Cell-Penetrating Peptides for Mitochondrial Targeting Carmine Pasquale Cerrato Academic dissertation for the Degree of Doctor of Philosophy in Neurochemistry with Molecular Neurobiology at Stockholm University to be publicly defended on Friday 1 June 2018 at 10.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B. Abstract Mitochondria have simply been known as the cell’s powerhouse for a long time, with its vital function of producing ATP. However, substantially more attention was directed towards these organelles once they were recognized to perform several essential functions having an impact in cell biology, pharmaceutics and medicine. Dysfunctions of these organelles have been linked to several diseases such as diabetes, cancer, neurodegenerative diseases and cardiovascular disorders. Mitochondrial medicine emerged once the relationship of reactive oxygen species and mutations of the mitochondrial DNA linked to diseases was shown, referred to as mitochondrial dysfunction. This has led to the need to deliver therapeutic molecules in their active form not only to the target cells but more importantly into the targeted organelles. In this thesis, cell-penetrating peptides (CPPs) used as mitochondrial drug delivery system and the pathways involved in the uptake mechanisms of a CPP are described. In particular, Paper I describes a novel cell-penetrating peptide targeting mitochondria with intrinsic antioxidant properties. Paper II expands upon this first finding and show that the same peptide can carry a glutathione analogue peptide with improved radical scavenging ability into cytoplasm and mitochondria. Paper III introduces mitochondrial targeting peptides for delivery of therapeutic biomolecules to modify mitochondrial gene expression. In Paper IV, the uptake mechanisms of the CPP delivery strategy has been investigated to gain a better understanding of the used transfection system. Overall, this thesis summarizes our current effort regarding cell-penetrating peptides delivery system to target mitochondria and the progress made towards a potential gene therapy. It contributes to the field of CPPs and drug delivery with a set of peptides with radical scavenging ability, a strategy to deliver oligonucleotides to mitochondria as proof-of- concept for mitochondrial gene therapy, and to help understanding the pathways involved in CPPs uptake. Keywords: Mitochondrial targeting, cell-penetrating peptides, antioxidant activity, scavenging ability, oligonucleotide delivery. Stockholm 2018 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-155156 ISBN 978-91-7797-230-3 ISBN 978-91-7797-231-0 Department of Biochemistry and Biophysics Stockholm University, 106 91 Stockholm
Transcript
Cell-Penetrating Peptides for MitochondrialTargetingCarmine Pasquale Cerrato
Academic dissertation for the Degree of Doctor of Philosophy in Neurochemistry withMolecular Neurobiology at Stockholm University to be publicly defended on Friday 1 June 2018at 10.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B.
AbstractMitochondria have simply been known as the cell’s powerhouse for a long time, with its vital function of producing
ATP. However, substantially more attention was directed towards these organelles once they were recognized to performseveral essential functions having an impact in cell biology, pharmaceutics and medicine. Dysfunctions of these organelleshave been linked to several diseases such as diabetes, cancer, neurodegenerative diseases and cardiovascular disorders.Mitochondrial medicine emerged once the relationship of reactive oxygen species and mutations of the mitochondrial DNAlinked to diseases was shown, referred to as mitochondrial dysfunction. This has led to the need to deliver therapeuticmolecules in their active form not only to the target cells but more importantly into the targeted organelles.
In this thesis, cell-penetrating peptides (CPPs) used as mitochondrial drug delivery system and the pathways involvedin the uptake mechanisms of a CPP are described. In particular, Paper I describes a novel cell-penetrating peptide targetingmitochondria with intrinsic antioxidant properties. Paper II expands upon this first finding and show that the same peptidecan carry a glutathione analogue peptide with improved radical scavenging ability into cytoplasm and mitochondria.Paper III introduces mitochondrial targeting peptides for delivery of therapeutic biomolecules to modify mitochondrialgene expression. In Paper IV, the uptake mechanisms of the CPP delivery strategy has been investigated to gain a betterunderstanding of the used transfection system.
Overall, this thesis summarizes our current effort regarding cell-penetrating peptides delivery system to targetmitochondria and the progress made towards a potential gene therapy. It contributes to the field of CPPs and drug deliverywith a set of peptides with radical scavenging ability, a strategy to deliver oligonucleotides to mitochondria as proof-of-concept for mitochondrial gene therapy, and to help understanding the pathways involved in CPPs uptake.
To my parents "Life does not end with death.What you pass on to othersremains. Immortality is not thebody, which will one day die.That does not matter… ofimportance is the message youleave to others. That isimmortality." Rita Levi-Montalcini
Department of Biochemistry and Biophysics, Stockholm University,Stockholm, Sweden Academic dissertation for the Degree of Doctor of Philosophy inNeurochemistry with Molecular Neurobiology at Stockholm University. SupervisorÜlo Langel, ProfessorDepartment of Biochemistry and BiophysicsStockholm University, Stockholm, Sweden Co-supervisorAnders Undén, Associate ProfessorDepartment of Biochemistry and BiophysicsStockholm University, Stockholm, Sweden Committee membersPontus Aspenström, Professor of Molecular Cell BiologyHead of Department of Microbiology, Tumor and Cell BiologyKarolinska Institutet, Stockholm, Sweden Ulf Göransson, Professor of PharmacognosyDepartment of Medicinal Chemistry, PharmacognosyUppsala Biomedicinska CentrumUppsala University, Uppsala, Sweden Amelie Eriksson Karlström, Professor of Molecular BiotechnologyDeputy Head of School of Engineering Sciences in Chemistry,Biotechnology, and HealthKTH Royal Institute of Technology, Stockholm, Sweden Reserve Committe memberDaniel Daley, Associate Professor Departement of Biochemistry and BiophysicsStockholm University, Stockholm, Sweden OpponentShana O. Kelley, Distinguished ProfessorFaculty of Arts and Sciences, Leslie Dan Faculty of Pharmacy, Faculty ofMedicine, Faculty of Engineering University of Toronto, Toronto, ON, Canada
I
Abstract
Mitochondria have simply been known as the cell’s powerhouse for a long time, with its vital function of producing ATP. However, substantially more attention was directed towards these organelles once they were recognized to perform several essential functions having an impact in cell biology, pharma-ceutics and medicine. Dysfunctions of these organelles have been linked to several diseases such as diabetes, cancer, neurodegenerative diseases and car-diovascular disorders. Mitochondrial medicine emerged once the relationship of reactive oxygen species and mutations of the mitochondrial DNA linked to diseases was shown, referred to as mitochondrial dysfunction. This has led to the need to deliver therapeutic molecules in its active form not only to the target cells but more importantly into the targeted organelles.
In this thesis, cell-penetrating peptides (CPPs) used as mitochondrial drug delivery system and the pathways involved in the uptake mechanisms of a CPP are described. In particular, Paper I describes a novel cell-penetrating peptide targeting mitochondria with intrinsic antioxidant properties. Paper II expands upon this first finding and show that the same peptide can carry a glutathione analogue peptide with improved radical scavenging ability into cytoplasm and mitochondria. Paper III introduces mitochondrial targeting peptides for delivery of therapeutic biomolecules to modify mitochondrial gene expression. In Paper IV, the uptake mechanisms of the CPP delivery strategy has been investigated to gain a better understanding of the used trans-fection system.
Overall, this thesis summarizes our current effort regarding cell-penetrating peptides delivery system to target mitochondria and the progress made to-wards a potential gene therapy. It contributes to the field with a set of peptides with radical scavenging ability, a strategy to deliver oligonucleotides to mito-chondria as proof-of-concept for mitochondrial gene therapy, and to help un-derstanding the pathways involved in CPPs uptake.
III
Populärvetenskaplig Sammanfattning
Mitokondrier har länge varit kända som cellens kraftverk med sin vitala funktion att producera energi. Under de senaste årtionden har man även upp-täckt att dessa organeller spelar en nyckelroll i flera medicinska processer. Forskning har exempelvis visat ett samband mellan mitokondriers dysfunkt-ion och vissa av dagens stora sjukdomar såsom diabetes och cancer, men även neurodegenerativa sjukdomar och hjärt- och kärlsjukdomar. Sambandet är ännu inte helt kartlagt, men bidragande faktorer till detta är bland annat mu-tationer i mitokondriellt DNA och en okontrollerbar halt av reaktiva syrearter. För att kunna justera dessa dysfunktioner finns det ett behov av att kunna le-verera terapeutiska molekyler in i cellerna och selektivt vidare genom dubbel-membranet in till mitokondrier för att rätta till det mitokondriella genuttrycket.
Denna avhandling beskriver utvecklingen av mitokondriella läkemedelsad-ministrationssystem genom användandet av cell-penetrerande peptider (CPP). Artikel I beskriver vår design och framställning av en ny CPP-kandidat med anti-oxidantegenskaper som har förmågan att ta sig genom cell-membranerna in till mitokondrier. Artikel II expanderar från den första upptäckten och visar att samma peptid tillsammans med ett glutation-derivat minskar halten av re-aktiva syrearter både i mitokondrierna samt i cellerna. Artikel III introducerar kombinationen av två CPPs med resulterande förmåga att kunna leverera te-rapeutiska biomolekyler in i mitokondrier för att kunna modifiera det mitokondriella genuttrycket. Artikel IV skildrar mekanismen för hur CPPs med eller utan terapeutiska biomolekyler tas upp och omsätts av cellerna.
Sammanfattningsvis återger denna avhandling vårt arbete inom forsknings-området cell-penetrerande peptider med fokus mot mitokondrier. Resultaten visar de framsteg som gjorts för utvecklingen av peptider med förmågan att ta sig in i mitokondrier och leverera terapeutiska biomolekyler, vilket öppnar upp för en framtida potentiell mitokondriell genterapi.
V
List of Publications
This thesis is based on the following four publications, in the text referred to as Paper I, II, III and IV, respectively.
I. Cerrato, C. P., Pirisinu, M., Vlachos, E. N. & Langel, Ü. Novel cell-penetrating peptide targeting mitochondria. FASEB J. 29, 4589–4599(2015).
II. Cerrato, C. P., Langel, Ü. Effect of a fusion peptide by covalent con-jugation of a mitochondria cell-penetrating peptide and glutathione an-alog peptide. Mol. Ther. Methods Clin. Dev. 5, 221-231 (2017).
III. Cerrato, C. P., Kivijärvi, T., Tozzi, R., Langel, Ü. Peptides targetingmitochondria for efficient delivery of therapeutic biomolecules. Manu-script (2018).
IV. Dowaidar, M., Gestin, M., Cerrato, C. P., Jafferali, M. H., Margus, H.,Kivistik, P. A., Ezzat, K., Hallberg, E., Pooga, M., Hällbrink, M., Langel,Ü. Role of autophagy in cell-penetrating peptide transfection model. Sci.Rep. 7, 1-14 (2017)
VI
Additional Publications
Publications not included in this thesis:
V. Lehto, T., Veiman, K-L., Margus, H., Cerrato, C. P., Pooga, M., Häll-brink, M., Langel, Ü. On the effects of changing the N-terminal carbon chain length of cell-penetrating peptide, PepFect14, in non-covalent plasmid delivery in vitro and in vivo. Manuscript (2018).
VI. Gestin, M., Cerrato, C. P., Dowaidar, M., Venit, T., Percipalle, P., Langel, Ü. Influence of the particle size of cell-penetrating peptides on the signaling pathways of the uptake. Manuscript (2018).
VII. Yap, J. L. Y., Tai, Y. K., Fröhlich, J., Pelczar, P., Beyer, C., Fong, C. H. H., Purnamawati, K., Casarosa, M., Cerrato, C. P., Yin, J. N., Ramanan S., Selvan, R. M. P., Bharathy, N., Degirmenci, U., Kala, M. P., Richards, P. J., Mirsaidi A., Xuan, T. G. R., Taneja, R., Egli, M., Wuertz-Kozak, K., Ferguson, S. J., Aguzzi, A., Monici, M., Drum, C. L., Sun, L., Lee, C. N., Franco-Obregón, A. Ambient and Exogenous Magnetic Fields Mod-ulate Myogenesis by Targeting TRPC1. Proc. Natl. Acad. Sci. USA. Sub-mitted (2018).
VIII. Cerrato C. P., Langel, Ü. Cell-Penetrating Peptides Targeting Mito-chondria. In: Oliveira P. (eds.) Mitochondrial Biology and Experimental Therapeutics. Springer Nature, Cham, pp 593-611 (2018).
IX. Cerrato, C. P., Künnapuu, K., Langel, Ü. Cell-penetrating peptides with intracellular organelles targeting. Expert Opin. Drug Deliv. 2, 1-11 (2016).
X. Cerrato, C. P., Veiman, K-L., Langel, Ü. Advances in peptide delivery. In: Kruger, H. G., Albericio, F. (eds.) Advances in the discovery and development of peptide therapeutics. Future Science, Future Science Group, London, pp 160-171 (2016).
XI. Cerrato, C. P., Lehto, T., Langel, Ü. Peptide-based vectors: recent de-velopments. Biomol. Concepts. 5, 479-88 (2014).
VII
XII. Cerrato, C. P., Cialdai, F., Sereni, F., Monici M. Effects of low fre-quency electromagnetic fields on SHSY5Y cells: a neuroblast model. En-ergy for Health. 8, 18-24 (2011).
Patent application
Langel Ü., Kurrikoff K., Cerrato C. P. Method of delivery of nucleic acid cargo into mammalian mitochondria. U.S. Patent. Filed March 15, 2018. No. US62/643,209 (2018).
Paper I in this thesis has previously been included in my licentiate thesis. ISBN 978-91-7649-312-0.
VIII
Table of Contents
Abstract ............................................................................................................ I
Populärvetenskaplig Sammanfattning ........................................................... III
List of Publications ......................................................................................... V
Additional Publications .................................................................................. VI
List of Figures ................................................................................................. X
List of Tables ................................................................................................. XI
Abbreviations ............................................................................................... XII
Acknowledgments ....................................................................................... XIV
1.1.1. Origin and Structure of Mitochondria ....................................................... 1 1.1.2. Functions and Dysfunctions of Mitochondria ............................................ 3 1.1.3. Mitochondria as a Target for Drug Discovery ........................................... 6 1.1.4. Agents Targeting Mitochondria ................................................................ 6
2. Aims ..................................................................................................... 27 2.1. Paper I ........................................................................................................ 27 2.2. Paper II ....................................................................................................... 28 2.3. Paper III ...................................................................................................... 28
IX
2.4. Paper IV ...................................................................................................... 28
3. Methods ............................................................................................... 29 3.1. Solid Phase Peptide Synthesis ................................................................... 293.2. Cell Cultures and Treatment ....................................................................... 323.3. Cell Viability Assays .................................................................................... 323.4. Isolation of Mitochondria ............................................................................. 333.5. Mitochondrial Membrane Potential Assay ................................................... 333.6. Determination Assay for Reactive Oxygen Species .................................... 343.7. Dynamic Light Scattering ............................................................................ 343.8. Circular Dichroism Spectroscopy ................................................................ 353.9. Luciferase ................................................................................................... 363.10. ASO delivery ............................................................................................... 373.11. SCO Delivery .............................................................................................. 373.12. Splice Correction Assay .............................................................................. 373.13. Determination Assay for Adenosine Triphosphate ...................................... 383.14. Fluorescence and Confocal Microscopy ...................................................... 383.15. Transmission Electron Microscopy .............................................................. 393.16. Western Blot Analysis ................................................................................. 39
4. Results and Discussion ....................................................................... 41 4.1. Paper I: mtCPP1, a Cell-Penetrating Peptide Targeting Mitochondria ......... 414.2. Paper II: The Scavenging Ability of mtgCPP for Reactive Oxygen Species. 434.3. Paper III: The Delivery of Therapeutic Biomolecules to Mitochondria .......... 444.4. Paper IV: Cellular Uptake Mechanism and Intracellular Pathway Modulation of CPP-Based Transfection System ............................................................................ 45
5. Concluding Remarks and Future Outlook ........................................... 47
Figure 1. Models for origins of mitochondria and electron micrographs……….2 Figure 2. Schematic representation of a mitochondrion showing the mitochon-
drial permeability transition pore and the respiratory chain…………..4 Figure 3. Schematic representation of a mitochondrion and the mode of action
of representative mitochondria targeting compounds……………….8 Figure 4. Chemical structures of featured mitochondria-targeting agents and
clinical drug candidates…………………………………………….10 Figure 5. Applications of cell-penetrating peptides as molecular delivery vehi-
cles.…………………………………………………………….….13 Figure 6. Statistical representations depicting the distribution of CPPs………14 Figure 7. Uptake and trafficking pathways of CPPs………………………….16 Figure 8. Number of gene therapy trials per year…………………………….22 Figure 9. General scheme of solid phase peptide synthesis…………………...30 Figure 10. Circular dichroism spectra of proteins and peptides with representa-
tive secondary structures…………………………………………36 Figure 11. Schematic presentation for the detection of proteins on the western
blot membrane by enhanced chemiluminescence (ECL)…………39 Figure 12. Structure of mtCPP1……………………………………………..42
XI
List of Tables
Table 1. Systems involved and clinical manifestations in patients with mitochondrial disorders…………………………………………………………………….5
Table 2. List of common reactive oxygen species. ………………………………….6 Table 3. Cell-penetrating peptides commonly used for delivery applications………15 Table 4. Peptide sequences used in this thesis………………………………………31
XII
Abbreviations
AMP antimicrobial peptide ASO antisense oligonucleotide ATP adenosine triphosphate Boc tert-butoxycarbonyl Cbz benzyloxycarbonyl CME clathrin mediated endocytosis COXII cytochrome c oxidase II CPP cell-penetrating peptide CRISPR clustered regularly interspaced short palindromic repeats DLS dynamic light scattering DMEM Dulbecco’s modified Eagle’s medium Dmt 2,6-L-dimethyltyrosine ESI electron spry ionisation EV extracellular vesicle ETC electron transport chain FAM 5(6)-carboxyfluorescein FBS fetal bovine serum FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone Fmoc 9-fluorenylmethyloxycarbonyl GSHPx glutathione peroxidases GSTs glutathione S-transferases HIV human immunodeficiency virus HPLC high performance liquid chromatography IMM inner mitochondrial membrane LHON Leber hereditary optic neuropathy LPL lipoprotein lipase deficiency MALDI-TOF matrix-assisted laser desorption/ionization - time of flight MAP model amphipathic peptide miRNA microRNA
XIII
mitoKATP mitochondrial ATP-regulated K+ channel
MR molar ratio mRNA messenger RNA mtDNA mitochondrial DNA MVBs microvesicular bodiesMTS mitochondrial targeting sequence Mtt 4-methyltritylnDNA nuclear DNAnt nucleotidesOMM outer mitochondrial membraneONs oligonuleotidesOXPHOS oxidative phosphorylation system pDNA plasmid DNA pVEC vascular endothelial-cadherin PTDs protein transduction domains r D-arginine ROS reactive oxygen species RNase H ribonuclease H SCARA scavenger receptor class A SCOs splice correcting oligonucleotides siRNA short interfering RNA SOD superoxide dismutaseTAT transactivator of transcription protein TFA tri-fluoroacetic acid TIM translocase of the inner membrane TIS triisopropylsilane TOM translocase of the outmembrane TPP triphenylphosphonium cation Y L-tyrosine YMe O-methyl-L-tyrosineYa 2,6-dimethyl-L-tyrosineΔΨp plasma membrane potentialΔΨm mitochondrial membrane potentialUCP2 uncoupling protein 2VDAC voltage-dependent anion channelζ potential zeta-potential
XIV
Acknowledgments
I would like to begin by thanking my supervisor, Prof. Ülo Langel for let-ting me be part of his group. Thank you for your advices and support, and for your inspiring way of looking forward. I am thankful for all the opportunities that were given to me, the freedom to choose my own projects and collabora-tors, and for your generosity to allow me to independently pursue my ideas. I have learnt much, directly and indirectly, about science and scientific life from our discussions.
A very big thank goes to the Swedish Research Council for Natural Sci-ences, the Swedish Research Council for Medical Research, the Swedish Can-cer Foundation, the Innovative Medicines Initiative Joint Undertaking from the European Union’s Seventh Framework Programme, the Estonian Ministry of Education and Research, the Estonian Research Council for funding the projects over these years; the Donationsstipendium, Sture Erikssons fond, W Bagge & E o H Rhodin stiftelse, Rhodins stiftelse, Wallenbergs Stipendie, CF Liljevalch och M Augustinssons resestipendier from Stockholm University as well as from several external foundations, Apotekarsocieteten, Kungliga Veternskapakademie, Wenner-Gren Stiftelserna, Lindhes Advokatbyra AB, ÅForsk, American Peptide Symposium, and Peptide Therapeutic Foundation for the funding received to participate at conferences.
I would like to thank my co-supervisors, Prof. Kerstin Iverfeldt at first and Assoc. Prof. Anders Undén later, and all the professors at the department for their discussions and input.
I wish to thank all of the members of the Langel group, past and present, for contributing to a very nice and enjoyable working atmosphere, and for their scientific insight and friendship. I do not like to list names, but it is duty-bound this time and I will do it in alphabetical order. I would like to thank Andrés, for his good sense of humour and for all the philosophical discussions. Daniel, for his physicochemical skills and as personal Porto wine courier. Henrik, who always had technical answers and for his true friendship. Jakob,
XV
for being helpful in the academia as for the social life, and for the nice time at conferences. Jonas, for his molecular biology advice and the nice time spent in the climbing gym. Kristin, for her kindness, smiles and organizer of group reunions. Mattias, for his ideas and motivation. Maxime, for the scientific dis-cussions as for the tips on travel in France. Moataz, for his pathways analysis and help as lab assistant in the peptide course. Staffan, for the nice discussions and advices. Tönis, for the synthesis discussion and for being a good company in the office. Ying, for her pharmaceutical and KLARA skills. I would also like to thank also all the students that I have supervised over these years. I have probably learned more from you than you have from my guidance.
Thank you, Marie-Louise and Sylvia, for your patience and great help an-swering my questions related to the bureaucracy, and not only, academic life. Everyone else working in the department for your presence and support at any time. I really appreciate the nice discussion with each and every one of you, thank you all. Time has flown, and maybe life goes like this, but it has been a pleasure to have all of you as travel-partners during the years at the Depart-ment of Neurochemistry, today Department of Biochemistry and Biophysics.
I would like to thank former supervisors: my BSc supervisor Prof. Monica Monici, Dr. Francesca Cialdai, Dr Giovanni Romano for introducing me to the life in science and guiding me through the first steps in the laboratory; my MSc supervisors Prof. Adriano Aguzzi, Assoc. Prof. Alfredo Franco-Obregon, Dr. Pawel Pelczar who contributed to my passion for cell and mo-lecular biology, and for the experience at the ETH and University of Zurich.
From a personal point of view, a big group of friends and family members have been a source of motivation, inspiration and a fount of new energies to overcome difficulties along this time. The contribution that each one of them gave me, in different ways, has been truly priceless.
I wish to deeply thank my parents and my sister. You have always sup-ported and encouraged me, regardless of the choices I have made. I am looking up to you for your accomplishments and for reminding me to be strong and make my way, step-by-step. Thank you for your love. Many thanks to my extended family here in Stockholm for welcoming me and being supportive at any time. Our recent vacation all together has been simply fantastic. Thank you for the input to Populärvetenskaplig Sammanfattning.
The love to science also brought me to meet a special person. I conclude by thanking my future wife Tove for her continuous support. Her love and dedication to science is a huge source of inspiration to me. Her help for this thesis as for other works has been critically important. I am looking forward to the future. Whatever it reserves for us, it is going to be great being together.
1
1. Introduction
1.1.1. Origin and Structure of Mitochondria Researchers have made a tremendous effort to elucidate the origin of en-
ergy in life, essential for the survival of the cells. To date, two theories about the origin of mitochondria have been formulated. The first hypothesis was de-scribed in 1926 by Ivan E. Wallin1 suggesting that a nucleus-bearing but am-itochondriate cell existed first, followed by the origin of mitochondria in a eukaryotic host2–4 (Figure 1a-d). The endosymbiotic hypothesis suggests that mitochondria originate from an ancient symbiosis that resulted when a nucle-ated cell engulfed an aerobic prokaryote5,6 (Figure 1e-g). The engulfed cell relied on the protective environment of the host cell and the host cell relied on the engulfed prokaryote for energy production. This engulfed prokaryote evolved over time into mitochondria. The first report of intracellular structure that could represent mitochondria dates back to the 1840s, and in 1857 the Swiss anatomist Rudolf von Koelliker described them as “sarcosomes” while studying human muscle. In 1890, Altmann called them “bioblasts” and de-scribed them as elementary organisms living inside cells and carrying out vital functions. Then, in 1898, Brenda coined the term “mitochondrion”, referring to the appearance of these organelle during spermatogenesis. The word mito-chondrion was derived from the Greek words “mitos” (thread) and “chondros” (granule).
It took more than 50 years from when mitochondria were recognized to the first high-resolution electron micrographs of mitochondria (Figure 1i-l). From these micrographs and the schematic representation of a mitochondrion (Fig-ure 2) it is possible to see the organizational structure of the organelle. Palade and Sjöstrand described the mitochondria as surrounded by a double limiting membrane, which gives form to different chambers or compartments7.
2
Figure 1. Models for origins of mitochondria and electron micrographs. Models that propose the origin of a nucleus-bearing but amitochondriate cell first, followed by the acquisition of mitochondria in a eukaryotic host (a-d). Models that propose the origin of mitochondria in a prokaryotic host, followed by the acquisition of eukaryotic-spe-cific features (e-g). Electron micrograph of kidney mitochondria from Palade (h) and Sjöstrand (i), 1953. Figure 1a-g8 and Figure 1h-I7 reprinted with permission.
The membranes of mitochondrion are distinguished by the outer (OMM) and inner mitochondrial membrane (IMM) and the space in between these two is referred to as intermembrane space. The space inside the IMM is referred to as the matrix, and the infold that forms ridges were named cristae mito-chondriales. The OMM separates the mitochondria from the cytosol. The pas-
h i
3
sage through the OMM of metabolites and nuclear-encoded proteins is regu-lated by the voltage-dependent anion channel (VDAC) and the translocase outer membrane (TOM)9,10. The translocase inner membrane (TIM) is instead present on the IMM and allows the passage of proteins in or out the matrix11. The cristae were originally described as simple invaginations of the IMM but extensive studies using electron tomography showed that they are fundamen-tal structures for mitochondria with bag-like structures to compartmentalize and limit the diffusion of molecules that are important for the oxidative phos-phorylation (OXPHOS) system12.
1.1.2. Functions and Dysfunctions of Mitochondria Mitochondria have a central role in cell life. They are a unique organelle
having their own DNA (mtDNA). mtDNA has independent origin from nu-clear DNA (nDNA) and is only maternally inherited. The human mtDNA is a circular double-strand DNA of 16,569 base pairs encoding for 37 genes (22 transfer and two ribosomal DNA and 13 proteins, including enzymes involved in the OXPHOS pathway for adenosine triphosphate (ATP) production). The number of mitochondria per cell vary based on the cell type/tissue and each mitochondrion contains between two and ten copies of mtDNA. The mtDNA sequence is identical in most cells, termed homoplasmic, but mutated and wild-type mtDNA can also coexist in the same mitochondrion, termed heter-oplasmic. The OXPHOS pathway occurs in the electron transport chain (ETC, also known as the respiratory chain) located on the IMM. The ETC consists of four complexes (complex I-IV) and ATP synthase that together contribute to the generation of the mitochondrial electrochemical gradient, the mitochon-drial membrane potential (ΔΨm), and ATP (Figure 2). The ΔΨm is normally in the range of 80 to 140 mV, with the optimal for ATP production within 100 to 120 mV. A ΔΨm over 140 mV is usually leading to increased reactive oxy-gen species (ROS) production at the expense of ATP production13.
In addition to their role in cell life controlling ATP production, OXPHOS process, intracellular calcium concentration and cellular metabolism, mito-chondria play an important role for cell death signaling. They can promote both necrotic and apoptotic cell death by increasing the permeability of the mitochondrial permeability transition (MPT) pore. This event leads to the dis-sipation of the proton electrochemical gradient with decreased ATP produc-tion, increased ROS production, calcium overload, and mitochondrial swell-ing14. The ATP depletion levels play a role in determining whether the necro-sis or apoptosis cell death pathway is activated. Damaged mitochondria and
4
the regulation of their number occurs via mitophagy, an organelle-specific au-tophagic elimination. This process occurs via ubiquitination of mitochondrial components to facilitate mitochondrial clearance15.
Figure 2. Schematic representation of a mitochondrion showing the mitochondrial permeability transition pore and the respiratory chain. Reprinted with permission16.
The tissues with high metabolic demand have the highest number of mito-chondria and are also the most susceptible to mitochondrial-driven diseases. These include brain, eye, liver, heart, and skeletal muscle and are linked to mitochondrial diseases such as mitochondrial myopathies, neuromuscular and neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease, Hun-tington disease and amyotrophic lateral sclerosis), diabetes, obesity and can-cer. The clinical expression of mitochondrial disorders can involve different systems (Table 1). The most common mitochondrial disease associated to mtDNA mutation is the Leber hereditary optic neuropathy (LHON), due to a degeneration of retinal ganglion cells and consequent visual failure.
5
Table 1. Systems involved and clinical manifestations in patients with mitochondrial disorders. System Clinical manifestations System Clinical manifestations Cardiovas-cular
Pulmonary
Neuro-logic
Renal
Hemato-logic
Endocrine
Heart failure Arrhythmias Murmurs Sudden death Left ventricular myocar-dial noncompaction Apical ballooning syn-drome
Muscle weakness with normal creatine kinase levels and normal electro-myographic and nerve-conduction studies Short stature Microcephaly Round face High forehead Low-set ears Short neck
1.1.3. Mitochondria as a Target for Drug Discovery The potential of selective drug delivery systems in modern drug therapies
has been the driving-force for developing therapeutic agents or imaging con-trast formulations towards greater targeting selectivity and better delivery ef-ficacy. One of the bottle-necks for this realization, is the inherent difficulties to penetrate mitochondrial membranes. A common approach is to increase the portion of drug accumulation in target cells versus normal cells or specific organelle in order to minimize the potential side effects and increase the ther-apeutic effects. The benefits of intracellular drug delivery and subcellular tar-geting range from significant reduction of the quantity of a therapeutic mole-cule for the desired effects, to decrease of the side effects.
1.1.4. Agents Targeting Mitochondria The pivotal role of mitochondria in controlling cell life and death make
them an attractive target for mitochondrial gene therapy and for the develop-ment of drugs that could treat mitochondrial related diseases. The ETC on the IMM is the major intracellular source of ROS, generated as by-products dur-ing mitochondrial electron transport. In addition, ROS are formed as neces-sary intermediates of metal catalysed oxidation reactions inside the cells. ROS such as anion superoxide, hydrogen peroxide, hydroxyl radical, hydroxyl ion, and nitric oxide (Table 2) are highly reactive with short half-life.
Table 2. List of common reactive oxygen species. ROS are found in normal and pathological tissues.
- Hydrogen peroxide H2O2 Singlet oxygen 1O2 Hypochlorous acid HClO The • designates an unpaired electron.
Mitochondria are continuously exposed to ROS and thus accumulate oxi-dative damage more rapidly than the rest of the cell17. High ROS levels can cause non-specific damage to lipids, proteins, and DNA, leading to alteration
7
or loss of cellular functions. Many studies have associated mitochondrial dys-function caused by ROS with both necrotic and apoptotic cell death18. The rate of mitochondrial ROS production can be altered by several physiological or pathological conditions. Several human pathologies like LHON, dystonia, and Leigh's disease19–21 are linked to oxidative damage by mitochondrial ROS. The onset mechanisms of diseases caused by mitochondrial oxidative stress are still an object of research. Drugs like paclitaxel22, etoposide23, betulinic acid24, lonidamine25, CD-43726, and ceramide have been clinically approved and used to initiate apoptosis in mitochondria. Different approaches have been used to successfully target the mitochondria. A schematic representation of a mitochondrion and the mode of action of representative mitochondria target-ing compounds is shown in Figure 3.
Vitamin E was the first small molecule to be used for specific targeting, acting also as an antioxidant against ROS. Vitamin E and analogues have been used for different models or diseases such as pro-apoptotic and anti-cancer activity27, breast cancer28, cardiovascular protection29, skin protection from UVB-irradiation30. Vitamin E has also been covalently coupled to a tri-phenylphosphonium (TPP) cation to have higher targeting efficacy compared to vitamin E alone31. The fluorescent lipophilic cation rhodamine 123 and other anticancer cyanine dyes have also been used as mitochondria-selective molecules for therapeutic purposes (anticancer or anti-apoptotic effects)32. Another strategy used to target mitochondria was based on mitochondrial tar-geting signal (MTS) sequences. Proteins that are not mitochondrial-encoded need to be imported into mitochondria. Most of these proteins are encoded from nuclear DNA, translated in the cytosol and imported into mitochondria for the presence of MTS at N-terminus. The majority of MTS are composed by positively charged and hydroxylated amino acids to form amphiphilic sec-ondary structure33. There are also some proteins that are lacking the MTS but that paradoxically can enter into mitochondria. The MTS interact with the TIM-TOM complexes and the terminal signal sequence is proteolytically re-moved by proteases present in the intramembrane space or in the matrix ac-cording to the final destination of the protein10,34,35. The MTS has been suc-cessfully used for the delivery of a variety of cargo molecules, including pro-teins36, nucleic acid37, and endonucleases38,39.
Another molecule/nanocarrier that has been used to target mitochondria is desqualinium chloride and several other compounds have been derived from it. DQAsome, a liposome-like vesicle based on desqualinium, was reported from Weissig et al. in 1998 as a novel potential drug and gene delivery sys-tem40. In 2001 Weissig and Torchilin extensively reviewed the development
8
of mitochondrial DNA delivery systems (DNA/DQAsome complex) towards mitochondrial gene therapy41. DQAsomes were able to bind plasmid DNA, form complexes between 70 and 700 nm, protect the DNA against nuclease attack, release DNA at mitochondria-like membranes, and have a cytotoxicity similar to Lipofectin and LipofectAmine.
Figure 3. Schematic representation of a mitochondrion and the mode of action of representative mitochondria targeting compounds. Cationic compounds (TPP-based agents, choline esters, SS peptides) are attracted by the negative potential of the IMM. Driven by their high affinity for IMM-specific phospholipids, gramicidin S (GS)-based antioxidants deliver the nitroxide ROS scavenger into the matrix. MTS can be utilized as vehicles to deliver metalloporphyrin superoxide dismutase (SOD)-mimics into the matrix. Alternatively, the mitochondrial agent can be encapsulated in a vesicle which undergoes fusion with the OMM. The filled circle represents the anti- or pro-oxidant payload. D-(KLAKLAK)2 and analogues are cationic amphipathic α-helical peptides able to disrupt mitochondrial membranes, hence triggering apoptosis. Other chemical agents target specific mitochondrial proteins. For instance, sulfonylureas block the mitochondrial ATP-regulated K+ channel (mitoKATP), benzothiazepines are inhibitors of the mitochondrial Na+-Ca2+ exchanger, and benzodiazepines are agonists or antagonists of the peripheral benzodiazepine receptor (PBR). ATP, adenosine tri-phosphate; ETC, electron transport chain; IMS, intermembrane space. Reprinted with permission42.
9
Sulfonylureas and potassium channel openers have been shown to interact with the mitochondrial adenosine triphosphate-dependent potassium (mito-KATP) channels and have cardio-protective effects43. Benzodiazepines and other peripheral benzodiazepine receptor ligands have been shown to be reg-ulator of the mitochondrial permeability transition pore and to have potential utility as anti-apoptotic or pro-apoptotic antitumor agents, based on agonist or antagonist effects44,45. Benzothiazepines derivatives have been reported to in-hibit the mitochondrial Na+-Ca2+ exchanger and to enhance glucose-stimu-lated insulin secretion in pancreatic β-cells46. D-(KLAKLAK)2 is a cationic amphipathic α-helical killer peptide derived from the sequence of membrane-disrupting antimicrobial peptides (AMP). Initially it was used as mitochon-dria-disruption peptides to trigger apoptosis of cancer cells47. It has been mod-ified exchanging the leucine with cyclohexylalanine to improve mitochondrial localization and efficacy48. Manganese metalloporphyrin conjugated to a sig-nal oligopeptide is a class of mitochondria-targeted SOD-mimics reported for the antioxidant properties49.
A peptide-based mitochondria-localizing antioxidant, termed SS-31, was developed and found to selectively target the inner mitochondrial mem-brane50,51. This tetra peptide exerts its function through the ROS-scavenging activity of 2,6-L-dimethyltyrosine (Dmt) residue found in its sequence (D-Arg-Dmt-Lys-Phe-NH2), which shares structural similarity with vitamin E. This compound was observed to display antioxidant activity and reduce cell death in two neuronal cell lines at nM concentrations, as well as decrease mi-tochondrial ROS production and prevent apoptosis-related events in isolated mouse liver mitochondria.52 It was also found that this antioxidant peptide prevents myocardial stunning that is associated with reperfusion in the is-chemic heart of an ex vivo guinea pig model. The chemical structures of fea-tured mitochondria-targeting agents and clinical drug candidates discussed herein are reported in Figure 4.
10
Figure 4. Chemical structures of mitochondria-targeting agents and clinical drug can-didates. For chimera molecules, substructures highlighted in dashed boxes represent the targeted bioactive components, and substructures highlighted in dashed circles represent the mitochondria-targeting cationic entities. Ph, phenyl; Me, methyl; Et, ethyl; Boc, tert-butoxycarbonyl; Cbz, benzyloxycarbonyl; Ac, acetyl. Reprinted with permission42.
11
Glutathione (GSH) is a water-soluble tripeptide (γ-Glu-Cys-Gly). It is the most prevalent low-molecular weight (307 Da) compound containing a sulfhydryl group in eukaryotic cells, and present in mM concentration range in various mammalian cells53,54. GSH is oxidized to glutathione disulphide (GSSG). Proteins or other molecules containing cysteine residues readily par-ticipate in thiol-disulphide exchange reactions with GSH. GSSG is usually rapidly reduced by glutathione reductase and maintained at less than 1% of the total glutathione pool55. GSSG and other glutathione-conjugates may also be excreted from cells. GSH has roles in cellular protection against oxidants and xenobiotics, and in signal transduction. In antioxidant defence, the major reaction of GSH is reduction of hydroperoxides by glutathione peroxidases (GSHPx) and at least one peroxiredoxins, which yields to GSSG. In redox signaling, GSH participates through both the removal of H2O2 and the reversal of thiolate oxidation. GSH is used for detoxification of several xenobiotics by glutathione S-transferases (GSTs), involved in the transport of nitric oxide. The glutathione system has become a drug target due to the large spectrum of bio-functionality of GSH in different pathological conditions.
Since glutathione itself cannot be administered clinically and having any effect, one strategy is to use chemically modified GSH analogues in order to mimic glutathione’s various physiologic and pharmacologic effects. Some ap-proaches aim to enhance the antioxidant activity while others to inhibit en-zymes with which GSH usually interacts. In both cases there are the conditions for a variety of molecules to be or enter clinical testing for therapeutic pur-poses.
1.2.1. Glutathione Analogues GSH analogues and GSH-like compounds could support parts of the gluta-
thione system and have an impact as an adjuvant therapeutic factor, for in-stance, in the case of oxidative stress when the production of the pro-oxidant GSSG is powerful. Different strategies have been used in order to maintain the functionality of GSH system. One of the main limiting factors for the de novo synthesis of GSH is the bioavailability of cysteine. Providing commer-cial dosage of cysteine would not be sufficient to support rates of synthesis that are adequate to sustain normal GSH concentrations. N-acetyl-L-cysteine has been used to avoid toxicity problems56–58. Esters derivatives of GSH were synthesized showing fast cellular uptake and subsequent de-esterification in-
12
side the cells providing the native GSH. This strategy has resulted in protec-tive effects against cerebral brain ischemia in rats59,60, in model of stroke and spinal cord injury61, Parkinson’s disease62, diabetic cataract63, LDL oxidative modification and liver perfusion injury64.
GSH analogues have also been developed for cancer therapies, due to the reported implication of GSH in cancer progression and chemoresistance65. One of the biochemical mechanisms reported to be responsible of drug re-sistance in cancer cells is the over expression of GST66. For this reason, some GSH analogues have been designed to inhibit different GST isoenzymes, such as phosphono analogues67 and peptidomimetic analogues of GSH68.
GSH analogues has been developed to overcome the stability problem to-wards peptidases and proteases. Cyclization has been one strategy; other strat-egies relied on the substitution of some amino acids or addition of more amino acids. The UPF1 analogue peptide followed this last strategy, adding the non-proteinogenic amino acid 4-metoxy-phenylalanine to GSH N-terminus. This modification showed to improve the antioxidant properties and to increase hy-drophobicity of the GSH derivative. A series of UPF analogue peptides were designed with variations including the replacement of the native gamma-glu-tamyl moiety in the GSH backbone with the alpha-glutamyl moiety, using D-amino acids instead of L-isomers and amidation of the terminal carboxyl group. This modification leaded to improved hydroxyl radical scavenging properties and increased antiradical efficacy69–71.
In 1988, the field of cell-penetrating peptides (CPPs) emerged from the
foundational work of Frankel and Pabo72 as well as Green and Loewenstein73. During the same time period, their laboratories discovered the transactivator of transcription (TAT) protein of human immunodeficiency virus (HIV) and described how the protein was able to cross cell membranes, efficiently inter-nalized by cells in vitro. In the following years, truncated versions of TAT were studied and a minimal sequence derived from TAT was identified to en-able cell entry74. A few years later, a 16 amino acid peptide derived from the amphiphilic Drosophila Antennapedia homeodomain, penetratin (pAntp), was discovered75. Since then, several other proteins and peptides that displayed translocation activity have successfully been reported such as VP2276, Trans-portan77, model amphipathic peptide (MAP)78, signal sequence-based pep-tides79, synthetic arginine-enriched sequences80.
13
CPPs can be described as short positively charged peptides varying from 4 to 40 amino acids in length. They are capable to cross cellular membranes and to be internalized into mammalian, plant, and bacterial cells. Furthermore, they can mediate the transport of a variety of biologically active molecules, cargos, and drugs with low or non-toxic effects81–83. Numerous CPPs have been developed within the fields of biology and medicine and several have been applied for a variety of applications, showing the utility of CPPs in the basic research as well as in clinic84–86. A variety of intracellular cargos have been used via direct conjugation, encapsulation or physical adsorption with CPPs (Figure 5).
Figure 5. Applications of cell-penetrating peptides as molecular delivery vehicles. This class of peptides has been demonstrated to successfully promote cellular inter-nalization for a wide array of biologically active molecules. Direct conjugation, en-capsulation, physical adsorption, or non-covalent complexation methods have been use to deliver imaging agents (silica nanoparticle, quantum dots, paramagnetic lan-thanide ions, gold nanoparticles, dextran-coated superparamagnetic-iron oxide nano-particles), carriers (polymeric particles, carbon nanotubes, dendrimers, micelles, solid lipid nanoparticles, liposomes), or cargoes such as peptides, proteins, drugs, plasmids, siRNA, miRNA, decoy DNA, antisense oligonucleotides into cells, nuclei or specific organelles. Reprinted with permission87.
These include imaging agents such as silica nanoparticles, quantum dots, paramagnetic lanthanide ions, gold nanoparticles, dextran-coated superpara-magnetic-iron oxide nanoparticles), carriers (polymeric particles, carbon
14
nanotubes, dendrimers, micelles, solid lipid nanoparticles, liposomes), or car-goes such as peptides, proteins, drugs, plasmids, siRNA, miRNA, decoy DNA, antisense oligonucleotides(ASO)87. CPPs have matured as a delivery platform technology to deliver agents, providing optimism for a wide range of therapeutic applications.
1.3.1. Design of CPPs Since the discovery and characterization of the protein transduction domain
of TAT in 198872,73, over 1000 individual CPPs have been reported and over 2500 papers have been published in this field to date. A statistical graphic representation of CPPs based on 1855 entries, of which 1699 are unique pep-tides, is shown in Figure 6 with various ways of categorizing CPPs. Most CPPs are linear peptides consisting of the natural abundant L-amino acids (Figure 6a and 6b) and about half of all CPPs are synthetically derived (Figure 6c). Examples of commonly used CPPs are included in Table 3.
Figure 6. Statistical representations depicting the distribution of CPPs reported in the literature based on (A) linear and cyclic conformation; (B) chirality/modifications, (C) origin and (D) type of cargoes delivered by CPPs in various in vitro and in vivo set-tings. Reprinted with permission88.
One can also categorize CPPs depending on their physico-chemical prop-erties, often into the sub-groups cationic, amphipathic and hydrophobic. Most
15
of the CPPs are cationic, and thus have a net positive charge at physiological pH (primarily due to arginine and lysine in the sequence). Amphipathic CPPs have the charge distribution originating primarily from lysine residues such as MAP77, transportan78, and Pep-189. The hydrophobic CPPs have charged and hydrophobic residues separated on the main chain, as in the vascular endothe-lial-cadherin (pVEC)90 and MPG peptides.
The number and order of amino acids in the peptide sequence is one of the factor determining the transduction properties of the CPP. Although CPPs can rapidly cross the cell membranes and be internalized, their physico-chemical properties, secondary structure, concentration, type of cargo, and cell line have all an effect on the mechanism of their internalization.
Table 3. Cell-penetrating peptides commonly used for delivery applications.
Cell-penetrating peptide Sequence Protein derived Penetratin RQIKIWFQNRRMKWKKTat (48–60) GRKKRRQRRRPPQpVEC LLIILRRRIRKQAHAHSK-NH2
1.3.2. Cellular Uptake Mechanisms Early studies of uptake mechanism suggested that CPPs could pass through
the cellular membrane by direct translocation, caused by favourable electro-static interactions and hydrogen bonding91,92. More recently, energy-inde-pendent processes are thought to occur when the peptide has characteristics that are compatible with the plasma bilayer or if the peptide sufficiently per-turb the structural integrity of the membrane77,93. However, recent evidence suggests that direct translocation plays a less important role, especially in large cargo delivery94. Instead, different energy-dependent endocytosis pathways have been proposed95–97 to be responsible for CPP-mediated intracellular de-livery of large molecules and nanoparticles (Figure 7).
16
Figure 7. Uptake and trafficking pathways of CPPs. CPPs use predominantly different sorts of endocytosis to gain access to the interior of cells. In the context of this, all major pathways for CPP uptake have been described, including clathrin- and caveo-lae-mediated endocytosis, as well as macropinocytosis. Less information is available for other less-defined pathways, such as several clathrin- and caveolae-independent endocytosis mechanisms. Uptake of the cargo molecule is followed by complex intra-cellular trafficking events towards early/sorting endosomes, late endosomes/microve-sicular bodies (MVBs), lysosomes or Golgi network. Typically, endo-/lysosomal mat-uration is characterized by gradual drop in the pH. Of note, recycling pathways can direct cargo also through late endosomes/MVBs for being released to extracellular milieu via extracellular vesicle (EV) release. In this case, the cargo could become incorporated into exosomes and subsequently be taken up by other cells (re-distribute) or by the same cell (reuptake). In addition to the more dominant endocytic pathways, some membrane active CPPs have also been reported to be taken up by direct trans-location over the cellular membrane, which potentially allows them to avoid endocytic pathways altogether. Reprinted with permission98.
These pathways are today considered as the predominant mechanisms of
transmembrane delivery of CPPs. Endocytosis can be divided into four path-ways: macropinocytosis, clathrin-mediated endocytosis (CME), caveolae/li-pid raft-mediated endocytosis, and clathrin/caveolae-independent endocyto-sis99. There are often conflicting results between studies of the exact internal-ization pathway of a given CPP but it is now evident that various CPPs and
17
CPP-cargo complexes can enter cells using different (single or multiple) en-docytotic mechanisms and therefore end up in different compartments into the cells100. This was shown for Antp, nona-arginine, and TAT where macropino-cytosis, clathrin-mediated endocytosis, and caveolae/lipid raft-mediated en-docytosis were used simultaneously100. Different results are sometimes due to different experimental setup such as choice of cell-line, CPP concentration used, type of cargo, method of link CPP to cargo molecule (covalently linked to- or complexed with the CPP).
Results from a large number of studies suggest that the mechanism of en-docytic uptake for a CPP is strongly dependent on the attached cargo78,97,101–
103. For example, Tat has been shown to use lipid raft-mediated endocytosiswhen conjugated to a protein and clathrin-dependent endocytosis when con-jugated to a fluorophore. Macropinocytosis has been implicated in the uptakeof a variety of CPP-cargo conjugates, suggesting that membrane ruffling aidsthe internalization of CPPs104,101. Additionally, the electrostatic interaction ofCPPs with surface proteoglycans has been shown to be responsible for theuptake of many CPPs96,97.
Independent of the initial interaction leading to endocytosis, the internal-ized CPP and its cargo end up into endosomes or lysosomes where they can stay for extended period of time, thus reducing bioavailability and activity. If the target of the delivered molecule is not the endocytic vesicles, the pep-tide/cargo complex has to be further transported to the target subcellular loca-tion (cytoplasm, nucleus, mitochondria) to exert its biological effect before being either transported back to the plasma membrane for recycling by exo-cytosis or fused to lysosomes for degradetion105. This process, called endoso-mal escape, is not completely understood but is considered a limiting factor for the success-rate of CPPs. The process of endosomal escape is in many cases not very efficient and the CPPs may deny the cargo from reaching the desired intracellular site 106,107. For this reason, efforts have been made to im-prove the ability of CPPs to exit from endosomes. Different chemical endoso-molytic agents have been use in vitro to induce osmotic swelling and rupture of endosomes. Examples of such chemicals are chloroquine108, sucrose109 and calcium ions110.
In 2012, the involvement of scavenger receptor class A (SCARA) in the uptake of non-covalent CPP/oligonucleotide (ON) complexes was shown for the first time111. It was previously shown that SCARA receptors bind and me-diate cellular uptake of a negatively charged molecule112, and the involvement of SCARA was shown for the CPP PF14/splice correcting oligonucleotides (SCO), which have negative zeta-potential (ζ-potential) and NF51/pDNA113.
18
The nature and secondary structure of the CPP, the ability to interact with cell surface and membrane lipid components, the nature, type, and active concen-tration of the cargo, the cell type, and the membrane composition are the pa-rameters that play a secondary role in the cellular uptake pathway.
1.3.3. Applications in Drug Delivery and Clinical Development of CPPs
Most drugs need to cross one or more cellular membranes in order to reach their target of interest and have any therapeutic effect. Since the passage through the plasma membrane is the limiting step, optimizing cellular delivery systems of therapeutics is an important priority of today’s research. One of the major hurdles to cure a disease lies in the low potency of current available drugs, which could partially be solved by using delivery vectors for specific targeting. CPP-based drug delivery has been explored to treat various diseases, including neuronal disease, asthma, ischemia, diabetes, and cancer89,114–116.
Despite the success reported through in vitro studies, only a few studies have shown treatment efficacy in animal models and no CPP or CPP conjugate has passed the FDA hurdle and reached the market. The first CPP clinical trial was done using oligoarginine to transport cyclosporine into cells throughout the epidermis and dermis of human skin. It has been discontinued in 2003 after Phase II clinical trials117. TATp has completed a Phase II clinical trial con-ducted by Revance Therapeutics and has been used for topical delivery of bot-ulinum toxin across the skin. Capstone Therapeutics has evaluated AZX-100 in Phase II trials, a cell-permeant peptide mimicking heat shock protein HSP20. Their strategy relied on bypassing the signaling pathway to smoothen muscle relaxation for prevention of dermal/keloid scarring. Protein Cdelta in-hibitor-TAT(47-57) conjugates, developed for myocardial infarction, pain, and cytoprotection/ischemia (KIA-9803, KIA-1678, and KIA-1455) are under evaluation by KIA Pharmaceuticals in Phase I/II. 6-aminohexanoic acid-spaced oligoarginine has been tested in vivo for splicing correction by Avi Biopharma118. A CPP-antisense peptide-morpholino (PMO) conjugated for aortocoronary bypass therapeutic application (AVI-5126) was terminated af-ter Phase II. Another CPP-PMO conjugate for Duchenne muscular dystrophy treatment is in preclinical development (AVI-5038). Istituto di Sanitá and No-vartis have a vaccine based on TAT-V2 deleted Env proteins in Phase I clini-cal trial. Multiple TAT peptide transduction domains (PTDs) linked to a dou-ble-strand RNA binding domain (DRBD) has been developed by Traversa Inc119,120. Diatos has developed the agent DTS-108, an active metabolite of the
19
anticancer drug irinotecan and the peptide DPV1047, for cancer treatment and in Phase I clinical trials in Europe. Data regarding clinical trials were retrieved from ClinicalTrials.gov, a database of privately and publicly funded clinical studies conducted around the world.
1.3.4. Targeting Intracellular Organelles Subcellular delivery implies the delivery of a molecule or drug in its active
form to its target site of action inside the cells. Peptides have facilitated the cellular uptake of a variety of cargoes into cells, but they have less often ex-erted the ability to specifically target organelles. Nevertheless, several pep-tides capable of localizing to specific subcellular compartments or organelles have already been demonstrated. For example, quantum dots derived from peptide JB858 and liposomes from bacteriorhodopsin helix C have been shown to localize into the membrane121,122; lysosomal targeting peptide, HIV-1 TAT, fibrinogen-derived ICAM-1 binding sequence, transportan10/siRNA to endosomes/lysosomes123–126; mitochondrial 3-oxoacyl-coenzyme-A, hybrid tumor homing/proapoptotic peptide, chemoselectively ligated cytochrome-C oxidase peptide to mitochondria122,127,128; TAT, polyarginine, influenza de-rived fusogenic peptide, nuclear localization sequence (NLS)-TAT to nu-clei129–131; endoplasmic reticulum(ER)-insertion signal, ligand for ER, ER tar-geting moiety like AAKKKAA to the ER132–134; dentin phosphophoryn, pep-tide derived from herpes simplex type 1 virus to the cytosol135,136.
Signal peptides have also been used to direct their cargo to the nucleus, endosomes, and other organelles. The plasma membrane is the first point of contact for peptide or complexes, thus membrane targeting using peptides as probes to localize onto the lipid bilayer is a valuable tool to understand the first critical interactions. The vesicles of the endosomal and lysosomal system are another desirable target for drug delivery due to disease or disease states associated with deficient enzymes in these pathways such as Fabry disease. The use of CPPs can be sufficient to target these compartments since they get entrapped and sequestrated by them as mentioned earlier. Nuclei, the site for genetic storage and gene transcription, are one of the primary organelles for targeting delivery. The Simian virus large T antigen nuclear localizing se-quence (SV40 NLS) has been commonly used for nuclear targeting137,138. Na-ked oligonucleotides have been shown to not be able to freely pass through membranes; for examples, only 0.1% of free plasmid from cytosol has been reported to translocate into the nucleus by crossing the nuclear membrane139.
Another effective strategy for nuclear targeting has been shown to be the
20
use of NLS. Macromolecules larger then 50kDa required the presence of NLS to be transported into the nucleus. The signal sequence is usually removed in the mature protein. Small peptides from viruses that show nuclear localization, such as KKKRKV peptide from SV40 have been used for nuclear delivery by non-viral strategy. Cationic lipids in complex with DNA, called lipoplex, and cationic polymers in complex with DNA, called polyplex, have also been re-ported to deliver DNA into the nucleus140,141. The NLS peptides have been demonstrated to guide uptake and nuclear localization of other species, includ-ing gold nanoparticles142, carboplatin-based anticancer therapeutics143, and green fluorescent protein (GFP)144.
Other organelles such as the Golgi and the endoplasmic reticulum have been targeted. Golgi was described more than a century ago by Camillo Golgi as an internal reticular apparatus. It has a central role for the cell secretory pathway and interacts with the ER. It carries out posttranslational modification of newly synthesized proteins and is involved in the synthesis of proteogly-cans and carbohydrate structures. Alterations of the Golgi-associated proteins and of the Golgi apparatus give rise to a variety of neurodegenerative disorders which include, for example, Alzheimer’s, Parkinson’s, Niemann-Pick disease. The ER is a network of folded membrane-enclosed tubules and cisternae that extend from the nuclear membrane throughout the cytoplasm. It facilitate the folding of secretory and membrane proteins, and is involved in calcium stor-age and signaling, as well as regulate apoptosis against disturbances in cal-cium homeostasis, ischemia, hypoxia, exposure to free radical, and oxidative stress145. For these reasons, ER-Golgi network has been considered a main target for anticancer therapy. Rapamycin, a regulator of mammalian cell growth and proliferation in response to environmental and nutritional condi-tions, has been delivered to target the mTOR pathway. In some cancer, mTOR is activated to enhance cell growth. Inhibition of mTOR pathway using ra-pamycin could decrease the anti-proliferative effect of mTOR and play a cen-tral role in fighting cancer146.
Mitochondria are the metabolic powerhouse of the cell and the control point for regulating programmed cell death. Dysfunctional mitochondria are implicated in a variety of diseases that range from metabolic disorders, to neu-rodegenerative diseases, to cardiovascular disease, to cancer147–149. It is not surprising that much attention has been dedicated to the targeting of these or-ganelles to treat such diseases. However, the difficulties to develop vectors for mitochondrial targeting has hindered the advancement of mitochondrial med-icine. Nonetheless, promising results within this area have emerged. For ex-
21
ample, it has been show that short peptide are also able to target mitochon-dria50,150 as well as mitochondrial targeting sequence fused to peptides151 or synthetic transporters that show promise for mito-specific delivery of bioac-tive cargos152,153.
CPPs with the ability to translocate into the cells and localize into mito-chondria could be highly beneficial for mitochondrial delivery applications. This require better understanding of structure and effects of CPPs on cells and organelles.
Gene therapy is still at its infancy even though the field arose in the 1960s. In 1970, Stanfield Roger proposed to use of “good DNA” to replace defective DNA in people with genetic disorders154. Two years later, Theodore Fried-mann and Richard Roblin published a paper in Science titled “Gene therapy for human genetic disease?” where they propose that a sustained effort should have been made to formulate a complete set of ethic-scientific criteria to guide the development and clinical application of gene therapy techniques155. Thirty years after, the first patient was treated with a gene therapy for a congenital disease called adenosine deaminase (ADA) deficiency which severely affects the immune system and the ability to fight infections. The infusion was per-formed using a retrovirus as a vector to carry the patient’s own corrected cells156. Since then, many other patients with ADA have been treated with improved gene therapy technology to achieve therapeutic benefits157.
The European Medicines Agency (EMA) defines a gene therapy medicinal product (GTMP) as a “biological medicinal product that contains an active substance which contains or consists of a recombinant nucleic acid used in or administrated to human to regulate, repair, replace, add or delate genetic se-quence and its therapeutic, prophylactic or diagnostic effect relates directly to the recombinant nucleic acid sequence it contains, or to the product of genetic expression of this sequence”158.
The Food and Drug Administration (FDA) defines gene therapy as agents “that mediate their effects by transcription and/or translation of transferred genetic material and/or by integrating into the host genome and that are ad-ministrated ad nucleic acid, viruses, or genetically engineered micro-organ-isms. The products may be used to modify cells in vivo or transferred to cells ex vivo prior to administration to the recipient”.159
22
Many hurdles have been and still need to be overcome in gene therapy such as safety, immunity and manufacturing but the logic of such treatment modal-ity could potentially take its place as a standard of care just as the use of phar-maceutical drugs.
Gene transfer has been complemented most recently by gene editing and potentially gene correction using technologies, such as engineering endonu-cleases, including CRISPR/Cas, and represent part of the exciting future of gene therapy. Gene therapies are promising for a broad range of diseases with the aim to radically treat the causes of the diseases instead of only relieving the symptoms. They may be effective on a wide range of previously untreated diseases such as haematological, ocular, neurodegenerative diseases, and sev-eral cancers160. In Figure 8, the number of gene therapy trials per year from 1989 to 2015 is reported, with a distinct increase over time. Adenovirus, ret-rovirus, and naked plasmid DNA were the most used vectors in the gene ther-apy trials, with respectively 22.14%, 18.76%, and 18.03% of the trials. Adeno-associated virus vectors were used in 6.63% of the trials, and vaccinia virus, lentivirus, and lipofection were used as vectors in around 5% of the trials161.
Figure 8. Number of gene therapy trials per year. Reprinted with permission161.
23
To date, eleven gene therapies have been approved. These are, in the order of their approval, Gendicine®, Oncorine®, Rexin-G®, Glybera®, Neovas-culagen®, Imlygic®, Strimvelis, Eteplirsen®, Zalmoxis®, Kymriah®, Yes-carta®, and Luxturna®. With particular interest are: Glybera, to treat adults with lipoprotein lipase deficiency (LPL), approved in 2014 by the EMA162; Strimvelis, to treat adenosine deaminase severe combined immune deficiency, approved by the EMA in 2016163; Eteplirsen, for Duchenne muscular dystro-phy (DMD), approved by the FDA in 2016164.
In 2017, researchers announced that a teenager had been cured of sickle-cell disease, an inherited blood disorder that affects 100000 people in the USA and millions around the world, after receiving an experimental gene therapy developed by Bluebird Bio. In the same year, FDA approved two pioneering treatments, Kymriah and Yescarta, that use the patient’s own immune cells to fight rare types of cancer. The first treats a bone marrow cancer that affects children and young adults, the second treats a type of lymphoma. Last Decem-ber, the FDA approved the first gene therapy for an inherited disease165. Lux-turna aims to correct a mutation responsible for a range of retinal diseases that make people gradually blind. More than twenty patients that were losing their sight have had their vision restored after the treatment.
There is hope also for haemophilia. In the same month, BioMartin company published early clinical trial results showing that nine patients who received its therapy saw substantial increases in the blood-clotting proteins absent in haemophilia166.
Oligonucleotide pharmacology and the first attempts to silence specific genes using antisense oligonucleotides (ASOs) date back to 1970s. An ASO is a short strand of deoxyribonucleotide analogue that hybridizes with com-plementary mRNA in a sequence-specific manner via Watson-Crick base pair-ing. While conventional drugs bind directly to proteins, formation of ASO-mRNA heteroduplex either triggers RNase H activity, leading to mRNA deg-radation, induces translational arrest by steric hindrance of ribosomal activity, interferes with mRNA maturation by inhibiting splicing or destabilizes pre-mRNA in the nucleus, resulting in the downregulation of target protein ex-pression.
In 1978, Paul Zamecnik et al. suggested that oligonucleotides could be used
24
therapeutically. In their pioneer work they reported that a 13-mer ASO con-taining a phosphodiester backbone complementary to the terminal repeat se-quences of the Rous sarcoma virus was able to inhibit its replication and cell transformation167. The success was not immediate and in the following years several chemical modifications were developed in order to increase stability, deliverability, potency, and improve pharmacological properties of ASO. Some of the most common modification are modification of the 2´-hydroxyl (OH) to 2´-O-methyl (O-Me), phosphotorhioate (PS), 2´-fluoro (F), 2´-meth-oxyethyl (MOE), bicyclics containing a 2´,4´-O-methylene bridge, called locked nucleic acid (LNA), and peptide nucleic acid (PNA).
Some ASOs have the ability to enter specific cell lines in naked form, a process called gymnosis168. However, the lack of efficient cell targeting and translocation into the desired cell type, organelle, or tissue by gymnosis have motivated researchers to investigate transfection methods, such as viral and non-viral vectors as well as liposome-mediated, for their delivery169–173. The first antisense oligonucleotide approved for marketing by the FDA, and suc-cessively by EMA, was Vitravene, also known as Formivirsen. This 21-mer phosphorothioate oligodeoxynucleotide targeting the mRNA that encode for the cytomegalovirus immediate-early-2 protein was developed by Iris Phar-maceuticals and Novartis Ophthalmics for the treatment of patients with cyto-megalovirus retinitis174. In 2013, Spinraza, an 18-mer phosphorothioate 2´-O-methoxyethoxy ASO, has been approved by FDA for the treatment of spinal muscular atrophy type 1, 2, and 3 in infants.
Additional strategies that recently are getting special attention from the pharmaceutical industry includes mRNA-based therapeutics and CRISPR-Cas9 genome editing machinery. As the subject of basic and applied research for more than five decades, mRNA has only recently come into the focus as a potentially powerful drug class able to deliver genetic information. It is based on cancer immunotherapies and infectious vaccines approaches such as in vivo delivery of mRNA to replace or supplement proteins, or mRNA-based induc-tion of pluripotent stem cells, or mRNA-assisted delivery of designed nucle-ases for genome engineering175. CRISPR-Cas9 technology, firstly described as an adaptive immune system in bacteria and archaea, have fast developed and become a popular tool for the targeted genome modification in many or-ganisms. The process needs two essential components: the Cas9 recombinase enzyme that cuts the DNA and a snippet of single guide RNA that guides the molecular scissors to the target sequence. These tools were shown to effi-ciently drive specific modification of mammalian genomes176.
ASO is a useful tool for protein target identification and validation, but also
25
a highly selective strategy for diseases with dysregulated protein expression. However, the alteration of animal or patient’s genomic DNA is also associated with ethical questions. The use of gene therapy appears to be closely related to factors such as economic difficulties, particularly with regard to wealth dis-tribution, political and cultural conflicts, as well as the scarcity of studies eval-uating the impacts of the use of gene therapy on human health177–180. In this thesis the use of oligonucleotides was limited to two types of therapeutic ONs: splice correcting oligonucleotides (SCOs) and ASO.
27
2. Aims
The experimental work conducted during this PhD focused on three main objectives:
• to develop cell-penetrating peptides targeting mitochondria for theirprimary characteristics as potential antioxidative molecules in orderto protect cells from oxidative stress;
• to target mitochondria as an intracellular target for drug delivery;• to understand uptake and transfection mechanisms of cell-penetrating
peptides alone or in conjugation with oligonucleotides (ONs).
The aims for each paper are detailed below.
The aim of Paper I was to develop a library of novel cell-penetrating pep-tides targeting mitochondria (mtCPPs peptides) with improved biological ef-ficiency. The peptides were designed to have a better ability to selectively accumulate in the mitochondria and to reduce the total level of reactive oxygen species in the cells. In this project we focused on the characterization of one of the new CPPs named mtCPP1. We investigated the effects of this peptide on intramitochondrial processes such as cytotoxicity, mitochondrial mem-brane potential and antioxidant activity. Fluorescent microscopy using car-boxyfluorescein-labelled peptides was used to determine cellular uptake and localization. The ultimate aim was to confirm the targeting ability of the pep-tide and for this reason localization experiments on isolated mitochondria and other cellular compartments were performed.
28
The aim of Paper II was to design and synthesise a fusion peptide by cova-
lent conjugation of the most promising mitochondrial antioxidative CPP from Paper I (mtCPP1) and a glutathione analogue peptide (UPF25), previously de-veloped in our laboratory. The purpose was to explore the potential to syner-gistically increase the treatment efficacy of the novel peptide (mtgCPP). Mi-tochondrial membrane potential and ATP production were investigated with the aim of assessing how these antioxidant peptides influences the mitochon-drial energy provision.
In the work of Paper III, the aim was to test a new synthesised library of
delivery vector complexed with nucleic acid cargo to form nanostructures able to penetrate cells and mitochondria. There are a number of diseases that orig-inate from mitochondrial dysfunctions. These dysfunctions can be reversed or alleviated by modifying and or regulating the expression of mitochondrial genes. The main aim was to develop a delivery method to transport nucleic acids into mitochondria and alter the mitochondrial gene expression.
The aim of Paper IV was to examine the intracellular trafficking after trans-
fection with an amphiphilic CPP, PF14 (used in Paper III), both alone or in complex with SCO using a transcriptomics-based approach to improve the understanding of their uptake mechanisms. Additionally, we aimed to unravel the pathways involved in fundamental cellular phenomena associated with up-take of PF14 transfection system and to use this knowledge to design more efficient CPPs.
29
3. Methods
The detailed description of all methods used for the work this thesis is built upon can be found in the included papers. In this section theoretical back-ground of the methods will be provided.
Solid phase peptide synthesis (SPPS) is the most common method to syn-thesise peptides for both research and therapeutic purposes. Robert Bruce Merrifield was awarded the Nobel Prize in chemistry in 1984 “for his devel-opment of methodology for chemical synthesis on a solid matrix”181. The pep-tide is attached via its C-terminus to a resin, representing the solid phase, which allows for removal of the excess soluble reagents used in each coupling step. In this work, the 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry has been used. This technique is mainly divided in two sequential steps, where cycles of addition of amino acid and removal of the Fmoc protecting group are repeated. In the coupling step, activators are used in order to convert the carboxyl group of the amino acid into a more reactive ester, susceptible for nucleophilic attack by the primary amine of the linker of the functionalized resin or the growing peptide. A schematic representation of the SPPS tech-nique is reported in Figure 9. The activators used for the work in this thesis are 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hex-afluorophosphate (HCTU) and 6-chloro-1-hydroxybenzotriazole dihydrate (6-Cl-HOBt) or ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma Pure®). The Fmoc group is protecting the α-amine of the amino acid in order to avoid un-specific reactions. Piperidine or piperazine are examples of weak bases used to easily remove this protecting group. The functional groups of the peptide side chains are also protected with acid labile groups. Protecting group with different characteristic can be used in order to continue the synthesis orthog-onally. When the synthesis of the whole peptide is complete, then the side chain protecting groups are removed and the unprotected peptide cleaved from
30
the solid support. In this work, all the side-chain modifications have been per-formed at a lysine protected with the acid labile 4-methyltrityl (Mtt)-group. The Mtt group can be removed with a solution containing 1% of tri-fluoroa-cetic acid (TFA), avoiding the cleavage of other protecting groups or the pep-tide from the resin.
All peptides were synthesized manually, using a room temperature parallel automated peptide synthesizer (Syro II, Multisyntech GmBH) or a fully auto-mated microwave peptide synthesizer (Alstra+, Biotage AB, Uppsala, Swe-den). 5(6)-Carboxyfluorescein was coupled manually or by using the Alstra+ synthesizer after removal of Fmoc protecting group from N-terminal amino acid of peptides. Stearic acid modification was also carried out either in a manual step after automated synthesis or coupled under microwave heating conditions in the Alstra+ synthesizer. All peptides were modified at the C-terminus by using a H-Rink-Amide-ChemMatrix resin (PCAS Biomatrix, St-Jean-sur-Richelieu (province of Quebec), Canada); this resin produces ami-dated peptides on the C-terminal. The peptides were cleaved from the resin, using 95 % TFA, 2.5 % triisopropylsilane (TIS) and 2.5 % H2O, precipitated in cold diethyl ether and lyophilized.
Figure 9. General scheme of Fmoc-solid phase peptide synthesis.
31
Crude peptides were purified using semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed using matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF) mass spec-trometry or by ultra-high performance liquid chromatography-mass spectrom-etry (UHPLC-MS). After purification, the peptides where lyophilized again and reconstituted in ultra-pure water (Milli Q, Merck Millipore) before use. Table 4 below lists the peptides used in this thesis.
For the experiments in this thesis, a diversity of cell types has been used.
HeLa cells, the first cell line that was propagated indefinitely in vivo from biopsy of a cervical tumour taken from the patience named Henrietta Lacks, were used in Paper IV. George O. Gay was the first researcher to use this cell line in the laboratory in 1951 and from then this cell line is the mostly common used human cell line due to rapid grow and easy transfection182.
The HeLa pLuc705 cell line is stably transfected with a luciferase-encoding gene interrupted by a mutated β-globin intron 2. These cells were used in all the papers included in this thesis. HeLa pLuc705 cells are a gift from Prof. Ryszard Kole (University of North Carolina, Chapel Hill, NC, USA). The bEnd.3 cell line is a mouse brain endothelial cell line and has been used for experiments in Paper I and II. The immortalized bEnd.3 cell line was estab-lished from an infection of the polyomavirus middle T antigene in a mice brain. The endothelial nature of these cells was confirmed by the observed expression of von Willebrand factor and uptake of fluorescently labelled low density lipoprotein (LDL)183,184. The U87 cell line is a human primary grade IV glioblastoma cell line which was derived from a malignant glioma by J. Ponten and associates in the 1960’s and reported to produce a malignant tumor in nude mice185. This cell line was used in Paper I and II.
The CHO cell line was derived from the Chinese hamster ovary in 1957 by T. Puck at the Boston Cancer Research Foundation. This cell line has become a staple source of cells due to their robust growth as adherent cells and usable for cell transfection186. CHO cell line has been used in Paper II.
Cells were grown at 37°C, 5 % CO2, in Dulbecco’s modified Eagle’s me-dium with glutamax supplemented with 0.1 mM non-essential amino acids, 10 % fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml strepto-mycin (Invitrogen, Stockholm, Sweden). Cells were seeded 24 h prior to ex-periments into 96-well plates. The cells were treated with peptides alone at different concentrations or mixed with ONs at different molar ratio (MR) in MilliQ-water in 10 % of the final treatment volume (i.e., 10 µl) and incubated for the appropriate time according to the experiments.
The translocation of CPPs across the plasma membrane can result in toxic
effects due to membrane perturbation, especially at high peptide concentra-tions. Exceeding the toxic threshold can result in irreversible damage to cells
33
which makes determination of cytotoxicity crucial. To find out if the activity of the CPPs alone or in complex with its cargo was associated with cytotoxi-city, cell viability was evaluated by conventional 2-(2-methoxy-4-nitro-phenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) toxicity assay (Roche Diagnostics Scandinavia AB, Bromma, Sweden) ac-cording to the manufacturer’s instructions.
This assay measures cell viability as a function of mitochondrial metabolic activity. The activity of the mitochondrial dehydrogenases is measured by their conversion of tetrazolium salts to formazan, cell-viability and cell-pro-liferation is directly correlated to the amount of formazan dye formed, more cells means more formazan dye. The dye can be elegantly quantified spectro-photometrically, using UV-absorption at 420 nm, more intense staining means more active mitochondrial dehydrogenase. The WST-1 reagent and the form-azan product are both water soluble, which means that measurements can be performed 1-4 h after addition, making it a very rapid and convenient assay compared to the commonly used MTT assay, which requires solubilisation of the formazan salt.
Mitochondria were isolated in order to detect the presence of peptide into the organelle after cell treatment. Mitochondria were extracted using the Mi-tochondria Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA) ac-cording to the manufacturer’s instruction. This assay allows the collection of a pellet containing the isolated mitochondria as well as for the cytosolic frac-tion and the membranes fraction.
Tetramethylrhodamine ethyl ester (TMRE) fluorescent probe was used to evaluate the mitochondrial membrane potential (ΔΨm). TMRE is a cellular positively charged permeant dye that readily accumulates in active mitochon-dria, both in the IMM and matrix space, due to its relative negative charge. Depolarized and/or non-functional mitochondria have decreased membrane potential and fail to sequester TMRE187. It was possible to evaluate the ΔΨm base on the fluorescence intensity of the dye sequestrated into mitochondria of cells treated with peptides versus untreated cells read on a fluorescence
34
plate reader (Flex Station II; Molecular Devices, Sunnyvale, CA, USA) with setting suitable for TMRE (Ex/Em = 549/575 nm).
MitoSOX Red mitochondrial superoxide indicator (Invitrogen Detection Technologies) fluorogenic dye was used to determine reactive oxygen species (ROS) production in mitochondria of live cells. MitoSOX Red reagent is rap-idly taken up and selectively targeted to mitochondria. Into mitochondria, Mi-toSOX Red reagent is oxidized by superoxide and exhibits red fluorescence. The selectivity of this probe is due to the oxidation by superoxide but not by other ROS- or reactive nitrogen species (RNS)-generating systems, and oxi-dation of the probe is prevented by superoxide dismutase. The oxidation prod-uct was read with a fluorescence reader (Flex Station II; Molecular Devices, Sunnyvale, CA, USA) with settings suitable for MitoSOX (excitation = 510 nm, emission = 580 nm).
In order to characterize the physiochemical properties of the peptides or
particles that formed when mixing the peptides with ONs, dynamic light scat-tering (DLS) was used. This method allows the determination of the size dis-tribution of small particles in solution. The Brownian (random) motion and the speed of the particle in solution is dependent on the size of the particles themselves. If particles are small compared to the wavelength of the laser used then the light scatters in all directions188. Due to the Brownian motion the dis-tance between the particles is constantly changing, therefore scattered light intensity fluctuates. The time-scale of the fluctuations is directly related to the translational diffusion coefficient of the scattering particles, which in turn is related to their size189. From this an autocorrelation function that relates the fluctuations in intensity to the size can be used for size determination. One drawback of this method is that in order to distinguish between two intensity peaks they need to be separated by a factor of two, or they will result in a single broader peak189. Another problem is that the method is more sensitive to larger species as they give higher intensity.
35
The DLS technique allows also the measurement of the electrical potential at the interface of the particle surface and the diffuse layer, known as ζ-poten-tial. The ζ-potential is the difference in potential between the layer of ions strongly bound to the charged particle surface (Stern layer) and that of the dispersion medium (diffuse layer). It can be determined by measuring the dif-ferential migration between two electrodes using laser Doppler velocimetry. This method allows for the measurement of the charge at the interface between the particle and the solvent in which is dispersed but not the actual particle charge. From this measurement is possible to get also an indication of the sta-bility of the particles in a solution, where potentials between -30 and 30 mV indicate low stability. In this work an instrument that can do both types of measurements was used; Zetasizer Nano ZS (Malvern Instruments, Malvert, UK).
Peptides or complexes were prepared as described in the respective papers and diluted in MQ-water or complete cell culture media DMEM. Samples were assessed in disposable low-volume cuvettes. Data were converted to rel-ative intensity plots, from which the mean hydrodynamic diameter was de-rived.
Circular dichroism (CD) spectroscopy is a method that gives information about the overall secondary structure of peptides or proteins in solution. CD analysis is based on light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light. The light is absorbed to different extents at specific wavelength depending on the differences in ex-tinction coefficients for the two polarized rays called circular dichroism. Cir-cular-polarized light rays will travel through an optically active medium with different velocities due to the different indices of refraction for right- and left-circularly polarized light, called optical rotation or circular birefrin-gence190,191.The variation of optical rotation as a function of wavelength is called optical rotary dispersion.
36
Figure 10. Circular dichroism spectra of proteins and peptides with representative secondary structures. a) CD spectra of poly-L-lysine in 1, alpha-helical, antiparallel beta-sheet in 2, extended disordered conformations of placental collagen in 3, native triple-helical in 4, and denaturated forms in 5. Reprinted with permission192.
It has been shown that CD spectra between 260 and 180 nm can be analyzed
for the different secondary structures: alpha helix, parallel and antiparallel beta sheet, turn, and others193. Representative CD spectra is shown in Figure 10. This method can be also used to accurately predict secondary structure using theoretically derived spectra194,195.
The characteristic glow in fireflies and click beetles is due to the luciferase.
Firefly luciferase is an enzyme responsible for a reaction that emits light. The enzyme catalyses the oxidation of firefly luciferin, requiring oxygen and ATP. The gene that encodes it can be used as a reporter gene in many different types of delivery assays. The light produced by the reaction is easy and convenient to measure, directly related to differences in gene expression. For example, delivering a luciferase encoding plasmid to a cell that does not normally pro-duce the enzyme gives a measurable increase light output that is proportional to the amount of luciferase present. Conversely, delivering siRNA to a cell that already expresses luciferase will result in a decrease in the amount of en-zyme and therefore a reduction in light output. The splice correcting assay
37
described below is another way to use luciferase as reporter gene in an ON assay.
In Paper III ASOs were delivered, using non-covalent complexation strat-egy with peptides, to HeLa pLuc705 cells. ASO targeting the uncoupling pro-tein 2 (ASO [UCP2]), D-arm modified ASO targeting UCP2 (D-arm ASO [UCP2]), and D-arm modified ASO targeting cytochrome c oxidase subunit II (D-arm ASO [COXII]). D-arm is a D stem-loop import signal of tRNA Tys(GUA) and has been shown to be efficiently imported into the mitochon-drion of Leishmania, a kinetoplastid protozoan, as well as into mitochondrial matrix in isolated mitochondria196–198. UCP2 is a mitochondrial anion carrier able to modulate the ROS and COXII is a mitochondrial protein that make up complex IV of the respiratory chain related to maintaining the mitochondrial membrane potential. The silencing of the mRNA coding for these two proteins yielded to significant changes of the mitochondrial membrane potential and of ROS levels.
In Papers III and IV SCOs were delivered, using non-covalent complexa-tion with CPPs, to cells that stably express an incorrectly spliced, and non-functional, luciferase. The successful delivery results in functional luciferase. A 96 well assay was used, seeding the cells 24 h before the treatment. The cell media was changed with fresh media and the CPP/SCO complexes then added.
The splice correction assay that was developed by Kole et al. provides an elegant quantitative assessment of cellular delivery efficiency of SCOs.199 The assay uses HeLa pLuc705 cells, a stably transfected cell line with a plasmid containing a luciferase-encoding gene interrupted by a mutated intron from a β-thalassemic globin gene. The intronic mutations activate a cryptic splice site that produces non-functional luciferase. Masking the mutated site with an an-tisense ON re-orients the splicing machinery to produce functional luciferase.
38
Subsequent luminescence measurement by a luminometer allows for quanti-fication of uptake.
In Papers I and II adenosine triphosphate (ATP) production levels were measured after peptides treatments on HeLa pLuc705. In order to measure ATP levels, the ATP determination assay was used. The quantitative determi-nation of ATP was assessed using recombinant firefly luciferase and its sub-strate D-luciferin. The assay was based on luciferase’s requirement for ATP to produce light. For the luciferase assay, a standard reaction solution was prepared. ATP was diluted serially in the standard reaction solution to gener-ate a standard curve. Cells were treated for 24 h with peptides and thereafter lysed. Standard reaction solution was added, and immediately light emission was acquired for 30 s using a GLOMAX 96 microplate luminometer (Promega, Stockholm, Sweden).
One of the experiments in Paper I and II uses fluorescence microscopy.
Confocal microscopy was used in Paper III. These techniques are essential tools in life sciences allowing for visualization and identification of cells, sub-microscopic cellular components and fluorescently labelled molecules. Fluo-rescence microscopy enables the study of single molecules as well as allowing for the identification of several target molecules simultaneously. Confocal mi-croscopy offers several advantages over conventional optical microscopy, in-cluding shallow depth of field, elimination of out-of-focus glare, and the abil-ity to collect serial optical sections from thick specimens. These techniques can be used for imaging of either fixed or living tissue/cells that have been labelled with one or more fluorescent probes. In this thesis, there techniques have been applied to study the cellular uptake of labelled CPPs and ON-cargo and to investigate the localization of fluoro-labelled peptides alone or peptides in complex with Alexa568-labelled SCO in live cells.
39
The transmission electron microscope (TEM) has become a very powerful tool in science due to the increased resolution than light microscopes. This allows the observation of ultrastructure of organelles, viruses and macromol-ecules. The TEM operated on the same basic principles as the light microscope but electrons are used instead of light. A high energy beam of electrons is accelerated through a very thin sample, and the interactions between the elec-trons and the atoms can be used to observe structures, shape, size density and quality of small entities (in some cases as small as individual atoms). In Paper IV we used TEM in combination with the application of autophagy inducers and gold-tagged plasmid DNA to examine the cellular delivery and uptake of nucleic acid complexed with PF14.
Western blot (WB) is a common method to detect and analyse proteins. In 1979, H. Towbin et al. described the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets where the original gel pattern was accurately obtained200. It is based on the separation of proteins by electropho-resis from the gel to a membrane where they can be visualized specifically.
Figure 11. Schematic presentation for the detection of proteins on the western blot membrane by enhanced chemiluminescence (ECL). ECL substrate is processed by the enzyme (peroxidase) tagged to the secondary antibody and generates chemilumines-cence where the primary antibody reacted with the antigen on the membrane. Re-printed with permission 201.
40
The setup consists of a standard of seven steps: sample preparation, gel electrophoresis, blotting to membrane, antibody probing, detection, imaging, and analysis. After the transfer, the membrane is blocked in order to prevent unwanted membrane-protein interaction in the following steps. To visualize the protein of interest the membrane is first probed using a primary protein-specific antibody followed by a labelled secondary antibody used for detec-tion. A schematic representation is shown in Figure 11. An image is then taken, and the result is analyzed.
41
4. Results and Discussion
Many human genetic, metabolic and degenerative diseases, along with nor-mal ageing, have been linked to mitochondrial dysfunction and defective as-sembly of the respiratory chain, which depends on expression of genes en-coded in both the nuclear and mitochondrial genomes.
This thesis describes the use of cell-penetrating peptides for their ability to cross cellular membranes and target organelles. It furthermore explores CPPs for drug development and therapeutic possibilities. The outcome of this work proposes CPPs targeting mitochondria with intrinsic antioxidant activity, complexes with ON cargoes for gene modulation, and pathway of uptake for this transfection system.
In this study we reported a series of short peptides targeting mitochondria. The design of the peptides was taking into consideration several characteris-tics: positively charged amino acids, lipophilicity, and alternating aromatic and basic residues. Rearrangement of the amino acid sequence had no effect on the scavenging properties of the peptides, but substitution of Dmt with phe-nylalanine resulted in complete loss of antioxidant activity. The resultant an-tioxidant action of the mtCPPs can thus be attributed to the Dmt residue, in line with previous studies. Tyrosine is known to scavenge oxy-radicals by for-mation of their corresponding relatively unreactive tyrosyl-radical. The radi-cal intermediate can either be capped by radical-radical coupling to give di-tyrosine202,203, or scavenged by glutathione and/or ascorbate204,205. Dmt bears many structural similarities to tyrosine, but also to vitamin E; the methylated phenolic structure50.
Unlike other antioxidants, mtCPPs are highly water-soluble and rapidly taken up by cells. Given to their positive net charge, they might be expected
42
to target the mitochondrial matrix in a potential-driven manner. By accumu-lating in the mitochondria, these peptides are localized to the site of ROS production. Treatment of cells with H2O2 causes increase of ROS levels.
Figure 12. Structure of mtCPP1. The most promising peptide candidate, mtCPP1 (Figure 12), demonstrated
antioxidative properties that selectively targeted mitochondria, thereby ena-bling scavenging of ROS at the site of production. The peptide showed a 2-fold reduction of ROS levels inside the cells compared to the SS31 peptide when H2O2 was added to induce an oxidative stress. As a consequence, pre-vention of mitochondrial depolarization could be achieved as well as preven-tion of cell death at the optimum concentration of 5µM. In contrast, most an-tioxidants require 100 µM to mM concentrations to prevent oxidative cell death206–208.
None of the peptides showed toxic effects on cells even at as high concen-tration as 100 µM, and this is consistent with their lack of effect on mitochon-drial potential and ATP production. The mitochondrial functionality was pro-tected by maintaining a constant ΔΨm at physiological level. The ATP meas-urement confirmed that mitochondria of cells treated with mtCPP1 were not affected from the treatment with no difference in ATP production compared to untreated cells.
Fluorescence microscopy showed 70 % higher uptake of mtCPP1 into the cells compared to the original peptide, based on relative quantification of the fluorescence intensity. Using isolated mitochondria, we showed the ability of mtCPP1 to pass the cellular membrane and to accumulate into mitochondria. The fluorescence intensity data from the isolated mitochondria showed a 25% increased up-take of mtCPP1 compared to the original peptide.
We designed CPPs targeting the site of ROS generation and protecting mi-tochondrial function with their intrinsic antioxidative properties. These anti-oxidant peptides may be beneficial in the treatment of ageing and diseases
43
associated with oxidative damage such as ischemia-reperfusion injury and neurodegeneration.
In this paper we took the advantage of mtCPP1 to cross the plasma mem-brane and to target the mitochondria to deliver a glutathione analogue peptide (UPF25) and study the antioxidant properties of the fused peptide. We hypoth-esized that the fused peptide, called mtgCPP, would have higher antioxidative and detoxifying effects than mtCPP1 alone.
The first finding from this work was the remarkably stronger superoxide anion scavenging ability of mtgCPP compared to mtCPP1 or UPF25 alone by 2- and 3-fold, respectively. The mitochondrial membrane potential and theATP production were investigated to assess the influence of peptide treatmenton mitochondrial energy provision. The fused peptide resulted to have similarproperties to mtCPP1, rescuing the mitochondrial membrane potential tophysiological levels when cells were insulted with H2O2 and ATP levels weresimilar to the one measured from untreated cells. We reasoned that mtgCPPwas capable of preventing damage to cellular compartment caused by ROSacting as GSH in the GSH metabolism.
Spectrofluorometric analysis was performed on three different subcellular fractions to investigate the subcellular uptake of the fluorescein-labelled pep-tides. The analysis showed that FAM-mtgCPP was mainly present into mito-chondria with a 10- and 33-fold higher intensity than that of FAM-mtCPP1 and FAM-UPF25, respectively. Interestingly, FAM-mtgCPP was shown to be present in the cytosolic fraction as well, with an 8- and 19-fold higher fluores-cence intensity than the other two fluoro-labelled peptides. The analysis also showed that the glutathione analogue peptide was mainly present in the mem-brane fraction.
DLS measurements showed that mtgCPP formed particles with sizes around 150 to 400 nm in diameter when dissolved in MilliQ water or in cell culture media at 5 µM final concentration, respectively. The ζ potential of mgtCPP was mainly electropositive at all concentrations and solutions used, as expected from the nature of the peptide consisting of positively charged residues.
The synergistic antioxidative activity might be the result of the ability of mtCPP1 to transport UPF25 fragments into the cells at higher concentrations.
44
The presence of the peptide in the cytosol and into mitochondria resulted in increased scavenging ability.
In this paper we developed a library of peptides generated from a combi-natorial covalent fusion of the previously used mitochondrial-penetrating pep-tide, mtCPP1, and PepFect14 (PF14) in order to deliver therapeutic biomole-cules to mitochondria. The first test was to elucidate the transfection ability and efficacy of the peptides complexed with SCO. Complexes were thus formed at different MRs to screen for the most optimal ratio for splice-correc-tion activity. This activity was found to be in a MRs-dependent manner. mit-Fect1/SCO at MR5 and MR10 had a transfection efficacy of 20- and 30-fold increase compared to PF14 at the respective MR. mtCPP1, PF14, and SCO mixture had a remarkably 20-fold increase in luminescence over PF14/SCO treatment.
To address the main question of this study, that is to be able to deliver bio-molecules to mitochondria, we evaluate the knockdown of mitochondrial mRNA using three different antisense oligonucleotides: ASO [UCP2], D-arm ASO [UCP2], and D-arm ASO [COXII]. Peptides efficiently delivered ASO [UCP2] to mitochondria affecting the mitochondrial membrane potential and ROS production. The 24 h treatment elicited higher changes in mitochondrial membrane potential and ROS production compared to the 6 h treatment. Pep-tides were able to efficiently deliver also D-arm ASO [UCP2] to mitochondria showing stronger effects on mitochondrial membrane potential. While there was a 1- to 4.5-fold increase compared to the treatment with ASO [UCP2], the mitochondria membrane potential was massively perturbed after 24 h treat-ment with D-arm ASO [UCP2], with 20-fold increase of TMRE uptake over untreated cells for mitFect4/antisense complex treatment. The delivery of D-arm ASO [COXII] complexed with peptides to mitochondria was also deliv-ered efficiently, and the mitochondrial functionality was affected. The 6 h treatment with D-arm ASO [COXII] resulted in a strong decrease of TMRE uptake, suggesting a depolarization of the mitochondrial membrane potential. The 24 h treatment showed to be less efficient, probably due to a recovery of the mitochondrial membrane potential over time. We also investigate the ef-fect of the ASO on the ROS production. We could observe an increase in the level of the superoxide anion.
45
The size and the ζ potential of the complexes were investigated. The ma-jority of the complexes were below 100 nm. A higher compaction degree was observed at increasing MR. The electrochemical potential resulted to switch from negative values at MR3 to positive values (~ 20 mV) at MR7 and MR10.
Based on these results, we concluded that the down regulation of the UCP2 or COXII protein levels after transfection of ASO [UCP2], D-arm ASO [UCP2], or D-arm ASO [COXII] by peptides resulted in an imbalance among the subunit in the Complex I or IV of the ETC, which affected mitochondrial functions such as maintaining the mitochondrial membrane potential and the production of ROS.
The uptake mechanisms for transfection with CPPs alone or in complexes with cargo molecules is still need of more clarifications. In this study, we per-formed RNA sequencing to study the effects of PF14 and PF14/SCO com-plexes on the gene expression of HeLa cells. PF14 alone and PF14/SCO re-sulted in significant effects on the gene expression with 292 and 934 genes differentially expressed, respectively. These treatments affected multiple pathway. We validated several of the most differentially expressed genes by qPCR. The genes were selected based on their potential to regulate autophagy due to preliminary data showing the possible involvement of autophagy path-way in the regulation of the transfection process. Expression of 26 autophagy-related genes were analyzed and several genes resulted to have a high fold change in gene expression. Genes like connective tissue growth factor (CTGF), nuclear receptor coactivator 7 (NCOA7), and apolipoprotein B re-ceptor (APOBR) resulted in 4-fold higher expression; whereas low-density lipoprotein receptor (LDLR) showed a 10-fold down-regulation in case of treatment with PF14. PF14/SCO treatment gave a completely different profile with mostly gene down-regulation. This difference can be due to the differ-ences between the peptide alone (or nanoparticles formed by positively charged molecule) and the PF14/SCO complex (formed by positively and neg-atively charged molecules).
We proceeded using small molecules to modulate autophagy pathway and investigate the effect of ON transfection. The ligands were chosen based on the selected genes from the pathway analysis in order to see if particular genes
46
could play a role in the uptake mechanism. CTGF inhibitor and focal adhesion kinase (FAK) inhibitor reduced 75% of the splice correcting activity; HMG-CoA reductase inhibitor and Prostaglandin E2 inhibited the activity by 60%. On the other hand, other inhibitors such as toll-like receptor 4 (TLR4) inhibi-tor, the nuclear transport receptor importin-beta inhibitor, hydrocarbon recep-tor (AhR) antagonist, the heat shock protein 70 (HSP70) inhibitor, and the beta-adrenoceptor antagonist increased the splice correction activity. The in-duction of autophagy was detected after 8 and 24 h treatment by confocal, fluorescence and electron microscopy. The autophagy induction in cells treated with PF14 or PF14/SCO was also confirmed by Western blot analysis. PF14 was able to induce autophagy to the same extent as in cells treated with the standard autophagy inducer, rapamycin. The co-treatment with ligand molecules influenced the cellular uptake of PF14/SCOAF568; for example, co-treatment with alprenolol resulted in 50% higher level of complex uptake.
This study demonstrates the autophagy induction and cellular effects trig-gered by CPP transfection system in basal cell signalling. We showed that the splice correction activity could be significantly upregulated by co-transfection with small ligand molecules able to suppress the autophagy process. This knowledge, in addition to other studies, can help understanding pathways in-volved in uptake mechanisms to optimize non-viral delivery systems for ther-apeutic and clinical applications.
47
5. Concluding Remarks and Future Outlook
Mitochondria play an important role in controlling both life and death of cells. As a consequence, malfunctions in this organelle leads to a range of human diseases. To circumvent these malfunctions, there is a need for better understanding of the molecular mechanisms responsible for mitochondria-linked disease processes. Oxidative stress has long been reported to mediate mitochondrial dysfunctions. For this reason, strategies for targeting mitochon-dria and delivery of antioxidants are being developed. There is a continuing search for better and more effective organelle targeting and effective antioxi-dants. During the last years, research efforts developing mitochondria-tar-geted antioxidants have increased steadily. Within this framework, our effort has consisted of the development of cell-penetrating peptides targeting mito-chondria with antioxidative properties. In Paper I we reported that mtCPP1 and other compounds studied protect mitochondria against oxidative insult. In Paper II we concluded that the fusion peptide mtgCPP, formed by covalent conjugation of mtCPP1 and a glutathione analogue peptide, resulted in syner-gistic antioxidative effects by scavenging H2O2 and lowering level of ROS production.
Due to the association between a range of human diseases and mutations in mitochondrial DNA, it is not surprising that mitochondria are an attractive target for gene therapy. Current therapeutic approaches are largely supportive rather than curative, and there is a lack in effective treatments for mitochon-drial disorders. Alternative strategies are required to develop innovative gene therapy vectors that can be produced in large scale in order to re-establish the normal mitochondrial function in mutated mitochondria or cells. Mitochon-drial gene therapy provides a new perspective to this; efficient and convenient cure due to higher targeting and prolonged duration of action. The strategy can be applied at different levels: targeting genes in the nucleus, targeting pro-tein import into the mitochondria, or targeting genes into the mitochondria. In Paper III we reported the ability of chemically modified CPPs to efficiently deliver antisense oligonucleotides into mitochondria to affect mitochondrial functions. The results from these peptides demonstrated a fruitful avenue that
48
may lead to clinically useful therapeutic formulations. New technologies such as RNA sequencing, super-resolution and cryo-electron microscopy could help to optimize the design of mitochondrial targeting CPPs.
Another bottleneck to bring gene therapy into clinical use have been the difficulties to deliver cargo molecules to cell nuclei. Genetic expression pro-files and intracellular signalling pathways controlling transfection efficacy can be helpful for the design of optimal delivery vectors. In order to elucidate the uptake mechanisms of CPPs for oligonucleotides transfection, we have investigated the effects of transfection by PF14 alone or in complex with oli-gonucleotide on gene expression. In Paper IV we reported the ability of PF14/ON complexes to induce autophagy-related genes, and consequentially the autophagy. The modulation of this process using small ligand molecules led to increased transfection efficacy. This finding opens possibilities to use autophagy modulators as combinatory strategy for future gene therapy.
Gene regulation after treatment with mitochondrial targeting CPPs is also of our interest. The finding in this thesis could reveal new insights useful for understanding the uptake mechanisms and pathways used to target mitochon-dria, contributing to the evolution of new suitable mitochondrial targeting pep-tide systems for mitochondrial gene therapy.
49
References
1. Wallin, I. E. Bacteria and the origin of species. Science 64, 173–175(1926).
2. Margulis, L., Dolan, M. F. . & Whiteside, J. H. . ‘Imperfections andOddities’ in the Origin of the Nucleus. Paleobiology 31, 175–191(2005).
3. Moreira, D. & López-García, P. Symbiosis between methanogenicarchaea and δ-proteobacteria as the origin of eukaryotes: Thesyntrophic hypothesis. J. Mol. Evol. 47, 517–530 (1998).
4. Cavalier-Smith, T. The phagotrophic origin of eukaryotes andphylogenetic classification on protozoa. Int. J. Syst. Evol. Microbiol.52, 297–354 (2002).
5. Martin, W. & Müller, M. The hydrogen hypothesis for the firsteukaryote. Nature 32, 37–41 (1998).
6. Vellai, T., Takács, K. & Vida, G. A new aspect to the origin andevolution of eukaryotes. J. Mol. Evol. 46, 499–507 (1998).
7. Ernster, L. & Schatz, G. Mitochondria : A Historical Review. J. CellBiol. 91, 227s–255s (1981).
8. Embley, T. M. & Martin, W. Eukaryotic evolution, changes andchallenges. Nature 440, 623–630 (2006).
9. Colombini, M., Blachly-Dyson, E. & Forte, M. in Ion Channels (ed.Narahashi, T.) 169–202 (Springer US, 1996).
10. Pfanner, N. & Wiedemann, N. Mitochondrial protein import: Twomembranes, three translocases. Curr. Opin. Cell Biol. 14, 400–411(2002).
11. Pfanner, N. & Meijer, M. Mitochondrial biogenesis: The Tom and Timmachine. Curr. Biol. 7, R100–R103 (1997).
12. Cogliati, S., Enriquez, J. A. & Scorrano, L. Mitochondrial Cristae:Where Beauty Meets Functionality. Trends Biochem. Sci. 41, 261–273(2016).
13. Hüttemann, M. et al. The multiple functions of cytochrome c and theirregulation in life and death decisions of the mammalian cell: Fromrespiration to apoptosis. Mitochondrion 11, 369–381 (2011).
14. Rodriguez-Enriquez, S., He, L. & Lemasters, J. J. Role of
50
mitochondrial permeability transition pores in mitochondrial autophagy. Int. J. Biochem. Cell Biol. 36, 2463–2472 (2004).
15. Lee, J. Y., Nagano, Y., Taylor, J. P., Lim, K. L. & Yao, T. P. Disease-causing mutations in Parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. J. Cell Biol. 189, 671–679 (2010).
16. Davis, R. E. & Williams, M. Mitochondrial Function and Dysfunction: An Update. J. Pharmacol. Exp. Ther. 342, 598–607 (2012).
17. Poljsak, B., Šuput, D. & Milisav, I. Achieving the balance between ROS and antioxidants: When to use the synthetic antioxidants. Oxid. Med. Cell. Longev. 2013, 1–11 (2013).
18. Kamogashira, T., Fujimoto, C. & Yamasoba, T. Reactive Oxygen Species, Apoptosis, and Mitochondrial Dysfunction in Hearing Loss. Biomed Res. Int. 2015, 1–7 (2015).
19. Wallace, D. C. Mitochondrial diseases in man and mouse. Science 283, 1482–1488 (1999).
20. Finkel, T. & Holbrook, N. J. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247 (2000).
21. Raha, S. & Robinson, B. H. Mitochondria, oxygen free radicals, disease and ageing. Trends Biochem. Sci. 25, 502–508 (2000).
22. Kidd, J. F. et al. Paclitaxel affects cytosolic calcium signals by opening the mitochondrial permeability transition pore. J. Biol. Chem. 277, 6504–6510 (2002).
23. Robertson, J. D., Gogvadze, V., Zhivotovsky, B. & Orrenius, S. Distinct pathways for stimulation of cytochrome c release by etoposide. J. Biol. Chem. 275, 32438–32443 (2000).
24. Andre, N. et al. Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells’. Cancer Res 60, 5349–5353 (2000).
25. Nancolas, B. et al. The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane Monocarboxylate Transporters. Biochem. J. 473, 929–936 (2016).
26. Costantini, P., Jacotot, E., Decaudin, D. & Kroemer, G. Mitochondrion as a Novel Target of Anticancer Chemotherapy. J. Natl. Cancer Inst. 92, 1042–1053 (2000).
27. Dong, L. F. et al. Mitochondrial targeting of vitamin E succinate enhances its pro-apoptotic and anti-cancer activity via mitochondrial complex II. J. Biol. Chem. 286, 3717–3728 (2011).
28. Cheng, G. et al. Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death. BMC Cancer 13, (2013).
29. Rocha, M. et al. Mitochondria-Targeted Vitamin E Antioxidant: An Agent for Cardiovascular Protection. Vasc. Dis. Prev. 6, 36–46 (2009).
30. Kim, W. S. et al. Mitochondria-targeted vitamin E protects skin from UVB-irradiation. Biomol. Ther. 24, 305–311 (2016).
51
31. Smith, R. A. J., Porteous, C. M., Coulter, C. V & Murphy, M. P.Selective targeting of an antioxidant to mitochondria. Eur. J. Biochem.263, 709–716 (1999).
32. Murphy, M. P. Selective targeting of bioactive compounds tomitochondria. Trends Biotechnol. 15, 326–330 (1997).
33. Abe, Y. et al. Structural basis of presequence recognition by themitochondrial protein import receptor Tom20. Cell 100, 551–560(2000).
34. Böhni, P. C., Daum, G. & Schatzl, G. Import of Proteins intoMitochondria. J. Biol. Chem. 258, 4937–4943 (1983).
35. Hawlitschek, G. et al. Mitochondrial protein import: Identification ofprocessing peptidase and of PEP, a processing enhancing protein. Cell53, 795–806 (1988).
36. Mukhopadhyay, A., Ni, L., Yang, C. S. & Weiner, H. Bacterial signalpeptide recognizes HeLa cell mitochondrial import receptors andfunctions as a mitochondrial leader sequence. Cell. Mol. Life Sci. 62,1890–1899 (2005).
37. Flierl, A. et al. Targeted delivery of DNA to the mitochondrialcompartment via import sequence-conjugated peptide nucleic acid.Mol. Ther. 7, 550–557 (2003).
38. Srivastava, S. & Moraes, C. T. Manipulating mitochondrial DNAheteroplasmy by a mitochondrially targeted restriction endonuclease.Hum. Mol. Genet. 10, 3093–3099 (2001).
39. Tanaka, M. et al. Gene therapy for mitochondrial disease by deliveringrestriction endonuclease Smal into mitochondria. J. Biomed. Sci. 9,534–541 (2002).
40. Weissig, V. et al. DQAsomes: a novel potential drug and gene deliverysystem made from dequalinium. Pharmaceutical Research 15, 334–337 (1998).
41. Weissig, V. & Torchilin, V. P. Towards mitochondrial gene therapy:DQAsomes as a strategy. J. Drug Target. 9, 1–13 (2001).
42. Frantz, M. C. & Wipf, P. Mitochondria as a target in treatment.Environ. Mol. Mutagen. 51, 462–475 (2010).
43. Szewczyk, A., Jarmuszkiewicz, W. & Kunz, W. S. Mitochondrialpotassium channels. IUBMB Life 61, 134–143 (2009).
44. Casellas, P., Galiegue, S. & Basile, A. S. Peripheral benzodiazepinereceptors and mitochondrial function. Neurochem. Int. 40, 475–486(2002).
45. Surinkaew, S., Chattipakorn, S. & Chattipakorn, N. Roles ofMitochondrial Benzodiazepine Receptor in the Heart. Can. J. Cardiol.27, 262.e3-262.e13 (2011).
46. Pei, Y. et al. Efficient syntheses of benzothiazepines as antagonists forthe mitochondrial sodium-calcium exchanger: Potential therapeuticsfor type II diabetes. J. Org. Chem. 68, 92–103 (2003).
47. Ellerby, H. M. et al. Anti-cancer activity of targeted pro-apoptotic
52
peptides. Nat. Med. 5, 1032–1038 (1999). 48. Horton, K. L. & Kelley, S. O. Engineered apoptosis-inducing peptides
with enhanced mitochondrial localization and potency. J. Med. Chem. 52, 3293–3299 (2009).
49. Asayama, S., Kawamura, E., Nagaoka, S. & Kawakami, H. Design of manganese porphyrin modified with mitochondrial signal peptide for a new antioxidant. Mol. Pharm. 3, 468–470 (2006).
50. Zhao, K. et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J. Biol. Chem. 279, 34682–34690 (2004).
51. Szeto, H. H. Mitochondria-targeted cytoprotective peptides for ischemia-reperfusion injury. Antioxid. Redox Signal. 10, 601–619 (2008).
52. Zhao, K., Luo, G., Giannelli, S. & Szeto, H. H. Mitochondria-targeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines. Biochem. Pharmacol. 70, 1796–1806 (2005).
53. Meister, A. & Anderson, M. E. Glutathione. Annu. Rev. Biochem. 52, 711–60 (1983).
54. Anderson, M. E. Glutathione and Glutathione Delivery Compounds. Adv. Pharmacol. 38, 65–78 (1997).
55. Forman, H. J. & Dickinson, D. A. Oxidative signaling and glutathione synthesis. BioFactors 17, 1–12 (2003).
56. Olney, J. W., Zorumski, C., Price, M. T. & Labruyere, J. L-Cysteine, a Bicarbonate-Sensitive Endogenous Excitotoxin. Science 248, 596–599 (1990).
57. Bernard, G. R. N-Acetylcysteine in experimental and clinical acute lung injury. Am. J. Med. 91, S54–S59 (1991).
58. Ortolani, O. et al. The Effect of Glutathione and N-Acetylcysteine on Lipoperoxidative Damage in Patients with Early Septic Shock. Am. J. Respir. Crit. Care Med. 161, 1907–1911 (2000).
59. Yamamoto, M. et al. Protective actions of YM737, a new glutathione analog, against cerebral ischemia in rats. Res. Commun. Chem. Pathol. Pharmacol. 81, 221–32 (1993).
60. Anderson, M. F., Nilsson, M. & Sims, N. R. Glutathione monoethylester prevents mitochondrial glutathione depletion during focal cerebral ischemia. Neurochem. Int. 44, 153–159 (2004).
61. GuÍzar-Sahagún, G. et al. Glutathione monoethyl ester improves functional recovery, enhances neuron survival, and stabilizes spinal cord blood flow after spinal cord injury in rats. Neuroscience 130, 639–649 (2005).
62. Zeevalk, G. D., Manzino, L., Sonsalla, P. K. & Bernard, L. P. Characterization of intracellular elevation of glutathione (GSH) with glutathione monoethyl ester and GSH in brain and neuronal cultures:
53
Relevance to Parkinson’s disease. Exp. Neurol. 203, 512–520 (2007). 63. Zhang, S., Chai, F.-Y., Yan, H., Guo, Y. & Harding, J. J. Effects of N-
acetylcysteine and glutathione ethyl ester drops on streptozotocin-induced diabetic cataract in rats. Mol. Vis. 14, 862–70 (2008).
64. Rajasekaran, N. S., Sathyanarayanan, S., Devaraj, N. S. & Devaraj, H.Chronic depletion of glutathione (GSH) and minimal modification ofLDL in vivo: its prevention by glutathione mono ester (GME) therapy.Biochim. Biophys. Acta - Mol. Basis Dis. 1741, 103–112 (2005).
65. Traverso, N. et al. Role of Glutathione in Cancer Progression andChemoresisstance. Oxid. Med. Cell. Longev. 2013, (2013).
66. Housman, G. et al. Drug resistance in cancer: an overview. Cancers 6,1769–92 (2014).
67. Kunze, T. & Heps, S. Phosphono analogs of glutathione: inhibition ofglutathione transferases, metabolic stability, and uptake by cancercells. Biochem. Pharmacol. 59, 973–981 (2000).
68. Burg, D. et al. Peptidomimetic Glutathione Analogues as Novel γGTStable GST Inhibitors. Bioorg. Med. Chem. 10, 195–205 (2002).
69. Ehrlich, K. et al. Design, synthesis and properties of novel powerfulantioxidants, glutathione analogues. Free Radic. Res. 41, 779–787(2007).
70. Ehrlich, K. et al. Characterization of UPF peptides , members of theglutathione analogues library , on the basis of their effects on oxidativestress-related enzymes. Free Radic. Res. 43, 572–580 (2009).
71. Kairane, C. et al. Diverse Effects of Glutathione and UPF Peptides onAntioxidant Defense System in Human Erythroleukemia Cells K562.Int. J. Pept. 2012, 1-5 (2012).
72. Frankel, A. D. & Pabo, C. O. Cellular uptake of the tat protein fromhuman immunodeficiency virus. Cell 55, 1189–1193 (1988).
73. Green, M. & Loewenstein, P. M. Autonomous functional domains ofchemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55, 1179–1188 (1988).
74. Vivès, E., Brodin, P. & Lebleu, B. A Truncated HIV-1 Tat ProteinBasic Domain Rapidly Translocates through the Plasma Membraneand Accumulates in the Cell Nucleus. J. Biol. Chem. 272, 16010–16017 (1997).
75. Derossi, D., Joliot, A. H., Chassaing, G. & Prochiantz, A. The thirdhelix of the Antennapedia homeodomain translocates throughbiological membranes. J. Biol. Chem. 269, 10444–10450 (1994).
76. Elliott, G. & Hare, P. O. Intercellular Trafficking and Protein Deliveryby a Herpesvirus Structural Protein. Cell 88, 223–233 (1997).
77. Pooga, M., Hällbrink, M., Zorko, M. & Langel, U. Cell penetration bytransportan. FASEB J. 12, 67–77 (1998).
78. Oehlke, J. et al. Cellular uptake of an alpha-helical amphipathic modelpeptide with the potential to deliver polar compounds into the cellinterior non-endocytically. Biochem. Biophys. Acta 1414, 127–139
54
(1998). 79. Lindgren, M., Hällbrink, M., Prochiantz, A. & Langel, Ü. Cell-
penetrating peptides. Trends Pharmacol. Sci. 21, 99–103 (2000). 80. Futaki, S. et al. Arginine-rich peptides. An abundant source of
membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276, 5836–5840 (2001).
81. Hudecz, F., Bánóczi, Z. & Csík, G. Medium-sized peptides as built in carriers for biologically active compounds. Med. Res. Rev. 25, 679–736 (2005).
82. Stewart, K. M., Horton, K. L. & Kelley, S. O. Cell-penetrating peptides as delivery vehicles for biology and medicine. Org. Biomol. Chem. 6, 2242–55 (2008).
83. Pooga, M. & Langel, Ü. in Cell-Peneetrating peptides: Methods in Molecular Biology (ed. Langel, Ü.) 3–28 (Springer, 2015).
84. Gupta, B., Levchenko, T. & Torchilin, V. P. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv. Drug Deliv. Rev. 57, 637–651 (2005).
85. Zhao, M. & Weissleder, R. Intracellular Cargo Delivery Using Tat Peptide and Derivatives. Med. Res. Rev. 24, 1–12 (2004).
86. Goun, E. A., Pillow, T. H., Jones, L. R., Rothbard, J. B. & Wender, P. A. Molecular Transporters: Synthesis of Oligoguanidinium Transporters and Their Application to Drug Delivery and Real-Time Imaging. ChemBioChem 7, 1497–1515 (2006).
87. Koren, E. & Torchilin, V. P. Cell-penetrating peptides: breaking through to the other side. Trends Mol. Med. 18, 385–393 (2012).
88. Agrawal, P. et al. CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides. Nucleic Acids Res. 44, 1098–1103 (2016).
89. Gros, E. et al. A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction. Biochim. Biophys. Acta - Biomembr. 1758, 384–393 (2006).
90. Elmquist, A., Lindgren, M., Bartfai, T. & Langel, Ü. VE-Cadherin-Derived Cell-Penetrating Peptide, pVEC, with Carrier Functions. Exp. Cell Res. 269, 237–244 (2001).
91. Herbig, M. E. et al. Membrane surface-associated helices promote lipid interactions and cellular uptake of human calcitonin-derived cell penetrating peptides. Biophys. J. 89, 4056–66 (2005).
92. Mai, J. C., Shen, H., Watkins, S. C., Cheng, T. & Robbins, P. D. Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate. J. Biol. Chem. 277, 30208–18 (2002).
93. Ter-Avetisyan, G. et al. Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis. J. Biol. Chem. 284, 3370–3378 (2009).
94. Rothbard, J. B., Jessop, T. C., Lewis, R. S., Murray, B. a. & Wender, P. a. Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells. J. Am. Chem. Soc. 126, 9506–9507 (2004).
55
95. Fischer, R., Fotin-Mleczek, M., Hufnagel, H. & Brock, R. Break onthrough to the other side-biophysics and cell biology shed light on cell-penetrating peptides. Chembiochem 6, 2126–2142 (2005).
96. Nakase, I. et al. Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organizationand macropinocytosis. Biochemistry 46, 492–501 (2007).
97. Richard, J. P. et al. Cellular Uptake of Unconjugated TAT PeptideInvolves Clathrin-dependent Endocytosis and Heparan SulfateReceptors. J. Biol. Chem. 280, 15300–15306 (2005).
98. Lehto, T., Ezzat, K., Wood, M. J. A. & EL Andaloussi, S. Peptides fornucleic acid delivery. Adv. Drug Deliv. Rev. 106, 172–182 (2016).
99. Conner, S. D. & Schmid, S. L. Regulated portals of entry into the cell.Nature 422, 37–44 (2003).
100. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R. & Brock,R. A Comprehensive Model for the Cellular Uptake of Cationic Cell-penetrating Peptides. Traffic 8, 848–866 (2007).
101. Jones, A. T. Macropinocytosis: Searching for an endocytic identity androle in the uptake of cell penetrating peptides. J. Cell. Mol. Med. 11,670–684 (2007).
102. Brugnano, J., McMasters, J. & Panitch, A. Characterization ofendocytic uptake of MK2-inhibitor peptides. J. Pept. Sci. 19, 629–38(2013).
103. McMahon, H. T. H. T. & Boucrot, E. Molecular mechanism andphysiological functions of clathrin-mediated endocytosis. Nat. Rev.Mol. Cell Biol. 12, 517–33 (2011).
104. EL Andaloussi, S., Johansson, H. J., Holm, T. & Langel, Ü. A NovelCell-penetrating Peptide, M918, for Efficient Delivery of Proteins andPeptide Nucleic Acids. Mol. Ther. 15, 1820–1826 (2007).
105. Räägel, H. et al. Cell-penetrating peptide secures an efficientendosomal escape of an intact cargo upon a brief photo-induction. Cell.Mol. Life Sci. 70, 4825–39 (2013).
106. Varkouhi, A. K., Scholte, M., Storm, G. & Haisma, H. J. Endosomalescape pathways for delivery of biologicals. J. Control. Release 151,220–8 (2011).
107. Lönn, P. et al. Enhancing Endosomal Escape for Intracellular Deliveryof Macromolecular Biologic Therapeutics. Sci. Rep. 6, 32301 (2016).
108. Ciftci, K. & Levy, R. J. Enhanced plasmid DNA transfection withlysosomotropic agents in cultured fibroblasts. Int. J. Pharm. 218, 81–92 (2001).
109. Abes, S. et al. Endosome trapping limits the efficiency of splicingcorrection by PNA-oligolysine conjugates. J. Control. Release 110,595–604 (2006).
110. Shiraishi, T., Pankratova, S. & Nielsen, P. E. Calcium ions effectivelyenhance the effect of antisense peptide nucleic acids conjugated tocationic tat and oligoarginine peptides. Chem. Biol. 12, 923–9 (2005).
56
111. Ezzat, K. et al. Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides. FASEB J. 26, 1172–1180 (2012).
112. Peiser, L. & Gordon, S. The function of scavenger receptorsexpressed by macrophages and their rolein the regulation of inflammation. Microbes Infect. 3, 149–159 (2001).
113. Arukuusk, P. et al. Differential endosomal pathways for radically modified peptide vectors. Bioconjug. Chem. 24, 1721–32 (2013).
114. Ezzat, K., EL Andaloussi, S., Abdo, R. & Langel, Ü. Peptide-Based Matrices as Drug Delivery Vehicles. Curr. Pharm. Des. 16, 1167–1178 (2010).
115. Dietz, G. P. H. & Bähr, M. Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol. Cell. Neurosci. 27, 85–131 (2004).
116. Snyder, E. L. & Dowdy, S. F. Recent advances in the use of protein transduction domains for the delivery of peptides, proteins and nucleic acids invivo. Expert Opin. Drug Deliv. 2, 43–51 (2005).
117. Kuai, R. et al. Targeted Delivery of Cargoes into a Murine Solid Tumor by a Cell-Penetrating Peptide and Cleavable Poly(ethylene glycol) Comodified Liposomal Delivery System via Systemic Administration. Mol. Pharm. 8, 2151–2161 (2011).
118. Abes, S. et al. Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents. J. Control. Release 116, 304–313 (2006).
119. Eguchi, A. et al. Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nat. Biotechnol. 27, 567–71 (2009).
120. Palm-Apergi, C., Eguchi, A. & Dowdy, S. F. in Cell-Penetrating peptides Methods in Molecular Biology (ed. Langel Ü.) 339–347 (Springer New York, 2011).
121. Boeneman, K. et al. Selecting Improved Peptidyl Motifs for Cytosolic Delivery of Disparate Protein and Nanoparticle Materials. 7, 3778–3796 (2013).
122. Delehanty, J. B. et al. Site-specific cellular delivery of quantum dots with chemoselectively-assembled modular peptides. Chem. Commun. 49, 7878 (2013).
123. Oh, E. et al. PEGylated Luminescent Gold Nanoclusters: Synthesis, Characterization, Bioconjugation, and Application to One- and Two-Photon Cellular Imaging. Part. Part. Syst. Charact. 30, 453–466 (2013).
124. Dekiwadia, C. D., Lawrie, A. C. & Fecondo, J. V. Peptide-mediated cell penetration and targeted delivery of gold nanoparticles into lysosomes. J. Pept. Sci. 527–534 (2012).
125. Garnacho, C., Serrano, D. & Muro, S. A fibrinogen-derived peptide provides intercellular adhesion molecule-1-specific targeting and
57
intraendothelial transport of polymer nanocarriers in human cell cultures and mice. J. Pharmacol. Exp. Ther. 340, 638–47 (2012).
126. Andaloussi, S. E. L. et al. Design of a peptide-based vector , PepFect6, for efficient delivery of siRNA in cell culture and systemically invivo. Nucleic Acids Res. 39, 3972–3987 (2011).
127. Salaklang, J. et al. Superparamagnetic Nanoparticles as a PowerfulSystems Biology Characterization Tool in the Physiological Context.Angew. Chemie Int. Ed. 47, 7857–7860 (2008).
128. Sharma, S. et al. Tumor-Penetrating Nanosystem Strongly SuppressesBreast Tumor Growth. Nano Lett. 17, 1356–1364 (2017).
129. Oh, E. et al. Cellular Uptake and Fate of PEGylated GoldNanoparticles Is Dependent on Both Cell-Penetration Peptides andParticle Size. ACS Nano 5, 6434–6448 (2011).
130. Oliveira, S., van Rooy, I., Kranenburg, O., Storm, G. & Schiffelers, R.M. Fusogenic peptides enhance endosomal escape improving siRNA-induced silencing of oncogenes. Int. J. Pharm. 331, 211–4 (2007).
131. Pan, L. et al. Nuclear-Targeted Drug Delivery of TAT Peptide-Conjugated Monodisperse Mesoporous Silica Nanoparticles. J. Am.Chem. Soc. 134, 5722–5725 (2012).
132. Matsuo, K. et al. Efficient generation of antigen-specific cellularimmunity by vaccination with poly(γ-glutamic acid) nanoparticlesentrapping endoplasmic reticulum-targeted peptides. Biochem.Biophys. Res. Commun. 362, 1069–1072 (2007).
133. Wang, G., Norton, A. S., Pokharel, D., Song, Y. & Hill, R. A. KDELpeptide gold nanoconstructs: promising nanoplatforms for drugdelivery. Nanomedicine Nanotechnology, Biol. Med. 9, 366–374(2013).
134. Sneh-Edri, H., Likhtenshtein, D. & Stepensky, D. IntracellularTargeting of PLGA Nanoparticles Encapsulating Antigenic Peptide tothe Endoplasmic Reticulum of Dendritic Cells and Its Effect onAntigen Cross-Presentation in Vitro. Mol. Pharm. 8, 1266–1275(2011).
135. Ravindran, S., Snee, P. T., Ramachandran, A. & George, A. Acidicdomain in dentin phosphophoryn facilitates cellular uptake:implications in targeted protein delivery. J. Biol. Chem. 288, 16098–109 (2013).
136. Falanga, A. et al. A peptide derived from herpes simplex virus type 1glycoprotein H: membrane translocation and applications to thedelivery of quantum dots. Nanomedicine Nanotechnology, Biol. Med.7, 925–934 (2011).
137. Morris, M. C., Vidal, P., Chaloin, L., Heitz, F. & Divita, G. A newpeptide vector for efficient delivery of oligonucleotides intomammalian cells. Nucleic Acids Res. 25, 2730–2736 (1997).
138. Kim, B. K. et al. Homodimeric SV40 NLS peptide formed by disulfidebond as enhancer for gene delivery. Bioorganic Med. Chem. Lett. 22,
58
5415–5418 (2012). 139. Pollard, H. et al. Polyethylenimine but not cationic lipids promotes
transgene delivery to the nucleus in mammalian cells. J. Biol. Chem. 273, 7507–11 (1998).
140. Li, S. & Huang, L. Nonviral gene therapy: promises and challenges. Gene Ther. 7, 31–34 (2000).
141. Nabel, G. J. et al. Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biologic activity, and lack of toxicity in humans. Proc. Natl. Acad. Sci. U. S. A. 90, 11307–11311 (1993).
142. Tkachenko, A. G. et al. Multifunctional gold nanoparticle-peptide complexes for nuclear targeting. J. Am. Chem. Soc. 125, 4700–4701 (2003).
143. Aronov, O. et al. Nuclear localization signal-targeted poly(ethylene glycol) conjugates as potential carriers and nuclear localizing agents for carboplatin analogues. Bioconjug. Chem. 15, 814–23 (2004).
144. Wagstaff, K. M., Glover, D. J., Tremethick, D. J. & Jans, D. a. Histone-mediated transduction as an efficient means for gene delivery. Mol. Ther. 15, 721–731 (2007).
145. Boelens, J., Lust, S., Offner, F., Bracke, M. E. & Vanhoecke, B. W. The Endoplasmic Reticulum: a target for new anticancer drugs. In Vivo 21, 215–226 (2007).
146. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: The next generation. Cell 144, 646–674 (2011).
147. Galluzzi, L., Larochette, N., Zamzami, N. & Kroemer, G. Mitochondria as therapeutic targets for cancer chemotherapy. Oncogene 25, 4812–4830 (2006).
148. Burchell, V. S. et al. Targeting mitochondrial dysfunction in neurodegenerative disease: Part I. Expert Opin. Ther. Targets 14, 497–511 (2010).
149. Burchell, V. S. et al. Targeting mitochondrial dysfunction in neurodegenerative disease: Part II. Expert Opin. Ther. Targets 14, 497–511 (2010).
150. Cerrato, C. P., Pirisinu, M., Vlachos, E. N. & Langel, Ü. Novel cell-penetrating peptide targeting mitochondria. FASEB J. 29, 4589–4599 (2015).
151. Shokolenko, I. N., Alexeyev, M. F., Ledoux, S. P. & Wilson, G. L. TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells. DNA Repair 4, 511–518 (2005).
152. Yousif, L. F., Stewart, K. M. & Kelley, S. O. Targeting mitochondria with organelle-specific compounds: Strategies and applications. ChemBioChem 10, 1939–1950 (2009).
153. Buondonno, I. et al. Mitochondria-Targeted Doxorubicin: A New Therapeutic Strategy against Doxorubicin-Resistant Osteosarcoma. Mol. Cancer Ther. 15, 2640–2652 (2016).
59
154. Friedmann, T. Stanfield Rogers: Insights into Virus Vectors andFailure of an Early Gene Therapy Model. Mol. Ther. 4, 285–288(2001).
155. Friedmann, T. & Roblin, R. Gene therapy for human genetic disease?Science 175, 949–55 (1972).
156. Kuska, B. Results From First Human Gene Therapy Clinical Trial.National Human Genome Research Institute 1 (1995).
157. Candotti, F. et al. Gene therapy for adenosine deaminase-deficientsevere combined immune deficiency: clinical comparison of retroviralvectors and treatment plans. Blood 120, 3635–46 (2012).
158. European Medicines Agency. Guideline on the quality, non-clinicaland clinical aspects of gene therapy medicinal products. (2015).
159. FDA. Guidance for Industry: Gene Therapy Clinical Trials –Observing Subjects for Delayed Adverse Events. (2006).
160. Kumar, S. R., Markusic, D. M., Biswas, M., High, K. A. & Herzog, R.W. Clinical development of gene therapy: results and lessons fromrecent successes. Mol. Ther. - Methods Clin. Dev. 3, 16034 (2016).
161. Hanna, E., Rémuzat, C., Auquier, P. & Toumi, M. Gene therapiesdevelopment: slow progress and promising prospect. J. Mark. accessHeal. policy 5, 1265293 (2017).
162. Dowdalls, J. $1-million price tag set for Glybera gene therapy. Nat.Biotechnol. 33, 217–218 (2015).
163. Thomas Scott, C. & DeFrancesco, L. Gene therapy’s out-of-bodyexperience. Nat. Biotechnol. 34, 600–607 (2016).
164. Dowling, J. J. Eteplirsen therapy for Duchenne muscular dystrophy:skipping to the front of the line. Nat. Rev. Neurol. 12, 675–676 (2016).
165. FDA. FDA approves novel gene therapy to treat patients with a rareform of inherited vision loss. (2017).
166. Rangarajan, S. et al. AAV5–Factor VIII Gene Transfer in SevereHemophilia A. N. Engl. J. Med. 377, 2519–2530 (2017).
167. Zamecnik, P. C. & Stephenson, M. L. Inhibition of Rous sarcoma virusreplication and cell transformation by a specific oligodeoxynucleotide.Proc. Natl. Acad. Sci. U. S. A. 75, 280–4 (1978).
168. Stein, C. A. et al. Efficient gene silencing by delivery of locked nucleicacid antisense oligonucleotides, unassisted by transfection reagents.Nucleic Acids Res. 38, e3 (2010).
169. Durzyńska, J. et al. Viral and other cell penetrating peptides as vectorsof therapeutic agents in medicine.
170. Phillips, M. I. Antisense inhibition and adeno-associated viral vectordelivery for reducing hypertension. Hypertens. 29, 177–87 (1997).
171. Hardee, C. L., Arévalo-Soliz, L. M., Hornstein, B. D. & Zechiedrich,L. Advances in Non-Viral DNA Vectors for Gene Therapy. Genes. 8,(2017).
172. Lehto, T., Kurrikoff, K. & Langel, Ü. Cell-penetrating peptides for thedelivery of nucleic acids. Expert Opin. Drug Deliv. 9, 823–36 (2012).
60
173. Shim, G., Kim, M.-G., Park, J. Y. & Oh, Y.-K. Application of cationic liposomes for delivery of nucleic acids. Asian J. Pharm. Sci. 8, 72–80 (2013).
174. Jiang, K. Biotech comes to its ‘antisenses’ after hard-won drug approval. Nat. Med. 19, 252–252 (2013).
175. Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014).
176. Mojica, F. J. M. & Montoliu, L. On the Origin of CRISPR-Cas Technology: From Prokaryotes to Mammals. Trends Microbiol. 24, 811–820 (2016).
177. Levin, A. V. Ethical considerations in gene therapy. Ophthalmic Genet. 37, 249–251 (2016).
178. Robillard, J. M. et al. Utilizing social media to study information-seeking and ethical issues in gene therapy. J. Med. Internet Res. 15, e44 (2013).
179. Ormandy, E. H., Dale, J. & Griffin, G. Genetic engineering of animals: ethical issues, including welfare concerns. Can. Vet. J. 52, 544–50 (2011).
180. Freire, J. E. da C. et al. Bioethical conflicts of gene therapy: a brief critical review. Rev. Assoc. Med. Bras. 60, 520–524 (2014).
181. Kaiser, E. T. The 1984 Nobel Prize in Chemistry. Science 226, 1151–1153 (1984).
182. Masters, J. R. HeLa cells 50 years on: the good, the bad and the ugly. Nat. Rev. Cancer 2, 315–319 (2002).
183. Williams, R. L. et al. Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment. Cell 57, 1053–1063 (1989).
184. Omidi, Y. et al. Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies. Brain Res. 990, 95–112 (2003).
185. Allen, M., Bjerke, M., Edlund, H., Nelander, S. & Westermark, B. Origin of the U87MG glioma cell line: Good news and bad news. Sci. Transl. Med. 8, 354re3 (2016).
186. Puck, T. T., Cieciura, S. J. & Robinson, A. Genetics of somatic mammalian cells. J. Exp. Med. 108, 945–956 (1958).
187. Scaduto, R. C. & Grotyohann, L. W. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys. J. 76, 469–477 (1999).
188. Nobbmann, U. et al. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol. Genet. Eng. Rev. 24, 117–128 (2007).
189. Strutt, H. J. W. On the scattering of light by small particles. Philisophical Mag. J. Sci. 41, 447–454 (1871).
190. Urnes, P. & Doty, P. Optical rotation and the conformation of
61
polypeptides and proteins. Adv. Protein Chem. 16, 401–544 (1961). 191. Urry, D. W. Protein conformation in biomembranes: optical rotation
and absorption of membrane suspensions. Biochim. Biophys. Acta 265,115–68 (1972).
192. Greenfield, N. J. Analysis of the kinetics of folding of proteins andpeptides using circular dichroism. Nat. Protoc. 1, 2891–9 (2006).
193. Greenfield, N. J. Using circular dichroism spectra to estimate proteinsecondary structure. Nat. Protoc. 1, 2876–2890 (2007).
194. Chou, P. & Fasman, G. Prediction of protein conformation.Biochemistry 13, 222–245 (1974).
195. Micsonai, A. et al. Accurate secondary structure prediction and foldrecognition for circular dichroism spectroscopy. Proc. Natl. Acad.Sci.U. S. A. 112, E3095–E3103 (2015).
196. Mahapatra, S. et al. The D arm of tRNA(Tyr) is necessary andsufficient for import into Leishmania mitochondria in vitro. NucleicAcids Res. 26, 2037–2041 (1998).
197. Nath Bhattacharyya, S., Chatterjee, S. & Adhya, S. MitochondrialRNA Import in Leishmania tropica: Aptamers Homologous toMultiple tRNA Domains That Interact Cooperatively orAntagonistically at the Inner Membrane. Mol. Cell. Biol. 22, 4372–4382 (2002).
198. Bhattacharyya, S. N., Mukherjee, S. & Adhya, S. Mutations in a tRNAimport signal define distinct receptors at the two membranes ofLeishmania mitochondria. Mol. Cell. Biol. 20, 7410–7417 (2000).
199. Kang, S.-H., Cho, M.-J. & Kole, R. Up-Regulation of Luciferase GeneExpression with Antisense Oligonucleotides: hImplications andApplications in Functional Assay Development. Biochemistry 37,6235–6239 (1998).
200. Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedureand some applications. Biotechnology 24, 145–149 (1979).
201. Hirano, S. in Nanotoxicity: Methods and Protocols (ed. Reineke, J.)87–97 (Humana Press, 2012).
202. Nakamura, M., Yamazaki, I., Ohtaki, S. & Nakamura, S.Characterization of One- and two-electron oxidations of glutathionecoupled with lactoperoxidase and thyroid peroxidase reactions. J. Biol.Chem. 261, 13923–13927 (1986).
203. Valoti, M., Tipton, K. F. & Sgaragli, G. P. Oxidative ring-coupling oftyrosine and its derivatives by purified rat intestinal peroxidase.Biochem Pharmacol 43, 945–951 (1992).
204. Nagy, P., Kettle, A. J. & Winterbourn, C. C. Superoxide-mediatedFormation of Tyrosine Hydroperoxides and Methionine Sulfoxide inPeptides through Radical Addition and Intramolecular OxygenTransfer. J. Biol. Chem. 284, 14723–14733 (2009).
205. Sturgeon, B. E. et al. The fate of the oxidizing tyrosyl radical in the
62
presence of glutathione and ascorbate: Implications for the radical sink hypothesis. J. Biol. Chem. 273, 30116–30121 (1998).
206. Pong, K., Doctrow, S. R., Huffman, K., Adinolfi, C. a & Baudry, M. Attenuation of staurosporine-induced apoptosis, oxidative stress, and mitochondrial dysfunction by synthetic superoxide dismutase and catalase mimetics, in cultured cortical neurons. Exp. Neurol. 171, 84–97 (2001).
207. Jauslin, M. L., Meier, T., Smith, R. a J. & Murphy, M. P. Mitochondria-targeted antioxidants protect Friedreich Ataxia fibroblasts from endogenous oxidative stress more effectively than untargeted antioxidants. FASEB J. 17, 1972–1974 (2003).
208. Pias, E. K. et al. Differential Effects of Superoxide Dismutase Isoform Expression on Hydroperoxide-induced Apoptosis in PC-12 Cells. J. Biol. Chem 278, 13294–301 (2003).
