Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates.

INTRODUCTION: Beyond the three-dimensional fibrin network, the mechanical and lytic stability of thrombi is supported by the matrix of neutrophil extracellular traps (NETs) composed of polyanionic DNA meshwork with attached proteins including polycationic histones. Polyphosphates represent another type of polyanions, which in their linear form are known to enhance the fibrin stabilizing effects of DNA and histones. However, in vivo polyphosphates are also present in the form of nanoparticles (PolyP-NP), the interference of which with the fibrin/NET matrix is poorly characterized. AIMS: To compare the effects of linear and nanoparticulate polyphosphates, and their combinations with relevant NET components (DNA, histone H3) on fibrin formation, structure, and lysis in in vitro assays focusing on histone-polyphosphate interactions. METHODS: Transmission electron microscopy and dynamic light scattering for stability of the PolyP-NP preparations. Turbidimetry for kinetics of fibrinogen clotting by thrombin and fibrin dissolution by tissue-type plasminogen activator/plasminogen. Scanning electron microscopy for fibrin structure. Surface plasmon resonance for strength of histone-PolyP interactions. RESULTS: Both linear PolyP and PolyP-NP accelerated the fibrin formation and slowed down its dissolution and these effects were strongly dependent on the number of individual PolyP particles and not on their size. Addition of DNA did not modify significantly the PolyP-NP effects on fibrin formation and lysis. Both linear and nanoparticulate PolyP counteracted the effect of histone in the acceleration of fibrinogen clotting by thrombin. PolyP-NP, but not linear PolyP enhanced the prolongation of lysis time in fibrin containing histone and caused more pronounced thickening of the fibrin fibers than the linear form. Finally, PolyP-NP bound weaker to histone than the linear form. CONCLUSIONS: The interaction of PolyP with histone was a stronger modulator of fibrin formation and lysis than its interaction with DNA. In addition, the PolyP nanoparticles enhanced the thrombus stabilizing effects of histone more effectively than linear PolyP.

Comparison of the Efficacy of Two Novel Antitubercular Agents in Free and Liposome-Encapsulated Formulations.

Tuberculosis is one of the top ten causes of death worldwide, and due to the appearance of drug-resistant strains, the development of new antituberculotic agents is a pressing challenge. Employing an in silico docking method, two coumaran (2,3-dihydrobenzofuran) derivatives-TB501 and TB515-were determined, with promising in vitro antimycobacterial activity. To enhance their effectiveness and reduce their cytotoxicity, we used liposomal drug carrier systems. Two types of small unilamellar vesicles (SUV) were prepared: multicomponent pH-sensitive stealth liposome (SUVmixed) and monocomponent conventional liposome. The long-term stability of our vesicles was obtained by the examination of particle size distribution with dynamic light scattering. Encapsulation efficiency (EE) of the two drugs was determined from absorption spectra before and after size exclusion chromatography. Cellular uptake and cytotoxicity were determined on human MonoMac-6 cells by flow cytometry. The antitubercular effect was characterized by the enumeration of colony-forming units on Mycobacterium tuberculosis H37Rv infected MonoMac-6 cultures. We found that SUVmixed + TB515 has the best long-term stability. TB515 has much higher EE in both types of SUVs. Cellular uptake for native TB501 is extremely low, but if it is encapsulated in SUVmixed it appreciably increases; in the case of TB515, quasi total uptake is accessible. It is concluded that SUVmixed + TB501 seems to be the most efficacious antitubercular formulation given the presented experiments; to find the most promising antituberculotic formulation for therapy further in vivo investigations are needed.

Comparison of light-induced formation of reactive oxygen species and the membrane destruction of two mesoporphyrin derivatives in liposomes.

The photodynamic effect requires the simultaneous presence of light, photosensitizer (PS) and molecular oxygen. In this process, the photoinduced damage of cells is caused by reactive oxygen species (ROS). Besides DNA, the other target of ROS is the membranes, separating internal compartments in living cells. Hence, the ability of ROS formation of porphyrins as PSs, in liposomes as simple models of cellular membranes is of outstanding interest. Earlier we compared the binding parameters and locations of mesoporphyrin IX dihydrochloride (MPCl) and mesoporphyrin IX dimethyl ester (MPE), in small unilamellar vesicles (SUV) made from various saturated phosphatidylcholines. In this study, we used the same kinds of samples for comparing the ROS forming ability. Triiodide production from potassium iodide because of light-induced ROS in the presence of molybdate catalyst was applied, and the amount of product was quantitatively followed by optical spectrometry. Furthermore, we demonstrated and carefully studied SUVs disruption as direct evidence of membrane destruction by the methods of dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS), applying unsaturated phosphatidylcholines as membrane components. Although the ROS forming ability is more pronounced in the case of MPCl, we found that the measured disruption was more effective in the samples containing MPE.

Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Comparison of binding ability and location of two mesoporphyrin derivatives in liposomes explored with conventional and site-selective fluorescence spectroscopy.

Application of porphyrins as photosensitizers is based on their light-triggered generation of reactive oxygen species (ROS) that may cause oxidative tissue damage and ultimately kill cells. Cellular membranes are the action grounds of many sensitizers due to their hydrophobic or amphiphilic character as well as the location of many of the targets attacked by ROS. Hence, the binding ability and location of porphyrins in liposomes as simple models of cellular membranes are of outstanding interest. Here we compare mesoporphyrin IX dimethyl ester (MPE) and its nonesterified form, mesoporphyrin IX dihydrochloride (MPCl). Monocomponent small unilamellar vesicles formed of various saturated phosphatidylcholines with incorporated mesoporphyrins were investigated. We determined the binding parameters and the inhomogeneous distribution functions (IDFs) by different fluorescence techniques. We found in general that the binding ability of MPE is considerably greater than that of MPCl. In the case of MPCl, the IDFs suggest that only one of the two binding site types identified earlier for MPE ("site II") exists; the other one ("site I") vanishes while a new one appears ("site III"). We can confirm that "site I" is located between the two lipid layers, "site II" is situated between the hydrocarbon chains, while the location of the novel "site III" is along the outer part of the hydrocarbon chains partially inserted between the lipid head groups.

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters.

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

Location of mesoporphyrin in liposomes determined by site-selective fluorescence spectroscopy.

Binding of photosensitizers to target cells is a crucial step during the photodynamic effect. Sensitizer distribution is a good indication of whether the chemical is a good candidate for perturbing cell membrane integrity. Hence, the photophysical properties of porphyrinoid sensitizers in microheterogeneous systems such as liposomes are of outstanding interest. Here we present a site-selective fluorescence study of liposome systems. Monocomponent, small unilamellar vesicles formed of different phosphatidylcholines with incorporated mesoporphyrin were investigated. The size distribution of liposomes was measured by dynamic light scattering after each step of the experiment. On the basis of fluorescence line narrowing spectra of mesoporphyrin, the inhomogeneous distribution function was determined in order to characterize the photosensitizer location. The dual character of the functions revealed two different locations. Decomposition of the inhomogeneous distribution functions into Gaussians and the analysis of the fit results suggest that one of the locations for mesoporphyrin is between the two lipid layers, and the other one is between the hydrocarbon chains of the lipid molecules.

Interaction of photosensitizers with liposomes containing unsaturated lipid.

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.

Interaction of tetraphenyl-porphyrin derivatives with DPPC-liposomes: an EPR study.

The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization and interaction with membrane lipids were investigated by electron paramagnetic resonance (EPR). For this purpose, two glycoconjugated tetraphenyl porphyrin derivatives were selected, respectively, symmetrically and asymmetrically substituted. Small unilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and spin labeled stearic acids were prepared. The spin probe was located at the 5th or 7th or 12th or 16th position of the hydrocarbon chain in order to monitor various regions of the lipid bilayer. EPR spectra of porphyrin-free and porphyrin-bound liposomes were recorded at various temperatures below and above the phase transition temperature of DPPC. The effect on membrane fluidity proved to be stronger with the asymmetrical porphyrin derivative than with the symmetrical one. The rigidity increased when the spin label was near lipid head groups. The difference observed between control and porphyrin-treated samples when measured below the main lipid transition temperature disappeared at higher temperature. When the spin label was near the end of the hydrophobic tails, the symmetrical porphyrin derivative caused increase in fluidity, while the asymmetrical one slightly decreased it. To explain this phenomenon we propose that the asymmetrical derivative exerts a stronger ordering effect caused by its fluorophenyl group located at the level of the lipid heads, which is attenuated to the hydrophobic tails. The perturbing effect of the symmetric derivative could not lead to similar extent of ordering at the head groups and looses the hydrocarbon chains deeper in the membrane.