Insight into the antibacterial resistance of graphdiyne functionalized by silver nanoparticles

Abstract Objectives Silver nanoparticles (AgNPs) tend to aggregate spontaneously due to larger surface‐to‐volume ratio, which causes decreased antibacterial activity and even enhanced antimicrobial resistance (AMR). Here, we aim to improve the stability of AgNPs by employing a growth anchor graphdiyne (GDY) to overcome these shortcomings. Materials and Methods Bacillus subtilis and Escherichia coli were selected to represent gram‐positive and gram‐negative bacteria, respectively. Transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), scanning electron microscopy (SEM)‐EDS mapping and inductively coupled plasma mass spectrometry (ICP‐MS) were carried out to characterize the physiochemical properties of materials. The antimicrobial property was determined by turbidimetry and plate colony‐counting methods. The physiology of bacteria was detected by SEM and confocal imaging, such as morphology, reactive oxygen species (ROS) and cell membrane. Results We successfully synthesized a hybrid graphdiyne @ silver nanoparticles (GDY@Ag) by an environment‐friendly approach without any reductants. The hybrid showed high stability and excellent broad‐spectrum antibacterial activity towards both gram‐positive and gram‐negative bacteria. It killed bacteria through membrane destruction and ROS production. Additionally, GDY@Ag did not induce the development of the bacterial resistance after repeated exposure. Conclusions GDY@Ag composite combats bacteria by synergetic action of GDY and AgNPs. Especially, GDY@Ag can preserve its bacterial susceptibility after repeated exposure compared to antibiotics. Our findings provide an avenue to design innovative antibacterial agents for effective sterilization.

nanomaterials show specific electric property, high catalytic activity, increased stability and biocompatibility, which have attracted considerable attention in biomedical applications including biosensor, cancer treatment and clinical infection therapeutics. For example, they have emerged as a new nanozyme with increased intrinsic catalytic capacities to kill bacteria by producing highly toxic radicals. 25,27,28 The disadvantage of nanozyme-related antibacterial strategy is that the reaction mostly requires additional hydrogen peroxide to trigger. In fact, Li's group have demonstrated that GDY and graphdiyne oxide (GDYO) can be used as photocatalytic antibacterial agents against bacteria. GDYO with good dispersion in the bacterial system showed stronger antibacterial activity than GDY both in dark and visible light irradiation. 29 The excellent inhibiting capacity of GDYO against bacteria is attributed to oxidative stress. However, in another study, GDY exhibits higher antimicrobial property than that of GDYO, and "physical" effects play a major role in the antibacterial process. 30 Although these conclusions are contradictory, it is sure that both GDY and GDYO are capable of hindering broad-spectrum bacterial growth.
Therefore, we postulate that the hybrid of GDY and AgNPs may not only solve the stability of Ag nanoparticles, but also provide a new bactericide with a synergistic antimicrobial effect.
In this work, we described a hybrid graphdiyne @ silver nanoparticles (GDY@Ag) as a high-performance bactericide. GDY@Ag was synthesized by simply mixing silver nitrate with GDY; surfactant was added to assist the growth of nanoparticles. The acetylenic groups in GDY acted not only as a reductant, but also as an anchor for AgNPs growth. The hybrid GDY@Ag showed exceptional broadspectrum antibacterial activity towards both gram-positive and gramnegative bacteria. Moreover, two bacterial strains did not develop the resistance to GDY@Ag after repeated exposure ( Figure 1). Our findings present an avenue to fabricate new antibacterial agents for effective bactericidal activity.

| Synthesis and characterization of GDY@Ag
The preparation of GDY powder was carried out according to previous work. 22 Then, GDY powder was dispersed into ultrapure water and ultrasonically agitated for 12 h to form a homogeneous solution with an ultrasonic cleaner (Kunshan ultrasonic instrument Co., Ltd., KQ-200KDE, frequency = 40KHz, input power = 200 W). GDY solution (1 mg/ml) was stored at room temperature.
GDY@Ag was prepared by a modified protocol published by Yuliang Li. 31 In brief, 500 μl of the above GDY solution was mixed with 4 ml of 5-mM AgNO 3 aqueous solution under vigorous magnetic stirring (700 rpm/min) at room temperature. After stirring for 10 min, 500 μl of 100-mM CTAB aqueous solution was added dropwise to the reaction mixture and continuously stirred for 20 min. The products were collected by centrifugation at 13,000 rpm for 15 min, followed by washing with ultrapure water four times.
The sample was dropped onto carbon film-coated copper grids and absorbed for 5 min and then washed with about 50-μl ultrapure water dropwise. The morphological structures of GDY and GDY@Ag were observed by transmission electron microscope (TEM) (FEI Tecnai G2 F20 S-TWIN, USA) and scanning electron microscope (SEM) (LEO 1530vp, Germany). 32 The energy dispersive spectroscopy (EDS) mapping (Oxford) was performed to analyse the element. The amount of silver element loaded on the GDY@Ag was quantified using an inductively coupled plasma mass spectrometer (ICP-MS) (NexION 300D, PerkinElmer, USA).

| Bacteria culture
Escherichia coli (GÀ) and Bacillus subtilis (G+) were employed to evaluate the antibacterial activity of GDY@Ag. The strains were grown overnight in fresh LB medium at 37 C in an incubator whilst shaking (220 rpm/min) and then harvested at the exponential growth phase.
The bacterial cells were washed twice and resuspended in sterile saline solution (0.9% NaCl). The bacterial concentration was quantified by measuring the optical density at 600 nm (OD 600 ). 33 The cultures can be stored at 4 C for a short time to be used in further experiments.

| Bacterial physiology analysis
The bacterial cells were treated with GDY@Ag for 2 h and washed with sterile saline solution by centrifugation at 13,000 rpm. On the one hand, the samples were stained according to the LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Thermo Fisher), and then, 5-μl stained bacterial suspension was trapped between a slide and a square coverslip. TCS SP8 laser confocal microscope (Leica, Germany) was used to analyse the bacterial survival. 34 On the other hand, the samples were fixed with the mixture of paraformaldehyde and glutaraldehyde for 3 h and then were dehydrated by gradient ethanol (35%, 50%, 75%, 85%, 95% and 100%) for 10 min each. Subsequently, 10-μl dehydrated suspension was dropped onto silicon wafers and dried naturally, then sputter coated with gold for SEM imaging.

| Bacterial resistance development test
The wild-type strains were incubated with sub-MIC of GDY@Ag at 37 C and 220 rpm/min for 24 h; ampicillin and chloramphenicol were used as negative control towards E. coli and B. subtilis, respectively.
The obtained culture was determined in the first passage. The MIC was tested as described above, and the passage was treated with the corresponding sub-MIC to acquire the second passage; 20 passages F I G U R E 1 Schematic of stable GDY@Ag with increased antimicrobial property and bacterial susceptibility compared to unstable AgNPs 16 were finally obtained in this way; MICs of every passage were recorded.

| Preparation and characterization of GDY@Ag
We prepared a hybrid graphdiyne @ Ag nanoparticles (GDY@Ag) according to the previously reported method. 31 Briefly, silver nitrate as the resource of Ag was mixed with the aqueous GDY solution sufficiently with vigorous stirring. Subsequently, aqueous surfactant CTAB was added to assist the seeding growth of the Ag species. 31,36 To obtain pure GDY@Ag, the mixture was centrifuged and washed with deionized water for several times to remove residual Ag + and CTAB. sheets. 37 No signal of N element derived from the quaternary ammonium group of CTAB was observed, suggesting that the local concentration of CTAB was far below the detection limit of 0.1% (w/w%) of EDS mapping. Due to benzene rings and alkyne units arranged in sphybridized atoms and a large conjugated surface, GDY has great advantages in the adsorption and immobilization of metal atoms. 23,38,39 Our results demonstrated that AgNPs as well as AgBrNPs were immobilized uniformly on the GDY sheets that we successfully prepared GDY@Ag composite.  (Figures 4f,g). The obtained growth curve showed that the exponential phase of both two bacterial species lagged for several hours and finally reached the peak anyway in the case of half MIC, whilst the proliferation was totally inhibited by GDY@Ag at MIC ( Figure 3). We could rationally infer that the remaining population in the half MIC case was temporarily tolerant that leads to the ultimate recovery, which calls the lagged phenomenon. 41

| Antimicrobial property of GDY@Ag
After confirming the antibacterial property of GDY@Ag, we set out to explore the mechanism of the bactericidal activity (Figure 5a). According to the most discussed "insertion" mechanism of 2D carbon nanomaterials, we speculated that GDY@Ag inactivates bacteria by It is well known that the repeated use of antibiotics leads to the decreased susceptibility of bacteria, that is, bacteria develop drug resistance. 42,43 Our results demonstrated GDY@Ag inactivated bacteria by physical and oxidative damage, which differed from the mechanism of antibiotics, 4 we thus hypothesized that GDY@Ag may not induce bacterial resistance. To test this hypothesis, we cultured 20 successive bacterial steps with two strains in media containing sub-inhibitory GDY@Ag and antibiotics. In detail, planktonic bacteria were inoculated to the bactericides at half MIC, and the obtained generation was defined as the first passage; the subsequent passages were derived by treating the former one with its corresponding half MIC. As shown in Figure 5h  Hitherto, the antibacterial mechanism of 2D carbon-based materials research refers to the following two mechanisms: (1) insertion of the sharp edge into the bacterial membrane, leading to the extraction of cellular contents, 46,47 (2) oxidative stress injury induced by excessive elevated ROS production. [48][49][50] The antimicrobial capacity of GDY is considered as a result of "physical" effects in combination with "chemical" actions. 30 The positively charged GDY is favourable to wrap bacterial surface with negative charge, which may prevent bacterial growth. With the increase in their interaction, GDY nanosheets can directly insert and disrupt the bacterial membranes. The zeta potential revealed that positively charged GDY@Ag contacted with negatively charged bacteria through electrostatic interaction (Figure 5f). The bacterial membrane destroyed by GDY@Ag was observed by SEM images (Figure 5d). Oxidative stress induced by GDY has a negligible effect on bactericidal activity without irradiation. 29,30 As we know, Ag + released from AgNPs and oxidative stress induced by it are two major antimicrobial factors. 51 Silver ions released from GDY@Ag were not detected by ICP-MS ( Figure S4), which could exclude the role of Ag ions in antibacterial activity. In fact, the level of cellular ROS significantly improved when bacteria exposed