Superoxide Formation in Cardiac Mitochondria and Effect of Phenolic Antioxidants.

Since mitochondria are the main cellular source of reactive oxygen species, it is important to study the effect of dietary phenolic compounds on the level of ROS in these organelles. Using the EPR spectroscopy and TIRON probe, the ability of the investigated phenols (quercetin, rutin, caffeic acid, curcumin, and resveratrol) to scavenge superoxide anion radicals generated by isolated heart mitochondria of Wistar rats under variable oxygen partial pressure was studied. It was shown that during a 10 min incubation, caffeic acid in concentrations of 10-500 µM most effectively scavenged superoxide radicals formed in the complex III of the mitochondrial respiratory chain. A comparable antioxidant effect of rutin under these experimental conditions was observed at higher concentrations of 1-10 mM. The antioxidant activity of quercetin in the concentration range of 10-500 µM during the first minutes of incubation was higher than that of caffeic acid. Of the phenolic compounds studied, curcumin had the least effect on the superoxide radicals.

Dinitrosyl iron complexes: Formation and antiradical action in heart mitochondria.

Mitochondria are widely known as a major source of reactive oxygen and nitrogen species for the cardiovascular system. Numerous studies established that superoxide anion radical production by heart mitochondria is only slightly suppressed under conditions of deep hypoxia, but is completely blocked under anoxia. It was found also that dinitrosyl iron complexes (DNIC) compare favourably with other physiologically active derivatives of nitric oxide (NO). DNIC with glutathione effectively scavenge superoxide radicals generated by mitochondria at different partial pressures of oxygen. Under conditions of simulated hypoxia, the synthesis of thiol-containing DNIC takes place in mitochondria and is concomitant with a significant decrease in the concentration of NO metabolites at the reoxygenation step. Free NO required for DNIC synthesis is generated in the reaction of S-nitrosothiols with superoxide or during single-electron oxidation of the nitroxyl radical (HNO) by coenzyme Q. Plausible mechanisms of antiradical effects of DNIC and their protective role in oxidative stress induced by hypoxia/reoxygenation of myocardial tissues are considered. © 2018 BioFactors, 44(3):237-244, 2018.

Selective nuclear localization of siRNA by metallic versus semiconducting single wall carbon nanotubes in keratinocytes.

BACKGROUND: The potential use of carbon nanotubes (CNTs) in gene therapy as delivery systems for nucleic acids has been recently recognized. Here, we describe that metallic versus semiconducting single-wall CNTs can produce significant differences in transfection rate and cellular distribution of siRNA in murine PAM212 keratinocytes. RESULTS/METHODOLOGY: The results of cell interaction studies, coupled with supportive computational simulations and ultrastructural studies revealed that the use of metallic single wall CNTs resulted in siRNA delivery into both the cytoplasm and nucleus of keratinocytes, whereas semiconducting CNTs resulted in delivery only to the cytoplasm. CONCLUSION: Using enriched fractions of metallic or semiconducting CNTs for siRNA complex preparation may provide specific subcellular targeting advantages.

Preclinical development and ocular biodistribution of gemini-DNA nanoparticles after intravitreal and topical administration: towards non-invasive glaucoma gene therapy.

Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering intraocular pressure. We have developed and characterized non-viral gemini surfactant-phospholipid nanoparticles (GL-NPs) for intravitreal and topical administration. Optimized GL-NPs (size range 150-180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells with >95% viability by PrestoBlue™ assay. GL-NPs carrying Cy5-labeled plasmid DNA demonstrated distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, GL-NPs localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva. GL-NPs were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, non-aggregated particles after both types of administration. FROM THE CLINICAL EDITOR: In this paper, the development and characterization of non-viral gemini surfactant-phospholipid nanoparticles is reported with the goal of establishing a gene delivery system that addresses glaucoma in a non-invasive fashion. The authors found that after topical application, the concentration of these nanoparticles was higher in anterior chamber-related components of the eye, whereas intra-vitreal administration resulted in accumulation in the retinal nerve fibre layer.

Effect of chemical permeation enhancers on stratum corneum barrier lipid organizational structure and interferon alpha permeability.

The outermost layer of the skin, known as the stratum corneum (SC), is composed of dead corneocytes embedded in an intercellular lipid matrix consisting of ceramides, free fatty acids, and cholesterol. The high level of organization within this matrix protects the body by limiting the permeation of most compounds through the skin. While essential for its protective functions, the SC poses a significant barrier for the delivery of topically applied pharmaceutical agents. Chemical permeation enhancers (CPEs) can increase delivery of small drug compounds into the skin by interacting with the intercellular lipids through physical processes including extraction, fluidization, increased disorder, and phase separation. However, it is not clear whether these same mechanisms are involved in delivery of biotherapeutic macromolecules, such as proteins. Here we describe the effect of three categories of CPEs {solvents [ethanol, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid], terpenes [menthol, nerol, camphor, methyl salicylate], and surfactants [Tween 80, SDS, benzalkonium chloride, polyoxyl 40 hydrogenated castor oil (Cremophor RH40), didecyldimethylammonium bromide (DDAB), didecyltrimethylammonium bromide (DTAB)]} on the lipid organizational structure of human SC as determined by X-ray scattering studies. Small- and wide-angle X-ray scattering studies were conducted to correlate the degree of structural changes and hydrocarbon chain packing in SC lipids caused by these various classes of CPEs to the extent of permeation of interferon alpha-2b (IFNα), a 19 kDa protein drug, into human skin. With the exception of solvents, propylene glycol and ethanol, all classes of CPEs caused increased disordering of lamellar and lateral packing of lipids. We observed that the highest degree of SC lipid disordering was caused by surfactants (especially SDS, DDAB, and DTAB) followed by terpenes, such as nerol. Interestingly, in vitro skin permeation studies indicated that, in most cases, absorption of IFNα was low and that an increase in SC lipid disorder does not correspond to an increase in IFNα absorption.

Challenges in neuroprotective nanomedicine development: progress towards noninvasive gene therapy of glaucoma.

Over the past decade the application of gene therapy of retinal diseases such as glaucoma has produced promising results. However, optic nerve regeneration and restoration of vision in patients with glaucoma is still far from reality. Neuroprotective approaches in the form of gene therapy may provide significant advantages, but are still limited by many factors both at the organ and cellular levels. In general, gene delivery systems for eye diseases range from simple eye drops and ointments to more advanced bio- and nanotechnology-based systems such as muco-adhesive systems, polymers, liposomes and ocular inserts. Most of these technologies were developed for front-of-the-eye ophthalmic therapies and are not applicable as back-of-the-eye delivery systems. Currently, only the invasive intravitreal injections are capable of successfully delivering genes to the retina. Here we review the challenges and possible strategies for the noninvasive gene therapy of glaucoma including the barriers in the eye and in neural cells, and present a cross-sectional view of gene delivery as it pertains to the prevention and treatment of glaucoma.

Pharmaceutical characterization of solid and dispersed carbon nanotubes as nanoexcipients.

BACKGROUND: Carbon nanotubes (CNTs) are novel materials with considerable potential in many areas related to nanomedicine. However, a major limitation in the development of CNT-based therapeutic nanomaterials is a lack of reliable and reproducible data describing their chemical and structural composition. Knowledge of properties including purity, structural quality, dispersion state, and concentration are essential before CNTs see widespread use in in vitro and in vivo experiments. In this work, we describe the characterization of several commercially available and two in-house-produced CNT samples and discuss the physicochemical profiles that will support their use in nanomedicine. METHODS: Eighteen single-walled and multi-walled CNT raw materials were characterized using established analytical techniques. Solid CNT powders were analyzed for purity and structural quality using thermogravimetric analysis and Raman spectroscopy. Extinction coefficients for each CNT sample were determined by ultraviolet-visible near infrared absorption spectroscopy. Standard curves for each CNT sample were generated in the 0-5 µg/mL concentration range for dispersions prepared in 1,2-dichlorobenzene. RESULTS: Raman spectroscopy and thermogravimetric analysis results demonstrated that CNT purity and overall quality differed substantially between samples and manufacturer sources, and were not always in agreement with purity levels claimed by suppliers. Absorbance values for individual dispersions were found to have significant variation between individual single-walled CNTs and multi-walled CNTs and sources supplying the same type of CNT. Significant differences (P < 0.01) in extinction coefficients were observed between and within single-walled CNTs (24.9-53.1 mL·cm(-1)·mg(-1)) and multi-walled CNTs (49.0-68.3 mL·cm(-1)·mg(-1)). The results described here suggest a considerable role for impurities and structural inhomogeneities within individual CNT preparations and the resulting spectroscopic properties of their dispersions. CONCLUSION: Raw CNT materials require thorough analytical workup before they can be used as nanoexcipients. This applies especially to the determination of CNT purity, structure, and concentration. The results presented here clearly demonstrate that extinction coefficients must be determined for individual CNT preparations prior to their use.

A quantitative evaluation of redox-active compounds in human blood lipids.

Endogenous low molecular weight redox active compounds (RACs) comprise antioxidants, pro-oxidants, transition metal cations and metal chelators. Traditional electrochemical methods of measuring RACs are limited to aqueous solutions, thus providing information of only hydrophilic RAC pools. In a large number of diseases associated with oxidative stress and/or with metal toxicity, redox states of hydrophilic as well as hydrophobic compartments are modified, and therefore development of methods for their detection is both necessary and important. The pools of lipid soluble RACs in reduced and oxidized forms in n-hexane extracts obtained from blood plasma, erythrocytes and whole blood of healthy donors were determined by spectrophotometric detection of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, which stoichiometrically interacts with hydrogen donors in non polar solutions. Measurements of RACs in extracts before and after treatment with NaBH(4) provided information about the levels of both reduced and oxidized RACs. Vitamin E was also determined using a fluorescence method. The results have shown that vitamin E is the major RAC in blood plasma lipids but not in blood cell lipids, where other phenols and quinones appear to predominate.

A comparative study on conformation and ligand binding of the neuronal uncoupling proteins.

Mitochondrial uncoupling proteins of the nervous system (UCPs 2, 4, and 5) have potential roles in the function and protection of the central nervous system (CNS). In the absence of structural information, conformations of the hexahistidine-tagged versions of all five human UCPs in liposomes were investigated for the first time, using far- and near-UV CD and fluorescence spectroscopy. Highly pure UCPs 1-5 were reconstituted in detergents and stable small unilamellar vesicles, appropriate for spectroscopic studies. All UCPs formed dominantly helical conformations in negatively charged phospholipid vesicles (palmitoyloleoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol, 7:3 molar ratio). UCPs 2 and 5 exhibited comparable helical conformations with possible association in lipid bilayers, whereas UCP4 had a different helical profile that can be related to its less associated form. Interaction of reconstituted UCPs with GDP and GTP, inhibitors of the prototypic UCP1, was detected by near-UV CD and fluorescence spectroscopy, utilizing the sensitivity of these techniques to microenvironments around Trp residues close to the nucleotide binding site. Binding of UCP4 to purine nucleotides was also different from other UCPs. Binding of fatty acids, activators of proton transport in UCPs, to UCPs could not be unambiguously detected, implying a nonbinding conformation/orientation of the proteoliposomes. Interaction of CoA with UCPs was comparable to nucleotide binding, suggesting a possible binding of this molecule at the nucleotide binding site. Despite dissimilar primary sequences, neuronal UCPs share common structural and functional properties with UCPs 1 and 3, supporting a common physiological role in addition to their specific roles in the CNS.

Iron chelators and free radical scavengers in naturally occurring polyhydroxylated 1,4-naphthoquinones.

The cardioprotective effect of polyhydroxylated 1,4-naphthoquinones on the experimental model of myocardial ischemia-reperfusion has been demonstrated previously. In this study, using different models, such as bulk organic phase, liposomes and sarcoplasmic reticulum (SR) vesicles, we have shown the ability of naturally occurring polyhydroxynaphthoquinones, echinochrome (Ech), spinochromes C, D and E (SpC, SpD and SpE) to inhibit free-radical oxidation induced by heme iron (hemin) or by free iron ions (in ferrous/ascorbate system). The polyhydroxy-1,4-naphthoquinones (PHNQs) were more effective in inhibiting the phosphatidyl choline liposome peroxidation induced by ferrous/ascorbate than that induced by hemin. The iron chelating ability of PHNQs was determined spectrophotometrically. Prevention of the ferrous/ascorbate-induced leakage of calcium by Ech was demonstrated in isolated SR vesicles from rabbit skeletal muscle. The PHNQs displayed high scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. We concluded that iron chelation predominates in the overall antioxidant potential of the polyhydroxynaphthoquinones.

A CD study of uncoupling protein-1 and its transmembrane and matrix-loop domains.

Conformations of the prototypic UCP-1 (uncoupling protein-1) and its TM (transmembrane) and ML (matrix-loop) domains were studied by CD spectroscopy. Recombinant, untagged mouse UCP-1 and a hexahistidine-tagged version of the protein were obtained in high purity following their overexpression in Escherichia coli. The TM and ML domains of hamster UCP-1 were chemically synthesized. Conformations of both recombinant UCP-1 proteins were dominantly helical (40-50%) in digitonin micelles. Binding of the purine nucleotides GDP and GTP to UCP-1, detected in the near-UV CD region, supported the existence of the functional form of the protein in digitonin micelles. All individual TM and ML peptides, except the third ML domain, adopted helical structures in aqueous trifluoroethanol, which implies that, in addition to six TM segments, at least two of the ML domains of the UCP-1 can form helical structures in membrane interface regions. TM and ML domains interacted with vesicles composed of the main phospholipids of the inner membrane of mitochondria, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, to adopt dominantly beta- and/or unordered conformations. Mixtures of UCP-1 peptide domains spontaneously associated in aqueous, phospholipid vesicles and digitonin micelle environments to form ordered conformations, which exhibited common features with the conformations of the full-length proteins. Thermal denaturations of UCP-1 and its nine-peptide-domain assembly in digitonin were co-operative but not reversible. Assembly of six TM domains in lipid bilayers formed ion-conducting units with possible helical bundle conformations. Consequently, covalent connection between peptide domains, tight domain interactions and TM potential are essential for the formation of the functional conformation of UCP-1.

Effect of group II metal cations on catecholate oxidation.

The unexpected effects of Ca(2+) on the free-radical chain reactions of dopamine, norepinephrine, isoproterenol, and pyrocatechol oxidation are studied using oxygen consumption measurements, EPR-spectroscopy, UV/VIS spectrophotometry, and by potentiometric titration. It is found that the formation of Ca(2+)-catecholate complexes is accompanied by an increase in the dissociation constants (K(ai) ) of their phenolic hydroxyls. At pH>pK(ai) and in the presence of alkaline-earth metal cations, the rate of catecholate oxidation increases (Ca(2+), Mg(2+)> Sr(2+), Ba(2+)), whereas on addition of Zn ions the rate decreases. The effects of Group II metal cations on catecholate autoxidation are concomitant with a transient increase of the EPR signal for metal-semiquinonate complexes. Therefore, the effects of Ca(2+) and other alkaline-earth metal cations on catecholate autoxidation can be defined as 1) additional deprotonation of catechol OH-groups involved in the formation of M(2+)-catecholate complexes, the latter exceeding catechols in the susceptibility to dioxygen-induced oxidation and 2) formation of relatively stable free-radical intermediates responsible for chain propagation.

Echinochrome, a naturally occurring iron chelator and free radical scavenger in artificial and natural membrane systems.

Echinochrome, or 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone, possesses cardioprotective activity, and diminishes the myocardial ischemia/reperfusion injury that is known to be accompanied by free-radical oxidative damage and calcium overload. In this study, we investigated the lipophilicity of echinochrome, its ability to inhibit free-radical oxidation both in the bulk organic phase and in an artificial membrane system (liposomes), and to prevent the ferrous/ascorbate-induced leakage of calcium from the isolated sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The experimentally-determined octanol/water partition coefficient (LogP) of echinochrome was +3.11, and the distribution coefficient (LogD) was +2.58 at pH 6.0 and -0.15 at pH 8.0. Echinochrome displayed high scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals with a stoichiometry of about 1:7. Echinochrome was more effective in inhibiting the phosphatidyl choline liposome peroxidation induced by Fe2+/ascorbate than that induced by hemin. The iron chelating ability of echinochrome was estimated spectrophotometrically. In isolated SR, echinochrome protected the ATP-dependent Ca2+-pump system from damage by Fe2+/ascorbate. It was concluded that iron chelation predominates in the overall antioxidant potential of echinochrome.

How do calcium ions induce free radical oxidation of hydroxy-1,4-naphthoquinone? Ca2+ stabilizes the naphthosemiquinone anion-radical of echinochrome A.

A surprising effect is the direct action of Ca(2+) on redox reactions of ortho-quinoid compounds. The effect of Ca(2+) on oxidation of the sea urchin pigment 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone (echinochrome A) has been studied by electron paramagnetic resonance (EPR) spectroscopy, by UV/VIS absorbance spectroscopy, and by measurement of oxygen consumption. Echinochrome A per se reacted with dioxygen only in an alkaline solution; 2,3-semiquinone anion-radical of echinochrome A and superoxide anion-radical were the intermediates of the oxidation. Addition of calcium ions sharply increased the rate of echinochrome A autooxidation at alkaline pH and provoked oxidation at neutral pH. To explain this phenomenon we have focused on changes of the acid-base properties of echinochrome A in the presence of calcium and on stabilization of 2,3-semiquinone anion-radical of echinochrome A by Ca(2+). Dissociation constants (pK(a1), pK(a2), and pK(a3)) of echinochrome A determined by potentiometric titration were 5.20, 6.78, and >10 in calcium-free solution and 5.00, 6.10, and 7.15 in the presence of Ca(2+). We have found that Ca(2+) forms an insoluble adduct with the 2,3-semiquinone anion-radical. Thus, the effect of redox-inert calcium on the free radical reactions could be explained (i) by additional deprotonation of echinochrome A and (ii) by formation of a Ca(2+)-naphtho-2,3-semiquinone complex (calcium semiquinonate). Additionally, we have shown that the dried red spines from Strongylocentrotus intermedius possess paramagnetic properties; the EPR signal of the natural spines was similar to that of calcium semiquinonate obtained in our artificial chemical system.