The Fabrication of Mechanobactericidal Coating and Its Application in Mechanical Sterilization

Bacteria are difficult to be eliminated because of their multi‐drug resistance, which brings significant threats to public health. Among the antibacterial methods, mechanobactericidal surfaces provide a possible approach to solving this problem. In this study, a generally used mechanical sterilization is developed by fabricating a nanostructured coating. The functional coating is simply fabricated by growing zeolitic imidazolate frameworks (ZIFs, ZIF‐8 mixed with ZIF‐67) nanospikes on the surface of the fabric substrate in situ. The fabricated ZIF nanospikes are stable and effective, with the sterilization efficiency approaching 99.9999%. This technique provides a fast, safe, and low‐cost antibacterial method without drug resistance, which can be potentially used in hospitals, emergency treatments, and superbug protection in the future.


Introduction
Antibiotics and antimicrobials are crucial in medical treatments. [1][2][3] They can kill bacteria by inhibiting bacterial cell wall synthesis or destroying DNA replication. [4,5] However, the abuse of antibiotics could seriously threaten human DOI: 10.1002/admi.202300208 nerves, [6] kidneys, [7] and the blood system. [8] Moreover, it could stimulate the growth and reproduction of insensitive bacteria, in the worst case, leading to drug resistance and the emergence of "superbugs." [9] According to recent reports by WHO, ≈700 000 people die of "super bacteria" each year, and even 230 000 newborns die earlier. It is estimated that the death may exceed 10 million by 2050, exceeding the annual death caused by cancers. [10] Recently, antibacterial methods using physical factors (such as surface topography) have been proposed as possible strategies to eliminate drug resistance. [11][12][13][14] Nanoarrays exhibit rapid mechanical bactericidal behavior. They provide a safe and effective alternative surface for the prevention of contamination and the spreading of bacteria. In the absence of a specific drug, it provides a meaningful way to control the rapid propagation of pathogen microorganisms. Inspired by patterned arrays of nanorods on the cicada, dragonfly wings, and gecko skin, which show excellent bactericidal effects, [15][16][17][18][19] various nanoarrays with high aspect ratio ratios, such as silicon, [20,21] titanium, [22] deacetylated chitosan, [23] stainless steel, [24] zinc oxide, [24,25] zinc phthalocyanine, [26] polymethyl methacrylate, [27] and carbon nanotubes, [28] are successfully fabricated. These reported results reinforce that the main antibacterial mechanism stems from physical structures rather than chemical interactions. The mechanobactericidal effect is a complex interaction between bacteria and substrate-dependent factors, including geometric, biological, electric, and physical interfacial characteristics. Once overlaid on the structures, bacteria are subjected to these interactions generated by extracellular polymeric substances (EPS). [16,[29][30][31] Hence the outer membrane of bacteria will be pierced, and then the bacteria are killed. [32] The physical antibacterial structures could be fabricated on different kinds of substrates. There are several reported research on using conventional filtered fabric as substrates, such as the survivability of microorganisms on the filtered fabric surface, [33] re-aerosolization of settled particles, [34] safe management and disposal of used fabric surfaces, [35] and fomite transmission. However, the large specific surface area, low air pressure, and excellent mechanical properties of fabric fiber make it suitable to be adopted as the substrate of mechanobactericidal coating. Furthermore, the fabric fibers can also prevent the skin from directly contacting the structures when considering practical applications. The fabrication of micro-and nano-structures using ZnO nanofibers has been reported. [36,37] The bionic urchin structures of ZnO nanofiber were fabricated on a rigid silicon substrate, which exhibited cell toxicity. [36] Similar cotton-like ZnO/SnO 2 nanocomposites have also been used in solar cell applications. [37] However, these flexible structures' of ZnO and ZnO/SnO 2 as well as the complex fabrication process have limited their applications in the antibacterial field. In this study, we developed a simple method to fabricate nanospikes on flexible substrates at a large scale, which would exhibit mechanical sterilization by the flexible substrate-structured surfaces. Cobalt 2methylimidazole (ZIF-67) and 2-Methylimidazole zinc salt (ZIF-8) were grown on PDA (polydopamine) in situ, obtaining ZIF nanospikes to mimic the bactericidal nanoscale pillar structures on the surfaces of cicada wings. These ZIF nanospikes exhibited excellent bactericidal performances, providing a safe and clean surface antibacterial without external chemical substances or drug resistance. The easy and large-scale fabrication of ZIF structure, with low cost, fast growth time, and longtime lifespan, make it possible to be widely used in many fields.

Surface Morphologies of ZIF-Coated PLA Fiber
Due to their diverse structures and customizable organic functions, ZIFs have been reported as multifunctional materials. [38,39] Nucleation and growth of the ZIFs layers can form uniform coatings spontaneously on the substrate by appropriate modification. In this study, the ZIF coatings are grown on the surfaces of polydopamine, forming dopamine layers on the fiber membrane by self-assembling. As has been reported, PDA exhibits an unparalleled adhesion as high as ≈71.62 mN m −1 , [40] which makes the three-layer structure of PLA fiber, PDA, and ZIF spikes stable (as shown in Figure S2, Supporting Information), And the strong adhesion force between PDA and the nano-metal frame material makes the ZIFs coatings difficult to fall off. The schematic fabrication process of nanospikes on the fibers is shown in Figure 1a, and the morphologies of the ZIFcoated PLA fiber membrane are shown in Figure 1b-e.
According to Figure 1b-e, it can be observed that the PLA fibers are uniformly covered with ZIFs layers, indicating that the PLA fiber membrane had successfully modified.

Modification of ZIFs Nanospike Coating
The size of structures of ZIF nanocrystals on PLA fiber membranes could be adjusted by changing the ratio of ZIF-8 and ZIF-67, which could directly influence bactericidal performances. Relative reports have reported that a smaller tip radius will induce a higher pressure on the bacterial membrane and hence improve the bactericidal effect. [32,41] Accordingly, the size of the effective bactericidal surface ranges from 100 nm to 1 μm in height, 10 to 300 nm in diameter, and horizontal space of <500 nm. [42,43] Different molar mass ratios of ZIF-8 and ZIF-67 could also obviously influence the surface structures. As shown in Figure 2, with the increase of the molar mass ratio of zinc sul-fate heptahydrate and cobalt sulfate heptahydrate, the aspect ratios of the microstructures increased and grew into nanospikes. Moreover, a smaller tip radius caused a higher pressure on the bacterial membrane and enhanced the bactericidal effect of the nanostructure surface.
When the molar mass ratio of zinc sulfate heptahydrate and cobalt sulfate heptahydrate was 1:1 (as shown in Figure 2a), the distribution of ZIFs structures was discontinuous with a relatively lower aspect ratio. With the increasing molar mass of ZIF-8 and ZIF-67 to 2:1, the aspect ratio increased, as shown in Figure 2b. However, the ZIFs tip of the columnar structure was not so sharp, which played a crucial role in sterilization. [44,45] When the molar mass ratio of ZIF-8 and ZIF-67 increased to 3:1, www.advancedsciencenews.com www.advmatinterfaces.de very sharp nanocolumnar structures with small tip radii were obtained, as shown in Figure 2c. The diameter of the nanocolumn was uniformly distributed, with a radius of ≈150 nm. However, when the molar mass ratio of ZIF-8 and ZIF-67 increased to 4:1, the diameter of the nanocollards was ≈500 nm, which was beyond the bactericidal diameter of 10-300 nm. Therefore, when the molar mass ratio of zinc sulfate heptahydrate and cobalt sulfate heptahydrate was 3:1, the sharpest structures were obtained successfully, which was supposed to bring the best bactericidal effects.

Antimicrobial Stability of Coatings
A schematic diagram of the nanospikes' antibacterial effects is shown in Figure 3a. When the bacteria contact with the nanostructured surfaces, very complex interactions occur between them. The antibacterial effects are influenced by a variety of factors of physical properties induced by micro-and nano-structures, such as the structural surface, the hydrophilicity, the electrostatic forces, the induction of oxidative stress, etc. Generally, the gravitation and hydrophilicity interactions usually occur between cell membranes and nanoarray surfaces, which are relatively weak and nonselective for different types of cell membranes. [43] The electrostatic force interaction between cells and surfaces depends on the type of bacteria and surface charges. It is well known that most bacterial cell membranes are negatively charged caused by the phospholipid layer. [46] When the nanostructured surface is positively charged, the microbial cell membrane exhibits a stronger electrostatic interaction with the structured surface. [47,48] When the cell membranes of bacteria are pierced by sharp spikes, the leakage of cytoplasm will lead to bacterial lysis and ultimately result in bacterial death. However, lethal stress will occur during the piercing process, triggering the production of ROS, which will induce the self-destruction of bacteria. These effects will lead to the inactivity or death of bacteria via ROS-mediated and oxidatively stressed effects. [49] Furthermore, the rough surfaces of nano-structured ZIF spikes will reduce the contact area between the surfaces and bacteria, which makes the bacteria easier to be inactive and even death. [50] These complicated interactions between the bacteria and the ZIFs nanospikes will facilitate to achieve the mechanobactericidal effects.
In order to observe the morphologies without deformation, the bacteria were fixed on the structures by glutaraldehyde when the solution was planted on the ZIFs nanospikes modified surfaces. As shown in Figure 3b, it could be observed that the morphologies and shape of E. coli show obvious deformation. Even some bacteria were pierced and cut off by ZIF spikes.
As has been discussed above, different morphologies of ZIFs coating exhibit obviously different bactericidal effects ( Figure S3, Supporting Information). The molar mass ratio of 3:1 of zinc sulfate heptahydrate to cobalt sulfate heptahydrate was adopted in the subsequent experiments. The quantitative characterizations of mechanobactericidal effects were carried out by gradient dilution of the bacteria solution for 24 h culturing in the dark environment. The results are shown in Figure 3d, and the E. coli colonies before and after sterilization were counted by CFU. Compared with the untreated E. coli with a number of ≈10 9 , E. coli on the ZIF-coated PLA surface reduced to ≈10 2 , indicating a bactericidal efficiency of >99.9999%. Moreover, the bactericidal efficiency of the ZIFs coating against other bacteria, such as Staphylococcus aureus and soft rot bacteria ( Figure S4, Supporting Information), can also reach 99.999%.
They are many factors, including the toxicity of ZIFs, the substrate, or even the release of Zn 2+ /Co 2+ , that could influence the antibacterial effects. In order to prove that the antibacterial effects were achieved by the nanospikes structures instead of the toxicity or the release of Zn 2+ /Co 2+ , relative experiments are conducted, and the results are shown in Figure S5 (Supporting Information). The E. coli in mixed solutions of ZIF-8 and ZIF-67 indicated that the possible toxicity has no obvious influence on the antibacterial activity, as shown in Figure S5b (Supporting Information). Similarly, the results shown in Figure S5c,d (Supporting Information) illustrated that releasing Zn 2+ /Co 2+ and the substrate (PLA) also had no obvious effects on the antibacterial activities. Therefore, it could be concluded that the antibacterial effects are attributed to the mechanobactericidal effects.

Antimicrobial Stability of Coatings
The stability of the ZIFs coatings is crucial when considering practical applications. As has been discussed above, the in situ growth of ZIF nanospikes would not be easy to fall off due to the higher adhesion forces between the three-layer structures of the PLA substrate, PDA, and the ZIF nanospikes. The stability experiments were conducted in liquid and air exposure, considering different application conditions, as shown in Figure 4. Compared with the original structures, no obvious variations of the morphologies of ZIFs coating after seven days' immersion in pure water, as shown in Figure 4b. Similarly, 120 days' exposure of these microstructures in air showed the same result, as shown in Figure 4c. A 50 times washing of ZIFs-coated PLA fibers was conducted with a water velocity of 5 mL s −1 and 30 s each time, and the result indicates that the fabricated nanospikes exhibited high stability, as shown in Figure 4d. Furthermore, the bactericidal effects of ZIFs coatings before and after 50 times washing were characterized respectively, and the bactericidal effects also must keep the same, Figure S6 (Supporting Information) conspicuously shows that the Y-axis remained virtually unchanged before and after washing, implying that the sterilization power had not deviated much. Subsequent to being washed 50 times, the ZIF-coated PLA's sterilization effect still surpassed 99.99%. These results indicate that the fabricated ZIFs coating has very high stability, which will facilitate a long-term life span and effective bactericidal performances.

Conclusion
To sum up, this study illustrates a general method of mechanobactericidal coating by in situ fabricating the biomimetic ZIFs nanospikes on the fabric substrate surfaces. The uniformly growing ZIFs nanospikes on fibers exhibit excellent mechanobactericidal effects for several kinds of bacteria. The characterizations indicate that the physically antibacterial ZIF nanospikes are effective and stable. The high sterilization efficiency, with simple and large-scale fabrication, and low cost and long-term life span provide a possible solution to drug resistance, which can be used in many fields, such as hospitals, emergency treatment, and superbug protection in the future.
The Fabrication of ZIF-8 and ZIF-67 Coatings: A 1.5 cm × 1.5 cm PLA fiber membrane was immersed in 35 mL dopamine hydrochloric acid solution (3 wt.%, pH 7.3) for 24 h. Then the fiber membrane was removed and cleaned repeatedly with deionized water, and then was immersed in a mixture of ZIF-8 and ZIF-67 for 4 h. The membrane was then removed, the coating was washed several times with ethanol to remove the residues on the surface of the membrane, and dried in an oven at 60°C. SEM Characterizations: The fibers' surface topographies before and after ZIFs (ZIF-8 mixed with ZIF-67) fabrications on the PLA fiber membrane were characterized by scanning electron microscopy (SEM, SU8000, Hitachi, Tokyo, Japan) to make sure that the PLA membrane was uniformly covered with ZIF coatings. The set parameters of SEM are 5 kV accelerate voltage, 10 μA current, and magnifications from 1000 to 50 000.
EDS Characterization: EDS accessories (Horiba 7021-H) were used to qualitatively analyze the components of the ZIF coatings by using local area mapping.
Colony Determination Experiment: The sterilized nutrient agar solution was poured into a hot Petri dish, cooled, and solidified naturally. The E. coli suspension was rinsed with PBS buffer three times, and the concentration was adjusted by diluting ten times based on an OD600 nm (optical density at 600 nm) value of 1. The bacterial turbidity was characterized by a spectrophotometer (Shimadzu UV3600). Four milliliters bacterial solution was put into a sample dish with a 15 mm diameter and cultured for 24 h. The diluting method was as follows: 0.5 mL bacteria solution was uniformly mixed with 4.5 mL PBS solution, and the ten times diluted bacterial solution was obtained. Different concentrations of dilution could be obtained by using the same diluting method. Each dilution with 200 μL bacteria solution was put into an agar plate respectively and uniformly coated on the agar plate using a sterilized coater. The coated petri dish was placed upside down under a constant temperature of 37°C for 24 h, and the number of colonies was counted by the Czone colony counting instrument. The average value of colonies between 30 and 300 was selected and multiplied by the dilution ratio, which was the number of colonies corresponding to the sterilization time.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.