Direct Determination of the Structure of Single Biopolymer Molecules Using Nanopore Sequencing.

This review highlights operational principles, features, and modern aspects of the development of third-generation sequencing technology of biopolymers focusing on the nucleic acids analysis, namely the nanopore sequencing system. Basics of the method and technical solutions used for its realization are considered, from the first works showing the possibility of creation of these systems to the easy-to-handle procedure developed by Oxford Nanopore Technologies company. Moreover, this review focuses on applications, which were developed and realized using equipment developed by the Oxford Nanopore Technologies, including assembly of whole genomes, methagenomics, direct analysis of the presence of modified bases.

Ultrasonic Film Rehydration Synthesis of Mixed Polylactide Micelles for Enzyme-Resistant Drug Delivery Nanovehicles.

A facile technique for the preparation of mixed polylactide micelles from amorphous poly-D,L-lactide-block-polyethyleneglycol and crystalline amino-terminated poly-L-lactide is described. In comparison to the classical routine solvent substitution method, the ultrasonication assisted formation of polymer micelles allows shortening of the preparation time from several days to 15-20 min. The structure and morphology of mixed micelles were analyzed with the assistance of electron microscopy, dynamic and static light scattering and differential scanning calorimetery. The resulting polymer micelles have a hydrodynamic radius of about 150 nm and a narrow size distribution. The average molecular weight of micelles was found to be 2.1 × 107 and the aggregation number was calculated to be 6000. The obtained biocompatible particles were shown to possess low cytotoxicity, high colloid stability and high stability towards enzymatic hydrolysis. The possible application of mixed polylactide micelles as drug delivery vehicles was studied for the antitumor hydrophobic drug paclitaxel. The lethal concentration (LC50) of paclitaxel encapsulated in polylactide micelles was found to be 42 ± 4 µg/mL-a value equal to the LC50 of paclitaxel in the commercial drug Paclitaxel-Teva.

Plasticity control of poly(vinyl alcohol)-graphene oxide nanocomposites.

Composite films containing poly(vinyl alcohol) filled with different amounts of graphene oxide (2 and 4 wt%) were prepared by the solution casting technique, and the mechanical properties of the resulting materials were modified with different amounts of glycerol as a plasticizer. Two series of pure poly(vinyl alcohol) and graphene oxide-loaded films with fixed amounts of water were used for modification with glycerol, since water can also serve as a plasticizer for poly(vinyl alcohol). The morphology and physical properties of the plasticized and non-plasticized composites were studied; tensile tests were performed to investigate and compare their mechanical properties. Glycerol addition does not affect the excellent compatibility of the filler with the polymer matrix and uniform distribution of graphene oxide in poly(vinyl alcohol). For poly(vinyl alcohol)/graphene oxide films an increase of the Young's modulus and yield stress was found with an increase of the filler content; the Young's modulus for poly(vinyl alcohol) filled with 4 wt% of graphene oxide is almost two times higher than that of the pure polymer. Simultaneously, a sharp decrease of the elongation at break from 80% for pure PVA to about 5% for the PVA/GO composite with 4 wt% of GO is observed, and the film's brittleness dramatically increases. It was shown that the addition of glycerol to the composite films leads both to the Young's modulus decrease and tensile energy at break increase; here the Young's modulus decreases by 18 times after addition of 20 wt% of glycerol to the poly(vinyl alcohol) film filled with 4 wt% of graphene oxide. Thus, the use of plasticizer results in a significant increase of the ductile properties of graphene oxide filled poly(vinyl alcohol) composite films, and the higher the water content in the composite film, the more drastic the increase of the ductile properties observed.

Nanodiamond-chymotrypsin and nanodiamond-papain conjugates, their synthesis and activity and visualization of their interaction with cells using optical and electron microscopy.

Two novel conjugates of detonation nanodiamonds (dNDs) with the proteolytic enzymes chymotrypsin and papain were synthesized. The synthesis was performed via functionalization of the dNDs' surface with acidic/alkali treatment followed by carbodiimide-mediated protein binding. Covalent binding of the enzymes was confirmed by Fourier transform infrared spectrographic analysis and high-performance liquid chromatography (HPLC) amino acid analysis. HPLC also proved the preservation of the enzymes' composition during synthesis. The same assay was used to determine the binding ratios. The ratios were 12% (mass to mass) for chymotrypsin and 7.4% for papain. The enzymatic activity of the conjugates was measured using chromogenic substrates and appeared to be approximately 40% of that of the native enzymes. The optimum pH values and stability under various conditions were determined. The sizes of resulting particles were measured using dynamic light scattering and direct electron microscopic observation. The enzyme conjugates were shown to be prone to aggregation, resulting in micrometer-sized particles. The ζ-potentials were measured and found to be positive for the conjugates. The conjugated enzymes were tested for biological activity using an in vitro model of cultured transformed human epithelial cells (HeLa cell line). It was shown that dND-conjugated enzymes effectively bind to the surface of the cells and that enzymes attack exposed proteins on the plasma membrane, including cell adhesion molecules. Incubation with conjugated enzymes results in morphological changes of the cells but does not affect cell viability, as judged by monitoring the cell division index and conducting ultrastructural studies. dNDs are internalized by the cells via endocytosis, being enclosed in forming coated vesicles by chance, and they accumulate in single membrane-bound vacuoles, presumably late endosomes/phagosomes, along with multimembranous onionlike structures. The authors propose a model of a stepwise conjugate binding to the cell membrane and gradual release of the enzymes.

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu.

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl-sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.