N-Halamine Functionalized Electrospun Poly(Vinyl Alcohol-co-Ethylene) Nanofibrous Membranes with Rechargeable Antibacterial Activity for Bioprotective Applications
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Developing bioprotective materials with bactericidal activity is of great significance since it can effectively keep healthcare workers from infection by emerging infectious diseases; however, this is still a big challenge. Herein, we fabricate a novel rechargeable N-halamine antibacterial material by functionalizing electrospun poly(vinyl alcohol-co-ethylene) (EVOH) nanofibers with dimethylol-5,5-dimethylhydantoin (DMDMH). The premise of the design is that the N-halamine compound, DMDMH, can be covalently grafted on the nanofibers, endowing the EVOH nanofibrous membranes (ENM) with rechargeable and durable bactericidal activity. The as-prepared DMDMH functionalized ENM (EDNM) render rechargeable chlorination capacity (> 2000 ppm), high inactivation efficacy against bacteria (> 99.9999% within 3 min), high filtration efficiency (> 99.5%) under low air resistance, and robust mechanical properties, which are due to the synergistic effect of the unique characters of N-halamines and electrospun nanofibrous architecture. The successful synthesis of the N-halamine antibacterial membranes can serve as a functional layer of protective equipment that capable of inactivating and intercepting pathogenic bioaerosols, providing new ways into the development of new-generation antibacterial bioprotective materials.
KeywordsElectrospinning Nanofibers Rechargeable Antibacterial Bioprotective applications
Emerging infectious diseases (EIDs) have been considered as a major global public health threat [1, 2]. EIDs, such as Middle East respiratory syndrome virus, pandemic H1N1, and Ebola virus disease, caused incalculable death and afflicted millions of people annually . By way of example, the outbreak of Ebola in West Africa caused nearly 30,000 confirmed patients and over 10,000 deaths, according to the data of the World Health Organization . Particularly, the healthcare workers (HCWs) who were committed to deliver care and services to infected patients suffered from a high risk of acquisition of work-related infectious illnesses . In the epidemic area, the protective equipment for HCWs played a critical role in preventing the transmission of pathogens, involving face shields, protective suits, and gloves . Whereas current protective equipment is typically a temporary physical interception, rather than thoroughly inactivation of pathogens . Accordingly, more effective and durable bioprotective materials to provide antibacterial protection are highly needed, especially for the emergency medical aid outdoors.
Currently, a variety of disinfectants are introduced to design and develop antibacterial materials, such as quaternary ammonium salt, silver nanoparticles, chitosan, and peptides, which can effectively prevent and control bacteria contamination [8, 9, 10, 11, 12]. However, these disinfectants experience various problems like irreversible consumption of disinfectants, slow inactivation rate, and poor durability, putting limitations on their practical use in bioprotective applications. Alternatively, N-halamine compounds that generated by the halogenation of nitrogen–hydrogen (N–H) bonds-containing precursors hold great promise as antibacterial materials in bioprotection field due to their integrated properties of rechargeable bactericidal activity, rapid inactivation rate, good human and environmental safety, and high durability [13, 14]. In view of their outstanding potential, several researches have been performed via directly using N-halamine precursor polymers that contained N–H bonds (such as polyamide and aramid) to develop N-halamine antibacterial materials, but the strong hydrogen bonds between the polymer chains inhibit the conversion of N–H groups to bactericidal N-halamine groups and the type of these polymers is limited [15, 16]. Besides, a few explorative works suggested that N-halamine antibacterial materials could be constructed by physically coating or blending N-halamine compound into substrate [17, 18, 19]; however, these compounds might be wiped off from the substrate when an external force was applied on the material, resulting in a dramatic decrease of the bactericidal activity. Therefore, it is still a challenge to fabricate covalently incorporated and structurally stable N-halamine materials with rechargeable antibacterial activity.
In addition to the powerful and rechargeable bactericidal activity, air filtration performance is of equal importance to the bioprotective materials, which can effectively decontaminate bioaerosols that containing pathogenic particles [20, 21]. More recently, benefiting from the high specific surface area, small pore size, tunable tortuous open-porous structures, and high porosity, electrospun nanofibrous materials showed great potential to construct bioprotective materials with excellent filtration performance [22, 23, 24, 25]. Taking electrospun nanofibrous membranes as the substrate and further coupling with N-halamine bactericidal groups has been considered as a promising approach to fabricate bioprotective materials with rechargeable antibacterial and excellent filtration performance.
Herein, we demonstrate an effective and facile strategy to create rechargeable antibacterial nanofibrous membranes by grafting dimethylol-5,5-dimethylhydantoin (DMDMH) onto poly(vinyl alcohol-co-ethylene) (EVOH) nanofibers. As far as we know, this is the first time that electrospun EVOH nanofibrous membranes (ENM) is functionalized with DMDMH to fabricate bactericidal membranes for bioprotective applications. The N-halamine precursor compound, DMDMH, is covalently grafted on EVOH nanofibers, ensuring the durable and rechargeable bactericidal activity of the membranes. The resulting DMDMH functionalized ENM (EDNM) exhibited the integrated properties of rechargeable chlorination capacity, excellent bactericidal activity, high particle removal efficacy, and robust breathability, which can serve as a scalable bactericidal and filtration layer of bioprotective equipment.
EVOH (ethylene content of 27 mol%) was obtained from Kuraray Co., Ltd., Japan. Aqueous solutions of DMDMH (solid content of 55 wt%) were purchased from Shanghai Wanhou Biotechnology Co., Ltd., China. Isopropyl alcohol (IPA), magnesium chloride (MgCl2), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were bought from Aladdin Chemistry Co., Ltd., China. A bleach solution with 3.4 wt% to 4.6 wt% of active chlorine content was supplied by Jiangsu aitefu group Co., Ltd., China. Iodine (I2) standardized solution (0.01 N), sodium thiosulfate (Na2S2O3) standard solution (0.1 N) were obtained from Acros Organics Co., Ltd., USA. Luriz-Bertani (LB) broth, LB agar and phosphate buffered saline (PBS) buffer were purchased from Shanghai Sangon Biotech Co., Ltd., China.
Preparation of ENM
An 8 wt% EVOH transparent solution was prepared by dissolving 3.2 g of the polymer in a 36.8 g of IPA/deionized water mixture (7/3, v/v) with stirring for 6 h in a water bath at 80 °C. Then the EVOH transparent solution was transferred into the syringes for the following spinning experiment, and the ENM were fabricated by an electrospinning setup, which was bought from SOF nanotechnology Co., China. The spinning process was operated with a voltage of 30 kV, a fixed speed of 4 mL h−1, and a tip-to-collector distance of 23 cm. The spinning environment temperature was 22 ± 3 °C and the humidity was 50 ± 5%.
Fabrication of EDNM
The EDNM were prepared by first dipping the ENM in an aqueous solution containing DMDMH and the catalyst (MgCl2 at 10% of the weight of DMDMH) for 30 min. Subsequently, the membranes were transferred into a drying oven for 1 h at 50 °C, and then the grafting reaction was carried out for 1 h at 140 °C. The modified membranes from various DMDMH concentration of 0.5, 1, and 3 wt% were named EDNM-0.5, EDNM-1, and EDNM-3, respectively.
Chlorination and Analytical Titration
Bacterial Culture and Antibacterial Testing
The E. coli (Gram-negative bacteria, ATCC 25922) were incubated in LB broth of 10 mL for 24 h at 37 °C, and then they were harvested and washed twice with PBS by centrifugation. Subsequently, the obtained bacteria were resuspended in diluted LB broth with chemical oxygen demand (COD) of 2000 or sterilized deionized water to get bacterial suspensions with concentration of 3 × 108 CFU mL−1 for the following experiments. For the antibacterial testing, the membranes were cut into a circle of 2 × 2 cm2 and put in a perforated plate, then each sample was subjected to 10 μL of the bacterial suspensions and incubated on an automated shaker at 37 °C with different contact time. At predetermined time points, a sample loaded with bacteria was taken out and immersed in a test tube that containing sterilized deionized water of 1 mL to obtain the bacterial suspension. Then the suspension was diluted serially (× 101, × 102, × 104, and × 106) and cultured on agar plates for enumeration.
Morphology Observation of Bacteria
The bacteria were first harvested from the membranes by vortexing and fixed with PBS solution that contained 2.5 wt% of glutaraldehyde for 1 h. Subsequently, the bacteria were dehydrated with a series of ethanol/water solution of 50%, 70%, and 90%. Finally, the bacterial suspension was dropped on fibrous membranes for SEM analysis.
The morphologies of the modified nanofibrous membranes and bacteria were investigated using scanning electron microscopy (SEM) (Tescan Vega 3, Czech). The chemical structures of the membranes before and after modification were performed by Fourier transform infrared (FT-IR) spectrometer (Thermo Scientific Nicolet iS10, USA). The dynamic water permeation process on the membranes was recorded by contact angle measurement (Kino SL200B, USA), and a droplet of 5 μL was used. The pore size distribution of the membranes was investigated by the capillary flow porometer (Porous Materials Inc. CFP-1100AI, USA). The tensile property of the samples was measured by using tensile tester (XQ-1C, China). The Brunauer–Emmett–Teller (BET) surface areas and N2 adsorption–desorption isotherms were investigated by physisorption analyzer (Micromeritics, ASAP 2020, USA). The chemical element composition of the membranes was tested through X-ray photoelectron spectroscopy (XPS) (Escalab 250Xi, USA). The filtration efficiency and pressure drop were characterized using a filter tester (Huada LZC-H, China).
Results and Discussion
Synthesis and Bactericidal Functions of EDNM
We designed the EDNM based on the following three principals: (1) the N-halamine moieties should be covalently bonded to nanofibrous membranes, (2) the membranes should be able to inactivate the bacteria and the bactericidal activity is rechargeable, and (3) the pathogenic particles must be effectively intercepted by the membranes. The first requirement can be satisfied by the grafting reaction between the EVOH nanofiber substrates and the N-halamine compound. To meet the other two requirements—the construction of rechargeable bactericidal nanofibrous filter—we combined the unique properties of N-halamine compound and electrospun nanofibrous architecture.
Morphologies and Structure of EDNM
To reveal the effect of the modification on the pore structure of the membranes, N2 adsorption–desorption measurements at 77 K were employed to study the porous structure of the membranes. As shown in Fig. 3c, the N2 adsorption–desorption isotherm curves represented typical IV type with tiny H3 hysteresis loops, implying the characteristics of mesopores within the ENM and EDNM [31, 32]. The inset of Fig. 3c demonstrated the BET surface areas of the membranes, which revealed slight decrease of the relevant surface area with the increasing DMDMH contents. This phenomenon illustrated the fact that the DMDMH played a critical role in deciding the surface area of the EDNM, which was ascribed to the increasing fiber diameter as a consequence of the adhesive structure. The membranes were further immersed into the chlorination solution, and then we utilized the iodometric titration method to determine the active chlorine content of the membranes. As we can see from Fig. 3d, the pristine ENM without DMDMH did not loaded with active chlorine due to the lack of N–H groups in EVOH polymer chains. The use of the DMDMH concentrations from 0.5 to 1 wt% presented a dramatic increase in active chlorine contents from 1830 to 2520 ppm, which was attributed to the increased N-Cl groups derived from more available N–H bonds. However, further increasing the concentration of DMDMH (3 wt%) resulted in a decline of active chlorine content. This phenomenon was ascribed to the decreased surface area that reduced the active sites of the nanofibers, revealing that the modification concentration of 1 wt% was enough to endow the EDNM with considerable chlorine content.
Rechargeable Chlorination Capability of EDNM-Cl
In order to understand the chemical composition changes of the membranes before and after chlorination, we analyzed the chemical composition of EDNM and EDNM-Cl by means of XPS (Fig. 4c). Compared with the EDNM, the chlorinated membranes represented a new characteristic peak (Cl 2p at 200.75 eV), indicating that the chlorine had been loaded on the membranes successfully [38, 39]. Additionally, Cl 2p core level spectrum was presented in Figure S2, which was on behalf of the N-Cl bonds. Owing to the renewable feature of N-halamine structure, the EDNM-Cl exhibited rechargeable chlorination capacity. As shown in Fig. 4d, the cyclic chlorination testing of EDNM-Cl was performed, the samples were chlorinated at pH of 5 for 1 h and then impregnated into an excess of Na2S2O3 solution to consume all active chlorine. After that, the membranes were washed several times with deionized water and immersed into chlorination solution for the next cycle. Interestingly, the active chlorine content displayed a slight decrease at the second cycle, and then it tended to be stable around 2000 ppm in the following cycles. The robust rechargeable chlorination capability was due to the stability of the N-halamine moieties. In addition, the SEM image of the membranes after 5 cycling chlorination tests was investigated. As we can see from Figure S3, there was no significant change in the morphology of the nanofibers after the cycling tests, suggesting the good stability of the nanofibrous architecture.
Bactericidal Activity and Filtration Performance of EDNM-Cl
To further evaluate the practical utilization of EDNM-Cl for bioprotective applications, we demonstrated the filtration performance of the membranes. Figure 5d exhibited the filtration efficiency and pressure drop of EDNM-Cl with virous basis weight, which were determined through feeding a given mass of aerosol containing sodium chloride particles with a diameter of around 0.3–0.5 μm under the flow of 32 L min−1. By increasing the basis weight of EDNM-Cl, the filtration efficiency exhibited distinct increase from 43.4 to 99.5% and the pressure drop showed an increased trend, revealing the critical role of the basis weight in enhancing the filtration performance of membranes . It was clearly visible that the filtration efficiency of EDNM-Cl could reach 99.1% with a low pressure drop of 105 Pa when the basis weight exceeded 3.2 g m−2, which was significantly higher than that of the N95 surgical masks (95%) used by healthcare workers. In addition, the SEM images of the top and bottom surface of EDNM-Cl after filtration were investigated. As we can see from Fig. 5e, almost all particles were trapped on the surface of the membranes, whereas no particle was observed on the bottom of EDNM-Cl, indicating a typical surface filtration for membrane filter media . Based on the above results, we believed that the EDNM-Cl was capable of resisting the invasion of bioaerosols that containing pathogenic bacteria and particles for HCWs, which was similar to the contaminated air in the epidemic area (Fig. 5f). When the bioaerosols passed through the materials, the fine particles and bacteria would be intercepted and inactivated by the nanofibers, yielding the clean air. Most importantly, as shown in Figure S4, a large-scale EDNM-Cl (60 × 70 cm2) was readily available, which made it possible for industrial application in bioprotective equipment.
In summary, we developed a novel, effective, and facile strategy for fabricating rechargeable antibacterial N-halamine nanofibrous membranes by combining the electrospun ENM and surface functionalization with DMDMH. The rechargeable N-halamine moieties were successfully grafted onto nanofibers, endowing the ENM with rechargeable high active chlorine content (> 2000 ppm). Benefiting from the three-dimensional nanofibrous architectures, rechargeable chlorination capacity, ease of industrialization, and robust mechanical properties, the resulting modified membranes exhibited 6 log reduction against typical pathogenic E. coli within 3 min of contact time; meanwhile, the membranes rendered high particle removal efficiency (99.1%) and low pressure drops (105 Pa) with the basis weight of 3.2 g m−2. These intriguing attributes enabled the EDNM-Cl to act as a functional layer of the protective equipment, which could intercept and inactivate the pathogenic air. The successful synthesis of the EDNM-Cl might also open new ways for the development of antibacterial materials for various applications.
This work was supported by the National Natural Science Foundation of China (Nos. 51773033, and 51673037), and the Shanghai Committee of Science and Technology (No. 18511109500).
Compliance with Ethical Standards
Conflict of interest
There are no conflicts to declare.
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