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. 2020 Jun 16;5(25):15290–15300. doi: 10.1021/acsomega.0c01335

Diverse Pathway to Obtain Antibacterial and Antifungal Agents Based on Silica Particles Functionalized by Amino and Phenyl Groups with Cu(II) Ion Complexes

Veronika V Tomina †, Iryna M Furtat §, Anastasia P Lebed §, Sofiya S Kotsyuda †,§, Hristo Kolev ∥, Maria Kanuchova ⊥, Dominika Marcin Behunova ‡, Miroslava Vaclavikova ‡, Inna V Melnyk †,‡,*
PMCID: PMC7331045  PMID: 32637802

Abstract

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Production of environmentally friendly multitasking materials is among the urgent challenges of chemistry and ecotechnology. The current research paper describes the synthesis of amino–/silica and amino–/phenyl–/silica particles using a one-pot sol–gel technique. CHNS analysis and titration demonstrated a high content of functional groups, while scanning electron microscopy revealed their spherical form and ∼200 nm in size. X-ray photoelectron spectroscopy data testified that hydrophobic groups reduced the number of water molecules and protonated amino groups on the surface, increasing the portion of free amino groups. The complexation with Cu(II) cations was used to analyze the sorption capacity and reactivity of the aminopropyl groups and to enhance the antimicrobial action of the samples. Antibacterial activities of suspensions of aminosilica particles and their derivative forms containing adsorbed copper(II) ions were assayed against Gram-positive (Staphylococcus aureus ATCC 25923) and Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853). Meanwhile, antifungal activity was tested against fungi (Candida albicans UCM Y-690). According to zeta potential measurements, its value could be depended on the suspension concentration, and it was demonstrated that the positively charged suspension had higher antibacterial efficiency. SiO2/–C6H5/–NH2 + Cu(II) sample’s water suspension (1%) showed complete growth inhibition of the bacterial culture on the solid medium. The antimicrobial activity could be due to occurrence of multiple and nonspecific interactions between the particle surfaces and the surface layers of bacteria or fungi.

Introduction

Recently, the scientific community has been facing a challenging task of creating antimicrobial agents and technologies without triggering undesirable microbial resistance.1,2 The solution of such a task requires multidisciplinary effort because the devices and materials designed for therapeutic purposes should also possess antimicrobial and/or antifungal properties. Moreover, the researchers are getting more interested in contact-active biocidal materials killing the bacteria via direct interaction with bacterial cell walls rather than releasing biocides.3

Among others, silica-based materials, especially, functionalized silica nanoparticles have attracted the attention of the researchers in terms of their antimicrobial properties.3,4 Such functionalized silica-based nanoparticles are widely used in numerous fields of science, including controlled delivery of organic and inorganic molecules and, what is also important, were shown to improve the antibacterial effect of most therapeutic agents.5 Their advantages include biocompatibility, low cytotoxicity, controllable particle size, and adjustable surface properties.6 There are several instruments for increasing the antibacterial action of nanoparticles. One of them is introducing specific functions onto their surface which would determine their physicochemical properties. According to Mathelié-Guinlet et al.,7 pure silica nanoparticles exhibit a microbicidal effect when their critical diameter is under 50 nm. However, the antibacterial action of functionalized particles, especially positively charged, is less size-dependent and mainly connected with their privileged electrostatic affinity to negatively charged bacterial cell walls. Numerous publications describe antibacterial properties of silica particles functionalized with N-containing groups, polymers,8−10 and antibiotics,11 determined by their positive surface charge. Antimicrobial performance can improve with the increasing number of active sites to interact bacterial cells. For example, Lee et al.8 showed that the state of the surface amino groups is a key factor in increasing kill-efficiency of the materials. In addition, amino groups from silica can be used for the following immobilization of antimicrobial compounds12 and ions, for example, of silver13 and copper.14,15 Regarding the latter, copper is known to possess excellent antimicrobial properties in various forms, being less studied than silver.16 Therefore, copper-containing materials have been recently probed for different applications as antimicrobial agents,16,17 and it was ascertained that incorporating complexes of copper ions is an effective strategy of increasing microbicidal18,19 and antifungal20 action of the materials.

Thus, in our research, we synthesized aminosilica particles with potential application in the treatment of sewage waters, including those contaminated with microorganisms. Therefore, we considered it appropriate to carry out screening analysis of the presence/absence of the antimicrobial effect for mono- and bifunctional aminosilica microspheres, NH2–/SiO2, and NH2–/C6H5–/SiO2, on the cells of strains of Gram-positive bacteria Staphylococcus aureus, Gram-negative bacteria Escherichia coli, and Pseudomonas aeruginosa, as well as eukaryotic microorganisms on the example of single-cell microscopic fungi Candida albicans. Such a choice of test cultures was caused by peculiarities of the chemical composition, structural organization, and cell wall architectonics of different microorganisms, each characterized by specific components. In particular, there are peptidoglycans (PGs), teichoic acids, and lipopolysaccharides in the surface layers of bacteria, while fungi contain mannan and glucan.21 Aminosilica particles were synthesized via a relatively simple one-stage technique, and additional surface phenyl groups were introduced as inherent to many antimicrobial agents capable of nonspecific interactions with bacterial and fungal cell walls.22 Derivatives with adsorbed copper(II) ions were also used.

Experimental Section

Sample Preparation

Reagents

Tetraethyl orthosilicate, Si(OC2H5)4 (TEOS, 98%, Aldrich), 3-aminopropyltriethoxysilane, NH2(CH2)3Si(OC2H5)3 (APTES, 99%, Aldrich), and phenyltriethoxysilane, C6H5Si(OC2H5)3 (PhTES, 98%, Aldrich) were purchased. The reaction was conducted in ethanol, C2H5OH, (96%, LLC Bio-pharma) and catalyzed with NH4OH (25%, Lach-Ner). The source of copper(II) ions was Cu(NO3)2·3H2O (analytical grade, ITES Vranov, Slovakia).

Procedure of Sample Production

The one-pot approaches described in ref (4, 23) were used to synthesize two types of samples: amino–/silica (NH2–/SiO2) and amino–/phenyl–/silica (NH2–/C6H5–/SiO2). Briefly, TEOS (0.018 mol) and APTES (0.006 mol) [or APTES (0.003 mol) with PhTES (0.003 mol)] were mixed with ethanol (100 mL). After opalescence observation (around 30–40 min of constant stirring), ammonia solution (2 mL) was added to the mixture, and the reaction continued for 60 min. The precipitate was separated by centrifugation (15 min, 6000 rpm), washed three times with ethanol to remove nonreacted reagents, and dried at 100 °C for 24 h. The characteristics of the samples are summarized in Table 1.

Table 1. Synthesis Conditions and Properties of Mono- and Bifunctional Silica Nanoparticles.
        concentration of NH2– groups (mmol g–1)
 concentration of C6H5– groups (mmol g–1)
  
sample N, mass (%) C, mass (%) H, mass (%) theor. data elem. anal. data titr. data theor. data elem. anal. data SC to Cu(II)(mmol g–1)
NH2–/SiO2 3.42 8.91 2.97 3.3 2.4 2.0 0 0 1.0
NH2–/C6H5–/SiO2 3.16 15.9 3.27 1.7 2.3 2.3 1.7 1.1 2.2
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To obtain samples with copper(II) ions (Cu(II) + NH2–/SiO2 and Cu(II) + NH2–/C6H5–/SiO2), a batch of each sample (0.025 g) was poured with 25 mL of 0.02 M copper(II) nitrate solution (pH ≈ 4.9, t = 25 °C. The sorption capacity (SC) of the samples was calculated from the difference in the Cu2+ concentration in the solution before and after sorption (Table 1). Before the preparation of test suspensions, the samples were washed with water and dried.

Characterization

The surface morphology and elemental composition of nanoparticles were measured by a field emission scanning electron microscope MIRA 3 (TESCAN, Czech Republic) equipped with a high-resolution cathode (Schottky field emitter), three-lens Wide Field Optics design, and energy-dispersive X-ray detector (EDX) (Oxford Instruments, UK). For analysis, the samples were mounted on a metal stub with adhesive carbon tape, coated with 15 nm of carbon. Conductive carbon coating is necessary to avoid charging artifacts. A method of photon cross-correlation spectroscopy (PCCS) using a Nanophox particle size analyzer (Sympatec, Germany) was employed to determine samples’ particle-size distributions.

The CHNS analysis was carried out by the elementary analyzer Vario MACRO cube (Elementar Analysensysteme GmbH, Germany) with a thermal conductivity detector. Helium and oxygen (both purity 99.995%) were used as the carrier and combustion gases, respectively, with the intake pressures of 2 bars. The combustion tube was set up at 1150 °C and reduction tube at 850 °C. Sulfanilamide C6H8N2O2S (Germany) was used as the CHNS standard. The content of amino groups (Camino, mmol g–1) was calculated from the mass fraction of the element N using the following formula: Camino = 1000ω/100ma = 10ω/ma (ω—content of nitrogen, %; ma—atomic mass of nitrogen, g mol–1). The content of phenyl groups (Cphenyl, mmol g–1) was calculated from the mass fraction of the element C that does not apply to aminopropyl groups: Cphenyl = (10ω/ma – 3Camino)/6 (ω—content of carbon, %; ma—atomic mass of carbon, g mol–1). The content of amino groups was also determined by acid–base titration.

Diffuse reflectance infrared Fourier transform (DRIFT) spectra were recorded for the samples ground with solid KBr (Spectranal, Aldrich) on a Thermo Nicolet Nexus FTIR spectrometer in the 400–4000 cm–1 range.

Zetasizer Nano ZS with DTS1070 cells (Malvern, Great Britain) was used for zeta potential measurements. Each sample (0.05 g) was dispersed into 0.001 M NaNO3 solution (5 mL) by ultrasonication for 10 min (1% suspension). Other concentrations were prepared from 1% suspensions. The results are the average of three independent measurements.

The copper(II) ion concentration in the solutions was measured by atomic absorption spectroscopy (AAS) using a Varian AA 240 FS atomic absorption spectrometer (Australia).

X-ray photoelectron spectroscopy (XPS) measurements were performed on the ESCALAB 2 spectrometer (VG Scientific, UK) with the base pressure inside the analysis chamber maintained at 5 × 10–10 mbar (2 × 10–8 mbar during measurements). The determination was conducted using a twin anode Mg Kα/Al Kα nonmonochromated X-ray source with excitation energies of 1253.6 and 1486.6 eV, respectively. The total instrumental resolution (measured with the FWHM of Ag 3d5/2 photoelectron line) was 1.06 and 1.18 eV for Mg Kα and Al Kα excitation sources, respectively. The data were analyzed by the software package SpecsLab2 CasaXPS (Casa Software Ltd.). To calibrate the energy scale, the C 1s line of hydrocarbons was normalized to 285.0 eV. The spectra processing included subtraction of X-ray satellites and Shirley-type background.24 Peak positions and areas were evaluated using the symmetrical Gaussian–Lorentzian curve fitting. The relative concentrations of different chemical species were determined by normalizing the peak areas to their photoionization cross-sections calculated by Scofield.25

Antibacterial and Antifungal Tests

Test Cultures of Microorganisms

For antibacterial and antifungal tests, we used S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and C. albicans UCM Y-690 grown in tryptone soya yeast extract agar (TSA, Himedia, India) for 24 h at 36 ± 1.0 °C in aerobic conditions. The microorganisms were suspended in 0.15 M NaCl solution to reach the turbidity equivalent of 0.5 McFarland standard units (1.5 × 108 cells mL–1 for S. aureus, E. coli, P. aeruginosa, and 1.5 × 106 cells mL–1 for C. albicans) using a densitometer DEN-1 (Biosan, Latvia). These initial standardized suspensions were diluted with saline solution to the concentration of 1.5 × 105 cells mL–1 for S. aureus, E. coli, and C. albicans and 1.5 × 104 cells mL–1 for P. aeruginosa.

Determination of Antimicrobial and Antifungal Activity

Determination of cell viability of S. aureus, E. coli, P. aeruginosa, and C. albicans after exposure to silica nanoparticles was performed by the method of macrocultivation seeding of samples on a dense TSA in Petri dishes.16,26 In order to reveal the antibacterial activity, 200 μL of the bacterial cell suspension was added to the test tubes, containing 1800 μL of the corresponding concentration of silica nanoparticles (1.0, 0.1, 0.01, or 0.001% v). Consequently, we obtained 1.5 × 104 cells mL–1 concentrations of S. aureus, E. coli, and C. albicans cells, and 1.5 × 103 cells mL–1 concentration of P. aeruginosa.

The contact time of the test culture with nanoparticles was τ = 60 and 120 min at 25 ± 1.0 °C (such temperature was chosen for possible future application of the particles in sewage treatment) and constant mixing with a EKROS 6300M shaker at a frequency of 32 min–1. After the contact, 100 μL of each sample was spread on the TSA of two Petri dishes and incubated for 24 h at 36 ± 1.0 °C in aerobic conditions. In 24 h, the grown CFUs (colony-forming units) were calculated, and relative cell viability [survival index (SI)] was estimated. The percentage of inhibited cells was determined from the difference between the living cells in the tested sample with silica nanoparticles and the control (microbial suspension without nanoparticles prepared and grown under the same conditions, set as 100%).

The number of CFUs was calculated as an average of two Petri dishes used in one experiment (n = 1). Each independent experiment and the control were repeated several times depending on the growth of microorganisms. The averaged numerical data were used to calculate average values, their mean square errors, and probability level (p). Because the obtained values did not correspond to the normal distribution and the selection size was small, we used the Kruskal–Wallis test for comparing groups which is a nonparametric alternative to the t-test for independent samples.27 The number of CFUs (N) was calculated using the formula

graphic file with name ao0c01335_m001.jpg

where R is the dilution from which cultivation was performed; V is the volume (mL) of the inoculum plated in a Petri dish; and M is the average number of grown colonies.28

The CFU (N) values calculated by the formula are hereinafter expressed as lg CFUs mL–1, and they were used to calculate SI (%)

graphic file with name ao0c01335_m002.jpg

where indexes Ne and Nc refer to the experimental and control groups (see exact numbers in Table S1).

Results and Discussion

Structure and Surface Chemistry of Aminosilica Spherical Particles

Amino–/silica and amino–/phenyl–/silica microparticles with and without adsorbed copper(II) ions were chosen for the research. These spherical particles were synthesized via a one-pot procedure in water–ethanolic media using the ammonia catalyst29 to provide similar particle sizes and amino group contents.

Elemental analysis and acid–base titration data confirm a similar amount of amino groups in both samples (Table 1). The divergence in elemental analysis and titration of NH2–/SiO2 may result from the inaccessibility of the groups or their washing off in the acidic medium. As previously reported,4,23 the presence of additional trifunctional silane with an organic group during the sol–gel transformations of TEOS and APTES affects the speed of co-condensation. There are simultaneous reactions of hydrolysis, condensation, and dissolution proceeding in the alkaline medium created by amino groups.30 The introduction of additional nonspecific organic groups displaces the equilibrium of the processes in the direction of condensation and formation of inorganic networks with organic groups. Therefore, despite the smaller quantity of APTES used for NH2–/C6H5–/SiO2 synthesis, both NH2–/SiO2 and NH2–/C6H5–/SiO2 contain identical amounts of amino groups. Also, according to elemental analysis, the groups are not hydrolyzed during the adsorption of copper(II) ions.

The DRIFT spectra of the samples (Figure S1) have all bands characteristic for silica materials with amino groups: two low-intensity absorption bands at 3295 and 3363 cm–1 referring to νs(NH) and νas(NH) stretches in amino groups; absorption band at 1593 cm–1 corresponding to the δ(NH2) vibrations; and the most intensive adsorption band in the region of 1000–1200 cm–1 related to ≡Si–O–Si≡ network stretching and bending vibrations. In the DRIFT spectra of amino–/phenyl–/silica samples, there is also a low-intensity adsorption band at 3060 cm–1 referring to ν(=C–H) vibrations of phenyl groups. The complex formation on the surfaces of the particles, between amino groups and copper(II) ions, is proved in the DRIFT spectra of the copper-containing samples by the disappearance of the absorption bands of stretching vibrations for amino groups in the region 3290–3370 cm–1, the band shift of the bending vibrations for amino groups to 1530 cm–1, and the presence of an absorption band at 1350 cm–1 corresponding to nitrate ions.

It also should be mentioned that the arrangement of amino groups on the surfaces of these samples varies and is favorable for the formation of complexes with copper(II) ions of different compositions, affecting the values of the static SC (Table 1). Thus, one copper(II) ion interacts with two surface amino groups of NH2–/SiO2 and one of NH2–/C6H5–/SiO2. It means that steric difficulties contribute to the simplification of the surface complex between Cu2+ and amino groups. The stability of the complex under the conditions of the experiment was confirmed by stirring the sample Cu(II) + NH2–/C6H5–/SiO2 in physiological saline solution for 2 h with verification of the copper(II) ion concentration by AAS. It was shown that under these conditions, the amount of desorbed copper(II) ions is 0.05 mg for 1% suspension.

The SEM images (Figure 1) of NH2–/SiO2 and NH2–/C6H5–/SiO2 showed spherical particles about 200 nm in size, and such a morphology remains after adsorption. Such particle’s size was confirmed by particle size distribution analysis (Figure S2). EDX spectra of all samples detected C, O, Si, and Cu (after Cu2+ adsorption) and indicate that NH2–/C6H5–/SiO2 uptakes twice more Cu2+ than NH2–/SiO2.

Figure 1.

Figure 1

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SEM images and EDX analysis of the amino–/silica (a) and amino–/phenyl–/silica (b) samples.

According to Mathelié-Guinlet et al.,7 positively charged nanoparticles disrupt bacterial membrane integrity and are more toxic than negatively charged nanoparticles. Therefore, we prepared suspensions with different concentrations of particles (used in the antibacterial tests) and measured their zeta potential and pH values (Table 2). There is a significant difference in the pH values of 1% particles suspensions. They change from alkaline to neutral with Cu2+ adsorption. However, the pH values of all diluted suspensions are about 7. Moreover, contrary to the zeta potential, the pH value changes linearly with the dilution of the suspension (Table 2).

Table 2. Values of pH and Zeta Potential in Function of Suspension Concentration.

    suspensions concentration
samples suspension parameters 1% 0.1% 0.01% 0.001%
NH2–/SiO2 pH 7.74 7.28 7.20 7.11
  ζ-potential (mV) 28.4 35.8 9.17 –5.2
Cu(II) + NH2–/SiO2 pH 6.24 6.76 6.98 7.04
  ζ-potential (mV) 37.1 26.1 0.6 –10.3
NH2–/C6H5–/SiO2 pH 7.91 7.13 7.04 6.93
  ζ-potential (mV) 17.7 25.1 –1.4 –7.2
Cu(II) + NH2–/C6H5–/SiO2 pH 5.74 6.67 6.91 6.96
  ζ-potential (mV) 52.7, 40.3 25.1 –1.2 –8.7
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Electrokinetic potential is a very complicated phenomenon depending on such factors as pH, ionic strength, valency of the ions, concentration, types of interactions on the surface, and so forth. The zeta potential value for the particles with both phenyl and amino groups may be somewhat lower compared to the particles with only amino groups because of the presence of hydrophobic sites and uneven distribution of the surface charge. In addition, a higher degree of amino group protonation (according to XPS data) results in a higher magnitude of positive charge and zeta potential for the monofunctional sample NH2–/SiO2 compared to NH2–/C6H5–/SiO2.

XPS analysis of the samples was conducted to analyze the effect of phenyl groups on the amine ones on the silica surface. The spectra (Figure 2) demonstrated that N 1s can be curve-fitted with two types of nitrogen with expected binding energy (BE): the peaks at lower BE (399.5 eV) is attributed to free amino groups (−NH2) and at higher BE (401.5 eV) is referred to protonated amino groups (NH3+).31,32 According to surface atomic concentrations data (at. %), the peak at 401.5 eV is by 12% smaller for NH2–/C6H5–/SiO2 compared to NH2–/SiO2. This may indicate a partially hydrophobic nature of the NH2–/C6H5–/SiO2 particle surface and exclusion of water molecules by phenyl groups. In addition, a higher degree of amino group protonation results in a higher magnitude of positive charge and zeta potential for a monofunctional sample NH2–/SiO2 compared to NH2–/C6H5–/SiO2.

Figure 2.

Figure 2

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XPS data of N 1s (a) and Cu 2p3/2 (b) for NH2–/SiO2 (1), NH2–/C6H5–/SiO2 (2), Cu(II) + NH2–/SiO2 (3), and Cu(II) + NH2–/C6H5–/SiO2 (4).

The recorded Cu 2p3/2 spectra of Cu(II) + NH2–/SiO2 and Cu(II) + NH2–/C6H5–/SiO2 (Figure 2) were curve-fitted using standard spectra of Cu (0, I, II). The peaks above 942 eV represent the satellite structure formed because of Cu-oxidation, different for Cu II and Cu I, and the intensity of the Cu I satellite is very low in the spectra. Thus, the pronounced peak above 942 eV is just a feature of the whole Cu II peak (main peak + satellite). The curve fitting shows two peaks, at 933.7 and 933.0 eV, matching Cu I and Cu II standards, which can be attributed to Cu(II) and Cu(I) ions, respectively. The Cu(II)/Cu(I) ratios for Cu(II) + NH2–/SiO2 and Cu(II) + NH2–/C6H5–/SiO2 are close to 70/30 and 90/10 (Table S2). Apparently, because of the presence of water and sample heating (45 °C) during the spectra recording, a part of Cu2+ can be restored (Jurado-López31 mentioned photo-reduction of copper during XPS measurements). Also, the percent of the recovered metal is higher for Cu(II) + NH2–/SiO2, containing more water on the surface (Figure 2). It explains lower zeta potential values of Cu(II) + NH2–/SiO2 suspensions compared to Cu(II) + NH2–/C6H5–/SiO2 (Table 2).

These data confirm our assumptions in the article4,23 that the introduction of hydrophobic groups reduces the number of hydrogen bonds of amino and silanol groups, or between amino groups through water molecules,33 which improves the kinetic characteristics of the bifunctional samples during the adsorption of copper(II) ions from water solutions.4

Antibacterial Properties of Amino–/Silica and Amino–/Phenyl–/Silica Particles with and without Adsorbed copper(II) Ions to Gram-Positive Bacteria S. aureus

S. aureus belongs to Gram-positive bacteria that possess a thick layer of PG in the cell wall and no outer membrane. This very thick PG layer protects Gram-positive bacteria against external stresses, and there is a great variation in the composition and structural arrangement of their PGs.34 The PG repeat unit of S. aureus consists of a disaccharide [N-acetyl-glucosamine and N-acetyl-muramic acid (NAM)], pentapeptide-stem (l-Ala-d-Glu-l-Lys-d-Ala-d-Ala, with l-Ala attached to NAM of disaccharide), and a bridge structure. The bridge structure in S. aureus is a pentaglycine attached to the ε nitrogen of l-Lys in the third position of the stem. A crosslink is formed between the N-terminus of the glycine bridge to the d-Ala (4th position) carbonyl carbon of the adjacent stem. In S. aureus, 85 to 90% of the polymerized disaccharides is between 3 and 10 disaccharide units (DU), with an average PG-chain length of only 6 DU.35 By comparison, other bacteria have much longer average chain lengths ranging from 30 to 60 DU in E. coli(36) to over 500 DU in Bacillus subtilis.37 Kim et al.38 conducted solid-state nuclear magnetic resonance analyses of the cell wall of S. aureus showing that PG chains are surprisingly ordered and densely packed because of the short PG chain lengths and high crosslinking degree. The diameter of the PG pores is about 23 Å. Thus, one of the possible mechanisms of antibiotic resistance is size exclusion, when bulky substances are unable to penetrate through the cell wall to reach the cytoplasmic membrane of bacteria.38

Different concentrations of Cu2+-loaded and pure NH2–/SiO2 and NH2–/C6H5–/SiO2 particles were put in contact with the S. aureus cell culture and tested for antimicrobial properties. Here, the mechanism of the antimicrobial effect involves the contact of positively charged particles with negatively charged surfaces of bacterial cells (the zeta potential of S. aureus is about −35.0 mV measured at neutral pH39). The inhibitory effect of NH2–/SiO2 on S. aureus ATCC 25923 cells (within 60 min of contact) was shown to strengthen with the increasing concentration and contact time. Thus, higher concentrations of NH2–/SiO2 (0.1 and 1%) caused a statistically significant decrease in SI to 47.4 and 42.4%, respectively, compared to the control (p = 0.038). A bactericidal effect was less significant at 1–2 order lower concentrations (Figure 3). The prolongation of the contact time of S. aureus cells with monofunctional microspheres to 120 min showed statistically significant (p = 0.029) increase in the antibacterial effect by 1.1–1.3 times as compared with the control (Figure 3). It may be explained by the key role of the particle’s positive surface charge (zeta potentials +35.8 and +28.4 mV of 1.0 and 0.1% suspensions) in establishing contact with negatively charged bacterial cells (−35 mV) and suppressing their activity. While in the beginning, this charge may be enough, but with the increasing contact time, it may be insufficient to affect new bacterial cells.

Figure 3.

Figure 3

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Dependence of the SI for S. aureus ATCC 25923 cells on the concentration of the silica particle suspensions and contact time (60 or 120 min) for amino–/silica (a) and amino–/phenyl–/silica (b) samples (blue − control, red − 60 min, green − 120 min).

Compared to monofunctional NH2–/SiO2, bifunctional NH2–/C6H5–/SiO2 microspheres with additional surface phenyl groups revealed the opposite dose dependence of the depressant effect on the concentration at the same exposure time (60 min) (Figure 3). That is, lower concentrations of NH2–/C6H5–/SiO2 showed a stronger S. aureus antibacterial effect. Thus, SI of S. aureus after contact with 1.0% NH2–/C6H5–/SiO2 was 84.7% and declined (approximately 0.8-fold) with a decrease in concentration showing values of 68.6, 43.7, and 34.3% (Figure 3). Such behavior may be explained by the dependence of antibacterial performance not so much on the particle’s zeta potential but on the protonation of amino groups in aqueous environment, observed by Lee at al.8 by testing silica particles coated with various amine functional polymers. The pH value of 0.001% water suspension of NH2–/C6H5–/SiO2 particles is 6.93, which is more favorable for the protonation of the surface amino groups than pH 7.91 created by 1% water suspension. The two times extension of the contact time of staphylococcal cells with NH2–/C6H5–/SiO2 (to 120 min) was accompanied by increasing the bactericidal effect. In particular, the percentage of surviving S. aureus cells decreased 1.8 times at a concentration of 1.0% (SI = 47.7%), 1.6 times at a concentration of 0.1% (SI = 42.7%), and 1.2 times at the concentrations of 0.01 and 0.001% (SI = 36.1 and 27.9%, respectively). Instead, monofunctional NH2–/SiO2 microspheres did not exert a similar effect. Thus, confirming previous observations,4 the introduction of hydrophobic groups in the surface layer of silica particles may increase the number of contact sites between the bacterial cells and the surface of the particles, slowing down the bacterial reproduction.40

The inclusion of copper ions into the nanoparticles is one of the most promising strategies for increasing the antibacterial activity of the latter. According to Tsao et al.41 and Azam et al.,42 ions of copper can induce changes in the lipid phase of the cytoplasmic membrane of microorganism cells. In addition, copper preparations introduced into the body of mammals in the form of nanoparticles are characterized by prolonged action, lower toxicity compared to salts, the ability to stimulate the microelement regulation mechanisms, and activity of antioxidant enzymes. Because of such characteristics, nanomaterials with introduced copper ions are now considered as promising candidates for creating alternative highly effective antimicrobials. Therefore, we also investigated the activity of silica microspheres with adsorbed Cu2+, Cu(II) + NH2–/SiO2, and Cu(II) + NH2–/C6H5–/SiO2 regarding the species of Gram-positive and Gram-negative bacteria.

It was determined that introduction of copper(II) ions into microspheres has significantly improved their activity against the cells of Gram-positive bacteria (Figure 3). So, after the 60 min contact of Cu(II) + NH2–/SiO2 with S. aureus ATCC 25923 strains, a statistically significant increase in their antibacterial activity was observed compared to NH2–/SiO2 at all tested concentrations (p ≤ 0.05). The number of viable cells decreased with increasing Cu(II) + NH2–/SiO2 suspension concentrations: SI values decreased from 54.2 to 40.5 and 32.4% for 0.001, 0.01, and 0.1% suspensions, respectively. Also, they reached the lowest value (SI = 28.3%) at the concentration of 1.0%. Under the conditions of prolongation of the contact of Cu(II) + NH2–/SiO2 with staphylococcal cells up to 120 min, the antibacterial activity reached a maximum (Figure 3).

Bactericidal activity of Cu(II) + NH2–/C6H5–/SiO2-type microspheres was more effective than those of Cu(II) + NH2–/SiO2. The 60 min contact of staphylococcal cells with Cu(II) + NH2–/C6H5–/SiO2 resulted in a decrease in the number of viable cells (Figure 3) following the dose-dependent pattern. Thus, the SI of staphylococcal cells were 57.0 and 16.0% at particles concentrations of 0.001 and 0.01%, decreasing to 2.7 and 2.2% with an increasing concentration to 0.1 and 1.0%, respectively. Such antibacterial activity can be explained by the influence of several factors: higher the content of copper(II) ions and zeta potential values, as well as the presence of centers for nonspecific interaction between bacteria and particles, mentioned above for NH2–/C6H5–/SiO2. Besides, the inhibited viability of staphylococcus cells in the presence of copper-containing microspheres may result not only from direct influence of Cu2+ but also from the change in the suspension pH (Table 2) because the acidification of the environment (optimal pH is 7.2–7.4) also adversely affects the bacteria growth and reproduction. In addition, according to XPS, materials with adsorbed copper contain both Cu+ and Cu2+, which could induce a Fenton-like reaction and enhance the degradation of the microbial cell membrane. Copper antibacterial activity could be attributed to its potential as a catalyst for oxidative damage to tissues through redox transformations between Cu(I) and Cu(II) with the participation of oxygen.43

Antibacterial Investigation of the Effect of Amino–/Silica Particles on the SI of Gram-Negative Bacteria E. coli and P. aeruginosa

Unlike Gram-positive, Gram-negative bacteria along with the cytoplasmic membrane and PG layer possess an outer membrane (consisting of the phospholipid bilayer that contains lipopolysaccharide molecules and proteins).44 We chose for our study the most well-known species of Gram-negative bacteria E. coli(45) and P. aeruginosa(46) because they are characterized by natural resistance to antibacterial agents, disinfectants, and other xenobiotics. The reason for this resistance is the low permeability of the outer cell membrane. Investigation of the effect of NH2–/SiO2 and NH2–/C6H5–/SiO2 on the cells of representatives of different species of Gram-negative bacteria after 60 min contact made it possible to conclude that compared to staphylococci, NH2–/SiO2 microspheres were less effective against Gram-negative bacteria (in particular, E. coli ATCC 25922), although the inhibitory activity remained dose-dependent (Figure 4). The SI of E. coli cells ranged from 60.9 to 84.5% depending on the concentration (p ≤ 0.05). Under similar conditions of 60 min contact, the most effective action of NH2–/C6H5–/SiO2 particles against E. coli cells was in the concentration of 0.01%. However, there was no statistically significant difference between the effects of different concentrations (p = 0.3). Mathelié-Guinlet et al.7 analyzed the influence of the surface charge and size of silica particles on their antibacterial effect against E. coli and reported different mechanisms of action. For pure SiO2 particles (with negative surface charge), the size is very important. The particles of 50 nm < d < 80 nm were shown to interact through hydrogen bonding with hydroxyl groups of the phospholipid bilayer and thus breaking down the cell clusters. Whereas APTES-modified silica particles (with positive zeta potential) having strong electrostatic affinity to negatively charged E. coli cells stick to them inducing drastic damages, such as extra-membrane aggregates and membrane invaginations.

Figure 4.

Figure 4

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Dependence of the SI for E. coli ATCC 25922 cells on the concentration of the silica particle suspensions for amino–/silica (a) and amino–/phenyl–/silica (b) samples (yellow − control, red − 60 min, green − 120 min).

The prolongation of the contact time to 120 min had no visual effect on the antibacterial activity of NH2–/SiO2 particles toward E. coli cells, whereas NH2–/C6H5–/SiO2 microspheres showed increase in the bactericidal effect (Figure 4). P. aeruginosa cells contrary to E. coli produce a number of enzymes and are highly resistant to antibiotics.47 Hence, contrary to E. coli, neither monofunctional (NH2–/SiO2) nor bifunctional (NH2–/C6H5–/SiO2) microspheres had an inhibitory effect on the cells of the P. aeruginosa strain ATCC 27853 during the exposure for 60 min. The absence of the antibacterial effect against P. aeruginosa ATCC 27853 can be primarily explained by their natural resistance to antimicrobials46 because of the low-permeability of the outer membrane in addition to secondary resistance mechanisms, among which systems of active pump-out of antimicrobial agents and xenobiotics play an important role.47

The suppressing effect of NH2–/C6H5–/SiO2 bifunctional microspheres on the cells of P. aeruginosa strains was indicative (Figure 5). Thus, while during the contact for 60 min, no microbicidal effect was observed, and the extension of the exposure to 120 min allowed registering the inhibition of vitality of P. aeruginosa cells. In particular, after the contact with NH2–/C6H5–/SiO2 particle concentration of 0.001% for 120 min, the number of surviving cells, compared with the control, decreased by half, while for concentrations of 1.0 and 0.1%, it was varying within the range of 28.3–39%. Hence, the prolongation of the contact time of NH2–/C6H5–/SiO2 with P. aeruginosa resulted in the improvement in their inhibitory activity against Gram-negative bacteria, which was observed for Gram-positive bacteria. It also should be mentioned that such necessity for a longer contact time for NH2–/C6H5–/SiO2 to reveal antibacterial properties may be partially connected with the introduction of phenyl groups. According to Song et al.40 the BE of bacteria with hydrophobic surfaces is weaker than with hydrophilic. So, on the one side the introduction of phenyl groups increases the portion of free amino groups (contact sites), and they could interact themselves with lipid components of the bacterial membrane, but it also increases the hydrophobicity of the sample. As a result, its antibacterial activity is revealed only during 2 h contact.

Figure 5.

Figure 5

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Dependence of the SI for P. aeruginosa ATCC 27853 cells on the concentration of the bifunctional silica particle suspensions (purple − control, red − 60 min, green − 120 min).

The introduction of Cu2+ in the composition of microspheres, especially bifunctional, allowed increasing their efficiency against the P. aeruginosa cells. Generally, the antibacterial efficiency of Cu2+ loaded samples against Gram-negative bacteria appeared lower compared to that against Gram-positive bacteria, although at Cu(II) + NH2–/C6H5–/SiO2 concentrations of 0.1 and 1% and a contact time of 120 min antibacterial activity reached 99 and 100%. Similarly, authors48 witnessed a bigger diameter of zone of growth inhibition of S. aureus when studying the antibacterial activity of copper-doped wollastonite particles against S. aureus (Gram-positive) and E. coli (Gram-negative) bacterial strains. Theivasanthi and Alagar49 also showed higher antibacterial activity (18–45% more) of copper nanoparticles against Gram-positive cells of S. albus, S. epidermidis, and S. aureus compared to Gram-negative cells of E. coli. Thus, after the contact of Cu(II) + NH2–/SiO2 type microspheres with E. coli cells for 60 min, similarly to Gram-positive bacteria, a dose-dependent increase in the suppressive effect was observed (Figure 4). When the concentration of Cu(II) + NH2–/SiO2 changed from 1.0 to 0.1%, the SI of the E. coli cells increased by 21 times. Lowering the particle concentration from 0.1 to 0.01% increased SI of E. coli 2.5 times and from 0.01 to 0.001%—1.3 times.

Similarly to the Cu(II) + NH2–/SiO2 microspheres, the antibacterial activity of Cu(II) + NH2–/C6H5–/SiO2 was found to be significant against the tested Gram-negative cultures and depended on the concentration. Against the cells of the strain E. coli ATCC 25922, the most effective of all analyzed Cu(II) + NH2–/C6H5–/SiO2 concentrations were 1.0 and 0.001%, for which practically no viable cells remained (SI = 0.5 and 0.3%) (Figure 4). Meanwhile, at a concentration of 0.1%, SI was 14.1%, whereas 0.01% had no inhibitory effect on E. coli. The latter concentration (0.01%) of Cu(II) + NH2–/C6H5–/SiO2 microspheres also turned out to be ineffective against P. aeruginosa cells ATCC 27853 (SI = 70.5%), whereas other investigated concentrations (1.0, 0.1, and 0.001%), significantly decreased the number of viable cells (SI = 3.2, 12.1, and 37.9%) at a contact time of 60 min.

The prolongation of the influence of Cu(II) + NH2–/SiO2 to 120 min almost completely inhibited the viability of E. coli cells, except for the 0.01% concentration, which appeared ineffective under these conditions. Significant suppression of the viability of E. coli cells was also observed after longer exposure to Cu(II) + NH2–/C6H5–/SiO2 (Figure 4). The latter sample was also effective against P. aeruginosa cells (SI = 1.0 and 11.0% at concentrations of 0.1 and 0.01%, respectively). However, similar to E. coli cells, the sensitivity of Pseudomonas cells to the lowest concentration was also negligible (SI = 88.7%).

Thus, as mentioned by other authors,40 we observe the synergistic effect of phenyl groups and antibacterial properties of silica particles having positive surface charge, hydrophobic groups, and copper(II) ions. These factors altogether lead to the damage of the bacterial cell membranes, violation of their surface charge, disorder of adhesive properties, and subsequent death.

Antifungal Activity of the Samples Against C. albicans

Candida species are among the major causes of healthcare-associated infections because they can survive on abiotic surfaces in hospitals.20 Copper is recognized for its antifungal activity and was shown to possess fungicidal activity against Candida.20 Therefore, we tested our copper-loaded and copper-free samples against C. albicans. Generally, fungicidal activity of copper is somewhat lower compared to its bactericidal activity because of the diverse chemical composition and structural organization of the yeast cell membranes.20

Among copper-free samples, only two highest used concentrations of amino–/silica (0.1 and 1.0%) showed an inhibitory effect against C. albicans after 60 min contact, SI = 65.8, and 34.5%, respectively (p ≤ 0.01) (Figure 6). Meanwhile, bifunctional microspheres NH2–/C6H5–/SiO2 revealed no fungicidal action against C. albicans after 60 min contact, similar to Gram-negative cells of P. aeruginosa.

Figure 6.

Figure 6

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Dependence of the SI for C. albicans UCM Y-690 cells on the concentration of the silica particle suspensions for amino–/silica (a) and amino–/phenyl–/silica (b) samples (grey − control, red − 60 min, green − 120 min).

Extending the exposure twice allowed increasing the fungicidal action of both monofunctional (NH2–/SiO2) and bifunctional (NH2–/C6H5–/SiO2) microspheres. Similar to the short-term contact, higher concentrations of NH2–/SiO2 were the most effective. Thus, at NH2–/SiO2 concentrations of 1.0 and 0.1%, the cell SI were 5.1 and 11.1%, respectively (p ≤ 0.026). No statistically significant difference was found in the case of low NH2–/SiO2 suspension concentrations. As for NH2–/C6H5–/SiO2, they exhibited a fungicidal effect solely in the case of prolongation of the contact (Figure 6). Therefore, in order to ensure the fungicidal action of the microspheres in question, there is a need for longer contact with the eukaryotic yeast cells.

The introduction of Cu2+ did not increase significantly the fungicidal activity of neither monofunctional (NH2–/SiO2) nor bifunctional (NH2–/C6H5–/SiO2) microspheres. For both types of microspheres, the statistically significant fungicidal effect, compared to control (p = 0.032), was observed only at a particle concentration of 1% after 120 min contact (the SI were 13.8 and 15.5%, respectively).

Conclusions

Two types of samples: monofunctional amino–/silica and bifunctional amino–/phenyl–/silica were synthesized and tested as antimicrobial agents. There were observed obvious advantages of the bifunctional sample already on the stage of characterization. Introduction of the hydrophobic component (phenyl groups) added to the hydrolytic stability of the functional layer. Phenyl groups prevented amino groups from back-bonding, making them available for adsorption; thus, the number of free sites able to interact with bacterial cells increased. Dilution of amino groups with phenyl also promoted simplification of the surface complex with copper(II) ions thus enhancing Cu2+ uptake. These advantages, consequently, resulted in improved antimicrobial performance of the bifunctional sample. Thus, the influence of microspheres on the test cultures of microorganisms and the effectiveness of the antimicrobial effect depend on the type of microspheres, their concentration, and the contact time with the cells of microorganisms, as well as taxonomic affiliation of the latter.

Monofunctional silica microspheres with amino groups showed the highest antimicrobial activity in the concentration of 1.0%. The SIs of the bacterial cells after 60 min contact with the particles were as follows: S. aureus—42.4%, E. coli—60.9%, and C. albicans—34.5%. The antibacterial effect of bifunctional silica microspheres with both amino and phenyl groups after 60 min of contact was witnessed only against Gram-positive bacteria. The SI of S. aureus after exposure to the lowest of tested concentrations, 0.001%, was 34.3%. Bifunctional silica microspheres (0.1% suspension) demonstrated antimicrobial action against Gram-negative test cultures and a fungicidal effect on microscopic yeast cells only after prolongation of the exposition term to 120 min. The SI of the test strains were: 1.8%—E. coli, 39.0%—P. aeruginosa and 43.7%—C. albicans. The introduction of Cu(II) ions into mono- and bifunctional silica microspheres has dramatically improved their antibacterial and fungicidal activity, which may be due to Fenton-like copper redox cycling resulting in membrane cell oxidation. Owing to the introduction of copper ions, the SI after contact with bifunctional microspheres for 120 min decreased by 36.7 times for S. aureus, 5000 times for E. coli, 2830 times for P. aeruginosa, and 2.9 times for the C. albicans strain. Thus, the synthesized materials with copper complexes on the surface can be highly effective disinfectants in sewage treatment.

Acknowledgments

The research was funded from the Marie Curie Programme H2020-MSCA-RISE-2016-NANOMED project no. 734641 and the project VEGA 2/0156/19.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01335.

  • Data on the resistance of cells of test cultures after contact with the adsorbed copper ions on the microsphere surface; XPS data of C 1s, O 1s, Si 2p, N 1s, and Cu 2p3/2 surface atomic concentrations and binding energies; DRIFT spectra for NH2–/SiO2, Cu(II) + NH2–/SiO2, NH2–/C6H5–/SiO2, and Cu(II) + NH2–/C6H5–/SiO2; and PCCS particle-size distributions of NH2–/SiO2 and NH2–/C6H5–/SiO2 (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c01335_si_001.pdf (752.4KB, pdf)

References

  1. Abd-El-Aziz A. S.; Agatemor C.; Etkin N. Antimicrobial resistance challenged with metal-based antimicrobial macromolecules. Biomaterials 2017, 118, 27–50. 10.1016/j.biomaterials.2016.12.002. [DOI] [PubMed] [Google Scholar]
  2. Regiel-Futyra A.; Dąbrowski J. M.; Mazuryk O.; Śpiewak K.; Kyzioł A.; Pucelik B.; Brindell M.; Stochel G. Bioinorganic antimicrobial strategies in the resistance era. Coord. Chem. Rev. 2017, 351, 76–117. 10.1016/j.ccr.2017.05.005. [DOI] [Google Scholar]
  3. Braun K.; Pochert A.; Lindén M.; Davoudi M.; Schmidtchen A.; Nordström R.; Malmsten M. Membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides. J. Colloid Interface Sci. 2016, 475, 161–170. 10.1016/j.jcis.2016.05.002. [DOI] [PubMed] [Google Scholar]
  4. Kotsyuda S. S.; Tomina V. V.; Zub Y. L.; Furtat I. M.; Lebed A. P.; Vaclavikova M.; Melnyk I. V. Bifunctional silica nanospheres with 3-aminopropyl and phenyl groups. Synthesis approach and prospects of their applications. Appl. Surf. Sci. 2017, 420, 782–791. 10.1016/j.apsusc.2017.05.150. [DOI] [Google Scholar]
  5. Grumezescu V.; Andronescu E.; Holban A. M.; Socol G.; Grumezescu A. M.; Ficai A.; Lazar V.; Chifiriuc M. C.; Trusca R.; Iordache F. Fabrication and characterization of functionalized surfaces with 3-aminopropyltrimethoxysilane films for anti-infective therapy applications. Appl. Surf. Sci. 2015, 336, 401–406. 10.1016/j.apsusc.2015.01.080. [DOI] [Google Scholar]
  6. Song J.; Kong H.; Jang J. Enhanced antibacterial performance of cationic polymer modified silica nanoparticles. Chem. Commun. 2009, 36, 5418–5420. 10.1039/b908060k. [DOI] [PubMed] [Google Scholar]
  7. Mathelié-Guinlet M.; Béven L.; Moroté F.; Moynet D.; Grauby-Heywang C.; Gammoudi I.; Delville M.-H.; Cohen-Bouhacina T. Probing the threshold of membrane damage and cytotoxicity effects induced by silica nanoparticles in Escherichia coli bacteria. Adv. Colloid Interface Sci. 2017, 245, 81–91. 10.1016/j.cis.2017.04.012. [DOI] [PubMed] [Google Scholar]
  8. Lee I.; Roh J.; Lee J.; Song J.; Jang J. Antibacterial performance of various amine functional polymers coated silica nanoparticles. Polymer 2016, 83, 223–229. 10.1016/j.polymer.2015.12.032. [DOI] [Google Scholar]
  9. Xu J.; Zhang Y.; Zhao Y.; Zou X. Antibacterial activity of N-halamine decorated mesoporous silica nanoparticles. J. Phys. Chem. Solid. 2017, 108, 21–24. 10.1016/j.jpcs.2017.04.008. [DOI] [Google Scholar]
  10. Song J.; Jung Y.; Lee I.; Jang J. Fabrication of pDMAEMA-coated silica nanoparticles and their enhanced antibacterial activity. J. Colloid Interface Sci. 2013, 407, 205–209. 10.1016/j.jcis.2013.06.016. [DOI] [PubMed] [Google Scholar]
  11. Gounani Z.; Asadollahi M. A.; Meyer R. L.; Arpanaei A. Loading of polymyxin B onto anionic mesoporous silica nanoparticles retains antibacterial activity and enhances biocompatibility. Int. J. Pharm. 2018, 537, 148–161. 10.1016/j.ijpharm.2017.12.039. [DOI] [PubMed] [Google Scholar]
  12. Hao N.; Jayawardana K. W.; Chen X.; Yan M. One-step synthesis of amine-functionalized hollow mesoporous silica nanoparticles as efficient antibacterial and anticancer materials. ACS Appl. Mater. Interfaces 2015, 7, 1040–1045. 10.1021/am508219g. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Quang D. V.; Sarawade P. B.; Hilonga A.; Kim J.-K.; Chai Y. G.; Kim S. H.; Ryu J.-Y.; Kim H. T. Preparation of amino functionalized silica micro beads by dry method for supporting silver nanoparticles with antibacterial properties. Colloids Surf., A 2011, 389, 118–126. 10.1016/j.colsurfa.2011.08.042. [DOI] [Google Scholar]
  14. Naz A.; Arun S.; Narvi S. S.; Alam M. S.; Singh A.; Bhartiya P.; Dutta P. K. Cu(II)-carboxymethyl chitosan-silane schiff base complex grafted on nano silica: Structural evolution, antibacterial performance and dye degradation ability. Int. J. Biol. Macromol. 2018, 110, 215–226. 10.1016/j.ijbiomac.2017.11.112. [DOI] [PubMed] [Google Scholar]
  15. Tahmasbi L.; Sedaghat T.; Motamedi H.; Kooti M. Mesoporous silica nanoparticles supported copper(II) and nickel(II) Schiff base complexes: Synthesis, characterization, antibacterial activity and enzyme immobilization. J. Solid State Chem. 2018, 258, 517–525. 10.1016/j.jssc.2017.11.015. [DOI] [Google Scholar]
  16. Sportelli M. C.; Picca R. A.; Cioffi N.. Nano-antimicrobials based on metals. In Novel Antimicrobial Agents and Strategies; Phoenix D. A., Harris F., Dennison S. R., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA, 2014; Vol. 8, pp 181–218. [Google Scholar]
  17. Vincent M.; Hartemann P.; Engels-Deutsch M. Antimicrobial applications of copper. Int. J. Hyg Environ. Health 2016, 219, 585–591. 10.1016/j.ijheh.2016.06.003. [DOI] [PubMed] [Google Scholar]
  18. Aktan E.; Gündüzalp A. B.; Özmen Ü. Ö. Structural, physicochemical characterization, theoretical studies of carboxamides and their Cu(II), Zn(II) complexes having antibacterial activities against E. Coli. J. Mol. Struct. 2017, 1128, 775–784. 10.1016/j.molstruc.2016.09.013. [DOI] [Google Scholar]
  19. Gritsch L.; Lovell C.; Goldmann W. H.; Boccaccini A. R. Fabrication and characterization of copper(II)-chitosan complexes as antibiotic-free antibacterial biomaterial. Carbohydr. Polym. 2018, 179, 370–378. 10.1016/j.carbpol.2017.09.095. [DOI] [PubMed] [Google Scholar]
  20. Ballo M. K. S.; Rtimi S.; Kiwi J.; Pulgarin C.; Entenza J. M.; Bizzini A. Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata. J. Photochem. Photobiol., B 2017, 174, 229–234. 10.1016/j.jphotobiol.2017.07.030. [DOI] [PubMed] [Google Scholar]
  21. Costerton J. W.; Ingram J. M.; Cheng K. J. Structure and function of the cell envelope of Gram-negative bacteria. Bacteriol. Rev. 1974, 38, 87–110. 10.1128/mmbr.38.1.87-110.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Armentano I.; Arciola C. R.; Fortunati E.; Ferrari D.; Mattioli S.; Amoroso C. F.; Rizzo J.; Kenny J. M.; Imbriani M.; Visai L. The interaction of bacteria with engineered nanostructured polymeric materials: a review. Sci. World J. 2014, 2014, 410423. 10.1155/2014/410423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tomina V. V.; Melnyk I. V.; Zub Y. L.; Kareiva A.; Vaclavikova M.; Seisenbaeva G. A.; Kessler V. G. Tailoring bifunctional hybrid organic–inorganic nanoadsorbents by the choice of functional layer composition probed by adsorption of Cu2+ ions. Beilstein J. Nanotechnol. 2017, 8, 334–347. 10.3762/bjnano.8.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shirley D. A. High-resolution X-ray photoemission spectrum of the valence bands of gold. Phys. Rev. B 1972, 5, 4709–4714. 10.1103/physrevb.5.4709. [DOI] [Google Scholar]
  25. Scofield J. H. Hartree-Slater subshell photoionization cross-sections at 1254 and 1487 eV. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129–137. 10.1016/0368-2048(76)80015-1. [DOI] [Google Scholar]
  26. NSAI Standards . Chemical Disinfectants and Antiseptics: Quantitative Suspension Test for the Evaluation of Fungicidal Activity for Instruments Used in Medical Area: Test Method and Requirements (Phase 2, Step 1); European Committee for Standardization: Brussels, 2003, EN 13624.
  27. Glantz S. A.Primer of Biostatistics, 7th ed., ISBN-13:978-0071781503; McGraw-Hill Education/Medical, 2011; p 320. [Google Scholar]
  28. Sutton S. Accuracy of plate counts. J. Validation Technol. 2011, 17, 42–46. [Google Scholar]
  29. Melnyk I. Aminosilica nano- and submicrospheres: analysis of factors influencing morphology, structure and properties. Chem. J. Mold. 2014, 9, 123–127. 10.19261/cjm.2014.09(1).16. [DOI] [Google Scholar]
  30. Brinker C. J. Hydrolysis and condensation of silicates: effects on structure. J. Non-Cryst. Solids 1988, 100, 31–50. 10.1016/0022-3093(88)90005-1. [DOI] [Google Scholar]
  31. Jurado-López B.; Vieira R. S.; Rabelo R. B.; Beppu M. M.; Casado J.; Rodríguez-Castellón E. Formation of complexes between functionalized chitosan membranes and copper: A study by angle resolved XPS. Mater. Chem. Phys. 2017, 185, 152–161. 10.1016/j.matchemphys.2016.10.018. [DOI] [Google Scholar]
  32. Vieira R. S.; Oliveira M. L. M.; Guibal E.; Rodríguez-Castellón E.; Beppu M. M. Copper, mercury and chromium adsorption on natural and crosslinked chitosan films: An XPS investigation of mechanism. Colloids Surf., A 2011, 374, 108–114. 10.1016/j.colsurfa.2010.11.022. [DOI] [Google Scholar]
  33. Zub Y. L.; Melnyk I. V.; White M. G.; Alonso B. Structural features of surface layers of bifunctional polysiloxane xerogels containing 3-aminopropyl groups and 3-mercaptopropyl groups. Ads. Sci. Technol. 2008, 26, 119–133. 10.1260/026361708786035440. [DOI] [Google Scholar]
  34. Schleifer K. H.; Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 1972, 36, 407–477. 10.1128/mmbr.36.4.407-477.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Boneca I. G.; Huang Z.-H.; Gage D. A.; Tomasz A. Characterization of Staphylococcus aureus cell wall glycan strands, evidence for a new β-N-acetylglucosaminidase activity. J. Biol. Chem. 2000, 275, 9910–9918. 10.1074/jbc.275.14.9910. [DOI] [PubMed] [Google Scholar]
  36. Glauner B.; Höltje J. V. Growth pattern of the murein sacculus of Escherichia coli. J. Biol. Chem. 1990, 265, 18988–18996. [PubMed] [Google Scholar]
  37. Hayhurst E. J.; Kailas L.; Hobbs J. K.; Foster S. J. Cell wall peptidoglycan architecture in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14603–14608. 10.1073/pnas.0804138105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kim S. J.; Chang J.; Singh M. Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR. Biochim. Biophys. Acta, Biomembr. 2015, 1848, 350–362. 10.1016/j.bbamem.2014.05.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kłodzińska E.; Szumski M.; Dziubakiewicz E.; Hrynkiewicz K.; Skwarek E.; Janusz W.; Buszewski B. Effect of zeta potential value on bacterial behavior during electrophoretic separation. Electrophoresis 2010, 31, 1590–1596. 10.1002/elps.200900559. [DOI] [PubMed] [Google Scholar]
  40. Song J.; Kong H.; Jang J. Bacterial adhesion inhibition of the quaternary ammonium functionalized silica nanoparticles. Colloids Surf., B 2011, 82, 651–656. 10.1016/j.colsurfb.2010.10.027. [DOI] [PubMed] [Google Scholar]
  41. Tsao N.; Luh T.-Y.; Chou C.-K.; Chang T.-Y.; Wu J.-J.; Liu C.-C.; Lei H.-Y. In vitro action of carboxyfullerene. J. Antimicrob. Chemother. 2002, 49, 641–649. 10.1093/jac/49.4.641. [DOI] [PubMed] [Google Scholar]
  42. Azam A.; Ahmed A. S.; Oves M.; Khan M.; Memic A. Size-dependent antimicrobial properties of CuO nanoparticles against Gram-positive and -negative bacterial strains. Int. J. Nanomed. 2012, 7, 3527–3535. 10.2147/ijn.s29020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pham A. N.; Xing G.; Miller C. J.; Waite T. D. Fenton-like copper redox chemistry revisited: Hydrogen peroxide and superoxide mediation of copper-catalyzed oxidant production. J. Catal. 2013, 301, 54–64. 10.1016/j.jcat.2013.01.025. [DOI] [Google Scholar]
  44. Miller S. I.; Salama N. R. The gram-negative bacterial periplasm: Size matters. PLoS Biol. 2018, 16, e2004935 10.1371/journal.pbio.2004935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Jang J.; Hur H.-G.; Sadowsky M. J.; Byappanahalli M. N.; Yan T.; Ishii S. Environmental Escherichia coli: ecology and public health implications-a review. J. Appl. Microbiol. 2017, 123, 570–581. 10.1111/jam.13468. [DOI] [PubMed] [Google Scholar]
  46. Chatterjee M.; Anju C. P.; Biswas L.; Anil Kumar V.; Gopi Mohan C.; Biswas R. Antibiotic resistance in Pseudomonas aeruginosa and alternative therapeutic options. Int. J. Med. Microbiol. 2016, 306, 48–58. 10.1016/j.ijmm.2015.11.004. [DOI] [PubMed] [Google Scholar]
  47. Schweizer H. P. Efflux as a mechanism of resistance to antimicrobials in Pseudomonas aeruginosa and related bacteria: unanswered questions. Genet. Mol. Res. 2003, 2, 48–62. [PubMed] [Google Scholar]
  48. Azeena S.; Subhapradha N.; Selvamurugan N.; Narayan S.; Srinivasan N.; Murugesan R.; Chung T. W.; Moorthi A. Antibacterial activity of agricultural waste derived wollastonite doped with copper for bone tissue engineering. Mater. Sci. Eng., C 2017, 71, 1156–1165. 10.1016/j.msec.2016.11.118. [DOI] [PubMed] [Google Scholar]
  49. Theivasanthi T.; Alagar M. Studies of Copper Nanoparticles Effects on Micro-organisms. Ann. Biol. Res. 2011, 2, 368–373. [Google Scholar]

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