Solid Xenon Carrier Based on α-Cyclodextrin: Properties, Preparation, and Application.

The inert gas xenon (Xe) is increasingly used in medicine as a universal anesthetic, a regulator of cellular metabolism, and a broad-spectrum organoprotector. Commonly utilized Xe inhalation requires expensive equipment that is not universally available. Here we describe the production process and physical characteristics of a solid, highly stable xenon carrier based on α-cyclodextrin (α-CD), developed for oral administration. It was found, that the interaction of α-CD with Xe in an aqueous solution and elevated pressure leads to precipitation of the α-CD-Xe complex. We have discovered three new properties of the resulting complex that promote long-term storage and oral delivery of Xe. (i) At temperatures below 0 °C, the precipitated α-CD-Xe complex containing water is so stable that it allows the removal of water by vacuum freeze-drying (lyophilization). (ii). Lyophilized α-CD-Xe remains stable for months at room temperature. (iii) Upon contact with water, α-CD-Xe rapidly releases gaseous Xe. As revealed in the forced swim test, after oral administration of lyophilized α-CD-Xe to rats, the duration of swimming was significantly increased. The obtained data open up prospects for the development of drugs based on the lyophilized α-CD-Xe complex suitable for storage, transportation, and medical use, including outside the hospital.

Hitchhiking into a cell: flavonoids may produce complexes with transition metals for transmembrane translocation.

Flavonoids are a group of food polyphenols that are delivered to the human body with plant foods. In recent years, these substances have attracted the attention of researchers due to their effectiveness in preventing a wide variety of diseases, including neurodegenerative, oncological, autoimmune, and cardiovascular. Similar pathologies may also occur with a lack of some first-row transition metals, including Cu(II), Zn(II), Mn(II), Fe(II/III). It is noteworthy that flavonoids are known as transition metal chelators. When a complex with these metals is formed, the therapeutic effect of flavonoids can be enhanced, assuming the possibility of synergy. Molecular models have shown that the lipophilicity of flavonoid-metal complexes can vary significantly depending on their binding stoichiometry. Therefore, a unique process of translocation of flavonoid-metal complexes of various lipophilicity through cell membranes is assumed, based on the possibility of their sequential association and dissociation, called "hitchhiking". It is expected that studies of the interaction of flavonoids with metals will improve the effectiveness of drugs based on flavonoids.

Pharmacological torpor prolongs rat survival in lethal normobaric hypoxia.

Resistance to hypoxia is one of the most prominent features of natural hibernation and is expected to be present in the pharmacological torpor (PT) that simulates hibernation. We studied resistance to lethal hypoxia (3.5% oxygen content) in rats under PT. To initiate PT, we used the previously developed pharmacological composition (PC) which, after a single intravenous injection, can induce a daily decrease in Tb by 7 °C-8 °C at the environmental temperature of 22 °C-23 °C. Half-survival (median) time of rats in lethal hypoxia was found to increase from 5 ± 0.8 min in anesthetized control rats to 150 ± 12 min in rats injected with PC, which is a 30-fold increase. Behavioral tests after PT and hypoxia, including the traveling distance, the number of rearing and grooming episodes, revealed that animal responses are significantly restored within a week. It is assumed that the discovered unprecedented resistance of artificially torpid rats to lethal hypoxia may open up broad prospects for the therapeutic use of PT for preconditioning to various damaging factors, treatment of diseases, and extend the so-called "golden hour" for lifesaving interventions.

Long-term pharmacological torpor of rats with feedback-controlled drug administration.

The maintenance of pharmacological torpor and hypothermia (body temperature 28 °C - 33 °C) in rats for a week is presented. For this purpose, our laboratory has developed a device (BioFeedback-2) for the feed-back controlled multiple injections of small doses of a pharmacological composition that we created earlier. On the 7th day, the rat spontaneously come out of the pharmacological torpor, the body temperature returned to normal, and on the 8th day, the animal could consume food and water. The proposed approach for maintaining multi-day pharmacological torpor can be applied in medicine, as well as for protecting astronauts during long missions in space.

Comparison of natural and pharmacological hypothermia in animals: Determination of activation energy of metabolism.

The constancy of the activation energy of metabolism (E) for all living organisms is one of the most impressive, though controversial, statements of the modern metabolic theory of evolution. According to WBE-theory suggested by West, Brown, and Enquist, E should be in the range from -0.6 to -0.7 eV. However, there are many examples of significant deviations of E from the predictions of the theory. Now we have conducted a study of this value using rats in different types of pharmacological hypothermia: 1. Short-term (for several hours) hypothermia induced by anesthetic xylazine; 2. Daily torpor-like state induced by the pharmacological composition developed in our previous study. It has been found that in pharmacological daily hypothermia E = -0.56 ± 0.03 eV, which was close to that in daily heterotherms found in literature, E = -0.57 ± 0.04 eV. In short-term hypothermia E was substantially lower, E = -0.17 ± 0.071 eV. Our analysis revealed that in short-term hypothermia, changes in body temperature may lag behind changes in metabolic rate for a period Δt, affecting E. We propose an approach for estimating Δt and obtaining an adjusted E = -0.68 ± 0.17 eV, which corresponds to theoretical predictions. We assume that a similar consideration of Δt should be done when calculating E of daily heterotherms. We assume that in ectotherms, when the ambient temperature changes rapidly, changes in metabolic rate may lag behind changes in body temperature for a period (-) Δt, that should also be considered in E calculations. The proposed approach may contribute to the further development of the metabolic theory of evolution and may be useful in comparing artificial and natural hypothermia, as well as in studying the energy transformations in ecosystems.

A pharmacological composition for induction of a reversible torpor-like state and hypothermia in rats.

AIMS: To initiate a state of artificial torpor we suggested a pharmacological multi-targeting strategy for simulation of the physiological pattern of natural hibernation including a significant reduction in heart rate, respiratory rate, body temperature and oxygen consumption as well as a decline in brain activity known as torpor. MATERIALS AND METHODS: We have developed a composition which initiates a pharmacologically induced torpor-like state (PITS-composition), made up of eight therapeutic agents, inert gas xenon and lipid emulsion served as a drug vehicle. KEY FINDINGS: After a single intravenous injection to rats, PITS-composition causes a rapid decline in heart rate followed by a steady decrease in body temperature from about 38.5 °C to 31.5 °C, at ambient temperature of 22 °C-23 °C. The hypothermic state may continue on average for 16-17 h with the subsequent spontaneous return of heart rate and body temperature to the initial values. In the open field test at torpor the motility, rearing and grooming were suppressed but 4-8 days later they were restored. SIGNIFICANCE: Suspended animation states, including natural hibernation or pharmacologically induced synthetic torpor are of special attention of medicine, since it may improve survival rate after cardiac arrest, brain hemorrhage and ischemia, and during long-term space traveling. The suggested here multi-targeting strategy made possible to develop the pharmacological composition able, after a single intravenous injection, to initiate long, stable and reversible hypothermia and torpor at room temperature. After the torpor, animals were able to spontaneously restore both physiological parameters, and behavioral reactions.

Flavonoids determine the rate of fibrillogenesis and structure of collagen type I fibrils in vitro.

Collagen fibrils are produced from collagen monomers not only in vivo, but also in vitro. The ability to have an influence on the structure and properties of fibrils may find medical application and can be useful for controlling the formation of collagen gels and sheets in tissue engineering. Here we investigated the influence of flavonoids, distinguished by the number of hydroxyl groups in the B-ring, on the formation of collagen fibrils. A correlation was found between the number of hydroxyl groups, lipophilicity of molecules and their ability to influence the fibril formation. The molecules with a smaller number of hydroxyls (flavone and kaempferol) were more lipophilic and accelerated the formation of fibrils, whereas molecules with a larger number of hydroxyls (quercetin, myricetin) were more hydrophilic and prevented the fibril formation. Among the studied substances, an exception was taxifolin, which accelerated the formation of fibrils in spite of the increased hydrophilicity of this compound. However, molecular modeling revealed that all investigated accelerators of the fibril formation, including taxifolin, were distinguished by the increased lipophilicity exactly in the B-ring. This suggests a critical role of the B-ring lipophilicity in the ability of the studied flavonoids to accelerate the formation of collagen fibrils.

Antipsychotic inductors of brain hypothermia and torpor-like states: perspectives of application.

Hypothermia and hypometabolism (hypometabothermia) normally observed during natural hibernation and torpor, allow animals to protect their body and brain against the damaging effects of adverse environment. A similar state of hypothermia can be achieved under artificial conditions through physical cooling or pharmacological effects directed at suppression of metabolism and the processes of thermoregulation. In these conditions called torpor-like states, the mammalian ability to recover from stroke, heart attack, and traumatic injuries greatly increases. Therefore, the development of therapeutic methods for different pathologies is a matter of great concern. With the discovery of the antipsychotic drug chlorpromazine in the 1950s of the last century, the first attempts to create a pharmacologically induced state of hibernation for therapeutic purposes were made. That was the beginning of numerous studies in animals and the broad use of therapeutic hypothermia in medicine. Over the last years, many new agents have been discovered which were capable of lowering the body temperature and inhibiting the metabolism. The psychotropic agents occupy a significant place among them, which, in our opinion, is not sufficiently recognized in the contemporary literature. In this review, we summarized the latest achievements related to the ability of modern antipsychotics to target specific receptors in the brain, responsible for the initiation of hypometabothermia.

Integration of Quercetin-Iron Complexes into Phosphatidylcholine or Phosphatidylethanolamine Liposomes.

It is well known that flavonoids can chelate transition metals. Flavonoid-metal complexes exhibit a high antioxidative and therapeutic potential. However, the complexes are frequently hydrophobic ones and low soluble in water, which restricts their medical applications. Integration of these complexes into liposomes may increase their bioavailability and therapeutic effect. Here, we studied the interaction of quercetin-iron complexes with dimyristoylphosphatidylcholine (DMPC) or palmitoyl-oleoyl phosphatidylethanolamine (POPE) multilamellar liposomes. Differential scanning calorimetry (DSC) and freeze-fracture electron microscopy revealed that quercetin-iron complexes did not interact with liposomes. Quercetin however could penetrate lipid bilayers, when added to liposomes at a temperature above lipid melting. Iron cations added later penetrated into the lipid bilayers and produced complexes with quercetin in the liposomes. The quercetin-iron entry in POPE liposomes was improved when the suspension was heated above the temperature of the bilayer-hexagonal HII phase transition of the lipid. The approach proposed facilitates the integration of quercetin-iron complexes into liposomes and may promote their use in medicine.

Flavonoid-membrane interactions: involvement of flavonoid-metal complexes in raft signaling.

Flavonoids are polyphenolic compounds produced by plants and delivered to the human body through food. Although the epidemiological analyses of large human populations did not reveal a simple correlation between flavonoid consumption and health, laboratory investigations and clinical trials clearly demonstrate the effectiveness of flavonoids in the prevention of cardiovascular, carcinogenic, neurodegenerative and immune diseases, as well as other diseases. At present, the abilities of flavonoids in the regulation of cell metabolism, gene expression, and protection against oxidative stress are well-known, although certain biophysical aspects of their functioning are not yet clear. Most flavonoids are poorly soluble in water and, similar to lipophilic compounds, have a tendency to accumulate in biological membranes, particularly in lipid rafts, where they can interact with different receptors and signal transducers and influence their functioning through modulation of the lipid-phase behavior. In this study, we discuss the enhancement in the lipophilicity and antioxidative activity of flavonoids after their complexation with transient metal cations. We hypothesize that flavonoid-metal complexes are involved in the formation of molecular assemblies due to the facilitation of membrane adhesion and fusion, protein-protein and protein-membrane binding, and other processes responsible for the regulation of cell metabolism and protection against environmental hazards.

Lipophilicity of flavonoid complexes with iron(II) and their interaction with liposomes.

We studied complex formation of flavonoids quercetin and taxifolin with iron(II) and the complex influence on phase transitions of phospholipid bilayer. UV-Vis spectroscopy revealed that the stoichiometry of flavonoid/iron complexes was equal to 3:2 and 2:1. Molecular modeling and experimental measurements demonstrated the increase of flavonoids lipophilicity after the complex formation. A considerable influence of quercetin-iron complex on Palmitoyl-Oleoyl-Phosphatidylethanolamine transitions from bilayer to hexagonal HII phase was detected by differential scanning calorimetry. The obtained data are related to flavonoid/iron complexes bioavailability, their influence on cell membrane functioning, and should be considered in designing liposomal vehicles for drug and gene delivery.

Calcium-dependent aggregation and fusion of phosphatidylcholine liposomes induced by complexes of flavonoids with divalent iron.

It was found that complexes of the flavonoids quercetin, taxifolin, catechin and morin with divalent iron initiated an increase in light scattering in a suspension of unilamellar 100nm liposomes. The concentration of divalent iron in the suspension was 10µM. Liposomes were prepared from 1-palmitoyl-2-oleoylglycero-3-phoshpatidylcholine. The fluorescent resonance energy transfer (FRET) analysis of liposomes labeled with NBD-PE and lissamine rhodamine B dyes detected a slow lipid exchange in liposomes treated with flavonoid-iron complexes and calcium, while photon correlation spectroscopy and freeze-fracture electron microscopy revealed the aggregation and fusion of liposomes to yield gigantic vesicles. Such processes were not found in liposomes treated with phloretin because this flavonoid is unable to interact with iron. Rutin was also unable to initiate any marked changes because this water-soluble flavonoid cannot interact with the lipid bilayer. The experimental data and computer calculations of lipophilicity (cLogP) as well as the charge distribution on flavonoid-iron complexes indicate that the adhesion of liposomes is provided by an iron link between flavonoid molecules integrated in adjacent bilayers. It is supposed that calcium cations facilitate the aggregation and fusion of liposomes because they interact with the phosphate moieties of lipids.

Rafts making and rafts braking: how plant flavonoids may control membrane heterogeneity.

Plant flavonoids are not only known as powerful antioxidants, but also as cell metabolism regulators. It has been postulated that they are able to control cell signal pathways by targeting receptors on the cell surface or by intercalating the lipid bilayer of membranes. Some flavonoids can increase lipid viscosity and decrease the cooperativity of hydrocarbon chain melting, while others can considerably decrease the lipid melting temperature, thus providing additional freedom for lipid diffusion. Here we discuss the ability of flavonoids to influence phase transition and lateral segregation of lipids, responsible for the formation of membrane compartments known as lipid rafts. The thermodynamic parameters of the bilayer determined by lipid packing characteristics and by lateral segregation of the bilayer are expected to depend on the location of flavonoid molecules in the bilayer. Flavonoid molecules preferably located in the hydrophobic region of the bilayer can initiate formation of raft-like domains (raft-making effect), while the molecules located in the polar interface region of the bilayer can fluidize membranes (raft-breaking effect), or initiate formation of interdigitated or micellar structures. Accordingly, we expect that in cellular membranes flavonoids can influence the appearance and development of rafts or raft-like membrane domains and thus influence the lateral diffusion of lipid molecules. Because rafts participate in cellular signal transduction, endocytosis and transmembrane translocation of different compounds, flavonoids may control cell metabolism by modulating the bilayer state.

Plant polyphenols in cell-cell interaction and communication.

Plant polyphenols including flavonoids and tannins are important constituent of our everyday diet and medical herbals. It is broadly accepted that polyphenols may protect us from toxins, carcinogens and pollutants though the mechanisms of the polyphenols action is still not clear. Here we discuss the ability of polyphenols and especially gallate rich compounds like tannins and catechin gallates to interact with proteins and lipids, establish binding between adjacent bilayer surfaces and initiate membrane aggregation. This phenomena discovered in model experiments could also influence lateral segregation and compartmentalization of cell surface compounds and assist the cell-cell interaction and signal transduction. The involvement of plant polyphenols in communication between cells could be an important factor responsible for anticarcinogenic, vascular and cardioprotective activity of these compounds and speculated to be implicated in the evolution of human brain and intelligence.

Lipoplex formulation of superior efficacy exhibits high surface activity and fusogenicity, and readily releases DNA.

Lipoplexes containing a mixture of cationic phospholipids dioleoylethylphosphatidylcholine (EDOPC) and dilauroylethylphosphatidylcholine (EDLPC) are known to be far more efficient agents in transfection of cultured primary endothelial cells than are lipoplexes containing either lipid alone. The large magnitude of the synergy permits comparison of the physical and physico-chemical properties of lipoplexes that have very different transfection efficiencies, but minor chemical differences. Here we report that the superior transfection efficiency of the EDLPC/EDOPC lipoplexes correlates with higher surface activity, higher affinity to interact and mix with negatively charged membrane-mimicking liposomes, and with considerably more efficient DNA release relative to the EDOPC lipoplexes. Observations on cultured cells agree with the results obtained with model systems; confocal microscopy of transfected human umbilical artery endothelial cells (HUAEC) demonstrated more extensive DNA release into the cytoplasm and nucleoplasm for the EDLPC/EDOPC lipoplexes than for EDOPC lipoplexes; electron microscopy of cells fixed and embedded directly on the culture dish revealed contact of EDLPC/EDOPC lipoplexes with various cellular membranes, including those of the endoplasmic reticulum, mitochondria and nucleus. The sequence of events outlining efficient lipofection is discussed based on the presented data.

DNA release from lipoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion.

DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugal separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells.

High temperature stabilization of DNA in complexes with cationic lipids.

The influence on the melting of calf thymus and plasmid DNA of cationic lipids of the type used in gene therapy was studied by ultraviolet spectrophotometry and differential scanning calorimetry. It was found that various membrane-forming cationic lipids are able to protect calf thymus DNA against denaturation at 100 degrees C. After interaction with cationic lipids, the differential scanning calorimetry melting profile of both calf thymus and plasmid DNA revealed two major components, one corresponding to a thermolabile complex with transition temperature, T(m(labile)), close to that of free DNA and a second corresponding to a thermostable complex with a transition temperature, T(m(stable)), at 105 to 115 degrees C. The parameter T(m(stable)) did not depend on the charge ratio, R(+/-). Instead, the amount of thermostable DNA and the enthalpy ratio Delta H((stable))/Delta H((labile)) depended upon R(+/-) and conditions of complex formation. In the case of O-ethyldioleoylphosphatidylcholine, the cationic lipid that was the main subject of the investigation, the maximal stabilization of DNA exceeded 90% between R(+/-) = 1.5 and 3.0. Several other lipids gave at least 75% protection in the range R(+/-) = 1.5 to 2.0. Centrifugal separation of the thermostable and thermolabile fractions revealed that almost all the transfection activity was present at the thermostable fraction. Electron microscopy of the thermostable complex demonstrated the presence of multilamellar membranes with a periodicity 6.0 to 6.5 nm. This periodic multilamellar structure was retained at temperatures as high as 130 degrees C. It is concluded that constraint of the DNA molecules between oppositely charged membrane surfaces in the multilamellar complex is responsible for DNA stabilization.