Janez Mravljak

The effect of lipid composition and liposome size on the release properties of liposomes-in-hydrogel

To study the release of liposome-associated drugs into hydrogels, we designed and synthesized two pH-sensitive rhodamine derivatives to use as model compounds of different lipophilicities. The dyes were fluorescent when in the free form released from liposomes into the chitosan hydrogel, but not when incorporated within liposomes. The effect of liposomal composition, surface charge and vesicle size on the release of those incorporated dyes was evaluated. The lipophilicity of the rhodamine derivatives affected both the amount and rate of release. While liposome size had only a minor effect on the release of dyes into the hydrogel, the surface charge affected the release to a greater extent. By optimizing the characteristics of liposomes we could develop a liposomes-in-hydrogel system for application in wound therapy. We further characterized liposomes-in-hydrogel for their rheological properties, textures and moisture handling, as well as their potential to achieve a controlled release of the dye. The polymer-dependent changes in the hydrogel properties were observed upon addition of liposomes. The charged liposomes exhibited stronger effects on the textures of the chitosan hydrogels than the neutral ones. In respect to the ability of the system to handle wound exudates, chitosan-based hydrogels were found to be superior to Carbopol-based hydrogels.

Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents.

Tremendous efforts have been dedicated to the development of effective therapeutics against Alzheimers disease, which represents the most common debilitating neurodegenerative disease. Multifunctional agents are molecules designed to have simultaneous effects on different pathological processes. Such compounds represent an emerging strategy for the development of effective treatments against Alzheimers disease. Here, we report on the synthesis and biological evaluation of a series of nitroxoline-based analogs that were designed by merging the scaffold of 8-hydroxyquinoline with that of a known selective butyrylcholinesterase inhibitor that has promising anti-Alzheimer properties. Most strikingly, compound 8g inhibits self-induced aggregation of the amyloid beta peptide (Aβ1-42), inhibits with sub-micromolar potency butyrylcholinesterase (IC50=215 nM), and also selectively complexes Cu(2+). Our study thus designates this compound as a promising multifunctional agent for therapeutic treatment of Alzheimers disease. The crystal structure of human butyrylcholinesterase in complex with compound 8g is also solved, which suggests ways to further optimize compounds featuring the 8-hydroxyquinoline scaffold.

In Silico Discovery of Novel Potent Antioxidants on the Basis of Pulvinic Acid and Coumarine Derivatives and Their Experimental Evaluation

A pigment from the edible mushroom Xerocomus badius norbadione A, which is a natural derivative of pulvinic acid, was found to possess antioxidant properties. Since the pulvinic acid represents a novel antioxidant scaffold, several other derivatives were recently synthetized and evaluated experimentally, along with some structurally related coumarine derivatives. The obtained data formed the basis for the construction of several quantitative structure-activity and pharmacophore models, which were employed in the virtual screening experiments of compound libraries and for the prediction of their antioxidant activity, with the goal of discovering novel compounds possessing antioxidant properties. A final prioritization list of 21 novel compounds alongside 8 established antioxidant compounds was created for their experimental evaluation, consisting of the DPPH assay, 2-deoxyribose assay, β-carotene bleaching assay and the cellular antioxidant activity assay. Ten novel compounds from the tetronic acid and barbituric acid chemical classes displayed promising antioxidant activity in at least one of the used assays, that is comparable to or even better than some standard antioxidants. Compounds 5, 7 and 9 displayed good activity in all the assays, and were furthermore effective preventers of oxidative stress in human peripheral blood mononuclear cells, which are promising features for the potential therapeutic use of such compounds.

Internalization and Accumulation in Dendritic Cells of a Small pH-Activatable Glycomimetic Fluorescent Probe as Revealed by Spectral Detection

DC‐SIGN, an antigen‐uptake receptor in dendritic cells (DCs), has a clear role in the immune response but, conversely, can also facilitate infection by providing entry of pathogens into DCs. The key action in both processes is internalization into acidic endosomes and lysosomes. Molecular probes that bind to DC‐SIGN could thus provide a useful tool to study internalization and constitute potential antagonists against pathogens. So far, only large molecules have been used to directly observe DC‐SIGN‐mediated internalization into DCs by fluorescence visualization. We designed and synthesized an appropriate small glycomimetic probe. Two particular properties of the probe were exploited: activation in a low‐pH environment and an aggregation‐induced spectral shift. Our results indicate that small glycomimetic molecules could compete with antigen/pathogen for binding not only outside but also inside the DC, thus preventing the harmful action of pathogens that are able to intrude into DCs, for example, HIV‐1.

Influence of cancerostatic perifosine on membrane fluidity of liposomes and different cell lines as measured by electron paramagnetic resonance.

Aim To test whether membrane fluidity and its changes are important for the sensitivity of cells to the action of perifosine (OPP), a new anticancer drug targeting cell membrane and not DNA. Method Influence of OPP on the membrane structure of OPP-resistant MCF7, and OPP-sensitive MT3 breast cancer cell lines, as well as of mouse fibroblasts (L929) cell lines, and model cells (liposomes) was investigated by electron paramagnetic resonance, using spin labeled derivative of OPP (P5) and 5-doxylpalmitoyl methylester (MeFASL(10,3)) as spin probes. Results OPP increased membrane fluidity of all cell lines at concentrations higher than 50 µM (on the level of P ≤ 0.05, t test). In cells, the differences were observed only by P5 and not by MeFASL(10,3). Average order parameter Seff decreased for about 12% in MCF7 and L929 and only for 8% in OPP-sensitive MT3 cells, showing that there was no correlation between membrane fluidity changes and sensitivity of cells to OPP. The only correlation we found was between OPP sensitivity and the cell growth rate. In liposomes, both spin probes were sensitive to the action of OPP. Seff decreased with increasing concentration of OPP. For MeFASL(10,3) a significant decrease was observed at 4 mol% OPP, while for P5 it was observed at 8 mol%. Conclusion Influence of OPP on plasma membrane fluidity of breast cancer cells is not the determining factor in the sensitivity of cells to OPP.

Microstructure evaluation of dermally applicable liquid crystals as a function of water content and temperature: Can electron paramagnetic resonance provide complementary data?

Insight into the microstructure of lyotropic liquid crystals (LCs) is of crucial importance for development of novel dermal delivery systems. Our aim was to evaluate the phase behaviour of dermally applicable LCs composed of isopropyl myristate/Tween 80/lecithin/water, along the dilution line, where phase transitions are predominantly driven by increased water content. Additionally, identification of LC temperature dependence is of great importance for skin application. Selected LCs were evaluated using electron paramagnetic resonance (EPR) plus conventionally used methods of polarization microscopy, small-angle X-ray scattering, differential scanning calorimetry, and rheological measurements. Depending on water content, LCs formed diverse microstructures, from (pseudo)hexagonal (LC1) and lamellar (LC2-LC7) liquid crystalline phases that possibly co-exist with rod-like micelles (LC4-LC7), to a transitional micellar phase (LC8). Furthermore, the LCs microstructure remained unaltered within the tested temperature range. EPR was shown to detect microstructural transitions of LCs and to provide complementary data to other techniques. These data thus confirm the applicability of EPR as a complementary technique for better understanding of LC microstructural transitions that are expected to contribute greatly to studies oriented towards the drug release characteristics from such systems.

Interaction of Alkylphospholipid Formulations with Breast Cancer Cells in the Context of Anticancer Drug Development

Alkylphospholipids have shown promising results in several clinical studies (Mollinedo 2007) and among them Perifosine (octadecyl(1,1-di-methyl-4-piperidinium-4-yl)phosphate, OPP), and miltefosine (hexadecylphosphatidylcholine (HPC)) seems to be most promising for breast cancer therapy (Fichtner, Zeisig et al. 1994). For this type of tumor, an antitumor effect was found only for hormone receptor negative tumors in vivo, while no effect was found for receptor positive tumors. The reason for this difference is not yet understood and requires further studies. The exact mechanism of action of alkylphospholipids on the molecular level is still not well known in detail. It is clear that they do not target DNA, but they insert into the plasma membrane and subsequently induce a broad range of biological effects, ultimately leading to cell death. Unfortunately, administration of free (micellar) alkylphospholipids results in unwanted side effects, reflected in gastrointestinal toxicity and hemolytic activity, which limits the application of higher doses of alkylphospholipids. To achieve better therapeutic effects of alkylphospholipids in vivo with less side effects, different liposomal formulations of alkylphospholipids have been tested and showed diminished hemolytic activity. On the other hand, in most cases, cytotoxic activity of liposomes was also lower as compared to free alkylphospholipids (Zeisig et al., 1998). For efficient application of liposomes as nanocarriers in breast cancer therapy it is not only necessary to investigate the properties of the nanocarrier, which has to transport the drug to the (target) cell, but also the properties of the target cell. The main difference between Perifosine (OPP) resistent MCF7 cells and OPP sensitive MT-3 cells is in the uptake of OPP liposomes by cells and the transport of OPP across plasma membrane. At physiological temperatures the rate of transfer of OPP across plasma membrane increases to greater extent in OPP resistant MCF7 cells, while the uptake of liposomal OPP formulations is lower for

Improved Yields in the Synthesis of Spin‐Labeled Fatty Acids

Abstract Paramagnetic amide side products (6a–g) have been isolated from the reaction mixture in the synthesis of spin‐labeled fatty acids of the doxyl type. After their hydrolysis to the corresponding acid, 7, the overall yield of spin‐labeled fatty acids is significantly increased compared with published procedures.

How perifosine affects liposome-encapsulated drug delivery across a cell barrier.

BACKGROUND The development of efficient drug delivery systems to transport therapeutics across barrier-forming cells remains a challenge. Recently it was shown that liposomes containing perifosine, a synthetic analog of lysophosphatidylcholine, efficiently deliver liposome encapsulated content across barrier-forming cells. METHODS To elucidate the mechanism of the delivery, fluorescent and spin labeled analog of perifosine were synthesized and their transport from liposomes to the barrier-forming MDKC cells was measured. RESULTS & CONCLUSION Perifosine analogs are rapidly transported from liposomes into cell membranes. The total amount of perifosine accumulated in plasma membranes seems to be the most important factor in efficient transepithelial transport of liposome-encapsulated substances. Lysolipid-containing liposomal formulations seem to be promising candidates as drug delivery systems in general.