Amperometric monitoring of quercetin permeation through skin membranes

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International Journal of Pharmaceutics xxx (2015) xxx–xxx

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Amperometric monitoring of quercetin permeation through skin membranes Jadwiga Rembiesaa,b , Hala Garia,b , Johan Engbloma,b , Tautgirdas Ruzgasa,b,* a b

Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, 205 06 Malmö, Sweden Biofilms—Research Center for Biointerfaces, Malmö University, 205 06 Malmö, Sweden

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 August 2015 Accepted 28 October 2015 Available online xxx

Transdermal delivery of quercetin (QR, 3,30 ,40 ,5,7-pentahydroxyflavone), a natural flavonoid with a considerable antioxidant capacity, is important for medical treatment of, e.g., skin disorders. QR permeability through skin is low, which, at the same time, makes the monitoring of percutaneous QR penetration difficult. The objective of this study was to assess an electrochemical method for monitoring QR penetration through skin membranes. An electrode was covered with the membrane, exposed to QR solution, and electrode current was measured. The registered current was due to electro-oxidation of QR penetrating the membrane. Exploiting strict current-QR flux relationships diffusion coefficient, D, of QR in skin and dialysis membranes was calculated. The D values were strongly dependent on the theoretical model and parameters assumed in the processing of the amperometric data. The highest values of D were in the range of 1.6–6.1 107 cm2/s. This was reached only for skin membranes pretreated with bufferethanol mixture for more than 24 h. QR solutions containing penetration enhancers, ethanol and Lmenthol, definitely increased D values. The results demonstrate that electrochemical setup gives a possibility to assess penetration characteristics as well as enables monitoring of penetration dynamics, which is more difficult by traditional methods using Franz cells. ã 2015 Elsevier B.V. All rights reserved.

Chemical compounds: Quercetin (PubChem CID: 5280343) L-menthol (PubChem CID: 16666) Ethanol (PubChem CID: 702) Methanol (PubChem CID: 887) Keywords: Quercetin Flavonoids Electrochemistry Skin permeation Penetration enhancers Diffusion coefficient

1. Introduction Skin is the largest organ of human body and it provides a protective function against external factors such as mechanical injuries, chemical toxins, ozone or UV radiation. To fulfill this role, skin is composed of two main parts: dermis and epidermis. Dermis is the inner part of the skin which contains blood capillaries, sweat glands, sebaceous glands, hair follicles and nerves. The outer part of the skin is called epidermis. It is built up of skin cells (keratinocytes) at different stage of differentiation: from active cells with the ability to divide, to the most outer layer of dead cells (corneocytes), which together with the extracellular lipids form the most impermeable part of the skin-the stratum corneum. Although stratum corneum is a tough barrier, it is challenged by topical application of drugs. Topically applied drugs may diffuse through or be trapped in epidermis, or penetrate to dermis and reach blood circulation. Transdermal drug transport is reported to

Abbreviations: CNP, carbon nanoparticles; EtOH, ethanol; PBS, phosphate buffer saline; QR, quercetin; MT, L-menthol. * Corresponding author at: Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, 205 06 Malmö, Sweden. Fax: +46 40 6658100. E-mail address: [email protected] (T. Ruzgas).

occur across the skin cells (transcellular route), lipids present in stratum corneum (intracellular route) or the hair follicles and sweat ducts (Moser et al., 2001; Barry, 2002). The skin, as an outer tissue of a body, is exposed to high risk of oxidative stress and increased amount of reactive oxygen species (ROS), which are one of the reasons of skin damage, photo-ageing and cancerous lesions. The generation of ROS in skin is related to external factors, especially to the UV irradiation, which penetrates through stratum corneum to deeper skin layers: UVB reaches mostly epidermis and UVA reaches both epidermis and dermis where the pathological changes starts (Farkas et al., 2002). Skin has its own protection mechanisms from ROS, such as p53 tumor suppressor gene (Yamaguchi et al., 2008) or creatine kinase system (Lenz et al., 2005), but in the conditions of excessive exposure to harmful agents it needs additional protection, e.g., topical application of antioxidants. Flavonoids, natural compounds found in plants, have strong antioxidant properties and, thus, are great agents to prevent the photooxidative stress in skin. QR is a flavonoid and is considered as one of the most powerful natural antioxidants (Bonina et al., 1996). It is widely used in medical treatment due to its anti-inflammatory (Lin et al., 2012), antibacterial (Rigano et al., 2007), antiviral (Ganesan et al., 2012), anticancer (Caltagirone et al., 2000) and

http://dx.doi.org/10.1016/j.ijpharm.2015.10.073 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

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antioxidative properties. It has been found that the amount of QR in apple skin increases with intensified exposure to sunlight as a possible protective reaction against UV-B radiation in plants (Solovchenko and Schmitz-Eiberger, 2003). The increased resistance to oxidative stress after QR application was observed in microorganisms (Belinha et al., 2007) and worms (Kampkötter et al., 2007). Casagrande et al. (2006) confirmed anti-inflammatory and anti-oxidative effect of QR on UVB-exposed skin of mice. The same was later confirmed on rats (Liu et al., 2013). From the discussion of QR effects on skin, it is obvious that topical application should be designed to optimize QR accumulation in epidermis or delivery to dermis and ultimately uptake in the blood. As concerns QR delivery to the blood, the main and most common route is oral administration. However, the bioavailability of QR from the gastrointestinal tract is limited. It was estimated that absorption of QR after oral application was less than 17% in rats (Khaled et al., 2003) and varied between 17 and 52% in human (Hollman et al., 1997). Topical QR delivery to the blood might, thus, be considered as an interesting alternative in the future. Despite the great anti-oxidative properties of QR, the drug is characterized by very low skin permeation and, thus, restricted percutaneous delivery (Saija et al., 1998; Liu et al., 2013; Lin et al., 2012). This was confirmed by a number of in-vitro penetration assays using Franz cells (Casagrande et al., 2007; Vicentini et al., 2008; Dal Belo et al., 2009; Bose et al., 2013). It is known that some compounds as alcohols, glycerides, fatty acids, terpenes or phospholipids are able to influence the transdermal permeation of drugs (for a review, see Sinha and Kaur, 2000). The use of penetration enhancers have been tested to improve QR permeation through skin. Some of them, inter alia D-limonene and lecithine had no effect (Saija et al., 1998), but others as QR-loaded lecithin– chitosan nanoparticles significantly increased the amount of QR accumulation in skin layers reaching 9 mg/cm2 and 3.3 mg/cm2 in epidermis and dermis, respectively (Tan et al., 2011). In all above mentioned investigations rather tedious methods to study QR penetration through skin membranes are used. The current study describes optimization and application of an electrochemical technique to monitor QR permeation through dialysis and skin membranes in the absence and the presence of penetration enhancers. To the best of our knowledge this is the first

report on application of skin membrane covered electrodes for amperometric in-vitro registration of QR penetration. General aspects of the methodology has been briefly introduced in our recent publication (Gari et al., 2015). This relatively simple, quick and highly convenient amperometric method allows the monitoring of QR permeation through membranes in real-time which is registered as an electric current. By knowing the mechanism of drug electro-oxidation at the electrode, the amperometric response, i.e., the electrode current, enables assessing the values of the flux and the diffusion coefficient of QR in skin membranes. 2. Material and methods 2.1. Materials Phosphate buffer saline (PBS) tablets, quercetin (QR), carbon nanopowder (CNP, with carbon particle size less than 50 nm), methanol, ethanol-(EtOH) and L-menthol (MT) were purchased from Sigma–Aldrich (St. Louise, USA). The solutions were prepared using water purified by Milli-Q system (Merck Millipore, Billerica, USA) with a resistivity of 18.2 MV cm. pH of solutions was adjusted by using 2.5 M HCl and 1.0 M NaOH. The stock solution of QR at concentration 0.01 M was prepared by diluting 15 mg of QR in 5 ml of methanol. The fresh stock solution was prepared weekly and was stored in the fridge at +4  C, in dark (wrapped in aluminum foil). For monitoring the penetration of QR through membranes, skin membranes were prepared from pig’s ears provided by a local abattoir. As a model membrane a dialysis tubing cellulose membrane with cut-off 12 kDa (Sigma–Aldrich, St. Louise, USA) was used. 2.2. Electrode preparation and experimental conditions Electrochemical measurements were performed using CompactStat potentiostat from IVIUM Technologies (Eindhoven, Netherlands). Three electrode electrochemical setup (Fig. 1(1)) was used. A platinum wire and an Ag/AgCl/(KCl saturated) electrode were used as a counter and reference electrodes, respectively. To enhance electro-oxidation of QR at the working electrode, a platinum disk electrode was modified with CNP

Fig. 1. (1) A schematic representation of the electrochemical cell for amperometric measurements (a) Ag/AgCl reference electrode, (b) working electrode covered with a membrane, (c) platinum mesh counter electrode, and (d) magnetic stirring. (2) A photo of the tip of the working electrode after different steps of the preparation (A) surface of platinum working electrode, dia. 0.2 mm, after polishing with emery paper, (B) surface of the working electrode after modification by CNP dispersion, dia. 2 mm, (C) working electrode covered with skin membrane, and (D) skin membrane covered working electrode after QR monitoring (yellow color is due to accumulation of QR in the membrane). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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dispersion (Fig. 1(2)). The surface of the working Pt electrode was washed with 70% ethanol, rinsed with water and gently polished using emery paper. A 5 ml droplet of CNP dispersion, prepared as described below, was put on the top of the Pt electrode and left to dry in the air for one hour. The CNP dispersion was prepared by mixing 3 mg of carbon nanopowder with 1 ml of distilled water in an Eppendorf tube using ultrasonic treatment for 15 min. This gave a homogenous dispersion, i.e., CNP ink. The CNP ink was then stored at room temperature until use. For monitoring QR penetration through membranes, the dialysis or the skin membrane was fixed on the surface of the working electrode modified with CNP by tightening it with a 100% polyester thread. The electroactive electrode diameter was 2 mm and the electrochemically measureable diffusion area was estimated to be 3.14  102 cm2. The thickness of the skin and the dialysis membranes was assumed to be 1 103 cm and 2.79  103 cm, respectively. During the electrochemical measurement, a defined amount of QR in methanol was pipetted into an electrochemical cell containing three electrodes in 20 ml of PBS solution, also containing some other compounds, e.g., penetration enhances. Immediately after QR injection it slowly penetrates through the membrane and reaches the surface of the working electrode. At the CNP modified surface of the working electrode QR undergoes an electro-oxidation, which is registered as an electrode current. During amperometric measurements the solution in the electrochemical cell was mixed with a magnetic stirrer. To avoid QR photo degradation the electrochemical cell was covered with aluminum foil minimizing light passage to the cell. All measurements were conducted at room temperature. After each measurement the electrochemical cell and electrodes were rinsed with 70% ethanol and water. 2.3. Preparation of skin membranes Skin membranes were prepared from pig ears, provided from a local abattoir and frozen at 80  C. Before measurements ears were defrosted, washed in cold water, shaved with a trimmer and cut into 2 cm width strips. Subsequently, the skin strips were peeled using the dermatome and cut to squared pieces (2 cm  2 cm) with scalpel. The squared skin pieces were then immersed in 60  C water for 90 s. After that the upper part of skin was carefully separated using scalpel and tweezers. Prepared membranes were placed on filter papers, put into petri dishes, moisturized with PBS (pH 7.4) and returned to 80  C until use. Before measurements skin membrane was thawed, hydrated with PBS (pH 7.4) or subjected (incubated) to a mixture of PBS (pH 7.4) and ethanol (7:3, v:v). For this procedure the membrane was placed on the receptor chamber of the Franz cell filled with said solution. The stratum corneum (outer) side of the skin faced the donor chamber, which was left empty. The Franz cell with assembled skin membrane was then closed with parafilm to prevent buffer evaporation through the empty donor chamber. Different incubation times were tested. Amperometric measurements on the membranes were conducted in PBS buffer, pH 7.4, containing ethanol (7:3; v:v) or PBS-ethanol mixture (7:3; v:v) with addition of 0.12% (w:w) L-menthol.

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relate the current to the QR flux the electrochemical redox conversion of QR at the electrode should be known and, thus, this reaction has been studied by cyclic voltammetry. As shown in Fig. 2, electrochemical oxidation and reduction of QR gives defined current peaks in CV, where the peak potentials strongly depend on the solution pH. The anodic and cathodic peak potentials shift to lower potential values at increasing pH. This behavior indicates that the redox process involves not only electrons, but also protons. Such redox process is consistent with the electrochemistry of QR at a glassy carbon electrode reported previously (Zare et al., 2005; Sokolova et al., 2012). 0 The formal potential, E0 , of the redox conversion of QR was calculated using Eq. (1) and its dependence on pH was analyzed (Fig. 2, insert). 0

E0 ¼

Epa  Epc 2

ð1Þ

where, Epa and Epc are anodic and cathodic peak potentials, respectively. As can be seen in Fig. 2 (insert) the formal potential s is linearly dependent on the pH of the buffer solution. The regression gives a slope equal to 74 mV pH 1 which being close to 60 mV pH 1 indicates that the same number of electrons and protons are involved in the QR oxidation/reduction process. The obtained value is in good agreement with the slope of 72 mV pH 1, reported by Sokolova et al. (2012), who concluded a two-electron and twoproton oxidation/reduction of QR at glassy carbon electrode at a pH range from 4.3 to 6.9. Obtained results are also in good agreement with previous reports (Brett and Ghica, 2003; Zare et al., 2005). Keeping this all in mind it can be concluded that electrooxidation of QR on a CNP modified electrode involves two electrons and two protons. Chronoamperometric measurements of QR were conducted to understand the electrode sensitivity and to determine the detection limit. Based on cyclic voltammetry data the applied potential was set to 0.5 V vs Ag/AgCl. The measurements were run in PBS buffer at pH of 4.5, 5.5 and 7.4. The concentration of QR in the electrochemical cell was increased gradually by pipetting specific amounts of QR stock solution resulting in a 2–20 mM concentration range in the electrochemical cell. After each QR injection the

3. Results and discussion 3.1. Characterization of QR electro-oxidation at the electrode surface QR penetration through skin membrane covering the electrode (Fig. 1) was monitored by measuring the current originating from electrooxidation of QR at the surface of CNP-modified electrode. To

Fig. 2. Cyclic voltammograms of QR recorded with CNP modified electrode in PBS buffer solution containing 0.1 mM QR at pH (A) 7.5 and (B) 4.5. Potential scan rate was 0.05 V/s. Inset shows the plot of formal potential (calculated by using Eq. (1)) versus pH.

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steady-state current was quickly reached (data not shown). Steady-state currents from the amperometric measurement versus QR concentration were linear up to 20 mM. The limit of detection (LOD) at each pH was calculated using the signal-to-noise ratio of 3 and it was equal 3.2 mM, 2.4 mM and 3.0 mM at pH 4.5, 5.5 and 7.4, respectively. For future studies of QR transdermal permeation PBS, pH 7.4, was chosen. Summarizing, the electrochemical experiments show that CNPmodified electrodes with applied potential of 0.5 V vs Ag/AgCl can be used for amperometric monitoring of QR at mM concentrations. For relating the electrode current to QR flux it is reasonable to accept that upon the electro-oxidation of QR at pH 7.4 two electrons and two protons are extracted from each QR molecule. 3.2. QR permeation studies The QR penetration studies were performed using a threeelectrode system where the working electrode was covered with skin or dialysis membrane as schematically shown in Fig. 1. QR permeation through skin membranes was compared to the permeation through dialysis membrane characterized by 12 kDa cut-off. The amperometric response to QR addition into the electrochemical cell containing membrane-free and the dialysis membrane covered CNP-modified working electrode are presented in Fig. 3. As can be seen the amperometric response obtained with membrane-free electrode appears immediately after injection and steady-state is quickly reached (Fig. 3, curve 1). During the measurement with electrode covered with dialysis membrane, current response is visible after 9 min from QR injection, and steady-state is reached after about 40 min from the injection (Fig. 3, curve 2). For skin membrane no amperometric response after QR injection was observed at these conditions, i.e., PBS with pH 7.4. This proves that the drug is not able to permeate through skin membrane and reach the electrode surface at the mentioned condition and the time frame investigated (2 h). However, the skin membrane acquired noticeable yellowish color suggesting that QR present in the electrochemical cell accumulated on or in the skin membrane probably with very slow transdermal transport of the drug. This is in agreement with previous studies of Dal Belo et al.

Fig. 3. Amperometric response obtained with CNP-modified electrode to injection (t = 0 min) of QR into electrochemical cell filled with PBS, pH 7.4. (1) Amperometric response to QR recorded with membrane-less and (2) dialysis membrane covered electrode. Final QR concentrations were 2 mM and 6 mM, respectively. Dialysis membrane with cut-off 12 kDa was used. The solution in the electrochemical cell was mixed with magnetic stirrer.

(2009) who demonstrated that during QR penetration studies using Franz cells QR from cosmetic formulations accumulates mostly in epidermis and stratum corneum and no QR was detected in dermis and receptor fluid. These results were also confirmed by other studies (Casagrande et al., 2007; Vicentini et al., 2008). Furthermore it has also been shown that stratum corneum is the main penetration barrier for QR (Lin et al., 2012). Due to the limited transdermal permeation of QR, penetration enhancers have been tested in subsequent experiments to improve QR transport through skin membranes. Up to now, a number of approaches have been used to facilitate QR permeation through skin, such as the use of chemical enhancers (Saija et al., 1998), nanoparticles (Tan et al., 2011), liposomes (Liu et al., 2013) and microneedles (Paleco et al., 2014). During this study two penetration enhancers, ethanol and L-menthol, have been tested to increase the QR permeability and allow for amperometric monitoring of the penetration. For QR penetration studies in the presence of enhancers, skin membranes were pretreated by incubating the membrane in PBS-ethanol mixtures for specific time (always more than 12 h). After that the skin membranes were mounted on the working electrode and the amperometric experiments were run. As can be seen from Fig. 4 pretreated membranes enable current response of the skin covered electrode after QR injection. A clear current response has been registered with electrodes covered with skin membrane pretreated for 24 h (Fig. 4, curve 2). Longer time (40 h) of membrane incubation in PBS-ethanol resulted in increased current response (Fig. 4, curve 1). It should be mentioned that skin membrane incubation for shorter times (e.g., 7.5 h) in PBS-ethanol mixture has not enabled QR transdermal penetration which could be detected by the amperometric measurement over 13 h (data not shown). Similar relationship between time of ethanol pretreatment and its penetration enhancing effect was concluded before (Kai et al., 1990). However, the results presented in Fig. 4 also shows penetration dynamics.

Fig. 4. Amperometric response to QR injection into the electrochemical cell obtained with CNP-modified electrodes covered with pretreated skin membranes. QR concentration in the cell was 0.5 mM. Electrolyte solution was PBS (pH7.4)— ethanol mixture (7:3, v:v). Current responses are for the electrodes covered with two different skin membranes incubated (pretreated) in PBS—ethanol mixtures for (1) 40 h and (2) 24 h before the amperometric measurements. The incubation was in PBS—ethanol mixture (7:3, v:v). Time t = 0 min corresponds to injection of QR into the electrochemical cell.

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It has been reported that treatment of skin membrane with ethanol can affect the lipids in stratum corneum, e.g., by increasing lipid fluidity (Van der Merwe and Riviere, 2005) or by lipid extraction (Kai et al., 1990; Bommannan et al., 1991). Additionally, it was assumed, that ethanol might also enhance drug permeability by increasing its solubility in the vehicle (Pershing et al., 1990). The pretreatment conditions for further measurements with skin membranes were chosen to be 24 h incubation in PBS-ethanol mixture. No pretreatment was done on dialysis membranes. For subsequent increase of QR permeability, the solution of PBSethanol-L-menthol has been tested. L-menthol is known as a good skin penetration enhancer for both hydrophilic and hydrophobic drugs. It modifies the integrity of the stratum corneum barrier by breaking the hydrogen bond network at lipid polar head groups and, thus, causes the lipid melting (Narishetty and Panchagnula, 2005). Moreover it is, probably, able to increase the solubility of drugs in tissue, thus increasing its permeability (Kaplun-Frischoff and Touitou, 1997). It has been noticed, that simultaneous use of ethanol and menthol in a mixture results in even better enhancement effect which is noticeable in increased flux values and decreased lag time (Kobayashi et al., 1994; Fujii et al., 2003). In our study, incubation of skin membranes in the PBS-ethanol mixture improved transdermal transport of QR which allowed for the amperometric detection of QR penetration through skin membranes (Fig. 5A). Addition of L-menthol to the solution improves QR penetration through skin, and resulted in higher current response (Fig. 5B). This is in agreement with Olivella et al. (2007), who observed up to 17-fold increase in QR transdermal permeation from a carbopol gel after menthol addition comparing to the control. During amperometric measurements of QR penetration through the dialysis membrane no significant difference was observed between PBS-ethanol solution without and with addition of menthol. This might suggest that the action of ethanol and Lmenthol in the experimental conditions of this work is mainly associated with its enhancement effect on skin due to structure change of the membrane. 3.3. Data evaluation for assessing flux and diffusion coefficient of QR in membranes To calculate QR flux through the membranes and to assess the diffusion coefficient of QR in the membranes, experimental amperometric current profiles (current vs time dependencies) of the membrane-covered electrodes were mathematically processed. Amperometric data (Fig. 5) enables very easy evaluation of steady-state flux, Jss, and lag-time, Tlag, for each measurement. These characteristics are presented in Table 1.

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As can be seen from Table 1 the characteristics describing QR penetration through skin membranes are considerably different from those recorded for dialysis membranes. Since these characteristics are dependent on experimental conditions, e.g., concentration, and thickness of the membrane, the diffusion coefficient of QR in the membranes was calculated. Theoretical diffusion coefficient (Db) for QR transdermal penetration was assessed to be 1.13  108 cm2/s using Eq. (2). The equation estimates diffusion coefficient of hydrophobic molecules with less than 400 Da molecular weight in lipid bilayers. Eq. (2) was recommended by Mitragotri (2003) as reasonable to estimate diffusion coefficients of drugs also in skin membranes:
 ð2Þ Db ¼ 2  105 exp 0:46r2 It should be noted that Eq. (2) approximates the penetrating substance as a sphere (Eq. (3)) with radius, r(Å). 4 3 pr ¼ 0:9087  Mw 3

ð3Þ

Amperometric measurement of penetration provides the dynamics of the process and thus provides data that can be processed by several methods to calculate diffusion coefficients, D. Those methods which can be used in relation to our experimental data were evaluated as described below. Calculated D values were then compared with the theoretical Db. Firstly, the amperometric current response obtained after QR injection was fitted to Eq. (4) (Kulys et al., 1998), which accounts for the relation between the electrode current and the second Fick’s law of diffusion. # " 2 1 I ðt Þ 4X ð1Þm ð2m þ 1Þ2 p2 ðD=d Þt ð4Þ exp ¼1 I1 pm¼02m þ 1 4 I(t) and I1 are electrode current at time t and at the infinite time, respectively. Thickness of membrane is d and diffusion coefficient, D, is obtained as a fitting parameter by fitting amperometric curves after injection of QR in the electrochemical cell. Calculation results are presented in Table 2, and will be discussed in comparison with D values obtained with other calculation methods. Secondly, to determine diffusion coefficients using the lag-time method, the cumulative flux versus time was plotted (not shown). At steady-state flux condition this plot becomes linear with the intercept on the time-axis giving the lag-time, Tlag. Diffusion coefficient is related to lag-time by Eq. (5) (Selzer et al., 2013). Calculation results are presented in Table 2. 2

T lag ¼

d 6D

ð5Þ

Fig. 5. Amperometric response to QR injection into electrochemical cell (resulting QR concentration was 0.5 mM) registered with the CNP-modified electrode covered with skin membrane. The measurements are conducted using four different skin membranes. Before the amperometric measurements the membranes were pretreated by incubation in PBS—ethanol mixture for 24 h. (A) Measurements done in PBS-ethanol (7:3, v:v) solution at pH 7.4; (B) measurements in PBS-ethanol (7:3, v:v) solution containing 0.12% of MT. The responses presented by (A) or by (B) are different due to the structural differences of the individual skin membrane. The lowest and the highest amperometric response, obtained in our studies of three skin membranes are presented. Time t = 0 min corresponds to the moment of QR injection into the cell.

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Table 1 Flux and lag-time values characterizing QR permeation through membranes from solution consisting of PBS and ethanol mixture (PBS + EtOH) and PBS-ethanol mixture containing menthol (PBS + EtOH + MT). Number of measurements, N, conducted using different membranes for each measurement. C is the concentration of QR in the measurement cell. Steady-state flux, mol/s cm2 Skin membrane

Dialysis membrane

PBS + EtOH (N = 2)

PBS + EtOH + MT (N = 3)

PBS + EtOH (N = 4)

PBS + EtOH + MT (N = 3)

1.4  0.7  1012 N = 2 C = 5  107 mol/ cm3

6.0  2.9  1012 N = 3 C = 5  107 mol/ cm3

9.6  1011 N = 1 C = 5  107 mol/cm3

6.3  1011 N = 1 C = 5  107 mol/cm3 0.9  0.7  1011 N = 2, C = 6  108mol/ cm3

1.4  0.6  1011 N = 3 C = 6  108 mol/ cm3 Lag-time, s 17,500  3500 N = 2 C = 5  107 mol/cm3 20,000  2900 N = 3 C = 5  107 mol/cm3 833 N = 1 C = 5  107 mol/cm3 860  140 N = 3 C = 6  108 mol/cm3

In the third method we exploited the relation between a steadystate condition and the first Fick’s law, i.e., the fact that the flux is proportional to the concentration, C, and the thickness, d, of the membrane (Eq. (6)). By knowing that electro-oxidation of each QR molecule contributes to the electrode current with two electrons the relation between the electrode current and QR flux is provided by Faraday’s law. In pharmaceutical science the steady-state flux is furthermore often defined through the permeability coefficient (Eq. (7)). By combining Eqs. (6) and (7) the diffusion coefficient from a steady-state experiment can be expressed by Eq. (8). KC J ss ¼ D d

ð6Þ

J ss ¼ K p  C

ð7Þ

D¼

d  Kp K

ð8Þ

Mitragotri (2003) has adopted this approach for estimating transdermal penetration of hydrophobic drugs with Mw < 400 Da, which are thought to be transported through skin via free-volume diffusion mode. Assuming that the major diffusion barrier is the 10–15 mm thick stratum corneum the model says that the total length of this pathway for transdermal drug penetration is approximately 3.6 cm. Partition coefficients in skin lipid-water can be approximated by exploiting known partition coefficients in octanol–water (Mitragotri, 2003). These considerations lead to that the diffusion coefficient at steady-state conditions can be expressed by Eq. (9). D¼

3:6  K p

ð9Þ

K 0;7 ow

1000 N = 1 C = 5  107mol/cm3 830  240 N = 2 C = 6  108 mol/cm3

The value of Kow was calculated to be 439 from known value of the log P = 1.82 (Rothwell et al., 2005). The values of the diffusion coefficient assessed by fitting the experimental data to Eqs. (4)–(9) are presented in Table 2. Comparing QR steady-state flux through (Table 1) and the diffusion coefficients (Table 2) in skin and dialysis membranes points to considerable differences. First, it should be noted that it was impossible to register any flux of QR through skin membranes which were not pretreated by incubating them in PBS—ethanol mixtures. After 24–40 h pretreatment of skin membranes the QR flux was still approximately 70 times lower than through dialysis membrane. The presence of menthol in the solution increased QR flux through skin membranes making the flux though skin vs dialysis membrane only approximately 10 times lower. These results confirm that the transdermal penetration of QR is highly restricted through skin membrane. Amperometric measurements, Figs. 3–5, demonstrate that QR penetration through any membrane proceeds with some lag-time, which is also an indication of slow QR diffusion in any membrane. Before concluding on the D values obtained in this study (Table 2) it would be beneficial to acquire some estimate about the values reported earlier. Zillich et al. (2013) estimated that the diffusion coefficient for QR is in the range (1.42–1.58)  107 cm2/s for skin membrane and (0.58–0.64)  107 cm2/s for dialysis membrane when the penetration was carried out from water/oil formulations. Their calculations were based on the lag-time method (Eq. (5)) and Fick’s first law for skin and dialysis membranes, respectively. In contrast, QR permeation studies from lipid formulations performed by Casagrande et al. (2007), showed no QR percutaneous transport and diffusion coefficient was not estimated, while for dialysis membrane the diffusion coefficient was in the range (7.5–14.9)  1012 cm2/s. The effect of different concentrations of L-menthol in a carbopol gel on QR skin

Table 2 Diffusion coefficients for QR in membranes calculated from amperometric measurements conducted in two different solutions: PBS-ethanol (7:3, v:v, pH 7.4), PBS + EtOH, and PBS-ethanol (7:3, v:v, pH 7.4) containing 0.12 % of MT, PBS + EtOH + MT. Amperometric measurements (example, Fig. 5) were processed by using appropriate equations as indicated. Diffusion coefficient is an average from the measurements using N different membranes. Other notations are as follows: K and Kow are partition coefficients of QR in membrane/solution and octanol/water, respectively; dskin,ddm, and dpp are assumed thickness of skin membrane, dialysis membrane and the thickness the penetration pathway in skin membrane, respectively. Skin membranes were pretreated by keeping them in PBS—ethanol mixtures for 24 h. Diffusion coefficient, cm2/s Method

Eq. (4) Eq. (5) Eq. (8) K = 1, dskin = 10 mm ddm = 27.9 mm Eq. (9) Kow, = 439, dpp = 3.6 cm

Skin membrane

Dialysis membrane

PBS + EtOH (N = 2)

PBS + EtOH + MT (N = 3)

PBS + EtOH (N = 4)

PBS + EtOH + MT (N = 3)

4.3  0.9  1013 9.7  2.0  1012 2.8  1.5  109

3.7  0.8  1013 9.3  1.6  1012 1.2  0.6  108

1.4  0.8  1011 1.6  0.2  109 6.1  2.4  107

2.4  1.2  1011 1.5  0.4  109 4.1  2.4  107

1.4  0.7  107

6.1  3.0  107

–

–

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penetration has been studied before (Olivella et al., 2007). The membranes were then prepared from abdominal part of pig skin. The diffusion coefficient without penetration enhancers was estimated based on the first Fick’s law and it was equal to 3.32  107 cm2/s. Olivella et al. (2007) concluded that menthol significantly increases the QR permeability and its most efficient concentration equals 1.95% which enables QR penetration with diffusion coefficient of 5.19  106 cm2/s. Concluding, it can be stated that available diffusion coefficient values for QR penetration through skin cover a very broad range, from 5  106 cm2/s to 7.5  1012 cm2/s. It is obvious that the value should be dependent on the skin and experimental conditions. However, the data summarized in Table 2 shows that the value obtained for the diffusion coefficient also strongly depends on the model, assumptions, and, thus, equations used to assess the diffusion coefficient. The diffusion coefficient values obtained by different methods during this study (Table 2) might be compared to the theoretical diffusion coefficient of QR in a lipid bilayer, Db = 1.13  108 cm2/s (Eq. (2)). The D values that are closest to this value are calculated by using steady-state flux data, i.e., Eqs. (8) and (9). It can be noticed that accounting for partition coefficient and full length of the penetration pathway give the highest D values in the range of 1.6– 6.1 107 cm2/s (Table 2, Eq. (9)). It is probably not so strange that approaches (Eqs. (4) and (5)) which do not account for full length of penetration pathway and do not take into consideration partition of QR give D values that are approximately 2–3 orders of magnitude lower. Addition of menthol in the formulation definitely increase the diffusion coefficient of QR in skin. 4. Conclusions In this study skin permeation of QR was investigated using a simple amperometric setup comprising of a working electrode modified with carbon nanoparticles. Skin or dialysis membrane was mounted on the electrode and the penetration of QR was registered as a current due to electro-oxidation of QR at the surface of the CNP-modified electrode. Due to the low transdermal permeation of QR from the PBS solution, the influence of two penetration enhancers, ethanol and L-menthol, has been assessed. It was found that amperometric detection of QR permeated through skin membranes is possible after 24 h pretreatment of skin membranes in PBS-ethanol (7:3; v:v) solution at pH 7.4. Further, addition of L-menthol increased the registered current response as well as reduced the lag-time for QR penetration. This can be translated into higher flux and diffusion coefficient of QR through skin in the presence of menthol. Fluxes and diffusion coefficients were estimated by four different data evaluation methods based on amperometric detection and Fick’s laws of diffusion. The diffusion coefficient estimated from steady-state measurements of QR penetration (Eq. (9)) gave the highest values in the range of 1.6– 6.1 107 cm2/s being comparable to the theoretical estimate of diffusion of QR in lipid bilayers (Db = 1.13  108 cm2/s) and to some high values of D reported previously. Acknowledgements We gratefully acknowledge support from the Swedish Research Council, the Knowledge Foundation, and the research center “Biofilms” at Malmö University. References Barry, B.W., 2002. Drug delivery routes in skin: a novel approach. Adv. Drug Deliv. Rev. 54 (1), S31–S40.

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Belinha, I., Amorim, M.A., Rodrigues, P., de Freitas, V., Moradas-Ferreira, P., Mateus, N., Costa, V., 2007. Quercetin increases oxidative stress resistance and longevity in Saccharomyces cerevisiae. J. Agric. Food Chem. 55, 2446–2451. Bommannan, D., Potts, R.O., Guy, R.H., 1991. Examination of the effect of ethanol on human stratum corneum in vivo using infrared spectroscopy. J. Control. Release 16, 299–304. Bonina, F., Lanza, M., Montenegro, L., Puglisi, C., Tomaino, A., Trombetta, D., Castelli, F., Saija, A., 1996. Flavonoids as potential protective agents against photooxidative skin damage. Int. J. Pharm. Sci. 145, 87–94. Bose, S., Du, Y., Takhistov, P., Michniak-Kohn, B., 2013. Formulation optimization and topical delivery of quercetin from solid lipid based nanosystems. Inter. J. Pharm. 441, 56–66. Brett, A.M.O., Ghica, M.-E., 2003. Electrochemical oxidation of quercetin. Electroanalysis 15 (22), 1745–1750. Caltagirone, S., Rossi, C., Poggi, A., Ranelletti, F.O., Natali, P.G., Brunetti, M., Aiello, F. B., Piantelli, M., 2000. Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential. Int. J. Cancer 87, 595–600. Casagrande, R., Georgetti, S.R., Verri Jr., W.A., Borin, M.F., Lopez, R.F., Fonseca, M.J., 2007. In vitro evaluation of quercetin cutaneous absorption from topical formulations and its functional stability by antioxidant activity. Inter. J. Pharm. 328, 183–190. Casagrande, R., Georgetti, S.R., Verri Jr., W.A., Dorta, D.J., dos Santos, A.C., Fonseca, M. J., 2006. Protective effect of topical formulations containing quercetin against UVB-induced oxidative stress in hairless mice. J. Photochem. Photobiol. B 84, 21–27. Dal Belo, S.E., Gaspar, L.R., Maia Campos, P.M., Marty, J.-P., 2009. Skin penetration of epigallocatechin-3-gallate and quercetin from green tea Ginkgo biloba extracts vehiculated in cosmetic formulations. Skin Pharmacol. Physiol. 22, 299–304. Farkas, B., Magyarlaki, M., Csete, B., Nemeth, J., Rabloczky, G., Bernath, S., Nagy, P.L., Sümegi, B., 2002. Reduction of acute photodamage in skin by topical application of a novel PARP inhibitor. Biochem. Pharm. 63, 921–932. Fujii, M., Takeda, Y., Yoshida, M., Utoguchi, N., Matsumoto, M., Watanabe, Y., 2003. Comparison of skin permeation enhancement by 3-l-menthoxypropane-1,2diol and L-menthol: the permeation of indomethacin and antipyrine though Yucatan micropig skin and changes in infrared spectra and X-ray diffraction patterns of stratum corneum. Inter. J. Pharm. 258, 217–223. Ganesan, S., Faris, A.N., Comstock, A.T., Wang, Q., Nanua, S., Hershenson, M.B., Sajjan, U.S., 2012. Quercetin inhibits rhinovirus replication in vitro and in vivo. Antiviral Res. 94, 258–271. Gari, H., Rembiesa, J., Masilionis, I., Vreva, N., Svensson, B., Sund, T., Hansson, H., Morén, A.K., Sjöö, M., Wahlgren, M., Engblom, J., Ruzgas, T., 2015. Amperometric in vitro monitoring of penetration through skin membrane. Electroanalysis 27, 111–117. Hollman, P.C.H., van Trijp, J.M.P., Mengelers, M.J.B., de Vries, J.H.M., Katan, M.B., 1997. Bioavailability of the dietary antioxidant flavonol quercetin in man. Cancer Lett. 114, 139–140. Kai, T., Mak, V.H.W., Potts, R.O., Guy, R.H., 1990. Mechanism of percutaneous penetration enhancement: effect of n-alkanols on the permeability barrier of hairless mouse skin. J. Control. Release 12, 103–112. Kampkötter, A., Nkwonkam, C.G., Zurawski, R.F., Timpel, C., Chovolou, Y., Wätjen, W., Kahl, R., 2007. Investigations of protective effects of the flavonoids quercetin and rutin on stress resistance in the model organism Caenorhabditis elegans. Toxicology 234, 113–123. Kaplun-Frischoff, Y., Touitou, E., 1997. Testosterone skin permeation enhancement by menthol though formation of eutectic with drug and interaction with skin lipids. J. Pharm. Sci. 86 (12), 1394–1398. Khaled, K.A., El-Sayed, Y.M., Al-Hadiya, B.M., 2003. Disposition of the flavonoid quercetin in rats after single intravenous and oral doses. Drug Dev. Ind. Pharm. 29 (4), 397–403. Kobayashi, D., Matsuzawa, T., Sugibayashi, K., Morimoto, Y., Kimura, M., 1994. Analysis of the combined effect of L-menthoL and ethanol as skin permeation enhancers based on a two-layer skin model. Pharm. Res. 11 (1), 96–103. Kulys, J., Drungiliene, A., Wollenberger, U., Scheller, F., 1998. Membrane covered carbon paste electrode for the electrochemical determination of peroxidase and microperoxidase in a flow system. Bioelectrochem. Bioenergetics 45, 227–232. Lenz, H., Schmidt, M., Welge, V., Schlattner, U., Wallimann, T., Elsässer, H.-P., Wittern, K.-P., Wenck, H., Stäb, F., Blatt, T., 2005. The creatine kinase system in human skin: protective effects of creatine against oxidative and UV damage in vitro and in vivo. J. Invest. Dermatol. 124, 443–452. Lin, C.-F., Leu, Y.-L., Al-Suwayeh, S.A., Ku, M.-C., Hwang, T.-L., Fang, J.-Y., 2012. Antiinflammatory activity and percutaneous absorption of quercetin and its polymethoxylated compound and glycosides: the relationships to chemical structures. Eur. J. Pharm. Sci. 47, 857–864. Liu, D., Hu, H., Lin, Z., Chen, D., Zhu, Y., Hou, S., Shi, X., 2013. Quercetin deformable liposome: preparation and efficacy against ultraviolet B induced skin damages in vitro and in vivo. J. Photochem. Photobiol. B 127, 8–17. Mitragotri, S., 2003. Modeling skin permeability to hydrophilic and hydrophobic solutes based on four permeation pathways. J. Control. Release 86, 69–92. Moser, K., Kriwet, K., Naik, A., Kalia, Y.N., Guy, R.H., 2001. Passive skin penetration enhancement and its quantification in vitro. Eur. J. Pharm. Biopharm. 52, 103– 112. Narishetty, S.T.K., Panchagnula, R., 2005. Effect of L-menthol and 1,8-cineole on phase behavior and molecular organization of SC lipids and skin permeation of zidovudine. J. Control. Release 102, 59–70.

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J. Rembiesa et al. / International Journal of Pharmaceutics xxx (2015) xxx–xxx

Olivella, M.S., Lhez, L., Pappano, N.B., Debattista, N.B., 2007. Effects of dimethylformamide and L-menthol permeation enhancers on transdermal delivery of quercetin. Pharm. Dev. Technol. 12, 481–484. Paleco, R., Vu9 cen, S.R., Crean, A.M., Moore, A., Scalia, S., 2014. Enhancement of the in vitro penetration of quercetin through pig skin by combined microneedles and lipid microparticles. Inter. J. Pharm. 472, 206–213. Pershing, L.K., Lambert, L.D., Knutson, K., 1990. Mechanism of ethanol-enhanced estradiol permeation across human skin in vivo. Pharm. Res. 7 (2), 170–175. Rigano, D., Formisano, C., Basile, A., Lavitola, A., Senatore, F., Rosselli, S., Bruno, M., 2007. Antibacterial activity of flavonoids and phenylpropanoids from Marrubium globosum ssp. libanoticum. Phytother. Res. 21, 395–397. Rothwell, J.A., Day, A.J., Morgan, M.R.A., 2005. Experimental Determination of Octanol-Water Partition Coefficients of Quercetin and Related Flavonoids. J. Agric. Food Chem. 53, 4355–4360. Saija, A., Tomaino, A., Trombetta, D., Giacchi, M., de Pasquale, A., Bonina, F., 1998. Influence of different penetration enhancers on in vitro skin permeation and in vivo photoprotective effect of flavonoids. Inter. J. Pharm. 175, 85–94. Selzer, D., Abdel-Mottaleb, M.M.A., Hahn, T., Schaefer, U., Neumann, D., 2013. Finite and infinite dosing: difficulties in measurements, evaluations and predictions. Adv. Drug Deliv. Rev. 65, 278–294. Sinha, V.R., Kaur, M.P., 2000. Permeation enhancers for transdermal drug delivery. Drug Dev. Ind. Pharm. 26 (11), 1131–1140.

Sokolova, R., Ramesova, S., Degano, I., Hromadova, M., Gal, M., Zabka, J., 2012. The oxidation of natural flavonoid quercetin. Chem. Commun. 48, 3433–3435. Solovchenko, A., Schmitz-Eiberger, M., 2003. Significance of skin flavonoids for UVB-protection in apple fruits. J. Exp. Bot. 54 (389), 1977–1984. Tan, Q., Liu, W., Guo, C., Zhai, G., 2011. Preparation and evaluation of quercetinloaded lecithin-chitosan nanoparticles for topical delivery. Inter. J. Nanomed. 6, 1621–1630. Van der Merwe, D., Riviere, J.E., 2005. Comparative studies on the effects of water, ethanol and water/ethanol mixtures on chemical partitioning into porcine stratum corneum and silastic membrane. Toxicol. In vitro 19, 69–77. Vicentini, F.T.M.C., Simi, T.R.M., del Ciampo, J.O., Wolga, N.O., Pitol, D.L., Iyomasa, M. M., Bentley, M.V.L.B., Fonseca, M.J.V., 2008. Quercetin in w/o microemulsions: in vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluation in vivo. Eur. J. Pharm. Biopharm. 69, 948–957. Yamaguchi, Y., Coelho, S.G., Zmudzka, B.Z., Takahashi, K., Beer, J.Z., Hearing, V.J., Miller, S.A., 2008. Cyclobutane pyrimidine dimer formation and p53 production in human skin after repeated UV radiation. Exp. Dermatol. 17, 916–924. Zare, H.R., Namazian, M., Nasirizadeh, N., 2005. Electrochemical behaviour of quercetin: experimental and theoretical studies. J. Electroanal. Chem. 584, 77– 83. Zillich, O.V., Schweiggert-Weisz, U., Hasenkopf, K., Eisner, P., Kerscher, M., 2013. Release and in vitro skin permeation of polyphenols from cosmetic emulsions. Inter. J. Cosm. Sci. 1–11.

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