用银纳米粒子作为抗菌剂装饰的基于氧化石墨烯的纳米复合材料

结果

GO 和 Ag-NPs 的特性

化学分析表明存在氮、碳、硫、氢和氧（表 1）。

GO 样品的物相分析（图 1）显示存在来自痕量石墨、硝酸钠和可能还原形式的氧化石墨烯的杂质。

GO粉末的X射线衍射图。 GO 样品的相分析表明存在来自痕量石墨、硝酸钠和可能还原形式的氧化石墨烯的杂质

拉曼光谱可以提供有关氧化石墨烯结构特征的信息。 D带归因于结构无序，而G带来自碳sp ² 的键拉伸 原子 [42]。额外的带（包括 D'、2D 和 D + G）源于碳材料石墨结构中存在的缺陷。 我 D/我 G 比（由 D 和 G 带的强度计算）可用于表征碳材料中石墨结构的无序。从图 2 中可以看出，由于石墨粉氧化过程中形成的结构中有许多官能团，GO 具有高度无序的结构。 D 波段的位置是 1351 cm ⁻¹ 和 G 波段 1590 cm ⁻¹ ; 我 D/我 G比为1.15。

氧化石墨烯的拉曼光谱，建议对 D、G、D'、2D 和 D + G 波段进行解卷积。由于石墨粉氧化过程中形成的结构中有许多官能团，GO具有高度无序的结构。 D 波段的位置是 1351 cm ⁻¹ 和 G 波段 1590 cm ⁻¹ ; 我 D/我 G比为1.15

在 ATR 模式下收集的氧化石墨烯的 FT-IR 光谱显示，GO 结构中存在大量官能团。最显着的峰值可以在 ~ 3500 cm ⁻¹ 处观察到 ，主要分配给水和羟基（图 3）。 1080 cm ⁻¹ 附近的一个非常密集的峰 也可以归因于羟基。 1600 cm ⁻¹ 附近的峰值 通常分配给石墨碳中存在的 C=C 键。然而，我们之前的 XPS 研究表明，氧化石墨烯中存在一些 C=C 键 [43]；因此，我们将此峰归因于仍然存在于氧化石墨烯中的大部分水。在 FT-IR 光谱上观察到的其他峰表明 GO 富含含有 C=O 键的基团（主要是羧基），峰位于 1720 cm ^-1 , 环氧树脂 (C–O–C) 可见峰在 1200 cm ^-1 , 和 C–H 键（峰值在 2800 cm ⁻¹ ）。 FT-IR 分析与对氧化石墨烯进行的 XPS 测量非常一致，其中还确定了羟基、羧基、环氧基和羰基 [44]。比较了 10 分钟超声均化后的 GO 和 GO（图 4 和 5），类似地，比较了 10 分钟超声均化后的 Ag-NP 和 Ag-NP（图 4 和 6）。为了避免化合物形态的变化，所有化合物用液氮快速冷却并在冻干机中干燥。图 5a、b 显示了 GO 薄片，而图 5c、d 显示了超声波对 GO 薄片的影响，GO 薄片经历了部分折叠和碎裂。图 6 还显示了 Ag-NP 的类似效果，其中材料形态的变化是可见的。图 6a、b 显示了干燥的聚乙烯醇，用于稳定 Ag-NP 的水悬浮液。超声均质的破坏作用显着，因为聚乙烯醇结构被分解成具有小球形开口的长异质部分（图 6c、d）。

氧化石墨烯的 FT-IR（ATR，衰减全反射）光谱以及 GO 中存在的官能团的建议分配。最显着的峰出现在 ~ 3500 cm ^-1 ,（归因于水和羟基），~ 1080 cm ⁻¹ （羟基），~ 1600 cm ⁻¹ （分配给存在于石墨碳中的 C=C 键）。在 FT-IR 光谱上观察到的其他峰表明 GO 富含含有 C=O 的基团（主要是羧基），峰值在 1720 cm ^-1 , 环氧树脂 (C–O–C) 可见峰在 1200 cm ^-1 , 和 C–H 键（峰值在 2800 cm ⁻¹ )

团聚的 GO 薄片 (a ), 超声波处理后的 GO 薄片 (b ), 聚集的 Ag-NPs (c ), 超声波处理后的 Ag-NPs (d ) 和 GO-Ag (e , f ）。超声处理后平均 GO 粒径的减小是由 GO 薄片的碎裂或折叠引起的。超声处理后平均银尺寸的减小是由银团块的碎裂引起的。注：箭头指向 Ag-NPs/团聚体

冻干 GO 薄片形貌的比较 (a , b ) 和超声波处理后的 GO 薄片 (c , d ) 使用扫描电子显微镜。超声波处理后GO平均粒径的减小是由于GO薄片的碎裂或折叠引起的

冻干 Ag-NP 混合物的形态比较 (a , b ) 和超声波处理后的 Ag-NP 混合物 (c , d ) 使用扫描电子显微镜

平均尺寸和 Zeta 电位

水悬浮液中平均颗粒/附聚物尺寸的结果列于表 2 中。对未进行超声均质化的浓缩悬浮液（原样）和稀释悬浮液进行平均尺寸分​​析。测试前稀释的悬浮液进行超声均质化，均质化参数与超声涂覆纳米材料层（Ag-NPs，GO）箔时使用的参数相同。对于 Ag-NP 悬浮液，超声波的作用导致平均粒径从 80 纳米增加到 218 纳米。悬浮液中超声均化后平均粒径增加的主要原因（除了 Ag-NP 团聚过程）是 Ag-NPs 被驱入用于悬浮液稳定的聚乙烯醇中。通过超声均质的 Ag-NP 样品的大标准偏差是由于松散的 Ag-NP 和 Ag-NP 的存在导致悬浮液中的聚（乙烯醇）。在 GO 悬浮液的情况下，经过超声匀浆的样品的平均粒径为 263 nm，比未经匀浆的样品的平均粒径小约 7.7 倍。所得结果与 SEM 测试（图 5）一致，显示了超声波对 GO 薄片的破坏作用。 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; e , f foil coated with Ag-NPs on which grid structures composed of polyvinyl alcohol and Ag-NPs are observed; and g , h 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; b Ag-NPs coated foil where characteristic and homogeneous grid structures are observed; c , d GO-Ag-coated foil; e 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; f 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 . 金黄色葡萄球菌 , 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 . 金黄色葡萄球菌 , 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 (b , c ), S . 金黄色葡萄球菌 (c , d ), S . epidermidis (e , f ), and C . albicans (g –i ) colonies is reduced after co-incubation with GO-Ag-coated foils at 37 °C for 24 h. 一 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 , g ) and foils coated with GO-Ag (b , d , f , h ) after incubation at 37 °C for 24 h. E . coli (a , b ), S . 金黄色葡萄球菌 (c , d ), S . epidermidis (e , f ), and C . albicans (g , h ) show decreased growth and deformed morphology after co-incubation with GO-Ag-coated foils

Foils coated with nanomaterials decrease E . coli , S . 金黄色葡萄球菌 , S . epidermidis , and C . albicans 可行性。 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 . 金黄色葡萄球菌 , 54.8% of S . epidermidis , and 48.5% of C . albicans .

Foils coated with nanomaterials decreased E . coli , S . 金黄色葡萄球菌 , 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 . 金黄色葡萄球菌 , 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 . 金黄色葡萄球菌 , S . epidermidis , E . coli , and C . albicans 生长; 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 . 金黄色葡萄球菌 , 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 . 金黄色葡萄球菌 , 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 . 金黄色葡萄球菌 和 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.赵等人。 [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.南达等人。 [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 . 金黄色葡萄球菌 和 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 . 金黄色葡萄球菌 和 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.

缩写

原子力显微镜：

原子力显微镜

Ag-NPs:

银纳米粒子

DLS：

动态光散射

开始：

氧化石墨烯

GO-Ag:

Graphene oxide decorated with silver nanoparticles

LDE:

Laser Doppler electrophoresis

LDH:

Lactate dehydrogenase

ROS：

活性氧

SEM：

扫描电镜

XRD：

X射线衍射