Polymer-Stabilized Silver (Gold)–Zinc Oxide Nanoheterodimer Structures as Antimicrobials

Abstract

A simple one-pot method is proposed for obtaining the colloidal nanohybrid structures of silver (gold) and zinc oxide as well as nanostructures doped with zinc ions. The copolymers of maleic acid were used for the stabilization of nanoheterostructures. To characterize the preparation, UV–Vis spectroscopy, TEM, FTIR, XPS, and XRD were used. The bactericidal properties of the nanoheterostructures were studied in relation to the fungus C. albicans and the bacteria E. coli and S. aureus, used in planktonic form. In general, the samples containing nanosilver were the most active, and the preparations containing gold nanoparticles were the least active. The minimum inhibitory concentrations (MICs) of the Ag/ZnO samples, based on all copolymers, were in the ranges of 1.4–1.7 μg/mL for C. albicans, 2.9–6.8 μg/mL for E. coli, and 23–27 μg/mL for S. aureus; the MIC values of Au/ZnO samples were 472 μg/mL for S. aureus and 945 μg/mL for C. albicans and E. coli. The additional introduction of zinc cations into heterodimers had practically no effect on the antimicrobial properties of the composites. For all prepared composites and all tested microorganisms, the fractional inhibitory concentration indexes were in the range of 0.5–2.2, which indicates a close-to-additive contribution of the bioactive components in the samples used in the bactericidal process.

1. Introduction

Due to their unique physicochemical properties, nanotechnological products are widely used in biotechnology [1,2,3,4]. In the antibiotics-resistant era, nano-antibiotics are potential nano-weapons in the fight against multidrug-resistant pathogens and even biofilms [5,6,7,8,9]. The results of using metal nanoparticles, their compounds, and their various combinations as antibacterial agents are very encouraging, since such structures are active against protozoa, fungi, bacteria, and viruses. Materials with antiseptic properties containing nanoparticles of silver or cations of copper, zinc, iron, manganese, etc., are known and widely used. Silver nanoparticles (AgNPs), known for their antimicrobial properties, are used as food additives (E 174). Silver, unlike conventional antibiotics, practically does not cause resistance to a wide range of pathogens [10]. Numerous reviews have been published on the production and use of AgNPs [11,12,13,14,15]. As for the mechanism of the toxic effect of AgNPs on microorganisms, a number of researchers believe that this occurs due to the release of silver ions from nanoparticles depositing corresponding cations [16,17]. Silver ions may bind to the phosphorous-containing groups of nucleic acids in microorganism cell nuclei or the sulfhydryl and carboxylic groups of bacterial enzymes. They can produce reactive oxygen species (ROSs) inside the pathogen cell, while disrupting the activity of the electron transport chain inside the cell. These factors are the cause of the suppression of vital activity and the colonization of bacteria [18,19,20]. Unlike silver nanoparticles, gold nanoparticles (AuNPs) are much less often used as bactericides. For example, AuNPs were found to have an almost negligible effect on E. coli, S. aureus, B. subtilis, and A. tumefaciens [21,22]. Greater bactericidal activity was observed for silver nanoparticles than for AuNPs [23,24,25,26,27].

Gold NPs exert their antibacterial activities in two predominant ways: one is to collapse the membrane potential, inhibiting ATPase activities to decrease the ATP level; the other is to inhibit the subunit of ribosome from binding to tRNA. Gold NPs enhance chemotaxis in an early-phase reaction. The actions of gold NPs do not include a reactive oxygen species (ROS)-related mechanism, which is the cause for cellular death induced by most bactericidal antibiotics and nanomaterials [28]. The disruption of the cell membrane of the P. aurigenosa and the uncontrolled leakage of cell DNA was reported after interacting with 4,6-dihydroxyl-2-pyrimidinethiol-functionalized cationic 3 nm AuNPs [29].

Gold nanoparticles were also tested against pathogenic microorganisms in combination with antibiotics. It was shown, for example, that vancomycin hybrid AuNPs showed better antibacterial activity than free vancomycin [30]. As for the toxicity of silver and gold nanoparticles, a comparative assessment was carried out on the adverse bioactivity of virtually equidimensional gold and silver nanoparticles that were administered to rats either intratracheally or intraperitoneally at equal mass doses. It was demonstrated that AgNPs were more toxic than AuNPs, notwithstanding the latter’s higher accumulation in RES-abundant organs [31]. In addition, gold has a low toxicity to biological systems, followed by silver, whether the systems are bacterial, animal, or human [32]. In other words, although silver nanoparticles have been shown to be more toxic than AuNPs, AgNPs have a relatively low toxicity to human cells in comparison with lower forms of life [31,33,34,35].

Zinc oxide NPs (ZnO NPs) and zinc cations are also among the most studied antibacterial agents. It is believed that ZnO NPs are biocompatible and nontoxic [36]. ZnO NPs are more active against S. epidermidis, S. pyogenes, S. aureus, and B. subtilis than MgO, TiO₂, Al₂O₃, CuO, and CeO₂ NPs [37]. It should be noted here that ZnO NPs have a minimal impact on human cells and have a highly specific toxicity against planktonic forms of bacteria and biofilms. The mechanism of the bacteriolytic action of ZnO NPs is explained by the formation of ROS, which damages the cell wall of the pathogen [38]. The use of ZnO micro- and nanoparticles in biotechnology, the food industry, and medicine is well known [39,40,41]. For example, they are used as a food additive and packaging component to protect against spoilage [42], in remedies for fungal infections or acne [43], and as a wound-healing stimulant that also keeps wounds moist and clean. Zinc cations are less bactericidal but less toxic compared to Ag cations and are also used in biomedicine [24]. Zinc sulfate and chloride are used in dental materials, such as cements, filling and impression materials, and dentifrice pastes [44]. The combination of zinc ions with a cholera vaccine containing a recombinant cholera toxin subunit had a positive effect on the course of both cholera and diarrhea in children [45]. The mechanism of action of ZnO NPs on microbial cells is similar to that of AgNPs. They can also be considered as a source of metal cations, which can be released under physiological conditions. Zinc ions released from ZnO NPs in the acidic environment of lysosomes interact with biomolecules in the bacterial cell, causing its death. Thus, these cations are bioactive, and the apoptosis of pathogen cells is associated with them [46,47]. Another hypothesis suggests that the death of microbial cells exposed to zinc oxide nanoparticles is due to the breakdown of proteins and lipids by ZnO NPs, which leads to the destruction of bacterial cells due to the leakage of intracellular contents [48].

It is known that zinc ions are essential nutrients for microbial growth [49]. However, under certain conditions, concentrations and combinations with a number of components, zinc cations are able to exhibit an antiviral and antibacterial effect [50,51,52]. Therefore, the combination of pyrithione (PT) and Zn²⁺ at concentrations of 2 μM Zn²⁺ and 2 μM PT inhibits the replication of RNA viruses: poliovirus, influenza virus, and SARS-CoV-2 (coronavirus). Zinc complexes with compounds of the fluoroquinolones such as levofloxacin and ciprofloxacin have higher bactericidal activity against K. pneumoniae, S. aureus, E. coli, and B. dysenteriae than original antibiotics [53,54]. Zinc-bound vancomycin implements were found to increase activity against bacteria which were resistant to vancomycin itself [55]. A combination of Ag⁺ and Zn²⁺ ions led to a synergistic antibacterial effect against E. faecalis [56].

When combining various antibacterial components, their additive or synergistic complicity is often revealed, which leads to a decrease in the therapeutic dose of the preparation and in some cases, to a cheaper drug due to the introduction of a cheaper bioactive component into the composites [57,58,59,60,61]. Similar effects were observed when using heterostructures, in particular Ag/ZnO composites. Various complexes based on Ag/ZnO have not only bactericidal [62,63,64,65] but also antiviral [66] activity.

As for the ways for obtaining such heterostructures, the one-pot method seems attractive, in which precursors (metal cations) are simultaneously introduced into the reaction system. One-pot syntheses of Au/ZnO hybrids at high temperature in the presence of hexamethylenetetramine, oleylamine, hydrazine hydrate, or in 1-dodecanol have been described previously [67,68,69,70,71].

Thus, the above-mentioned processes for the formation of Ag(Au)/ZnO heterodimer systems are quite laborious: high temperature and pressure, and a long synthesis time are used. As a rule, when using these methods, spontaneous formation of cluster structures occurs and, as a result, water-insoluble composites are obtained. These factors pose serious limitations for the wide application of such composites in biomedicine, laboratory settings, and at industrial scales especially.

For practical applications as bactericidal and antibiotic drugs, the simplicity of their preparation and their ability to dissolve in water are important properties. Thus, taking into account the potential use of Ag(Au)/Zn complexes as bactericides, a facile efficient and inexpensive route for obtaining such colloidal water-dispersed nanoheterostructures is still required. Nanoparticles of metals, their oxides, and complexes have a natural tendency to agglomerate in colloidal solutions. Surface protection of nanoparticles using various stabilizers increases their kinetic stability [72]. Polymers are most often used to stabilize nanoparticles. In particular, organic polymers can act as stabilizers of bioactive nanoparticles, preventing their aggregation by reducing their surface energy [59]. Water-soluble polymers can promote the binding of metal nanoparticles to the pathogen cell due to highly active potential interactions between repeating polymer units and multivalent surface properties of bacteria [73]. Previously, polyvinylpyrrolidone was used to obtain heterostructures, but through a two-step process [74]. This polymer was used to promote the deposition of Zn oxide onto AuNPs.

Growing oxide shells on seed nanoparticles requires the control of several processes: (a) the nucleation and growth of the shell material; (b) the “wetting” of the shell material on the seeds; and (c) the aggregation of nanoparticles [75]. Synthesis of a ZnO-Ag heterostructure was carried out by a precipitation method with cellulose nanofibers as a stabilizer (with the formation of Ag nanoparticles on ZnO particles) [76]. Using dextran as a reducing agent and stabilizer, ZnO-Au NPs composites with petal-like morphology were synthesized [77].

In comparison with a number of natural and synthetic polymers, maleic acid (anhydride) copolymers have a complex of positive properties. Maleic acid residues contained copolymers have a regular structure; they are water soluble, amphiphilic, available, and nontoxic. Previously, such copolymers were used to stabilize silver nanoparticles [78]. It was shown that such nanocomplexes can be used as antimicrobial agents in combination with traditional antibiotics [57] or silver cations [79]. AgNPs have also been incorporated in a phenol-containing polymer [60] and into preparations containing specific lectin markers on the surface of pathogen cells [80].

The purpose of this study was to develop a convenient synthesis of silver (gold)/ZnO colloidal nanostructure which are stabilized by maleic acid copolymers, and to dope these nanostructures with zinc ions for use as antimicrobials.

2. Materials and Methods

2.1. Materials

Poly(N-vinyl-pyrrolidone-alt-maleic anhydride) (VM-anh.) M = 40,000 was synthesized according to [81]. Poly(ethylene-alt-maleic anhydride) (EM-anh.) M = 25,000 (Monsanto, Saint Louis, MO, USA) and poly(styrene-alt-maleic anhydride) (SM-anh.) M = 50,000 (Sterlitamak chemical plant, Sterlitamak, Russia) were purchased. The copolymers of maleic acid were obtained after hydrolysis of copolymers of maleic anhydride and designated VM, EM, and SM, respectively. NaBH₄, HAuCl₄·3H₂O, and ZnSO₄·7H₂O were the products of Sigma Aldrich; and AgNO₃, NaOH are Reahim products. All reagents were of an analytical grade and were used without purification. The tested microorganisms were as follows (“BD MicrotrolTM”, Becton Dickinson, Diagnostic Systems Product Catalog for Industrial Microbiology, Heidelberg, Germany): Escherichia coli (E. coli) NCTC 11954/ATCC 35218—Gram-negative, facultative anaerobic bacteria; Staphylococcus aureus MRSA (S. aureus MRSA) NCTC 12973/ATCC 43300—Gram-positive bacteria, resistant to oxacillin; Candida albicans (C. albicans) NCPF3255/ATCC 10231—yeast-like fungi.

2.2. Instrumentation

Bench meters (Hanna, Vöhringen, Germany) HI 2211 were used for pH and temperature registration. UV-Vis spectra were measured with Hitachi U-5100 spectrophotometer (Japan). LEO 912 AB microscope (Omega, Karl Zeiss, Jena, Germany) operated at voltage of 100 kV was used to record transmission electron microscopy (TEM) micrographs. To obtain a TEM micrograph, a drop of the investigated solution was evaporated on Formvar-coated copper grid. Fourier transform infrared spectra (FTIR) of the solid samples (KBr) were recorded using a Magna IR-720 Fourier spectrometer Magna IR-720 (Nicolet, Parsons W118, USA). Compositions of the preparations were determined in the laboratory for microanalysis of INEOS RAS. Powder X-ray diffractometry (XRD) phase analysis of the samples placed on flat holders was carried out with a diffractometer D8 Advance (Bruker AXS, San Jose, CA, USA) in the Bragg–Brentano focusing geometry using CuK_α radiation, the scan rate was 0.5° min⁻¹ and angular step was 0.02°. Powder patterns were processed with DIFFRACplus EVA (Bruker AXS GmbH DIFFRAC.EVA, Bruker AXS GmbH, Karlsruhe, Germany, 2011), and Bruker AXS software version number 6.0.0.6; the search/match procedure was implemented. The L_vol-IB formalism was used for evaluating the values of mean crystalline size [82]. The content of crystal phases of the heterodimers was calculated using DIFFRAC TOPAZ software (Coelho, A. TOPIC 5.0, Bruker AXS GmbH, Karlsruhe, Germany, 2012; Bruker AXS uses Rietveld refinement of collected X-ray data).

The samples in powder form were characterized by X-ray photoelectron spectroscopy (XPS) in an Axis Ultra DLD (Kratos Analytical, Manchester, UK) spectrometer with monochromatic Al K_α (hν = 1486.6 eV) radiation source operated at 150 W at room temperature under ultrahigh vacuum conditions (<10⁻⁸ Torr). High-resolution spectra were measured with 40.0 eV pass energy and 0.1 eV step. The analysis area was 300 μm × 700 μm. The energy scale of the spectrometer was calibrated using the following values: Au 4f_7/2–83.96 eV, Ag 3d_5/2–368.21 eV, and Cu 2p_3/2–932.62 eV. An electron neutralizer was used to compensate for the surface charging. For an internal charge reference, the C 1s spectra were fitted to identify the C–C/C–H state which was assigned a binding energy of 285.0 eV.

2.3. Methods

2.3.1. Preparation of Polymer-Stabilized AgNPs, AuNPs, and Heterodimers

Preparation of VM/Ag⁰, EM/Ag⁰, and SM/Ag⁰

Synthesis of nanosized silver was described elsewhere [78]. Briefly: To the solution of copolymer of maleic acid (0.01 M, here the molar concentration of copolymer refers to the maleic acid units) in deionized (DI) water at pH 7 (titration with NaOH solution, 5%), a freshly prepared solution of AgNO₃ (0.1 M) (for the ratio of reagents 1:1 (mol.)) was added with vigorous stirring. Then, after about 5 min, a freshly prepared solution of NaBH₄ (0.1 M, 2-fold molar excess with respect to silver ions) in DI water was added to the reaction solution with vigorous stirring. The reaction mixture was then stirred for 24 h at room temperature. The samples of polymer stabilized silver nanoparticles were dried using lyophilization (0.05 mbar, −55 °C) after ultrafiltration (YM5 membrane (“DIAFLO”, AMICON CORPORATION, Palo Alto, CA, USA).

Preparation of VM/Au⁰

Nano-sized gold was synthesized by the reduction of gold ions at ~20 °C with NaBH₄ in the presence of copolymer of maleic acid following the procedure [83]. Briefly: To the solution of the VM (0.007 M) in DI water at pH 6 (titration with water solution of NaOH, 5%), the freshly prepared solution of HAuCl₄·3H₂O (0.01 M) (for the ratio 1:5 of reagents, respectively) was added with vigorous stirring. Then, after about ten minutes, a freshly prepared solution of NaBH₄ in DI water (0.1 M, in 3-fold molar excess with respect to metal ions) was added to the reaction solution with vigorous stirring. The reaction mixture was then stirred for 24 h at room temperature. The dried sample was obtained using lyophilization (0.05 mbar, −55 °C) after ultrafiltration (or dialysis) of the reaction solution.

Synthesis of VM/Au⁰/ZnO

A measure of 20 mL of VM solution (0.007 M, pH 8), 0.322 mL ZnSO₄·7H₂O (0.1 M), 2.1 mL HAuCl₄·3H₂O (0.01 M) in DI water, and 0.4 mL NaOH (1 M) were mixed in glass vial with a screw-caps tightly closed. The pH of the reaction was 12. Then, the test vial was kept in a boiling water bath for 3 h. The molar ratio of units of maleic acid residues of copolymer to gold cations and zinc cations was 1/0.15/0.23. After cooling to room temperature, the solution was left overnight at this temperature and then was dialyzed against DI water and lyophilized (0.05 mbar, −55 °C).

Synthesis of VM (EM, SM)/Ag⁰/ZnO

The solutions of ZnSO₄·7H₂O (0.262 mL, 0.1 M), VM (25 mL, 0.007 M), AgNO₃ (0.52 mL, 0.1 M), all in DI water, and NaOH (0.325 mL, 1 M) were mixed in glass vial with a screw-caps tightly closed. After shaking (the pH of solution was 12), the test vial was kept in a boiling water bath for 6 h. The molar ratio of units of maleic acid residues of copolymer to silver cations and zinc cations was 1/0.3/0.15. The samples EM/Ag⁰/ZnO and SM/Ag⁰/ZnO were synthesized at the same temperature and at the same molar ratio of reagents. The reaction time for these preparations was 7 h. Samples containing zinc ions were prepared with the addition of a zinc salt to the copolymer- or heterodimer-contained copolymer. The samples were dialyzed against DI water or ultrafiltered (YM5 membrane, “DIAFLO”, Amicron Corporation, Palo Alto, CA, USA) and then lyophilized (–55 °C, 0.05 mbar).

2.3.2. Antimicrobial Activity Tests

The determination of the minimum inhibitory concentrations (MICs) of the preparations in relation to the strain of microorganisms used was carried out using a serial microdilution method in accordance with the standard procedure [84]. Initial concentrations of VM/Ag⁰, SM/Ag⁰, and EM/Ag⁰ were 1.35, 1.35, and 1.5 mg/mL, respectively; VM/Ag⁰/ZnO, SM/Ag⁰/ZnO, and EM/Ag⁰/ZnO were 1.9, 1.9, and 1.4 mg/mL, respectively; and VM/Au⁰ and VM/Au⁰/ZnO were 1.7 and 1.8 mg/mL, respectively. The description of the experiment is given in the protocol published elsewhere [79]. Briefly: The tests were carried out in 0.2 mL volume using sterile 96-well standard polystyrene plates. The final concentration of the investigated microorganism is 105 CFU/mL. Trypticase-soy broth (Bio-Rad Laboratories, Hercules, CA, USA) was poured into 0.1 mL in each well of the plate. The number of wells was determined by the necessary dilution range, the last well was left for setting negative control. The solutions of studied composites in fourfold ratio to the above-mentioned concentrations in the volume of 0.1 mL were pipetted into the first well containing 0.1 mL of broth.

After mixing, 0.1 mL of this mixture was pipetted to the second well, also initially containing 0.1 mL of broth and so on. The procedure was repeated until a sufficient number of dilutions were made. For inoculation, a microbial suspension of test microorganisms prepared on 0.45% saline solution was used. A measure of 0.1 mL of the inoculum was introduced into testing wells with the appropriate dilution of the composite, as well as in the last well with a nutrient broth without the test preparation (positive control). The plates covered with a sterile film were incubated at 35 °C overnight (48 h for C. albicans). The culture growth in the presence of the composites was compared with the reference wells without the composites (positive control) under visual control (wells in plates were viewed in transmitted light). The MICs of the samples were determined by the lowest concentrations of preparations in the well, which suppress the apparent growth of the microorganisms used. Statistical processing of data based on the results of 3 measurements was carried out.

3. Results

Colloidal nanoheterostructures containing silver (or gold) and zinc oxide were prepared using the one-pot method and were used as potential bactericidal agents (Scheme 1). Maleic-acid-containing alternating copolymers with N-vinyl-2-pyrrolidone (VM), ethylene (EM), and styrene (SM) were used as matrices for binding precursor metal ions and stabilizing the resulting nanoparticles. The transformation of metal cations into nanoparticles was carried out by the hydrothermal method in an alkaline medium. For the production ZnO/Ag⁰ and ZnO/Au⁰ composites, the numbers of entered metal cations in terms of each maleic acid residue in the copolymers were practically equivalent: Zn²⁺/Ag⁺ = 0.15/0.3 and Zn²⁺/Au³⁺ = 0.23/0.15 (mol/mol).

The amount of zinc cations introduced into the composite was limited by the solubility of the polymer salt obtained and depends on the structure of the copolymer used. In addition, this limit turned out to be different for the initial copolymers and polymer complexes containing AgNPs. In the first case, the limit of salt stability is reached when 0.25 mol of zinc cations is introduced onto the copolymer link. When zinc cations are introduced into a solution of silver-containing nanocomplexes, this limit increases, and the ratio reaches 0.5 for VM and SM, and 1.0 for EM. This is due to the different conformation of the initial copolymers and nanocomplexes.

In our previous study [85], using the EM copolymer, we showed by diffuse light scattering (DLS) that this copolymer was characterized by a wide and bimodal particle size distribution (hydrodynamic radius, Rh = 50–300 nm, depending on ionic strength and pH) and an intermolecular association. In the study of the complex of this copolymer with EM/Ag⁰ silver nanoparticles by DLS, well-formed and narrowly dispersed (Rh = 105 nm) micelles were detected [85].

Simultaneous production of silver (gold) and zinc oxide nanoparticles was recorded by heating an alkaline solution containing noble metal and zinc salts. The course of conversion of metal cations to reaction products was monitored by UV-visible spectra. Metal oxide and noble metal nanoparticles have specific absorption bands in the visible region [86,87,88]. Figure 1 shows UV-Vis spectra of reaction products. The broad absorption band at 365–375 nm indicates the presence of ZnO; the peak at 406 nm and the band at 520 nm appeared due to surface plasmon resonance corresponding to the Ag⁰ and Au⁰ states, respectively.

In the presence of copolymer stabilizer VM, Au, and Zn cations under the conditions used, the formation of nanoparticles was almost complete after 2 h. In the case of use of Ag and Zn cations, and the VM copolymer, the reaction was practically completed in 6 h; for EM and SM, the reaction was practically completed after 7 h. Figure S1a–c (Supplementary Materials) shows FTIR spectra of polymer-stabilized heterostructures in comparison with those of the initial copolymers. This method allows you to make sure qualitatively that the preparations contain zinc oxide. The Zn-O stretching mode was observed in the 400–700 cm⁻¹ range for ZnO crystals [87,89,90,91,92], so the region of 400–800 cm⁻¹ is presented in detail. The bands at 625, 624, 551, and 544 cm⁻¹ are stretching vibrations of Zn–O and Ag–ZnO bonds [87,89,90,91,92].

TEM micrographs of colloidal solutions of all prepared Au⁰/ZnO and Ag⁰/ZnO composites show relatively monodispersed nanoparticles, predominantly 6–20 nm in size with a shape close to a sphere (Figure 2). The size and shape of the particles shown in the micrographs did not depend on the nature of the polymers used as stabilizers of nanoparticles.

Previously, similar images were recorded for heterodimers obtained in a different way [76]. In addition to the TEM, the XRD gives a more detailed qualitative and quantitative composition of the crystal phases of the heterodimers. SAED patterns demonstrate concentric rings with intermittent dots, indicating that these nanoparticles are crystalline in nature.

Table 1. Composition of the nanoheterodimers.

Sample	Phases	Number of Crystalline Phases, % (Deviation)	Size of Crystalline Phases, nm (Deviation)
VM/Ag0/ZnO	Silver, Zincite	79.5 (6), 20.5 (6)	8.64 (8), 16.2 (8)
EM/Ag0/ZnO	Silver, Zincite	80.8 (7), 19.2 (7)	7.76 (8), 12.9 (7)
SM/Ag0/ZnO	Silver, Zincite	91.8 (10), 8.2 (10)	6.04 (10), 18 (4)
VM/Au0/ZnO	Gold, Zincite	77.5 (17), 22.5 (17)	6.57 (6), 20.7 (16)

contains the XRD characteristics of the composites. The crystal structures of metal-containing components in the obtained noble metal–zinc oxide heterostructures stabilized by a copolymer were studied by powder XRD (Table 1, Figure S2).

The X-ray examination of heterodimer samples showed the presence of the following crystalline phases: the cubic phase related to either silver or gold and the hexagonal phase of zincite (ZnO) with the average sizes of 6–9 and 16–20 nm (Table 1), respectively. The mean crystallite sizes of the nanoparticles were calculated from the broadening of the peaks using the Scherrer formula (Supplementary Materials).

The peaks corresponding to the Ag crystal phase are at ~38.1 and 44.2°; those corresponding to the Au crystal phase are at ~38.2 and 44.3°; those corresponding to the ZnO (zincite) phase are at ~31.8, 34.4, 36.1, 47.5, and 56.7. Their positions correspond to the Miller indexes: 111 and 002 for Ag(Au), and 100, 002, 101, 102, and 110 for ZnO (Figure S2).

The content of AgNPs in the crystalline phase of the corresponding samples VM/Ag⁰/ZnO, EM/Ag⁰/ZnO, and SM/Ag⁰/ZnO was 80–91% and zinc oxide was 8–20%. The gold-containing composite VM/Au⁰/ZnO contained 77.5% AuNPs and 22.5% zincite.

The chemical compositions of the samples VM/Ag⁰/ZnO and VM/Au⁰/ZnO were studied by XPS. The Au 4f_7/2, Zn 2p_3/2 a, and Zn 3p spectra are given in Figure 3 and Figure 4. In view of the overlap of the photoelectron spectra of Zn 3p and Au 4f core levels in the spectrum of the VM/Au⁰/ZnO composite and the presence of a satellite peak characteristic of the Zn 3p spectrum, the analysis of the spectra was started from the Zn 3p core-level of Ag⁰/ZnO nanoheterodimer stabilized with VM. The spectrum is presented as a sum of two Gaussian peaks at 89.4 and 92.25 eV, which are related to 3p_3/2 and 3p_1/2 sublevels with the 3p_3/2/3p_1/2 branching ratio of 0.5 and the satellite peak at 86.39 eV (subtracted for clarity). The latter describes a low-energy region and may be assigned to a specific energy loss [93,94,95]. The corresponding characteristics of the photoelectron spectrum given in Table S1 were used for approximation of the Zn 3p-Au 4f energy region of the VM/Au⁰/ZnO sample. For better perception, the satellite peaks associated with the energy losses of photoelectrons have been subtracted in Figure 3. The spectrum of Zn 3p core-level spectrum of the VM stabilized Au⁰/ZnO nanoheterodimer was approximated with two spin–orbit doublets and with the same constraints.

The Zn 2p_3/2 XPS spectra of VM/Ag⁰/ZnO and VM/Au⁰/ZnO samples (Figure 4) are close each other and may be presented as a sum of two Gaussian profiles at 1022.19 and 1024.20 eV, and 1022.20 and 1023.83 eV, respectively, with the same Gaussian width of 1.44 eV. As for the Au 4f spectrum, it was approximated with two 4f_7/2–4f_5/2 spin–orbit doublets with spin–orbit splitting of 3.65 eV and branching ratio of 1.33. The binding energies of 83.88 and 84.80 eV are attributed to the Au⁰ and Au⁺ states related to Au-Zn-O and Au-O-Zn structures, respectively [96,97,98]. Table S1 presents the characteristics of the Zn 3p and Au 4f spectra.

The Ag 3d XPS spectrum (Figure 5) is characterized with two main symmetric Ag 3d_5/2 and Ag 3d_3/2 peaks at 368.14 and 374.14 eV and two satellite peaks shifted relative to the main peaks by 3.4 eV. The latter are descriptors of Ag⁰ state. A negative CLS by 0.13 eV relative to the reference value of 368.27 eV may be attributed to Ag-Zn bonds [96]. The binding energies of 367.6 and 373.6 eV recorded for Ag-doped ZnO were assigned to Ag 3d_5/2 and Ag 3d_3/2 core levels and attributed to Ag-O bonds [93]. The change in the energy interval between the photoelectron peaks of various elements in the spectrum of a compound and elementary solids can serve as a measure of their interaction with each other. Such an interval does not depend on the charge reference and surface charging. Comparing the energy intervals between the Zn 2p_3/2 and Ag 3d_5/2 peaks for VM/Ag⁰/ZnO and Ag-doped ZnO [93], which are quite close, 654.2 and 654.1 eV, respectively, one can conclude that the similar chemical states of Ag atoms are similar. The corresponding O 1s, C 1s and N 1s XPS spectra are presented in Supplementary Materials (Figure S3).

Thus, the TEM, XPS, and XRD data indicate the formation of polycrystalline heterodimeric materials with a core–shell structure.

Table 2. Antimicrobial properties of the samples (MIC—minimum inhibitory concentrations; FIC—fractional inhibitory concentration; FICI—fractional inhibitory concentration index).

Sample	Content, %				MIC (μg/mL)			FIC			FICI
	Ag0(Au0)	ZnO	Zn2+	Sample a	Ag0 (Au0)	ZnO	Zn2+	Ag0(Au0)	ZnO	Zn2+
S. aureus MRSA
EM/Ag0	42.8	-	-	27.3	11.5	-	-	-	-	-	-
EM/Zn2+	-	-	4.4	259.8	-	-	11.4	-	-	-	-
EM/Ag0/Zn2+	38.0	-	11.1	28.1	10.7	-	3.0	0.91	-	0.26	1.17
EM/Ag0/ZnO	13.8	5.9	-	44.0	6.1	2.6	-	0.52	0.02	-	0.54
EM/Ag0/ZnO/Zn2+	14.4	5.5	4.2	45.5	6.55	2.5	1.89	0.57	0.02	0.16	0.59 c 0.73 d
ZnO b	-	100	-	125.0	-	125.0	-	-	-	-	-
VM/Ag0	32.0	-	-	23.0	7.4	-	-	-	-	-	-
VM/Zn2+	-	-	6.4	419.3	-	-	26.8	-	-	-	-
VM/Ag0/Zn2+	24.6	-	8.7	49.4	12.1	-	4.3	1.05	-	0.16	1.21
VM/Ag0/ZnO	9.8	4.2	-	122.5	12.0	5.1	-	1.62	0.04	-	1.66
VM/Ag0/ZnO/Zn2+	10.4	4.0	3.1	62.6	6.5	2.5	1.92	0.88	0.02	0.07	0.90 c 0.95 d
SM/Ag0	32.4	-	-	23.1	7.5	-	-	-	-	-	-
SM/Zn2+	-	-	6.7	405.0	-	-	27.0	-	-	-	-
SM/Ag0/Zn2+	29.5	-	8.9	50.8	15.0	-	4.52	2.0	-	0.17	2.17
SM/Ag0/ZnO	11.0	4.2	-	59.8	6.6	2.5	-	0.88	0.02	-	0.90
VM/Au0	15.25	-	-	920.0	140.3	-	-	-	-	-	-
VM/Au0/ZnO	14.65	3.9	-	472.5	69.2	18.7	-	0.49	0.15	-	0.63
E. coli
EM/Ag0	42.8	-	-	6.8	2.9	-	-	-	-	-	-
EM/Zn2+	-	-	4.4	519.5	-	-	22.8	-	-	-	-
EM/Ag0/Zn2+	38.0	-	11.1	7.0	2.7	-	0.78	0.92	-	0.002	0.92
EM/Ag0/ZnO	13.8	5.9	-	11.0	1.5	0.65	-	0.51	0.0013	-	0.51
EM/Ag0/ZnO/Zn2+	14.4	5.5	4.2	22.8	3.3	1.25	0.96	1.13	0.0025	0.042	1.13 c 1.17 d
ZnO b	-	100	-	500.0	-	500.0	-	-	-	-	-
VM/Ag0	32.0	-	-	5.7	1.8	-	-	-	-	-	-
VM/Zn2+	-	-	6.4	419.3	-	-	26.8	-	-	-	-
VM/Ag0/Zn2+	24.6	-	8.7	6.2	1.5	-	0.54	0.83	-	0.02	0.85
VM/Ag0/ZnO	9.8	4.2	-	30.6	3.0	1.28	-	1.66	0.0030	-	1.66
VM/Ag0/ZnO/Zn2+	10.4	4.0	3.1	31.3	3.3	1.25	0.94	1.8	0.0025	0.035	1.80 c 1.83 d
SM/Ag0	32.4	-	-	2.9	0.9	-	-	-	-	-	-
SM/Zn2+	-	-	6.7	519.5	-	-	22.8	-	-	-	-
SM/Ag0/Zn2+	29.5	-	8.9	3.2	0.9	0.28	-	1.0	-	0.001	1.00
SM/Ag0/ZnO	11.0	4.2	-	14.9	1.6	0.63	-	1.7	0.0013	-	1.70
VM/Au0	15.25	-	-	920.0	140.3	-	-	-	-	-	-
VM/Au0/ZnO	14.65	3.9	-	945.0	138.4	37.33	-	0.99	0.075	-	1.06
C. albicans
EM/Ag0	42.8	-	-	1.7	0.73	-	-	-	-	-	-
EM/Zn2+	-	-	4.4	520.0	-	-	22.8	-	-	-	-
EM/Ag0/Zn2+	38.0	-	11.1	1.75	0.67	-	0.19	0.91	-	0.008	0.90
EM/Ag0/ZnO	13.8	5.9	-	2.7	0.38	0.16	-	0.52	0.0008	-	0.52
EM/Ag0/ZnO/Zn2+	14.4	5.5	4.2	2.8	0.41	0.16	0.12	0.56	0.0008	0.005	0.56 c 0.565 d
ZnO e	-	100	-	200.0	-	200.0	-	-	-	-	-
VM/Ag0	32.0	-	-	1.4	0.46	-	-	-	-	-	-
VM/Zn2+	-	-	6.4	839.0	-	-	53.7	-	-	-	-
VM/Ag0/Zn2+	24.6	-	8.7	1.75	0.67	-	0.19	1.45	-	0.0035	1.45
VM/Ag0/ZnO	9.8	4.2	-	3.8	0.38	0.16	-	0.82	0.0008	-	0.82
VM/Ag0/ZnO/Zn2+	10.4	4.0	3.1	3.9	0.40	0.156	0.12	0.87	0.0008	0.002	0.87 c 0.87 d
SM/Ag0	32.4	-	-	1.45	0.47	-	-	-	-	-	-
SM/Zn2+	-	-	6.7	405.0	-	-	26.97	-	-	-	-
SM/Ag0/Zn2+	29.5	-	8.9	1.75	0.67	-	0.19	1.42	-	0.007	1.43
SM/Ag0/ZnO	11.0	4.2	-	3.7	0.41	0.16	-	0.87	0.0008	-	0.87
VM/Au0	15.25	-	-	920.0	140.3	-	-	-	-	-	-
VM/Au0/ZnO	14.65	-	3.9	945.0	138.4	37.33	-	0.98	0.19	-	1.16

^a These data include averages of three repeats. ^b Data from [99] (for size of ZnO nanoparticles 20–30 nm). ^c FICI was calculated for Ag⁰/ZnO-active components of system. ^d FICI was calculated for Ag⁰/Zn²⁺-active components of system. ^e Data from [100].

presents the bactericidal properties of the obtained heterostructures in relation to Gram-positive S. aureus, Gram-negative E. coli bacteria, and C. albicans fungus used in planktonic form.

All the samples obtained were active against the studied microbial strains. The MICs of samples containing AgNPs and zinc oxide for S. aureus were in the range of 44–122, for E. coli they were in the range of 11–31; for C. albicans they were in the range of 2.7–3.8 μg/mL. Thus, all preparations acted better on C. albicans and E. coli but not on S. aureus, as in publication [63], although there is evidence of a reverse trend [64,91]. The differences are associated with different ratios of the metal-containing components in the samples and the nature of the copolymer stabilizers. In general, the samples containing AgNPs were the most active, and the preparations containing gold nanoparticles were the least active. Polymer zinc salts also showed low activity. However, ionic zinc in the copolymer EM turned out to be more active against S. aureus than against E. coli, as it was shown for the complex of these cations with ε-poly-L-lysine [101], and showed almost identical activity in the case of maleic acid copolymers with other co-monomers. Apparently, the low activity of zinc cations is associated with the rapid inactivation of divalent cations when binding to cellular components. The increase in the MIC of heterodimers samples compared to samples containing only AgNPs is explained by a significant decrease in the content of a more expensive component, silver, due to the introduction of cheap and affordable zinc-containing components into its composition. Nevertheless, when studying the bactericidal properties of the obtained heterostructures, it was possible to achieve noticeable activity, especially in the case of using the EM copolymer as a stabilizer of nanoparticles. Moreover, this trend was observed for complex preparations that included zinc oxide in addition to nanometals. For samples containing AgNPs and zinc oxide, the MIC values under using different copolymers-stabilizers were close: 23–27 for S. aureus, 2.9–6.8—for E. coli, and 1.4–1.7 μg/mL—for C. albicans. In addition to the MICs of the samples, the MICs of the bioactive components present in the sample are given in terms of their content in the composite; this makes it possible to account for their mutual influence on the activity of the drug.

The degree of mutual influence of antimicrobial components—nanometals and zinc oxide (X and Y, respectively)—in their conjugates can be estimated by calculations of the fractional inhibitory concentration (FIC) and the fractional inhibitory concentration index (FICI).

$$FIC(X)=\frac{MIC(X,Y)}{MIC(X)}$$

where MIC(X,Y) is the MIC for X in combination with Y and MIC(X) is the MIC for X alone.

$$FIC(Y)=\frac{MIC(Y,X)}{MIC(Y)}$$

where MIC(Y,X) is the MIC for Y in combination with X and MIC(Y) is the MIC for Y alone.

$$FICI=FIC(X)+FIC(Y)$$

For our preparations, FICI values were calculated using the following equation:

$FICI=FIC({Me}^0)+FIC(ZnO)$ or $FICI=FIC({Me}^0)+FIC({Zn}^{2+})$, where

$$FIC({Me}^0)=\frac{MIC({Me}^0) in preparation}{MIC({Me}^0)}$$

Me⁰ is Ag⁰ or Au⁰

$$FIC(ZnO)=\frac{MIC(ZnO) in sample}{MIC(ZnO) alone}$$

$$FIC(ZnO)=\frac{MIC({Zn}^{2+}) in sample}{MIC({Zn}^{2+}) alone}$$

The FICI values are interpreted as follows: if FICI ≤ 0.5, then the interaction of the active components of the system is defined as synergistic; if 0.5 < FICI < 1.0, then the interaction of the active components of the system is defined as additive [102]. According to other interpretation, FICI > 2.0 indicates antagonistic effects, while values from 0.5 to 2.0 characterize additive effects [103].

The revealed FICI values for all the studied objects and for all the strains used are mainly in the 0.5–2.2 range. This indicates a close-to-additive participation of bioactive components of the systems in the bactericidal process. These circumstances, apparently, can be explained by a similar mechanism of action the bactericides present in the samples on the cells of pathogens. The mechanism of antimicrobial action at the molecular level for composite Ag/ZnO was considered earlier [91].

It should be stressed here that the addition of ZnO NPs into the AgNP-containing system led, in most cases, to better results in comparison with the activity of the AgNPs preparations with added zinc cations (the FICI values were lower in the first case). This might be due to different mechanisms of action of zinc cations and zinc oxide, or the prolongation of the action of cations released from deposited in zinc oxide. Testing was carried out in the dark. It was previously shown that the inactivation of bacteria by zinc oxide follows different mechanisms in the dark and under illumination. In the dark, the action of Zn²⁺ ions were manifested, while under illumination, mainly the process of photocatalytic injection of electrons took place [104]. For composites containing an EM copolymer, the joint action of the metal-containing components of the system was close to synergistic, and the FICI values were close to 0.5 for all microorganisms used. Apparently, this can be explained by the difference in the structure of the polymer chains of the copolymers used. With the additional introduction of zinc cations into the Ag/ZnO heterostructures, the FICI in terms of Ag/ZnO and Ag/Zn²⁺ pairs practically did not change, which indicates that there is no expediency of introducing a third bactericidal component into the system.

4. Conclusions

Water-soluble complex nanostructures of silver (gold) with zinc oxide, stabilized by biocompatible nontoxic copolymers of maleic acid, and nanostructures doped with zinc ions were obtained through the use of a one-pot method without using a high-temperature regime. The proposed method is easy to implement; it is carried out in an alkaline environment in the absence of difficult-to-remove organic solvents and additionally introduced reagents. The obtained samples can be stored in dry form. Our preparations turned out to be interesting not only in terms of their formation, but also as antimicrobial agents in relation to representatives of conditionally pathogenic microorganisms. AuNP-containing preparations were noticeably less active than Ag-containing composites. It was possible to achieve noticeable activity with a decrease in the composition of the more expensive component of silver (gold) due to the introduction of nontoxic, affordable zinc derivatives into the composition. The calculated FICI values for all synthesized composites in relation to all tested pathogens were mainly in the range of 0.5–2.2; this indicates additional interactions of metal-containing components in the preparations. For the composites which contained the copolymer of maleic acid with ethylene as stabilized agent, the participation of the metal-containing components of the system was close to synergistic (the FICI values were close to 0.5 for all microorganisms used). This is explained by the specific structure of macromolecules of this copolymer.