Materials Science and Engineering C 44 (2014) 380–385
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Iontophoresis with gold nanoparticles improves mitochondrial activity and oxidative stress markers of burn wounds Paulo C.L. Silveira b, Mirelli Venâncio a, Priscila S. Souza b, Eduardo G. Victor a, Frederico de Souza Notoya a, Carla S. Paganini b, Emilio L. Streck c, Luciano da Silva a, Ricardo A. Pinho b, Marcos M.S. Paula a,⁎ a b c
Laboratory of Synthesis of Multifunctional Complexes, PPGCS, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil Laboratory of Physiology and Biochemistry of Exercise, PPGCS, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil Laboratory of Experimental Physiopathology, PPGCS, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil
a r t i c l e
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Article history: Received 8 May 2014 Received in revised form 22 July 2014 Accepted 6 August 2014 Available online 27 August 2014 Keywords: Microcurrent Gold nanoparticles Burn wounds Respiratory chain Oxidative damage Antioxidant defence
a b s t r a c t The aim of this study was to analyse the effects of microcurrent and gold nanoparticles on oxidative stress parameters and the mitochondrial respiratory chain in the healing of skin wounds. Thirty 60-day old male Wistar rats (250–300 g) were divided into five groups (N = 6): Control; Burn wounds; Microcurrent (MIC); Gold nanoparticle gel (GNP gel) and Microcurrent + Gold nanoparticle gel (MIC + GNP gel). The microcurrent treatment was applied for five consecutive days at a dose of 300 μA. The results demonstrate a significant decrease in the activity of complexes I, II–III and IV in the Burn Wounds group compared to the control, and the MIC + GNP gel group was able to reverse this inhibition in complexes I, III and IV. Furthermore, a significant reduction in oxidative damage parameters and a significant increase in the levels of antioxidant defence enzymes were induced in the MIC + GNP gel group compared to the Burn Wounds group. The data strongly indicate that the group receiving treatment with MIC + GNP gel had improved mitochondrial functioning and oxidative stress parameters, which contributed to tissue repair. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Skin burns have high mortality rates and are responsible for severe long-term incapacitation. Broadly speaking, the healing time of skin injury is an important determining factor in the progression of further complications, amongst which are infections and functional sequelae [1]. Disruption of the skin generally leads to increased fluid loss, infection, hypothermia, scarring, compromised immunity and changes in body image [2]. Burn wound healing is a complex process involving several mechanisms such as coagulation, inflammation, matrix synthesis and deposition, angiogenesis, epithelialisation, fibroplasia, contraction and remodelling. Growth factors are polypeptides that control the growth, differentiation and metabolism of cells and regulate the process of tissue repair. The wound healing process of skin burns can drastically increase the levels of reactive oxygen species (ROS) and reduce enzymatic antioxidant defence mechanisms, subjecting the burn patient to oxidative stress. Skin tissue without the presence of lesions shows low levels of ROS, but injury exacerbates oxidative stress, leading to perpetuation of the inflammatory response and to a decrease in metabolic activity [3]. ⁎ Corresponding author at: Av. Universitária, 1105 Universitário, P.O. Box 3167, 88806-000 Criciúma, SC, Brazil. Tel.: +55 48 3431 2775; fax: +55 48 3344 3877. E-mail address: [email protected] (M.M.S. Paula).
http://dx.doi.org/10.1016/j.msec.2014.08.045 0928-4931/© 2014 Elsevier B.V. All rights reserved.
Mitochondria are intracellular organelles that play a crucial role in wound healing; they produce adenosine triphosphate (ATP), which is essential in the healing process. ATP is the main source of energy for the cell and is required to control the main functions of primary cells such as adjusting the levels of sodium, potassium and magnesium. It has been demonstrated that injured tissues have ATP disorders [4]. Technological advances have permitted the emergence of a wide variety of wound healing treatments. The application of low-current electrical stimuli modifies the healing process in living organisms, especially factors that delay or impair this process, [5]. Animal studies using a variety of wound models and electrical stimulation protocols have reported an enhancement in some aspects of wound healing [6]. In this respect, microcurrent (MIC) stimulation with up to 20 mA, which induces the flow of electrons into the skin and subcutaneous injury, has been shown to affect wound healing [7]. Gold nanoparticles (GNP) are used medically to enhance the therapeutic potential of drugs by altering the pharmacokinetics, biodistribution, or cellular uptake. GNPs have been in clinical use as an antioxidant and anti-inflammatory agent for a long time [8]. Gold has its own importance because of its relatively nonreactive and comparatively safe nature. In Ayurveda, the traditional system of Indian medicine, gold has been in clinical use for centuries and is termed “Swarna Bhasma” for various ailments [9]. Furthermore, in allopathic medicine, gold salts are used to treat inflammation/arthritis [10]. Recently, gold nanoparticles have been used for diagnostics and as drug
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carriers [10]. Nanostructures are defined as materials with overall dimension under 100 nm and can be synthesised in a variety of ways [11]. Attempts have been made to achieve therapeutically relevant concentrations of a drug by transdermal introduction into the tissues with the use of microcurrent. There are reports that after topical application, drug accumulation can be enhanced in various subjacent structures such as the underlying muscle and synovium [12,13] Thus, the purpose of this paper was investigate the effects of applying microcurrent and gold nanoparticles alone or in combination (iontophoresis) on the activity of the mitochondrial respiratory chain and oxidative stress parameters in the wound healing process of burns. 2. Materials and methods 2.1. Animals Thirty male Wistar Rats (250–300 g) were obtained from the breeding colony of Universidade do Extremo Sul Catarinense. Animals were kept in cages in a room at 23 ± 1 °C with ad libitum access to water and food under 12-h dark:light cycles. Animals were divided into five groups (N = 6): Control (skin without injury); Burn wounds; injury treated with microcurrent (MIC); injury treated with gold nanoparticle gel (GNP gel) and injury treated with a combination of microcurrent plus gold nanoparticle gel (MIC + GNP gel). 2.2. Skin injury protocol Animals were anaesthetised with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (20 mg/kg). Then, the dorsal region was shaved. Burns were inflicted using a 20 × 10 × 10 mm copper plate as previously described [14] held at 100 °C, which was measured using a digital thermometer. Total contact time between the plate and the skin was 15 s. Rats were given dipyrone (80 mg/kg) every 8 h for pain control; the control animals underwent the same anaesthetic protocol but were not challenged with burns. 2.3. Preparation and characterisation of gold nanoparticles Gold nanoparticles were synthesised as described [15] with minor modifications. Tetrachloroauric acid (HAuCl4) was acquired from Sigma-Aldrich (St. Louis, MO, USA), and sodium citrate (Na3C6H5O7.2H2O), a reducing agent and stabiliser, was acquired from Nuclear (Diadema, SP, Brazil. The electronic spectra were obtained with a Shimadzu instrument (model UV-1800; Shimadzu Corp., Kyoto, Japan). X-ray diffraction analysis was performed with a Shimadzu LABX model XDR-6000 diffractometer with Cu Kα radiation (λ = 1.54056 Â, 30 kV, 30 mA). The scan rate was 2°/min from 20 to 80°. It was possible to calculate the mean particle diameter using the Scherrer equation on the signal from the X-ray diffractogram at 2θ = 38° (major relative intensity) and by transmission electron microscopy (TEM) analysis, which was performed using a JEOL Titan 80–300 kV [16]. 2.4. Microcurrent therapy Animals were anesthetised with ether to allow the correct placement of electrodes on the injury site. Microcurrent was applied using an electrical stimulation device (Versalite AF7, Tonederm, Caxias do Sul, RS, Brazil). Eight-minute microcurrent sessions were carried out daily for five days as described in the literature [17]. The current applied using the described electrode was 300 μA/cm2. A gel impregnated with a solution of gold nanoparticles at a concentration of 40 mg/L and a particle size of 25 nm was used as a conducting agent for the electric current. The first treatment session started 24 h after the burn protocol.
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2.5. Tissue and homogenate preparation Twenty-four hours after the last treatment session, the animals were killed by decapitation and the tissue around the burn was surgically removed and immediately processed. The tissue was homogenised (1:10, w/v) in SETH buffer, pH 7.4 (250 mM sucrose, 2 mM EDTA, 10 mM Trizma base, 50 IU/ml heparin). The homogenates were centrifuged at 800 ×g for 10 min, and the supernatants were stored at −70 °C until being used for the determination enzyme activity and oxidative damage. All biochemical assays were performed within 30 days [18]. 2.6. Activities of mitochondrial respiratory chain enzymes The samples were frozen and thawed in hypotonic assay buffer three times to fully expose the enzymes to the substrates and to achieve maximal activity. NADH dehydrogenase (complex I) was evaluated according to the method described [19] based on the rate of NADH-dependent ferricyanide reduction at 420 nm. The activities of succinate:DCIP oxidoreductase (complex II) and succinate:cytochrome c oxidoreductase (complex III) were determined according to the methods described [20]. Complex II activity was measured by the decrease in absorbance at 600 nm due to the reduction in 2,6-DCIP levels. Complex III activity was measured by cytochrome c reduction by succinate. The activity of cytochrome c oxidase (complex IV) was assayed following the method described [21] and was measured as the decrease in absorbance at 550 nm due to the oxidation of previously reduced cytochrome c. The activities of the mitochondrial respiratory chain complexes were expressed as nmol/min mg protein of epithelial tissue. 2.7. Oxidative damage The levels of 2-thiobarbituric acid reactive species (TBARS) were measured as an index of lipoperoxidation according to a previously described method [22]. Briefly, a portion of the epithelial tissue was mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid. Subsequently, the mixture was heated in a boiling water bath for 15 min. The amount of TBARS was determined by measuring the absorbance at 532 nm, and the results were given as nmol TBARS/mg protein. Protein carbonylation was determined according to a method described in the literature [23]. Protein carbonyl content was measured by labelled protein-hydrazone derivatives using 2,4-dinitrophenylhydrazide (DNPH). These derivatives were extracted with 10% (vol/vol) trichloroacetic acid. The extraction procedure was followed by treatment with a 1:1 (vol/vol) mixture of ethanol/ethyl acetate and re-extraction with 10% trichloroacetic acid. The resulting precipitate was dissolved in 6 M urea hydrochloride. The difference in the spectrum against a 2,4dinitrophenylhydrazideprotein blank was used to calculate the concentration of 2,4-dinitrophenylhydrazide incorporated per mg of protein. Incorporation was measured as the absorbance at 370 nm using a spectrophotometer. 2.8. Antioxidant enzymes Superoxide dismutase (SOD) activity was measured by adrenalin oxidation inhibition adapted from the method of [24]. Skin samples were homogenised in glycine buffer. Volumes of the homogenate of 5, 10 and 15 μL were retrieved, to which 5 μL catalase (0.0024 mg/mL distilled water), 175–185 μL glycine buffer (0.75 g in 200 mL distilled water at 32 °C, pH 10.2), and 5 μL adrenalin (60 mM in distilled water + 15 μL/mL fuming HCl) were added. Readings were conducted for 180 s at 10-s intervals and measured with an ELISA reader at 480 nm. Values were expressed as SOD units per milligram of protein (U/mg protein). Catalase (CAT) activity was determined based on the hydrogen peroxide (H2O2) decomposition rate of the enzyme present in the sample using a 10 mM H2O2 solution in potassium phosphate buffer (50 mM) pH 7.0 [25]. One millilitre of the H2O2 solution and 20 μL of sample
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were placed in a quartz cuvette. After homogenisation, the maximum H2O2 decomposition rate was measured in a spectrophotometer at 240 nm, and the values were expressed as catalase units/mg protein.
compared to the Burn wounds group (p b 0.01). Additionally, the activity was significantly higher in complexes I, III and IV in MIC + GNP gel compared to MIC (p b 0.01). No statistically significant differences were observed between MIC + GNP gel and the control group.
2.9. Protein content 3.3. Oxidative damage markers The protein content from epithelial tissue homogenates was assayed using bovine serum albumin as a standard according to the method described [26]. Folin phenol reagent (phosphomolybdic-phosphotungstic reagent) was added to the protein homogenate. The bound reagent was slowly reduced, changing from yellow to blue. The absorbance was read at 750 nm. 2.10. Statistical analysis Data were expressed as the mean ± SEM, and differences amongst groups were determined by one-way ANOVA, followed by Tukey's post-hoc test when the ANOVA results were significant; p values less than 0.01 were considered to be statistically significant. The software used for data analysis was the Statistical Package for the Social Sciences (SPSS) version 18.0 for Microsoft Windows. 3. Results 3.1. GNP characterisation The electronic spectra of gold nanoparticle solution showed a SPR (surface plasmon resonance) band with λmax at 525 nm. This value is in accord with previous values observed [27]. The microstructural analysis was performed by TEM and showed that GNPs were spheroid, with a mean diameter of 25 nm, as shown in Fig. 1. 3.2. Complexes I, II, III and IV When compared to the control, the activities of complexes I, II, III and IV were significantly lower in the Burn wounds, MIC and GNP gel groups (p b 0.01) (Fig. 2A–D). However, significantly higher activity was observed in complexes I, III and IV in MIC + GNP gel when
Fig. 1. Gold nanoparticle TEM micrograph from aqueous media.
Increases in the TBARS level and protein oxidation were observed in the Burn wounds, MIC and GNP gel groups compared to the control (p b 0.01) (Fig. 3A and B). Both parameters were significantly lower in MIC, GNP gel and MIC + GNP gel when compared to Burn wounds (p b 0.01). Additionally, both parameters were significantly lower in MIC + GNP gel when compared to MIC (p b 0,01). However, no significant difference was observed between the control group and the MIC + GNP gel group. 3.4. Antioxidant enzymes (SOD and CAT) SOD and CAT activity were lower in the Burn wounds, MIC and GNP gel groups when compared to the control group (p b 0.01) (Fig. 3C and D). In addition, the SOD activity was significantly higher in the MIC + GNP gel group when compared to Burn wounds (p b 0.01). However, the CAT activity was significantly higher in MIC and MIC + GNP gel when compared to Burn wounds (p b 0.01). Additionally, the SOD and CAT activity was significantly higher in MIC + GNP gel when compared to MIC (p b 0.01). However, no significant difference was observed between the control group and the MIC + GNP gel group. 4. Discussion We used a model of burn wounds in rats to evaluate the influence of iontophoresis with gold nanoparticles on the activity of the mitochondrial respiratory chain and oxidative stress parameters five days after injury. Oxidative phosphorylation in intact cells is a process that requires the action of five enzyme complexes arranged in the electron transport chain. Electrons from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) through the Krebs cycle and other reactions catalysed by dehydrogenases are transferred to the electron transport chain and have molecular oxygen as the end acceptor. This process is coupled to proton translocation across the inner mitochondrial membrane and the endergonic synthesis of ATP through four enzyme complexes that employ as the driving force energy stored as an electrochemical gradient of protons [28]. Burn wounds induce alterations in mitochondrial function and impair the utilisation of oxygen by epithelial cells. This causes a reduction in the electron transport chain and consequently a reduction in the generation of ATP, which may compromise cellular metabolic activity and induce biochemical dysfunctions in the skin [29]. In line with this idea, our results show that the mitochondrial respiratory chain activity (Fig. 2) in the burn wounds caused a significant reduction in the activity of the four respiratory chain complexes, and the group that received iontophoresis with gold nanoparticles (MIC + GNP gel) reversed this effect in complexes I, III and IV. The relationship between endogenous electrical activity and wound healing has been investigated in several areas of clinical practice and has been well documented [30,31]. Microcurrents have been used to re-establish the endogenous bioelectrical activity of damaged tissues. This process is called biostimulation because it has been compatible with endogenous currents that act in the organism at the cellular level [13,32] The therapeutic application of microcurrents increases protein inflow to the lymphatic system, speeding up the absorption of liquids into the interstitial space and contributing to faster tissue repair [32, 33]. Additionally, other studies revealed that microcurrents may promote mitochondrial stability by increasing ATP production [32,34–36].
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Fig. 2. Effect of microcurrent (MIC) combined with gold nanoparticle gel (GNP gel) on the activity of mitochondrial respiratory chain complexes I, II, III and IV in epithelial tissue after burn injury (5 days). Data are expressed as the mean ± SEM for six animals. *: p b 0.01, different from control; #: p b 0.01, different from Burn wounds, and &: p b 0.01, different from MIC (Tukey's test).
The therapeutic action of microcurrents is enhanced when the technique is combined with GNP gel (iontophoresis). We believe that microcurrents increase the electron flux in the presence of GNP. Studies
show that the GNPs may improve the active transport of proteins and amino acids or even stabilise some of these proteins, preventing oxidative stress that may lead to DNA and mitochondrial damage
Fig. 3. Effect of microcurrent (MIC) combined with gold nanoparticle gel (GNP gel) on thiobarbituric acid reactive substances (TBARS) (A), protein carbonyl (B), superoxide dismutase (C) and catalase (D) in epithelial tissue after burn injury (5 days). Data are expressed as the mean ± SEM for six animals. *: p b 0.01, different from control; #: p b 0.01, different from Burn wounds, and &: p b 0.01, different from MIC (Tukey's test).
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[11,37,38]. Moreover, some previous studies have shown that gold nanoparticles catalyse the oxidation of NADH to NAD+. Complex I in the mitochondrial electron transport chain not only oxidises NADH to NAD + but also reduces ubiquinone (CoQ) to ubiquinol (CoQH2) [39–41]. We believe that due to the effects mentioned above, iontophoresis with gold nanoparticles improves mitochondrial function. The mitochondrial respiratory chain, which constitutes the main intracellular source of ROS in most tissues, contains several redox centres that may leak electrons to oxygen, constituting the primary source of O−• 2 in most tissues [42]. The steady-state concentration of these oxidants is maintained at nontoxic levels by a variety of antioxidant defences and repair enzymes. The delicate balance between antioxidant defences and ROS production may be disrupted by deficient antioxidant defences, inhibition of electron flow or exposure to xenobiotics. This imbalance appears as a common denominator in various pathological processes in which the resulting oxidative insult causes tissue damage and, eventually, cell death [43]. ROS are generated in inflammatory processes of skin injuries mainly in mitochondria and via NADPH oxidase enzymes [44]. Studies show that in lesions, the combination of an increase in ROS and a decrease in antioxidants generates delays in wound healing that should therefore be restored rapidly [3,30,45]. As our results show, the oxidative damage to lipids and proteins significantly increased in the Burn wounds group compared to the control group. Moreover, there was also a significant decrease in the activity of the antioxidant enzymes SOD and CAT. These results are likely the consequence of the acute inflammation induced by burn wounds, in which the excessive production of ROS and inhibition of antioxidant enzymes occur. Along these lines, a study reported the efficiency of microcurrent therapy to improve tissue regeneration by reducing ROS levels [35]. Our results show that this effect occurred only in the MIC + GNP gel group, most likely from the combined effects of the two therapies and because the conduction of GNPs is higher when stimulated by an electric current. The reduced ROS production in inflammatory processes favours to changes in the release of inflammatory mediators and activates growth and regeneration factors promoting a quick regeneration of the injured tissue. GNPs have been actively investigated in a wide variety of biomedical applications due to their biocompatibility and easy conjugation to biomolecules [46,47]. According to [48] GNPs are anti-oxidative agents that inhibit the formation of ROS and scavenge free radicals, thus improving the anti-oxidant enzyme defences. Moreover, GNPs are effective in quenching reactive oxygen species in a dose-dependent manner [49]. GNPs enhanced the antioxidant activity of vitamin E to decrease reactive oxygen species induced in a hepatoma cell line [50]. Moreover, GNPs inhibited osteoclast formation induced by the receptor activator of NF-κB ligand in bone marrow derived macrophages [51]. In another study with a wound healing model, GNPs decreased CD68 expression and increased SOD1 expression around the wound area, suggesting the occurrence of anti-inflammatory and antioxidative effects [52]. 5. Conclusion We conclude that the combination of electrical current and GNPs showed beneficial effects on markers of mitochondrial function and oxidative stress in a model of burn injury. Thus, iontophoresis may be a promising application for use in the recovery of skin lesions; however, further studies are needed to elucidate their precise mechanism of action. Acknowledgements This work was supported by grants from the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) and CAPES (Contract number:
477892/2013-2). The authors would also like to thank CNPq and UNESC for fellowship support.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Iontophoresis with gold nanoparticles improves mitochondrial activity and oxidative stress markers of burn wounds Paulo C.L. Silveira b, Mirelli Venâncio a, Priscila S. Souza b, Eduardo G. Victor a, Frederico de Souza Notoya a, Carla S. Paganini b, Emilio L. Streck c, Luciano da Silva a, Ricardo A. Pinho b, Marcos M.S. Paula a,⁎ a b c
Laboratory of Synthesis of Multifunctional Complexes, PPGCS, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil Laboratory of Physiology and Biochemistry of Exercise, PPGCS, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil Laboratory of Experimental Physiopathology, PPGCS, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil
a r t i c l e
i n f o
Article history: Received 8 May 2014 Received in revised form 22 July 2014 Accepted 6 August 2014 Available online 27 August 2014 Keywords: Microcurrent Gold nanoparticles Burn wounds Respiratory chain Oxidative damage Antioxidant defence
a b s t r a c t The aim of this study was to analyse the effects of microcurrent and gold nanoparticles on oxidative stress parameters and the mitochondrial respiratory chain in the healing of skin wounds. Thirty 60-day old male Wistar rats (250–300 g) were divided into five groups (N = 6): Control; Burn wounds; Microcurrent (MIC); Gold nanoparticle gel (GNP gel) and Microcurrent + Gold nanoparticle gel (MIC + GNP gel). The microcurrent treatment was applied for five consecutive days at a dose of 300 μA. The results demonstrate a significant decrease in the activity of complexes I, II–III and IV in the Burn Wounds group compared to the control, and the MIC + GNP gel group was able to reverse this inhibition in complexes I, III and IV. Furthermore, a significant reduction in oxidative damage parameters and a significant increase in the levels of antioxidant defence enzymes were induced in the MIC + GNP gel group compared to the Burn Wounds group. The data strongly indicate that the group receiving treatment with MIC + GNP gel had improved mitochondrial functioning and oxidative stress parameters, which contributed to tissue repair. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Skin burns have high mortality rates and are responsible for severe long-term incapacitation. Broadly speaking, the healing time of skin injury is an important determining factor in the progression of further complications, amongst which are infections and functional sequelae [1]. Disruption of the skin generally leads to increased fluid loss, infection, hypothermia, scarring, compromised immunity and changes in body image [2]. Burn wound healing is a complex process involving several mechanisms such as coagulation, inflammation, matrix synthesis and deposition, angiogenesis, epithelialisation, fibroplasia, contraction and remodelling. Growth factors are polypeptides that control the growth, differentiation and metabolism of cells and regulate the process of tissue repair. The wound healing process of skin burns can drastically increase the levels of reactive oxygen species (ROS) and reduce enzymatic antioxidant defence mechanisms, subjecting the burn patient to oxidative stress. Skin tissue without the presence of lesions shows low levels of ROS, but injury exacerbates oxidative stress, leading to perpetuation of the inflammatory response and to a decrease in metabolic activity [3]. ⁎ Corresponding author at: Av. Universitária, 1105 Universitário, P.O. Box 3167, 88806-000 Criciúma, SC, Brazil. Tel.: +55 48 3431 2775; fax: +55 48 3344 3877. E-mail address: [email protected] (M.M.S. Paula).
http://dx.doi.org/10.1016/j.msec.2014.08.045 0928-4931/© 2014 Elsevier B.V. All rights reserved.
Mitochondria are intracellular organelles that play a crucial role in wound healing; they produce adenosine triphosphate (ATP), which is essential in the healing process. ATP is the main source of energy for the cell and is required to control the main functions of primary cells such as adjusting the levels of sodium, potassium and magnesium. It has been demonstrated that injured tissues have ATP disorders [4]. Technological advances have permitted the emergence of a wide variety of wound healing treatments. The application of low-current electrical stimuli modifies the healing process in living organisms, especially factors that delay or impair this process, [5]. Animal studies using a variety of wound models and electrical stimulation protocols have reported an enhancement in some aspects of wound healing [6]. In this respect, microcurrent (MIC) stimulation with up to 20 mA, which induces the flow of electrons into the skin and subcutaneous injury, has been shown to affect wound healing [7]. Gold nanoparticles (GNP) are used medically to enhance the therapeutic potential of drugs by altering the pharmacokinetics, biodistribution, or cellular uptake. GNPs have been in clinical use as an antioxidant and anti-inflammatory agent for a long time [8]. Gold has its own importance because of its relatively nonreactive and comparatively safe nature. In Ayurveda, the traditional system of Indian medicine, gold has been in clinical use for centuries and is termed “Swarna Bhasma” for various ailments [9]. Furthermore, in allopathic medicine, gold salts are used to treat inflammation/arthritis [10]. Recently, gold nanoparticles have been used for diagnostics and as drug
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carriers [10]. Nanostructures are defined as materials with overall dimension under 100 nm and can be synthesised in a variety of ways [11]. Attempts have been made to achieve therapeutically relevant concentrations of a drug by transdermal introduction into the tissues with the use of microcurrent. There are reports that after topical application, drug accumulation can be enhanced in various subjacent structures such as the underlying muscle and synovium [12,13] Thus, the purpose of this paper was investigate the effects of applying microcurrent and gold nanoparticles alone or in combination (iontophoresis) on the activity of the mitochondrial respiratory chain and oxidative stress parameters in the wound healing process of burns. 2. Materials and methods 2.1. Animals Thirty male Wistar Rats (250–300 g) were obtained from the breeding colony of Universidade do Extremo Sul Catarinense. Animals were kept in cages in a room at 23 ± 1 °C with ad libitum access to water and food under 12-h dark:light cycles. Animals were divided into five groups (N = 6): Control (skin without injury); Burn wounds; injury treated with microcurrent (MIC); injury treated with gold nanoparticle gel (GNP gel) and injury treated with a combination of microcurrent plus gold nanoparticle gel (MIC + GNP gel). 2.2. Skin injury protocol Animals were anaesthetised with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (20 mg/kg). Then, the dorsal region was shaved. Burns were inflicted using a 20 × 10 × 10 mm copper plate as previously described [14] held at 100 °C, which was measured using a digital thermometer. Total contact time between the plate and the skin was 15 s. Rats were given dipyrone (80 mg/kg) every 8 h for pain control; the control animals underwent the same anaesthetic protocol but were not challenged with burns. 2.3. Preparation and characterisation of gold nanoparticles Gold nanoparticles were synthesised as described [15] with minor modifications. Tetrachloroauric acid (HAuCl4) was acquired from Sigma-Aldrich (St. Louis, MO, USA), and sodium citrate (Na3C6H5O7.2H2O), a reducing agent and stabiliser, was acquired from Nuclear (Diadema, SP, Brazil. The electronic spectra were obtained with a Shimadzu instrument (model UV-1800; Shimadzu Corp., Kyoto, Japan). X-ray diffraction analysis was performed with a Shimadzu LABX model XDR-6000 diffractometer with Cu Kα radiation (λ = 1.54056 Â, 30 kV, 30 mA). The scan rate was 2°/min from 20 to 80°. It was possible to calculate the mean particle diameter using the Scherrer equation on the signal from the X-ray diffractogram at 2θ = 38° (major relative intensity) and by transmission electron microscopy (TEM) analysis, which was performed using a JEOL Titan 80–300 kV [16]. 2.4. Microcurrent therapy Animals were anesthetised with ether to allow the correct placement of electrodes on the injury site. Microcurrent was applied using an electrical stimulation device (Versalite AF7, Tonederm, Caxias do Sul, RS, Brazil). Eight-minute microcurrent sessions were carried out daily for five days as described in the literature [17]. The current applied using the described electrode was 300 μA/cm2. A gel impregnated with a solution of gold nanoparticles at a concentration of 40 mg/L and a particle size of 25 nm was used as a conducting agent for the electric current. The first treatment session started 24 h after the burn protocol.
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2.5. Tissue and homogenate preparation Twenty-four hours after the last treatment session, the animals were killed by decapitation and the tissue around the burn was surgically removed and immediately processed. The tissue was homogenised (1:10, w/v) in SETH buffer, pH 7.4 (250 mM sucrose, 2 mM EDTA, 10 mM Trizma base, 50 IU/ml heparin). The homogenates were centrifuged at 800 ×g for 10 min, and the supernatants were stored at −70 °C until being used for the determination enzyme activity and oxidative damage. All biochemical assays were performed within 30 days [18]. 2.6. Activities of mitochondrial respiratory chain enzymes The samples were frozen and thawed in hypotonic assay buffer three times to fully expose the enzymes to the substrates and to achieve maximal activity. NADH dehydrogenase (complex I) was evaluated according to the method described [19] based on the rate of NADH-dependent ferricyanide reduction at 420 nm. The activities of succinate:DCIP oxidoreductase (complex II) and succinate:cytochrome c oxidoreductase (complex III) were determined according to the methods described [20]. Complex II activity was measured by the decrease in absorbance at 600 nm due to the reduction in 2,6-DCIP levels. Complex III activity was measured by cytochrome c reduction by succinate. The activity of cytochrome c oxidase (complex IV) was assayed following the method described [21] and was measured as the decrease in absorbance at 550 nm due to the oxidation of previously reduced cytochrome c. The activities of the mitochondrial respiratory chain complexes were expressed as nmol/min mg protein of epithelial tissue. 2.7. Oxidative damage The levels of 2-thiobarbituric acid reactive species (TBARS) were measured as an index of lipoperoxidation according to a previously described method [22]. Briefly, a portion of the epithelial tissue was mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid. Subsequently, the mixture was heated in a boiling water bath for 15 min. The amount of TBARS was determined by measuring the absorbance at 532 nm, and the results were given as nmol TBARS/mg protein. Protein carbonylation was determined according to a method described in the literature [23]. Protein carbonyl content was measured by labelled protein-hydrazone derivatives using 2,4-dinitrophenylhydrazide (DNPH). These derivatives were extracted with 10% (vol/vol) trichloroacetic acid. The extraction procedure was followed by treatment with a 1:1 (vol/vol) mixture of ethanol/ethyl acetate and re-extraction with 10% trichloroacetic acid. The resulting precipitate was dissolved in 6 M urea hydrochloride. The difference in the spectrum against a 2,4dinitrophenylhydrazideprotein blank was used to calculate the concentration of 2,4-dinitrophenylhydrazide incorporated per mg of protein. Incorporation was measured as the absorbance at 370 nm using a spectrophotometer. 2.8. Antioxidant enzymes Superoxide dismutase (SOD) activity was measured by adrenalin oxidation inhibition adapted from the method of [24]. Skin samples were homogenised in glycine buffer. Volumes of the homogenate of 5, 10 and 15 μL were retrieved, to which 5 μL catalase (0.0024 mg/mL distilled water), 175–185 μL glycine buffer (0.75 g in 200 mL distilled water at 32 °C, pH 10.2), and 5 μL adrenalin (60 mM in distilled water + 15 μL/mL fuming HCl) were added. Readings were conducted for 180 s at 10-s intervals and measured with an ELISA reader at 480 nm. Values were expressed as SOD units per milligram of protein (U/mg protein). Catalase (CAT) activity was determined based on the hydrogen peroxide (H2O2) decomposition rate of the enzyme present in the sample using a 10 mM H2O2 solution in potassium phosphate buffer (50 mM) pH 7.0 [25]. One millilitre of the H2O2 solution and 20 μL of sample
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were placed in a quartz cuvette. After homogenisation, the maximum H2O2 decomposition rate was measured in a spectrophotometer at 240 nm, and the values were expressed as catalase units/mg protein.
compared to the Burn wounds group (p b 0.01). Additionally, the activity was significantly higher in complexes I, III and IV in MIC + GNP gel compared to MIC (p b 0.01). No statistically significant differences were observed between MIC + GNP gel and the control group.
2.9. Protein content 3.3. Oxidative damage markers The protein content from epithelial tissue homogenates was assayed using bovine serum albumin as a standard according to the method described [26]. Folin phenol reagent (phosphomolybdic-phosphotungstic reagent) was added to the protein homogenate. The bound reagent was slowly reduced, changing from yellow to blue. The absorbance was read at 750 nm. 2.10. Statistical analysis Data were expressed as the mean ± SEM, and differences amongst groups were determined by one-way ANOVA, followed by Tukey's post-hoc test when the ANOVA results were significant; p values less than 0.01 were considered to be statistically significant. The software used for data analysis was the Statistical Package for the Social Sciences (SPSS) version 18.0 for Microsoft Windows. 3. Results 3.1. GNP characterisation The electronic spectra of gold nanoparticle solution showed a SPR (surface plasmon resonance) band with λmax at 525 nm. This value is in accord with previous values observed [27]. The microstructural analysis was performed by TEM and showed that GNPs were spheroid, with a mean diameter of 25 nm, as shown in Fig. 1. 3.2. Complexes I, II, III and IV When compared to the control, the activities of complexes I, II, III and IV were significantly lower in the Burn wounds, MIC and GNP gel groups (p b 0.01) (Fig. 2A–D). However, significantly higher activity was observed in complexes I, III and IV in MIC + GNP gel when
Fig. 1. Gold nanoparticle TEM micrograph from aqueous media.
Increases in the TBARS level and protein oxidation were observed in the Burn wounds, MIC and GNP gel groups compared to the control (p b 0.01) (Fig. 3A and B). Both parameters were significantly lower in MIC, GNP gel and MIC + GNP gel when compared to Burn wounds (p b 0.01). Additionally, both parameters were significantly lower in MIC + GNP gel when compared to MIC (p b 0,01). However, no significant difference was observed between the control group and the MIC + GNP gel group. 3.4. Antioxidant enzymes (SOD and CAT) SOD and CAT activity were lower in the Burn wounds, MIC and GNP gel groups when compared to the control group (p b 0.01) (Fig. 3C and D). In addition, the SOD activity was significantly higher in the MIC + GNP gel group when compared to Burn wounds (p b 0.01). However, the CAT activity was significantly higher in MIC and MIC + GNP gel when compared to Burn wounds (p b 0.01). Additionally, the SOD and CAT activity was significantly higher in MIC + GNP gel when compared to MIC (p b 0.01). However, no significant difference was observed between the control group and the MIC + GNP gel group. 4. Discussion We used a model of burn wounds in rats to evaluate the influence of iontophoresis with gold nanoparticles on the activity of the mitochondrial respiratory chain and oxidative stress parameters five days after injury. Oxidative phosphorylation in intact cells is a process that requires the action of five enzyme complexes arranged in the electron transport chain. Electrons from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) through the Krebs cycle and other reactions catalysed by dehydrogenases are transferred to the electron transport chain and have molecular oxygen as the end acceptor. This process is coupled to proton translocation across the inner mitochondrial membrane and the endergonic synthesis of ATP through four enzyme complexes that employ as the driving force energy stored as an electrochemical gradient of protons [28]. Burn wounds induce alterations in mitochondrial function and impair the utilisation of oxygen by epithelial cells. This causes a reduction in the electron transport chain and consequently a reduction in the generation of ATP, which may compromise cellular metabolic activity and induce biochemical dysfunctions in the skin [29]. In line with this idea, our results show that the mitochondrial respiratory chain activity (Fig. 2) in the burn wounds caused a significant reduction in the activity of the four respiratory chain complexes, and the group that received iontophoresis with gold nanoparticles (MIC + GNP gel) reversed this effect in complexes I, III and IV. The relationship between endogenous electrical activity and wound healing has been investigated in several areas of clinical practice and has been well documented [30,31]. Microcurrents have been used to re-establish the endogenous bioelectrical activity of damaged tissues. This process is called biostimulation because it has been compatible with endogenous currents that act in the organism at the cellular level [13,32] The therapeutic application of microcurrents increases protein inflow to the lymphatic system, speeding up the absorption of liquids into the interstitial space and contributing to faster tissue repair [32, 33]. Additionally, other studies revealed that microcurrents may promote mitochondrial stability by increasing ATP production [32,34–36].
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Fig. 2. Effect of microcurrent (MIC) combined with gold nanoparticle gel (GNP gel) on the activity of mitochondrial respiratory chain complexes I, II, III and IV in epithelial tissue after burn injury (5 days). Data are expressed as the mean ± SEM for six animals. *: p b 0.01, different from control; #: p b 0.01, different from Burn wounds, and &: p b 0.01, different from MIC (Tukey's test).
The therapeutic action of microcurrents is enhanced when the technique is combined with GNP gel (iontophoresis). We believe that microcurrents increase the electron flux in the presence of GNP. Studies
show that the GNPs may improve the active transport of proteins and amino acids or even stabilise some of these proteins, preventing oxidative stress that may lead to DNA and mitochondrial damage
Fig. 3. Effect of microcurrent (MIC) combined with gold nanoparticle gel (GNP gel) on thiobarbituric acid reactive substances (TBARS) (A), protein carbonyl (B), superoxide dismutase (C) and catalase (D) in epithelial tissue after burn injury (5 days). Data are expressed as the mean ± SEM for six animals. *: p b 0.01, different from control; #: p b 0.01, different from Burn wounds, and &: p b 0.01, different from MIC (Tukey's test).
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[11,37,38]. Moreover, some previous studies have shown that gold nanoparticles catalyse the oxidation of NADH to NAD+. Complex I in the mitochondrial electron transport chain not only oxidises NADH to NAD + but also reduces ubiquinone (CoQ) to ubiquinol (CoQH2) [39–41]. We believe that due to the effects mentioned above, iontophoresis with gold nanoparticles improves mitochondrial function. The mitochondrial respiratory chain, which constitutes the main intracellular source of ROS in most tissues, contains several redox centres that may leak electrons to oxygen, constituting the primary source of O−• 2 in most tissues [42]. The steady-state concentration of these oxidants is maintained at nontoxic levels by a variety of antioxidant defences and repair enzymes. The delicate balance between antioxidant defences and ROS production may be disrupted by deficient antioxidant defences, inhibition of electron flow or exposure to xenobiotics. This imbalance appears as a common denominator in various pathological processes in which the resulting oxidative insult causes tissue damage and, eventually, cell death [43]. ROS are generated in inflammatory processes of skin injuries mainly in mitochondria and via NADPH oxidase enzymes [44]. Studies show that in lesions, the combination of an increase in ROS and a decrease in antioxidants generates delays in wound healing that should therefore be restored rapidly [3,30,45]. As our results show, the oxidative damage to lipids and proteins significantly increased in the Burn wounds group compared to the control group. Moreover, there was also a significant decrease in the activity of the antioxidant enzymes SOD and CAT. These results are likely the consequence of the acute inflammation induced by burn wounds, in which the excessive production of ROS and inhibition of antioxidant enzymes occur. Along these lines, a study reported the efficiency of microcurrent therapy to improve tissue regeneration by reducing ROS levels [35]. Our results show that this effect occurred only in the MIC + GNP gel group, most likely from the combined effects of the two therapies and because the conduction of GNPs is higher when stimulated by an electric current. The reduced ROS production in inflammatory processes favours to changes in the release of inflammatory mediators and activates growth and regeneration factors promoting a quick regeneration of the injured tissue. GNPs have been actively investigated in a wide variety of biomedical applications due to their biocompatibility and easy conjugation to biomolecules [46,47]. According to [48] GNPs are anti-oxidative agents that inhibit the formation of ROS and scavenge free radicals, thus improving the anti-oxidant enzyme defences. Moreover, GNPs are effective in quenching reactive oxygen species in a dose-dependent manner [49]. GNPs enhanced the antioxidant activity of vitamin E to decrease reactive oxygen species induced in a hepatoma cell line [50]. Moreover, GNPs inhibited osteoclast formation induced by the receptor activator of NF-κB ligand in bone marrow derived macrophages [51]. In another study with a wound healing model, GNPs decreased CD68 expression and increased SOD1 expression around the wound area, suggesting the occurrence of anti-inflammatory and antioxidative effects [52]. 5. Conclusion We conclude that the combination of electrical current and GNPs showed beneficial effects on markers of mitochondrial function and oxidative stress in a model of burn injury. Thus, iontophoresis may be a promising application for use in the recovery of skin lesions; however, further studies are needed to elucidate their precise mechanism of action. Acknowledgements This work was supported by grants from the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) and CAPES (Contract number:
477892/2013-2). The authors would also like to thank CNPq and UNESC for fellowship support.
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