Electrochemical Analysis in a Liposome Suspension Using Lapachol as a Hydrophobic Electro Active Species

Abstract

This study demonstrated that the electro-chemical analysis of hydrophobic quinones can be performed in liposome suspension systems. We prepared and analyzed liposome suspensions containing lapachol, which is a quinone-based anti-tumor activity compound. In this suspension system, a simple one redox couple of lapachol is observed. These results are quite different from those obtained in organic solvents. In addition, the pH dependence of redox behaviors of lapachol could be observed in multilamellar vesicle (MLV) suspension system. This MLV suspension system method may approximate the electrochemical behavior of hydrophobic compounds in aqueous conditions. A benefit of this liposome suspension system for electrochemical analysis is that it enables to observe water-insoluble compounds without using organic solvents.

Electrochemical analyses of organic compounds with cyclic voltammetry or pulse voltammetry provide us general information about the redox property of those compounds. Commonly, organic solvents are used for the analysis of organic compounds with an excessive amount of supporting electrolytes such as tetraalkyl ammonium salts. The combination of an organic solvent with an electrolyte enables it to analyze electrochemical behaviors of organic compounds. However, the results highly depend on the stabilization energy from organic solvents and electrolytes, especially redox potentials are very sensitive to electrolyte concentrations.^1,2) This mutual dependence sometimes causes problems in understanding the meaning of the potentials.

On the other hand, quinones play an important role in biological systems and serve as active sites for quinoenzymes in biological functions. It is presumed that the redox properties of quinones are controlled by hydrogen bonding with surrounding proteins or solvents. As a result, hydrogen-bonding interactions have been the primary focused of most studies.^3–5) Most studies are performed using quinone compounds and proton donors in coexistence with organic solvents, so as to increase the solubility. However, it is unclear whether the results of an electrochemical measurement using organic solvents reflect real in vivo behavior.

We propose that liposome suspensions might serve as a substitute for organic solvents. Liposomes have been studied as an artificial biomembranes and also play important roles as drug delivery systems. The possibility of liposomal use diverges into many scientific branches. Recently, an interesting study reported that electrochemical measurements were performed using a liposome-modified electrode.^6,7) On the basis of these studies, we attempted to perform electrochemical measurements using a liposome suspension in place of organic solvents.

Results and Discussion

Preparation of a Liposome Suspension Using a Hydrophobic Electro-Active Compound

First, we tried to prepare electro-active liposome suspensions. A chloroform solution containing soy lecithin (200 mg) is taken in a round-bottom flask and evaporated under reduced pressure to remove the solvent. Then, a thin film of lecithin is obtained on the surface of the round-bottom flask. Next, 0.1 M (pH 7) phosphate buffer (20 mL) is poured into the round-bottom flask and mixed using a vortex mixer warming at ca. 50°C. From this, we obtain a white colored multilamellar vesicle (MLV) suspension.⁸⁾ The MLV suspension is very stable and does not precipitate throughout the day. Figure 1 shows an optical microscope photograph of the MLV suspension, prepared by the method mentioned above. It illustrates spherical MLVs, which have an average diameter of ca. 5 µm. In similar manner, liposome suspensions are prepared, which contain a hydrophobic compound in the buffer solution. Lapachol, a water-insoluble quinone, is selected as a hydrophobic compound.⁹⁾ Lapachol is a quinone-based anti-tumor compound derived from natural products.^10–12) Most quinone system anticancer agents show activity by an active oxygen species produced by redox processes. Because of this, the electrochemical behavior of lapachol has been extensively studied since 2002.^13–15) Similar to the method mentioned above, chloroform solutions containing both soy lecithin (200 mg) and lapachol (0.02 mmol) are poured into a round-bottom flask and evaporated. Then 0.1 M (pH 7) phosphate buffer (20 mL) is added and a MLV-lapachol suspension is obtained.¹⁶⁾ The MLV-lapachol suspension is slightly orange and appears stable throughout the day. Therefore this stable sample solution was selected for electrochemical analysis.

Fig. 1. Photograph of a MLV Suspension by an Optical Microscope

The scale in the photograph represents 24.6 µm.

Electrochemical Measurements of MLV Suspensions

Cyclic voltammograms (CVs) of the MLV suspension in the absence (MLV) and presence of lapachol (MLV-lapachol) are shown in Figs. 2a and b, respectively. The lapachol is easily reduced and shows a simple one-step redox couple in the MLV suspension (Fig. 2b). However it is known that lapachol exhibits complicated redox behavior in aprotic solvents.^13–15)

Fig. 2. Cyclic Voltammograms of a MLV Suspension in the Absence (Dotted Line (a)) of Lapachol and a MLV-Lapachol Suspension (Bold Line (b): [Lapachol]=1 mM¹⁶⁾) in 0.1 M Phosphate Buffer (pH 7)

Both are recorded with GC electrode at 0.1 V s⁻¹. (c) Dependence of the cathodic peak current of the MLV suspension including 0.5 mM lapachol on the square root of the sweep scan rate.

Next, we mixed lapachol (0.02 mmol) in MLV suspension which was already prepared (20 mL, pH 7). After that, CV measurement was carried out for the suspension. But the lapachol in MLV shows no electrochemical responses as same as background CV of Fig. 2a. It is indicated that the step of making a thin film of lecithin with lapachol is necessary to prepare the electro active suspension (MLV-lapachol).

In addition, CV measurements of MLV-lapachol at various scan rates provide a linear relationship between the values of the cathodic peak current and square of the scan rates. This linear relationship indicates that the redox reaction progresses via diffusion of the MLV-lapachol particles to the glassy carbon (GC) electrode. To confirm whether the MLV-lapachol particles adsorb on a GC electrode surface, a GC electrode is immersed in the suspension for 10 min; after which, the electrode is rinsed with distilled water, and a CV measurement is performed in a pH 7 phosphate buffer solution using the same electrode. The result shows a similar to the background CV shown in Fig. 2a. This indicates that the absorption of MLV particles on the electrode is negligible.

The redox reaction progresses via diffusion of the MLV-lapachol particles to the electrode, therefore the observed current value depends on the average value of the diffusion coefficient of MLV-lapachol. The diffusion coefficient of MLV-lapachol particles is not uniform because of these non-uniform sizes, however, the influence on CV measurement could be ignored.

Next, we made thin films of lecithin (200 mg) with various amount of lapachol, and then prepared several MLV-lapachol suspensions. Figure 3 shows the CVs of MLV-lapachol suspensions that include different amounts of lapachol. With increasing lapachol concentrations,¹⁶⁾ the cathodic peak current increases. A linear relationship between the peak current and the lapachol concentration is apparent. However, the cathodic peak current is smaller than expected from the lapachol concentration. One reason for this is that the diffusion coefficient of MLV-lapachol is much smaller than lapachol itself. Furthermore, it is believed that lapachol exists in the inner layer of the MLV, indicating that the distance to the electrode is too far for redox reactions to occur.

Fig. 3. Cyclic Voltammograms of a MLV-Lapachol Suspension Including Various Amounts of Lapachol in 0.1 M Phosphate Buffer (pH 7) at 100 mV s⁻¹

[Lapachol]/mM¹⁶⁾: (1) 0.12, (2) 0.25, (3) 0.50, (4) 0.74. A linear relationship between the cathodic peak current and the concentration of lapachol is shown in this figure.

The pH Dependence of CVs of MLV-Lapachol Suspensions vs. CVs of Lapachol in CH₃CN with Acetic Acid

Figure 4 shows the changes in CVs of the MLV-lapachol suspension with the addition of different amount of 10% NaOH solutions. The CV, represented as a solid line, corresponds to MLV-lapachol in pH 7 phosphate buffer. When 10% NaOH is added, the pH increases, and the redox wave is shifted in a negative direction. This behavior indicates that protons participate in the redox reaction. Figure 4 shows a relationship between the cathodic peak potential and pH of the suspensions. The dashed line is the linear regression analysis and the slope shows −57.7 mV/pH. It suggested that the cathodic peak potential moves negative direction according to the Nernst equation. In the case of around pH 10, it was difficult to do experiment because of the less buffering ability. On the other hand, by increasing the pH value, the color of the MLV-lapachol suspension becomes orange red. This color change indicates the dissociation of the OH group, and the stability of the MLV-lapachol suspension decreased for pH over 11. Regardless, the stability during the measurement did not cause a problem.

Fig. 4. Cyclic Voltammograms of a MLV-Lapachol Suspension ([Lapachol]=0.5 mM) in 0.1 M Phosphate Buffer (pH 7) with the Addition of Various Amounts of 10% NaOH, Recorded by a GC Electrode at 100 mV s⁻¹

The pH of each MLV-lapachol suspension: (1) 7.0 (bold line), (2) 8.0, (3) 10.0, (4) 10.8, (5) 11.3, (6) 11.5, (7) 11.9. Changes in the total volume of the suspension were negligible. The pH dependence of the cathodic peak potential is shown in this figure.

Next, we measured CVs of lapachol in organic solvent mixed with proton donors, and the results were compared with those of MLV-lapachol. Figure 5 shows the changes in CVs of lapachol with increasing concentrations of acetic acid in dry CH₃CN. In the absence of acetic acid, lapachol shows complicated redox behaviors (Fig. 5(1)). These are one electron (the first wave) and two electron (the second wave) reactions.^13–15) Increasing the concentration of acetic acid shifts the second wave in a positive direction and merges it into the first wave. When the concentration of acetic acid reaches 10 mM (Fig. 5(5)), the cathodic current value is three times larger than the first cathodic peak current of Fig. 5(1). Furthermore, when acetic acid is added, the redox couple is shifted positively, and the cathodic current is decreased. In the presence of excess acetic acid (ca. 5 M), the CV shows a 2e⁻/2H⁺ reaction wave. When comparing the results of MLV-lapachol (Fig. 2b) with the CV of lapachol in CH₃CN containing 5 M acetic acid (Fig. 5(8)), these shape of CV are similar but not same.

Fig. 5. Cyclic Voltammograms of 0.5 mM Lapachol in the Absence and Presence of Acetic Acid in Dry Acetonitrile with 0.1 M TPAPF₆, Recorded with a GC Electrode at a Scan Rate of 50 mV s⁻¹

[Acetic acid]/mM: (1) 0.0 (opened circles), (2) 1, (3) 2, (4) 5, (5) 10, (6) 100, (7) 1000, (8) 5000.

The electrochemical behavior of lapachol has been studied in organic solvent because of the solubility. In this work, we prepared MLV-lapachol suspensions and performed electrochemical measurements of those, and then it was shown that lapachol is easily reduced in pH 7 buffer solution. In addition, the pH dependence of redox behaviors of lapachol could be observed in MLV suspension system. This MLV suspension system method may approximate the electrochemical behavior of hydrophobic compounds in aqueous conditions. Also a benefit of this suspension system for electrochemical analysis is that it enables to observe water-insoluble compounds without using organic solvents.

Experimental

Lecithin from soybeans, chloroform (99%) and acetic acid (99%) are commercially available (Nacalai Tesque, Inc.) and are used as received without further purification. Lapachol and dehydrated CH₃CN of the best available grade from Aldrich Chemical Co. are used. Cyclic voltammograms are recorded at room temperature (ca. 25°C) under nitrogen atmosphere with an ALS 600C model electroanalytical system (BAS, Inc.) using a three electrode system consisting of a GC disk electrode with a diameter of 3 mm as a working electrode, a platinum wire as a counter electrode, and a Ag/Ag⁺ reference electrode (BAS, Inc.). Further details of our electrochemical measurements are described in a previous paper.⁵⁾

Acknowledgment

This study was supported by JSPS KAKENHI a Grant-in-Aid for Young Scientists (B) No. 21790048, and a Research Grant from Kinjo Gakuin University.

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