Daniel Horák

Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.

New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly( L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 ( L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLL-modified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLL-modified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in vitro and in vivo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.

Reactive poly(glycidyl methacrylate) microspheres prepared by dispersion polymerization

Poly(glycidyl methacrylate) [poly(GMA)] microspheres of narrow size distribution were prepared in a simple one-step procedure by dispersion radical polymerization. Depending on the solvent used, poly(GMA) particle size could be controlled in the range of 0.5–4 μm by changing the solubility parameter of the reaction mixture. In N,N′-dimethylformamide (DMF)/methanol mixture, the particle size increased and the size distribution broadened with decreasing initial solubility parameter. While in the DMF/methanol solvent system, hydroxypropyl cellulose (HPC) or cellulose acetate butyrate (CAB) were taken as steric stabilizers of the dispersion polymerization, poly(vinylpyrrolidone) (PVP) was used in alcoholic media. Contrary to the DMF/methanol system, narrow particle size distributions were obtained with PVP-stabilized polymerizations in ethanolic, methanolic, propan-1-olic or butan-1-olic medium. Both the particle size and polydispersity were reduced with increasing stabilizer concentration. If lower molecular-weight PVP was used, larger microspheres were obtained. Poly(GMA) samples prepared in a neat alcoholic medium virtually quantitatively retained oxirane group content after the polymerization. Reactivity of the poly(GMA) microspheres was confirmed by their hydrolysis and aminolysis.

Hydrogels in endovascular embolization. I. Spherical particles of poly(2-hydroxyethyl methacrylate) and their medico-biological properties

Spherical macroporous particles based on poly(2-hydroxyethyl methacrylate) with defined porosity, swelling and morphology have been developed, and are suitable for endovascular occlusion of various organs. Unlike cylindrical particles, spherical particles are specifically suited for transcatheteral introduction. The method chosen for the preparation of such particles was suspension radical polymerization, where the monomers were dissolved in a mixture of higher-boiling alcohols, and the solution dispersed in water. Physicochemical and medico-biological properties of spherical particles were examined. The residual amounts of monomers and other low-molecular compounds were checked; haematological analyses showed that the value 10(-5) g/g of the polymer was not toxic and contributed to an irreversible aggregation of thrombocytes. The occlusion effect in the vascular lumen was stable. The histomorphological results fully demonstrated the perfect biocompatibility of artificial spherical emboli. The latter met the requirements of application to clinical practice.

Hydrogels in endovascular embolization. III. Radiopaque spherical particles, their preparation and properties

The synthesis and properties of spherical radiopaque hydrogel particles designed for endovascular occlusion are reported. These particles were prepared by the hydroxyl acylation of low crosslinked poly (2-hydroxyethyl methacrylate) beads with a nontoxic radiopaque compound based on triiodobenzoic acid, without affecting their properties which are advantages in medical practice. The effect of the iodine content on the size of dry and swollen particles is discussed. It has been found that an iodine content of about 25-30 wt% is desirable in order to obtain an easily recognizable X-ray image. These particles make the immediate control of embolus application easy and enable periodical inspection of the polymer to check the successful blockage of the vessel. They also open up the method of endovascular occlusion to further improvement.

Hydrogels in endovascular embolization: VI. Toxicity tests of poly(2-hydroxyethyl methacrylate) particles on cell cultures

Cytotoxicity of poly(2-hydroxyethyl methacrylate) [poly(HEMA)] hydrogel spherical particles, prepared by radical suspension polymerization and designed for endovascular occlusion, was studied in vitro on cell cultures. Testing methods included a direct contact test and extraction test. No inhibition of growth of cells surrounding the poly(HEMA) beads and a very low inhibition of cell viability, only in concentrated extracts in long-term contact, were observed. As a result, poly(HEMA) beads can be considered non-toxic.

Hydrogels in endovascular embolization. II. Clinical use of spherical particles

In this study we report the results of clinical experiments, obtained with spherical particles made from poly(2-hydroxyethyl methacrylate) used in the embolization of arteriovenous anastomoses, in the suppression of pulmonary haemorrhage and haemoptysis and in the occlusion of some other arteries. So far we have used these particles in the treatment of 187 patients. It must be stressed that the advantage of spherical particles consists in the simplicity of their introduction into the blood vessel through a catheter, while in the blood vessel itself the particle swells in blood still more, when compared with the particle size in saline. This results in an immediate and permanent haemostatic effect. No revascularization occurs.

Nanopaper as an Optical Sensing Platform

Bacterial cellulose nanopaper (BC) is a multifunctional material known for numerous desirable properties: sustainability, biocompatibility, biodegradability, optical transparency, thermal properties, flexibility, high mechanical strength, hydrophilicity, high porosity, broad chemical-modification capabilities and high surface area. Herein, we report various nanopaper-based optical sensing platforms and describe how they can be tuned, using nanomaterials, to exhibit plasmonic or photoluminescent properties that can be exploited for sensing applications. We also describe several nanopaper configurations, including cuvettes, plates and spots that we printed or punched on BC. The platforms include a colorimetric-based sensor based on nanopaper containing embedded silver and gold nanoparticles; a photoluminescent-based sensor, comprising CdSe@ZnS quantum dots conjugated to nanopaper; and a potential up-conversion sensing platform constructed from nanopaper functionalized with NaYF4:Yb(3+)@Er(3+)&SiO2 nanoparticles. We have explored modulation of the plasmonic or photoluminescent properties of these platforms using various model biologically relevant analytes. Moreover, we prove that BC is and advantageous preconcentration platform that facilitates the analysis of small volumes of optically active materials (∼4 μL). We are confident that these platforms will pave the way to optical (bio)sensors or theranostic devices that are simple, transparent, flexible, disposable, lightweight, miniaturized and perhaps wearable.

Oriented immobilization of galactose oxidase to bead and magnetic bead cellulose and poly(HEMA-co-EDMA) and magnetic poly(HEMA-co-EDMA) microspheres

In order to obtain an active and stable oxidation reactor for daily use in biochemical laboratory we decided to immobilize galactose oxidase orientedly through a carbohydrate chain to the magnetic carriers. We used hydrazide derivatives of non-magnetic and magnetic bead cellulose and of magnetic and non-magnetic poly(HEMA-co-EDMA) microspheres. Activation of the enzyme molecules was done by sodium periodate in the presence of supplements (fucose, CuSO4, catalase). Orientedly immobilized galactose oxidase presents high storage stability and lower susceptibility to inappropriate microenvironmental conditions. Reactor reactivated by three pulses of D-galactose retained practically 100% of its native activity after 6 months. The positive properties of both magnetic carriers were entirely confirmed.

Effect of reaction parameters on the particle size in the dispersion polymerization of 2‐hydroxyethyl methacrylate

Poly(2-hydroxyethyl methacrylate) particles in the micron size range were obtained by the dispersion polymerization. Cellulose acetate butyrate and dibenzoyl peroxide were used as steric stabilizer and initiator, respectively. The ultimate particle size could be adjusted by the selection of a suitable polymerization medium consisting of an alcohol added to toluene and by varying their relative amounts. The particle size increased with increasing solubility parameter of the mixture, i.e., by decreasing the toluene/2-methylpropan-1-ol, toluene/butan-2-ol, and toluene/3-methylbutan-1-ol ratio. The particle size decreased with increasing concentration of the stabilizer and/or initiator. At the same time, the particle size distribution became narrower. Particles prepared from polymerization mixtures purged with nitrogen before the start of polymerization were smaller, and of narrower distribution, than those prepared from nitrogen-non-purged mixtures. Equilibrium swelling of particles in toluene decreased with the decreasing content of toluene in the polymerization mixture.

Enzymes immobilized on magnetic carriers: efficient and selective system for protein modification

In order to obtain an economical, efficient and selective system for glycoprotein modification we prepared reactors with immobilized neuraminidase or (and) galactose oxidase. High storage and operational stability of the enzyme reactors was obtained by their immobilization through the carbohydrate parts of the enzyme molecules to hydrazide-modified supports. Magnetic and non-magnetic forms of bead cellulose and poly(HEMA-co-EDMA) microspheres were used for immobilization. These reactors can be used almost universally for the activation of ligands and for labelling of substances having a carbohydrate moiety.