Glycopolymer N-halamine-modified biochars with high specificity for Escherichia

Qinggele Borjihan,Quanfu Yao,Huihui Qu,Haixia Wu,Ying Liu3,,Alideertu Dong

1 College of Chemistry and Chemical Engineering,Inner Mongolia University,Hohhot 010021,China

2 Engineering Research Center of Dairy Quality and Safety Control Technology,Ministry of Education,Inner Mongolia University,Hohhot 010021,China

3 College of Life and Environmental Sciences,Minzu University of China,Beijing 100081,China

4 College of Chemistry and Environment,Hohhot Minzu College,Hohhot 010051,China

Keywords:Biochars N-halamine Glycopolymer Antibacterial coating Specific killing

ABSTRACT Antibiotic-resistant bacteria contamination in environments imposes great threats to human life health.This research aims to develop novel targeted antibacterial biochars for achieving high selectivity to kill pathogenic Escherichia coli (E.coli).The glycopolymer N-halamine-modified biochars (i.e.,BCPMA-Cl)were synthesized by the modification of biochars with poly[2-(methacrylamido) glucopyranose-coacrylamide] (P(MAG-co-AM),followed by chlorination treatment.Based on the results of FTIR,turbidity,XPS,and UV–vis,BCPMA-Cl was successfully synthesized and demonstrated to be able to eliminate Staphylococcus aureus (S.aureus) and E.coli.Especially,BCPMA-Cl possessed extremely potent to specific-killing 104 CFU∙ml-1 of E.coli with lower hemolytic activity(<5%).Additionally,the antibacterial mechanisms of BCPMA-Cl against bacteria were contact-killing and release-killing contributed by active chlorine (i.e.,Cl+).Therefore,this work provided a cost-effective and facile approach for preparation of functional biochars used for bacteria-specific therapeutic applications via livestock pollutants as well as showing a promising strategy to avoid bacterial resistance.

1.Introduction

The abuse of antibiotics in agriculture and biomedical fields has created multi-drug resistant bacteria that poses a serious threat to community health worldwide [1,2].Therefore,various antibacterial materials have been created and achieved great success in fighting against bacterial infections,including two-dimensional nanosheets,quaternary ammonium salts,peptides,povidoneiodine,and N-halamines [3–7].However,broad-spectrum antimicrobials attack bacteria indiscriminately and may result in the emergence of many strains of multi-drug resistant pathogens along with undesirable health concerns.In order to combat pathogens without causing drug-resistance,it could be a smart strategy to develop the pathogen-specific-killing technology through incorporating bacteria-targeted group into the antibacterial system.

Escherichia coli(E.coli)is an important part of a healthy human intestinal tract.Actually,E.coli consists of a diverse subgroups,most of which are harmless,however some are pathogenic.Pathogenic E.coli arises varied kinds of damages and pathological responses in the gastrointestinal tract[8,9].It is worthy to develop E.coli-specific-killing agents for highly targeted killing the E.coli.Glycopolymers with long chains monomers are effective in targeting E.coli attributing to good affinity between glycopolymer and FimH protein of E.coli [10–12].N-halamines have many superior functional features,including non-toxicity and effectiveness against pathogens,high stability and durability as well as the renewable characteristic of their antibacterial groups [13–16].The antibacterial mechanism of N-halamine involves that the active chlorines penetrate bacterial cells and attack intracellular contents through oxidation,as a result denaturing proteins.Since N-halamines are able to nonspecifically interact easily with important bacterial proteins,they are unlikely to induce drug resistance of bacteria [17–18].Accordingly,a collaboration between Nhalamine and glycopolymer endows E.coli-specific recognition and binding ability will significantly improve the E.coli-killing effciency.

The past several decades have witnessed the great development of carbon materials in chemistry and material science[19–20].Carbon nanotube (CNTs) [21],graphene oxide (GO) [22],graphdiyne(GDY) [23],and biochars (BC)[24] are the most typical ones.In comparison,the former three commonly were separated from refined and sophisticated carbon templates via a harsh process,however,biochar is well-sourced and available in a mild condition.As a highly aromatic carbon-rich solid matter,biochar is produced easily by slow pyrolysis of plant or animal wastes in hypoxic environments[25,26],Biochar is recognized as an ideal carrier material owing to its large surface area,high stability,and chemically inertness [27].More significantly,abundant oxygenated functional groups(-COOH,-OH,-COOR,etc.)on the surface of biochar offer versatile possibilities for covalently functionalization[28].Furthermore,it is well known that biochar is a highly efficient and environmentally friendly adsorbent [29].To date,biochar has been succeed in water purification of various organic and inorganic pollutants,such as antibotics,dyes,heavy metals and so on [30–32].However,few research have considered on combating pathogenic bacteria pollution by using biochars.

Herein,we developed an effective E.coli-specific-killing agent(BCPMA-Cl) by modifying biochar with glycopolymer Nhalamines.In this work,the glycopolymer poly[2-(methacryla mido)glucopyranose] (PMAG) acts as the especially recognizer of E.coli pili protein,and N-halamine group of copolymers functions as an oxidative chlorine (Cl+)-bearing agent to inactivate E.coli.The antibacterial activities of the as-designed BCPMA-Cl against E.coli and S.aureus as well as the E.coli-specific-killing,were evaluated.Also the hemolysis of BCPMA-Cl was examined.We believe this strategy promises a biocompatible and environment-friendly antibacterial agent to fight against pathogens.

2.Materials and Methods

2.1.Materials

All chemical reagents were of analytical grade and purchased from Aladdin Biochemical Technology Co.,Ltd.or Tianjin Beilian Fine Chemicals Development Co.,Ltd.,China.All these chemicals were used as received without further purification.

2.2.Characterization

The Fourier transform infrared (FT-IR) spectra were recorded using a FT-IR spectrometer (NICOLET6700,Thermo Fisher,USA).The UV–vis spectra were observed by a UV–vis spectrophotometer(U-3900).XPS spectra were performed on an ESCALAB 250Xi XPS system (Thermo Fisher,USA).The morphology and elemental information were obtained with a field emission scanning electron microscope (SEM) (SSX-550,Shimadzu) at 15.0 kV.

2.3.Synthesis

2.3.1.Biochars

The biochars were prepared by using a reported method [33].Briefly,after being air-dried and grinded,livestock wastes were pyrolyzed in a tube furnace at 600 °C for 5 h at a heat rate of 10 °C.min-1under nitrogen circulation condition.

2.3.2.2-(Methacrylamido)glucopyranose (MAG)

MAG was synthesized according to a reported method [34].Typically,D-(+)-glucosamine hydrochloride (5 g,23.2 mmol) and K2CO3(5 g,36.2 mmol)were magnetic stirred in anhydrous methanol at 0 °C for 30 min,then dropwise addition of methacryloyl chloride (20.7 mmol) into above reaction mixture.Kept at 0 °C and continued to react for another 4 h,and the residue was purified with silica-gel column chromatography (elute:dichloromethane/anhydrous methanol=4:1) to give a white solid MAG (2.05 g,41% yield).

2.3.3.P(MAG-co-AM)-modified biochars (BCPMA)

According to literature,briefly,BC (0.2 g),acrylamide (AM)(0.4 g),and MAG (0.4 g) were immersed into 50 ml of DMF.Then,0.05 g of KPS was added into the reaction mixture to start the polymerization process at 75°C for 12 h under nitrogen atmosphere to achieve BCPMA.As control,polyacrylamide-modified biochars(BCPA) were prepared in a similar procedure.

2.3.4.BCPMA-Cl

Typically,BCPMA(0.2 g) was dispersed in 20 ml of commercial NaClO solution(4.5%,by mass)under mechanical agitation at room temperature.The chlorination treatment was conducted for different aging periods to give crude products of BCPMA-Cl,then they were purified by repeated washing with distilled water to remove residual free chlorine from the surface of samples and vacuum drying for 2 h.Meanwhile,BCPA was also treated with chlorination in similar approach to obtain BCPA-Cl as control.

2.4.Determination of oxidative halogen content

The percentage of active chlorine of BCPMA-Cl obtained by treatment of different chlorination time was determined by the iodometric/thiosulfate titration method.

2.5.Chlorine release testing

The suspension of BCPMA-Cl in distilled water was placed into a dialysis bag(100 Da) and immersed into distilled water.To examine the amount of Cl+released from BCPMA-Cl over time,the dialysate solutions during a period of time were took and measured via the iodometric/thiosulfate titration method.

2.6.Antibacterial performance

Two model strains(S.aureus and E.coli)were mixed at radio of 1 :1 at a density of 1 × 104colony-fomring units (CFU) per ml,which was added into BCPA-Cl and BCPMA-Cl(10 mg.ml-1)for different contact periods,respectively.Then the bacterial survival and density were determined and quantified by the colony counting assay in order to evaluate the antibacterial activity of BCPA-Cl and BCPMA-Cl.

2.7.Hemolysis studies

Fresh mice blood was separated by centrifugation at 5000 r.min-1for 15 min,and washed with PBS buffer (pH 7.2).BC,BCPMA,BCPMA-Cl,trition-100 (1%) (negative control),and PBS(positive control),were diluted by the erythrocyte suspension and incubated for 2 h,respectively,then centrifuged at 5000 r.min-1for 15 min.Hemolytic activity was measured by OD450using a microplate reader.The hemolysis percentage(%)was calculated using Eq.(1) below:

where Asis the absorbance value of examined samples,A-is the absorbance value of negative control (red blood cells in trition-100),and A+is the absorbance value of positive control (PBS).

Fig.1.Schematic illustration of synthesis of BCPMA-Cl for E.coli-specific-killing.

3.Results and Discussion

As schematically illustrated in Fig.1,the E.coli-specific-killing biochars (i.e.,BCPMA-Cl) were syethesised via following steps:(i) biochars production,(ii) polymer loading,and (iii) chlorination treatment.The construction of BCPMA-Cl was confirmed by a series of characterizations,including FT-IR,XPS,UV–vis and SEM analysis.The FT-IR spectra of BC,PMAG,BCPA,and BCPMA are shown in Fig.2.The peak around 1660 cm-1was assigned to C=O in amide from AM and MAG,and the intensities of the C=O bonds in BCPMA were greater than that of BCPA (Fig.2A) [35].As can be seen in the second derivative spectra (Fig.2B),a double peak of -NH2from AM could be observed at 3440 and 3377 cm-1in BCPA,and BCPMA,indicating that P(MAG-co-AM) copolymer was successfully loaded onto biochars [36].

Fig.2.FT-IR spectra (A) and the second order derivative spectra (B,C) of BC,PMAG,BCPA,and BCPMA,respectively.

Fig.3.(A) Photographs of dispersions of BC and BCPMA after standing for 0,5,15,30,and 60 min,respectively.(B) Turbidity of BC and BCPMA.

Fig.4.(A–C) SEM images of BC,BCPMA,and BCPMA-Cl,respectively.(D) SEM-mapping,(E) SEM-line scanning,and (F) EDS images of BCPMA-Cl respectively.

Fig.5.(A)XPS survey scan,(B)N 1s spectrum,and(C)Cl 2p spectrum of BC,BCPMA,and BCPMA-Cl.(D)C 1s peak of BC,(E)C 1s peak of BCPMA.(F)Mole percentage of the separated peaks for C 1s of BC and BCPMA.

Fig.6.(A)UV–vis spectra of BC,BCPMA,and BCPMA-Cl after react with KI/H+.(B)The relationship between active chlorine mass content of BCPMA-Cl and chlorination time of BCPMA-Cl.(C) Photographs of color changes of BCPMA-Cl by iodometric titration.

Meanwhile,the turbidity,XPS and SEM elemental mapping were conducted to further demonstrate the loading of copolymer on biochars.As seen in Fig.3,the dispersity of BC and BCPMA in water was tested by monitoring their turbidities.The turbidity of BCPMA is higher than that of BC,which is owing to the hydrophilic copoymer P(MAG-co-AM) loading on the surface of BC.SEM imaging was used to monitor the morphology difference of biochars before and after polymer loading.As shown in Fig.4A and B,the pristine BC offers an irregular morphology with smooth surface and sharp edges,while the BCPMA exhibits rough surface on the schistose BC,demonstrating that copolymer based on Nhalamines were loaded on the surface of biochars.After chlorination treatment,the as-synthesized BCPMA-Cl (Fig.4C) displays a similar morphology to that of unchlorinated precursor BCPMA.The SEM elemental mappings show uniform distributions of C,N,O,and Cl elements [28],confirming that all elements are well matched with their corresponding morphologies,and indicate that P(MAG-co-AM) could scatter uniformly on the surface of BC.

Additionally,we carried out XPS study on BCPMA-Cl,BCPMA and BC.The peaks of N 1s arise from P(MAG-co-AM) in BCPMA-Cl and BCPMA(Fig.5A and B),and compared to BCPMA,the signal of Cl 2p at 199.6 eV appears in BCPMA-Cl,suggesting the success of the N -H →N -Cl transformation by chlorination (Fig.5C)[37,38].The XPS spectra of tested samples all appear two characteristic peaks attributing to C 1s and O 1s[39].Moreover,the signal intensity of O 1s rises significantly after P(MAG-co-AM)loading on the BC (Fig.5A).When the signal of C 1s in BC and BCPMA are deconvoluted (Fig.5D and E),BC show four separate binding energy peaks at 284.4,284.8,286.2.5 and 288.1 eV,which are corresponding to C-C sp2,C-C sp3,C-O,and C=O,respectively[39,40].BCPMA present five separate binding energy peaks at 284.4,284.9,285.4,286.0 and 287.9 eV,belonging to C-C sp2,C-C sp3,C-N,C-O,and C=O,respectively.As shown in Fig.5F,the molar percentages of C-C sp3,C-O,C-N and C=O bound of BCPMA in the C 1s peak are significantly higher than that of BC owing to the P(MAG-co-AM) loading on BC.

Fig.7.(A) Schematic illustration of specific E.coli-eradicating;Antibacterial kinetic assay against E.coli and S.aureus of BCPMA-Cl (B) and BCPA-Cl (C);(D) Illustration of culture plates of E.coli and S.aureus.(E)Photographs of the bacterial culture plates of E.coli and S.aureus after different aging time exposure to BCPMA-Cl and BCPA-Cl;(F)Illustration of chlorine release testing process;(G)Photographs of color changes of released chlorines’iodometric/thiosulfate titration;(H)The relationship between chlorine mass content released from BCPMA-Cl and released time.

Fig.8.The hemolysis photographs (A) and hemolysis rates (B) of PBS,Trition-100,BC,BCPMA,and BCPMA-Cl,respectively.

In Fig.4C–E,the appearances of elemental signal of the Cl in EDX spectrum and elemental mapping of BCPMA-Cl demonstrate the success of chlorine loading on BCPMA,which is consistent with the result based on XRD spectrum of BCPMA-Cl.Additionally,the UV–vis absorption spectrum of BCPMA-Cl appeares two peaks at 293 nm and 351 nm when BCPMA-Cl is titrated with KI in the presence of H+,confirming the presence of oxidative halogen (Cl+)(Fig.6A) [41].The amounts of active Cl+released from N-Cl covalent bonds of BCPMA-Cl treated with different chlorination time were evaluated using an iodometric titration method.N-halamines first oxidize iodide ions to produce iodine,which is exhausted by titrated thiosulfate to color fade.It is no surprising that the chlorination time relates to the content of active Cl+of BCPMA-Cl,a sharp increasing trend of chlorine loading mass content from 0% to 0.35% is detected within a lower aging period(i.e.,below 2 h) (Fig.6B).When the aging period further extends to 4 h,the increasing trend slows down gradually and moves towards balance.

Antibacterial activities of modified biochars were investigated by colony counting.As illstrated in Fig.7A,the mixture of two model bacterial (104CFU.ml-1) was determined upon 10 mg∙ml-1of samples (BCPA-Cl and BCPMA-Cl) for a certain aging time to assess the target specificity of modified biochars.Fig.7E show photographs of culture plates,with the surviving E.coli and S.aureus appearing respectively as big and small white dots,as schematically shown in Fig.7D.BCPA-Cl has equal antibacterial efficiency on two types of model strain.However,the antibacterial avtitity of BCPMA-Cl on E.coli is higher than that on S.aureus (Fig.7B and C and E),which suggests that the selectivity binding between BCPMA-Cl and E.coli can indeed enhance the killing ability of BCPMA-Cl against E.coli,attributing to the existence of MAG.Glyco-sensitive fimbriae in E.coli mediates binding to receptor structures to allow bacteria to adhere to BCPMA-Cl.Thus,BCPMA-Cl killing E.coli mainly depend on contact killing and combine with released Cl+ions,which result in a stronger antibacterial ability.In contrast,MAG on the BCPMA-Cl cannot selectively bind to S.aureus cells,resulting a poor antibacterial ability.

To examine the active chlorine release mechanism of BCPMA-Cl,a dialysis test was carried out using the dialysis bag (100 Da) test(Fig.7F).Specificly,the dialysis bag containing BCPMA-Cl was immersed into distilled water for 3 h,and then active chlorine concentration in the outer water system was measured.The titrated reactions and corresponding color changes are shown in Fig.7G.The results proved that a small amount of chlorine could be released out of the dialysis bag to kill S.aureus and E.coli.Therefore,specific affinity of BCPMA-Cl for the pili of E.coli allows fully contacting with a large amount of unreleased Cl+,which lead to the improves of targeted bacteria-killing capacity (Fig.7H).

Hemolysis is an important index on safety for biomedical applications.In our hemolysis test,we used 1%of triton-100 as negative control.In the BC,BCPMA and BCPMA-Cl centrifuge tubes,the blood deposits at the bottom,and the supernatant is as clear as the PBS group (Fig.8A).As shown in Fig.8B,all tested samples show low hemolytic activity (<5%) after being incubated for 1 h.The results reflect that BC,BCPMA and BCPMA-Cl all have low toxicity,importantly,the modified biochars have obvious enhanced ability to inhibit bacteria but the hemolysis still keep lower.

4.Conclusions

In summary,this work provided a facile strategy to develop novel biochars based on glycopolymer N-hamamine for specific killing of E.coli by introducing specific targeting groups to antibacterial N-halamine system using biocompatible and environmentfriendly biochars as a support template.The as-synthesized modified biochars display higher specific killing of E.coli owing to specific adhesion between MAG with glycol-sensitive fimbriae of E.coli compared to that of S.aureus without glycol-sensitive fimbriae.Additionally,the modified biochars have low hemolytic activity during sterilization.On the basis of these results,we believe the modified biochars could be applied as targeted antibacterial agents for microorganism decontamination.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China(21304044,51663019,and 22062017),the Natural Science Foundation of Inner Mongolia Autonomous Region(2015MS0520,2019JQ03 and 2019BS02004),the State Key Laboratory of Medicinal Chemical Biology(201603006 and 2018051),the State Key Laboratory of Polymer Physics and Chemistry(2018-08),and the Program of Higher-Level Talents of Inner Mongolia University (30105-125136)