1. Introduction
Biocompatible agents containing copper in an inorganic form or in the context of metal–organic complexes are widely used in a variety of applications, largely as antibacterial and antifungal drugs, as well as in tumor treatment [
1,
2,
3,
4,
5]. Copper oxide-based materials varying from sub-10 nm to 40–60 nm (nanoparticles; NPs) and fine (<10 µM) particles have demonstrated cytotoxicity against prokaryotic, yeast, mammalian cell lines and zebrafish embryos [
6,
7,
8,
9]. Coordination of Cu
2+ or Cu
1+ with complex organic scaffolds has yielded a number of perspective multi-targeting chemotypes with differential antitumor properties [
10,
11,
12,
13,
14]. Thus, copper has proved its therapeutic value in a wide variety of applications.
Copper’s unique sensitivity to the electrochemical status makes it particularly advantageous in situations where the biological effects are mechanistically dependent on reactive oxygen species (ROS) [
15,
16,
17,
18,
19]. These studies have highlighted the opportunity of using Cu-containing compounds alone or in combinations with conventional and novel agents. However, undesired toxicity of metal-containing drugs may limit their practical use. Zheng et al. [
20] showed that the combination of CuCl
2 and the antioxidant N-acetylcysteine (NAC) induced a sizeable cytotoxic effect in cancer cell lines via the production of hydroperoxide. We are interested in determining whether Cu-containing NPs or Cu–organic complexes at non-toxic concentrations become potent against tumor cells if combined with non-toxic doses of NAC. Here, we demonstrate that these combinations triggered oxidative stress within the initial few hours followed by a complex mode of cell death regardless of the species and tissue origin. The effect of potentiation was limited to Cu-containing compounds since NAC did not change the cytotoxicity of other tested metals. Importantly, electrochemical experiments showed that the ability to synergize with Cu-containing compounds was attributed not to the antioxidant property of NAC but to a rapid reduction of Cu
2+ to Cu
1+. This mechanism was demonstrated for CuO NPs and Cu-coordinated organic complexes and represents a means of copper reduction in inorganic and organic compounds as an approach to oxidative burst-mediated purge of pleural or abdominal cavities from metastatic tumor cells.
3. Discussion
We demonstrated that the combinations of copper-containing inorganic compounds (CuO NPs or Cu salts) as well as Cu–organic complexes with NAC or ascorbate were remarkably cytotoxic for human tumor cell lines, including the variants with the determinants of anticancer drug resistance. Importantly, each component alone evoked little-to-no activity, whereas together, the effect of potentiation was dramatic. Death as a result of the combinations was observed for a variety of cell lines regardless of the tissue origin and was accompanied by rapid (within the initial hours) ROS generation followed by plasma membrane perturbations, such as disappearance of the phosphatidylserine gradient across the membrane (annexin V reactivity) and loss of integrity (PI influx). Cell death was independent of caspase activation or PARP cleavage, strongly suggesting that the plasma membrane (and perhaps other cellular membranes) was the key target of the combinations. These findings are in line with the recently demonstrated efficacy of the combination of Cu (II)-containing compounds and ascorbate in inducing cell cycle perturbation and death via oxidative damage of biomacromolecules [
19].
Among a panel of tested metals (Zn, Mo, Fe, and Co), Cu was the only one that evoked cytotoxicity in combination with NAC or cysteine. Phenylalanine, an amino acid that lacks the SH group at the C3 position, was without effect, suggesting the critical role of sulfur. Importantly, the S atom required a specific molecular context, because the S-methyl containing the amino acid methionine did not synergize with CuO. Nevertheless, the S atom was not mandatory since the sulfur-free ascorbate had similar effect to that of NAC, despite being somewhat less pronounced. Altogether, these results point to Cu2+ transformation as a mechanism of cell killing.
Electrochemical studies revealed that NAC or ascorbate reduced divalent copper into a monovalent and then into an uncharged state. In cell-free systems, this process depended on the ratio of Cu2+ and the reducing agent; 40–100 min were sufficient for the complete transition of the Cu atom’s electric charge to zero. One may hypothesize that reduction took place in the coordination sphere of copper and included the substitution of the SH group for Cl- followed by intramolecular electron transfer from S to Cu atoms and elimination of the S-centered radical from the Cu atom. At the final step, this radical dimerized into disulfide; S atoms became no longer available for copper reduction.
Furthermore, NAC reduced the charge of Cu cations in the context of copper–organic complexes. The presence of the metal was necessary for tumor cell sensitization since NAC showed a weak, if any, effect in combination with Cu-free organic ligands. Importantly, this efficacy significantly differed depending on the molecular environment of the copper ion. As shown in
Table 2, the effect of the combination of NAC and compound
1 was much stronger than that of compound
2 + NAC or compound
5 + NAC. This difference can be attributed to the +2 oxidation state of Cu ion in
1 coordinated by monodentate chloride anions in addition to the organic ligand. Complexes
3 and
5 contain copper ions in the oxidation states +1 and +1.5, respectively. Furthermore, in
6 and
8, copper is tightly coordinated by the tetradentate porphyrin ligand. It can be assumed that, to trigger ROS upon Cu reduction, the coordination compound must fit the following structural requirements: (1) a Cu ion in the complex must be in the +2 oxidation state, and (2) at least one monodentate ligand capable of maintaining the form of a stable anion after reduction must be present.
Electrochemical modifications of Cu ions are mechanistically relevant to the generation of oxidative stress via combinations of inorganic or organic Cu-containing compounds with reducing agents. In the presence of oxygen, Cu
+1 complexes are capable of generating a superoxide anion that can induce ROS formation in a Fenton-like reaction (Cu
+1L + O
2 → O
2−. + Cu
+2L) [
30]. We demonstrated intracellular ROS formation within a few hours of exposure to CuO NPs and NAC. In addition to these results, we did not exclude the possibility of the generation of extracellular ROS by these combinations. Indeed, intracellular accumulation of CuO NPs during 24 h cell treatment was as low as ~5% of the initial input as determined by the measurement of copper content in cell lysates by atomic force spectroscopy (S.A.T., V.K.Karandashev and A.A.S., unpublished). Moreover, in the experiments with artificial lipid bilayers, we found no significant change in the membrane permeability caused by CuO NPs in the absence of NAC. The latter agent alone increased the membrane conductance; however, CuO NPs did not augment this effect. We therefore hypothesized that extracellular ROS may represent a major source of oxidative species for cell damage. This suggestion is in line with the molecular ordering of cell death-associated events in which rapid loss of the plasma membrane integrity was the leading factor. Cell-penetrating Cu–organic complexes might be advantageous over CuO NPs in terms of water solubility and intracellular accumulation.
Vulnerability to oxidative stress makes the plasma membrane (as well as membrane organelles) an attractive target for elimination of “intractable” tumor cells otherwise irresponsive to apoptotic stimuli [
31,
32]. We found that CuO NPs + NAC efficiently killed the K562/4 (P-glycoprotein mediated MDR), SCOV-3/CDDP (resistance to cisplatin; [
24] and HCT116p53KO (altered response to DNA damaging agents; [
25] sublines; the potency of the combination was similar among these sublines and their parental counterparts. However, non-malignant fibroblasts were sensitive to the combination of CuO NPs and NAC, indicating no cell-type selectivity of the combination.
Nevertheless, the combinations analyzed herein can be applied to advanced disease where conventional treatments usually fail, e.g., for the purge of thoracic or peritoneal cavities from metastatic cells. In these situations, the efficacy of therapies aimed at induction of apoptosis is unlikely; instead, salvage treatment strategies should be considered. The escape of metastatic cells cannot be solely attributed to an impairment of individual death signaling pathways. Mechanisms such as an altered intracellular metal transport can also be advantageous for tumor cell survival. In this scenario, Cu reduction and ROS generation in the extracellular milieu are perspective for potent and irreversible damage of metastatic cells.
Recently, Wu et al. [
33] reported a tumor-specific “non-toxicity-to-toxicity” transition strategy based on hollow mesoporous silica NPs as the carrier. The particles doped with Cu
2+ and the FDA-approved drug disulfiram released these components under acidic conditions of the tumor microenvironment. The in situ chelation reaction between the co-released Cu
2+ ions and disulfiram generated toxic products. Concurrently, Fenton-like reactions between the generated Cu
+ ions and H
2O
2 resulted in ROS production. This strategy was efficacious against tumor xenografts. Thus, biocompatible materials for targeting tumors with locally controllable cytocidal oxidative burst provide a valuable approach to eradicating intractable malignancies.
In the present study, we used undecorated CuO NPs, proving their efficacy. Future developments should address the targeted delivery of Cu-containing materials using surface coating with antibodies to identify tumor-specific antigens. Furthermore, given the ability of sulfur to reduce copper in different molecular contexts, the design of supramolecular systems to guide Cu2+ to critical cysteine residues for oxidative destruction of targeted proteins has emerged as a practically important problem.
4. Materials and Methods
4.1. Metal (II)-Containing Agents
Iron chloride hexahydrate (FeCl
3∙6H
2O), iron (II) sulfate (FeSO
4∙7H
2O), and cobalt nitrate hexahydrate (Co(NO
3)
2∙6H
2O), as well as other reagents unless specified otherwise, were purchased from Sigma-Aldrich. CuO NPs were synthesized using the precipitation method [
21]. Briefly, 0.5 mg of CuSO
4∙5H
2O (Reachim, Saint-Petersburg, Russia) was dissolved in 100 mL of deionized water to obtain a 10 mM solution, which was heated at 98 °C. Sodium hydroxide (1 M; LenReactiv, Saint-Petersburg, Russia) was added to the mixture under vigorous stirring. The instantly formed black precipitate was washed twice with ethanol and twice with deionized water by pelleting at 12,000 g for 10 min. The size of the formed NPs was determined by dynamic light scattering (DLS) using a Photocor EPM/Photocor Compact-Z analyzer (Photocor, Moscow, Russia). ZnO NPs were synthesized using the same procedure as that for CuO NPs. A solution of 1.5 g Zn(NO
3)
2∙6H
2O in 60 mL water was heated to 91 °C followed by the addition of 1 mL 1 M NaOH. Formation of white color indicated the completion of the reaction. Magnetite (Fe
3O
4) NPs were synthesized as described [
34]. In addition, divalent metal salts Co(NO
3)2
*6H
2O and MoS
2 (Alfa Aesar, Ward Hill, MA, USA) were tested. Materials were stored at 4 °C as dry powders.
4.2. Cu (II)-Containing Organic Complexes
Synthesis of Cu-containing organic complexes [(Z)-3-(2-fluorophenyl)-2-methylthio-5-(pyridin-2-ylmethylene)-3,5-dihydro-4H-imidazol-4-one]copper(II) dichloride (
1;
Scheme 1), [(Z)-3-(2-fluorophenyl)-2-methylthio-5-(pyridin-2-ylmethylene)-3,5-dihydro-4H-imidazol-4-one]copper(I) chloride (
3) and bis[(Z)-3-(2-fluorophenyl)-2-thio-5-(pyridin-2-ylmethylene)-3,5-dihydro-4H-imidazol-4-one]copper(+1.5) µ-chloride (
5), and corresponding organic ligands [(Z)-3-(2-fluorophenyl)-2-methylthio-5-(pyridin-2-ylmethylene)-3,5-dihydro-4H-imidazol-4-one (
2) and (Z)-3-(2-fluorophenyl)-2-thio-5-(pyridin-2-ylmethylene)-3,5-dihydro-4H-imidazol-4-one (
4) has been reported elsewhere [
10].
Synthesis of the Cu complex of 2-(3′-maleimido)-5,10,15,20-tetraphenylporphyrin
6 and the metal free 2-(3′-maleimido)-5,10,15,20-tetraphenylporphyrin
7 has been reported in [
24]. The copper complex of 5-[
p-(3′-maleimido)phenyl]-10,15,20-triphenylporphyrin; compound
8) was synthesized as follows: a solution of 50 mg (0.25 mmol) Cu(OAc)
2·H
2O in methanol (5 mL) was added to a solution 50 mg (0.07 mmol) 5-[
p-(3′-maleimido)phenyl]-10,15,20-triphenylporphyrin
9 in methylene chloride (5 mL). The resulting mixture was stirred for 1.5 h at room temperature with TLC monitoring (CHCl
3–hexane 1:1). Then, the reaction mixture was poured into water and extracted with methylene chloride. The organic layer was dried over Na
2SO
4, and the solvent was removed under reduced pressure. Yield 50 mg (93%, dark red solid). UV–Vis (CH
2Cl
2) λ
max, (ε × 10
−3) nm: 419 (253), 541 (10.7), 577 (2.4). IR (KBr) ν
max, cm
−1: 1719 (C=O), 1597 (C=C). The metal-free 5-(
p-(3′-maleimido)phenyl)-10,15,20-triphenylporphyrin (compound
9) was previously synthesized by us [
29]. Chemical structures of compounds
1–
9 are shown in
Scheme 1.
4.3. Cell Lines and Treatment
The panel of tumor cell lines included human K562, KU-812, and MOLM-6 chronic myelogenous leukemia; HCT116 colon, SKOV-3 ovarian, MCF-7, and MDA-MB-231 breast carcinomas; and B16F10 murine melanoma (all from American Type Culture Collection (Manassas, VA, USA)). In addition, the isogenic sublines with the molecular determinants of altered drug response were multidrug resistant
MDR1/P-glycoprotein overexpressing K562/4 subline, HCT116p53KO (non-functional p53) and SKOV-3/CDDP (a 4-fold resistance to cisplatin; gift of G.A.Posypanova, National Research Center ‘The Kurchatov Institute’, Moscow, Russia) [
24,
25,
35]. Non-malignant hFB-hTERT6 skin fibroblasts were obtained via a lentiviral transduction of the full-length
TERT gene under a cytomegalovirus promoter (gift of E. Dashinimaev, Engelhardt Institute of Molecular Biology, Moscow). Cells were maintained in RPMI-1640 (K562, K562/4, KU-812, and MOLM-6) or Dulbecco mediated Eagle’s medium (other cell lines) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 50 μg/mL gentamicin at 37 °C, 5% CO
2 in a humidified atmosphere. The freshly prepared aqueous suspensions of inorganic materials (see above) were added to the cell-free systems or cell cultures. Copper organic complexes and the respective metal-free organic ligands were dissolved in dimethyl sulfoxide (10 mM). Aqueous solutions were prepared on the day of experiments. N-acetylcysteine (NAC) and cysteine were dissolved in culture media at 50 mM stock solutions, and then pH was adjusted to 7.2–7.4 with 1 M NaOH. Stock solutions of sodium ascorbate (500 mM) and α-tocopherol (250 mM) were prepared in water or used as oil, respectively. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-FMK) was from Selleckchem, Houston, TX, USA. The cytotoxicity of metal NPs and Cu–organic complexes alone or in combinations with NAC, ascorbate, or α-tocopherol was determined in MTT assays [
36].
4.4. Cell Fluorescence
Cell associated fluorescence was recorded on a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA). At least 10,000 events were collected per sample. Data were analyzed using CytExpert software (Beckman Coulter).
4.5. Time Course of Cell Death
CuO NPs (0.1 µg/mL) and NAC (1 mM) were added to the K562 cells in 24-well plates (5 × 104 per well in 1 mL medium). Every hour, cell aliquots were collected and incubated with propidium iodide (PI; 10 µg/mL) for 2 min in the dark. The intensity of fluorescence was measured in the phycoerythrin (PE) channel (585/42 nm).
4.6. Annexin V-FITC/PI Staining
K562 cells were plated into 6-well plates (Eppendorf; 2 × 105 cells/well) and treated with the combination of CuO NPs (0.1 µg/mL) and NAC (1 mM) for 4–24 h. Then, cells were stained with annexin V-FITC for 15 min at room temperature, washed with PBS, and counterstained with 10 µg/mL PI (Dead Cell Apoptosis Kit for flow cytometry; Thermo Fisher Sci., Waltham, MA, USA). Fluorescence intensity was detected in FITC (525/40 nm) and PE channels.
4.7. Cell Cycle Distribution
K562 cells (2 × 105) were plated into 6-well plates overnight and then treated with 0.1 µg/mL CuO NPs and 1 mM NAC for 6 h or 24 h. Cells were pelleted and lysed in the buffer containing 50 µg/mL PI, 0.1% sodium citrate, 100 µg/mL RNase A, and 0.3% NP-40 for 30 min in the dark. Fluorescence was detected in the PE channel.
4.8. Mitochondrial Transmembrane Potential
The MitoTracker™ Red CMXRos (Thermo Fisher Sci.) was used to monitor the mitochondrial potential. K562 cells were treated with a combination of CuO NPs (0.1 µg/mL) and 1 mM NAC for 4–24 h. The MitoTracker (final concentration 85 nM) was added to the cells 30 min prior to the completion of treatment. Then, the cells were washed with cold PBS and analyzed in the allophycocyanin (APC) channel (660/10 nm).
4.9. ROS Detection
The 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) dye (Thermo Fisher Sci.) was used to detect intracellular ROS. K562 cells (2 × 105 in 1 mL of medium) were treated with 5 µM carboxy-H2DCFDA for 30 min, washed, and resuspended in fresh medium. CuO (1 µg/mL), NAC (1 mM), or their combination was added to corresponding tubes, and cells were incubated at 37 °C, 5% CO2 for 1–4 h. Cell associated fluorescence of carboxy-H2DCFDA was measured by flow cytometry in the FITC channel. Ten thousand events were collected per sample. Values of mean fluorescence intensities were normalized by the respective values in untreated (DCFDA alone) cells.
Laser scanning confocal microscopy was used to visualize the morphology of mitochondria and to monitor the integrity of the plasma membrane upon treatment with CuO NPs and NAC. The HCT116 cells were plated on 35 mm dishes with the glass bottom (SPL Life Sci., Gyeonggi-do, Korea) and incubated for 72 h at 37 °C, 5% CO
2 to reach 50% confluence. Then, the cells were incubated with CuO NPs (1 µg/mL) and NAC (1 mM) for 1–24 h followed by washing with PBS. Photolon (10 µM, 3 h, illumination at 660 nm, 33 J/cm
2) was a reference compound for light-activated oxidative stress and rapid organelle damage, a property of chlorin e6 derivatives [
26,
37]. For labeling mitochondria and nuclei, dihydrorhodamine 123 (DHR 123; (488 nm/510–570 nm)) and Hoechst 33342 (405 nm/415–470 nm), respectively, were used as recommended by the manufacturer (Thermo Fisher Sci.). The entry of PI (488 nm/550–600 nm) was used to monitor the plasma membrane integrity. Images were analyzed on a Leica TCS SPE 5 laser scanning confocal microscope with LAS AF software (Leica Microsystems GmbH, Wetzlar, Germany).
4.10. Immunostaining
K562 cells were treated with 0.1 µg/mL CuO NPs in the absence or presence of 1 mM NAC for 6 h and 24 h. After fixation in 0.75% paraformaldehyde for 15 min and permeabilization in ice cold methanol for 30 min, cells were washed with PBS and incubated with rabbit antibodies against cleaved caspase 3 and PARP (Cell Signaling, Danvers, MA, USA; 1:1000) for 1 h. After washing with PBS, the secondary goat anti-rabbit antibodies conjugated with AlexaFluor 488 (Thermo Fisher Sci.; 1:1000) were added, and cells were incubated for 1 h and analyzed by flow cytometry in the FITC channel.
4.11. Immunoblotting
The K562 and HCT116 cells in 60 mm Petri dishes (80% confluency) were treated with CuO NPs (0.1 µg/mL) and NAC (1 mM) for 24 h at 37 °C, 5% CO2. Dox (0.2 µM) was used as a reference compound. After the completion of treatment, the cells were lysed in the buffer containing 150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris base pH 8.0, 2 mM phenylmethylsulfonyl fluoride, and protein inhibitor cocktail (Roche) for 30 min on ice. The protein concentration was evaluated using Bradford reagent. Cell lysates (35 µg of total protein) were resolved in 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) at 120 V for 1.5 h and transferred onto 0.2 µm nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). After blocking with skimmed milk, the membranes were incubated with rabbit antibodies against human cleaved PARP and total or cleaved caspase 3 (Cell Signaling) at 4 °C overnight, washed, and incubated with secondary goat anti-rabbit antibodies conjugated with horseradish peroxidase (Cell Signaling). Proteins were visualized using the enhanced chemiluminescence reagent and a ChemiDoc MP gel imaging system (BioRad, Hercules, CA, USA).
4.12. Planar Lipid Bilayer Setup, Recording System, and Mode of Calculations
Synthetic 1-palmitoyl-2-oleyl-
sn-glycero-3-phosphocholine (POPC) and1-palmitoyl-2-oleyl-
sn-glycero-3-phospho-(1′-glycerol) (POPG) were obtained from Avanti Polar Lipids, Inc. (Pelham, AL, USA). KCl solution (0.1 M) was buffered with 5 mM HEPES, pH 7.4. Bilayer lipid membranes were prepared from POPC or POPG using a monolayer-opposition technique [
38] on a 50-µm-diameter aperture in a 10-µm-thick Teflon film separating
cis- and
trans-compartments of the Teflon chamber. Experiments were performed in the chambers containing 0.1 M KCl, 5 mM HEPES-KOH pH 7.4. NAC (1 mM) and CuO NPs (1–4 µg/mL) (each agent alone or in combinations) were added to the
cis-chamber to mimic the physiologically relevant conditions. Ag/AgCl electrodes with 1.5% agarose/2 M KCl bridges were used to apply the transmembrane voltage (
V) and measure the transmembrane current (
I). “Positive voltage” refers to the case in which the
cis-side compartment is positive with respect to the
trans-side. All experiments were performed at room temperature.
Measurements of electric current were carried out using an Axopatch 200B amplifier (AutoMate Scientific Inc., Berkeley, CA, USA) in the voltage-clamp mode. Data acquisition was performed with a 5 kHz sampling frequency and low-pass filtering at 100 Hz. The current tracks were processed through an 8-pole Bessel 100 kHz filter. Data were digitized by Digidata 1440A and analyzed using pClamp 10 (AutoMate Sci.) and Origin 8.0 (OriginLab Corp., Northampton, MA, USA).
4.13. Calcein Release from Liposomes
Large unilamellar vesicles were made from POPC or POPG and loaded with the fluorescent dye calcein (35 mM) using a mini-extruder (Avanti Polar Lipids, Alabaster, AL, USA). At this concentration, calcein fluorescence inside the liposomes is self-quenched. The increase in fluorescence of free calcein in the surrounding media is a measure of the disturbance of membrane integrity in the absence and presence of NAC (1 mM), CuO NPs (1–4 µg/mL), or their combinations. The fluorescence of released calcein was determined on a Fluorat-02-Panorama spectrofluorometer (Lumex, Saint-Petersburg, Russia) at λex = 490 nm, λem = 520 nm. The detergent Triton X-100 (at final concentration of 1%) was added at the end of experiments for complete disruption of liposomes (referred to full disengagement of the marker from vesicles).
The relative intensity of calcein fluorescence (
IF, %) was calculated as follows:
where
I and
I0 are the calcein fluorescence intensities in the presence and absence of NAC (1 mM), CuO NPs (1–4 µg/mL), or their combinations, respectively, and
Imax is the maximal fluorescence after treatment of liposomes with Triton X-100. A factor of 0.9 was introduced to account for sample dilution by Triton X-100.
4.14. Differential Scanning Microcalorimetry (DSC) of Liposomal Suspensions
Experiments were performed on a μDSC 7 EVO microcalorimeter (Setaram, France). Giant unilamellar vesicles were formed from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-phospho-1′-glycerol (DPPG) (Avanti Polar Lipids, Inc. (Pelham, AL, USA) by the electroformation method (standard protocol, 3V, 10 Hz, 1 h, 55 °C) using the Nanion vesicle prep pro device (Nanion Technologies, Munich, Germany). Final concentrations were 3 mM, 1 mM, and 1–4 µg/mL of DPPC/DPPG, NAC and CuO NPs, respectively. The liposomal suspension was heated at a constant rate of 0.2 C/min. Reversibility of thermal transitions was assessed by reheating the sample immediately after the cooling step from the previous scan. The temperature dependence of the excess heat capacity was analyzed using Calisto Processing (Setaram, Caluire-et-Cuire, France). The thermograms were characterized by temperature of the main phase transition of DPPC or DPPG (Tm), and the half-width of main peak (T1/2) that characterizes the size of the cooperative lipid unit.
4.15. Electrochemical Measurements of Cu Redox State
The electrochemical behavior of CuO NPs and compounds 1–5 was studied using cyclic voltammetry (CV) and rotating disk electrode (RDE) techniques. An IPC Pro M potentiostat was used for electrochemical studies. The working electrode was a glassy carbon disk (d = 2 mm), and the reference electrode was Ag/AgCl/KCl (sat.). The auxiliary electrode was a platinum plate, and the supporting electrolyte was 0.1 M Bu4NClO4 solution in dimethylformamide. The potential sweep rates were 100 mV/s (CV method) and 20 mV/s (RDE method). Samples were dissolved in an air-free solvent. All measurements were carried out in a dry argon atmosphere.
4.16. Mass Spectrometry
For HPLC analysis, a system with a Shimadzu Prominence LC-20 (Shimadzu Corporation, Kioto, Japan) column and a convection fraction collector connected with a single quadrupole mass spectrometer Shimadzu LCMS-2020 (Shimadzu Corporation, Kioto, Japan) with dual ionization source DUIS-ESI-APCI were used. The analytical and preparative column was Phenomenex Luna 3u C18 100A.
4.17. Measurement of Thiol Group Content
The 5,5-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman’s reagent) (Thermo Fisher Sci.) was used to determine the sulfhydryl groups. The solution of 2 mM DTNB in 50 mM sodium acetate was mixed with 1 M Tris base pH 8.0 (1:2 v/v) to obtain the working solution. The reaction mixture (400 µL) contained 100 ng/mL or 1 µg/mL CuO NPs alone, 1 mM NAC alone or the combinations of each concentration of NPs with NAC. An aliquot (10 µL) was immediately transferred into a 96-well plate (Eppendorf), and then 190 µL of the working solution was added. Values of optical density (OD) at 412 nm were measured on a Tecan Spark 10M spectrophotometer (Tecan, Mennedorf, Switzerland).
4.18. Statistical Analysis
Experiments were carried out in 3–4 replicates. Data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 7 and Microsoft Office Excel software.