Influence of fluorophore and linker length on the localization and trafficking of fluorescent sterol probes.

Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.

Hydrogels based on low-methoxyl amidated citrus pectin and flaxseed gum formulated with tripeptide glycyl-l-histidyl-l-lysine improve the healing of experimental cutting wounds in rats.

Hydrogels based on natural and modified polysaccharides represent growing group of suitable matrices for the construction of effective wound healing materials. Bioactive tripeptide glycyl-l-histidyl-l-lysine and amino acid α-l-arginine are known to accelerate wound healing and skin repair. In this study, hydrogels based on low-methoxyl amidated citrus pectin or flaxseed gum were prepared and used for the transport of these healing agents to the experimental cutting wounds affected by extensive skin damage. Fourier-transform infrared spectroscopy, rheology, differential scanning calorimetry, scanning electron microscopy, swelling and release tests confirmed that these hydrogels differed in structure and physical properties. The cationic tripeptide was found to bind to carboxylic groups in LMA pectin, and the C3OH hydroxyl and ring oxygen O5 are involved in this interaction. The pectin hydrogel showed high viscosity and strong elastic properties, while the flaxseed gum hydrogel was characterised as a viscoelastic system of much lower viscosity. The former hydrogel released the drugs very slowly, while the latter hydrogel demonstrated zero order releasing kinetics optimal for drug delivery. In the in vivo wound healing testing on rats, both polysaccharide hydrogels improved the healing process mediated by the mentioned biomolecules. The tripeptide applied in the hydrogels showed significantly higher healing degree and lower healing time than in the control animals without treatment and when it was applied in an aqueous solution. Despite the absence of a synergistic effect, the mixture of the tripeptide and α-l-arginine in the hydrogels was also quite effective in wound healing. According to histological analysis, complete healing was achieved only when using the tripeptide in the flaxseed gum hydrogel. These observations might have an important prospect in clinical application of polysaccharide hydrogels.

New multimodal stationary phases prepared by Ugi multicomponent approach.

Eight different stationary phases based on two aminopropyl silicas of different brands suitable for multimodal chromatography applications have been prepared by a four-component Ugi reaction. The intention was to synthesize stationary phases significantly differing in their properties hereby demonstrating flexibility of the Ugi synthetic protocol. Diverse functional groups including a nonpolar long aliphatic chain, phenyl moiety, cholic acid scaffold, phenylboronic and monosaccharide units, charged betaine, and arginine moieties were immobilized on a silica surface. The novel sorbents were extensively characterized by elemental analysis, Raman spectroscopy, and chromatography. Considering the anchored chemical structures covalently bonded to the silica surface, reversed-phase, hydrophilic, and ion-exchange separation modes were expected. The chromatographic evaluation was performed directed to map the potential of the individual columns specifically in the mentioned chromatographic modes. The Ugi synthetic protocol has proven to be a simple, feasible, and versatile tool for the synthesis of sorbents of variable properties. The newly prepared stationary phases differed considerably in hydrophobicity and ion-exchange ability. A significant influence of the supporting aminopropyl silica on the final chromatographic behavior was observed. Finally, one practical example confirming applicability of the newly prepared sorbents was demonstrated in separation of cytarabine.

Formaldehyde Reacts with Amino Acids and Peptides with a Potential Role in Acute Methanol Intoxication.

Methanol, an aliphatic alcohol widely used in the industry, causes acute and chronic intoxications associated with severe long-term health damage, including permanent visual impairment, brain damage, mainly necrosis of the basal ganglia and high mortality due to cancer. However, the role of formaldehyde, an intermediate metabolite of methanol oxidation, in methanol toxicity remains unclear. Thus, we studied the reactivity of several amino acids and peptides in the presence of formaldehyde by identifying products by direct infusion electrospray high-resolution mass spectrometry (MS) and matrix-assisted laser desorption-ionization MS. Cysteine, homocysteine and two peptides, CG and CGAG, provided cyclic products with a +12 amu mass shift with respect to the original compounds. The proposed structures of the products were confirmed by high-resolution tandem MS. Moreover, the formation of the products with +12 amu mass shift was also shown for two biologically relevant peptides, fragments of ipilimumab, which is a human IgG1 monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4. Overall, our experimental results indicate that formaldehyde reacts with some amino acids and peptides, yielding covalently modified structures. Such chemical modifications may induce undesirable changes in the properties and function of vital biomolecules (e.g., hormones, enzymes) and consequently pathogenesis.

Coumarin Tröger's base derivatives with cyanine substitution as selective and sensitive fluorescent lysosomal probes.

The fluorescent probes based on Tröger's base motive with both coumarin and cyanine substitution 11-13 have been synthesized by multi-step synthesis in high overall yields. Intracellular localization of prepared probes have been tested using four different cell lines (HF-P4, BLM, U-2 OS and A-2058). Prepared probes have intensive green and red fluorescence. Co-localization with commercial lysosome specific marker LysoTracker Blue DND 22 has been confirmed that all prepared fluorescent probes labeled lysosomal compartment with high selectivity and probes show excellent brightness at low concentration.

A Cyclic Pentamethinium Salt Induces Cancer Cell Cytotoxicity through Mitochondrial Disintegration and Metabolic Collapse.

Cancer cells preferentially utilize glycolysis for ATP production even in aerobic conditions (the Warburg effect) and adapt mitochondrial processes to their specific needs. Recent studies indicate that altered mitochondrial activities in cancer represent an actionable target for therapy. We previously showed that salt 1-3C, a quinoxaline unit (with cytotoxic activity) incorporated into a meso-substituted pentamethinium salt (with mitochondrial selectivity and fluorescence properties), displayed potent cytotoxic effects in vitro and in vivo, without significant toxic effects to normal tissues. Here, we investigated the cytotoxic mechanism of salt 1-3C compared to its analogue, salt 1-8C, with an extended side carbon chain. Live cell imaging demonstrated that salt 1-3C, but not 1-8C, is rapidly incorporated into mitochondria, correlating with increased cytotoxicity of salt 1-3C. The accumulation in mitochondria led to their fragmentation and loss of function, accompanied by increased autophagy/mitophagy. Salt 1-3C preferentially activated AMP-activated kinase and inhibited mammalian target of rapamycin (mTOR) signaling pathways, sensors of cellular metabolism, but did not induce apoptosis. These data indicate that salt 1-3C cytotoxicity involves mitochondrial perturbation and disintegration, and such compounds are promising candidates for targeting mitochondria as a weak spot of cancer.

Strategy for improved therapeutic efficiency of curcumin in the treatment of gastric cancer.

Gastric cancer is a common oncological disease. Although enormous efforts have been expended, possible therapeutic modalities are still limited. For this reason, new therapeutic approaches and agents are highly requested and intensively developed. One strategy is the application of natural agents, such as curcumin, with proven anticancer effects and low toxicity for patients. Therefore, this review discusses the potential application of curcumin in the therapy of gastric cancer and its potential incorporation in therapeutic regimens. Because one of the largest impediments for widespread curcumin application is its limited bioavailability (caused mainly by its very low water solubility), studied strategies (drug delivery systems and curcumin derivatization) aimed to solve this obstacle are discussed in more detail.

Hydrazones as novel epigenetic modulators: Correlation between TET 1 protein inhibition activity and their iron(II) binding ability.

Ten-eleven translocation protein (TET) 1 plays a key role in control of DNA demethylation and thereby of gene expression. Dysregulation of these processes leads to serious pathological states such as oncological and neurodegenerative ones and thus TET 1 targeting is highly requested. Therefore, in this work, we examined the ability of hydrazones (acyl-, aroyl- and heterocyclic hydrazones) to inhibit the TET 1 protein and its mechanism of action. Inhibitory activity of hydrazones 1-7 towards TET 1 was measured. The results showed a high affinity of the tested chelators for iron(II). The study clearly showed a significant correlation between the chelator's affinity for iron(II) ions (represented by the binding constant) and TET 1 protein inhibitory activity (represented by IC50 values).

Versatile fluorophores for bioimaging applications: π-expanded naphthalimide derivatives with skeletal and appendage diversity.

Four novel fluorescent cores bearing a transformable functional group based on a π-expanded naphthalimide including a fused pyranone or furan ring have been prepared. Fluorescent probes LysoSers 13-16 for lysosomal targeting have been tested. Co-localization with a commercial lysosome specific marker confirmed that the LysoSers labeled the lysosomal compartment with high selectivity. The LysoSers show excellent brightness and low toxicity.

Pigments from Filamentous Ascomycetes for Combination Therapy.

Filamentous ascomycetes (Neurospora and Monascus) have been studied for a long time because of their production of secondary metabolites such as microbial pigments. The ascomycetes represent an interesting group of compounds with high potential for medicinal applications. Many recent studies have shown their efficacy in the treatment of serious pathological states such as oncological diseases, neurodegenerative diseases and hyperlipidaemia. Nevertheless, the clinical usability of ascomycetes is still limited. However, this problem can be solved by the use of these compounds with combinations of other therapeutic agents. This strategy can suppress their side effects and improve their therapeutic efficacy. Moreover, their co-application can significantly enhance conventional therapies that are used. This review summarizes and discusses the general principles of this approach, introduced and supported by numerous examples. In addition, the prediction of the future potential application of this methodology is included.

Pentamethinium salts as ligands for cancer: Sulfated polysaccharide co-receptors as possible therapeutic target.

A series of pentamethinium salts with benzothiazolium and indolium side units comprising one or two positive charges were designed and synthesized to determine the relationships among the molecular structure, charge density, affinity to sulfated polysaccharides, and biological activity. Firstly, it was found that the affinity of the pentamethinium salts to sulfated polysaccharides correlated with their biological activity. Secondly, the side heteroaromates displayed a strong effect on the cytotoxicity and selectivity towards cancer cells. Finally, doubly charged pentamethinium salts possessing benzothiazolium side units exhibited remarkably high efficacy against a taxol-resistant cancer cell line.

Interleukin-6: a molecule with complex biological impact in cancer.

Interleukin-6 is a multifaceted cytokine, usually reported as a pro-inflammatory molecule. However, certain anti-inflammatory activities were also attributed to IL-6. The levels of IL-6 in serum as well as in other biological fluids are elevated in an age-dependent manner. Notably, it is consistently reported also as a key feature of the senescence-associated secretory phenotype. In the elderly, this cytokine participates in the initiation of catabolism resulting in, e.g. sarcopenia. It can cross the blood-brain barrier, and so it is in causal association with, e.g. depression, bipolar disorder, schizophrenia, and anorexia. In the cancer patient, IL-6 is produced by cancer and stromal cells and actively participates in their crosstalk. IL-6 supports tumour growth and metastasising in terminal patients, and it significantly engages in cancer cachexia (including anorexia) and depression associated with malignancy. The pharmacological treatment impairing IL-6 signalling represents a potential mechanism of anti-tumour therapy targeting cancer growth, metastatic spread, metabolic deterioration and terminal cachexia in patients.

Recent advances in mixed-mode chromatographic stationary phases.

Mixed-mode phases have become very popular in the last decade, and the number of new mixed/multi-mode sorbents is growing fast. Unlike single-mode stationary phases, perfectly suited for the separation of the analytes possessing similar physicochemical properties, for instance reversed-phase chromatography for hydrophobic solutes, mixed-mode sorbents providing multimodal interactions can render better separation selectivity for complex mixtures of solutes differing significantly in their physicochemical characteristics. The most frequent modern mixed-mode stationary phases are di/tri-mode sorbents embracing the following interactions, hydrophobic, electrostatic (coulombic), and hydrophilic. According to their structures, it is possible to distinguish silica-based, polymer-based, hybrid, and monolithic mixed-mode stationary phases. Herewith, newly synthesized mixed-mode sorbents developed within the last two and half years are categorized, discussed, and summarized. The main attention is devoted to the description of the synthetic routes and characterization methods applied for the new stationary phases.

Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders.

The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.

Epigenetic agents in combined anticancer therapy.

In the last decade, epigenetic drugs (such as inhibitors of DNA methyltransferases and histone deacetylases) have been intensively used for cancer treatment. Their applications have shown high anticancer effectivity and tolerable side effects. However, they are unfortunately not effective in the treatment of some types and phenotypes of cancers. Nevertheless, several studies have demonstrated that problems of drug efficacy can be overcome through the combined application of therapeutic modulates. Therefore, combined applications of epigenetic agents with chemotherapy, radiation therapy, immunotherapy, oncolytic virotherapy and hyperthermia have been presented. This review summarizes and discusses the general principles of this approach, as introduced and supported by numerous examples. In addition, predictions of the future potential applications of this methodology are included.

Metallomics for Alzheimer's disease treatment: Use of new generation of chelators combining metal-cation binding and transport properties.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting tens of million people. Currently marketed drugs have limited therapeutic efficacy and only slowing down the neurodegenerative process. Interestingly, it has been suggested that biometal cations in the amyloid beta (Aß) aggregate deposits contribute to neurotoxicity and degenerative changes in AD. Thus, chelation therapy could represent novel mode of therapeutic intervention. Here we describe the features of chelators with therapeutically relevant mechanism of action. We have found that the tested compounds effectively reduce the toxicity of exogenous Aß and suppress its endogenous production as well as decrease oxidative stress. Cholyl hydrazones were found to be the most active compounds. In summary, our data show that cation complexation, together with improving transport efficacy may represent basis for eventual treatment strategy in AD.

Silica-based nanoparticles are efficient delivery systems for temoporfin.

BACKGROUND: Drug targeting using functionalized nanoparticles to advance their transport to the dedicated site became a new standard in novel anticancer methods Anticancer photodynamic therapy also takes benefit from using nanoparticles by means of increasing targeting efficiency and decreased side effect. With this in mind, the silica-based nanoparticles, as drug delivery systems for the second-generation photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (temoporfin) were developed. METHODS: In order to determine the stability and therapeutic performance of the selected nanomaterials in physiological fluids, their physicochemical properties (i.e. size, polydispersity, zeta potential) were measured by dynamic light scattering technique and the diameter and the morphology of the individual particles were visualized by a transmission electron microscopy. Their efficacy was compared with commercial temoporfin formulation in terms of in vitro phototoxicity in 4T1 (murine mammary carcinoma) and of in vivo anticancer effect in Nu/Nu mice bearing MDA-MB-231 tumors. RESULTS AND CONCLUSIONS: The two types of silica nanoparticles, porous and non-porous and with different surface chemical modification, were involved and critically compared within the study. Their efficacy was successfully demonstrated and was shown to be superior in comparison with commercial temoporfin formulation in terms of in vitro phototoxicity and cellular uptake as well as in terms of in vivo anticancer effect on human breast cancer model. Temoporfin-loaded silica nanoparticles also passed through the blood-brain barrier showing potential for the treatment of brain metastases.

Separation of oligopeptides, nucleobases, nucleosides and nucleotides using capillary electrophoresis/electrochromatography with sol-gel modified inner capillary wall.

The aim of this article is to study the modification of an inner capillary wall with sol-gel coating (pure silica sol-gel or silica sol-gel containing porphyrin-brucine conjugate) and determine its influence on the separation process using capillary electrophoresis/electrochromatography method. After modification of the inner capillary surface the separation of analytes was performed using two different phosphate buffers (pH 2.5 and 9.0) and finally the changes in electrophoretic mobilities of various samples were calculated. To confirm that the modification of the inner capillary surface was successful, the parts of the inner surfaces of capillaries were observed using scanning electron microscopy. The analytes used as testing samples were oligopeptides, nucleosides, nucleobases and finally nucleotides.

Immobilized strychnine as a new chiral stationary phase for HPLC.

A new ion-exchanger type chiral stationary phase for high-performance liquid chromatography was prepared. The synthetic protocol is based on derivatization of silica with (3-iodopropyl)trimethoxysilane in the first step followed by immobilization of strychnine via quaternization of nitrogen atom of the alkaloid strychnine. The synthesized chiral stationary phase was chromatographically characterized. The main effort was headed towards the evaluation of the enantioselectivity of the novel sorbent. For that purpose a set of suitable chiral probes, specifically, binaphthyl derivatives, was employed. The influence of methanol content, concentration of aqueous ammonium acetate buffer, and its pH on retention factors, separation selectivity, and resolution of the atropoisomers of the mentioned chiral solutes was studied in detail. It was demonstrated that the new chiral stationary phase was capable to separate atropoisomers of four out of seven testing compounds. Despite the strong influence of the above mentioned variables on retention, their impact on selectivity and resolution was rather moderate. Concerning retention mechanism, it seems that electrostatic interaction between the positively charged quaternary nitrogen of the chiral stationary phase and anionic solute participates significantly in the retention process.

Optical probes and sensors as perspective tools in epigenetics.

Modifications of DNA cytosine bases and histone posttranslational modifications play key roles in the control of gene expression and specification of cell states. Such modifications affect many important biological processes and changes to these important regulation mechanisms can initiate or significantly contribute to the development of many serious pathological states. Therefore, recognition and determination of chromatin modifications is an important goal in basic and clinical research. Two of the most promising tools for this purpose are optical probes and sensors, especially colourimetric and fluorescence devices. The use of optical probes and sensors is simple, without highly expensive instrumentation, and with excellent sensitivity and specificity for target structural motifs. Accordingly, the application of various probes and sensors in the recognition and determination of cytosine modifications and structure of histones and histone posttranslational modifications, are discussed in detail in this review.