Use of Micronization and Complex Coacervation to Preserve Antioxidant Properties of Flavonoids.

The plant flavonoids taxifolin and rutin are among the best known and best studied antioxidants. In addition to their antioxidant properties, other pharmacobiological properties have been established for these substances. At the same time, taxifolin and rutin are chemically labile. They are prone to oxidative degradation and have poor water solubility. Under conditions of their real consumption, all this can lead to a significant reduction or complete loss of bioactivity of these flavonoids. Flavonoid modification and encapsulation techniques can be used to overcome these barrier factors. The use of micronization process for taxifolin and rutin allows changing the lipophilicity values of antioxidants. For micronized taxifolin, the log P value is 1.3 (1.12 for the control forms), and for rutin, it was 0.15 (-0.64 for the control forms). The antioxidant activity of micronized flavonoids has increased about 1.16 times compared to control forms. The present study evaluates the possibility of using encapsulation of premyconized flavonoids by complex coacervation, in order to preserve their antioxidant properties. The results of an in vitro digestion study show that the encapsulated forms of antioxidants retain their bioactivity and bioavailability better than their original forms. The bioavailability indices for the encapsulated forms of flavonoids are more than 1.6 times higher than for their original forms. The digested fractions of the encapsulated properties reveal better antioxidant properties than their original forms in in vitro tests evaluating the antioxidant properties on cultures of the protozoan Paramecium caudatum and human neuroblastoma SH-SY5Y cells. Encapsulated rutin indicates the highest activity, 0.64 relative to PMA. Thus, the studies represent the feasibility of using encapsulation to protect flavonoids during digestion and ensure the preservation of their antioxidant properties.

Photoinduced Antibacterial Activity and Cytotoxicity of CdS Stabilized on Mesoporous Aluminosilicates and Silicates.

Inactivation of bacteria under the influence of visible light in presence of nanostructured materials is an alternative approach to overcome the serious problem of the growing resistance of pathogenic bacteria to antibiotics. Cadmium sulfide quantum dots are superefficient photocatalytic material suitable for visible light transformation. In this work, CdS nanoparticles with size of less than 10 nm (QDs) were synthesized on the surface of natural and synthetic mesoporous aluminosilicates and silicates (halloysite nanotubes, MCM-41, MCM-41/Halloysite, SBA-15). Materials containing 5-7 wt.% of CdS were characterized and tested as agents for photocatalytic bacteria degradation of Gram-positive S. aureus and Gram-negative E. coli with multiple antibiotic resistance. Eukaryotic cell viability tests were also conducted on the model cancer cells A 459. We found that the carrier affects prokaryotic and eukaryotic toxicity of CdS quantum dots. CdS/MCM-41/HNTs were assumed to be less toxic to eukaryotic cells and possess the most prominent photocatalytic antibacterial efficiency. Under visible light irradiation, it induced 100% bacterial growth inhibition at the concentration of 125 µg/mL and the bacteriostatic effect at the concentration of 63 µg/mL. CdS/MCM-41/HNTs showed 100% E. coli growth inhibition in the concentration of 1000 µg/mL under visible light irradiation.

The Effect of Ultrasonic Water Treatment on the Change in the Microstructure of Wheat Grain, Dough, and Wheat Flour Bread.

Visualization of the microstructure of the food matrix of both raw materials and the final product is one of the keys to understanding the processes occurring during its formation. It is the fixation of the results at the microlevel that allows us to form a hypothesis and then confirm it with the obtained array of experimental data. The presented study is aimed at studying the effect of ultrasonic water treatment on the change in the microstructure of wheat grain during its humidification. The article also presents the results of studying the microstructure of dough and wheat flour bread obtained using water after ultrasonic water treatment and the intensity of the processes of staling of finished bread in storage. The object of the study was grain of soft spring white wheat (Triticum aestivum L.), varieties of Lubava, harvest 2014-2018, Russia (the protein content was 12.5 ± 0.3 g/100 g in terms of humidity); dough and bread made from wheat flour (ash content 0.55%, mass fraction of gluten 28.5%), produced using the technology of plain bread, a classic recipe without improvers. Ultrasound-treated water with an exposure frequency of 22 ± 1.65 kHz and with a power variation of 252-630 W/l was used in test technology. The experimental data obtained made it possible to establish the intensification of the processes of swelling of wheat grain during soaking. In the experimental samples, after 8 hours of soaking, the loosened structure of the endosperm and evenly swollen components of the grain were observed, and the loop of the groove was closed. Activation of the processes of dough science was established, and gluten flour in the dough formed a single monolithic frame, in which the swollen starch grains are tightly packed. The interstitial walls of the crumb of the prototypes consisted of a solid mass of protein coagulated during baking, inside of which swollen gelatinized starch grains are interspersed, they are closely adjacent to the mass of coagulated protein with their entire surface, and therefore, there is no sharp, clearly visible boundary between them. The most pronounced changes in the structure of the dough and bread crumb were noted when using water, after ultrasonic water treatment at a power of 504 and 630 W/l. This method of exposure can be recommended as the best for obtaining good quality bread with less pronounced staling during storage.

Forskolin-Loaded Halloysite Nanotubes as Osteoconductive Additive for the Biopolymer Tissue Engineering Scaffolds.

Here we report the use of forskolin-modified halloysite nanotubes (HNTs) as a dopant for biopolymer porous hydrogel scaffolds to impart osteoinductive properties. Forskolin is a labdane diterpenoid isolated from the Indian Coleus plant. This small molecule is widely used as a supplement in molecular biology for cell differentiation. It has been reported in some earlier publications that forskolin can activate osteodifferentiation process by cyclic adenosine monophosphate (c-AMP) signalling activation in stem cells. In presented study it was demonstrated that forskolin release from halloysite-doped scaffolds induced the osteodifferentiation of equine mesenchymal stem cells (MSCs) in vitro without addition of any specific growth factors. The reinforcement of mechanical properties of cells and intercellular space during the osteodifferentiation was demonstrated using atomic force microscopy (AFM). These clay-doped scaffolds may find applications to accelerate the regeneration of horse bone defects by inducing the processes of osteodifferentiation of endogenous MSCs.

Revisiting the Cytotoxicity of Cationic Polyelectrolytes as a Principal Component in Layer-by-Layer Assembly Fabrication.

Polycations are an essential part of layer-by-layer (LbL)-assembled drug delivery systems, especially for gene delivery. In addition, they are used for other related applications, such as cell surface engineering. As a result, an assessment of the cytotoxicity of polycations and elucidation of the mechanisms of polycation toxicity is of paramount importance. In this study, we examined in detail the effects of a variety of water-soluble, positively charged synthetic polyelectrolytes on in vitro cytotoxicity, cell and nucleus morphology, and monolayer expansion changes. We have ranked the most popular cationic polyelectrolytes from the safest to the most toxic in relation to cell cultures. 3D cellular cluster formation was disturbed by addition of polyelectrolytes in most cases in a dose-dependent manner. Atomic force microscopy allowed us to visualize in detail the structures of the polyelectrolyte-DNA complexes formed due to electrostatic interactions. Our results indicate a relationship between the structure of the polyelectrolytes and their toxicity, which is necessary for optimization of drug and gene delivery systems.

Selective Cytotoxic Activity of Prodigiosin@halloysite Nanoformulation.

Prodigiosin, a bioactive secondary metabolite produced by Serratia marcescens, is an effective proapoptotic agent against various cancer cell lines, with little or no toxicity toward normal cells. The hydrophobicity of prodigiosin limits its use for medical and biotechnological applications, these limitations, however, can be overcome by using nanoscale drug carriers, resulting in promising formulations for target delivery systems with great potential for anticancer therapy. Here we report on prodigiosin-loaded halloysite-based nanoformulation and its effects on viability of malignant and non-malignant cells. We have found that prodigiosin-loaded halloysite nanotubes inhibit human epithelial colorectal adenocarcinoma (Caco-2) and human colon carcinoma (HCT116) cells proliferative activity. After treatment of Caco-2 cells with prodigiosin-loaded halloysite nanotubes, we have observed a disorganization of the F-actin structure. Comparison of this effects on malignant (Caco-2, HCT116) and non-malignant (MSC, HSF) cells suggests the selective cytotoxic and genotoxic activity of prodigiosin-HNTs nanoformulation.

Halloysite Nanoclay/Biopolymers Composite Materials in Tissue Engineering.

Biocompatible materials for the fabrication of tissue substitutes are crucially important in the advancement of modern medicinal biotechnology. These materials, to serve their function, should be similar in physical, chemical, biological, and structural properties to native tissues which they are aimed to mimic. The porosity of artificial scaffolds is essential for normal nutrient transmission to cells, gas diffusion, and cell attachment and proliferation. Nanoscale inorganic additives and dopants are widely used to improve the functional properties of the polymer materials for tissue engineering. Among these inorganic dopants, halloysite nanotubes are arguably the most perspective candidates because of their biocompatibility and functional properties allowing to enhance significantly the mechanical and chemical stability of tissue engineering scaffolds. Here, this vibrant field of biotechnology for regenerative medicine is overviewed.

Spatial manipulation of magnetically-responsive nanoparticle engineered human neuronal progenitor cells.

Here we report a detailed investigation of the interaction of neuronal progenitor cells and neurons with polyelectrolyte-stabilized magnetic iron oxide nanoparticles. Human neuronal progenitor and neurons were differentiated in vitro from fibroblast-derived induced pluripotent stem cells. The cytotoxic effects of poly(allylamine hydrochloride) were determined on human skin fibroblasts and neuronal progenitor cells. Immunocytochemical staining of lamins A/C and B in cells treated separately with poly(allylamine hydrochloride) and magnetic nanoparticles allowed to exclude these nuclear components as targets of toxic effects. We demonstrate that magnetic nanoparticles accumulated in cytoplasm and on the surface of neuronal progenitor cells neither interacted with the nuclear envelope nor penetrated into the nuclei of neuronal cells. The possibility of guidance of magnetically functionalized neuronal progenitor cells under magnetic field was demonstrated. Magnetization of progenitor cells using poly(allylaminehydrochloride)-stabilized magnetic nanoparticles allows for successful managing their in vitro localization in a monolayer.

Cryogel composites based on hyaluronic acid and halloysite nanotubes as scaffold for tissue engineering.

We present here preparation of mechanically strong and biocompatible cryogel composites based on hyaluronic acid (HA) and halloysite nanotubes (HNTs) of various compositions, and their applications as scaffold for different cell growing media. Uniaxial compression tests reveal that the incorporation of HNTs into HA cryogels leads to a ~2.5-fold increase in their Young moduli, e.g., from 38 ±â€¯1 to 99 ±â€¯4 kPa at a HA:HNTs weight ratio of 1:2. Although HA:HNTs based cryogels were found to be blood compatible with 1.37 ±â€¯0.11% hemolysis ratio at a HA:HNTs weight ratio of 1:2, they trigger thrombogenic activity with a blood clotting index of 17.3 ±â€¯4.8. Remarkably, HA:HNTs cryogel composites were found to be excellent scaffold materials in the proliferation of rat mesenchymal stem cells (MSC), human cervical carcinoma cells (HeLa), and human colon cancer cells (HCT116). The cell studies revealed that an increased amount of HNT embedding into HA cryogels leads to an increase of MSC proliferation.

Paclitaxel Encapsulated in Halloysite Clay Nanotubes for Intestinal and Intracellular Delivery.

Naturally formed halloysite tubules have a length of 1 µm and lumens with a diameter of 12-15 nm which can be loaded with drugs. Halloysite's biocompatibility allows for its safe delivering to cells at a concentration of up to 0.5 mg/mL. We encapsulated the anticancer drug paclitaxel in halloysite and evaluated the drug release kinetics in simulated gastric and intestinal conditions. To facilitate maximum drug release in intestinal tract, halloysite tubes were coated with the pH-responsive polymer poly(methacrylic acid-co-methyl methacrylate). Release kinetics indicated a triggered drug release pattern at higher pH, corresponding to digestive tract environment. Tablets containing halloysite, loaded with paclitaxel, as a compression excipient were formulated with drug release occurring at a sustained rate. In vitro anticancer effects of paclitaxel-loaded halloysite nanotubes were evaluated on human cancer cells. In all the treated cell samples, polyploid nuclei of different sizes and fragmented chromatin were observed, indicating a high therapeutic effect of halloysite formulated paclitaxel.

Species-specific differences in peroxisome proliferation, catalase, and SOD2 upregulation as well as toxicity in human, mouse, and rat hepatoma cells induced by the explosive and environmental pollutant 2,4,6-trinitrotoluene.

2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017.

Clay nanotube-biopolymer composite scaffolds for tissue engineering.

Porous biopolymer hydrogels doped at 3-6 wt% with 50 nm diameter/0.8 µm long natural clay nanotubes were produced without any cross-linkers using the freeze-drying method. The enhancement of mechanical strength (doubled pick load), higher water uptake and thermal properties in chitosan-gelatine-agarose hydrogels doped with halloysite was demonstrated. SEM and AFM imaging has shown the even distribution of nanotubes within the scaffolds. We used enhanced dark-field microscopy to visualise the distribution of halloysite nanotubes in the implantation area. In vitro cell adhesion and proliferation on the nanocomposites occur without changes in viability and cytoskeleton formation. In vivo biocompatibility and biodegradability evaluation in rats has confirmed that the scaffolds promote the formation of novel blood vessels around the implantation sites. The scaffolds show excellent resorption within six weeks after implantation in rats. Neo-vascularization observed in newly formed connective tissue placed near the scaffold allows for the complete restoration of blood flow. These phenomena indicate that the halloysite-doped scaffolds are biocompatible as demonstrated both in vitro and in vivo. The chitosan-gelatine-agarose doped clay nanotube nanocomposite scaffolds fabricated in this work are promising candidates for tissue engineering applications.

Enzyme-activated intracellular drug delivery with tubule clay nanoformulation.

Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.

Nano-labelled cells-a functional tool in biomedical applications.

Nanotechnology offers an unprecedented number of opportunities for biomedical research, utilizing the unusual functionalities of nanosized materials. Here we describe the recent advances in fabrication and utilization of nanoparticle-labelled cells. We present a brief overview of the most promising techniques, namely layer-by-layer polyelectrolyte assembly on cells and intracellular and extracellular labelling with magnetic nanoparticles. Several important practical application of nanofucntionalized cells, including tissue engineering and tumour therapy, are reviewed.

Surface-modified magnetic human cells for scaffold-free tissue engineering.

We report the magnetically-facilitated scaffold-free assembly of lung tissue mimicking two-layered multicellular clusters. Polymer-stabilized magnetic nanoparticles were deposited on surfaces of viable human cells (A549 and skin fibroblasts), allowing the formation of two-layered porous tissue prototypes.