Electrochemical Studies of the Interaction of Phospholipid Nanoparticles with dsDNA

Abstract

The effect of phospholipid nanoparticles with different contents of phosphatidylcholine (PhNP80 and PhNP100) on dsDNA was studied by means of the electrochemical method. Changes in the electrochemical behavior of heterocyclic bases guanine, adenine and thymine in the range of potentials of 0.2–1.2 V in the presence of PhNPs were used for the assessment of the binding mechanism of the ligand–DNA interaction. Comparative analysis of the effect of PhNPs with different contents of phosphatidylcholine showed a more pronounced effect on the dsDNA of the PhNP100 nanosystem. From the obtained experimental data on the decrease in the amplitude of the nucleobases’ electrochemical oxidation currents, the electrochemical coefficient of the toxic effect was calculated as the ratio of the electrooxidation currents of dsDNA and dsDNA in the presence of phospholipid nanoparticles. PhNP80/100 (up to 11.4 mg/mL) does not influence dsDNA, PhNP80/100 (14.3–28.5 mg/mL) has a moderate toxic effect on dsDNA, PhNP80/100 at concentrations above 28.5 mg/mL already have a toxic effect, significantly reducing the maximum amplitude of the heterocyclic bases’ electrochemical oxidation current. Peak potentials of electrochemical oxidation of nucleobases did not shift in the presence of PhNP80 and PhNP100 (in the concentration range of 2.3–42.2 mg/mL), which could be possible through a groove-binding mode of phospholipid nanoparticle interaction with dsDNA.

1. Introduction

In the last decade, significant attention has been shown not only to the development of new biologically active substances but also to the development of new delivery systems. Drug delivery systems can improve transport in the body and increase bioavailability and the effectiveness of therapeutic action, reducing adverse side effects. PhNPs are often used for this purpose. PhNPs are inherently biocompatible, provide improved bioavailability for poorly soluble drugs, and can enable a reduced dose level due to sustained drug release. The unique properties of PhNPs, including their biocompatibility, biodegradability, amphiphilicity, low toxic effect, and ability to be loaded with biologically and pharmacologically active compounds, have found applications in medicine, especially for the delivery of biologically active substances [1,2,3,4,5,6].

Phospholipid nanoparticles can be modified with target molecules to provide directional delivery. Phospholipids are natural components of the biomembranes of all living cells, as well as blood lipoproteins. Soy phospholipids, as components of the proposed nanosystem, differing from animal cell phospholipids in fatty acid composition, are nevertheless able to integrate into cell membranes and effectively contribute to the restoration of the structure and function of damaged animal and human membranes. In this regard, soy phospholipids with a high content of phosphatidylcholine (PC from 75 to 98%) are of particular interest. As a source of phosphatidylcholine, they can be involved in many vital processes of the body, such as building and restoring the cell wall and membrane and hemostasis. According to modern concepts, the nanoscale form of such a transport system makes it possible to overcome biological barriers and deliver the substance built into it into the cell [6,7,8,9,10]. The efficiency of PhNPs, especially provided by their size of 10–30 nm [6,7,8,9,10] as a drug delivery system, helps to improve the pharmacokinetics of a drug.

DNA plays a key role in transcription, translation, division and replication. From a pharmacological point of view, DNA is the most important target for many cytotoxic and therapeutic agents. The interaction of biologically active compounds with DNA is an important direction for pharmacogenomics [11,12]. The binding of small molecules to DNA can drastically change the properties of DNA, leading to its destruction or to a change in the functioning of the entire genome. Investigation and understanding of ligand–DNA interaction is an important part of the rational design of biologically active compounds, predicting their impact on genomic processing, as well as pharmacogenomics studies.

The effect of PhNPs as potential drug delivery systems with a phosphatidylcholine content of 74.5% (PhNP80) and 96.2% (PhNP100) on DNA has not been studied before. The aim of this work was to conduct studies on the effect of phospholipid nanoparticles on dsDNA using the electrochemical method.

2. Materials and Methods

The following reagents were used in the work: monosubstituted potassium phosphate (Reakhim, Moscow, Russia), sodium chloride (Reakhim, Moscow, Russia), single-walled carbon nanotubes 0.4 wt.%, stabilized with carboxymethylcellulose 0.6 wt.% (Tuball Batt H₂O 0.4 wt.%, OCSIAL Ltd., Oksial Additives NSK LLC, Novosibirsk, Russia, https://ocsial.com (accessed on 1 October 2021), double-stranded fish sperm DNA (dsDNA) was bought from Sigma-Aldrich (D3159, Product of Japan).

PhNPs were obtained by successive suspension of soy phospholipids (Lipoid GmbH, Germany) in an aqueous solution of maltose, homogenization of the resulting emulsion at high pressure (800–1500 bar), followed by lyophilization [13]. The lyophilized powder of PhNPs with different content of phosphatidylcholine (96.2% in Lipoid S100 substance and 74.5% in Lipoid S80 substance), which is a composition of soy phospholipids (500 mg) and maltose (2000 mg), retained its physicochemical properties during rehydration.

The nanoparticle size and ζ-potential were determined in aqueous solution of PhNPs using a Zetasizer Nano ZS photon correlation spectrophotometer (Malvern, UK). In each case, three measurements were made and the result of the polydispersity distribution of particles was averaged by volume. Stock solutions of PhNPs were prepared in deionized (18 MΩ) Milli-Q water.

Electrochemical measurements were performed using a PalmSens potentiostat (PalmSens BV, The Netherlands, Holland) with PSTrace software (version 5.8). We used three-contact SPEs (ColorElectronics, Moscow, Russia, http://www.colorel.ru (accessed on 1 January 2022); with working and auxiliary graphite electrodes, silver/silver chloride reference electrode. Working electrode diameter is 0.2 cm (area 0.0314 cm²). All potentials are given relative to a silver/silver chloride reference electrode (vs. Ag/AgCl).

Solutions of analytes were prepared in 0.1 M potassium phosphate buffer (pH 7.4) containing 0.05 M NaCl; freshly prepared solutions were used in electrochemical experiment.

The electrodes were modified with 2 μL of 1 mg/mL a freshly prepared water dispersion of single-walled carbon nanotubes (SPE/CNT) Tuball Batt H₂O 0.4 wt.% (preliminarily diluted in distilled water 6 times) and dried at room temperature for 25 min. After that, the modified electrodes were scanned in the potential range of 0.2–1.2 V (4 scans) in 0.1 M potassium phosphate buffer (pH 7.4) containing 0.05 M NaCl, and then 60 μL of the analyte was applied, incubated for 10 min on the electrode (for the formation of dsDNA–PhNP complex) and electrochemical measurements by differential pulse voltammetry (DPV) were carried out in the potential range of 0.2–1.2 V, potential step 0.005 V, pulse amplitude 0.025 V, pulse duration 0.05 s, scanning speed 0.05 s. The experiments were performed under aerobic conditions at room temperature (25 ± 3 °C) using a Faraday cell (Metrohm Autolab BV, The Netherlands, Holland). To assess the reproducibility of the results for each concentration, at least 3 electrodes were used and the standard deviation was calculated.

The electrochemical coefficient of the toxic effect can be estimated at each PhNP concentration as a value of the percentage of the G, A or T peak height change (S%) using Equation (1) as reported in [14,15,16,17]:

S = (Ss/Sb) × 100%,

(1)

where Ss is the maximum value of the currents of the oxidative signals of the nucleobase after interaction with the PhNPs and Sb (before) is the maximum value of the currents of the oxidative signals of the nucleobase before the interaction [14,15,16,17]. To calculate the maximum peak current of the nucleobase’s electrochemical oxidation signals, a baseline correction was carried out using the PSTrace software package (version 5.8).

3. Results

The phospholipid nanoparticles’ (PhNPs) size and the value of the ζ-potential were determined by dynamic light scattering. It was shown that PhNP100 and PhNP80 demonstrate a 30.7 ± 1.6 nm and 8.7 ± 1.2 nm size, respectively (Figure S1, Table S1). The value of the ζ-potential was determined, which amounted to −(3.9 ± 1.1) mV (PhNP100) and −(35.7 ± 3.0) mV (PhNP80) (Figure S2, Table S1).

Disposable electrodes produced by screen printing are currently most actively used (Scheme 1) [18]. Screen-printed electrodes possess two types of electrochemical modes, such as vertical (in the volume of several ml), or a horizontal or planar regimen, which permits the use of small volumes of analyte (60 μL). This type of electrode makes it possible to work with biological objects without the stage of sensor regeneration. Modification of the electrode surface with nanoparticles (electrode nanostructuring) makes it possible to select the optimal conditions, adjust the sensor to the selected electrochemical reaction, the substance to be determined, and provide the necessary analytical characteristics of the method [19,20,21,22,23,24,25]. In this work, we used electrodes modified with single-walled carbon nanotubes, stabilized with carboxymethylcellulose. The broad potential window 0.2–1.2 V permits the registration of the electrochemical oxidation of three nucleobases of dsDNA (guanine, adenine, and thymine).

Differential pulse voltammograms for the electrochemical oxidation of dsDNA (1 mg/mL) under aerobic conditions were presented in Figure 1A–C. In the first scan, three well-defined peaks were recorded at potentials of 0.59 ± 0.01 V, 0.89 ± 0.01 V, and 1.12 ± 0.01 V (vs. Ag/AgCl) for guanine (G), adenine (A) and thymine (T), respectively (Figure 1A). The electrochemical oxidation potentials of the nucleobases correlate well with the previously reported results on DNA detection and the development of DNA sensors [22,25]. It should be noted that SPE/CNT permits the registering of electrochemical oxidation of not only guanine and adenine but also thymine (inset in Figure 1A), which makes it possible to analyze dsDNA based on three bases, such as G, A, and T. Optimum incubation time of dsDNA–PhNP complex formation was chosen as 10 min (Figure S3). In the presence of PhNPs (19 and 38 mg/mL), the amplitudes of the oxidation currents of three nucleobases decrease but the oxidation potentials do not shift significantly (Figure 1B,C).

A linear decrease in the maximum amplitude of the DPV peaks current of guanine, adenine and thymine in the presence of PhNPs in the concentration range of 14.3–42.2 mg/mL was registered. Figure 2A,B show the concentration dependences of nucleobase signals calculated as ΔI = I(dsDNA) − I(dsDNA/PhNP). The sensitivity estimated from the slope of the calibration curves for PhNP100 (Figure 2A) demonstrated a 1.4–2-times higher value when compared to PhNP80 (Figure 2B), which indicates a more pronounced effect of these phospholipid nanoparticles on dsDNA when compared to PhNP80 (Figure 2B).

No significant effect on the dsDNA oxidation signal of DPV was observed using 2.3—11.4 mg/mL PhNP80/100, as shown in the histograms of Figure 3A,B.

Based on the analysis of the decrease in the intensity of the electrooxidation signals of guanine, adenine and thymine, the electrochemical coefficient of the toxic effect of PhNP100 (

Table 1. The influence of PhNP100 on DPV oxidation signals of guanine (G), adenine (A) and thymine (T). S%—electrochemical coefficient of toxic effect.

[PhNP100], mg/mL	S (G), %	S (A), %	S (T), %
2.3	100	100	100
11.4	100	100	100
14.3	95	81	80
19.0	92	75	78
28.5	74	57	60
38.0	24	18	14
42.2	18	11	8

) and PhNP80 (

Table 2. The influence of PhNP80 on DPV oxidation signals of guanine (G), adenine (A) and thymine (T). S%—electrochemical coefficient of toxic effect.

[PhNP80], mg/mL	S (G), %	S (A), %	S (T), %
2.3	100	100	100
11.4	100	100	100
14.3	80	76	81
19.0	80	75	79
28.5	55	47	50
38.0	44	30	30
42.2	36	23	18

) was calculated using Equation (1) [14]. In accordance with [14,15,16,17], a sample does not have a toxic effect if the S value is above 85%; has a moderate toxic effect if the value of S ≈ 50–85%; and if S is less than 50% then the compound has a toxic effect.

Based on the values of electrochemical coefficient of the toxic effect (S), it is possible to conclude that PhNP80 and PhNP100 at concentrations up to 11.4 mg/mL do not have a toxic effect on dsDNA (S was taken as 100% since the signal change in the presence of PhNPs was insignificant). In the concentration range of 14.3–28.5 mg/mL PhNP80/100 shows a moderate toxic effect on dsDNA (S ≈ 50–85%) and PhNP80/100 at concentrations above 28.5 mg/mL already has a toxic effect. PhNP80/100 at concentrations of 38.0 and 42.2 mg/mL (highlighted in color in Table 1 and Table 2) shows a decrease in the maximum amplitudes of nucleobase oxidation currents by more than 50% and a greater toxic effect on dsDNA was observed in the presence of PhNP100 compared to PhNP80 (S_PhNP100 < S_PhNP80).

The advantage of the electrochemical method of dsDNA analysis is the registration of three GCOs (guanine, adenine, and thymine). This approach makes it possible to register the selective effect of PhNP on purine and pyrimidine bases. As follows from Table 1 and Table 2, the most intense effect of PhNP100 at concentrations of 14.3 and 19.0 mg/mL is manifested in the analysis of changes in the intensity of the signals of the electrochemical oxidation of adenine and thymine, while the effect of PhNP100 on guanine is manifested to a lesser extent.

The DP voltammograms presented in Figure 4 demonstrated a comparative analysis of the effect of PhNP 80/100 on dsDNA at concentrations of (A) 19 and (B) 38 mg/mL.

As can be seen from Figure 4A,B, as well as Table 1 and Table 2, the effect on dsDNA signals is more intense for PhNP100 compared to PhNP80 in the concentration range of 19.0—42.2 mg/mL. Such an effect may be due to the high content of phosphatidylcholine in PhNP100—96.2% compared to PhNP80—74.5%).

With the intercalation (hydrophobic) nature of the interaction of ligands with DNA, a positive shift of oxidation potentials of nucleobases is observed; negative shifts reflect the formation of hydrogen bonds and/or electrostatic interactions in the DNA–ligand complex [26,27,28,29,30]. The PhNP80/100 studied in the concentration range of 2.3–42.2 mg/mL did not have a significant effect on the shift of DNA oxidation potentials. This observation allows us to conclude that a dsDNA–PhNP80/100 complex of a non-intercalation type, is localized, as a rule, in the grooves of DNA due to the formation of hydrogen bonds [26,27,28,29,30]. A long chain in the structure of PC (Scheme 2), which is the main composition compound of PhNP, permits us to confirm the mode of binding as groove interaction.

Since PhNPs do not contain any aromatic ring to facilitate the intercalating, the classical intercalative interaction may be precluded [31].

4. Discussion

This study represents electrochemical voltammetric detection of the interaction of phospholipid nanoparticles with different contents of PC as a potential drug delivery system with dsDNA. Single-use disposable SPEs are widely used in DNA and ligand–DNA interaction studies due to their commercial availability, ease of modification, reproducibility and relatively low cost. Modification of SPE with the dispersion of carbon nanotubes stabilized by carboxymethyl cellulose permits us to register oxidation peak current and peak potentials of guanine, adenine and thymine at 0.59 ± 0.01 V, 0.89 ± 0.01 V, and 1.12 ± 0.01 V (vs. Ag/AgCl), respectively. Such a multi-parametric assay has shown that the sensitivity of adenine and thymine nucleobases for PhNP80 and 100 are more pronounced in comparison with guanine residue (Figure 2A,B; slopes of the curves). The effect of PhNP based on soy phospholipids with different contents of phosphatidylcholine, PC (PhNP80—74.5% PC, PhNP100—96.2% PC) on dsDNA was also studied. A comparative analysis of the effect of phospholipid nanoparticles with different contents of phosphatidylcholine showed a more pronounced effect with the PhNP100 nanosystem, probably due to the higher content of PC (96.2%) and greater size dimension of nanoparticles (Table S1). The sensitivity for PhNP100 (Figure 2A; slopes of the curves) demonstrated a 1.4–2-times higher value when compared to PhNP80 (Figure 2B; slopes of the curves). These results indicate a more pronounced effect of PhNP100 nanoparticles on dsDNA when compared to PhNP80.

From the experimental data, the electrochemical coefficient of the toxic effect was calculated. The results clearly showed that PhNP80/100 at concentrations of up to 11.4 mg/mL does not have a toxic effect, at concentrations of 14.3–28.5 mg/mL PhNP80/100 has a moderately toxic effect on dsDNA, and PhNP80/100 at concentrations above 28.5 mg/mL already has a toxic effect, registered by a decrease in the current of electrochemical oxidation of dsDNA. We can emphasize that the effects of PhNP80 or PhNP100 on DNA as an intracellular target, are not innocent, especially at concentrations higher than 28.5 mg/mL. These data must be kept in mind when developing PhNP-based drug-delivery systems. PC as the main component of phospholipid nanoparticles possesses hydrophobic chains and ionic parts of molecules (Scheme 2), therefore it is possible to predict different types of interactions with DNA. A positive shift of the oxidation potential of the nucleobase reflects the intercalation mode of drug–DNA interactions, which is a thermodynamically less favorable electrochemical process due to the shielding of nucleobases; negative shifts of oxidation potential reflect the formation of hydrogen bonds and/or electrostatic interactions in the DNA–ligand complex [25,26,27,28,29] with easier electron transfer processes. However, in our experiments, the shift of dsDNA guanine, adenine and thymine oxidation potentials were not statistically registered in the presence of PhNP80 or PhNP100. Therefore, the intercalative mode of PhNPs–DNA interaction should be excluded and interaction could be possible through a groove-binding mode. It is a well-known viewpoint that small molecules, such as drugs, organic dyes, etc., bind with DNA in a minor groove-binding mode for their small size, while the macromolecules, such as protein molecules, bind with DNA in a major groove-binding mode [31]. Accordingly, based on our experimental data, it was assumed that phospholipid nanoparticles interact with dsDNA minor grooves. Exploring the PhNPs–DNA interactions is a key parameter for the construction of a drug-delivery system based on phospholipid nanoparticles. Unlike the spectroscopic approach, where only one parameter, such as absorbance at 260 nm, can be registered, the electrochemical oxidation of purine and pyrimidine nucleobases expands the understanding of an interactions’ specificity for pharmacogenomics studies and constrictions of new drug delivery systems.

5. Conclusions

DNA sensors have the potential for monitoring biologically active substances or chemotherapeutic–DNA interactions. Investigation of the ligand–DNA interplay is key for pharmacogenomics studies. Modification of screen-printed electrodes by means of single-walled carbon nanotubes significantly improved the dsDNA electrochemical signature, with the registration of electrooxidation of guanine, adenine and thymine nucleobases. Electrochemical monitoring of interactions between DNA and biologically active compounds revealed the mechanism of binding events and the range of nontoxic concentrations. The results of the DPV analysis of the PhNP–DNA interactions revealed the range of the safe concentrations of phospholipid nanoparticles, which was 2.3–11.4 mg/mL. PhNP80/100 at concentrations above 38.0 mg/mL manifested a toxic effect. To the best of our knowledge, it is the first example of a pharmacogenomic study with the evaluation of three heterocyclic bases for the assessment of the influence of chemotherapeutics on DNA performed with an electrochemical technique. Electrochemical analysis of dsDNA oxidation peak current and peak potentials of guanine, adenine and thymine was performed for the comparative analysis of DNA–PhNP complexes. The evaluation of the role of phospholipid nanoparticles in the interaction with dsDNA based on an electrochemical approach by means of DPV analysis permits us to propose minor groove-binding and rule out the involvement of the intercalation mode.