Нанокомпозиты на основе оксида графена, украшенные наночастицами серебра в качестве антибактериального агента

Результаты

Характеристики ГО и Ag-НП

Химический анализ показал присутствие азота, углерода, серы, водорода и кислорода (таблица 1).

Фазовый анализ образца GO (рис. 1) выявил присутствие примесей из следовых количеств графита, нитрата натрия и, вероятно, восстановленной формы оксида графена.

Рентгенограммы порошков GO. Фазовый анализ образца GO выявил наличие примесей из следовых количеств графита, нитрата натрия и, вероятно, восстановленной формы оксида графена

Рамановская спектроскопия может дать информацию о структурных особенностях оксида графена. Полоса D связана со структурным беспорядком, а полоса G связана с растяжением связей углерода sp ² атомы [42]. Дополнительные полосы (включая D ', 2D и D + G) возникают из-за дефектов, присутствующих в графитовой структуре углеродного материала. Я _D / Я _G соотношение (рассчитанное по интенсивности полос D и G) может быть использовано для характеристики беспорядка графитовой структуры в углеродных материалах. Как видно на рис. 2, GO имеет сильно разупорядоченную структуру из-за множества функциональных групп в структуре, образовавшихся в процессе окисления графитового порошка. Положение полосы D составляет 1351 см ⁻¹ и полоса G 1590 см ⁻¹ ; Я _D / Я _G коэффициент 1,15.

Рамановский спектр оксида графена с предполагаемой деконволюцией полос D, G, D ', 2D и D + G. GO имеет сильно разупорядоченную структуру из-за множества функциональных групп в структуре, образовавшихся в процессе окисления графитового порошка. Положение полосы D составляет 1351 см ⁻¹ и полоса G 1590 см ⁻¹ ; Я _D / Я _G коэффициент 1,15

ИК-Фурье-спектр оксида графена, полученный в режиме НПВО, показал, что GO имеет много функциональных групп, присутствующих в структуре. Наиболее заметный пик наблюдается на ~ 3500 см ⁻¹ . , который относится в основном к воде и гидроксильным группам (рис. 3). Очень интенсивный пик около 1080 см ⁻¹ также можно отнести к гидроксильным группам. Пик около 1600 см ⁻¹ обычно приписывается связям C =C, присутствующим в графитовом углероде. Однако наши предыдущие исследования XPS показывают, что в оксиде графена есть несколько связей C =C [43]; следовательно, мы приписываем этот пик в основном воде, все еще присутствующей в оксиде графена. Другие пики, наблюдаемые в спектре FT-IR, показывают, что GO богат группами, содержащими связи C =O (в основном карбоксильные группы), пик около 1720 см ^-1 , эпоксидная смола (C – O – C) с видимым пиком около 1200 см ⁻¹ , и связи C – H (пик около 2800 см ⁻¹ ). Анализ FT-IR хорошо согласуется с измерениями XPS, выполненными для оксида графена, где также были идентифицированы гидроксильные, карбоксильные, эпоксидные и карбонильные группы [44]. GO и GO после 10-минутной ультразвуковой гомогенизации сравнивали (рис. 4 и 5), и аналогичным образом сравнивали Ag-NP с Ag-NP после 10-минутной ультразвуковой гомогенизации (рис. 4 и 6). Чтобы избежать изменения морфологии соединения, все соединения быстро охлаждали жидким азотом и сушили в лиофилизаторе. На рис. 5а, б представлены чешуйки ГО, а на рис. 5в, г показано влияние ультразвука на хлопья ГО, которые претерпевают частичное сворачивание и фрагментацию. На рисунке 6 также показан аналогичный эффект для Ag-NP, где видно изменение морфологии материала. На рис. 6а, б показан высушенный поливиниловый спирт, использованный для стабилизации водной суспензии НЧ Ag. Деструктивный эффект ультразвуковой гомогенизации был заметен, поскольку структура поливинилового спирта была разбита на длинные неоднородные части с небольшими сферическими отверстиями (рис. 6c, d).

FT-IR (НПВО, ослабленное полное отражение) спектр оксида графена с предполагаемым назначением функциональных групп, присутствующих в GO. Наиболее заметные пики наблюдались при ~ 3500 см ^-1 . , (отнесено к воде и гидроксильным группам), ~ 1080 см ⁻¹ (гидроксильные группы), ~ 1600 см ^-1 (присвоено связям C =C, присутствующим в графитном углероде). Другие пики, наблюдаемые в спектре FT-IR, показывают, что GO богат группами, содержащими C =O (в основном карбоксильные группы), пик около 1720 см ^-1 , эпоксидная смола (C – O – C) с видимым пиком около 1200 см ⁻¹ , и связи C – H (пик около 2800 см ⁻¹ )

ПЭМ-изображения агломерированных хлопьев GO ( a ), Хлопья ГО после ультразвуковой обработки ( б ), агломерированные Ag-NP ( c ), Ag-НЧ после ультразвуковой обработки ( d ) и GO-Ag ( e , f ). Уменьшение среднего размера частиц GO после ультразвуковой обработки было вызвано дефрагментацией или складыванием хлопьев GO. Уменьшение среднего размера Ag после ультразвуковой обработки вызвано дефрагментацией агломератов Ag. Примечание:стрелки указывают на Ag-NP / агломераты

Сравнение морфологии лиофилизированных хлопьев GO ( a , b ) и хлопья GO после ультразвуковой обработки ( c , d ) с помощью сканирующей электронной микроскопии. Уменьшение среднего размера частиц GO после ультразвуковой обработки было вызвано дефрагментацией или складыванием хлопьев GO

Сравнение морфологии лиофилизированной смеси Ag-NP ( a , b ) и смесью Ag-NP после ультразвуковой обработки ( c , d ) с помощью сканирующей электронной микроскопии

Средний размер и дзета-потенциал

Результаты среднего размера частиц / агломератов в водных суспензиях представлены в таблице 2. Анализы среднего размера были выполнены для концентрированных суспензий, которые не подвергались ультразвуковой гомогенизации (в том виде, в котором они были получены), и для разбавленных суспензий. Разбавленные суспензии перед испытанием подвергали ультразвуковой гомогенизации с параметрами гомогенизации, идентичными тем, которые использовались при ультразвуковом покрытии фольги слоями наноматериалов (Ag-NPs, GO). Для суспензии Ag-NP воздействие ультразвука вызывало увеличение среднего размера частиц с 80 до 218 нм. Основная причина увеличения среднего размера частиц после ультразвуковой гомогенизации в суспензии (помимо процесса агломерации Ag-NP) заключалась в том, что Ag-NP были введены в поливиниловый спирт, который использовался для стабилизации суспензии. The large standard deviation of the Ag-NP sample homogenized by ultrasound resulted from the presence of both loose Ag-NPs and Ag-NPs driven into the poly(vinyl alcohol) in the suspension. In the case of GO suspension, the average particle size of the sample subject to ultrasonic homogenization was 263 nm and was ca 7.7 times smaller than the average particle size of the sample that was not subject to homogenization. The obtained results are convergent with the SEM tests (Fig. 5), which show the destructive effect of ultrasounds on GO flakes. The decrease of the average GO particle size was caused by defragmentation or folding of the GO flakes. However, it should be emphasized that the results of the average particle size of GO suspension samples involve an error related to the nanomaterial shape. The results obtained by the DLS method are a hydrodynamic average that is calculated based on the shape of a sphere that has the same diffusion coefficient as the measured particles; however, the shape of GO was flakes, which was confirmed by SEM images.

Test results of the zeta potential analysis of samples are provided in Table 3. The zeta potential of Ag-NPs in a water suspension was merely − 5.9 mV, which resulted in a lack of electrostatic stability of the sample. The sample of Ag-NP suspension was stabilized sterically by preserving Ag-NP distances through poly(vinyl alcohol) addition, which prevented agglomeration/aggregation of Ag-NPs. The zeta potential of the GO suspension sample, in turn, was − 41 mV, which gave a moderate electrostatic stability to the sample. A moderate electrostatic stability of a sample is characterized by slow sedimentation with virtually negligible change of particle size in the period of declared fitness of the suspension. The zeta potential result of the mixture of Ag-NPs and GO was − 7.1 mV, which potentially means that during the action of the ultrasounds, the GO flakes were coated by poly(vinyl alcohol) and Ag-NPs. The obtained zeta potential result of the mixture of Ag-NPs and GO sample in the water suspension implied that electrostatic stability was not present.

Foil Characteristics

In order to determine the morphology of the created layers, four types of foil samples were compared (Fig. 7):pure polyurethane foil (A, B), GO-coated polyurethane foil (C, D), Ag-NP-coated polyurethane foil (E, F), and GO-Ag mixture-coated polyurethane foil (G, H).

Scanning electron microscopy images of a , b non-coated polyurethane foil with a smooth surface with single impurities; c , d foil coated with GO, the broken-down GO flakes deposited on the polyurethane foil surface; е , f foil coated with Ag-NPs on which grid structures composed of polyvinyl alcohol and Ag-NPs are observed; and g , ч foil coated with GO-Ag, which was mixed under the influence of ultrasounds and deposited on the foil surface

Figure 7a, b shows an uncoated polyurethane foil with a smooth surface with single impurities. In Fig. 4c, d, the broken-down GO flakes deposited on the polyurethane foil surface are noticeable. Figure 7e, f shows the foil surface coated with Ag-NPs on which grid structures composed of polyvinyl alcohol and Ag-NPs are observable. Figure 7g, h presents a mixture of GO-Ag composition, which was mixed under the influence of ultrasounds and deposited on the foil surface.

AFM Analysis and Surface Free Energy

AFM and LFM were used to complement the information about the surface morphology investigated by SEM. The investigation confirmed evolution of surface morphology by sonication of the polyvinyl alcohol with Ag-NP, GO, and GO-Ag NP solutions on the foils. Pure polyurethane foil was used as a reference foil in relation to foils coated by the ultrasonic method. The images in Fig. 8 are the AFM phase contrast images made in AC; additionally, cross sections of the GO flakes are attached under the corresponding images. Figure 8a is an image of pure polyurethane film; Fig. 7b depicts the Ag-NP-coated film, where characteristic and homogeneous grid structures are observable, being similar to those in Fig. 8e, f. Figure 8c, d shows GO-Ag-NP-coated film. Figure 8e shows the surface of the foil almost entirely covered with GO flakes; the phase contrast image helps to observe these two phases, the darker area is GO and the lighter area is polymer foil. It was noticed that the morphology of the foils has changed after Ag-NP coating comparing to the not-coated foils. The GO-Ag-NP coating differs from the previous one because it contains also small amounts of GO flakes seen as small black spots on the image, as it was mentioned earlier. Figure 8f depicts magnification of one GO flake made in LFM. The reduced friction confirms that it is, in fact, a GO flake.

AFM phase contrast images and cross sections topographic images of graphene flakes:a non-coated foil polyurethane foil; б Ag-NPs coated foil where characteristic and homogeneous grid structures are observed; c , d GO-Ag-coated foil; е GO-coated foil, the surface of the foil almost entirely covered with GO flakes; the phase contrast image helps to observe these two phases, the darker area is GO and the lighter area is polymer foil; е LFM image of graphene flake. Red marks, area of cross section

The polar component for the GO-coated foil increased in relation to pure foil, from 2.3 ± 0.6 to 68.9 ± 2.8 mJ/m ² , while the dispersion component decreased from 34.4 ± 1.3 to 8.2 ± 1.2 mJ/m ² . SFE increased from 36.7 ± 1.4 to 77.0 ± 3.4 mJ/m ² . A similar effect was not observed on foil surfaces coated with Ag-NPs and GO-Ag mixture. SFE of foil samples coated with Ag-NPs and GO-Ag mixture does not differ statistically (Table 4).

Antibacterial Properties

The antibacterial activity of the different foils coated with GO, Ag-NPs, and GO-Ag were tested with E . coli , S . aureus , S . epidermidis , and C . albicans. Results showed that after co-incubation with bacteria at 37 °C for 24 h, foils inhibited the growth of all tested microorganisms but to various degrees. The maximum antibacterial effect against all tested microorganisms was with foil coated with the GO-Ag nanocomposite. The bacterial growth of the cells treated with foils coated with GO and Ag-NPs was slightly lower than that of cells in the control group whereas the growth of bacterial cells treated with foils coated with GO-Ag was greatly inhibited, 88.6% of E . coli , 79.6% of S . aureus , 76.5% of S . epidermidis , and 77.5% of C . albicans (Figs. 9, 10, and 11).

Antimicrobial properties of GO-Ag coated foils. The growth of E . coli ( б , c ), S . aureus ( c , d ), S . epidermidis ( е , f ), and C . albicans (g - я ) colonies is reduced after co-incubation with GO-Ag-coated foils at 37 °C for 24 h. а Representative agar plate with GO-Ag-coated foils. Notes:Black arrows point to GO-Ag coated foils; arrowheads point to colonies of microorganisms

Morphology of microorganisms after co-incubation with GO-Ag-coated foils. Scanning electron microscopy images of bacteria and yeast in the control foils (a , c , e , г ) and foils coated with GO-Ag (b , d , f , ч ) after incubation at 37 °C for 24 h. E . coli ( а , b ), S . aureus ( c , d ), S . epidermidis ( е , f ), and C . albicans (g , ч ) show decreased growth and deformed morphology after co-incubation with GO-Ag-coated foils

Foils coated with nanomaterials decrease E . coli , S . aureus , S . epidermidis , and C . albicans viability. Viability of bacteria and yeast after incubation on foils coated with Ag-NPs, GO, and GO-Ag at 37 °C for 24 h was assessed with Presto Blue assay. C control group (foil without nanoparticles), Ag foil coated with silver nanoparticles, GO foil coated with graphene oxide, GO-Ag foil coated with composite of graphene oxide and silver nanoparticles. Note:The columns with different letters (a–d) indicate significant differences between the concentrations (P  = 0.001); error bars are standard deviations

Membrane Integrity

In cases where the cell membrane is damaged, intracellular LDH molecules could be released into the culture medium. The LDH level outside the cells demonstrates the cell membrane integrity. Foils coated with GO, Ag-NPs, and GO-Ag disrupted cell membrane functionality and integrity with significant differences between control groups and the Ag-NPs and GO-Ag groups (Fig. 12). The highest disruption of cell membranes was observed in the GO-Ag groups, 66.3% of E . coli , 59.4% of S . aureus , 54.8% of S . epidermidis , and 48.5% of C . albicans .

Foils coated with nanomaterials decreased E . coli , S . aureus , S . epidermidis , and C . albicans membrane integrity. Membrane integrity of bacteria and yeast after incubation on foils coated with Ag-NPs, GO, and GO-Ag at 37 °C for 24 h was assessed with LDH assay. C control group (foil without nanoparticles), Ag foil coated with silver nanoparticles, GO foil coated with graphene oxide, GO-Ag foil coated with composite of graphene oxide and silver nanoparticles. Note:The columns with different letters (a–c) indicate significant differences between the concentrations (P  = 0.000); error bars are standard deviations

ROS Production

Low levels (or optimum levels) of ROS play an important role in signaling pathways. However, when ROS production increases and overwhelms the cellular antioxidant capacity, it can induce macromolecular damage (by reacting with DNA, proteins, and lipids) and disrupt thiol redox circuits. Foils coated with Ag-NPs and GO-Ag (P  < 0.05) increased the ROS production of all tested microorganisms compared to the control group. Foils coated with GO only increased the ROS production of C . albicans . The highest ROS production was observed in the GO-Ag group (Fig. 13).

Effect of foils coated with nanomaterials on the production of reactive oxygen species. E . coli , S . aureus , S . epidermidis , and C . albicans were cultured on foils coated with Ag-NPs, GO, and GO-Ag at 37 °C for 24 h. Production of reactive oxygen species was assessed with DCFDA, Cellular Reactive Oxygen Species Detection Assay Kit. C control group (foil without nanoparticles), Ag foil coated with silver nanoparticles, GO foil coated with graphene oxide, GO-Ag foil coated with composite of graphene oxide and silver nanoparticles. Note:The columns with different letters (a–d) indicate significant differences between the concentrations (P  = 0.000); error bars are standard deviations

Discussion

The discovery of antibiotics, natural products produced by microorganisms that are able to prevent the growth of bacteria and thus cure infectious diseases, revolutionized medical therapy; however, the overuse and misuse of antibiotics have been key factors contributing to antibiotic resistance. Now, the era of antibiotics is coming to an end, and new antibacterial agents are needed. In recent years, studies have reported nanoparticles as a promising alternative to antibacterial reagents because of their antibacterial activity in several biomedical applications [19, 45]. Nanoparticles can be an effective way to control many pathogenic and antibiotic-resistant microorganisms. Among many metal nanoparticles, Ag-NPs have been intensely studied because of the distinct properties of their antibacterial activity [7]. Ag-NPs have been proved effective against over 650 microorganisms including bacteria (both gram-positive and negative), fungi, and viruses; however, the precise mechanism of antimicrobial action is not understood completely [46]. Ag-NP exposure to microorganisms could cause adhesion of nanoparticles to the peptidoglycan and the cell membrane [47], penetration inside the cell [48], induction of ROS [49], and damaging of intracellular structures [50]. However, bare Ag-NPs aggregate when they come into contact with bacteria; thus, they lose their active surface area and show weaker antibacterial activity [51]. To overcome this problem, nanocomposites composed of graphenic materials and Ag-NPs or other metal nanoparticles could be fabricated. GO with oxygen-containing functional groups is water soluble and therefore more biocompatible than pristine graphene. As a result, Ag-based GO nanocomposites may be used as antibacterial agents. However, the information about antimicrobial properties of graphene-based composites is limited, and mechanisms of toxicity or lack of toxicity are not fully explained.

The aim of this work was to study the action of graphene oxide-based nanocomposites decorated with Ag nanoparticles on S . aureus , S . epidermidis , E . coli , and C . albicans growth; membrane integrity; and ROS production. After co-incubation with the bacterial and yeast cells for 24 h, foils coated with GO-Ag nanocomposite inhibited the growth of all tested microorganisms with varying degrees, 88.6% of E . coli , 79.6% of S . aureus , 76.5% of S . epidermidis , and 77.5% of C . albicans . This action is most probably due to an increase in cell membrane and wall penetration by the nanoparticles. Some researchers suggest that the antimicrobial activity of graphene-based nanocomposites may be due to the disruption of cell membrane integrity and oxidative stress [52].

Foils coated with GO, Ag-NPs, and GO-Ag disrupted cell membrane functionality and integrity with significant differences between the control group and the Ag-NPs and GO-Ag groups. The highest disruption of cell membranes was observed in the GO-Ag groups, 66.3% of E . coli , 59.4% of S . aureus , 54.8% of S . epidermidis , and 48.5% of C . albicans . However, foils coated with bare Ag-NPs also disrupted cell membranes. It has been proposed that Ag-NPs are able to interact with bacterial membranes by increasing permeability and changing the structure of membranes, which finally leads to cell death [50]. Ag-NPs can cause direct damage to the bacterial cell membrane. Bacteria may be killed by the combined bactericidal effects of the released Ag ⁺ ions and Ag nanoparticles. Additionally, the antimicrobial potential of Ag-NPs is also influenced by the thickness of the cell wall of the microorganisms [53]. The wall of gram-positive cells contains a thick layer (20–80 nm) of peptidoglycan, which is attached to teichoic acids. In gram-negative bacteria, the cell wall comprises a thin (7–8 nm) peptidoglycan layer and contains an outer membrane. The thicker peptidoglycan layer in gram-positive bacteria, such as S . aureus and S . epidermidis , may explain why these bacteria are more resistant to the antibacterial effects of GO-Ag.

Many studies have sought to establish a mechanism of action of antibacterial activity exhibited by silver in both its colloidal and ionic form. A disruption of membrane functionality from an interaction between released Ag ⁺ ions and the cell membrane and extensive cell membrane damage caused by the formation of ROS ultimately causes damage to the cell due to oxidative stress. Additionally, Ag ⁺ ions could cause dysfunction of the respiratory electron transport chain by uncoupling it from oxidative phosphorylation by inhibiting respiratory chain enzymes [54]. Foils coated with Ag-NPs and GO-Ag increased the ROS production of all tested microorganisms compared to the control group. The biological targets are DNA, RNA, proteins, and lipids. Lipids are one major target during oxidative stress. Free radicals can directly attack polyunsaturated fatty acids in bacterial and yeast membranes and activate peroxidation of lipids. A fundamental effect of lipid peroxidation is a decrease in membrane fluidity, which can significantly disrupt membrane-bound proteins. DNA is also a main target. Mechanisms of DNA damage involve abstractions and addition reactions by free radicals leading to carbon-centered sugar radicals and OH- or H-adduct radicals of heterocyclic bases. The sugar moieties producing single- and double-strand breaks in the backbone, adducts of base and sugar groups, and cross-links to other molecules can block replication. Foils coated with GO increased the ROS production at very low levels. However, Hu et al. [55] demonstrated that GO had a detrimental effect on E . coli due to decreased production of ATP and increased ROS production. Zhao et al. [56] reported the antibacterial activity of GO and reduced GO. Also, Gurunathan et al. [57] presented that GO and reduced GO showed significant antibacterial activity in a concentration- and time-dependent manner. Their results demonstrated that oxidative stress is a key mechanism for the antibacterial activity of GO and reduced GO through ROS generation. Nanda et al. [53] reported the effect of cystamine-conjugated GO against E . coli , S . typhimurium , E . faecalis , and B . subtilis with ROS production and high antibacterial activity.

Kurantowicz et al. [20] confirmed that bacteria could adhere to the GO surface, which results in the highest antibacterial activity. GO is characterized by a high degree of oxygenated functional groups:carbonyl, carboxylate, and hydroxyl. We hypothesize that these groups can be attractive groups for bacterial and yeast attachment. These groups are present on a large range of nutrients (amino acids, fatty acids) which are commonly recognized by microorganisms. In the present study, foils coated with GO induced membrane disruption and ROS production at a lower level than the Ag-NP and Ag-GO groups; however, cell viability was decreased, which is likely connected to the smaller active surface of GO after ultrasonic modifications.

Conclusions

Ag-NPs, GO, and Ag-GO nanocomposites demonstrated the antibacterial activity that is stronger against gram-negative bacteria (E . coli ) versus gram-positive bacteria (S . aureus and S . epidermidis ) and yeast (C . albicans ). The results showed that the decoration of GO with Ag-NPs promotes a synergistic effect and reduces dramatically the concentrations required to inhibit all tested bacterial and yeast strains. The antimicrobial potential of Ag-GO is also influenced by the thickness of the cell wall of the microorganisms. The thicker peptidoglycan layer in gram-positive bacteria, such as S . aureus and S . epidermidis , may explain why these bacteria are more resistant to the antibacterial effects of GO-Ag. A disruption of membrane functionality from an interaction between released Ag nanoparticles/Ag ⁺ ions and the cell membrane and extensive cell membrane damage caused by the formation of ROS ultimately caused damage to the cell due to oxidative stress. In order to explain the mechanism of ROS production, additional studies are needed. Our research indicates the potential applicability of GO-Ag as an antimicrobial agent.

Сокращения

AFM:

Атомно-силовая микроскопия

Ag-NPs:

Silver nanoparticles

DLS:

Динамическое рассеяние света

GO:

Оксид графена

GO-Ag:

Graphene oxide decorated with silver nanoparticles

LDE:

Laser Doppler electrophoresis

LDH:

Lactate dehydrogenase

ROS:

Reactive oxygen species

SEM:

Сканирующая электронная микроскопия

XRD:

Рентгеновская дифракция