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Mitochondrial gene therapy: Advances in mitochondrial gene cloning, plasmid production and nanosystems targeted to mitochondria Eduarda Coutinho, Cátia Batista, Fani Sousa, João Queiroz, and Diana Costa Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00823 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 17, 2017

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Molecular Pharmaceutics

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Mitochondrial gene therapy: Advances in mitochondrial gene cloning, plasmid

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production and nanosystems targeted to mitochondria

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Eduarda Coutinho, Cátia Batista, Fani Sousa, João Queiroz and Diana Costa

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CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante

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D. Henrique, 6200-506 Covilhã, Portugal

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Corresponding author:

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Diana Rita Barata Costa

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Universidade da Beira Interior

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6201-001 Covilhã

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Portugal

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E-mail address: [email protected]

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Keywords: mitochondrial gene cloning; non-viral vectors; nanoparticles; targeted delivery; mitochondrial gene therapy.

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Abstract

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Mitochondrial gene therapy seems to be a valuable and promising strategy to treat

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mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA,

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along with its affinity to the site of mitochondria can be considered a powerful tool in

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the reestablishment of normal mitochondrial function. In line with this and for the first

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time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in

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multi-copy pCAG-GFP plasmid, used to transform E. coli. This mitochondrial gene

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based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization

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of nanoparticles with polymers, such as, cellulose or gelatin enhances their overall

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properties and performance for gene therapy. The fluorescence arising from rhodamine

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nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-

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ND1 based nanoparticles cell internalization and mitochondria targeting. The

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quantification of GFP expression strongly supports this finding. This work highlights

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the viability of gene therapy based on mitochondrial DNA instigating further in vitro

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research and clinical translation.

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Introduction

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Human mitochondrion is a mobile and dynamic cytoplasmic organelle in virtue of

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frequent fusion and fission cycle in response to cell needs and environment.

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Mitochondrial fusion contributes to regulate mitochondrial function and avoid the

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accumulation of mitochondrial mutations during aging while mitochondrial fission acts

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in the elimination of damaged organelles through autophagy process. Therefore, these

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processes contribute for normal mitochondrial function and optimize bioenergetic

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metabolism.1,2 Mitochondria are involved in cellular signaling, ion homeostasis, in the

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metabolism of aminoacids, lipids, steroids, cholesterol and nucleotides, as well as, in the

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control of both cell cycle and cell growth.3,4 It has a major role in the conversion of food

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energy into chemical energy (ATP) by the use of mitochondrial respiratory chain, which

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consists in four enzymatic complexes.5,6 Complexes I, III and IV are energy coupling

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centers. These complexes, the complex V and the electron carriers ubiquinone and

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cytochrome c, make the oxidative phosphorylation system which provides ATP

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according to cell needs. Besides this, the active role of mitochondria in apoptosis is well

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recognized in mammals.7,8 This programmed form of cell death involves the activation

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of caspase proteases that dismantle cells and signal efficient phagocytosis of apoptotic

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bodies. To date, two main pathways for apoptosis are well known: the extrinsic or death

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receptor pathway and the intrinsic or mitochondrial pathway, the latter being activated

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in response to death stimuli, including DNA damage, chemotherapeutic agents, serum

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starvation and UV radiation. All of these stimuli can introduce alterations in the inner

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mitochondrial membrane leading to the opening of the mitochondrial permeability

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transition (MPT) pore and to the loss of the mitochondrial transmembrane potential;

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both, promoting the delivery of pro-apoptotic proteins from the intermembrane site into

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the cytosol. Moreover, both the control and regulation of these apoptotic mitochondrial

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phenomena are intimately related with the action of B-cell lymphoma-2 (BCL-2) family

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of proteins.9

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Mitochondrial DNA (mtDNA) is a double stranded and circular molecule with

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approximately 16 kbp and contains 37 genes encoding 13 polypeptides that take part in

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the oxidative phosphorylation chain, 2 rRNAs and 22 tRNAs, all exclusive to the

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mitochondria. Seven proteins (ND1 to ND6 and ND4L) are integrated in subunits of

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complex I, one is involved in complex III, three are included in the complex IV and two

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are part of ATP synthase. The other genes left contain information related with the

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translational machinery of the mitochondrial genome.10,11 In the nucleus are encoded the 3 ACS Paragon Plus Environment

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genes responsible for the control of mitochondria. Although accounting for only 1% of

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total DNA, mutations in this genome have been linked to a large variety of metabolic

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and neuromuscular degenerative syndromes that involves tissues requiring high levels

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of energy, such as, heart and the brain, and the endocrine and nervous systems.12,13

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Mutations involving complex I encoding genes frequently cause mitochondrial

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disorders characterized by diverse clinical phenotypes related with severe childhood

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metabolic dysfunctions, such as progressive cardiomyopathy, encephalopathy,

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leukodystrophy, Leigh´s syndrome or ragged red fibbers syndrome and premature age-

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related symptoms.14-17 Furthermore, mutations and/or the great polymorphism

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occurrence in complex I genes are associated with Parkinson and Alzheimer´s diseases,

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diabetes and even the propensity for cancer.18-23

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Therefore, due to its relevant role in several cellular processes ranging from apoptosis,

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bioenergetics and redox metabolism to diseases related with mtDNA mutations,

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mitochondria revealed to be a promising therapeutic target.9,24-26

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Considering, in particular, pathologies arising from mtDNA mutations current

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therapeutic approaches are largely supportive rather than curative, being innefective.27

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There is, thus, a clear requirement for alternative strategies as can be the development of

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an innovative gene therapeutic vector that can be produced and distributed in a large

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scale for the reestablishment of normal mitochondrial function in mutated cells. Gene

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therapy brings a new perspective of cure, is more economical and convenient because it

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provides higher targeting and prolonged duration of action.28-31 Mitochondrial gene

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therapy can be applied through different strategies. The first considers the expression of

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a gene in the nucleus, followed by its synthesis in the cytosol and, thereafter, targeting

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and protein imported into the site of mitochondrion. Some groups, in most cases using

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viral vectors, developed this strategy and relevant progresses have been made with

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clinical translation, namely concerning the treatment of Leber hereditary optic

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neuropathy (LHON),32-34 and led to the creation of suitable animal models for mtATP6

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mutations.35 Although powerful technique, the allotopic expression can have some

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limitations such as, the difficulty of mitochondria import of more hydrophobic

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proteins36 and apparent complementation attributed to forced revertants of the original

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mtDNA mutations.37 Therefore, gene-to-gene variability can occur and limit the success

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of allotopic expression. Additionally, besides the great transfection efficiency achieved

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by using viral systems, their antigenicity, oncogenic effects and instability of storage

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limited this therapy. Considering these drawbacks, the direct transfection of 4 ACS Paragon Plus Environment

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mitochondria, envisioned and pioneered by V. Weissig and co-workers,38-43 appears as a

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promising alternative tool for mitochondrial gene therapy. For the viability of the direct

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delivery of therapeutic genes into the mitochondria, the formulation of a suitable DNA

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carrier with mitochondrial targeting ability is imperative. In this context, the

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development of a human mitochondrial gene vector has appeared as a very challenging

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step. Some groups devoted their attention to the mitochondrial genome cloning with

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limited success. Progresses have been made in the cloning of mouse mitochondrial

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genome in Escherichia coli;44-46 in particular, the use of homologous recombination in

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Saccharomyces cerevisiae to obtain a suitable clone before shuttling back to E. coli

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gives rise to stable full-sized clones.45 An interesting approach, using in vitro

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transposition reaction, showed to be appropriate for engineering mitochondrial genomes

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and for additional in organello analysis.47 More recently, Bigger et al. found that human

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mitochondrial DNA is clonable in yeast in a single-copy centromeric plasmid.48

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Moreover, to assure the intracellular access, protection and bioavailability of the

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produced mitochondrial gene vector, its encapsulation into nanocarriers is mandatory. In

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this sense, non-viral therapy arises as a remarkable improvement due to the absence of

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immune response, ease and variability of preparation and unlimited DNA-carrying

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capacity of synthetic vehicles. The design of mtDNA based delivery systems also

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brought enormous challenges. Although poorly studied area, some outstanding

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researchers increased our knowledge in the area of mitochondrial therapy, of which,

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perhaps the contribution of Weissig´s research team was the most significant as it opens

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an entire new route of possibilities for therapies centered in this organelle.38-43 Weissig

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formulated advanced mitochondria-targeted delivery systems, such as dequalinium-

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based liposome-like vesicles,43 for the release of plasmid DNA and drugs to

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mitochondria. Another set of interesting studies also contributed to the evolution of this

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field;49-51 we added to this topic a report using model plasmids encapsulated into

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nanoparticles with mitochondria affinity.52 Moreover, Lyrawati et al. progressed in the

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expression of GFP in mammalian mitochondria by using an artificial mitochondrial

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genome where GFP has been recoded.53

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In this work we bring relevant novelty by cloning, for the first time, the mitochondrial

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gene ND1 (mitochondrially encoded NADH dehydrogenase 1 protein) in E. coli, being

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stably maintained in pCAG-GFP plasmid. Mitochondrial affinity pCAG-GFP-ND1

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based nanoparticles have been designed and conceived by a co-precipitation method.

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The pCAG-GFP-ND1 nanoparticles allowed the cellular uptake and successfully 5 ACS Paragon Plus Environment

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targeted mitochondria of neuronal N2a cells. This finding has been well corroborated by

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fluorescence confocal microscopy. The findings reported herein are a strong

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achievement to extend and intensify the research in mitochondrial gene therapy

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implementation through an integrative approach that conjugates the production of a

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mitochondrial gene plasmid with the transfection efficiency of a new pDNA delivery

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system for the translation into clinical applications, bringing novel perspectives of cure

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for mitochondrial disorders.

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Materials and Methods

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Materials. Anhydrous magnesium chloride, anhydrous potassium chloride, anhydrous

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sodium chloride, anhydrous calcium chloride, anhydrous sodium carbonate of analytical

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grade, α cellulose powder (MW: 162.4 g mol-1), ethylenediamine tetra acetic acid

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(EDTA),

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dimethylsulfoxide (DMSO), IGEPAL, gelatin and rhodamine 123 were obtained from

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Sigma-Aldrich (St Louis, MO, USA). Agarose and GreenSafe Premium were obtained

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from NZYTech Lda. (Lisbon, Portugal). All solutions were freshly prepared using water

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ultra-pure grade, purified with a Milli-Q system from Millipore (Billerica, MA, USA).

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Normal Human Dermal Fibroblast (NHDF) adult donor cells, Ref. C-12302

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(cryopreserved cells), mouse brain neuroblastoma cells (N2a) and HeLa cells were

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purchased from PromoCell, Invitrogen and ATCC (Middlesex, UK), respectively.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT),

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Extraction of human DNA with enrichment of mtDNA. 5 ml of peripheral human

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blood from donors were collected in EDTA vacutainers and a method adapted from

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Ahmad et al. was followed.54 The blood was transferred to a centrifuge tube and 5 mL

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of solution TKM1 (10 mM Tris-HCl pH 7.6, 10 mM MgCl2, 2 mM EDTA) was added.

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To that 100 µL of IGEPAL was added, mixed in the vortex and incubated for 10

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minutes at room temperature for complete lysis of erythrocytes. We then proceeded

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with centrifugation at 800 g for 20 min. The obtained pellet was resuspended in TKM1

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buffer (5 mL) with 100 µL of IGEPAL and centrifuged again. The supernatant from the

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first and the second centrifugation was then retained in a sterile 50 mL centrifuge tube 6 ACS Paragon Plus Environment

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Molecular Pharmaceutics

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and centrifuged at 15 000 g, at 4 ºC for 30 min to sediment the mitochondrial pellet.

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This pellet was washed twice with TKM1, transferred to a 2 mL Eppendorf tube and

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suspended in 500 mL of TKM2 buffer (Tris-HCl 10 mM pH 7.6, 10 mM KCl, 10 mM

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MgCl2, 0.4 M NaCl and 2 mM EDTA). Thereafter, 100 µL of SDS 10% was added and

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incubated at 55 ºC overnight. Salting out of proteins was promoted by addition of 200

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µL of NaCl 6 M and centrifugation at 12 000 g for 20 min. The supernatant was

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transferred to Falcon tubes and twice volume of 100% ethanol was added, and

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centrifuged at 12 000 g for 5 min for complete precipitation of mtDNA pellet. This

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pellet was washed twice with 70% ethanol, dried and hydrated with 200 µL of TE

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buffer.

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Amplification and sequencing of mitochondrial DNA enriched fraction. In order to

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confirm the enriched mitochondrial fraction of the DNA samples, a master fragment of

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2544 bp including the mtND1 gene sequence, was amplified by polymerase chain

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reaction (PCR) together with another sample of human genomic DNA extracted with a

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standard method55 that does not promote the enrichment in the mtDNA fraction. In

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brief, 100–200 ng of mtDNA were used in 15µL reactions containing 25 pmol of each

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primer, 1 U of DreamTaq Green DNA Polymerase (Thermo Fisher Scientific, Waltham,

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MA, USA), 200 µM of each dNTP, and 2.0 mM MgCl2 and reactions were performed in

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a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc, Hercules, California, USA ). Due

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to its larger size, this master fragment was subdivided into 4 internal fragments by

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nested-PCR using previously described primers,56 this strategy allowed sequencing of

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the total mtDNA master fragment and increased the specificity of the template

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amplified. The PCR products were analysed by electrophoresis in 1% agarose gel

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stained with GreenSafe Premium (NZYTech, Lda. Lisbon, Portugal) and were cleaned

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up prior to sequencing using the enzymatic purification method (FastAP™ and Exo I,

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Thermo Fisher Scientific, Waltham, MA, USA). Direct sequencing of both strands of

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the PCR products was carried out on a GenomeLabTM GeXP sequencer, using the CEQ

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Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Fullerton, CA,

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USA). Sequence data were analysed with GenomeLab System Beckman Coulter version

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10.2 software and matched with MITOMAP reference sequence (NC_012920,

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GenBank). The mtND1 gene was amplified by PCR directly from the mtDNA sample,

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using the primers mt_MF_F4_Fw and mtND1_Rv4 listed in Table 1 of Supporting

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Information (Table 1 SI), resulting in a fragment of 1127 bp. This fragment was 7 ACS Paragon Plus Environment

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sequenced by Sanger sequencing, as previously described, with the same pair of primers

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to confirm the correct sequence of the mtND1 gene and discard the presence of any

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mutation introduced by PCR amplification.

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Cloning of mtND1 into pGEM®-T Easy Vector. DH5α, XL1-B and JM109 E. coli

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strains were made competent using an adapted protocol by Inoue et al..57 The mtND1

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PCR product was purified with GRS PCR & Gel Band Purification Kit (GRiSP, Porto,

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Portugal), quantified on a NanoPhotometer™ (Implen, Inc; Westlake Village, CA,

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USA) and directly ligated into pGEM®-T Easy Vector System I (Promega, Madison,

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Wisconsin, USA) accordingly with the manufacturer’s instructions. The ligation

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reaction was used to transform 100 µL of JM109 competent cells. Shortly, the mixtures

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were incubated on ice for 30 min, then heat shocked at 42ºC during 45 sec and

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incubated on ice for 2 min. After this, 200 µL of LB-Broth medium was added and the

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cells were incubated during 2h at 37ºC with orbital shaking of 250 rpm. The total

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volume was spread on LB-agar/Ampicillin plates (100 µg/mL) and incubated at 37ºC

16

overnight. A negative control was performed with JM109 cells only. Some of the

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colonies obtained were picked and grown in 3 mL LB/Ampicillin (100 µg/mL) during

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24 h at 37ºC and 250 rpm. The cells were then harvested and the recombinant plasmids

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were purified using Wizard Plus SV Minipreps DNA Purification System (Promega,

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Madison, Wisconsin, USA). The proper insertion of mtND1 gene into pGEM®-T

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plasmid was verified by electrophoretic analysis. The recombinant plasmid with the

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expected length were amplified by PCR using T7 and SP6 specific primers and

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nucleotide sequence of recombinant insert was confirmed by automated DNA

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sequencing, as previously described (data not shown).

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Cloning of mtND1 into pCAG-GFP vector. After cloning the mtND1 gene into

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pGEM®-T Easy Vector was transferred into a mammalian expression vector, the

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pCAG-GFP that was a gift from Connie Cepko (Addgene plasmid # 11150).58 The

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mtND1 gene was amplified by PCR, as previously described, but now with specific

30

primers including the SmaI and XbaI restriction enzymes recognition sites (Table 1 SI).

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After purification with GRS PCR & Gel Band Purification Kit (GRiSP, Porto,

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Portugal), the PCR product was sequentially digested with both enzymes, as well the

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pCAG-GFP vector, accordingly with the supplier recommendations (Takara Bio Inc.,

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Otsu, Japan). After digestion, the products were again purified and quantified on a 8 ACS Paragon Plus Environment

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NanoPhotometer™ (Implen, Inc; Westlake Village, CA, USA). For the ligation of the

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constructs different insert:vector ratios (1:3; 1:1 and 3:1) were tested and the reaction

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was performed with the DNA Ligation Kit (Takara Bio Inc., Otsu, Japan) during 4 h at

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room temperature. After this time, the ligation products were used to transform 100 µL

5

of JM109 competent cells by the heat shock method as described above. The

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transformed cells were plated in LB-agar/Ampicillin plates (100 µg/mL) and incubated

7

at 37ºC overnight. The isolated colonies were picked and incubated in 3mL of liquid

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LB/ampicillin for 24 h, then the cells were harvested and the recombinant plasmid

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purified with the Wizard Plus SV Minipreps DNA Purification System (Promega,

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Madison, Wisconsin, USA). In order to evaluate the presence of the mtND1 gene into

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the pCAG-GFP plasmid, we performed a PCR using a forward primer of the insert

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(mt_Fw4) and a reverse primer of the vector (EGFP-N), the expected product has 1127

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bp. The plasmids with positive amplification of mtND1 were then sequenced by Sanger

14

sequencing method carried out on a GenomeLabTM GeXP sequencer, using the CEQ

15

Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Fullerton, CA,

16

USA). The data were analysed with GenomeLab System Beckman Coulter version 10.2

17

software and matched with MITOMAP reference sequence (NC_012920, GenBank) in

18

order to confirm the correct sequence of mtND1 gene inserted in the mammalian

19

expression plasmid pCAG-GFP. These positive plasmids were also digested with SmaI

20

and XbaI restriction enzymes to confirm the excision of the insert with the same

21

molecular weight of the mtND1 PCR product.

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Plasmid DNA production studies. To evaluate the strain efficiency to produce higher

24

pCAG-GFP-ND1 yields, the plasmid was produced in E. coli DH5α, XL1B and JM109

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strains in 125 mL of Terrific Broth (TB) medium (20 g/L tryptone, 24 g/L yeast extract,

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4 mL/L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) in 500 mL Erlenmeyer

27

supplemented with 100 µg/mL ampicillin and 50 µg/mL nalidixic acid at 37 ºC in an

28

orbital shaker at 250 rpm. As E. coli DH5α, XL1-B and JM109 strains are constitutively

29

resistant to nalidixic acid, its use guarantees the growth of desired cells preventing

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contamination with other cell types. The fermentations were carried out for 9.5 h and

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samples of the culture media with an OD of 0.4 were taken every one and half hour. The

32

samples were centrifuged at 13,000 rpm for 10 min in a Mikro 20 centrifuge (Hettich

33

Centrifuges, UK), the supernatant was removed and cells were stored at -20 ºC for

34

following purification. Three independent studies were made for each strain. The 9 ACS Paragon Plus Environment

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purification of plasmid DNA samples for the yield studies was carried out using the

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GeneJet Plasmid Miniprep Kit (Thermo Scientific, Waltham, MA USA). This kit is

3

suitable for a rapid and economic small-scale preparation and ensures high quality

4

plasmid DNA from recombinant E. coli cultures.

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Purification of pCAG-GFP-ND1 for nanoparticles production. NZYtech maxiprep

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(NZYTech, Lda. Lisbon, Portugal) kit is designed for the rapid, large-scale preparation

8

of highly pure plasmid DNA from recombinant E. coli strains. Previously harvested

9

cells were ressuspended and a SDS/alkaline lysis was promoted. A neutralization

10

solution was added and the sample was centrifuged for 30 min at 20.000 x g at 4ºC. The

11

supernatant was transfered to a new tube and a 15 min centrifugation was made. The

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lysate was applied to the NZYTech Maxi Colunm and the elution occurred by gravity

13

flow. The column was washed to remove contaminants and finally the pDNA was

14

eluted. Precipitation of the eluted pDNA was promoted by adding 0.7 volumes of room-

15

temperature isopropanol. A 20 min centrifugation at 15 000 g was made and then pDNA

16

was ressuspended in a pH 7.5 TE buffer. The plasmid yield was determined by

17

spectrophotometry at 260 nm and its integrity was confirmed by agarose gel

18

electrophoresis. 500 µL aliquots of pDNA at 100 µg/mL were prepared and stored at -

19

80ºC.

20 21

Agarose gel electrophoresis. Agarose gel electrophoresis was performed to evaluate

22

the size and conformation of the purified pCAG-GFP plasmid, to evaluate the size of

23

mtND1 gene, the ligation product and to verify the enzymatic digestion of the ligation

24

product. The electrophoresis was carried out using a gel with 1% agarose and 1 µg/mL

25

GreenSafe Premium and it was run at 150 V for 30 min in TAE buffer (40 mM Tris

26

base, 20 mM acetic acid and 1 mM EDTA, pH 8.0). The gel visualization was made in

27

UVItec Gel documentation system under UV light (UVItec Limited, Cambridge, United

28

Kingdom).

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Preparation of ND1 plasmid DNA nanoparticles. Nanoparticles were synthesized by

31

using a co-precipitation protocol. Plasmid DNA solution containing 5 or 10 µg of

32

pCAG-GFP-ND1, 120 µL of CaCl2 solution (0.03 g mL-1) and 7.5 or 15 µL rhodamine

33

123 (Rho123) were mixed and then diluted with deionized water to make a solution A

34

with a total volume of 290 µL. 255 µL of Na2CO3 solution (0.0425 mg mL-1) was mixed 10 ACS Paragon Plus Environment

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Molecular Pharmaceutics

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together with 1 mg mL-1 of cellulose or 1 mg mL-1 of gelatin and then diluted with

2

deionized water to make solution B with a total volume of 260 µL. Solution A was

3

added dropwise with a micropipette to solution B to form the nanoparticles. The final

4

solution, solution C, was centrifuged at 10 000 rpm for 15 min and the pellet contained

5

the ND1 pDNA based nanoparticles.

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Morphology, size and surface charges of nanoparticles. The morphology of pDNA

8

based particles was investigated by Scanning Electron Microscopy (SEM). After

9

formation, nanoparticles were centrifuged (10.000 g, 20 min., 25 ºC) and the resultant

10

pellet recovered and suspended in a solution containing 20 µL deionized water with 20

11

µL tungsten. This solution was set in roundly shaped cover-slip and dried overnight at

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25 ºC. The samples were mounted on aluminum supports and sputter coated with gold

13

using an Emitech K550 (London, England) sputter coater. An Hitachi S-2700 (Tokyo,

14

Japan) scanning electron microscope, operating at an accelerating voltage of 20 kV at

15

various magnifications, was employed to analyze the samples of nanoparticles.

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The average particle size and the zeta potential of pDNA particles were determined, at

17

25 ºC, using a Zetasizer nano ZS. Dynamic light scattering using a He-Ne laser 633 nm

18

with non-invasive backscatter optics (NIBS) and electrophoretic light scattering using

19

M3-PALS laser technique (Phase analysis Light Scattering) were applied for particle

20

size and surface charges evaluation, respectively. The Malvern zetasizer software v 6.34

21

was used. The average values of size and zeta potential were calculated with the data

22

obtained from three measurements ± SD.

23 24

Determination of encapsulation efficiency. After synthesis, nanoparticles were

25

centrifuged for 10 min and the obtained supernatant was recovered. The amount of non-

26

bound pDNA was determined spectrophotometrically measuring the absorbance at 260

27

nm using a NanoPhotometer™ (Implen, Inc; Westlake Village, CA, USA). To

28

determine the encapsulation efficiency, the following equation was applied:

29

EE(%) = [(Total Amount of pDNA –Non-bound pDNA)/ Total amount of pDNA] x100

30 31

In vitro transfection studies. Mouse N2a neuroblastoma cells were incubated at 37˚C

32

in an atmosphere containing 5% CO2 and maintained in DMEM containing 10% fetal

33

calf serum, 1 mM glutamine, penicillin G (100 U mL-1) and streptomycin (100 µg mL-

34

1

). Cells were seeded in 75 cm3 T-flasks until confluence (~ 90 %) was achieved. The 11 ACS Paragon Plus Environment

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1

cells were then sub-cultivated on 0.18% trypsin (1:250) with 5 mM EDTA. For

2

transfection, the cells were cultivated in 24 well-plates at a density of 4 x 104 cells/well

3

in 1mL of DMEM and incubated for one day. The complete medium was replaced by

4

500 µL of medium supplemented with 10% FBS and without antibiotic. All cells were

5

then incubated for a period of 8 h and then supplemented with DMEM medium. On the

6

day of transfection, confluent neuroblastoma cells were transfected with pDNA based

7

vectors. The pCAG-GFP-ND1/Rho/cellulose or gelatin based nanoparticles (50 µL)

8

were added to each well and the cells incubated at 37 ˚C for further studies.

9 10

Isolation of Mitochondria. Mitochondria have been isolated from N2a cells after

11

transfection occurred. The Mitochondria Isolation Kit for Cultured Cells (#89874,

12

Thermo Fisher Scientific Inc., Rockford, USA) was employed. This experimental

13

protocol consists in a method based on reagents and on differential centrifugation,

14

separating the mitochondrial and cytosolic fractions. Briefly, 800 µL of Mitochondria

15

Isolation Reagent A was added to N2a cells (3 x 106) and incubated on ice for exactly 2

16

min. Then, 10 µL of Mitochondria Isolation Reagent B was added and cells were

17

vortexed at maximum speed for 5 sec, incubated on ice for 5 min and vortexed at

18

maximum speed every minute. Then, Mitochondria Isolation Reagent C (800 µL) was

19

added, the samples were centrifuged at 700 g for 10 min at 4˚C and the supernatant was

20

centrifuged at 3 000 g for 15 min at 4˚C. The supernatant contains the cytosolic fraction

21

and the pellet consists of the intact mitochondria. Reagent C (500µL) was, then, added

22

to the pellet and after centrifugation at 12 000 g for 5 min, the supernatant was

23

discarded. The pellets containing the mitochondria were resuspended in 50 µL of ice-

24

cold PBS, mixed with 500 µL of carbonate buffer (fresh cold 0.1 M Na2CO3) and used

25

in subsequent work.

26 27

Rhodamine based nanoparticles mediated internalization. The fluorescence

28

intensity of rhodamine 123 was assessed by the use of a microplate reader, considering

29

485 and 528 nm as excitation and emission wavelengths, respectively. The levels of this

30

dye in each well were quantified through the use of a calibration curve.

31 32

Quantification of green fluorescent protein (GFP). After in vitro transfection with

33

pCAG-GFP-ND1/cellulose or gelatin based rhodamine nanoparticles or pCAG-GFP-

34

ND1/cellulose or gelatin nanoparticles without rhodamine (n = 3), GFP content has 12 ACS Paragon Plus Environment

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Molecular Pharmaceutics

1

been quantified by a GFP ELISA kit (MitoSciences, ab 117992, Abcam, United

2

Kingdom). This consists in an enzyme immunoassay that allows the sensitive detection

3

and determination of GFP in cells. Transfected cell lysates were prepared from

4

detergent lysis (lysis buffer-tissue, 50:50 vol vol-1) in lysis buffer (1% Triton X-100 and

5

0.1% SDS in PBS, pH 7.4 and proteinase inhibitor cocktail) and homogenization. The

6

method uses a specific GFP antibody coated onto a well plate strips and the GFP

7

included in the samples can then be bounded to the wells by this antibody. After

8

washing the wells, a primary anti-GFP detector antibody is added. After new washing

9

step, HRP-conjugated secondary detector antibody specific for the primary detector

10

antibody is added to the wells. A TMB substrate is also added to the wells and the

11

presence of GFP leads to the development of a blue color. The concentration of GFP

12

can be quantified by measuring the absorbance at 600 nm in a spectrophotometer. The

13

assays were repeated three times in triplicate. The experimental data was statistically

14

analysed by using the Student´s t-test and the results were shown as mean ± standard

15

deviation. The p values were considered statistical significant at p < 0.05.

16 17

Fluorescence confocal microscopy. The microcopy study was performed on HeLa

18

cells. The cells (45,000 cells) were seeded into a 48 well plate containing round shaped

19

lamella for a period of one day to promote adhesion. HeLa cells were incubated in the

20

presence of pCAG-GFP-ND1/Rho/cellulose or gelatin nanoparticles for 3 hours at 37ºC

21

to induce transfection. Thereafter, cells were stained with 200 nM of Mitotracker

22

Orange CMTMROS for 50 minutes at 37ºC. Followed mitochondria staining, HeLa

23

cells were fixed with paraformaldehyde (PFA) 4%. Cell nucleus has been labeled by

24

incubation of cells with 1 µM Hoescht 33342 for a period of 10 minutes. Entellan

25

solution was applied to facilitate the mounting procedure of placing the round lamellas

26

in a lamina for confocal microscopy (ZEISS LSM 710, Oberkochen, Germany)

27

visualization. In order to maintain the efficacy of the dyes, all experiments after

28

transfection with pDNA nanoparticles, were performed in the absence of light.

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1

Results and Discussion

2 3

Cloning of ND1 mitochondrial gene into a plasmid. In this study, it is proposed the

4

cloning of a human mitochondrial gene in a mammalian expression vector for the

5

development of a new mitochondrial gene therapy approach. To our knowledge, this is

6

the first time that a human mitochondrial gene is cloned and produced in E. coli strains.

7

The first step was to obtain a human DNA sample enriched in the mtDNA fraction and

8

confirm that enrichment by PCR with specific primers for the mtDNA. The PCR

9

products were directly sequenced to confirm the correct sequence of the gene of interest,

10

the mtND1 gene. First the mtND1 gene was cloned into the standard cloning vector

11

pGEM®-T and once this was successfully achieved, it was transferred to a mammalian

12

expression vector, the pCAG-GFP. The mtDNA was successfully extracted from human

13

peripheral blood samples as demonstrated by the amplification of the mtDNA master

14

fragment of 2544 bp and the increased intensity of the PCR product in the samples

15

extracted by the mtDNA enrichment method (Figure 1A). In order to be sequenced, this

16

bigger fragment was divided by nested PCR in four smaller and overlapping fragments

17

of 860 bp, 774 bp, 737 bp and 709 bp, as presented in Figure 1B. The sequences of the

18

resulting fragments were confirmed and compared with MITOMAP reference sequence

19

(NC_012920, GenBank). Once the insertion of the mtND1 gene into pGEM®-T cloning

20

vector was successfully achieved, it was mandatory to insert the gene of interest into a

21

mammalian expression vector that would allow us to establish a new therapeutic

22

protocol on mitochondrial gene therapy. The pCAG-GFP was the chosen expression

23

vector for this purpose. The mtND1 gene was amplified with primers containing the

24

recognition sites for the restriction enzymes SmaI and XbaI used to digest both the

25

plasmid and the mtND1 PCR product (Figure 1C), and thus allow the ligation of the

26

insert to the vector.

27

JM109 E. coli strain was transformed with the constructs and grown in LB-

28

agar/ampicillin plates for 24 h. After the incubation period, some isolated colonies were

29

picked and cultured in liquid LB/ampicillin medium for posterior extraction of the

30

plasmids. In Figure 2A it is possible to observe the plasmids recovered from the

31

transformed cells. Samples 1, 3 and 8 present a different mobility on the agarose gel

32

what indicates that the mtND1 gene is inserted. So, we performed a PCR with a forward

33

primer of the insert and a reverse primer of the plasmid, and as expected, we confirmed

34

the amplification of a product with a similar length to the insert (Figure 2B). 14 ACS Paragon Plus Environment

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Molecular Pharmaceutics

1

Once three pCAG-GFP-ND1 recombinant plasmids were successfully constructed, it

2

was necessary to confirm if the sequence of the mitochondrial gene ND1 was correctly

3

inserted into pCAG-GFP plasmids. These three plasmids (1, 3 and 8) were directly

4

sequenced and the obtained sequence was matched with the mtND1 gene sequence

5

registered at MITOMAP reference sequence (NC_012920, GenBank). Figure 3A shows

6

a part of one of the electropherograms collected during Sanger sequencing of pCAG-

7

GFP-ND1 constructs and panel B is the acquired sequence compared to the MITOMAP

8

reference. It was also confirmed the presence of the insert digesting the constructs with

9

SmaI/XbaI, the same restriction enzymes used for the cloning process, as presented in

10

Figure 4.

11

Our finding is impressive and opens an incredible new route of great possibilities for the

12

biotechnological production of mitochondrial genes based plasmids that can found

13

innovative clinical applications in mitochondrial gene therapy. In the next step, we

14

describe an example of a delivery system based on the produced plasmid that can be

15

easily internalized into cells and targets mitochondria.

16 17

ND1 gene plasmid production and development of plasmid loaded nanoparticles.

18

After obtaining pCAG-GFP-ND1 recombinant plasmid, both the growth and plasmid

19

yield using three different E. coli strains (DH5α, XL1-B and JM109) were studied. Cell

20

banks were prepared for all the strains and the strains were transformed with the pCAG-

21

GFP-ND1 plasmid using the heat shock method. Growth curves were obtained for all

22

the strains and three different times for the yield studies (1.5, 4.5 and 7.5 hours) were

23

selected. For the plasmid purification, the GeneJet Plasmid Miniprep Kit was employed

24

and plasmid concentration was measured on a NanoPhotometerTM. For a more accurate

25

calculation of pCAG-GFP-ND1 specific yields, the cell dry weight (CDW) was

26

determined for each one of the strains by performing fermentations in the same

27

conditions. The growth curve profiles were very similar between strains, all reaching

28

approximately an OD of 4 in 9.5 hours of fermentation. However, concerning the

29

plasmid specific yield, some differences arise (Figure 5). JM109 revealed to have

30

slightly better plasmid specific yields, and was the strain selected for plasmid

31

production.

32

Once the plasmid was produced, our interest relied on the encapsulation of the ND1

33

gene plasmid vector into a suitable formulation for mitochondrial gene delivery. ND1

34

plasmid based particles were synthesized by a co-precipitation approach based on 15 ACS Paragon Plus Environment

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1

calcium carbonate. The nanoparticles are produced by co-precipitation of Ca2+ with

2

pDNA in the presence of CO32-. This is an attractive method of particles production

3

since it is a very simple, fast and economical technique, with facilitated control of both

4

the size and composition of the nano-system. To direct the nanoparticles to

5

mitochondria, rhodamine 123 (Rho), a cationic fluorescent compound, has also been

6

included in the protocol of pDNA nanoparticles formation. Rhodamine 123 is a

7

mitochondria dye that preferentially accumulates in the mitochondria of live cells.59,60

8

Furthermore, we found that the main properties of pDNA based vectors could be

9

controlled and manipulated by the incorporation of polymers such as cellulose or

10

gelatin. Not only, these molecules can reduce the size of nanoparticles but they also

11

seem to play a role in the stability of the pDNA system in aqueous solution and

12

contribute for improved transfection efficiency. Additionally, cellulose, a natural

13

polysaccharide produced by linking D-glucose units, and gelatin, a natural polymer

14

derived from collagen, exhibit remarkable biocompatibility and biodegradability being

15

quite suitable to be incorporated in gene delivery formulations. These polymers can be

16

viewed as efficient stabilizers, because they prevent the precipitation of inorganic

17

elements and retard the growth of calcium carbonate co-precipitates. Cellulose and

18

gelatin bind Ca2+ ions, and this will induce a significant reduction in the electrostatic

19

repulsion between hydroxyl and carboxyl groups in cellulose and gelatin chains,

20

respectively. Therefore, the cellulose or gelatin chains in the Ca2+ domains become

21

more condense, leading to the formation of stable pDNA nanoparticles at the surface

22

layer.

23

Figure 6 shows the images obtained from the scanning electron micrograph study of

24

rhodamine based nanoparticles for 5 µg and 10 µg ND1 plasmid in the absence of

25

polymers and with cellulose or gelatin functionalization. All the synthesized systems are

26

spherical or oval presenting diameter sizes ranging from 140 to 280 nm, approximately,

27

what make them suitable for gene delivery aims. Moreover, the introduction of cellulose

28

or gelatin into the formulation leads to a decrease in particle size. Along with the size

29

decrease, other characteristics of nanoparticles such as their zeta potential are modified

30

by the inclusion of these polymers.

31

Table 1 shows the average size and zeta potential for 5 µg and 10 µg ND1 gene plasmid

32

based nanoparticles and of the same nanoparticles functionalized with cellulose or

33

gelatin. We do confirm the decrease in particle size when cellulose or gelatin is present

34

in the system. This property is also dependent on the pDNA initial loading amount, with 16 ACS Paragon Plus Environment

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Molecular Pharmaceutics

1

particles formulated with higher pDNA amounts exhibiting lower sizes suggesting a

2

stronger electrostatic interaction as the negative charges of pDNA are increasing.

3

Besides the size displayed by nanoparticles, the surface charge they carry is also a

4

relevant parameter to analyse. Most of the particulate vectors show positive zeta

5

potential values ranging from +12 to +52 mV, approximately. The exception is found in

6

the pCAG-GFP-ND1/Rho/cellulose nanoparticles with 10 µg pDNA loading amount

7

which present a negative zeta potential, in consequence of both the high negative charge

8

of the pDNA and the presence of cellulose which, at the pH used to promote particle

9

formation (pH 7), has a negative charge. The cell surface is rich in anionic

10

proteoglycans, therefore, vectors exhibiting positive charges are easily cell attached by

11

electrostatic interactions and the transportation, through endocytosis mechanism to the

12

desired cell organelle, is facilitated. In line with this, the addition of gelatin, which

13

presents a positive zeta potential, in particle formation protocol can be considered an

14

improved approach in the formulation of more suitable carriers for pDNA delivery.

15

Furthermore, and as expected, an alteration in the zeta potential values was observed

16

with the increases in pDNA content in the nanoparticle system. An increase in the levels

17

of pDNA in the nano-systems directly leads to an increment in the negative charges

18

present at the surface of the carrier.

19

Moreover, other properties displayed by the pDNA based nanoparticles, such as the

20

pDNA loading amount and the encapsulation efficiency, must be taken into account

21

when designing appropriate vectors for gene therapy. Table 2 SI, available in the

22

Supporting Information, presents the pDNA and rhodamine 123 efficiencies for pCAG-

23

GFP-ND1/rhodamine,

24

ND1/Rho/gelatin nanosystems, prepared with 5 µg and 10 µg of pDNA. High pDNA

25

encapsulation efficiencies were achieved for all pDNA systems, with slightly higher

26

values obtained for the 10 µg pDNA systems. It seems that the encapsulation efficiency

27

increases with an increment in the initial pDNA loading amount in the nanoparticle

28

formulation. The presence of gelatin in the complex also improves the efficiency of

29

pDNA encapsulation. The same trend has been observed when considering the presence

30

of rhodamine 123 into the formulations. In addition, high values of rhodamine EE were

31

found for all systems. The cytotoxic analysis was performed by using the 3-[4,5-

32

dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) based colorimetric

33

assay. The nanoparticles show to be not toxic to cells since every system is able to

pCAG-GFP-ND1/Rho/cellulose

17 ACS Paragon Plus Environment

and

pCAG-GFP-

Molecular Pharmaceutics

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1

induce dehydrogenase activity ensuring normal cell growth and function (data not

2

shown).

3 4 5

Targeting mitochondria. Plasmid DNA release profiles have been investigated in vitro

6

at two different pH values, as presented in Figure 1 of Supporting Information. The

7

kinetics of pDNA release demonstrated that, in acidic conditions, pDNA can be released

8

from nanoparticles in a great extent (Figure 1 SI). The phenomena of

9

pDNA/Rho/cellulose or gelatin nanoparticles cell uptake and internalization have been

10

investigated by the quantification of the rhodamine fluorescence intensity in both, the

11

cytosolic fraction and isolated mitochondria of N2a cells. Figure 7 shows the cell-

12

associated fluorescence intensity of rhodamine 123, when transfection was mediated by

13

pCAG-GFP-ND1/Rho/cellulose or gelatin nanoparticles at one and two days. Untreated

14

cells have been used as control. In addition, neuroblastoma cells have been incubated

15

with only rhodamine 123 and the dye fluorescence intensity in the cytosol and

16

mitochondria of these cells has also been determined. The greater mitochondrial affinity

17

of rhodamine has been confirmed by the observation of its higher fluorescence intensity

18

in the mitochondria of N2a cells, in contrast with the very low fluorescence that can be

19

detected in the cytosolic fraction. Similar trend, and even in a more pronounced extent,

20

is found when cellular transfection is mediated by pDNA/Rho/cellulose or gelatin

21

nanoparticles. In both cases, larger fluorescence intensity was quantified in the

22

mitochondrial fraction of N2a cells; the cytosolic fraction exhibits very low levels of

23

rhodamine 123 fluorescence intensity. Additionally, we also monitored the rhodamine

24

fluorescence intensity when the transfection is mediated by pCAG-GFP-ND1/Rho

25

nanoparticles. A decrease in the fluorescence intensity was found when compared with

26

the systems where these polymers are present (data not shown); this may illustrate the

27

enhanced properties of cellulose and gelatin based vectors for in vitro transfection.

28

Compared to the rhodamine 123 solution, the cellular uptake and internalization of

29

pDNA/Rho/cellulose or gelatin nanoparticles is more efficient resulting in nanoparticles

30

targeting mitochondria, with higher rhodamine fluorescence intensity detection. This

31

phenomenon observed in isolated mitochondria cannot be due to GFP expression, as

32

GFP based plasmid used was not recoded to adapt to the codon code of mitochondria

33

and, therefore, it is unable to mitochondrial production of green fluorescent protein.

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Molecular Pharmaceutics

1

Thus, the fluorescence intensity comes from rhodamine nanoparticles targeted to

2

mitochondria.

3

From this study, the uptake of pDNA based carriers into N2a cells after 24 hours of

4

transfection can be stated. Moreover, at day two, the fluorescence intensity increases

5

demonstrating the cell internalization of nanoparticles, with subsequent targeting of

6

mitochondria. A comparison between the two systems leads to the observation of a

7

larger rhodamine fluorescence intensity when the transfection is mediated by pCAG-

8

GFP-ND1/Rho/gelatin based carrier. At this point, however, we should hypothesized,

9

although in a very lower extent, the nucleus targeting during transfection mediated by

10

the formulated rhodamine nanoparticles resulting in some nuclear GFP expression and

11

consequent protein production. Based on this, one can consider the presence of GFP in

12

the cytosolic fraction what can also contribute for the observed higher fluorescence

13

intensity when using the pDNA/Rho/cellulose or gelatin particles compared to the study

14

where cells were only incubated with rhodamine 123. Therefore, we investigated the

15

GFP expression in both cytosolic fraction and mitochondria of neuronal cells, after

16

transfection with pCAG-GFP-ND1/Rho/cellulose or gelatin nanoparticles at 24 and 48

17

hours of transfection. The GFP content has been quantified through the use of the

18

described GFP ELISA kit. The results are shown in Table 2. Small amounts of GFP

19

were found in cytosolic fraction when transfection is mediated by pDNA/Rho/cellulose

20

or gelatin nanoparticles, while in mitochondria no GFP expression occurred. Because

21

mitochondria possess a different codon system, GFP cannot be expressed in this

22

organelle. As the main focus of the present report was the formulation of a suitable

23

mitochondrial gene plasmid based carrier able to target mitochondria, we went further

24

and performed another experiment to demonstrate the mitochondrial targeting ability of

25

pCAG-GFP-ND1/Rho/cellulose or gelatin vectors. Free rhodamine pCAG-GFP-

26

ND1/cellulose or gelatin nanoparticles have been considered and the expression of GFP

27

has been evaluated in cytosol and mitochondria after 24 and 48 hours of N2a cells

28

transfection (Table 2). Considerable higher amounts of green fluorescent protein can be

29

produced in cytosol, with protein levels considerably increasing after two days of

30

transfection. The data revealed that free rhodamine nanoparticles can be easily uptaked

31

by cells, internalized most probably through the endocytosis mechanism and are able to

32

deliver bioactive pDNA into the cytoplasm. To be incorporated into the nucleus, where

33

it can be expressed, this pDNA must cross the nuclear barrier, which only appears to be

34

more permeable during mitosis. As cancer cells present a higher rate of division, this 19 ACS Paragon Plus Environment

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1

step becomes facilitated enhancing transfection and gene expression. This correlates

2

well with the minimal content of GFP found in the cytosol when pCAG-GFP-

3

ND1/Rho/cellulose or gelatin vectors mediate the transfection and strongly supports the

4

mitochondrial targeting assumption.

5 6

In addition to the previous study, a fluorescence confocal microscopy investigation was

7

also carried out to confirm the ability of the formulated carriers for mitochondria

8

targeting. The main cell organelles, as nucleus and mitochondria, have been labeled

9

with Hoescht 33342 and Mitotracker Orange dye. Additionally, and for comparison,

10

mitochondrion has also been stained with rhodamine 123. The images are presented in

11

Figure 8. Image A exhibits the blue color arising from stained nucleus, where B reveals

12

that pCAG-GFP-ND1/Rho/cellulose nanoparticles can enter the cell showing a green

13

stain, due to the rhodamine incorporated into the particles. The poor labelling of

14

mitochondria with rhodamine 123 (C), in comparison to the use of Mitotracker Orange

15

dye (D) evidences the inefficacy of this fluorescent compound for cell stain after

16

fixation.61 The incorporation of rhodamine 123 into nanoparticles (B) and the use of a

17

Mitotracker dye (D) are valuable alternatives to overcome this drawback.

18

Additionally, the images revealed that rhodamine was efficiently encapsulated into the

19

pDNA systems and transfection actually takes place, therefore, demonstrating that the

20

green color observed in image B corresponds to the nanoparticles and it is not due to

21

free

22

nanoparticles can be observed in the yellow color present in image E, which

23

corresponds to the merged picture of stained mitochondria and green fluorescent

24

nanoparticles captured in the same field. From this result, and as nucleus is blue stained,

25

it can be stated that the formulated nanocarrier is targeted to mitochondria. Moreover, a

26

three-dimensional deep analysis conducted on Z plane supports the accumulation of

27

pCAG-GFP-ND1/Rho/cellulose carrier into mitochondria (F and G). Similar results

28

were obtained for pCAG-GFP-ND1/Rho/gelatin nanoparticles mediated transfection

29

(data not shown).

30

At this stage, we are aware that, as mitochondrial targeting has been achieved, research

31

should evolve in order to clarify all aspects related with both gene delivery and protein

32

expression into mitochondria.

rhodamine.

Mitochondrial

targeting

of

pCAG-GFP-ND1/Rho/cellulose

33 20 ACS Paragon Plus Environment

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Molecular Pharmaceutics

1

Conclusion

2

For the first time, it was successfully cloned the mitochondrially NADH dehydrogenase

3

1 protein encoded gene (mtND1) in E. coli and it remained stable in multi-copy pCAG-

4

GFP plasmid. This step represents a huge advance in the development of a gene

5

delivery vector for human mitochondrial gene therapy. Following this achievement,

6

biocompatible cellulose or gelatin functionalized pCAG-GFP-ND1 rhodamine

7

nanoparticles have been formulated by a co-precipitation method. These vehicles can be

8

easily cell internalized and preferentially localized into mitochondria. The expression of

9

green fluorescent protein in cytosol, but not in mitochondria, further supports the

10

mitochondrial targeting of these rhodamine/pDNA carriers.

11

The present report is a remarkable contribution for the creation of an adequate

12

mitochondrial targeted vector towards effective treatments for mitochondrial DNA

13

disorders, due to mutations in Complex I genes. This study brings a new insight into

14

mitochondrial gene cloning, plasmid production and formulation of mitochondrial

15

targeting systems contributing to the evolution in the design of suitable non-viral

16

systems for mitochondrial gene therapy.

17 18

Competing interests

19

The authors declare that they have no competing interests.

20 21

Contributions

22

Conceived and designed the experiments: E. Coutinho, F. Sousa and D. Costa.

23

Performed the experiments: E. Coutinho, C. Batista and D. Costa. Analyzed the data,

24

discussion of the results, corrections and revision of the manuscript: all authors.

25

Contributed reagents/materials/analysis tools: E. Coutinho, F. Sousa, D. Costa and J.

26

Queiroz. Wrote the paper: E. Coutinho, C. Batista and D. Costa.

27 28

Acknowledgments

29

We are grateful for financial support from Fundação para a Ciência e a Tecnologia

30

(FCT), (SFRH/BPD/103592/2014) and from PEst-OE/SAU/UI0709/2014 project. The

31

authors acknowledge to Connie Cepko for the pCAG-GFP Addgene plasmid, to Eng.

32

Ana Paula Gomes for acquiring SEM images, to Filomena Silva for providing DH5α E.

21 ACS Paragon Plus Environment

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1

coli strain and to Patricia Pereira and Augusto Pedro for their aid in both gene cloning

2

and plasmid production.

3 4 5

References

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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(36) Claros, M. G.; Perea, J.; Shu, Y.; Samatey, S.; Popot, J. L.; Jacq, C. Limitations to in vivo import of hydrophobic proteins into yeast mitochondria-the case of a cytoplasmically synthesized apocytochrome b. Eur. J. Biochem. 1995, 228, 762-771. (37) Perales-Clemente, E.; Fernandez-Silva, P.; Acin-Perez, R.; Perez-Martos, A.; Enriquez, J. A. Allotopic expression of mitochondrial-encoded genes in mammals: achivied goal, undemonstrated mechanism or impossible task? Nucl. Acids Res. 2010, published September 7, 1-10. (38) Weissig, V.; Lizano, C.; Torchilin, V. P. Selective DNA release from DQAsome/DNA complexes at mitochondria-like membranes. Drug. Deliv. 2000, 7, 1-5. (39) Weissig, V.; Torchilin, V. P. Towards mitochondrial gene therapy: DQAsomes as a strategy. J. Drug Target 2001, 9, 1-13. (40) Weissig, V.; D´Souza, G. G.; Torchilin, V. P. DQAsome/DNA complexes release DNA upon contact with isolated mouse liver mitochondria. J. Control Release 2001, 75, 401-408. (41) D´Souza, G. G. M.; Boddapati, S. V.; Weissig, V. Gene therapy of the other genome: the challenges of treating mitochondrial DNA defects. Pharmaceut. Res. 2007, 24, 228-238. (42) Edeas, M.; Weissig V. Targeting mitochondria: strategies, innovations and challenges: the future of medicine will come through mitochondria. Mitochondrion 2013, 13, 389-390. (43) Weissig V. DQAsomes as the prototype of mitochondria-targeted pharmaceutical nanocarriers: preparation, characterization and use. Methods Mol. Biol. 2015, 1265, 111. (44) Wheeler, V. C.; Aitken, M.; Coutelle, C. Modification of the mouse mitochondrial genome by insertion of an exogenous gene. Gene 1997, 198, 203-209.

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(47) Yoon, Y. G.; Koob, M. D. Efficient cloning and engineering of entire mitochondrial genomes in Escherichia coli and transfer into transcriptionally active mitochondria. Nucleic Acids Res. 2003, 31, 1407-1415. (48) Bigger, B.; Liao, A.; Sergijenko, A.; Coutelle, C. Trial and error: how the unclonable human mitochondrial genome was cloned in yeast. Pharm. Res. 2011, 28, 2863-2870. (49) Furukawa, R.; Yamada, Y.; Kawamura, E.; Harashima, H. Mitochondrial delivery of antisense RNA by MITO-Porter results in mitochondrial RNA knockdown, and has a functional impact on mitochondria. Biomaterials 2015, 57, 107-115. (50) Yasuzaki, Y.; Yamada, Y.; Ishikawa, T.; Harashima, H. Validation of mitochondrial gene delivery in liver and skeletal muscle via hydrodynamic injection using an artificial mitochondrial reporter DNA vector. Mol. Pharmaceutics 2015, 12, 4311-4320. (51) Cardoso, A. M.; Morais, C. M.; Cruz, A. R.; Cardoso, A. L.; Silva, S. G.; Vale, M. L.; Marques, E. F.; Lima, C. P.; Jurado, A. Gemini Surfactants Mediate Efficient Mitochondrial Gene Delivery and Expression. Mol. Pharmaceutics 2015, 12, 716-730. (52) Santos, J.; Sousa, F.; Queiroz, J.; Costa, D. Rhodamine based plasmid DNA nanoparticles for mitochondrial gene therapy. Colloids Surf. B 2014, 121, 129-140. (53) Lyrawati, D.; Trounson, A.; Cram, D. Expression of GFP in mitochondrial compartment using DQAsome-mediated delivery of an artificial mini-mitochondrial genome. Pharm. Res. 2011, 28, 2848-2862. (54) Ahmad, S.; Ghosh, A.; Nair, D. L.; Seshadri, M. Simultaneous extraction of nuclear and mitochondrial DNA from human blood. Genes Genet. Syst. 2007, 82, 429432. (55) Miller, S. A.; Dykes, D. D.; Polesky, H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988, 16, 1215. (56) Ramos, A.; Santos, C.; Alvarez, L.; Nogués, R.; Aluja, M. P. Human mitochondrial DNA complete amplification and sequencing: a new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification. Electrophoresis 2009, 30, 1587-1593. (57) Inoue, H.; Nojima, H.; Okayama, H. High efficiency transformation of Escherichia coli with plasmids. Gene 1990, 96, 23-28. (58) Matsuda, T.; Cepko, C. L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA 2004, 101, 16-22.

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(59) Modica-Napolitano, J. S.; Aprille, J. R. Basis for the selective cytotoxicity of rhodamine 123. Cancer Res. 1987, 47, 4361-4365. (60) Snyder, D. S.; Small, P. L. C. Staining of cellular mitochondria with LDS-751. J. Immunol. Method 2001, 257, 35-40. (61) Johnson, L. V.; Walsh, M. L.; Chen, L. B. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci USA 1980, 77, 990-994.

Figure Captions Figure 1. Amplification of the mitochondrial master fragment of 2544 bp. M: 1 kb DNA Ladder (New England Biolabs, Inc, Ipswich, MA, USA); N: negative control; G: Human Genomic DNA not enriched in the mtDNA fraction; 1 and 2: Human DNA samples enriched in mtDNA fraction (A). Nested PCR of the 4 mtDNA internal fragments. M: 100 bp DNA Ladder (New England Biolabs, Inc, Ipswich, MA, USA); N: negative control; 1 and 2: Human DNA samples enriched in mtDNA fraction (B). mtND1 and pCAG-GFP digested with SmaI and XbaI after purification. M: 1 kb DNA Ladder (New England Biolabs, Inc, Ipswich, MA, USA); I: Insert; V: Vector (C).

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Figure 2. Recombinant plasmids (1–10) extracted from JM109 cells after growth in liquid culture of the transformed single colonies (A). PCR products indicating the presence of the insert in constructs 1, 3 and 8. M: 1 kb DNA Ladder (New England Biolabs, Inc, Ipswich, MA, USA) (B).

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Figure 3. Representative sequencing electropherogram of pCAG-GFP-mtND1 constructs (A). Alignment of the acquired sequence with the MITOMAP reference sequence (NC_012920, GenBank) (B).

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Figure 4. Digestion of pCAG-GFP-mtND1 constructs with SmaI/XbaI. M: 1 kb DNA Ladder (New England Biolabs, Inc, Ipswich, MA, USA); V: Vector; I: Insert; 1, 3 and 8: Recombinant plasmids with positive amplification of mtND1 gene.

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Figure 5. Growth curve of E. coli JM109 (A), DH5α (B) and XL1B (C) transformed with pCAG-GFP-ND1. Plotted is the optical density at 600 nm as a function of time in hours. pDNA specific yield was measured at 1.5, 4.5 and 7.5 hours. Data was obtained from three independent measurements.

37 38 39 40

Figure 6. Scanning electron micrograph of 5 µg (A) and 10 µg (B) pCAG-GFPND1/Rho based nanoparticles, 5 µg (C) and 10 µg (D) pCAG-GFP-ND1/Rho/gelatin based nanoparticles and 5 µg (E) and 10 µg (F) pCAG-GFP-ND1/Rho/cellulose based nanoparticles.

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Figure 7. Rhodamine fluorescence intensity in neuroblastoma N2a cancer cells after 24 and 48 hours incubation with rhodamine 123 or after transfection with pCAG-GFPND1/Rho/cellulose (A) or pCAG-GFP-ND1/Rho/gelatin based nanoparticles (B). The data were obtained by calculating the average of 3 experiments. The respective errors 27 ACS Paragon Plus Environment

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were determined and were below 0.05%. One-way Anova analysis was performed followed by Bonferroni´s multiple comparison test that indicated that differences of control versus pCAG-GFP-ND1/Rho/cellulose or pCAG-GFP-ND1/Rho/gelatin nanoparticles and differences of rhodamine 123 versus pCAG-GFP-ND1/Rho/cellulose or pCAG-GFP-ND1/Rho/gelatin nanoparticles were statistically significant (P ˂ 0.05). Figure 8. Fluorescence confocal microscopy study. (A) Nucleus stained blue by Hoescht 33342, (B) pCAG-GFP-ND1/Rho/cellulose nanoparticles stained green due to the presence of Rho123, (C) Mitochondria stained green by Rho 123, (D) Mitochondria stained orange by Mitotracker Orange and (E) Merged image. (F) and (G) represent three-dimensional Z-plane stacks of the co-localization of mitochondria and pCAGGFP-ND1/Rho/cellulose nanoparticles.

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1

Figures

2 3 4 5 6 7 8 9 10 11

Figure 1.

12 13 14 15 16 17 18 19 20 21 22 23

Figure 2.

24 25 26 27 28

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Figure 3.

11 12

13 14

Figure 4.

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A

1

B

C

2 3

Figure 5.

4 A

C

B

D

E

F

5 6

Figure 6.

7 8

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(a) Rho 123 pDNA/Rho/Cellulose nanoparticles

Fluorescence Intensity (a.u.)/µg Protein

4.0 3.5 3.0

Mitochondria

2.5 2.0 1.5 1.0

Cytosol

0.5 0.0 24

48

24

48

Time (hr.)

1 2 (b)

Rho 123 pDNA/Rho/gelatin nanoparticles

4.0 Fluorescence Intensity (a.u.)/µg Protein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Mitochondria

3.5 3.0 2.5 2.0 1.5 Cytosol

1.0 0.5 0.0 24

48

24 Time (hr.)

3 4 5

Figure 7.

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Figure 8.

4 5 6 7 8 9 10

Tables Table 1. Average zeta potential and size of rhodamine pCAG-GFP-ND1 and rhodamine pCAGGFP-ND1/cellulose or gelatin based nanoparticles with 5 µg and 10 µg pDNA loading amount. Average zeta potential values of the pCAG-GFP-ND1, free rhodamine 123, cellulose and gelatin were also presented. The values of zeta potential and size were calculated with the data obtained from three independent measurements (mean ± SD, n = 3). Size (nm) System

5 µg

10 µg

pCAG-GFP-ND1

Zeta Potential (mV) 5 µg

10 µg

-102.3 ± 5.4

-198 ± 12.2

pCAG-GFP-ND1/Rho

338 ± 2.1

281 ± 4.4

+40.1 ± 1.9

+12.4 ± 0.2

pCAG-GFP-ND1/Rho/Cellulose

282 ± 9.2

212 ± 8.9

+29.7 ± 0.5

-10.1 ± 2.2

pCAG-GFP-ND1/Rho/Gelatin

196 ± 14.9 141 ± 10.6

+51.4 ± 9.6

+39.8 ± 4.1

Zeta Potential (mV) Rhodamine 123

+62.3 ± 1.1

Cellulose

-89.6 ± 1.4

Gelatin

+71.1 ± 8.9

11

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Table 2. Quantification of GFP expression in N2a cells using a GFP ELISA kit, after 24 and 48 hours of transfection with pCAG-GFP-ND1/cellulose or gelatin and pCAG-GFPND1/Rho/cellulose or gelatin nanoparticles. The values are calculated with the data obtained from three independent measurements (mean ± SD, n = 3).

5 GFP (ng/mL) Cytosol System

Mitochondria

24 hr

pCAG-GFP-ND1/Cellulose

48 hr

24 hr

48 hr

67 ± 1.9

83 ± 2.8

0

0

pCAG-GFP-ND1/Gelatin

198 ± 3.1

274 ± 7.3

0

0

pCAG-GFP-ND1/Rho/Cellulose

0.4 ± 0.02

0.6 ± 0.07

0

0

pCAG-GFP-ND1/Rho/Gelatin

0.7 ± 0.04

1 ± 0.1

0

0

6 7 8 9 10 11 12 13 14 15 16 17

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