ROS-producing nanomaterial engineered from Cu(I) complexes with P2N2-ligands for cancer cells treating.

The work presents core-shell nanoparticles (NPs) built from the novel Cu(I) complexes with cyclic P2N2-ligands (1,5-diaza-3,7-diphosphacyclooctanes) that can visualize their entry into cancer and normal cells using a luminescent signal and treat cells by self-enhancing generation of reactive oxygen species (ROS). Variation of P- and N-substituents in the series of P2N2-ligands allows structure optimization of the Cu(I) complexes for the formation of the luminescent NPs with high chemical stability. The non-covalent modification of the NPs with triblock copolymer F-127 provides their high colloidal stability, followed by efficient cell internalization of the NPs visualized by their blue (⁓450 nm) luminescence. The cytotoxic effects of the NPs toward the normal and some of cancer cells are significantly lower than those of the corresponding molecular complexes, which correlates with the chemical stability of the NPs in the solutions. The ability of the NPs to self-enhanced and H2O2-induced ROS generation is demonstrated in solutions and intracellular space by means of the standard electron spin resonance (ESR) and fluorescence techniques correspondingly. The anticancer specificity of the NPs toward HuTu 80 cancer cells and the apoptotic cell death pathway correlate with the intracellular level of ROS, which agrees well with the self-enhancing ROS generation of the NPs. The enhanced level of ROS revealed in HuTu 80 cells incubated with the NPs can be associated with the significant level of their mitochondrial localization.

Lipid-dependent regulation of neurotransmitter release from sympathetic nerve endings in mice atria.

Neurotransmitter release from sympathetic terminals is a key avenue for heart regulation. Herein, presynaptic exocytotic activity was monitored in mice atrial tissue using a false fluorescent neurotransmitter FFN511, a substrate for monoamine transporters. FFN511 labeling had similarity with tyrosine hydroxylase immunostaining. High [K+]o depolarization caused FFN511 release, which was augmented by reserpine, an inhibitor of neurotransmitter uptake. However, reserpine lost the ability to increase depolarization-induced FFN511 unloading after depletion of ready releasable pool with hyperosmotic sucrose. Cholesterol oxidase and sphingomyelinase modified atrial membranes, changing in opposite manner fluorescence of lipid ordering-sensitive probe. Plasmalemmal cholesterol oxidation increased FFN511 release upon K+-depolarization and more markedly potentiated FFN511 unloading in the presence of reserpine. Hydrolysis of plasmalemmal sphingomyelin profoundly enhanced the rate of FFN511 loss due to K+-depolarization, but completely prevented potentiating action of reserpine on FFN511 unloading. If cholesterol oxidase or sphingomyelinase got access to membranes of recycling synaptic vesicles, then the enzyme effects were suppressed. Hence, a fast neurotransmitter reuptake dependent on exocytosis of vesicles from ready releasable pool occurs during presynaptic activity. This reuptake can be enhanced or inhibited by plasmalemmal cholesterol oxidation or sphingomyelin hydrolysis, respectively. These modifications of plasmalemmal (but not vesicular) lipids increase the evoked neurotransmitter release.

Sympathetic Innervation and Endogenous Catecholamines in Neuromuscular Preparations of Muscles with Different Functional Profiles.

Influence of the sympathetic nervous system on the work of skeletal muscles contractile apparatus is now beyond doubt. However, until recently there was no evidence that the endings of sympathetic nerves can be located in close proximity to the neuromuscular synapses, and there is also no reliable data on how much endogenous adrenaline and noradrenaline can be contained near the synaptic contact in skeletal muscles. In this research, using fluorescent analysis, immunohistochemical and enzyme immunoassays the isolated neuromuscular preparations of three skeletal muscles of different functional profiles and containing different types of muscle fibers were examined. Close contact between the sympathetic and motor cholinergic nerve endings and the presence of tyrosine hydroxylase in this area were demonstrated. Concentrations of endogenous adrenaline and noradrenaline in the solution perfusing the neuromuscular preparation were determined under different modes of its functioning. The effects of α and ß adrenoreceptor blockers on the processes of acetylcholine quantal secretion from the motor nerve endings were compared. The data obtained provide evidence for the presence of endogenous catecholamines in the neuromuscular junction region and their role in modulation of the synaptic function.

Structure impact on photodynamic therapy and cellular contrasting functions of colloids constructed from dimeric Au(I) complex and hexamolybdenum clusters.

Electrostatically driven self-assembly of [Au2L2]2+ (L is cyclic PNNP ligand) with [{Mo6I8}(L')6]2- (L' = I-, CH3COO-) in aqueous solutions is introduced as facile route for combination of therapeutic and cellular contrasting functions within heterometallic colloids (Mo6-Au2). The nature of L' affects the size and aggregation behavior of crystalline Mo6-Au2 aggregates, which in turn affect the luminescence of the cluster units incorporated into Mo6-Au2 colloids. The spin trap facilitated electron spin resonance spectroscopy technique indicates that the level of ROS generated by Mo6-Au2 colloids is also affected by their size. Both (L' = I-, CH3COO-) Mo6-Au2 colloids undergo cell internalization, which is enhanced by their assembly with poly-DL-lysine (PL) for L' = CH3COO-, but remains unchanged for L' = I-. The colloids PL-Mo6-Au2 (L' = CH3COO-) are visualized as huge crystalline aggregates both outside and inside the cell cytoplasm by confocal microscopy imaging of the incubated cells, while the smaller sized (30-50 nm) PL-Mo6-Au2 (L' = I-) efficiently stain the cell nuclei. Quantitative colocalization analysis of PL-Mo6-Au2 (L' = CH3COO-) in lysosomal compartments points to the fast endo-lysosomal escape of the colloids followed by their intracellular aggregation. The cytotoxicity of PL-Mo6-Au2 differs from that of Mo6 and Au2 blocks, predominantly acting through apoptotic pathway. The photodynamic therapeutic effect of the PL-Mo6-Au2 colloids on the cancer cells correlates with their intracellular trafficking and aggregation.

Mitochondria-targeted cationic liposomes modified with alkyltriphenylphosphonium bromides loaded with hydrophilic drugs: preparation, cytotoxicity and colocalization assay.

The purpose of this work was to obtain cationic liposomes based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine noncovalently modified using alkyltriphenylphosphonium bromides (TPPB-n) with different lengths of hydrocarbon tail for targeted delivery to mitochondria. The hydrodynamic diameter and electrokinetic potential of hybrid liposomes depending on the lipid/surfactant ratio were monitored in time with the aim to optimize the composition with sufficient stability and positive charge for mitochondria-targeted delivery. It was found that increasing the alkyl tail length of the surfactant (up to TPPB-14) leads to an increase in the positive charge of the liposomes. The most optimal results of stability were obtained for hybrid liposomes based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and TPPB-12, TPPB-14. The obtained modified liposomes were loaded with hydrophilic substrates (a model probe Rhodamine B and medicines metronidazole and doxorubicin). This is one of the first examples of fabrication of liposomes noncovalently modified using an amphiphilic TPP cation, with the alkyl tail length of surfactant and TPP/lipid ratio optimized in terms of stability of the liposomes and the binding/release behavior of hydrophilic probes. Using the confocal microscopy method, it was shown that modification of liposomes with a triphenylphosphonium cation results in targeted delivery of encapsulated compounds to mitochondria.