Electrochemical Evaluation of Aminoguanidine Hydrazone Derivative with Potential Anticancer Activity: Studies of Glassy Carbon/CNT and Gold Electrodes Both Modified with PAMAM

Aminoguanidine hydrazones (AGHs) are a class of compounds that have interesting pharmacological activities. They are derived from the same chemical group as aminoguanidine, so it has mixed properties (receptor and donor) in the formation of hydrogen bonds. Its anticancer agent properties were recently highlighted, but the molecules of this class have solubility in aqueous solutions that can be considered low. The identification of this class, by a simple, sensitive and low-cost technique, such as electrochemistry, which also allows the evaluation of its solubilization process through agents such as PAMAM dendrimer is the main objective of the work described here. The electrochemical response of the LQM10 (AGH derivative) was evaluated, as well as its behavior in different electrochemical sensors. Electrochemical experiments were performed in buffered (phosphate at pH 7.02 and acetate at 4.5). LQM10 has a reversible oxidation peak with a potential of +0.22 V. It was efficiently detected in different electrodes tested (glass carbon/CNT, glass carbon/CNT/PAMAM), which proves the viability of the electrodes for various analyses and has the determination of the apparent constant association, indicating its interaction with the analysis that is higher in the presence of the PAMAM encapsulating agent. This was corroborated by the results for the modified gold electrode with MUA and PAMAM. The sum of the results shows the possibility of electrochemically evaluating the Aminoguanidine hydrazone derivative, the viability of electrodes employed and the greater soHow to cite this paper: da Silva, M.P.G., de Oliveira, Y.M., Candido, A.C.L., de Araújo-Júnior, J.X., da Silva Rodrigues, É.E., Monteiro, K.L.C., de Aquino, T.M. and de Abreu, F.C. (2020) Electrochemical Evaluation of Aminoguanidine Hydrazone Derivative with Potential Anticancer Activity: Studies of Glassy Carbon/CNT and Gold Electrodes Both Modified with PAMAM. Journal of Biomaterials and Nanobiotechnology, 11, 33-48. https://doi.org/10.4236/jbnb.2020.111003 Received: November 1, 2019 Accepted: December 2, 2019 Published: December 5, 2019 Copyright © 2020 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/

The activity anticancer of Aminoguanidine hydrazine (AGH) of this class of compound a drug of considerable pharmacological interest. They are derived from the same chemical group as aminoguanidine, so it has mixed properties (receptor and donor) in the formation of hydrogen bonds. Its anticancer agent properties were recently highlighted, but the molecules of this class have solubility in aqueous solutions that can be considered low [8]. Studies the association of this class the compound with some carriers as cyclodextrin, liposomes or linear polymers were few reported [9].
In last years, polymer-based nanomedicine has received increasing attention because of its ability to improve therapeutic efficacy in cancer treatment [10] [11] [12]. Dendritic scaffold has been found to be suitable carrier for a variety of drugs including anticancer, anti-viral, anti-bacterial, anti-tubercular, with capacity to improve solubility and bioavailability of poorly soluble drugs [10] [13]. A promising alternative to solubilize AGH in aqueous media is the use of dendrimers, which are highly branched polymers and that their physicochemical properties, such as high control in their structure, size, shape, density and surface groups with many functionalities, they are ideal carriers in biomedical applications such as drug transport at specific sites in the biological system [11] [14] [15] [16]. Among the available dendrimers, the polyamidoamine dendrimer (PAMAM) already studied with several antitumor drugs and the first to present its complete series, that is, from generation 0 to 10 (G0 -G10), the lowest generation (G0 -G3) almost has no cytotoxicity [17] [18].
Electrochemical technique in association with PAMAM, has already been used for numerous applications, including as an immobilized substrate for glassy carbon electrodes [19] [20] [21] and/or modification of the surface of gold electrodes [21]. Recently our group related dendrimers derivatives for the construction of chemical sensors [22] and inclusion complexation of surface-confined with PAMAM on electrodes using cyclic voltammetry [23] [24]. These methodologies will also be used here to evaluate the formation of inclusion complexes between LQM10 and dendrimers through electrochemistry to investigate the Journal of Biomaterials and Nanobiotechnology type of interaction between Aminoguanidine hydrazone and generation 3 PAMAM dendrimer, immobilized on a gold electrode and vitreous carbon/Carbon Nanotubes.
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were performed using a conventional undivided three-electrode cell and an AutolabPGSTAT-30 potentiostat (Eco Chemie, Utrecht, Netherlands) coupled to a microcomputer interfaced by GPES 4.9 software. The working electrodes were a glassy carbon (GC-diameter = 3 mm), gold bead modified electrode with β-CDSH and PAMAM 3G, an Ag|AgCl, Cl − (saturated) reference electrode and a Pt wire as the counter electrode. The GC was cleaned by polishing with alumina on a polishing felt. The gold bead working electrode was prepared by annealing the tip of a gold wire (99.999%, 0.5 mm diameter) in an oxygen gas flame and the voltammetric response of this electrode was established as 0.2 mol/L Na 2 SO 4 after modification. Inert gas was used to degas the solution and the solution was covered with a nitrogen blanket during some experiments. The pH was measured (QUIMIS). All experiments were conducted at room temperature (25˚C ± 2˚C) The solution used in the protic media was performed using a phosphate buffer with pH 7.02 (ionic strength 0.2) or ethanol (EtOH) at 5% and acetate buffer with pH 4.5 (ionic strength 0.2). PAMAM generation 3 (Aldrich) was also added to the phosphate buffer in order to evaluate its interaction with LQM10 over time.

Preparation of Carbon Nanotubes Modified Glassy Carbon Electrode (CNT)
The glass carbon electrode was modified with CNT in two different ways. Method 1: 1.0 mg CNT was suspended in 1.0 mL of DMF and dispersed on the ultrasound for 2 h before being deposited on the surface of GC electrodes. 10 μL were added to the solution (1 μL added at a time). The electrode was taken to the stove at 80˚C for 10 min (at each addition).
Method 2: The procedure of Method 1 was repeated and then a 10 μL aliquot of PAMAM 3G was added to the surface of the CNT-modified electrode and dried over N 2 gas flow before proceeding with the analysis. Journal of Biomaterials and Nanobiotechnology

Electrochemical Behavior of LQM10
LQM10 (Figure 1), as well as the other Aminoguanidine hydrazones, did not have its electrochemical profile determined until then, so the initial analyzes sought to verify its behavior by cyclic voltammetry in GCE, in a protic medium, phosphate buffer with pH 7.02 in 10% of Ethanol PA, due to its low solubility in aqueous medium. The first scanning was performed from 0 to +1.2 V (oxidation) and the second from −1.2 to 0 V (reduction), the last being performed in an atmosphere of N 2 gas to avoid any interference of O 2 in the reaction ( Figure   2).
The compound being oxidized shows a pair of anodic peaks, Ep a1 and Ep c1 , at +0.267 V and +0.239 V, respectively (Figure 2(a)), at 0.050 V/s. Already to be reduced, no signal was observed on the voltammogram, indicating that the LQM10 does not undergo reduction process (Figure 2(b)).
The analysis of the electrochemical parameters, anodic peak currents, obtained in the LQM10 studies showed that the mass transport to the electrode surface is controlled by diffusional process (spontaneous movement of the chemical species due to the formation of a concentration gradient of the analyte of interest), in accordance with the linearity between the anodic peak currents (Ip a1 ) as a function of the square root of the sweep velocity (ѵ 1/2 ). When evaluating the values of anodic and cathodic currents it is concluded that the oxidation process represented in the voltammogram is reversible, according to diagnostic tests defined in the specific literature [26].
The electrochemical mechanism for oxidation process may be associated probably the oxidation of the guanidine Hydrazone group generating the quinone methide derivative after oxidation process of the 2e − following the deprotonation step as showed in Scheme 1.
In this work, we evaluated the electrochemical behavior of the LQM10 against two differently modified electrodes, whose results will be presented in the following sessions and are summarized in Figure 3. Journal of Biomaterials and Nanobiotechnology Scheme 1. Probable oxidation mechanism of LQM10.

GCE Modified with CNT (GCE-CNT)
There is a clear indication, in the literature, of the applicability of CNT as an efficient modifier of the glassy carbon surface due to its chemical and physical properties such as surface area, the possibility of adhering many functional groups to the surface and increasing the transfer speed of electron at the interface of the electrode [27] [28] [29].
In GCE-CNT, it is possible to notice the increase in the current of the characteristic peaks of the LQM10, with displacement to more positive potential of Ep a1 and to potentials closer to 0V in the case of Ep a2 , after 50 consecutive sweeps, under the same conditions and with concentration of LQM10 in the constant medium (Figure 4(a)). Indicating the durability of the electrode and the facility of the oxidation process when modified with CNT [29].
It was possible to construct a calibration curve for LQM10 in phosphate buffer of 0.2 mol/L, pH 7.02, using the GCE-CNT sensor, varying the concentration of LQM10 (10 −6 to 8 × 10 −6 mol/L) shown in Figure 4  times higher than the one presented by Silva et al. [24] for a nitro compound and 100 times higher than the electrode modified with CNT alone, which ratifies the hypothesis of the improvement in solubility in the presence of the dendrimer.
Comparing the two calibration curves (Figure 6), the higher values of currents in the presence of PAMAM are again evidenced.
For GCE-CNT-PAMAM G3 the values of LD and LQ were respectively 0.06 × 10 −6 mol/L and 0.2 × 10 −6 mol/L, these values compared to those obtained by GCE-CNT electrode, ratify the higher sensitivity in the presence of PAMAM G3.

Electrochemical Behavior of LQM10 Interaction with PAMAM G3 Immobilized on a Gold Electrode
By evaluating the structure of the PAMAM dendrimer, its inner, terminal groups with hydrophobic regions, it is concluded that the stoichiometry of the inclusion complex formed with it will probably involve more than one molecule of the drug for each mole of PAMAM [39]. In the literature, the Benesi-Hildebrand equation is applicable to interactions with 1:1 or 1:2 stoichiometry to calculate the equilibrium constant of a complex [40] [41]. In a previous work [24], based on the data of BOBROVNIK [42] and BUCZKOWSKI and collaborators [43], a methodology has been developed that shows that more active PAMAM sites are available for interaction, so that the new calculated constant takes all these factors into account.  The first step is to evaluate the oxidation process of LQM10 on the MUA-functionalized gold electrode (AU/MUA) in aqueous-ethanol medium (10% of ethanol P.A.), as can be observed in Figure 7(a). The concentration of LQM10 in solution ranged from 10 −6 to 8 × 10 −6 mol/L and the peak currents Ep a2 obtained are considered to refer to a non-encapsulated substance. Since in Figure 7(b) it is observed the voltammograms of LQM10 at the same concentrations as previously, however, in this case, the PAMAM G3 is immobilized on the surface of the gold electrode already functionalized with MUA (AU/MUA/ PAMAM), as described in the methodology [24]. The difference between the peak currents Ep a2 of the two electrodes corresponds to the LQM10 that interacted with the PAMAM. Then, with this information, the binding of the number of molecules of associated ligands per 1 mole of combined receptor and the concentration of the substance added to the medium have a hyperbolic character(Equation (2) [ ]   for quinone β-lapachone [23].
The LQM10 can be detected using CNT-modified glassy carbon electrode and when the PAMAM G3 dendrimer was also adsorbed on its surface, the response was more sensible due to observed current increase. Applying the methodology of modifying the surface of the gold electrode with PAMAM, it was possible to evaluate the complex LQM10:PAMAM and a formation constant can be calculated.