the last decade, Ficin, a proteolytic enzyme extracted from the latex sap of the wild fig tree, has been widely investigated as a promising tool for the treatment of microbial biofilms, wound healing, and oral care. Here we report the antibiofilm properties of the enzyme immobilized on soluble carboxymethyl chitosan (CMCh) and CMCh itself. Ficin was immobilized on CMCh with molecular weights of either 200, 350 or 600 kDa. Among them, the carrier with a molecular weight of 200 kDa bound the maximum amount of enzyme, binding up to 49% of the total protein compared to 19-32% of the total protein bound to other CMChs. Treatment with pure CMCh led to the destruction of biofilms formed by Streptococcus salivarius, Streptococcus gordonii, Streptococcus mutans, and Candida albicans, while no apparent effect on Staphylococcus aureus was observed. A soluble Ficin was less efficient in the destruction of the biofilms formed by Streptococcus sobrinus and S. gordonii. By contrast, treatment with CMCh200-immobilized Ficin led to a significant reduction of the biofilms of the primary colonizers S. gordonii and S. mutans. In model biofilms obtained by the inoculation of swabs from teeth of healthy volunteers, the destruction of the biofilm by both soluble and immobilized Ficin was observed, although the degree of the destruction varied between artificial plaque samples. Nevertheless, combined treatment of oral Streptococci biofilm by enzyme and chlorhexidine for 3 h led to a significant decrease in the viability of biofilm-embedded cells, compared to solely chlorhexidine application. This suggests that the use of either soluble or immobilized Ficin would allow decreasing the amount and/or concentration of the antiseptics required for oral care or improving the efficiency of oral cavity sanitization.

Peptidomimetics based on ammonium decasubstituted pillar[5]arenes: Influence of the alpha-amino acid residue nature on cholinesterase inhibition.

Cholinesterase inhibitors are a group of medicines that are widely used for the treatment of cognitive impairments accompanying Alzheimer's disease as well as for the treatment of pathological muscle weaknesses syndromes such as myasthenia gravis. The search for novel non-toxic and effective cholinesterase inhibitors for creating neuroprotective and neurotransmitter agents is an urgent interdisciplinary problem. For the first time, the application of water-soluble pillar[5]arenes containing amino acid residues as effective cholinesterase inhibitors was shown. The influence of the nature of aliphatic and aromatic alpha-amino acid residues (glycine, l-alanine, l-phenylalanine and l-tryptophan) on self-assembly, aggregate's stability, cytotoxicity on A549 and LEK cells and cholinesterase inhibition was studied. It was found that the studied compounds with aliphatic amino acid residues showed a low inhibitory ability against cholinesterases. It was established that the pillar[5]arene containing fragments of l-phenylalanine is the most promising inhibitor of butyrylcholinesterase (IC50 = 0.32 ± 0.01 µM), the pillar[5]arene with l-tryptophan residues is the most promising inhibitor of acetylcholinesterase (IC50 = 0.32 ± 0.01 µM). This study has shown a possible application of peptidomimetics based on pillar[5]arenes to inhibit cholinesterase, as well as control the binding affinity to a particular enzyme in a structure-dependent manner.

Towards Protection of Nucleic Acids from Herbicide Attack: Self-Assembly of Betaines Based on Pillar[5]arene with Glyphosate and DNA.

Herbicides are one of the main parts of pesticides used today. Due to the high efficiency and widespread use of glyphosate-based herbicides, the search for substances reducing their genotoxicity is an important interdisciplinary task. One possible approach for solving the problem of herbicide toxicity is to use compounds that can protect DNA from damage by glyphosate derivatives. For the first time, a method for developing DNA-protecting measures against glyphosate isopropylamine salt (GIS) damage was presented and realized, based on low-toxicity water-soluble pillar[5]arene derivatives. Two- and three-component systems based on pillar[5]arene derivatives, GIS, and model DNA from salmon sperm, as well as their cytotoxicity, were studied. The synthesized pillar[5]arene derivatives do not interact with GIS, while GIS is able to bind DNA from salmon sperm with lgKa = 4.92. The pillar[5]arene betaine derivative containing fragments of L-phenylalanine and the ester derivative with diglycine fragments bind DNA with lgKa = 5.24 and lgKa = 4.88, respectively. The study of the associates (pillar[5]arene-DNA) with GIS showed that the interaction of GIS with DNA is inhibited only by the betaine pillar[5]arene containing fragments of L-Phe (lgKa = 3.60). This study has shown a possible application of betaine pillar[5]arene derivatives for nucleic acid protection according to its competitive binding with biomacromolecules.

Brightfield vs Fluorescent Staining Dataset-A Test Bed Image Set for Machine Learning based Virtual Staining.

Differential fluorescent staining is an effective tool widely adopted for the visualization, segmentation and quantification of cells and cellular substructures as a part of standard microscopic imaging protocols. Incompatibility of staining agents with viable cells represents major and often inevitable limitations to its applicability in live experiments, requiring extraction of samples at different stages of experiment increasing laboratory costs. Accordingly, development of computerized image analysis methodology capable of segmentation and quantification of cells and cellular substructures from plain monochromatic images obtained by light microscopy without help of any physical markup techniques is of considerable interest. The enclosed set contains human colon adenocarcinoma Caco-2 cells microscopic images obtained under various imaging conditions with different viable vs non-viable cells fractions. Each field of view is provided in a three-fold representation, including phase-contrast microscopy and two differential fluorescent microscopy images with specific markup of viable and non-viable cells, respectively, produced using two different staining schemes, representing a prominent test bed for the validation of image analysis methods.

Toward Pathogenic Biofilm Suppressors: Synthesis of Amino Derivatives of Pillar[5]arene and Supramolecular Assembly with DNA.

New amino derivatives of pillar[5]arene were obtained in three stages with good yields. It was shown that pillar[5]arene containing thiaether and tertiary amino groups formed supramolecular complexes with low molecular weight model DNA. Pillar[5]arene formed complexes with a DNA nucleotide pair at a ratio of 1:2 (macrocycle/DNA base pairs), as demonstrated by UV-visible and fluorescence spectroscopy. The association constants of pillar[5]arene with DNA were lgKass1:1 = 2.38 and lgKass1:2 = 5.07, accordingly. By using dynamic light scattering and transmission electron microscopy, it was established that the interaction of pillar[5]arene containing thiaether and tertiary amino groups (concentration of 10-5 M) with a model nucleic acid led to the formation of stable nanosized macrocycle/DNA associates with an average particle size of 220 nm. It was shown that the obtained compounds did not exhibit a pronounced toxicity toward human adenocarcinoma cells (A549) and bovine lung epithelial cells (LECs). The hypothesis about a possible usage of the synthesized macrocycle for the aggregation of extracellular bacterial DNA in a biofilm matrix was confirmed by the example of St. Aureus. It was found that pillar[5]arene at a concentration of 10-5 M was able to reduce the thickness of the St. Aureus biofilm by 15%.

Fibrin-Rhamnogalacturonan I Composite Gel for Therapeutic Enzyme Delivery to Intestinal Tumors.

Therapy of colorectal cancer with protein drugs, including targeted therapy using monoclonal antibodies, requires the preservation of the drug's structure and activity in the gastrointestinal tract or bloodstream. Here, we confirmed experimentally the fundamental possibility of creating composite protein-polysaccharide hydrogels based on non-degrading rhamnogalacturonan I (RG) and fibrin as a delivery vehicle for antitumor RNase binase. The method is based on enzymatic polymerization of fibrin in the presence of RG with the inclusion of liposomes, containing an encapsulated enzyme drug, into the gel network. The proposed method for fabricating a gel matrix does not require the use of cytotoxic chemical cross-linking agents and divalent cations, and contains completely biocompatible and biodegradable components. The process proceeds under physiological conditions, excluding the effect of high temperatures, organic solvents and ultrasound on protein components. Immobilization of therapeutic enzyme binase in the carrier matrix by encapsulating it in liposomes made from uncharged lipid made it possible to achieve its prolonged release with preservation of activity for a long time. The release time of binase from the composite carrier can be regulated by variation of the fibrin and RG concentration.

Fluorescein-Labeled Thiacalix[4]arenes as Potential Theranostic Molecules: Synthesis, Self-Association, and Antitumor Activity.

In this paper, a series of thiacalix[4]arenes were synthesized as potential theranostic molecules for antitumor therapy. We propose an original strategy for the regioselective functionalization of thiacalix[4]arene with a fluorescent label to obtain antiangiogenic agent mimetics. The aggregation properties of the synthesized compounds were determined using the dynamic light scattering. The average hydrodynamic diameter of self-associates formed by the macrocycles in 1,3-alternate conformation is larger (277-323 nm) than that of the similar macrocycle in cone conformation (185-262 nm). The cytotoxic action mechanism of the obtained compounds and their ability to penetrate into of human lung adenocarcinoma and human duodenal adenocarcinoma cells were established using the MTT-test and flow cytometry. Thiacalix[4]arenes in 1,3-alternate conformation did not have a strong toxic effect. The toxicity of macrocycles in cone conformations on HuTu-80 and A549 cells (IC50 = 21.83-49.11 µg/mL) is shown. The resulting macrocycles are potential theranostic molecules that combine both the pharmacophore fragment for neoplasmas treatment and the fluorescent fragment for monitoring the delivery and biodistribution of nanomedicines.

Alginate-Based Hydrogel as Delivery System for Therapeutic Bacterial RNase.

To deliver therapeutic proteins into a living body, it is important to maintain their target activity in the gastrointestinal tract after oral administration. Secreted ribonuclease from Bacillus pumilus (binase) has antitumor and antiviral activity, which makes it a promising therapeutic agent. This globular protein of small molecular weight (12.2 kDa) is considered as a potential agent that induces apoptosis of tumor cells expressing certain oncogenes, including colorectal and duodenum cancer. The most important problem of its usage is the preservation of its structure and target activity, which could be lost during oral administration. Here, we developed alginate microspheres reinforced with divalent cations and analyzed the enzyme release from them. Using methods of scanning electron microscopy, measurements of fluorescence, enzyme catalytic activity, and determination of viability of the duodenum adenocarcinoma tumor cell line, we characterized obtained microspheres and chose calcium as a biogenic ion-strengthening microsphere structure. Among such modified additivities as beta-casein, gelatin, and carbon nanotubes introduced into microspheres, only gelatin showed a pronounced increase in their stability and provided data on the prolonged action of enzyme release from microspheres into tumor cell culture medium during 48 h in an amount of about 70% of the loaded quantity.

Self-Healing Thiolated Pillar[5]arene Films Containing Moxifloxacin Suppress the Development of Bacterial Biofilms.

Polymer self-healing films containing fragments of pillar[5]arene were obtained for the first time using thiol/disulfide redox cross-linking. These films were characterized by thermogravimetric analysis and differential scanning calorimetry, FTIR spectroscopy, and electron microscopy. The films demonstrated the ability to self-heal through the action of atmospheric oxygen. Using UV-vis, 2D 1H-1H NOESY, and DOSY NMR spectroscopy, the pillar[5]arene was shown to form complexes with the antimicrobial drug moxifloxacin in a 2:1 composition (logK11 = 2.14 and logK12 = 6.20). Films containing moxifloxacin effectively reduced Staphylococcus aureus and Klebsiella pneumoniae biofilms formation on adhesive surfaces.

Water-soluble pillar[5]arene sulfo-derivatives self-assemble into biocompatible nanosystems to stabilize therapeutic proteins.

Pillar[5]arenes containing sulfonate fragments have been shown to form supramolecular complexes with therapeutic proteins to facilitate targeted transport with an increased duration of action and enhanced bioavailability. Regioselective synthesis was used to obtain a water-soluble pillar[5]arene containing the fluorescent label FITC and nine sulfoethoxy fragments. The pillar[5]arene formed complexes with the therapeutic proteins binase, bleomycin, and lysozyme in a 1:2 ratio as demonstrated by UV-vis and fluorescence spectroscopy. The formation of stable spherical nanosized macrocycle/binase complexes with an average particle size of 200 nm was established by dynamic light scattering and transmission electron microscopy. Flow cytometry demonstrated the ability of macrocycle/binase complexes to penetrate into tumor cells where they exhibited significant cytotoxicity towards A549 cells at 10-5-10-6 M while maintaining the enzymatic activity of binase.

Towards new nanoporous biomaterials: self-assembly of sulfopillar[5]arenes with vitamin D3 into supramolecular polymers.

Novel water-soluble, deca-substituted pillar[5]arenes containing thiasulfate and thiacarboxylate fragments were synthesized and characterized. UV-vis, 2D 1H-1H NOESY and DOSY NMR spectroscopy revealed the ability of pillar[5]arenes containing thiasulfate fragments to form an inclusion complex with cholecalciferol (vitamin D3) in a 1 : 2 ratio (lg Kass = 2.2). Using DLS and SEM it was found that upon concentration and/or evaporation of the solvent, the supramolecular polymer (pillar[5]arene/vitamin D3 (1 : 2)) forms a porous material with an average wall diameter of 53 nm. It was shown that the supramolecular polymer is stable during photolysis by UV radiation (k1 = 1.7 × 10-5 s-1).

Nanoparticles based on the zwitterionic pillar[5]arene and Ag+: synthesis, self-assembly and cytotoxicity in the human lung cancer cell line A549.

For the first time, stable pillar[5]arene/Ag+ nanoparticles, consisting of water-soluble pillar[5]arene containing γ-sulfobetaine fragments and Ag+ ions without Ag-Ag bonds, were synthesized and characterized. The pillar[5]arene/Ag+ (ratio 1:10) nanoparticles obtained were cubic with a rib length of 100 nm and are less cytotoxic than Ag+ ions. The survival of the A549 model cells in the presence of pillar[5]arene/Ag+ (1:10) nanoparticles at a concentration of 30 and 40 µM was 76% and 55%, while in the absence of pillar[5]arene, the cell survival for free Ag+ ions at the same concentration was 30% and 10%, respectively. The results can be used to create new antibacterial materials and 2D biomedical coatings.

Zeolites as Carriers of Antitumor Ribonuclease Binase.

Natural and synthetic zeolites have many applications in biomedicine and nutrition. Due to its properties, zeolites can absorb therapeutically active proteins and release them under physiological conditions. In this study we tested the clinoptilolite, chabazite, and natrolite ability to be loaded by antitumor ribonuclease binase and the cytotoxicity of the obtained complexes. We found the optimal conditions for binase loading into zeolites and established the dynamic of its release. Cytotoxic effects of zeolite-binase complexes toward colorectal cancer Caco2 cells were characterized after 24 and 48 h of incubation with cells using MTT-test. Zeolites were toxic by itselfs and reduced cells viability by 30% (clinoptilolite), 40% (chabazite), and 70% (natrolite) after 48 h of incubation. Binase complexes with clinoptilolite as well as chabazite always demonstrated enhanced toxicity (up to 57 and 60% for clinoptilolite and chabazite, respectively) in comparison with binase and zeolites separately. Our results contribute to the perspective development of binase-based complexes for therapy of colorectal cancer for or the treatment of malignant skin neoplasms where the complexes can be used in pasty form.

Tiny Rare-Earth Fluoride Nanoparticles Activate Tumour Cell Growth via Electrical Polar Interactions.

Localised extracellular interactions between nanoparticles and transmembrane signal receptors may well activate cancer cell growth. Herein, tiny LaF3 and PrF3 nanoparticles in DMEM+FBS suspensions stimulated tumour cell growth in three different human cell lines (A549, SW837 and MCF7). Size distribution of nanoparticles, activation of AKT and ERK signalling pathways and viability tests pointed to mechanical stimulation of ligand adhesion binding sites of integrins and EGFR via a synergistic action of an ensemble of tiny size nanoparticles (< 10 nm). While tiny size nanoparticles may be well associated with the activation of EGFR, integrin interplay with nanoparticles remains a multifaceted issue. A theoretical motif shows that, within the requisite pN force scale, each ligand adhesion binding site can be activated by a tiny size dielectric nanoparticle via electrical dipole interaction. The size of the active nanoparticle stayed specified by the amount of the surface charges on the ligand adhesion binding site and the nanoparticle, and also by the separating distance between them. The polar component of the electrical dipole force remained inversely proportional to the second power of nanoparticle's size, evincing that only tiny size dielectric nanoparticles might stimulate cancer cell growth via electrical dipole interactions. The work contributes towards recognising different cytoskeletal stressing modes of cancer cells.

Consequences of chitosan decomposition by nitrous acid: Approach to non-branched oligochitosan oxime.

It is well known that chitosan degradation by nitrous acid leads to oligochitosan (oligoCHIt-ahm) bearing reactive 2,5-anhydromannose (3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-aldehyde) units at the new reducing ends of macromolecules. Standard protocol requires reduction of oligoCHIt-ahm with NaBH4 to corresponding oligoCHIt-hml bearing unreactive hydroxymethyl group instead of reactive aldehyde group. For the first time, HP SEC as well as UV and CD spectroscopy methods have revealed that the reduction leads to an indefinite side modification and the formation of a branched oligoCHIt-hml with increased molecular weight. Here, it is shown that the branching and modification can be prevented by means of the simple and reproducible reaction of oligoCHIt-ahm with hydroxylamine that allows preparation of a stable linear oligochitosan oxime, oligoCHIt-oxm. Cytotoxicity tests show that oligoCHIt-ahm, oligoCHIt-hml and oligoCHIt-oxm are non-toxic at concentration below 2.5 mg/ml, and the cytotoxicity is concentration dependent and decreases in the order oligoCHIt-ahm > oligoCHIt-hml > oligoCHIt-oxm at higher concentrations both before and after long shelf-storage. The elaborated approach and cytotoxicity data give an opportunity to use the non-branched oligoCHIt-oxm for biomedical applications.