Synthesis and biophysical evaluation of carbosilane dendrimers as therapeutic siRNA carriers.

Gene therapy presents an innovative approach to the treatment of previously incurable diseases. The advancement of research in the field of nanotechnology has the potential to overcome the current limitations and challenges of conventional therapy methods, and therefore to unlocking the full potential of dendrimers for use in the gene therapy of neurodegenerative disorders. The blood-brain barrier (BBB) poses a significant challenge when delivering therapeutic agents to the central nervous system. In this study, we investigated the biophysical properties of dendrimers and their complexes with siRNA directed against the apolipoprotein E (APOE) gene to identify an appropriate nanocarrier capable of safely delivering the cargo across the BBB. Our study yielded valuable insights into the complexation process, stability over time, the mechanisms of interaction, the influence of dendrimers on the oligonucleotide's spatial structure, and the potential cytotoxic effects on human cerebral microvascular endothelium cells. Based on our findings, we identified that the dendrimer G3Si PEG6000 was an optimal candidate for further research, potentially serving as a nanocarrier capable of safely delivering therapeutic agents across the BBB for the treatment of neurodegenerative disorders.

Selection of collagen IV fragments forming the outer sphere of the native protein: Assessment of biological activity for regenerative medicine.

The aim of this research was to select the fragments that make up the outer layer of the collagen IV (COL4A6) protein and to assess their potential usefulness for regenerative medicine. It was expected that because protein-protein interactions take place via contact between external domains, the set of peptides forming the outer sphere of collagen IV will determine its interaction with other proteins. Cellulose-immobilized protein fragment libraries treated with polyclonal anti-collagen IV antibodies were used to select the peptides forming the outer sphere of collagen IV. In the first test, 33 peptides that strongly interacted with the polyclonal anti-collagen IV antibodies were selected from a library of non-overlapping fragments of collagen IV. The selected fragments of collagen IV (cleaved from the cellulose matrix) were tested for their cytotoxicity, their effects on cell viability and proliferation, and their impact on the formation of reactive oxygen species (ROS). The studies used RAW 264.7 mouse macrophage cells and Hs 680.Tr human fibroblasts. PrestoBlue, ToxiLight™, and ToxiLight 100% Lysis Control assays were conducted. The viability of fibroblasts cultured with the addition of increasing concentrations of the peptide mix did not show statistically significant differences from the control. Fragments 161-170, 221-230, 721-730, 1331-1340, 1521-1530, and 1661-1670 of COL4A6 were examined for cytotoxicity against BJ normal human foreskin fibroblasts. None of the collagen fragments were found to be cytotoxic. Further research is underway on the potential uses of collagen IV fragments in regenerative medicine.

Flavonol and A-type procyanidin-rich extracts of Prunus spinosa L. flower exhibit anticoagulant activity through direct thrombin inhibition, but do not affect platelet aggregation in vitro.

Background: Blackthorn flower (Prunus spinosa L.) is a traditional herbal remedy recommended for treating cardiovascular diseases (CVDs). Aim: This in vitro study investigates the effects of flavonol and A-type procyanidin-rich blackthorn flower extracts on the hemostatic system, including the blood plasma coagulation cascade and platelet aggregation. Methods: Six distinct extracts, characterized through various techniques, including LC-MS/MS, were assessed at in vivo-relevant levels (1-50 µg/mL) for their antithrombotic activity. The thrombin, prothrombin, and activated partial thromboplastin times were measured. Additionally, the thrombin enzymatic activity was tested using the chromogenic substrate S-2238 and fibrinogen as the physiological substrate of the enzyme. To gain insights into the mechanism of action, the interactions between the primary extracts' constituents, their potential metabolites, and thrombin were examined in silico. The computational analyses were complemented by in vitro experiments and circular dichroism spectroscopy. The platelet aggregation in human platelet-rich plasma was assessed after ADP or collagen stimulation. Furthermore, the extracts' biocompatibility was tested on human peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs). Results: The extracts slightly prolonged the prothrombin and thrombin times and effectively inhibited the thrombin's enzymatic activity, reducing its amidolytic and proteolytic functions at 50 µg/mL by 91.2% and 74.8%, respectively. In silico molecular docking demonstrated a strong binding affinity of the examined polyphenols and their metabolites to thrombin. Most analytes bound exclusively within the enzyme active site; however, afzelin, kaempferitrin, and procyanidin A2 revealed the affinity to additional binding sites, including exosite I. The structure-activity relationship of flavonols as thrombin inhibitors was studied in vitro. Circular dichroism spectroscopy confirmed that the interactions between thrombin and the compounds (even at 1 µg/mL) induce alterations within the α-helices' secondary structure, resulting in noticeable changes in the enzyme's CD spectrum. On the other hand, the extracts did not influence platelet aggregation. Eventually, their cellular biocompatibility with PBMCs and RBCs was confirmed. Conclusion: The extracts directly inhibit thrombin, a critical serine protease in hemostasis and a prime anticoagulant drug target, and do not exhibit antiplatelet effects. This study enhances the knowledge of the biological activity of blackthorn flowers and supports their traditional use in CVDs.

The Anti-Aggregative Potential of Resolvin E1 on Human Platelets.

Resolvin E1 is a metabolite of eicosapentaenoic acid (EPA) which is one of the omega-3 polyunsaturated fatty acids (omega-3 PUFAs). The antiplatelet properties of omega-3 PUFAs are well known, but the effect of resolvin E1 on platelets via the collagen receptors is extremely poorly reported. We investigated the effect of resolvin E1 on collagen-induced platelet aggregation, activation, and reactivity, and also platelet membrane fluidity. The ultimate and statistically significant results showed that resolvin E1 may inhibit platelet reactivity due to the reduction of collagen-induced platelet aggregation in platelet-rich plasma and isolated platelets, but not in whole blood. Also, resolvin E1 significantly reduced P-selectin exposure on collagen-stimulated platelets. Moreover, we demonstrated that resolvin E1 can maintain platelet membrane structure (without increasing membrane fluidity). The association between platelet reactivity and membrane fluidity, including resolvin E1 and collagen receptors requires further research. However, the goal of this study was to shed light on the molecular mechanisms behind the anti-aggregative effects of resolvin E1 on platelets, which are still not fully clarified. We also indicate an innovative research direction focused on further analysis and then use of omega-3 PUFAs metabolites as antiplatelet compounds for future applications in the treatment and prevention of cardiovascular diseases.

The effects of non-functionalized polystyrene nanoparticles with different diameters on human erythrocyte membrane and morphology.

In this study, the potential toxicity of non-functionalized polystyrene nanoparticles (PS-NPs) in human erythrocytes has been assessed. The effect of PS-NPs with different diameters (∼30 nm, ∼45 nm, ∼70 nm) on fluidity of erythrocytes membrane, red blood cells shape, as well as haemolysis of these cells has been investigated. Erythrocytes were incubated for 24 h with non-functionalized PS-NPs in concentrations ranging from 0.001 to 200 µg/mL in order to study haemolysis and from 0.001 to 10 µg/mL to determine other parameters. Fluidity was estimated by electron paramagnetic resonance (EPR) and the fluorimetric method. It has been shown that PS-NPs induced haemolysis, caused changes in the fluidity of red blood cells membrane, and altered their shape. Non-functionalized PS-NPs increased the membrane stiffness in the hydrophobic region of hydrocarbon chains of fatty acids. The observed changes in haemolysis and morphology were dependent on the size of the nanoparticles. The smallest PS-NPs of ∼30 nm (with the smallest absolute value of the negative zeta potential -29.68 mV) induced the greatest haemolysis, while the largest PS-NPs of ∼70 nm (with the highest absolute value of the negative zeta potential -42.00 mV) caused the greatest changes in erythrocyte shape and stomatocytes formation.

Pegylated Gold Nanoparticles Conjugated with siRNA: Complexes Formation and Cytotoxicity.

Drug delivery systems such as dendrimers, liposomes, polymers or gold/silver nanoparticles could be used to advance modern medicine. One significant pharmacological problem is crossing biological barriers by commonly used drugs, e.g., in the treatment of neurodegenerative diseases, which have a problem of the blood-brain barrier (BBB) restricting drug delivery. Numerous studies have been conducted to find appropriate drug carriers that are safe, biocompatible and efficient. In this work, we evaluate pegylated gold nanoparticles AuNP14a and AuNP14b after their conjugation with therapeutic siRNA directed against APOE4. This genetic risk factor remains the strongest predictor for late-onset Alzheimer's disease. The study aimed to assess the biophysical properties of AuNPs/siAPOE complexes and to check their biological safety on healthy cells using human brain endothelial cells (HBEC-5i). Techniques such as fluorescence polarization, circular dichroism, dynamic light scattering, ζ-potential measurements and gel retardation assay showed that AuNPs form stable complexes with siRNA. Subsequently, cytotoxicity assays proved the biological safety of formed conjugates. Obtained results enabled us to find effective concentrations of AuNPs when complexes are formed and non-toxic for healthy cells. One of the studied nanoparticles, AuNP14b complexed with siRNA, displayed lower cytotoxicity (MTT assay, cells viability -74.8 ± 3.1%) than free nanoparticles (44.7 ± 3.6%). This may be promising for further investigations in nucleic acid delivery and could have practical use in treating neurodegenerative diseases.

Glassy-like Metal Oxide Particles Embedded on Micrometer Thicker Alginate Films as Promising Wound Healing Nanomaterials.

Micrometer-thicker, biologically responsive nanocomposite films were prepared starting from alginate-metal alkoxide colloidal solution followed by sol-gel chemistry and solvent removal through evaporation-induced assembly. The disclosed approach is straightforward and highly versatile, allowing the entrapment and growth of a set of glassy-like metal oxide within the network of alginate and their shaping as crake-free transparent and flexible films. Immersing these films in aqueous medium triggers alginate solubilization, and affords water-soluble metal oxides wrapped in a biocompatible carbohydrate framework. Biological activity of the nano-composites films was also studied including their hemolytic activity, methemoglobin, prothrombin, and thrombine time. The effect of the films on fibroblasts and keratinocytes of human skin was also investigated with a special emphasis on the role played by the incorporated metal oxide. This comparative study sheds light on the crucial biological response of the ceramic phase embedded inside of the films, with titanium dioxide being the most promising for wound healing purposes.

Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells Cultured in 3D Spheroids.

Cationic dendrimers are effective carriers for the delivery of siRNA into cells; they can penetrate cell membranes and protect nucleic acids against RNase degradation. Two types of dendrimers (CBD-1 and CBD-2) and their complexes with pro-apoptotic siRNA (Mcl-1 and Bcl-2) were tested on MCF-7 cells cultured as spheroids. Cytotoxicity of dendrimers and dendriplexes was measured using the live-dead test and Annexin V-FITC Apoptosis Detection Kit (flow cytometry). Uptake of dendriplexes was examined using flow cytometry and confocal microscopy. The live-dead test showed that for cells in 3D, CBD-2 is more toxic than CBD-1, contrasting with the data for 2D cultures. Attaching siRNA to a dendrimer molecule did not lead to increased cytotoxic effect in cells, either after 24 or 48 h. Measurements of apoptosis did not show a high increase in the level of the apoptosis marker after 24 h exposure of spheroids to CBD-2 and its dendriplexes. Measurements of the internalization of dendriplexes and microscopy images confirmed that the dendriplexes were transported into cells of the spheroids. Flow cytometry analysis of internalization indicated that CBD-2 transported siRNAs more effectively than CBD-1. Cytotoxic effects were visible after incubation with 3 doses of complexes for CBD-1 and both siRNAs.

Synthesis and Hemostatic Activity of New Amide Derivatives.

Eight dipeptides containing antifibrinolytic agents (tranexamic acid, aminocaproic acid, 4-(aminomethyl)benzoic acid, and glycine-natural amino acids) were synthesized in a three-step process with good or very good yields. DMT/NMM/TsO- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate) was used as a coupling reagent. Hemolysis tests were used to study the effects of the dipeptides on blood components. Blood plasma clotting tests were used to examine their effects on thrombin time (TT), prothrombin time (PT), and the activated partial thromboplastin time (aPTT). The level of hemolysis did not exceed 1%. In clotting tests, TT, PT, and aPTT did not differentiate any of the compounds. The prothrombin times for all amides 1-8 were similar. The obtained results in the presence of amides 1-4 and 8 were slightly lower than for the other compounds and the positive control, and they were similar to the results obtained for TA. In the case of amide 3, a significantly decreased aPTT was observed. The aPTTs observed for plasma treated with amide 3 and TA were comparable. In the case of amide 6 and 8, TT values significantly lower than for the other compounds were found. The clot formation and fibrinolysis (CFF) assay was used to assess the influence of the dipeptides on the blood plasma coagulation cascade and the fibrinolytic efficiency of the blood plasma. In the clot formation and fibrinolysis assay, amides 5 and 7 were among the most active compounds. The cytotoxicity and genotoxicity of the synthesized dipeptides were evaluated on the monocyte/macrophage peripheral blood cell line. The dipeptides did not cause hemolysis at any concentrations. They exhibited no significant cytotoxic effect on SC cells and did not induce significant DNA damage.

Insight into Factors Influencing Wound Healing Using Phosphorylated Cellulose-Filled-Chitosan Nanocomposite Films.

Marine polysaccharides are believed to be promising wound-dressing nanomaterials because of their biocompatibility, antibacterial and hemostatic activity, and ability to easily shape into transparent films, hydrogels, and porous foams that can provide a moist micro-environment and adsorb exudates. Current efforts are firmly focused on the preparation of novel polysaccharide-derived nanomaterials functionalized with chemical objects to meet the mechanical and biological requirements of ideal wound healing systems. In this contribution, we investigated the characteristics of six different cellulose-filled chitosan transparent films as potential factors that could help to accelerate wound healing. Both microcrystalline and nano-sized cellulose, as well as native and phosphorylated cellulose, were used as fillers to simultaneously elucidate the roles of size and functionalization. The assessment of their influences on hemostatic properties indicated that the tested nanocomposites shorten clotting times by affecting both the extrinsic and intrinsic pathways of the blood coagulation system. We also showed that all biocomposites have antioxidant capacity. Moreover, the cytotoxicity and genotoxicity of the materials against two cell lines, human BJ fibroblasts and human KERTr keratinocytes, was investigated. The nature of the cellulose used as a filler was found to influence their cytotoxicity at a relatively low level. Potential mechanisms of cytotoxicity were also investigated; only one (phosphorylated microcellulose-filled chitosan films) of the compounds tested produced reactive oxygen species (ROS) to a small extent, and some films reduced the level of ROS, probably due to their antioxidant properties. The transmembrane mitochondrial potential was very slightly lowered. These biocompatible films showed no genotoxicity, and very importantly for wound healing, most of them significantly accelerated migration of both fibroblasts and keratinocytes.

Thermodynamic Studies of Interactions between Sertraline Hydrochloride and Randomly Methylated ß-Cyclodextrin Molecules Supported by Circular Dichroism Spectroscopy and Molecular Docking Results.

The interaction between sertraline hydrochloride (SRT) and randomly methylated ß-cyclodextrin (RMßCD) molecules have been investigated at 298.15 K under atmospheric pressure. The method used-Isothermal Titration Calorimetry (ITC) enabled to determine values of the thermodynamic functions like the enthalpy (ΔH), the entropy (ΔS) and the Gibbs free energy (ΔG) of binding for the examined system. Moreover, the stoichiometry coefficient of binding (n) and binding/association constant (K) value have been calculated from the experimental results. The obtained outcome was compared with the data from the literature for other non-ionic ßCD derivatives interacting with SRT and the enthalpy-entropy compensation were observed and interpreted. Furthermore, the connection of RMßCD with SRT was characterized by circular dichroism spectroscopy (CD) and complexes of ßCD derivatives with SRT were characterized through the computational studies with the use of molecular docking (MD).

Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson's Disease Patient-Specific Dopamine Neurons.

Accumulation of misfolded α-synuclein (α-syn) is a hallmark of Parkinson's disease (PD) thought to play important roles in the pathophysiology of the disease. Dendritic systems, able to modulate the folding of proteins, have emerged as promising new therapeutic strategies for PD treatment. Dendrimers have been shown to be effective at inhibiting α-syn aggregation in cell-free systems and in cell lines. Here, we set out to investigate the effects of dendrimers on endogenous α-syn accumulation in disease-relevant cell types from PD patients. For this purpose, we chose cationic carbosilane dendrimers of bow-tie topology based on their performance at inhibiting α-syn aggregation in vitro. Dopamine neurons were differentiated from induced pluripotent stem cell (iPSC) lines generated from PD patients carrying the LRRK2G2019S mutation, which reportedly display abnormal accumulation of α-syn, and from healthy individuals as controls. Treatment of PD dopamine neurons with non-cytotoxic concentrations of dendrimers was effective at preventing abnormal accumulation and aggregation of α-syn. Our results in a genuinely human experimental model of PD highlight the therapeutic potential of dendritic systems and open the way to developing safe and efficient therapies for delaying or even halting PD progression.

Thermoresponsive chimeric nanocarriers as drug delivery systems.

Chimeric or mixed nanosystems belong to the class of advanced therapeutics. Their distinctive characteristic compared with other types of nanoparticles is that they combine two or more different classes of biomaterials. These platforms have created a promising and versatile field of nanomedicine, incorporating materials that are biocompatible, such as lipids, but also functional, such as stimuli-responsive polymers. In the present work, thermoresponsive chimeric nanocarriers composed of l-α-phosphatidylcholine (Egg, Chicken) (EPC) phospholipids and poly(N-isopropylacrylamide)-b-poly(lauryl acrylate) (PNIPAM-b-PLA) block copolymers were designed and developed. Initially, model lipid bilayers with incorporated polymers and drug molecule TRAM-34 were built and studied for their thermodynamics, in order to assess the stability and functionality of the systems. Chimeric nanoparticles of EPC and PNIPAM-b-PLA were then developed and evaluated for their physicochemical properties in different medium conditions, as well as for their morphology. Polymer incorporation led to alterations in the properties and morphology of the nanoparticles, while interactions with serum proteins were absent. TRAM-34 was also incorporated inside the developed nanocarriers, followed by incorporation and release studies, which revealed the functionality of the system in elevated temperature conditions. Finally, in vitro studies on normal cells suggest the biocompatibility of these nanosystems. The proposed platforms are promising for further studies and applications in vitro and in vivo.

The Interaction of Heptakis (2,6-di-O-Methyl)-ß-cyclodextrin with Mianserin Hydrochloride and Its Influence on the Drug Toxicity.

One tetracyclic antidepressant, mianserin hydrochloride (MIA), has quite significant side effects on a patients' health. Cyclodextrins, which are most commonly used to reduce the undesirable features of contained drugs within their hydrophobic interior, also have the potential to alter the toxic behavior of the drug. The present paper contains investigations and the characteristics of interaction mechanisms for MIA and the heptakis (2,6-di-O-methyl)-ß-cyclodextrin (DM-ß-CD) system, and evaluated the effects of the complexation on MIA cytotoxicity. In order to assess whether there was an interaction between MIA and DM-ß-CD molecules, isothermal titration calorimetry (ITC) have been chosen. Electrospray ionization mass spectrometry (ESI-MS) helped to establish the complex stoichiometry, and circular dichroism spectroscopy was used to describe the process of complex formation. In order to make a wider interpretative perspective, the molecular docking results have been performed. The viability of Chinese hamster cells were investigated in the presence of DM-ß-CD and its complexes with MIA in order to estimate the cytotoxicity of the drug and the conjugate with the chosen cyclodextrin. The viability of B14 cells treated with MIA+DM-ß-CD is lower (the toxicity is higher) than with MIA alone, and no protective effects have been observed for complexes of MIA with DM-ß-CD in any ratio.

Dendrimeric HIV-peptide delivery nanosystem affects lipid membranes structure.

The aim of this study was to evaluate the nature and mechanisms of interaction between HIV peptide/dendrimer complexes (dendriplex) and artificial lipid membranes, such as large unilayered vesicles (LUV) and lipid monolayers in the air-water interface. Dendriplexes were combined as one of three HIV-derived peptides (Gp160, P24 and Nef) and one of two cationic phosphorus dendrimers (CPD-G3 and CPD-G4). LUVs were formed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) or of a mixture of DMPC and dipalmitoyl-phosphatidylglycerol (DPPG). Interactions between dendriplexes and vesicles were characterized by dynamic light scattering (DLS), fluorescence anisotropy, differential scanning calorimetry (DSC) and Langmuir-Blodgett methods. The morphology of formed systems was examined by transmission electron microscopy (TEM). The results suggest that dendriplexes interact with both hydrophobic and hydrophilic regions of lipid bilayers. The interactions between dendriplexes and negatively charged lipids (DMPC-DPPG) were stronger than those between dendriplexes and liposomes composed of zwitterionic lipids (DMPC). The former were primarily of electrostatic nature due to the positive charge of dendriplexes and the negative charge of the membrane, whereas the latter can be attributed to disturbances in the hydrophobic domain of the membrane. Obtained results provide new information about mechanisms of interaction between lipid membranes and nanocomplexes formed with HIV-derived peptides and phosphorus dendrimers. These data could be important for the choosing the appropriate antigen delivery vehicle in the new vaccines against HIV infection.

Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells.

The application of siRNA in gene therapy is mainly limited because of the problems with its transport into cells. Utilization of cationic dendrimers as siRNA carriers seems to be a promising solution in overcoming these issues, due to their positive charge and ability to penetrate cell membranes. The following two types of carbosilane dendrimers were examined: CBD-1 and CBD-2. Dendrimers were complexed with pro-apoptotic siRNA (Mcl-1 and Bcl-2) and the complexes were characterized by measuring their zeta potential, circular dichroism and fluorescence of ethidium bromide associated with dendrimers. CBD-2/siRNA complexes were also examined by agarose gel electrophoresis. Both dendrimers form complexes with siRNA. Moreover, the cellular uptake and influence on the cell viability of the dendrimers and dendriplexes were evaluated using microscopic methods and XTT assay on MCF-7 cells. Microscopy showed that both dendrimers can transport siRNA into cells; however, a cytotoxicity assay showed differences in the toxicity of these dendrimers.

Chimeric Stimuli-Responsive Liposomes as Nanocarriers for the Delivery of the Anti-Glioma Agent TRAM-34.

Nanocarriers are delivery platforms of drugs, peptides, nucleic acids and other therapeutic molecules that are indicated for severe human diseases. Gliomas are the most frequent type of brain tumor, with glioblastoma being the most common and malignant type. The current state of glioma treatment requires innovative approaches that will lead to efficient and safe therapies. Advanced nanosystems and stimuli-responsive materials are available and well-studied technologies that may contribute to this effort. The present study deals with the development of functional chimeric nanocarriers composed of a phospholipid and a diblock copolymer, for the incorporation, delivery and pH-responsive release of the antiglioma agent TRAM-34 inside glioblastoma cells. Nanocarrier analysis included light scattering, protein incubation and electron microscopy, and fluorescence anisotropy and thermal analysis techniques were also applied. Biological assays were carried out in order to evaluate the nanocarrier nanotoxicity in vitro and in vivo, as well as to evaluate antiglioma activity. The nanosystems were able to successfully manifest functional properties under pH conditions, and their biocompatibility and cellular internalization were also evident. The chimeric nanoplatforms presented herein have shown promise for biomedical applications so far and should be further studied in terms of their ability to deliver TRAM-34 and other therapeutic molecules to glioblastoma cells.

Antimicrobial Effect of Chitosan Films on Food Spoilage Bacteria.

Synthetic materials commonly used in the packaging industry generate a considerable amount of waste each year. Chitosan is a promising feedstock for the production of functional biomaterials. From a biological point of view, chitosan is very attractive for food packaging. The purposes of this study were to evaluate the antibacterial activity of a set of chitosan-metal oxide films and different chitosan-modified graphene (oxide) films against two foodborne pathogens: Campylobacter jejuni ATCC 33560 and Listeria monocytogenes 19115. Moreover, we wanted to check whether the incorporation of antimicrobial constituents such as TiO2, ZnO, Fe2O3, Ag, and graphene oxide (GO) into the polymer matrices can improve the antibacterial properties of these nanocomposite films. Finally, this research helps elucidate the interactions of these materials with eukaryotic cells. All chitosan-metal oxide films and chitosan-modified graphene (oxide) films displayed improved antibacterial (C. jejuni ATCC 33560 and L. monocytogenes 19115) properties compared to native chitosan films. The CS-ZnO films had excellent antibacterial activity towards L. monocytogenes (90% growth inhibition). Moreover, graphene-based chitosan films caused high inhibition of both tested strains. Chitosan films with graphene (GO, GOP, GOP-HMDS, rGO, GO-HMDS, rGOP), titanium dioxide (CS-TiO2 20:1a, CS-TiO2 20:1b, CS-TiO2 2:1, CS-TiO2 1:1a, CS-TiO2 1:1b) and zinc oxide (CS-ZnO 20:1a, CS-ZnO 20:1b) may be considered as a safe, non-cytotoxic packaging materials in the future.

Spheroids as a Type of Three-Dimensional Cell Cultures-Examples of Methods of Preparation and the Most Important Application.

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell-cell and cell-extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications.

Evaluation of the Effect of Selected Brominated Flame Retardants on Human Serum Albumin and Human Erythrocyte Membrane Proteins.

Brominated flame retardants (BFRs) have been using to reduce the flammability of plastics contained in many products, such as household articles, furniture, mattresses, textiles or insulation. Considering the fact that these compounds may be released into the environment leading to the exposure of living organisms, it is necessary to study their possible effects and mechanisms of action. Proteins play a crucial role in all biological processes. For this reason, a simple model of human serum albumin (HSA) was chosen to study the mechanism of BFRs' effect on proteins. The study determined interactions between selected BFRs, i.e., tetrabromobisphenol A (TBBPA), tetrabromobisphenol S (TBBPS), 2,4-dibromophenol (2,4-DBP), 2,4,6-tribromophenol (2,4,6-TBP) and pentabromophenol (PBP), and HSA by measurement of fluorescence of intrinsic tryptophan and absorbance of circular dichroism (CD). In addition, in order to understand the possible effect of these compounds in their native environment, the effect of BFRs on membrane proteins of human erythrocytes (red blood cells, RBCs) was also assessed. Among bromophenols, PBP had the strongest oxidative effect on RBC membrane, and 2,4-DBP demonstrated the weakest fluorescence-quenching effect of both membrane tryptophan and HSA. By contrast to PBP, 2,4-DBP and 2,4,6-TBP caused spatial changes of HSA. We have observed that among all analyzed BFRs, TBBPA caused the strongest oxidation of RBC membrane proteins and the model HSA protein, causing reduction of fluorescence of tryptophan contained in them. TBBPA also changed albumin conformation properties, leading to impairment of the α-helix structure. However, TBBPS had the weakest oxidative effect on proteins among studied BFRs and did not affect the secondary structure of HSA.