Pharmaceutical Research (v.28, #11)
Mitochondrial Delivery of Biologically Active Molecules by Volkmar Weissig (2633-2638).
is Professor of Pharmacology and Interim Chair of the Department of Pharmaceutical Sciences at Midwestern University College of Pharmacy Glendale in Glendale, Arizona, USA. He received his B.S., M.S., and Ph.D. degrees in Chemistry and his postdoctoral Sc.D. degree (the German “Habilitation”) in Biochemistry and Pharmaceutical Biotechnology from the Martin-Luther University in Halle (Germany). Combined he completed several years of postdoctoral fellowships at the Cardiology Research Center in Moscow (Russia); the Academic Department of Medicine at the Royal Free Hospital School of Medicine in London (UK); the Institute of Organic Chemistry at the Czechoslovakian Academy of Science in Prague (CSFR); the College of Pharmacy and the College of Medicine at the University of Florida in Gainesville, FL, and at Harvard Medical School and Massachusetts General Hospital in Boston, MA. Before joining the faculty at Midwestern University, he was an Assistant Professor of Pharmaceutical Sciences at Northeastern University in Boston, MA. Since the beginning of the 1980s, Dr. Weissig has been working in the field of Liposome Technology. Toward the end of the 1990s, he turned his focus on Mitochondrial Pharmaceutics, an area he actively has been pioneering for the last 10 years.Dr. Weissig holds 16 patents and has published over 80 research papers, review articles and book chapters, mostly in the area of nano drug delivery systems. Several of his book chapters have been translated into Chinese, German, and Polish. He has also edited four books. He serves as the Associate Editor of the Journal of Liposome Research and is a member of several other Editorial Boards. In 2004, Dr. Weissig was elected as the Vice Chair and the following year Chair of the American Association of Pharmaceutical Sciences Northeast Regional Discussion Group (AAPS-NERDG). He also is an active member of the International Liposome Society, Mitochondrial Research Society, American Association of Pharmaceutical Sciences, and American Society for Advancement of Science. In 2002, Dr. Weissig was elected as member of the Rho Chi Honor Society Beta Tau Chapter at Northeastern University in Boston, MA, and in 2011 he was inducted into the Kappa Psi Pharmaceutical Fraternity Delta Sigma Chapter at Midwestern University. In July 2009, Dr. Weissig was elected as a Fellow of the World Technology Network.
Transduction of Human Recombinant Proteins into Mitochondria as a Protein Therapeutic Approach for Mitochondrial Disorders by Lefkothea C. Papadopoulou; Asterios S. Tsiftsoglou (2639-2656).
Protein therapy is considered an alternative approach to gene therapy for treatment of genetic-metabolic disorders. Human protein therapeutics (PTs), developed via recombinant DNA technology and used for the treatment of these illnesses, act upon membrane-bound receptors to achieve their pharmacological response. On the contrary, proteins that normally act inside the cells cannot be developed as PTs in the conventional way, since they are not able to “cross” the plasma membrane. Furthermore, in mitochondrial disorders, attributed either to depleted or malfunctioned mitochondrial proteins, PTs should also have to reach the subcellular mitochondria to exert their therapeutic potential. Nowadays, there is no effective therapy for mitochondrial disorders. The development of PTs, however, via the Protein Transduction Domain (PTD) technology offered new opportunities for the deliberate delivery of human recombinant proteins inside eukaryotic subcellular organelles. To this end, mitochondrial disorders could be clinically encountered with the delivery of human mitochondrial proteins (engineered via recombinant DNA and PTD technologies) at specific intramitochondrial sites to exert their function. Overall, PTD-mediated Protein Replacement Therapy emerges as a suitable model system for the therapeutic approach for mitochondrial disorders.
Keywords: mitochondrial disorders; protein therapy; protein transduction; PTDs
From Serendipity to Mitochondria-Targeted Nanocarriers by Volkmar Weissig (2657-2668).
This review illustrates how a random observation at the laboratory bench has helped pave the way towards the development of organelle-targeted pharmaceutical nanocarriers. A fortuitous discovery in the mid 1990s involving the self-assembly of a molecule, known to accumulate inside mitochondria, has lead to the development of subcellular nanocarriers suited for the selective delivery of biologically active molecules to mitochondria inside living mammalian cells. Applications for mitochondria-specific drug and DNA delivery are described, the current state-of-the-art of mitochondrial drug targeting technology is reviewed, and its future perspectives are discussed.
Keywords: apoptosis; dequalinium; DQAsomes; gene therapy; mitochondria; mitochondria-specific liposomes; mitochondria-targeted nanocarriers; STPP liposomes; triphenylphosphonium
Novel Therapies Targeting Inner Mitochondrial Membrane—From Discovery to Clinical Development by Hazel H. Szeto; Peter W. Schiller (2669-2679).
Mitochondrial oxidative stress and dysfunction have been implicated in the aging process and in numerous chronic diseases. The need for therapies that can protect and/or improve mitochondrial function is obvious. However, the development of mitoprotective drugs has been hampered by a number of challenges, and there are at present no approved therapies for mitochondrial dysfunction. This article describes the original discovery, preclinical development, and clinical development of a novel class of small peptide molecules that selectively target the inner mitochondrial membrane and protect mitochondrial function. These compounds have the potential to be a paradigm-shifting approach to the treatment of mitochondrial dysfunction, which underlies many common diseases, including cardiorenal, neurologic, and metabolic disorders.
Keywords: ischemia-reperfusion injury; metabolic disorders; neurodegenerative diseases; oxidative stress; Szeto-Schiller peptides
Hormetics: Dietary Triggers of an Adaptive Stress Response by Marc Birringer (2680-2694).
A series of dietary ingredients and metabolites are able to induce an adaptive stress response either by generation of reactive oxygen species (ROS) and/or via activation of the Nrf2/Keap1 stress response network. Most of the molecules belong to activated Michael acceptors, electrophiles capable to S-alkylate redox sensitive cysteine thiols. This review summarizes recent advances in the (re)search of these compounds and classifies them into distinct groups. More than 60 molecules are described that induce the Nrf2 network, most of them found in our daily diet. Although known as typical antioxidants, a closer look reveals that these molecules induce an initial mitochondrial or cytosolic ROS formation and thereby trigger an adaptive stress response and hormesis, respectively. This, however, leads to higher levels of intracellular glutathione and increased expression levels of antioxidant enzymes such as glutathione peroxidase, thioredoxin reductase, and superoxide dismutase. According to this principle, the author suggests the term hormetics to describe these indirect antioxidants.
Keywords: adaptive response; hormesis; hormetics; Nrf2; nutrition; oxidative stress; ROS
Inhibitors of Succinate: Quinone Reductase/Complex II Regulate Production of Mitochondrial Reactive Oxygen Species and Protect Normal Cells from Ischemic Damage but Induce Specific Cancer Cell Death by Stephen J. Ralph; Rafael Moreno-Sánchez; Jiri Neuzil; Sara Rodríguez-Enríquez (2695-2730).
Succinate:quinone reductase (SQR) of Complex II, occupying a unique central point in the mitochondrial respiratory system as a major source of electrons driving reactive oxygen species (ROS) production, is an ideal pharmaceutical target for modulating ROS levels in normal cells to prevent oxidative stress-induced damage or increase ROS in cancer cells, inducing cell death. Value of drugs like diazoxide to prevent ROS production, protecting normal cells, while vit. E analogues promote ROS in cancer cells to kill them, is highlighted. As pharmaceuticals, agents may prevent degenerative disease; their modes of action are being fully explored. Evidence that SDH/Complex II is tightly coupled to NADH/NAD+ ratio in all cells, impacted by available supplies of Krebs cycle intermediates as essential NAD-linked substrates, and NAD+-dependent regulation of SDH/Complex II are reviewed, as are links to NAD+-dependent dehydrogenases, Complex I and E3 dihiydrolipoamide dehydrogenase to produce ROS. We collate and discuss diverse sources of information relating to ROS production in different biological systems, focussing on evidence for SQR as main source of ROS production in mitochondria, particularly its relevance to protection from oxidative stress and to mitochondrial-targeted anticancer drugs (mitocans) as novel cancer therapies.
Keywords: mitocans; mitochondria; ROS production; SDH/Complex II; superoxide
Mitochondrial Metabolism Inhibitors for Cancer Therapy by Emma E. Ramsay; Philip J. Hogg; Pierre J. Dilda (2731-2744).
Cancer cells catabolise nutrients in a different way than healthy cells. Healthy cells mainly rely on oxidative phosphorylation, while cancer cells employ aerobic glycolysis. Glucose is the main nutrient catabolised by healthy cells, while cancer cells often depend on catabolism of both glucose and glutamine. A key organelle involved in this altered metabolism is mitochondria. Mitochondria coordinate the catabolism of glucose and glutamine across the cancer cell. Targeting mitochondrial metabolism in cancer cells has potential for the treatment of this disease. Perhaps the most promising target is the hexokinase-voltage dependent anion channel-adenine nucleotide translocase complex that spans the outer- and inner-mitochondrial membranes. This complex links glycolysis, oxidative phosphorylation and mitochondrial-mediated apoptosis in cancer cells. This review discusses cancer cell mitochondrial metabolism and the small molecule inhibitors of this metabolism that are in pre-clinical or clinical development.
Keywords: adenine nucleotide translocase; cancer; hexokinase; metabolism; mitochondria
Quantitative Glucose and ATP Sensing in Mammalian Cells by Dania C. Liemburg-Apers; Hiromi Imamura; Marleen Forkink; Marco Nooteboom; Herman G. Swarts; Roland Brock; Jan A. M. Smeitink; Peter H. G. M. Willems; Werner J. H. Koopman (2745-2757).
The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic dysfunction plays an important role in many human diseases, including diabetes, cancer, inherited mitochondrial disorders, and metabolic syndrome. The monosaccharide glucose constitutes a key source of cellular energy. Following its import across the plasma membrane, glucose is converted into pyruvate by the glycolysis pathway. Pyruvate oxidation supplies substrates for the ATP-generating mitochondrial oxidative phosphorylation (OXPHOS) system. To gain cell-biochemical knowledge about the operation and regulation of the cellular energy metabolism in the healthy and diseased state, quantitative knowledge is required about (changes in) metabolite concentrations under (non) steady-state conditions. This information can, for instance, be used to construct more realistic in silico models of cell metabolism, which facilitates understanding the consequences of metabolic dysfunction as well as on- and off-target effects of mitochondrial drugs. Here we review the current state-of-the-art live-cell quantification of two key cellular metabolites, glucose and ATP, using protein-based sensors. The latter apply the principle of FRET (fluorescence resonance energy transfer) and allow measurements in different cell compartments by fluorescence microscopy. We further summarize the properties and applications of the FRET-based sensors, their calibration, pitfalls, and future perspectives.
Keywords: ATeam; fibroblast; GLUT; systems biology
Mitochondria and Trypanosomatids: Targets and Drugs by Lianet Monzote Fidalgo; Lars Gille (2758-2770).
The family Trypanosomatidae, flagellated parasitic protozoa, is responsible for important infectious diseases in humans: sleeping sickness, Chagas diseases and leishmaniasis. Currently, development of effective vaccines against these parasites remains an unrealized goal, and clinical management is based on chemotherapeutics. Cost, toxicity and resistance problems of conventional drugs result in an urgent need to identify and develop new therapeutic alternatives. The sound understanding of parasites, biology is key for identifying novel lead structures and new drug targets. This article reviews current knowledge about mitochondrial drug targets and existing drugs against Trypanosoma and Leishmania. In the past, several targets in trypanosomatid mitochondria (electron transport chain, kDNA and topoisomerases, tRNA import and fatty acid synthesis) have been identified. It has been suggested that inhibition of certain targets is involved in triggering apoptosis by impairment of mitochondrial membrane potential and/or production of reactive oxygen species. The inhibitory mechanism of approved drugs, such as pentamidine, nifurtimox, artemisinin and atovaquone, is described in parallel with others products from preclinical studies. In spite of the large amount of genetic information, the analysis of the phenotype of the trypanosomatid mitochondrion in different life stages will remain a useful tool to design new active compounds with selective toxicity against these parasites.
Keywords: drug; Leishmania ; mitochondria; target; Trypanosoma
Strategies to Target Mitochondria and Oxidative Stress by Antioxidants: Key Points and Perspectives by Marvin Edeas (2771-2779).
For several decades, many antioxidants studies have emphasized the marked disparity between the beneficial effect of the antioxidants shown in preclinical studies and their inability to show beneficial effects in clinical trials. Besides, it is not uncommon to find highly contradictory clinical results, which may explain why consumers are less enthusiastic for antioxidant uses. This perspective article aims to highlights the critical role of Reactive Oxygen Species (ROS) and antioxidants, the potential mechanisms that might account for these discrepancies in clinical trials and some strategies to target oxidative stress and mitochondria by antioxidants. We need urgently to set up standard methods to evaluate antioxidants and oxidative stress in human and in particular at mitochondria level. The determination of what the basal level of ROS is in normal human may be used to identify pathologic ROS levels in patients and ultimately guide antioxidants treatment.
Keywords: antioxidant; mitochondria; oxidative stress; standardization methods
Multiple Triphenylphosphonium Cations as a Platform for the Delivery of a Pro-Apoptotic Peptide by Netanel Kolevzon; Uriel Kuflik; Miriam Shmuel; Sandrine Benhamron; Israel Ringel; Eylon Yavin (2780-2789).
Triphenyl phosphonium cations (TPPs) are delocalized lipophilic cations that accumulate in the mitochondria of cells. We have explore the effect of increasing the number of TPPs on delivery of a cell-impermeable pro-apoptotic peptide to intact cells.The pro-apoptotic peptide D-(KLAKLAK)2 (KLA) was extended with 0–3 L-Lysines modified at their ε-amine with TPP. Peptides were studied in HeLa cells to determine their cytotoxic activity and cellular uptake.In HeLa cells, the increased cytotoxicity correlates with the number of TPPs; the peptide with 3 TPP molecules (3-KLA) exerts the highest cytotoxic activity. This FITC-labeled peptide is found to accumulate in intact HeLa cells, whereas peptides with 0–2 TPPs are not detected at the same peptide concentration. Mitochondria-dependent apoptosis of HeLa cells in the presence of 3-KLA was followed by propidium iodide, Annexin-V and DiOC fluorescence by FACS.A facile synthetic methodology has been presented for the delivery of a biologically active peptide into mitochondria of intact cells by attaching multiple TPP moieties to the peptide. This approach was shown to dramatically increase biological activity of the peptide as a pro-apoptotic agent.
Keywords: antimicrobial peptide; apoptosis; mitochondria; tripenylphosphonium
The Utility of an Isolated Mitochondrial Fraction in the Preparation of Liposomes for the Specific Delivery of Bioactives to Mitochondria in Live Mammalian Cells by Mayura A. Wagle; Laura E. Martinville; Gerard G. M. D’Souza (2790-2796).
To develop and evaluate liposomal formulations prepared with an isolated mitochondrial fraction as a mitochondria-specific delivery vehicleLiposomes were prepared with either a crude mitochondrial fraction extracted from cells or lipids extracted from the crude mitochondrial fraction and were then characterized by determining their size and zeta potential. The cell uptake of the liposomes and loaded bioactive was studied using flow cytometry and confocal microscopy. The cytotoxicity of the formulations was tested by MTS cytotoxicity assay.Liposomes prepared with the mitochondrial extracts were non-toxic and colocalized with mitochondria in F98 cells. Addition of DOTAP to the liposomes facilitated DNA complexation and the DNA delivered intracellularly co-localized with mitochondria.The results from this study establish the potential of using a mitochondrial fraction isolated from cells to prepare liposomes capable of delivering biologically active molecules to mitochondria of live mammalian cells.
Keywords: DNA delivery; liposomes; mitochondria; sub-cellular; targeting
Dimeric Cationic Amphiphilic Polyproline Helices for Mitochondrial Targeting by Iris M. Geisler; Jean Chmielewski (2797-2807).
Efficient delivery of therapeutic biopolymers across cell membranes remains a daunting challenge. The development of cell-penetrating peptides (CPPs) has been useful; however, many CPPs are found trapped within endosomes, limiting their use as delivery agents. We optimize a class of CPPs, cationic amphiphilic polyproline helices (CAPHs), for direct transport into cells with mitochondrial localization through dimerization.The CAPH P11LRR used for this study has been found to enter cells by two distinct pathways: an endocytotic pathway was favored at low concentrations; internalization by direct transport was observed at higher concentrations. CAPH was dimerized to probe if direct transport within cells may be enhanced through increased association of CAPH with the membrane and through the association of individual peptides within the membrane.The dimerization of the CAPH was found to significantly increase cellular uptake over its monomeric counterpart, with a concomitant lowering of the concentration threshold favoring direct transport. Evidence for direct transport within cells and mitochondrial localization was observed.CAPH cellular uptake efficiency can be significantly enhanced through peptide dimerization while favoring cell entry via direct transport at low concentration with low cell toxicity.
Keywords: cationic amphiphilic polyproline helices; cell penetrating peptides; dimerization; mitochondrial targeting
Development of Novel Peptides for Mitochondrial Drug Delivery: Amino Acids Featuring Delocalized Lipophilic Cations by Shana O. Kelley; Kelly M. Stewart; Rida Mourtada (2808-2819).
To create a new class of mitochondria-penetrating peptides (MPPs) that would facilitate drug delivery into the organelle through the inclusion of delocalized lipophilic cations (DLCs) in the peptide sequence.We synthesized two novel amino acids featuring DLCs and incorporated them into peptides. Systematic studies were conducted to compare peptides containing these residues to those with natural cationic amino acids. Diastereomers were compared to determine the most advantageous arrangement for these peptides. Peptide lipophilicity, cellular uptake and mitochondrial specificity were compared for a variety of peptides.Synthetic DLC residues were found to increase mitochondrial localization of MPPs due to higher overall hydrophobicity. MPP stereochemistry was important for cellular uptake rather than subcellular localization. This study reaffirmed the importance of uniform overall charge distribution for mitochondrial specificity.DLCs can be incorporated into synthetic peptides and facilitate mitochondrial drug delivery. Lipophilicity and charge distribution must be carefully balanced to ensure localization within mitochondria.
Keywords: cell-penetrating peptides; drug delivery; mitochondria; mitochondria-penetrating peptides
New Fluorescent Probes Targeting the Mitochondrial-Located Translocator Protein 18 kDa (TSPO) as Activated Microglia Imaging Agents by Nunzio Denora; Valentino Laquintana; Adriana Trapani; Hiromi Suzuki; Makoto Sawada; Giuseppe Trapani (2820-2832).
To evaluate the utility of new Translocator protein 18 kDa (TSPO)-targeted fluorescent probes for in vivo molecular imaging of activated microglia.Compounds 2–4 were synthesized; their stability and affinity for TSPO were determined. Compounds 2–4 were incubated both with Ra2 cells in the presence of LPS, a potent activator of microglia, and with tissue sections of normal and chemically injured brains. Compounds 2–4 were injected into carotid artery or directly in striatum of mice. Cells and tissue sections from these in vitro and in vivo studies were observed by fluorescence microscopy after histochemical treatments.Compounds 2–4 are stable in both buffer and physiological medium and showed high affinity for TSPO and were found to stain live Ra2 microglial cells effectively. Double staining with Mito Tracker Red suggested that binding sites of compounds 2 and 3 may exist on mitochondria. In vivo studies showed that compounds 2–4 may penetrate in part into brain; moreover, cells in mouse striatum were stained with compounds 2–4 and microglial marker CD11b.Compounds 2–4 can fluorescently label activated microglia in vitro and in vivo.
Keywords: activated microglia imaging; fluorescence microscopy.; imidazopyridine-compounds; TSPO
Brain Mitochondrial Drug Delivery: Influence of Drug Physicochemical Properties by Shelley A. Durazo; Rajendra S. Kadam; Derek Drechsel; Manisha Patel; Uday B. Kompella (2833-2847).
To determine the influence of drug physicochemical properties on brain mitochondrial delivery of 20 drugs at physiological pH.The delivery of 8 cationic drugs (beta-blockers), 6 neutral drugs (corticosteroids), and 6 anionic drugs (non-steroidal anti-inflammatory drugs, NSAIDs) to isolated rat brain mitochondria was determined with and without membrane depolarization. Multiple linear regression was used to determine whether lipophilicity (Log D), charge, polarizability, polar surface area (PSA), and molecular weight influence mitochondrial delivery.The Log D for beta-blockers, corticosteroids, and NSAIDs was in the range of −1.41 to 1.37, 0.72 to 2.97, and −0.98 to 2, respectively. The % mitochondrial uptake increased exponentially with an increase in Log D for each class of drugs, with the uptake at a given lipophilicity obeying the rank order cationic>anionic>neutral. Valinomycin reduced membrane potential and the delivery of positively charged propranolol and betaxolol. The best equation for the combined data set was Log % Uptake = 0.333 Log D + 0.157 Charge – 0.887 Log PSA + 2.032 (R2 = 0.738).Drug lipopohilicity, charge, and polar surface area and membrane potential influence mitochondrial drug delivery, with the uptake of positively charged, lipophilic molecules being the most efficient.
Keywords: brain delivery; lipophilicity; membrane potential; mitochondrial delivery; polar surface area
Expression of GFP in the Mitochondrial Compartment Using DQAsome-Mediated Delivery of an Artificial Mini-mitochondrial Genome by Diana Lyrawati; Alan Trounson; David Cram (2848-2862).
We describe a novel strategy for expression of GFP in mammalian mitochondria.The key components of the strategy were an artificially created mitochondrial genome pmtGFP and a DQAsome transfection system.Using immunofluorescence and a combination of immunohistochemical and molecular based techniques, we show that DQAsomes are capable of delivering the pmtGFP construct to the mitochondrial compartment of the mouse macrophage cell line RAW264.7, albeit at low efficiency (1–5%), resulting in the expression of GFP mRNA and protein. Similar transfection efficiencies were also demonstrated in a range of other mammalian cell lines.The DQAsome-transfection technique was able to deliver the exogenous DNA into the cellular mitochondria and the pmtGFP was functional. Further optimization of this strategy would provide a flexible and rapid way to generate mutant cells and useful animal models of mitochondrial disease.
Keywords: DQAsome; GFP expression; mini-mitochondrial genome; mitochondrial gene delivery; transfection
Trial and Error: How the Unclonable Human Mitochondrial Genome was Cloned in Yeast by Brian W. Bigger; Ai-Yin Liao; Ana Sergijenko; Charles Coutelle (2863-2870).
Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNAThr gene.We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination.Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNAThr were both stable and unmutated.This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.
Keywords: gene therapy; human mitochondrial DNA; mitochondrial disease; unclonable; yeast
DNA Delivery to Mitochondria: Sequence Specificity and Energy Enhancement by Noha Ibrahim; Hirokazu Handa; Anne Cosset; Milana Koulintchenko; Yuri Konstantinov; Robert N. Lightowlers; André Dietrich; Frédérique Weber-Lotfi (2871-2882).
Mitochondria are competent for DNA uptake in vitro, a mechanism which may support delivery of therapeutic DNA to complement organelle DNA mutations. We document here key aspects of the DNA import process, so as to further lay the ground for mitochondrial transfection in intact cells.We developed DNA import assays with isolated mitochondria from different organisms, using DNA substrates of various sequences and sizes. Further import experiments investigated the possible role of ATP and protein phosphorylation in the uptake process. The fate of adenine nucleotides and the formation of phosphorylated proteins were analyzed.We demonstrate that the efficiency of mitochondrial uptake depends on the sequence of the DNA to be translocated. The process becomes sequence-selective for large DNA substrates. Assays run with a natural mitochondrial plasmid identified sequence elements which promote organellar uptake. ATP enhances DNA import and allows tight integration of the exogenous DNA into mitochondrial nucleoids. ATP hydrolysis has to occur during the DNA uptake process and might trigger phosphorylation of co-factors.Our data contribute critical information to optimize DNA delivery into mitochondria and open the prospect of targeting whole mitochondrial genomes or complex constructs into mammalian organelles in vitro and in vivo.
Keywords: DNA import; mitochondrial disease; mitochondrial plasmid; organelle transfection; protein phosphorylation
Novel Mitochondria-Targeted Antioxidants: Plastoquinone Conjugated with Cationic Plant Alkaloids Berberine and Palmatine by Konstantin G. Lyamzaev; Antonina V. Pustovidko; Ruben A. Simonyan; Tatyana I. Rokitskaya; Lidia V. Domnina; Olga Yu. Ivanova; Inna I. Severina; Natalia V. Sumbatyan; Galina A. Korshunova; Vadim N. Tashlitsky; Vitaly A. Roginsky; Yuriy N. Antonenko; Maxim V. Skulachev; Boris V. Chernyak; Vladimir P. Skulachev (2883-2895).
To develop effective mitochondria-targeted antioxidants composed entirely of natural constituents.Novel mitochondria-targeted antioxidants were synthesized containing plant electron carrier and antioxidant plastoquinone conjugated by nonyloxycarbonylmethyl residue with berberine or palmatine, penetrating cations of plant origin. These compounds, SkQBerb and SkQPalm, were tested in model planar phospholipid membranes and micelles, liposomes, isolated mitochondria and living cells.SkQBerb and SkQPalm penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in mitochondria isolated or in living human cells in culture. Reduced forms of SkQBerb and SkQPalm as well as C10Berb and C10Palm (SkQBerb and SkQPalm analogs lacking plastoquinol moiety) revealed radical scavenging activity in lipid micelles and liposomes, while oxidized forms were inactive. In isolated mitochondria and in living cells, berberine and palmatine moieties were not reduced, so antioxidant activity of C10Berb and C10Palm was not detected. SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations; their prooxidant effect was observed at 1,000 times higher concentrations. In human cell cuture, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide.This is the first successful attempt to construct mitochondria-targeted antioxidants composed entirely of natural components, namely plastoquinone, nonyl, acetyl and berberine or palmatine residues.
Keywords: antioxidant; berberine; mitochondria; palmatine; plastoquinone; SkQ
An Acetate Prodrug of a Pyridinol-Based Vitamin E Analogue by Omar M. Khdour; Jun Lu; Sidney M. Hecht (2896-2909).
To investigate of an approach to stabilize a novel pyridinol based α–tocopherol analogue (1) as a prodrug by acetylation of its phenol moiety.Biochemical indicators of oxidative stress in mitochondria were utilized to gain insight into the cytoprotective mechanism(s) of compound 1 acetate. Oxygen free radical scavenging activity was measured using DCF probe in a cultured cell model system that had been placed under oxidative stress. Lipid peroxidation was examined both in a cell-free system and in oxidatively stressed cultured cells. The bioenergetic parameters of mitochondria were evaluated by measuring mitochondrial membrane potential (Δψm) and the MPT.The present results suggest strongly that the antioxidant efficacy of compound 1 can be improved by using it as a prodrug. The tested prodrug has shown to be activated as a function of time, presumably due to susceptibility to enzymatic hydrolysis, and exhibits an antioxidant effect in time-dependent manner, providing a compound that is more effective than α-tocopherol acetate with regard to all protective properties studied.An effective approach to stabilize compound 1 was realized by using its acetate as a prodrug.
Keywords: analogue; antioxidant; prodrug; pyridinol; reactive oxygen species (ROS); stability; α-tocopherol acetate
Mitochondrial Antioxidants Alleviate Oxidative and Nitrosative Stress in a Cellular Model of Sepsis by Nadezda Apostolova; Remedios Garcia-Bou; Antonio Hernandez-Mijares; Raul Herance; Milagros Rocha; Victor M. Victor (2910-2919).
Mitochondrial dysfunction plays a key role in sepsis.We used a sepsis model of human endothelial cells (HUVEC) to study mitochondrial function during normoxic (21% O2) and hypoxic (1% O2) conditions.When stimulated with a LPS cocktail, HUVEC displayed an increase of nitric oxide (NO) in normoxic and hipoxic conditions, being higher at 21% O2. LPS-activation for 24 h at 1% O2 increased ROS production, which was reversed with the mitochondrial antioxidant Mitoquinone (MQ) and Glutathione Ethyl Ester (GEE). Activated cells displayed diminished mitochondrial O2 consumption with specific inhibition of Complex I, accompanied by increase in tyrosine nitration and Type II NOS protein expression, effects which were recovered by antioxidants and/or with L-NAME. These parameters varied with O2 environment, namely inhibition of respiration observed in both O2 environments at 24 h was very similar, whereas O2 consumption rate fell earlier in 1% O2-exposed cells. While no significant differences were detected at earlier time points, at 24 h tyrosine nitration was higher in normoxic vs. hypoxic cells.Mitochondria are heavily implicated in sepsis. Mitochondrial antioxidants provide a mechanistic model for the development of potential therapies.
Keywords: hypoxia; mitochondria; mitoquinone; reactive oxygen species; sepsis
Time-Correlated Single Photon Counting For Simultaneous Monitoring Of Zinc Oxide Nanoparticles And NAD(P)H In Intact And Barrier-Disrupted Volunteer Skin by Lynlee L. Lin; Jeffrey E. Grice; Margaret K. Butler; Andrei V. Zvyagin; Wolfgang Becker; Thomas A. Robertson; H. Peter Soyer; Michael S. Roberts; Tarl W. Prow (2920-2930).
There is a lack of relevant, non-animal alternatives for assessing exposure and toxicity of nanoparticle-containing cosmetics, e.g. sunscreens. Our goal was to evaluate timecorrelated single photon counting (TCSPC) for simultaneous monitoring of zinc oxide nanoparticles (ZnO-NP) and the metabolic state of volunteer skin.We separated the fluorescence lifetime signatures of endogenous fluorophore signals (i.e. nicotinamide adenine dinucleotide phosphate, NAD(P)H and keratin) and the ZnO-NP signal using advanced TCSPC to simultaneously determine ZnO-NP penetration profiles and NAD(P)H changes in subjects with altered barrier function, including tape-stripped skin and in psoriasis or atopic dermatitis lesions.We detected no ZnO-NP penetration into viable human skin in any group. ZnO-NP signal was significantly increased (p < 0.01) on the surface of tape-stripped and lesional skin after 4 and 2 h of treatment, respectively. Free NAD(P)H signal significantly increased in tape-stripped viable epidermis treated for 4 h of ZnO-NP compared to vehicle control. No significant NAD(P)H changes were noted in the lesional study.TCSPC techniques enabled simultaneous, real-time quantification of ZnO-NP concentration and NAD(P)H via non-invasive imaging in the stratum corneum and viable epidermis of volunteers.
Keywords: human skin; metabolism; multiphoton microscopy; sunscreen; zinc oxide nanoparticle
Gold Nanoparticle Penetration and Reduced Metabolism in Human Skin by Toluene by Hagar I. Labouta; David C. Liu; Lynlee L. Lin; Margaret K. Butler; Jeffrey E. Grice; Anthony P. Raphael; Tobias Kraus; Labiba K. El-Khordagui; H. Peter Soyer; Michael S. Roberts; Marc Schneider; Tarl W. Prow (2931-2944).
To measure penetration and metabolic effects of ion-stabilized, polar, 15 nm gold nanoparticles in aqueous solution (AuNP-Aq) and sterically stabilized, non-polar, 6 nm gold nanoparticles in toluene (AuNP-TOL) on excised human skin.Gold nanoparticles were characterized with dynamic light scattering and transmission electron microscopy (TEM). Skin penetration studies were done on frozen or fresh excised skin using static Franz diffusion cells. Viable treated skin was assessed by dermoscopy, reflectance confocal microscopy (RCM), multiphoton tomography (MPT) with fluorescence lifetime imaging microscopy (FLIM), and TEM.Dermoscopy and RCM showed large aggregates in the furrows of AuNP-Aq-treated skin. Treatment of thawed and viable skin only showed enhanced permeability to nanoparticles in the AuNP-TOL group with MPT and FLIM imaging to stratum spinosum of epidermis. TEM analysis revealed gold nanoparticles within AuNP-treated stratum corneum. FLIM analysis of NAD(P)H showed a significant decrease in total NAD(P)H in all toluene-treated groups.Gold nanoparticles, 15 nm, in aqueous solution aggregated on the skin surface. Toluene treatment eliminated skin metabolism; skin treated with toluene/gold nanoparticles (6 nm) for 24 h, but not at 4 h, showed increased nanoparticle permeability. These results are of value to nanotoxicology.
Keywords: confocal reflectance microscopy; fluorescence lifetime; multiphoton microscopy; nanoparticle; skin
Improvement of Cerebral Metabolism Mediated by Ro5-4864 is Associated with Relief of Intracranial Pressure and Mitochondrial Protective Effect in Experimental Brain Injury by Jean F. Soustiel; Eugene Vlodavsky; Felix Milman; Moshe Gavish; Menashe Zaaroor (2945-2953).
To investigate the possible impact of reduction of mitochondrial membrane permeabilization by modulation of the 18 kDa translocator protein mediated by Ro5-4864 over post-traumatic cerebral edema and metabolic crisis.Cerebral microdialysis and intracranial pressure (ICP) monitoring were performed in Sprague–Dawley rats treated by intraperitoneal injection of either dimethylsulfoxide (vehicle) or Ro5-4864 following cortical contusion and further correlated with quantitative assessment of mitochondrial damage, water content in the injured tissue, modified neurological severity score, and lesion size.Ro5-4864 resulted in a profound decrease in ICP that correlated with improved cerebral metabolism characterized by significantly higher glucose and pyruvate and lower lactate concentrations in the pericontusional area in comparison with vehicle-treated animals. Reduced ICP correlated with reduced water content in the injured tissue; improved metabolism was associated with reduced mitochondrial damage evidenced by electron microscopy. Both effects were associated with a profound and significant reduction in glycerol release and lesion size, and correlated with improved neurological recovery.The present study shows that Ro5-4864 has a favorable effect on the fate of injured brain, presumably mediated by improvement of metabolism. It further suggests that improvement of metabolism may contribute to ICP relief.
Keywords: 18 kDa translocator protein; intracranial pressure; microdialysis; mitochondria; traumatic brain injury
A Clinician’s Commentary: Mitochondria and Revolution by Stephen P. Hersh (2954-2957).
Keywords: energy; evolution; mitochondria; revolution; timing
AAPS Connection (2958-2960).