Electrochemical Behaviour of a Novel Mitochondria-targeted

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Electrochemical Behaviour of a Novel Mitochondria-targeted Antioxidant at an ITIES: an Alternative Approach to Study Lipophilicity José A Ribeiro, Sofia Benfeito, Fernando Cagide, José Teixeira, Paulo J. Oliveira, Fernanda Borges, António F. Silva, and Carlos M Pereira Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00787 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Analytical Chemistry

Electrochemical

Behaviour

of

a

Novel

Mitochondria-targeted

Antioxidant at an ITIES: an Alternative Approach to Study Lipophilicity José A. Ribeiroa,*, Sofia Benfeitoa, Fernando Cagidea, José Teixeiraa,b, Paulo J. Oliveirab, Fernanda Borgesa, António F. Silvaa, Carlos M. Pereiraa,* a

CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of

Porto, Porto 4169-007, Portugal b

CNC – Center for Neuroscience and Cell Biology, University of Coimbra, UC-Biotech

Building, Biocant Park, Cantanhede 3060-197, Portugal Fax: +351 220402659; Tel: +351 220402641; *E-mail: [email protected]; [email protected]

ABSTRACT In this work, we report for the first time the accumulation activity by energised rat heart mitochondria and the ionic transfer process at a liquid-liquid interface of a novel mitochondria-targeted antioxidant, named as AntiOxCIN4, which is structurally based on a hydroxycinnamic acid. Lipophilicity studies conducted at the water/1,6-dichlorohexane (DCH) interface allowed the building up of ionic partition diagram of AntiOxCIN4 in accordance with electrochemical data obtained. The partition coefficients of both, positively charged (2.3) and zwitterionic (0.2) forms of the antioxidant, were determined. This study contributed to gaining an insight about the ability of the synthesized antioxidants to cross biomembranes barriers by using ITIES as model system. Keywords: Mitochondria-targeted antioxidant; rat heart mitochondria; ITIES; Interface; Lipophilicity; Partition coefficient.

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INTRODUCTION Mitochondria are sub-cellular organelles described as the powerhouses of the cells1, as 95% of total energy requirements by cells are produced in mitochondria.2 These organelles coordinate several metabolic pathways producing essential metabolites in cell life and death.3,4 Despite that, mitochondria are particularly vulnerable to oxidative damage, and the continuously metabolism of molecular oxygen to produce adenosine triphosphate (ATP) may lead to oxidative stress and mitochondrial dysfunction. Mitochondrial alterations leading to disease can be divided in: 1) primary events, characterized by a mutation in a gene encoded by mitochondrial DNA (mtDNA) or a nuclear-encoded gene for a mitochondrial protein, or from a mitochondrial toxin and 2) secondary mitochondrial dysfunction, because of pathological events originated outside mitochondria.5 Factors such as 1) increased generation of reactive oxygen species (ROS), 2) disruption of mitochondrial calcium homeostasis, including storage, and 3) defective mitochondrial of ATP production are often related with secondary mitochondrial dysfunction.6,7 Because of the critical role of mitochondria in cell metabolism and redox signalling8, targeting mitochondria with small molecules using the triphenylphosphonium (TPP) lipophilic cation as a carrier is a very hot topic in medicinal chemistry area and has been used as an effective strategy in the context of cardiovascular, neurodegenerative and hepatic diseases, as well as in cancer.8 This strategy takes advantage of the negative charge generated in polarized mitochondria, resulting from the oxidation of substrates coupled to proton translocation from the matrix to the inter-membrane space 9. The charge of the TPP cation is spread over a large hydrophobic surface area, allowing it to cross easily the mitochondrial membrane and accumulate within mitochondrial matrix in response to the membrane potential.10,11 The most studied mitochondrial-targeted antioxidant

MitoQ

([10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-

yl)decyl]triphenylphosphonium), schematically represented in Fig. 1A, consists in the conjugation of an antioxidant quinone moiety (coenzyme Q) covalently attached to a lipophilic TPP cation. MitoQ has been tested for different pathologies, including neurodegenerative diseases, but clinical assays have failed to show beneficial effects over placebo.10,11 Meanwhile another mitochondrial-targeted antioxidant based on plastoquinone (SkQ1, Fig. 1B), a quinone involved in the electron transfer chain of


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chloroplasts, has entered in phase II clinical trials for treatment of dry eye syndrome.12,13 Fig. 1 During the last years, several other mitochondria-targeted antioxidants have been synthesized, mostly based on naturally occurring molecules.14 Within this framework, a novel mitochondria-directed antioxidant based on caffeic acid (Fig. 1C), named as AntiOxCIN1, was previously developed by our group.15 A follow-up AntiOxCIN1 optimization process led to an innovative and potent mitochondriotropic antioxidant, AntiOxCIN4 (Fig. 1D). Evidences have been acquired to validate AntiOxCIN4 as a potent mitochondriotropic antioxidant, able to prevent rat liver mitochondria from oxidative damage and hepatic cells from iron- and hydrogen peroxide-induced damage without associated toxicity to mitochondrial morphology and functions properties.16 Drug-membrane interaction studies play an essential role for better understanding drug biological action and toxic effects and predict their movement across membranes in the body (drug disposition)17. In fact, lipophilicity, i.e. the affinity of a compound for a lipid environment, is of paramount importance in medicinal chemistry and molecular toxicology since it is a key property governing biomolecules passive transport through biomembranes, drug-target interactions and ADMET properties.17-19 Lipophilicity of a solute is commonly measured by its partition coefficient (P) in a biphasic system20, usually between water and n-octanol, and several analytical techniques (chromatography, simple shake flask, etc.) were employed in these measurements.21-23 Although the estimation of the partition coefficients of neutral molecules has been an important subject from the early years of the 20th century, the use of ion transfer across electrified interfaces between two immiscible electrolyte solutions (ITIES) is one of few methods that allows the determination of the partition coefficients of ionic solutes. Accounting that many drugs (˃70%) are in its ionic form under physiological conditions, ITIES is considered to be a very useful technique for the reliable determination of the partition coefficients of molecules in its ionic and neutral forms.17 The voltammetric evaluation of lipophilicity of small molecules (neutral or ionic) at ITIES has the advantage of potentiostatic control of interfacial transfer of the species in different pre-established experimental conditions (aqueous pH, applied potential, etc.).24-27 In addition, the partitioning properties at the ITIES can be further correlated to their behaviour at the n-octanol-water partitioning system.17,28,29 3 ACS Paragon Plus Environment


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Thus, electrochemistry at ITIES can be considered as a simple model system mimicking the transfer of (bio)molecules across membranes and provide valuable information about their biological activity.26,28,30-33 As part of our current research mission to develop mitochondriotropic antioxidants, here we measured AntiOxCIN4 uptake by rat heart mitochondria as well as its lipophilicity and partition behaviour in a mimic transfer system by using four-electrode voltammetry at the water/1,6-dichlorohexane (DCH) interface. The gathered information about mitochondria targeted-antioxidants will be a contribute for a better understanding of their biological behaviour (see Scheme 1). Scheme 1

EXPERIMENTAL SECTION

Synthesis of AntiOxCIN4 The synthetic strategy and procedures used in the synthesis of the mitochondriotropic antioxidant AntiOxCIN4 have been previously described (see reference 16).

Evaluation of AntiOxCIN4 heart mitochondria uptake Isolation of rat heart mitochondria and measurement of mitochondria uptake were achieved as described elsewhere (see experimental details in references 34 and 35, respectively). Mitochondrial modulators rotenone (>95%, Sigma, Barcelona, Spain) succinate (SUC, >98%, Sigma, Barcelona, Spain) and valinomycin (VAL, >98%, Aldrich, Barcelona, Spain) were used in the experiments. All reagents used were of the highest grade of purity commercially available (analytical grade or superior).

Evaluation of AntiOxCIN4 lipophilicity The electrochemical cell used with arrays of micro liquid-liquid interfaces (µITIES; PET microporous membrane with an interfacial area of 5.2×10-5 cm2) was described previously in reference 36. The four-electrode system used is formed by two counter electrodes of Pt, one in each phase, and two Ag/AgCl references electrodes, one in the 4 ACS Paragon Plus Environment

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aqueous phase and the other in the organic phase reference solution (solidified as a gel to improve mechanical stability). Furthermore, the following experimental setup was used to study the transfer of AntiOxCIN4 ions from water to DCH. Scheme 2 1,6-dichlorohexane (DCH, 98%) was obtained from Aldrich and was purified according to previously described procedure.37 The organic electrolyte salt selected for the experiments,

bis(triphenylphosphoranylidene)ammonium

tetrakis(4-

chlorophenyl)borate (BTPPA.TPBCl), was synthesized from a equimolar mixture of bis(triphenylphosphoranylidene)ammonium chloride (BTPPACl, 97%, Aldrich) and potassium

tetrakis(4-chlorophenyl)borate

(KTPBCl,

98%,

Fluka).

Sodium

methanesulfonate (98%) was purchased from Sigma-Aldrich. The aqueous supporting electrolyte solutions used in the studies were: hydrochloric acid 2 - 10 mM for pH between 2.0 – 2.7, lithium acetate/acetic acid buffer 10 mM for pH between 4.0 - 6.1, Tris-HCl buffer 10 mM for pH between 7.2 - 8.7, lithium hydrogen carbonate/lithium carbonate 10 mM for pH between 9.0 - 9.5 and lithium hydroxide between 2 - 10 mM for pH between 11.3 - 12.0. All aqueous solutions were prepared using water purified with a Milli-RO3 Plus and Milli-Q purification systems (resistivity ≥ 18 MΩ cm-2). The stock solutions of AntiOxCIN4 were prepared by dissolving the antioxidant in pure water and were stored at 4 ºC. Aliquots from stock AntiOxCIN4 solution were added to the aqueous phase (V = 4.0 mL) in order to obtained the desired final concentration. Tetrabutylammonium chloride (TBACl, purum, Aldrich) was selected as reference in the half-wave potential measurements. The value of ∆ wDCH φ 0 (TBA + ) = − 0.193 V was used.38 Electrochemical measurements were performed using a potentiostat Autolab PGSTAT302N (Eco Chemie B.V., Utrecht, Netherlands) computer controlled by the General Purpose Electrochemical System (GPES) version 4.9 software package. Cyclic voltammetry (CV) was performed at a scan rate of 50 mV s−1 and differential pulse voltammetry (DPV) operated under the following conditions: step potential of 4 mV, pulse amplitude of 50 mV and scan rate of 8 mV s−1. All electrochemical experiments were carried out at room temperature. The entire cell setup was placed inside a Faraday cage to reduce background noise. 5 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION Mitochondrial pharmacology is a feasible strategy because therapies that impact on a few common cellular damaging pathways can treat patients with a wide range of primary and secondary mitochondrial disorders. As mitochondria are key sites for the ATP production, it is not surprising that most mitochondrial drug targets are related and involved in energetic metabolism, and mostly focused on mechanisms regulating mitochondrial biogenesis, ROS and respiration. Nowadays, the central focus on finding mitochondrial-targeted drugs is related with the discovery and development of antioxidants able to block mitochondrial oxidative damage with therapeutically benefits in multiple context.39 Following this, a rational design of mitochondriotropic antioxidants based on a dietary antioxidant (caffeic acid) was performed by our group using as a smart carrier a triphenylphosphonium cation. In this work, AntiOxCIN4 mitochondrial accumulation in response to the transmembrane electric potential (∆Ψ) was assessed in isolated rat heart mitochondria (RHM). Non-energized RHM supplemented with rotenone, a complex I inhibitor used to prevent ∆Ψ formation, were used. Five consecutive AntiOxCIN4 additions were then made to calibrate the electrode response. The addition of complex II substrate succinate resulted in ∆Ψ generation and consequent AntiOxCIN4 translocation to mitochondria. The ∆Ψ generated caused a decrease in the extramitochondrial AntiOxCIN4 concentration (from 5 µM to 1.7 µM) and in an accumulation ratio of ≈ 2600-fold vs. outside mitochondrial space (see Fig. 2). Once the ∆Ψ was abolished by the K+ionophore valinomycin the accumulated AntiOxCIN4 were then released from mitochondria, clearly indicating a ∆Ψ-dependent uptake of AntiOxCIN4. The result also shows that the TPP cationic moiety is responsible for the increased mitochondrial translocation of the dietary antioxidant. Fig. 2 As stated before, solute lipophilicity strongly affects the routes of passive drug transfer through

membranes

barriers

and

consequently

its

biological

activity

and

pharmacokinetics.16 In fact, lipophilicity is a physicochemical property, usually 6 ACS Paragon Plus Environment

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expressed as log P, which encrypts information on the network of inter- and intramolecular forces affecting drug interactions with lipid structures as well as with the target protein. Log P is as measure of the distribution of a compound in a biphasic system and is one criterion to determine lipophilicity. However, in the case of TPPbased mitochondriotropic antioxidants the direct methods either computational prediction or octanol/water distribution procedures cannot be applied due their ionic and surfactant character. So, to better understand AntiOxCIN4 ability to cross membrane barriers a simple and reliable voltammetric methodology based on ion transfer at ITIES was performed in this work. The voltammetric behaviour of the AntiOxCIN4 transfer at the water/DCH microinterface is shown in Fig. 3. Fig. 3 The peak current observed at 0.7 V during the forward scan (towards more positive potentials) is due to positively charged antioxidant transferring from aqueous phase to organic phase while the return peak current, at around 0.45 V, is due to the reverse ion transfer from oil to water phase. When AntiOxCIN4 was not added to the aqueous phase the residual current measured arises from supporting electrolyte solutions double layer. Liquid-liquid interfaces can be considered as a simple model for studying transport processes through biologic membranes. H-bond interactions, the main intermolecular forces governing the partitioning in the ITIES (i.e., the ability of a solute to leave or not the favourable hydrogen-bonding environment of the aqueous phase),40-42 is also described as an important factor for the passive diffusion across biomembranes.43-45 Ordered lipid layers provide hydrogen bonding groups located exclusively in the head group region of the lipids. To partition into the hydrocarbon region of the bilayer, the solute must be lipophilic enough to overcome the energy losses that occur in breaking the hydrogen bonds with water or the lipid head groups.43-45 In this work, the synthesized mitochondria-targeted antioxidant showed to be highly active at the liquidliquid interface with the hydrophilic part (pyrogallol group) of the antioxidant being present on the water side due to favourable hydrogen bonds with water molecules while the long lipophilic alkyl chain linked to the TPP group being surrounded by organic solvent molecules. Furthermore, the TPP carrier is expected to rule the antioxidant migration from water to DCH phase (see Scheme 1). Although the positively charged 7 ACS Paragon Plus Environment


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antioxidant does not spontaneously migrate to the organic phase its transfer across the water/DCH interface occurs when an external potential difference is applied. Notably, the ion transport occurs within the available potential window. Thus, considering the electrochemical behaviour of AntiOxCIN4 at the water/DCH interface it is foreseen that the mitochondria-targeted antioxidant can also be able to cross biological membranes. Complementary electrochemical experiments focused on the AntiOxCIN4 counter-ion, the methanesulfonate anion, were also performed to test its ability to cross the liquidliquid interface. In absence of the antioxidant, only a residual transfer of the anion through the interface was observed within the potential limits (see Fig. S1, Supporting Information). ITIES ionic partition diagrams (Pourbaix-type diagrams) indicates the molecules behaviour as a function of the interfacial Galvani potential difference and the pH.46 In this work, an electrochemical study was performed to assess the evolution of standard ion transfer potential upon changes of aqueous pH aiming to draw the ionic partition diagram of mitochondriotropic antioxidant AntiOxCIN4 at the water/DCH interface. Complementary experiments allowed to conclude that ion transfer potentials were fairly insensitive to ionic strength changes of the aqueous electrolyte solutions at different pH values for the concentration range applied in the experiments. DPV was selected for these experiments because it provides more accurate estimation of the transfer potentials comparing to CV.26 For each experiment performed at different pH, MitoCIN4 (C = 0.26 mM) and tetrabutylammonium chloride (TBA+; C = 0.13 mM) were added to the aqueous phase, following by electrochemical measurement. TBA+ was selected among others tetraalkylcations to the experiments to avoid the overlap of both tetraalkylcation and AntiOxCIN4 voltammetric responses and worked as internal reference ion for all halfwave potential measurements to transpose the experimental transfer potentials to the absolute Galvani potential scale on the basis of the following assumption: 47

(

)

(

E p (antioxidan t ) − ∆woφ 0 (antioxidan t ) = E p TBA + − ∆woφ 0 TBA +

)

(1)

where ∆ ow φ 0 (TBA + ) is the standard Galvani potential for the transfer of TBA+ from water to DCH38 and Ep(antioxidant) and Ep(TBA+) are the peak potentials obtained experimentally for the antioxidant and TBA+ transfer reactions, respectively. 8 ACS Paragon Plus Environment

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Fig. 4 shows the DPV evolution of AntiOxCIN4 ion transfer across the liquid-liquid interface for each experiment carried out at different pH (pH values ranging from 2.0 to 12.0) under experimental conditions depicted in Scheme 2. Fig. 4 The transfer potential at which the mitochondria-targeted antioxidant cross the liquidliquid interface strongly depends on its dissociation constant (pKa). AntiOxCIN4 pKa values is expected to be close to the ones found for similar systems, namely caffeic48 and gallic49 acids, shown in Fig 5A. Likely, for AntiOxCIN4 it is expected the occurrence of two dissociation constants coming from the deprotonation of the OHs (pOH and m-OH) present at the pyrogallol system. In this work, a value of pKa1 (p-OH) = 8.0 was obtained for the AntiOxCIN4 by using a pH titration method (see Fig. S2, Supporting Information). The value obtained is in good agreement with the values reported in the literature for caffeic and gallic acids (from 8.5 to 8.7, Fig. 5A) since the synthesized antioxidant combines effects from the physicochemical properties of these two acids. Fig. 5 The standard transfer potential remained unchanged (within experimental error) for pH values below AntiOxCIN4 pKa1 (p-OH, 8.0) and corresponds to the transfer of positively charged AntiOxCIN4 from water to DCH phase. For pH values higher than pKa1, AntiOxCIN4 is zwitterionic and shifts in standard transfer potential to higher values were observed. In the voltammograms obtained at pH 8.0 and 9.9, a high transfer current at the more positive potentials of the potential window was observed probably due to the assisted transfer of the anion composing the aqueous electrolyte by the positively charged triphenylphosphonium group of AntiOxCIN4 adsorbed at the interface (ion-pair formation), decreasing the available potential window. For pHs above AntiOxCIN4 pKa2, the predominant species in solution is expected to be negatively charged due to the second deprotonation of the m–OH group of the pyrogallol moiety. However, this transfer process was not observed in our experiments probably due to the pronounced transfer of the aqueous supporting electrolyte observed at extreme pH values, which reduced considerably the available potential window. 9 ACS Paragon Plus Environment


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For construction of the ionic partition diagram of MitoCIN4 let us consider the partition of a lipophilic monobasic compound B between two immiscible phases, where the boundary lines defining the domain of predominance species involved are given by the following equations (equiconcentration convection):46

line 1:

∆ φ w

o

=

∆ φ w

0

o

BH

(2)

+

0

line 2 : pH = pK a − log PB line 3:

∆

w o

φ =

∆ φ w

0

o

BH

+

+

(3)

2 . 3 RT 2 . 3 RT (log PB0 − pK aw ) + pH F F

(4)

0

where ∆ ow φ is the Galvani potential difference between the aqueous and the organic phases,

∆

w o

0 is the standard potential of cation BH+ and logPB0 is the standard φ BH +

partition coefficient of the monobase B. In accordance, the DPV experimental results attained at different pHs have been used to build the AntiOxCIN4 ionic partition diagram, as represented in Fig. 6. For pH values below the pKa1 (p-OH) of AntiOxCIN4 the transfer potential of the positively charged antioxidant remains constant upon changes in pH of the aqueous phase and the boundary line 1 is parallel to the pH axis. For pH values higher than the pKa1, AntiOxCIN4 is zwitterionic and boundary line 3 is oblique with a potential increase of 2.3RT/F, i.e. 59 mV/pH unit, at 25 ºC. Dotted line 3 in ionic partition diagram represents the transfer process for pH values > pKa2, where the AntiOxCIN4 antioxidant is expected to be predominantly negatively charged and its transfer across the interface at the less positive potentials of the potential window can also occur. Fig. 6 Ionic partition diagrams also give access to the apparent pKa which shifts from the effective pKa of the lipophilic compound by a quantity related to the partition coefficient (logPB0).46 Thus, after comparing the AntiOxCIN4 measured dissociation constant (pKa1 = 8.0, dashed line in Fig. 6) with the calculated dissociation constant (pKa1 = 7.8) from ionic partition diagram, a value of 0.2 was obtained for the logP0 of the zwitterionic form of the antioxidant. 10 ACS Paragon Plus Environment

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The Gibbs energy of the transfer ( ∆ G tr0 , w → o ), a highly important thermodynamic data, is a measure of lipophilicity allowing to assess relevant parameters like membrane permeability and intra- and intermolecular interactions of biomolecules.50 Knowing the standard Gibbs energy of the transfer it is possible to estimate the partition coefficient of the positively charged form of the compound (logP0BH+), according to the following relationships:26

∆ φ w

0

o

BH

0 BH +

log P

+

=

∆ G tr0 , w → o zF

(5)

∆Gtr0 , w→o =− 2.3 RT

(6)

From the standard transfer potential obtained by DPV data, the Gibbs energy of transfer (13.1 kJ mol-1) and the partition coefficient of the positively charged antioxidant (logPBH+0 = -2.3) have been obtained from eqs. 5 and 6. The calculated thermodynamic data are resumed in Table 1. Table 1 The measurement of AntiOxCIN4 partition coefficients (P, expressed as logP0) at the water/DCH interface allowed to understand its lipophilic properties, namely the different affinity of its charged and zwitterionic forms towards the organic phase. Although our studies showed that both antioxidant forms can cross the liquid-liquid interface, the positively charged AntiOxCIN4 form is more hydrophilic than the correspondent zwitterionic form due to the existence of favourable hydrogen bonds with water molecules. After ionization (pH > pKa1), the zwitterionic form is formed, which is stabilized by the formation of intramolecular H-bonds assisted by resonance-electron delocalization throughout the cinnamic moiety (see Fig. 5B).24 So, the pH diagram clearly showed that the acid-base properties of the antioxidant, ruled out by the pH of medium, influence its partition at the liquid-liquid interface. Under physiological conditions AntiOxCIN4 is positively charged due to the TPP cation and its transfer across the liquid-liquid interface was observed is response to the applied external electric potential difference. Although the relatively hydrophilic nature of 11 ACS Paragon Plus Environment


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positively charged antioxidant (measured partition coefficient value of -2.3) may suggest unfavourable permeability in the context of mitochondria membranes, the results obtained by electrochemistry at the ITIES are in good agreement with the pharmacological behaviour observed in biological assays, where the positively charged AntiOxCIN4 succeeds to cross the mitochondrial membrane due to the large negative potential (-180 to -200 mV), leading to the selective accumulation of the lipophilic cation to mitochondria. Also, the electric potential at the plasma membrane (of -30 to 60 mV) can increase the concentration of charged compounds in intact cells51 (see Scheme 1). Moreover, only in mitochondrial matrix where the pH is slightly alkaline (pH ≈ 8.0), AntiOxCIN4 can suffer ionization, meaning that this mitochondriotropic antioxidant reaches its target (mitochondria) in its active form (reduced form).16

CONCLUSIONS The extent of uptake of a TPP+ derivative anchoring to the mitochondrial inner membrane is dependent upon their hydrophobicity, the length of the linker unit and the functionalization of the bioactive molecule. Within mitochondria, it can elicit beneficial effects by diverse mechanisms, namely by scavenging of reactive radicals and preventing membrane lipid peroxidation and/or control mitochondrial redox signalling. The results obtained in this work reinforce previous data as it was shown that AntiOxCIN4 can accumulate inside heart mitochondria with an accumulation ratio of ≈ 2600-fold vs. outside mitochondrial space. The electrochemistry at ITIES has shown to be a powerful tool for assessing the passive transfer and partition of mitochondria-targeted antioxidant AntiOxCIN4 in a polarized water/DCH interface. Electrochemical data collected was used to draw the ionic partition diagram which allow envisaging which specie (positively charged or zwitterionic) is involved in the transfer across the water/oil interface upon variation of potential and/or aqueous pH. In addition, important thermodynamic data was obtained, such as the partition coefficients of the positively charged (-2.3) and zwitterionic (0.2) forms of the MitoOxCIN4, using DPV technique. The data gathered so far revealed that the mitochondria-targeted antioxidants, in particular AntiOxCIN4, can cross (bio)membranes. This work showed that AntiOxCIN4 is positively charged at physiological pH and that it can suffer a subsequent ionization process, which is dependent of the pH of the cellular compartment, meaning that this mitochondriotropic 12 ACS Paragon Plus Environment

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antioxidant can reach its target (mitochondria) in its active form (reduced form). The data obtained at the liquid-liquid interface is of utmost importance to understand ion passive transport across membranes and bioavailability (pharmacokinetics). The determination of the pH-lipophilicity profile of drugs could be considered as an interesting alternative to the traditionally used water/n-octanol system in the search of simple models for biological membranes, especially for compounds having ionic and surfactant properties. Also, H-bond ability, which is identified as an important factor for biomolecules passive transfer across biomembranes, are the main intermolecular forces governing the partitioning in the ITIES system, enhancing the contribution of the ITIES in ion drug disposition and pharmacokinetics studies.

ACKNOWLEDGEMENTS This work was carried out under FEDER funds through the Operational Programme Competitiveness Factors - COMPETE and national funds by FCT – Foundation for Science and Technology under research grants (QUI/UI0081/2013, NORTE-01-0145FEDER-000028, POCI-01-0145-FEDER-007440, POCI-01-0145-FEDER-016659 and PTDC/DTP-FTO/2433/2014). J. Ribeiro (SFRH/BPD/105395/2014) and S. Benfeito (SFRH/BD/99189/2013) are supported by FCT Postdoctoral and Doctoral grants under QREN – POPH – Advanced Training, subsidized by European Union and national MEC funds. F. Cagide and J. Teixeira grants are from NORTE-01-0145-FEDER-000028.

ADDITIONAL INFORMATION The authors declare no competing financial interest. AntiOxCIN4 synthesis and application are under patent (NPAT20161000075435). PJO and FB are co-founders of spin-off MitoTAG and the mentors of MitoDIETS project. This start-up did not provide any funding for the work.

REFERENCES (1) Moreira, P. I.; Zhu, X.; Wang, X.; Lee, H.-g.; Nunomura, A.; Petersen, R. B.; Perry, G.; Smith, M. A. Mitochondria: A Therapeutic Target in Neurodegeneration.



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Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 2010, 1802, 212220. (2) Murphy, Michael P. How Mitochondria Produce Reactive Oxygen Species. Biochemical Journal 2008, 417, 1-13. (3) Valcarcel-Jimenez, L.; Gaude, E.; Torrano, V.; Frezza, C.; Carracedo, A. Mitochondrial Metabolism: Yin and Yang for Tumor Progression. Trends in Endocrinology & Metabolism 2017, 28, 748-757. (4) Ignazio, G.; Stefan, R.; Catia, D.; Leonilde, B.; Paulo, J. O.; David, Q. H. W. a. P. P. Mitochondria in Chronic Liver Disease. Current Drug Targets 2011, 12, 879-893. (5) Pfeffer, G.; Majamaa, K.; Turnbull, D. M.; Thorburn, D.; Chinnery, P. F. Treatment for Mitochondrial Disorders. Cochrane Database of Systematic Reviews 2012, 4, Article number: CD004426. (6) Alfadda, A. A.; Sallam, R. M. Reactive Oxygen Species in Health and Disease. Journal of Biomedicine and Biotechnology 2012, 2012, 1-14. (7) Malty, R. H.; Jessulat, M.; Jin, K.; Musso, G.; Vlasblom, J.; Phanse, S.; Zhang, Z.; Babu, M. Mitochondrial Targets for Pharmacological Intervention in Human Disease. Journal of Proteome Research 2015, 14, 5-21. (8) Nunnari, J.; Suomalainen, A. Mitochondria: in Sickness and in Health. Cell 2012, 148, 1145-1159. (9) Nicholls, D. G. Mitochondrial ion circuits. Essays In Biochemistry 2010, 47, 25-35. (10) Murphy, M. P. Targeting Lipophilic Cations to Mitochondria. Biochimica et Biophysica Acta (BBA) - Bioenergetics 2008, 1777, 1028-1031. (11) Oyewole, A. O.; Birch-Machin, M. A. Mitochondria-targeted Antioxidants. The FASEB Journal 2015, 29, 4766-4771. (12) Boris, A. F.; Vladimir, P. S. Cellular and Molecular Mechanisms of Action of Mitochondria-Targeted Antioxidants. Current Aging Science 2017, 10, 41-48. (13) Isaev Nickolay, K.; Stelmashook Elena, V.; Genrikhs Elisaveta, E.; Korshunova Galina, A.; Sumbatyan Natalya, V.; Kapkaeva Marina, R.; Skulachev Vladimir, P. Neuroprotective Properties of Mitochondria-targeted Antioxidants of the SkQ-type. Reviews in the Neurosciences 2016, 27, 849-855. (14) Filomena, S. G. S.; Rui, F. S.; Renata Couto and Paulo, J. O. Targeting Mitochondria in Cardiovascular Diseases. Current Pharmaceutical Design 2016, 22, 5698-5717.


Page 15 of 29 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


(15) Teixeira, J.; Soares, P.; Benfeito, S.; Gaspar, A.; Garrido, J.; Murphy, M. P.; Borges, F. Rational Discovery and Development of a Mitochondria-targeted Antioxidant Based on Cinnamic Acid Scaffold. Free Radical Research 2012, 46, 600611. (16) Teixeira, J.; Cagide, F.; Benfeito, S.; Soares, P.; Garrido, J.; Baldeiras, I.; Ribeiro, J. A.; Pereira, C. M.; Silva, A. F.; Andrade, P. B.; Oliveira, P. J.; Borges, F. Development of a Mitochondriotropic Antioxidant Based on Caffeic Acid: Proof of Concept on Cellular and Mitochondrial Oxidative Stress Models. Journal of Medicinal Chemistry 2017, 60, 7084-7098. (17) Gulaboski, R.; Cordeiro, M. N. D. S.; Milhazes, N.; Garrido, J.; Borges, F.; Jorge, M.; Pereira, C. M.; Bogeski, I.; Morales, A. H.; Naumoski, B.; Silva, A. F. Evaluation of the Lipophilic Properties of Opioids, Amphetamine-like Drugs, and Metabolites Through Electrochemical Studies at the Interface Between Two Immiscible Solutions. Analytical Biochemistry 2007, 361, 236-243. (18) van De Waterbeemd, H. The History of Drug Research: From Hansch to the Present. Quantitative Structure-Activity Relationships 1992, 11, 200-204. (19) Lill, M. A. Multi-dimensional QSAR in Drug Discovery. Drug Discovery Today 2007, 12, 1013-1017. (20) Leo, A.; Hansch, C.; Elkins, D. Partition Coefficients and Their Uses. Chemical Reviews 1971, 71, 525-616. (21) Berthod, A.; Carda-Broch, S. Determination of Liquid-liquid Partition Coefficients by Separation Methods. Journal of Chromatography A 2004, 1037, 3-14. (22) Volkov, A. G. Liquid Interfaces In Chemical, Biological And Pharmaceutical Applications; Taylor & Francis, 2001. (23) Sangster, J. Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry; Wiley, 1997. (24) Bouchard, G.; Carrupt, P.-A.; Testa, B.; Gobry, V.; Girault, H. H. Lipophilicity and Solvation of Anionic Drugs. Chemistry – A European Journal 2002, 8, 3478-3484. (25) Reymond, F.; Carrupt, P.-A.; Testa, B.; Girault, H. H. Charge and Delocalisation Effects on the Lipophilicity of Protonable Drugs. Chemistry – A European Journal 1999, 5, 39-47. (26) Lam, H.-T.; Pereira, C. M.; Roussel, C.; Carrupt, P.-A.; Girault, H. H. Immobilized pH Gradient Gel Cell to Study the pH Dependence of Drug Lipophilicity. Analytical Chemistry 2006, 78, 1503-1508. 15 ACS Paragon Plus Environment


Page 16 of 29

(27) Reymond, F.; Steyaert, G.; Carrupt, P.-A.; Testa, B.; Girault, H. Ionic Partition Diagrams: A Potential−pH Representation. Journal of the American Chemical Society 1996, 118, 11951-11957. (28) Alemu, H. Voltammetry of Drugs at the Interface Between Two Immiscible Electrolyte Solutions*. Pure and Applied Chemistry 2004, 76, 697-705. (29) Volkov, A. G.; Deamer, D. W. Liquid-Liquid InterfacesTheory and Methods; Taylor & Francis, 1996. (30) Ribeiro, J. A.; Silva, F.; Pereira, C. M. Electrochemical Study of the Anticancer Drug Daunorubicin at a Water/Oil Interface: Drug Lipophilicity and Quantification. Analytical Chemistry 2013, 85, 1582-1590. (31) Ortuño, J. A.; Gil, A.; Serna, C.; Molina, A. Voltammetry of some Catamphiphilic Drugs with Solvent Polymeric Membrane Ion Sensors. Journal of Electroanalytical Chemistry 2007, 605, 157-161. (32) Kim, H. R.; Pereira, C. M.; Han, H. Y.; Lee, H. J. Voltammetric Studies of Topotecan Transfer Across Liquid/Liquid Interfaces and Sensing Applications. Analytical Chemistry 2015, 87, 5356-5362. (33) Reymond, F.; Fermı́n, D.; Lee, H. J.; Girault, H. H. Electrochemistry at Liquid/Liquid Interfaces: Methodology and Potential Applications. Electrochimica Acta 2000, 45, 2647-2662. (34) Pereira, S. P.; Pereira, G. C.; Pereira, C. V.; Carvalho, F. S.; Cordeiro, M. H.; Mota, P. C.; Ramalho-Santos, J.; Moreno, A. J.; Oliveira, P. J. Dioxin-induced Acute Cardiac Mitochondrial Oxidative Damage and Increased Activity of ATP-sensitive Potassium Channels in Wistar Rats. Environmental Pollution 2013, 180, 281-290. (35) Teixeira, J.; Oliveira, C.; Amorim, R.; Cagide, F.; Garrido, J.; Ribeiro, J. A.; Pereira, C. M.; Silva, A. F.; Andrade, P. B.; Oliveira, P. J.; Borges, F. Development of Hydroxybenzoic-based Platforms as a Solution to Deliver Dietary Antioxidants to Mitochondria. Scientific Reports 2017, 7, Article number: 68426842. (36) Ribeiro, J. A.; Miranda, I. M.; Silva, F.; Pereira, C. M. Electrochemical Study of Dopamine and Noradrenaline at the Water/1,6-Dichlorohexane Interface. Physical Chemistry Chemical Physics 2010, 12, 15190-15194. (37) Katano, H.; Tatsumi, H.; Senda, M. Ion-transfer Voltammetry at 1,6Dichlorohexane|Water and 1,4-Dichlorobutane|Water Interfaces. Talanta 2004, 63, 185193.


Page 17 of 29 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


(38) Katano, H.; Senda, M. Voltammetry at 1,6-Dichlorohexane/Water interface. Analytical Sciences 2001, 17, 1027-1029. (39) Smith, R. A. J.; Hartley, R. C.; Cochemé, H. M.; Murphy, M. P. Mitochondrial Pharmacology. Trends in Pharmacological Sciences 2012, 33, 341-352. (40) Steyaert, G.; Lisa, G.; Gaillard, P.; Boss, G.; Reymond, F.; Hubert H, G.; Carrupt, P.-A.; Testa, B. Intermolecular Forces Expressed in 1,2-Dichloroethane–Water Partition Coefficients. Journal of the Chemical Society, Faraday Transactions 1997, 93, 401406. (41) Osakai, T.; Ebina, K. Non-Bornian Theory of the Gibbs Energy of Ion Transfer between Two Immiscible Liquids. The Journal of Physical Chemistry B 1998, 102, 5691-5698. (42) Osakai, T.; Ogata, A.; Ebina, K. Hydration of Ions in Organic Solvent and Its Significance in the Gibbs Energy of Ion Transfer between Two Immiscible Liquids. The Journal of Physical Chemistry B 1997, 101, 8341-8348. (43) Mälkiä, A.; Murtomäki, L.; Urtti, A.; Kontturi, K. Drug Permeation in Biomembranes: In Vitro and In Silico Prediction and Influence of Physicochemical Properties. European Journal of Pharmaceutical Sciences 2004, 23, 13-47. (44) Vaes, W. H. J.; Urrestarazu Ramos, E.; Verhaar, H. J. M.; Cramer, C. J.; Hermens, J. L. M. Understanding and Estimating Membrane/Water Partition Coefficients: Approaches to Derive Quantitative Structure Property Relationships. Chemical Research in Toxicology 1998, 11, 847-854. (45) Goodwin, J. T.; Conradi, R. A.; Ho, N. F. H.; Burton, P. S. Physicochemical Determinants of Passive Membrane Permeability: Role of Solute Hydrogen-bonding Potential and Volume. Journal of Medicinal Chemistry 2001, 44, 3721-3729. (46) Gobry, V.; Ulmeanu, S.; Reymond, F.; Bouchard, G.; Carrupt, P.-A.; Testa, B.; Girault, H. H. Generalization of Ionic Partition Diagrams to Lipophilic Compounds and to Biphasic Systems with Variable Phase Volume Ratios. Journal of the American Chemical Society 2001, 123, 10684-10690. (47) Ferreira, E. S.; Garau, A.; Lippolis, V.; Pereira, C. M.; Silva, F. Electrochemistry of 2,8-Dithia[9],(2,9)-1,10-phenanthrolinophane (L) at the Polarized Water/1,2Dichloroethane Interface: Evaluation of the Complexation Properties Towards Transition and Post-transition Metal Ions. Journal of Electroanalytical Chemistry 2006, 587, 155-160.



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(48) Silva, F. A. M.; Borges, F.; Guimarães, C.; Lima, J. L. F. C.; Matos, C.; Reis, S. Phenolic Acids and Derivatives: Studies on the Relationship Among Structure, Radical Scavenging Activity, and Physicochemical Parameters. Journal of Agricultural and Food Chemistry 2000, 48, 2122-2126. (49)

Tam,

K.

Y.;

Takács-Novák,

K.

Multi-wavelength

Spectrophotometric

Determination of Acid Dissociation Constants: A Validation Study. Analytica Chimica Acta 2001, 434, 157-167. (50) Komorsky-Lovrić, Š.; Riedl, K.; Gulaboski, R.; Mirčeski, V.; Scholz, F. Determination of Standard Gibbs Energies of Transfer of Organic Anions across the Water/Nitrobenzene Interface. Langmuir 2002, 18, 8000-8005. (51) Porteous, C. M.; Logan, A.; Evans, C.; Ledgerwood, E. C.; Menon, D. K.; Aigbirhio, F.; Smith, R. A. J.; Murphy, M. P. Rapid Uptake of Lipophilic Triphenylphosphonium Cations by Mitochondria In Vivo Following Intravenous Injection: Implications for Mitochondria-specific Therapies and Probes. Biochimica et Biophysica Acta (BBA) - General Subjects 2010, 1800, 1009-1017.


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List of Figures and Schemes Figure 1. Structures of A) MitoQ, B) SKQ1H, C) caffeic acid and D) AntiOxCIN4. Scheme 1. The transfer of the targeted-antioxidant AntiOxCIN4 at the liquid-liquid interface in response to the applied potential (∆V) under pre-established experimental conditions (left) can be useful to predict the ability of the penetration cation to cross biological membranes (right) due to transmembrane electrical potential difference (∆ψ). Scheme 2. Schematic representation of the electrochemical cell used throughout this study. Figure 2. AntiOxCIN4 uptake by energised rat heart mitochondria, as measured by using a TPP-selective electrode. Figure 3. CVs obtained at the water/DCH interface in the absence (---) and presence (— ) of 0.34 mM of AntiOxCIN4 at pH 7.0. Figure 4. DPVs representing the transfer of 0.26 mM AntiOxCIN4 at the water/DCH micro-interface at pH 4.0 (black), pH 6.0 (red), pH 7.2 (blue), pH 8.0 (green) and pH 9.9 (pink). [TBA+] = 0.13 mM. Figure 5. A) Dissociation constants (pKa) of caffeic and gallic acids at 25 ºC. B) Phenolate stabilization by intramolecular hydrogen bonding and negative charge delocalization in AntiOxCIN4. Figure 6. Ionic partition diagram of AntiOxCIN4 at the water/DCH interface. The dashed line represents the pKa1 of AntiOxCIN4. Lines 1, 2 and 3 respectively correspond to eqs 2, 3 and 4.



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Fig. 1


Page 21 of 29 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


Scheme 1



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Scheme 2

Ag

AgCl

NaCl 2 mM

BTPPATPBCl

x mM AntiOxCIN4

BTPPACl 2 mM

1 mM

Buffer solution pH 2.0 – 12.0

(Water)

(DCH)

(Water)

AgCl

Ag’


Page 23 of 29

Fig. 2

SUC

↓

VAL

↓

[ AntiOxCIN 4 ]

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


MIT

↓

Time



Page 24 of 29

Fig. 3


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Fig. 4



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Fig. 5


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Fig. 6



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List of Tables Table 1. Thermodynamic data obtained for the transfer of AntiOxCIN4 at the water/DCH micro-interface ∆0wφ0 /V

∆Gtr0, w→DCH

logP0DCH

logP0DCH

(in DCH)

/kJ mol-1

(positively charged)

(zwitterionic)

0.135

13.1

-2.3

0.2


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Electrochemical Behaviour of a Novel Mitochondria-targeted

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