Assessment of the influence of gold nanoparticles stabilized with PAMAM dendrimers on HUVEC barrier cells.

Civilization diseases, cancer, frequent mutations of viruses and other pathogens constitute the need to look for new drugs, as well as systems for their targeted delivery. One of the promising way of using drugs is supplying them by linking to nanostructures. One of the solution for the development of nanobiomedicine are metallic nanoparticles stabilized with various polymer structures. In this report, we present the synthesis of gold nanoparticles, their stabilization with polyamidoamine (PAMAM) dendrimers with ethylenediamine core and the characteristics of the obtained product (AuNPs/PAMAM). The presence, size and morphology of synthesized gold nanoparticles were evaluated by ultraviolet-visible light spectroscopy, transmission electron microscopy and atomic force microscopy. The hydrodynamic radius distribution of the colloids was analyzed by dynamic light scattering technique. Additionally, the cytotoxicity and changes in mechanical properties of human umbilical vein endothelial cell line (HUVEC) cells caused by AuNPs/PAMAM were assessed. The results of studies on the nanomechanical properties of cells suggest a two-step changes in cell elasticity as a response to contact with nanoparticles. When using AuNPs/PAMAM in lower concentrations, no changes in cell viability were observed and the cells were softer than untreated cells. When higher concentrations were used, a decrease in the cells viability to about 80 % were observed, as well as non-physiological stiffening of the cells. The presented results may play a significant role in the development of nanomedicine.

Investigation of HUVEC response to exposure to PAMAM dendrimers - changes in cell elasticity and vesicles release.

The aim of this study is to assess the effect of PAMAM dendrimers of second, fourth, and seventh generations on human umbilical vein endothelial cells. Primary endothelial cells were exposed to PAMAM dendrimers for 24 h, using concentrations reducing cellular viability to the levels of 90, 75, and 50%. We assumed, that changes in mechanical properties reflect toxicity of PAMAM dendrimers. The mechanical properties were investigated using atomic force spectroscopy (AFS) technique with the use of two approaches for measuring cell elasticity: global, where the tests were performed using a micrometer-hemispherical probe, and local, where a nanometer-sized probe was used. For the sharp probe, a reduction in the elasticity modulus was observed in comparison to untreated control cells, that is related to the depolymerization of the cytoskeleton and the processes leading to cell apoptosis. In the case of the hemispherical probe, cell softening was also observed in comparison to control cells, but with increasing PAMAM concentrations, the modulus of elasticity increases. It is related to the sensing of numerous intracellular vesicles with the use of this probe, e.g. endosomal and empty plasmalemmal which can also alter cell elasticity. The presence of external and intracellular vesicles was confirmed by scanning and transmission electron microscopy. The relationship between the elasticity of HUVEC cells exposed to PAMAM dendrimers of selected generations and their toxic effects was presented herein for the first time. In the transmission electron microscopy images of the cells exposed to PAMAM dendrimers, we have also observed distinctive vesicles with regular multilayer arranged structure.

Dysfunction of endothelial cells exposed to nanomaterials assessed by atomic force spectroscopy.

The study of the impact of nanomaterials on endothelial cell elasticity with the atomic force spectroscopy (AFS) can be a significant model for assessing nanomaterials toxic effects in vitro. The mechanical properties of cells exposed to nanostructures can provide information not only about cellular nano and micro-structure, but also about cell physiology. The toxicity of nanostructures is an important issue which must be carefully considered when the optimal nanomaterial is defined. There are no universal properties characterizing such a nanomaterial, i.e. depending on the intended use, the requirements can be diverse. For example, for biomedical use a nanomaterial should not negatively affect the cells or should cause the expected therapeutic or diagnostic effects in justified cases. The present study was devoted to the effects of silver nanoparticles (SNPs), multi-walled carbon nanotubes (MWCNTs) and poly(amidoamine) (PAMAM) dendrimers of 4th generation on functioning of endothelial cells. Immortalized endothelial cells were exposed for 24 h to the tested nanomaterials used in concentrations reducing cellular viability to the levels of 90 % and 75 %. The innovative nature of our work is the comparison of cell elasticity performed with various AFS probes, which enabled detection of local and global elasticity alteration caused by the nanostructures. The obtained results demonstrated changes in elasticity of endothelial cell induced by the nanostructures, which were closely correlated with the level of cellular viability, forming of actin stress fibres and elevated levels of reactive oxygen species. Trend of changes in local and global elasticity of cells exposed to nanostructures was similar, but the magnitude of the response was dependent on the selected probe. SNPs and MWCNTs evoked cells stiffening, which was correlated with changes in production levels of reactive oxygen species (ROS) and the cytoskeletal alteration. Softening of cells exposed to PAMAM dendrimers correlated with increased number of apoptotic cells and ROS production levels. Based on the obtained results we conclude, that the structure and the type of nanostructure (nanoparticle) is essential for their localization inside the cells and for the toxic effect on the endothelial cells.

Synthesis and physicochemical characterization of chitin dihexanoate--A new biocompatible chitin derivative--In comparison to chitin dibutyrate.

Chitin dihexanoate (DHCH) is the novel biocompatible and technologically friendly highly substituted chitin diester. Here we described optimization of DHCH and chitin dibutyrate (dibutyryl chitin, DBC) synthesis conditions (temperature and reaction time) to obtain desired polymers with high reaction yield, high substitution degree (close to 2) and appropriately high molecular weights. A two-step procedure, employing acidic anhydrides (hexanoic or butyric anhydride) as the acylation agent and methanesulfonic acid both as the catalyst and the reaction medium, was applied. Chemical structures of DBC and DHCH were confirmed by NMR ((1)H and (13)C) and IR investigations. Mechanical properties, thermogravimetric analysis, differential scanning calorimetry and biocompatibility (Neutral red uptake assay, Skin Sensitization and Irritation Tests) were assessed. Both polymers proved highly biocompatible (non-cytotoxic in vitro, non-irritating and non-allergic to skin) and soluble in several organic solvents (dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetone, ethanol and others). It is worth emphasizing that DHCH and DBC can be easily processed by solvent casting method and the salt-leaching method, what gives the opportunity to prepare highly porous structures, which can be further successfully applied as the material for wound dressings and scaffolds for tissue engineering.

Chitin dipentanoate as the new technologically usable biomaterial.

In this article, the synthesis of novel biopolymer, chitin dipentanoate (Di-O-Valeryl Chitin, DVCH) has been described. DVCH is a chitin derivative esterified with two valeryl groups at positions 3 and 6 of the N-acetylglucosamine units and it is soluble in common organic solvents like ethanol, methanol, acetone, dichloromethane, 1,2-dichloroethane, N,N-dimethylmethanamide, N,N-dimethylacetamide and ethyl acetate. Highly efficient synthesis (degree of esterification close to 2) of DVCH was achieved by employing a huge excess of valeric anhydride used as both the acylation agent and the reaction medium in the presence of perchloric acid as catalyst. Studies on the DVCH synthesis were aimed at finding optimal conditions (temperature, reaction time) to obtain DVCH with high reaction yield and desirable physicochemical properties. Biological data demonstrate that DVCH is non-cytotoxic in vitro and doesn't exert irritating or allergic effects to animal skin. Thanks to its filmogenic properties, it can be used to manufacture threads, foils, foams and non-woven materials. Moreover, DVCH can be easily processed by salt-leaching method to prepare highly porous structures exhibiting open-cell architecture, that can be further employed in wound dressing therapies and scaffolds for tissue engineering.