Zuzana Kroneková

Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells

BackgroundDuring functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences.ResultsWe found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways.ConclusionsOur observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells.

Synthesis and bioimmunological efficiency of poly(2-oxazolines) containing a free amino group

Novel amphiphilic copolymers on the basis of 2-oxazolines containing a free amino group were prepared. The copolymers were synthesized by the living cationic polymerization of 2-ethyl-2-oxazoline (ETOX) and 2-(4-aminophenyl)-2-oxazoline (APOX). The main goal of this work was the synthesis of water soluble polymer material with the defined number of functional groups necessary for the attachment of proteins and polysaccharides. A high concentration of free amino groups allows immobilization of various biosubstances, e.g. drugs, proteins or polysaccharides. Thermal properties have been studied with respect to the composition of the copolymers. Cytotoxicity and the bioimmunological efficiency of the selected copolymer were studied.

Immunomodulatory efficiency of poly(2-oxazolines)

Poly(2-oxazolines) represent promising polymer materials for biomedical applications. The activation of mouse lymphoid macrophage line P388.D1 (clone 3124) by two selected representatives of poly(2-oxazolines), namely poly(2-ethyl-2-oxazoline) (PETOX100) and poly[2-(4-aminophenyl)-2-oxazoline-co-2-ethyl-2-oxazoline] (AEOX10), was assessed in vitro. The immunomodulatory efficacy of both polymers was evaluated via the induced release of pro-inflammatory cytokines (TNF-α, IL-1α and IL-6) and the acceleration of reactive free radicals. The present study revealed effective structure-immunomodulating associations of AEOX10 and PETOX100, which are desirable in biomedical and pharmaceutical applications of aliphatic and aromatic poly (2-oxazolines) in vivo.

Ex Vivo and In Vitro Studies on the Cytotoxicity and Immunomodulative Properties of Poly(2-isopropenyl-2-oxazoline) as a New Type of Biomedical Polymer

Poly(2-alkenyl-2-oxazoline)s are promising functional polymers for a variety of biomedical applications, such as drug delivery systems, peptide conjugates, or gene delivery. In this study, poly(2-isopropenyl-2-oxazoline) (PIPOx) is prepared through free-radical polymerization initiated with azobisisobutyronitrile. Reactive 2-oxazoline units in the side chain support an addition reaction with different compounds containing a carboxylic group, which facilitates the preparation of polymers labeled with two different fluorescent dyes. The cytotoxicities of 2-oxazoline monomers, PIPOx, and fluorescently labeled PIPOx are evaluated in vitro using an 3-(4,5-Dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and ex vivo using a cell proliferation assay with adenosine triphosphate bioluminescence. The cell uptake of labeled PIPOx is used to determine the colocalization of PIPOx with cell organelles that are part of the endocytic pathway. For the first time, it is shown that poly(2-isopropenyl-2-oxazoline) is a biocompatible material and is suitable for biomedical applications; further, its immunomodulative properties are evaluated.

Biosensor for calcium based on a hydrogel optical waveguide with integrated sensor proteins

We present a new optical biosensor concept based on hydrogel waveguides with integrated fluorescent proteins developed as a platform for implantable sensors. In this prototype the sensor detects the presence or absence of calcium through changes in fluorescence resonance energy transfer (FRET) induced by conformational changes of a FRET protein. The protein is immobilized in a synthetic hydrogel matrix with cylindrical shape, which serves simultaneously as an optical waveguide for the excitation light. The specificity of the protein determines which molecule can be detected.

Carbonyl iron coated with a sulfobetaine moiety as a biocompatible system and the magnetorheological performance of its silicone oil suspensions

In this study, surface modification of carbonyl iron (CI) particles with sulfobetaine moieties (SBE) was performed by the silanization of activated CI to form stable CI–SBE particles. The modification led to a significant improvement of the thermo-oxidation stability and a negligible suppression of the magnetization of the particles, as revealed by thermogravimetric analysis and vibrating sample magnetometry, respectively. The effect of a magnetic field and temperature on the magnetorheological performance of particle suspensions was investigated using a rotational rheometer in order to clarify the suitability of these systems for the local embolization of blood veins. The suspension based on CI–SBE exhibited a pseudoplastic behaviour and a tunable yield stress in a range from 0.3–4 kPa at the normal human body temperature. Moreover, cell viability for fibroblasts and macrophages was examined via MTT assay, which revealed their suitability for the intended applications for the local embolization of blood veins.

Chitosan–silver nanocomposites: New functional biomaterial for health-care applications

ABSTRACT Chitosan–silver nanocomposites (CS-HDA-AgNCs) was prepared using chitosan, biogenic silver nanocomposites, and crosslinker, hexamethylene 1,6-di(amino carboxysulfonate) (HDA). The film is flexible and transparent. Its physical, mechanical, thermal, hydrophilicity, and swelling properties were improved by HDA (2.5%). The antimicrobial activity of CS-HDA-AgNCs were not displayed any remarkable zone of inhibition but showed toxic effect in the presence of normal 3T3 fibroblasts and cancer HeLa cells. It decreases to ca. 5–7% for both cell lines. In conclusion, it can be mentioned that the CS-HDA-AgNCs, a kind of new functional biomaterial, could be useful for health-care applications. GRAPHICAL ABSTRACT

Poly(2-oxazoline) hydrogels by photoinduced thiol-ene “click” reaction using different dithiol crosslinkers

Fabrication of a library of biocompatible hydrogels by photoinduced thiol-ene “click” reaction is described. The hydrogels are based on two different statistical copolymers consisting of 2-butenyl-2-oxazoline (EnOx) with either 2-methyl-2-oxazoline (MeOx) or 2-ethyl-2-oxazoline (EtOx). Crosslinking is achieved via pendant vinyl groups of EnOx in the copolymers with selected dithiol crosslinkers, namely dithiothreitol, 1,3-propanedithiol, 1,6-hexanedithiol, and 1,9-nonanedithiol by thiol-ene photocoupling. On the basis of different polarity and flexibility of particular dithiols and three different molar ratios to double bonds, it is possible to control swelling and mechanical characteristics of resulting hydrogels. Biocompatibility of all hydrogel types is verified by determination of toxicity of hydrogel extracts and contact direct toxicity towards mice 3T3 fibroblast cell line. The resulting low toxicity of all prepared hydrogels predetermines its utilization in biomedicine.

Bioactive polymeric formulations for wound healing

Ricinoleic acid (RA) has potential to promote wound healing because of its analgesic and anti-inflammatory properties. This study investigates the synthesis and characterization of RA liposomes infused in a hydrogel for topical application. Lecithin liposomes containing RA were prepared and incorporated into a chitosan solution and were subsequently cross-linked with dialdehyde β-cyclodextrin (Di-β-CD). Chitosan/Di-β-CD concentrations and reaction temperatures were varied to alter gelation time, water content, and mechanical properties of the hydrogel in an effort to obtain a wide range of RA release profiles. Hydrogel cross-linking was confirmed by spectroscopy, and liposome and carrier hydrogel morphology via microscopy. Chitosan, Di-β-CD, and liposome concentrations within the formulation affected the extent of matrix swelling, mechanical strength, and pore and overall morphology. Higher cross-linking density of the hydrogel led to lower water uptake and slower release rate of RA. Optimized formulations resulted in a burst release of RA followed by a steady release pattern accounting for 80% of the encapsulated RA over a period of 48 hours. However, RA concentrations above 0.1 mg/mL were found to be cytotoxic to fibroblast cultures in vitro because of the oily nature of RA. These formulations promoted wound healing when used to treat full thickness skin wounds (2 cm2) in Wister male rats. The wound contraction rates were significantly higher compared to a commercially available topical cream after a time period of 21 days. Histopathological analysis of the RA-liposomal chitosan hydrogel group showed that the epidermis, dermis, and subcutaneous skin layers displayed an accelerated yet normal healing compared to control group.

Green synthesis of silver nanoparticles and biopolymer nanocomposites: a comparative study on physico-chemical, antimicrobial and anticancer activity

The current report was intended towards comparative study of green-synthesized biogenic Rhubarb silver nanoparticles (RS-AgNPs) and chitosan crosslinked silver nanocomposites (CSHD-AgNCs). The physico-chemical characterization was done by UV–visible, FTIR, scanning electron microscopy (SEM), transmission electron microscopy (TEM), EDX, TGA, XRD and zeta potential (