Increased endocytosis rate and enhanced lysosomal pathway of silica-coated superparamagnetic nanoparticles into M-HeLa cells compared with cultured primary motor neurons.

The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30 s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1 h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.

A supramolecular assembly of sulfobutyl ether-ß-cyclodextrin and viologen calix[4]resorcinol as the master key to sustainable and eco-friendly catalyst for paraoxon hydrolysis.

The search for effective ways of paraoxon (POX) degradation becomes an extremely urgent problem, which can be solved by creating effective bioscavengers in the form of three-dimensional macrocycles. In this work, supramolecular interactions in an aqueous medium were studied between (4-sulfobutyl)-ß-cyclodextrin, the hydrophobic cavity of which is capable of binding POX, and viologen calix[4]resorcinol, the cationic groups of which are able to facilitate the nucleophilic attack on the phosphorus atom of the pesticide. A complex of physicochemical methods revealed the nature of the interactions between these cyclodextrin and calix[4]resorcinol, as a result of which the spontaneous formation of nanoparticles occurs. The kinetics of POX hydrolysis reaction using these nanoparticles was studied at room temperature in aqueous Tris-buffer medium by spectrophotometric method. Pure cyclodextrin does not exhibit catalytic activity in the POX hydrolysis, but its presence in a mixture with calix[4]resorcinol leads to a fivefold increase in the hydrolysis rate constant compared to pure calix[4]resorcinol.

A supramolecular catalyst based on sodium alginate and viologen calix[4]resorcinol for the room temperature hydrolysis of paraoxon.

Properties of paraoxon, such as poor water solubility, low rate of natural decomposition, ability to accumulate in soil and wastewater, lead to the fact that paraoxon is found in various agricultural products and textiles. In this regard, the search for effective ways of paraoxon degradation becomes an extremely urgent problem, which can be solved by creating catalysts by mimicking paraxonase. In this work, a complex of physicochemical methods was used to study the supramolecular interactions of sodium alginate, which has a calcium-binding ability similar to paraxonase, with viologen calix[4]resorcinol and to reveal the nature of the intermolecular interactions between them resulting in the spontaneous formation of nanoparticles. Before proceeding to the investigation of the binding ability of obtained nanoparticles to paraoxon, the encapsulating effect of nanoparticles on a number of model substrates of different solubility (doxorubicin hydrochloride, quercetin and oleic acid) was studied. The kinetics of paraoxon hydrolysis reaction using these nanoparticles was studied at room temperature in an aqueous medium by spectrophotometric method. The rate of this reaction increases with increasing concentration of stable nanoparticles having hydrophobic domains that ensure paraoxon immobilization. The results obtained allow considering the supramolecular polysaccharide/calixarene system as an effective biomimetic catalyst.

Phase Behavior of Aqueous Mixtures of Sodium Alginate with Fish Gelatin: Effects of pH and Ionic Strength.

The phase behavior of aqueous mixtures of fish gelatin (FG) and sodium alginate (SA) and complex coacervation phenomena depending on pH, ionic strength, and cation type (Na+, Ca2+) were studied by turbidimetric acid titration, UV spectrophotometry, dynamic light scattering, transmission electron microscopy and scanning electron microscopy for different mass ratios of sodium alginate and gelatin (Z = 0.01-1.00). The boundary pH values determining the formation and dissociation of SA-FG complexes were measured, and we found that the formation of soluble SA-FG complexes occurs in the transition from neutral (pHc) to acidic (pHφ1) conditions. Insoluble complexes formed below pHφ1 separate into distinct phases, and the phenomenon of complex coacervation is thus observed. Formation of the highest number of insoluble SA-FG complexes, based on the value of the absorption maximum, is observed at рHopt and results from strong electrostatic interactions. Then, visible aggregation occurs, and dissociation of the complexes is observed when the next boundary, pHφ2, is reached. As Z increases in the range of SA-FG mass ratios from 0.01 to 1.00, the boundary values of рНc, рHφ1, рHopt, and рHφ2 become more acidic, shifting from 7.0 to 4.6, from 6.8 to 4.3, from 6.6 to 2.8, and from 6.0 to 2.7, respectively. An increase in ionic strength leads to suppression of the electrostatic interaction between the FG and SA molecules, and no complex coacervation is observed at NaCl and CaCl2 concentrations of 50 to 200 mM.

Green-step assembly of the supramolecular amphiphile constructed by sodium carboxymethyl cellulose and calixarene for facile loading of hydrophobic food bioactive compounds.

The use of biologically active compounds is often limited due to their poor aqueous solubility, which generally reduces their bioavailability and useful efficacy. In this regard, a wide search is currently underway for colloidal systems capable of encapsulating these compounds. In the creation of colloidal systems, long-chain molecules of surfactants and polymers are mainly used, which in an individual state do not always aggregate into homogeneous and stable nanoparticles. In the present work, cavity-bearing calixarene was used for the first time to order polymeric molecules of sodium carboxymethyl cellulose. A set of physicochemical methods demonstrated the spontaneous formation of spherical nanoparticles by non-covalent self-assembly contributed by macrocycle and polymer, and formed nanoparticles were able to encapsulate hydrophobic quercetin and oleic acid. The preparation of nanoparticles by supramolecular self-assembly without use of organic solvents, temperature and ultrasound effects can be an effective strategy for creating water-soluble forms of lipophilic bioactive compounds.

Emergence of Nanoscale Drug Carriers through Supramolecular Self-Assembly of RNA with Calixarene.

Supramolecular self-assembly is a powerful tool for the development of polymolecular assemblies that can form the basis of useful nanomaterials. Given the increasing popularity of RNA therapy, the extension of this concept of self-assembly to RNA is limited. Herein, a simple method for the creation of nanosized particles through the supramolecular self-assembly of RNA with a three-dimensional macrocycle from the calixarene family was reported for the first time. This self-assembly into nanoparticles was realized using cooperative supramolecular interactions under mild conditions. The obtained nanoparticles are able to bind various hydrophobic (quercetin, oleic acid) and hydrophilic (doxorubicin) drugs, as a result of which their cytotoxic properties are enhanced. This work demonstrates that intermolecular interactions between flexible RNA and rigid calixarene is a promising route to bottom-up assembly of novel supramolecular soft matter, expanding the design possibilities of nanoscale drug carriers.

Cataleptogenic Effect of Haloperidol Formulated in Water-Soluble Calixarene-Based Nanoparticles.

In this study, a water-soluble form of haloperidol was obtained by coaggregation with calix[4]resorcinol bearing viologen groups on the upper rim and decyl chains on the lower rim to form vesicular nanoparticles. The formation of nanoparticles is achieved by the spontaneous loading of haloperidol into the hydrophobic domains of aggregates based on this macrocycle. The mucoadhesive and thermosensitive properties of calix[4]resorcinol-haloperidol nanoparticles were established by UV-, fluorescence and CD spectroscopy data. Pharmacological studies have revealed low in vivo toxicity of pure calix[4]resorcinol (LD50 is 540 ± 75 mg/kg for mice and 510 ± 63 mg/kg for rats) and the absence of its effect on the motor activity and psycho-emotional state of mice, which opens up a possibility for its use in the design of effective drug delivery systems. Haloperidol formulated with calix[4]resorcinol exhibits a cataleptogenic effect in rats both when administered intranasally and intraperitoneally. The effect of the intranasal administration of haloperidol with macrocycle in the first 120 min is comparable to the effect of commercial haloperidol, but the duration of catalepsy was shorter by 2.9 and 2.3 times (p < 0.05) at 180 and 240 min, respectively, than that of the control. There was a statistically significant reduction in the cataleptogenic activity at 10 and 30 min after the intraperitoneal injection of haloperidol with calix[4]resorcinol, then there was an increase in the activity by 1.8 times (p < 0.05) at 60 min, and after 120, 180 and 240 min the effect of this haloperidol formulation was at the level of the control sample.

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.

The dielectric response of hydrated water as a structural signature of nanoconfined lichen melanins.

Lichens are unique symbiotic organisms from a mutually beneficial alliance of fungi and algae/cyanobacteria that successfully survive extreme temperatures and drought conditions. Most probably such extraordinary vitality of lichens is underlain by melanins, one of the main structural and chemical lichen components, and their mutual relationship with residual water. In this paper, we propose mechanisms, which allow lichens to store up the extra water in their structure. Melanins that are constituents of the cortical lichen layer and presumably contribute to unique water-lichen interactions are chosen for physical experiments in a wide temperature domain. Two melanin pigments extracted from different lichens are studied here - eumelanin from Lobaria pulmonaria and allomelanin from Cetraria islandica. To investigate the inner melanin structure and water-melanin interactions, FTIR and BDS techniques are applied. The BDS technique was used in a wide temperature region of 123-293 K for melanins with various hydration levels. The relaxation processes related to the confinement of supercooled water - in melanins are observed and discussed in details. At medium and high hydration levels, the relaxation process in two melanins of different chemical compositions and supramolecular structures exhibits a well-known crossover that was already observed in many types of confinements. The analysis of FTIR and BDS results helps to clarify the lichen-water interaction processes.

Supramolecular Self-Assembly of Porphyrin and Metallosurfactant as a Drug Nanocontainer Design.

The combined method of treating malignant neoplasms using photodynamic therapy and chemotherapy is undoubtedly a promising and highly effective treatment method. The development and establishment of photodynamic cancer therapy is closely related to the creation of sensitizers based on porphyrins. The present study is devoted to the investigation of the spectroscopic, aggregation, and solubilization properties of the supramolecular system based on 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TSPP) and lanthanum-containing surfactant (LaSurf) in an aqueous medium. The latter is a complex of lanthanum nitrate and two cationic amphiphilic molecules of 4-aza-1-hexadecylazoniabicyclo[2.2.2]octane bromide. The mixed TSPP-LaSurf complexes can spontaneously assemble into various nanostructures capable of binding the anticancer drug cisplatin. Morphological behavior, stability, and ability to drug binding of nanostructures can be tailored by varying the molar ratio and the concentration of components. The guest binding is shown to be additional factor controlling structural rearrangements and properties of the supramolecular TSPP-LaSurf complexes.

Modulation of Molecular Structure and Mechanical Properties of κ-Carrageenan-Gelatin Hydrogel with Multi-Walled Carbon Nanotubes.

Hydrogels, three-dimensional hydrophilic water-insoluble polymer networks having mechanical properties inherent for solids, have attracted continuous research attention over a long time period. Here, we studied the structure and properties of hydrogel based on gelatin, κ-carrageenan and CNTs using the combination of SAXS, PXRD, AFM microscopy, SEM and rheology methods. We have shown that the integration of polysaccharide and protein in the composite hydrogel leads to suppression of their individual structural features and homogenization of two macromolecular components into a single structural formation. According to obtained SAXS results, we observed the supramolecular complex, which includes both polysaccharide and protein components associated with each other. It was determined that hydrogel structure formed in the initial solution state (dispersion) retains hydrogel supramolecular structure under its cooling up to gel state. The sizes of dense cores of these polyelectrolyte complexes (PEC) slightly decrease in the gel state in comparison with PEC water dispersion. The introduction of CNTs to hydrogel does not principally change the type of supramolecular structure and common structural tendencies observed for dispersion and gel states of the system. It was shown that carbon nanotubes embedded in hydrogel act as the supplementary template for formation of the three-dimensional net, giving additional mechanical strengthening to the studied system.

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-Assembling Metallocomplexes of the Amphiphilic 1,4-Diazabicyclo[2.2.2]octane Derivative as a Platform for the Development of Nonplatinum Anticancer Drugs.

New 1-cetyl-4-aza-1-azoniabicyclo[2.2.2]octane bromide complexes with copper(II) bromide and lanthanum(III) nitrate were characterized using dynamic light scattering and transmission electron microscopy, with self-assembly and the morphological behavior elucidated. For the lanthanum(III) nitrate complex, the 3D crystal structure was characterized using X-ray diffractometry. These metallosurfactants were tested as antitumor agents, and a high cytotoxic effect comparable with doxorubicin was revealed against the M-HeLa and A-549 cell lines. Both complexes were 2 times more active toward the MCF-7 cell line than the breast cancer drug tamoxifen. The cytotoxic mechanism of complexes is assumed to be related to the induction of apoptosis through the mitochondrial pathway.

The protealysin operon encodes emfourin, a prototype of a novel family of protein metalloprotease inhibitors.

Protealysin is a Serratia proteamaculans metalloproteinase of the M4 peptidase family and the prototype of a large group of protealysin-like proteases (PLPs). PLPs are likely involved in bacterial interaction with plants and animals as well as in bacterial pathogenesis. We demonstrated that the PLP genes in bacteria colocalize with the genes of putative conserved proteins. In S. proteamaculans, these two genes form a bicistronic operon. The putative S. proteamaculans protein that we called emfourin (M4in) was expressed in Escherichia coli and characterized. M4in forms a complex with protealysin with a 1:1 stoichiometry and is a potent slow-binding competitive inhibitor of protealysin (Ki = 52 ± 14 pM); besides, M4in is not secreted from S. proteamaculans constitutively. A comparison of amino acid sequences of M4in and its homologs with those of known inhibitors suggests that M4in is the prototype of a new family of protein inhibitors of proteases.

Supraamphiphilic Systems Based on Metallosurfactant and Calix[4]resorcinol: Self-Assembly and Drug Delivery Potential.

Metallic amphiphiles are used as building blocks in the construction of nanoscale superstructures, where the hydrophobic effects induce the self-assembly of the nanoparticles of interest. However, the influence of synergizing multiple chemical interactions on an effective design of these structures mostly remains an open question. In this regard, supraamphiphilic systems based on flexible surfactant molecules and rigid macrocycles are being actively developed, but there are few works on the interaction between metallosurfactants and macrocycles. In the present work, the self-assembly and biological properties of a metallosurfactant with calixarene were studied for the first time. The metallosurfactant, a complex between lanthanum nitrate and two 4-aza-1-hexadecylazoniabicyclo[2.2.2]octane bromide units, and calix[4]resorcinol containing sulfonate groups on the upper rim were used to form a novel supraamphiphilic composition. The system formed was studied using a variety of physicochemical methods, including spectrophotometry, NMR, XRF, and dynamic and electrophoretic light scattering. It was found that the most optimal tetraanionic calix[4]resorcinol to dicationic metallosurfactant molar ratio, leading to mixed aggregation upon ion pair complexation, is 2:3. The mixed aggregates formed in the pentamolar concentration range were able to encapsulate hydrophilic substrates, including the anticancer drug cisplatin, the pure form of which is more cytotoxic toward healthy cells than toward diseased cells. Interestingly, the drug loaded into the macrocycle-metallosurfactant particles was less cytotoxic to a healthy Chang liver cell line and more cytotoxic to tumor M-HeLa cells. This selectivity depends on the amount of cisplatin added. The more drug is added to the macrocycle-metallosurfactant composition, the greater the biological activity against cancer cells. Taking into account that the appearance of resistance of cancer cells to drugs, especially to cisplatin, is one of the most important problems in treatment, the results of this work envisage the potential application of a mixed macrocycle-metallosurfactant system for the design of therapeutic cisplatin compositions.

Direct interaction of fibrinogen with lipid microparticles modulates clotting kinetics and clot structure.

Extensive studies revealed the role of blood lipid microparticles (liposomes, microvesicles) in activation of coagulation cascade. The direct interaction of fibrinogen/fibrin with lipid surfaces and its consequence for hemostasis received much less attention. We observed pronounced changes in both clot morphology and kinetics of fibrin clotting in the presence of artificial liposomes. The evidence was obtained that lipid microparticles per se present a diffusion barrier to the three-dimensional fibril assembling and pose spatial restrictions for fiber elongation. On the other hand, fibrinogen adsorption results in its high local concentration on liposome surface that accelerates fibrin polymerization. Adsorption induces Fg secondary structure alterations which may contribute to the abnormal clot morphology. In dependence on lipid composition and size of microparticles, the interplay of all the outlined mechanisms determines functionally important changes of clot morphology. The obtained results contribute to the knowledge of clotting mechanisms in the presence of artificial and natural lipid microparticles.

Protealysin is not Secreted Constitutively.

BACKGROUND: Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published. OBJECTIVE: Here, the protealysin cellular localization was studied for the first time using immunochemical methods. METHODS AND RESULTS: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm. CONCLUSION: The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis.

Pectobacterium atrosepticum exopolysaccharides: identification, molecular structure, formation under stress and in planta conditions.

In the present study, we identified exopolysaccharides of the harmful phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 and characterized the molecular structure of these polymers. The synthesis of the target polysaccharides was shown to be induced under starvation conditions. Moreover, intensive accumulation of exopolysaccharides occurred during the colonization by bacteria of the xylem vessels of infected plants, where microorganisms formed specific 3D "multicellular" structures-bacterial emboli. Thus, the identified polymers are likely to be involved in the adaptation and virulence of bacteria of Pectobacterium genus.

A Ni(iii) complex stabilized by silica nanoparticles as an efficient nanoheterogeneous catalyst for oxidative C-H fluoroalkylation.

We have developed Ni(III)-doped silica nanoparticles ([(bpy)xNi(III)]@SiO2) as a recyclable, low-leaching, and efficient oxidative functionalization nanocatalyst for aromatic C-H bonds. The catalyst is obtained by doping the complex [(bpy)3Ni(II)] on silica nanoparticles along with its subsequent electrooxidation to [(bpy)xNi(III)] without an additional oxidant. The coupling reaction of arenes with perfluoroheptanoic acid occurs with 100% conversion of reactants in a single step at room temperature under nanoheterogeneous conditions. The catalyst content is only 1% with respect to the substrates under electrochemical regeneration conditions. The catalyst can be easily separated from the reaction mixture and reused a minimum of five times. The results emphasize immobilization on the silica support and the electrochemical regeneration of Ni(III) complexes as a facile route for developing an efficient nanocatalyst for oxidative functionalization.

Aspen Tension Wood Fibers Contain ß-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls.

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). ß-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. ß-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, ß-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high ß-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.