ZHANG Huan ,ZHAO Qinghua ,ZHONG Yaping ,CAI Ruiyan ,LIU Ke ,WANG Dong ,LU Zhentan＊

(1. Key Laboratory of Textile Fiber and Products, Wuhan Textile University, Ministry of Education, Hubei International Scientific and Technological Cooperation Base of Intelligent Textile Materials &Application, School of Materials Science and Engineering, Wuhan Textile University, Wuhan 430200, China; 2. Guangdong Engineering Technology Research Center of Functional Fibers for Clothing, Guangdong Bobaolon Co., Ltd., Puning 515300, China)

Abstract: The work is dedicated to develop a one-step eco-friendly method to prepare antibacterial polyethylene terephthalate (PET).We report a one-step eco-friendly method to manufacture antibacterial PET via on-line amination reaction by melt coextrusion.Beside evenly mixing of poly(hexamethylene guanidine)(PHMG) and PET in the melt coextrusion procedure,the amination reaction also occurred between PHMG and PET under high temperature (230-270 ℃).The antibacterial ability of composite PET showed obvious PHMG concentration dependence,and antibacterial activity reached more than 99% when PHMG content was 2.5 wt%.Moreover,LIVE/DEAD fluorescence test further confirmed that the composite PET could kill bacteria quickly and effectively (within 30 min);while negligible cytotoxicity was observed to HSF and HUVEC cells.Onestep eco-friendly fabrication of composite antibacterial PET was accomplished by on-line melt coextrusion.The composite antibacterial PET has potential use in multiple fields to combat with pathogenic including textiles,packaging materials,decoration materials and biomedical devices, etc.

Key words: antibacterial polyester;one-step eco-friendly method;on-line amination reaction;melt coextrusion;cytocompatibility

1 Introduction

Polyethylene terephthalate (PET),as commodity polymer,are widely used in industrial,household and medical fields,which could be made into textiles,packaging materials,decoration materials and biomedical devices,etc[1-6].The proliferation of pathogenic on PET could result in unfavorable change to mechanical properties and appearance,and also bring serious harm to public health[7,8].Antibacterial PET could significantly improve public health,which attracted growing attentions in both industry and academia field[9,10].Green and low-carbon short process technology are the new trend in industry,including textiles and polymer processing[11].Therefore,developing one-step eco-friendly method for preparing antibacterial PET meet the needs of today’s industry.

Currently,the fabrication strategies of antibacterial PET are mainly divided into three categories:(1) melt blending with inorganic antibacterial nanoparticles;(2) loading antibacterial agent after hydrolysis or aminolysis;(3) surface chemical modification or physical adsorption antibacterial agent.The first strategy,melt blending is the most effective and simple method to manufacture antibacterial composite materials.PET could obtain excellent antibacterial ability after melt blending with various metal nanoparticles.However,inorganic antibacterial nanoparticles will aggregate during melt blending with polymers,which cause the decline in the performance of polymer nanocomposites.And,metal antibacterial agents also have the heavy metal toxicity[12,13].Zhouet alprepared antibacterial polyester based on organic-inorganic hybrid strategy,which could avoid the aggregation of antibacterial nanoparticles effectively[14,15].However,the synthesis of micro-nano hydrid antibacterial nanoparticles was complicated,requiring multiple chemical process.No active groups on molecular skeleton are the main reason why PET is difficult to functionalize.The second strategy of hydrolysis and aminolysis can give PET a large number of active groups,which is conducive to obtain the antibacterial ability,and could improve its hydrophilicity[16,17].However,the hydrolysis and aminolysis strategies require the participation of a variety of chemical reagents.The third strategy of surface chemical modification or physical adsorption antibacterial agent has no effect on the molecular structure of PET skeleton.Chenet alprepared multifunctional antibacterial PET fabrics by surface chemical modification[18].Zhanget alprepared antibacterial PET fabrics by surface adsorption graphene[19].Despite these methods achieved considerable advances compared with previous work,it is still not optimal owing to the using of chemical reagents being harmful to the environment and the multi-step operation consuming a lot of energy.

Organic antibacterial agents have no heavy metal toxicity and accumulation,and it has good structural compatibility with PET,easy to disperse evenly.Melt blending is an eco-friendly method,which does not use any harmful organic solvents.Unfortunately,most of organic antibacterial agents have poor heat resistance,and are not suitable for melt blending to manufacture composite antibacterial PET.Guanidine,as cationic board spectrum antibacterial agent,was widely used in preparing antibacterial polymers,because it has good thermal stability and biocompatibility[20].Caoet alprepared antibacterial PET by blending with guanidine grafted polypropylene[21].However,the guanidine,as antibacterial agent,need be chemically modified before blending and the multi-step melt blending process will consume a lot of energy.Herein,we proposed an ecofriendly one-step on-line melt coextrusion method to prepare antibacterial composite PET,and covalent bonding was achieved between PHMG and PET via the amination reaction under high temperature (230-270℃).The prepared composite PET showed excellent antibacterial ability against both Gram-positive and Gram-negative bacterial,as well as good cellcompatibility.The developing one-step eco-friendly green method is suitable for large-scale production and meet the requirements of low-carbon.

2 Experimental

2.1 Materials

Polyethylene glycol terephthalate (PET) was purchased from Sinopec Yizheng Chemical Fiber Co.,Ltd.(Melting point 249 ℃,density 1.4±0.006 g/m3).Poly(iminocarbonimidoyliminocarbonimidoyl imino-1,6-hexanediyl) hydrochloride (PHMG) was purchased from Aladdin.Tryptone and yeast extract were purchased from Beijing Aobox.NaCl,agar,25% glutaraldehyde aqueous solution,Na2HPO3,KH2PO3were purchased from Sinopharm Chemical Reagent Co.,Ltd.Acridine orange and ethidium bromide were purchased from Shanghai Macklin.Staphylococcus aureus ATCC 29213 (S.aureus) and Escherichia coli ATCC 25922 (E.coli) were purchased from Nanjing Bianzhen Biotechnology Co.,Ltd.Human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVEC) cells were purchased from Shanghai MINGJING Biology.Fetal bovine serum was purchased from Clarkbio,dulbecco’s modified eagle medium was purchased from Vutiva,and penicillinstreptomycin solution and trypsin-EDTA solution were purchased from Thermo Fisher.

2.2 Preparation of composite antibacterial PET

The amination reaction was controlled by coextrusion temperature,screw extrusion speed and the ratio of PHMG and PET.PET and PHMG were premixed,and the mass ratios of PHMG are 5%,2.5%,1%,0.5%,0.1%,0.05%,which was used to regulate the antibacterial ability of the composite PET.The premixed samples were dried gradient in vacuum under 80 ℃ for 3 h and 130 ℃ for 8 h.PET and PHMG after drying were transferred into screw extruder to prepared the antibacterial functional PET (PET@PHMG) (Fig.1).The screw 5 heating zone temperatures were 230,255,260,260 and 270 ℃,and the screw extrusion speed is 10 rpm/min.The preparation process is presented in Fig.1(a).Antibacterial PET chips could be directly obtained by the melt co-extrusion process.Using antibacterial PET chips as raw materials could further prepare antibacterial PET fibers and membrane,and PET fiber were prepared to further evaluate its antibacterial ability and cell compatibility.

Fig.1 Fabrication of the PET@PHMG: (a) Schematic illustration showing the fabrication process of PET@PHMG;(b) Schematic illustration of amidation reaction between PHMG and PET under 230-270 ℃

The composite PETs were characterized by fourier transform infrared spectrometer (FT-IR,Vertex70,Bruker,Germany),thermal gravimetric analyzer (TG,209F1,Netzsch,Germany) and contact angle measuring instrument (KRUSS,DSA30S).Before testing its infrared spectrum,PET was dried under infrared lamp for 30 min.For detecting thermal stability,PET was heated in N2from room temperature to 800 ℃ at a rate of 10 ℃/min.

2.3 Antibacterial ability

The antibacterial efficiency of the prepared composite antibacterial polymers was evaluated by the plate colony assay according to GB/T20944.3-2008 and published literature[22],S.aureusandE.coilas model bacteria.Firstly,the bacterial suspension was adjusted to achieve a concentration of 1.0 × 104-1.0 × 105CFU/mL.Then,3 mL of bacterial suspension and 100 mg of composite antibacterial PET were added into centrifuge tube,and these samples were incubated at 120 rpm for 18 h at 37 ℃.The bacterial suspension was taken from centrifuge tube,and the diluted bacterial suspension was co-cultured uniformly onto a solid plate.The bacterial concentration was counted after incubation at 37 ℃ for 24 h.Each sample was repeated in triplicates.

The antibacterial kinetic process of the composite PET was monitored by fluorescent staining,and the staining method was according to the published method[23].The fluorescent dyes solution was prepared by mixing 10 mg of acridine orange and 10 mg of ethidium bromide in 10 mL of phosphate buffer solution (PBS,0.01 mol/L,pH 7.4).The bacterial suspension (3 mL,1.0 × 108CFU/mL) in PBS was co-cultured with composite polyester (100 mg) for 5 and 30 min.The bacteria were stained with 100 μL of fluorescent dyes for 15 min.After rinsing with PBS for 3 times,the samples were re-dispersed and placed on a glass slide.Then,the samples were observed under fluorescence microscope.The negative control group was performed without composite polyester treatment.

The morphological change of bacteria was investigated by SEM,bacteria strains were incubated with PBS and composite polyester for 5 and 30 min,respectively.The bacteria were fixed with 2.5% glutaraldehyde for 5 h.The fixed bacteria were then dehydrated successively by 30%,50%,70%,90% and 100% ethanol,then these samples were freeze-dried for 12 h.The dried samples were used to observe bacterial morphology by SEM.

2.4 Cytotoxicity test

Cytotoxicity of composite PET was evaluated by CCK-8 assay,HSF and HUVEC cells as model cells.The cells were cultured in Dulbecco’s modified eagles medium (DMEM) with 10% fetal bovine serum,and the cells were dispersed about 1.0 × 105cells/mL.100 μL cells per well were seeded into 96-well plates and further incubated for 24 h at a humidified atmosphere(5% CO2,95% O2and 37 ℃).Composite PET (100 mg) was incubated in pH 7.4 PBS (5 mL) for 24 h under 37 ℃,and then the extracts were filtrated and diluted to 50%,25% and 12.5% with DMEM.The medium in the 96-well plates were replaced with the composite polyester extracts (100 μL).Then,the plates were again placed in the humidified atmosphere for 24 h.100 μL 10% CCK-8 solution was added in each well and incubated for 2 h.The supernatant was transfer to blank 96-well plate,and the optical density at 450 nm was read on a microplate reader.The cells without the polyester extracts were used as the control and the cell viability was set at 100%.

3 Results and discussion

3.1 Structure characterization of the composite PET

Guanidine salt has been used to prepare antibacterial materials by melt processing under 200℃,however it required multi-step operation and chemical pre-modification[21].True eco-friendly green manufacturing did not achieve.Herein,true ecofriendly green manufacturing was achieved to fabricate composite antibacterial polyester by high temperature melt extrusion.Beside achieving uniform mixing of PHMG and PET in the melt coextrusion procedure,PET also undergone amidation reaction with PHMG under high temperature (230-270 ℃,Fig.1(a)).PHMG is rich in amine groups (Fig.1B(b)).The FTIR spectra of PET@PHMG was shown at Fig.2(a),where C=O stretching vibration of PET appeared at 1 712 cm-1,and 1 238 cm-1was assigned to stretching vibration of C-O;1 089 cm-1was para-substitution vibration of benzene ring;721 cm-1was C-H bending vibration of benzene ring.Meanwhile,new bands emerging at 1 640 and 1 285 cm-1.1 640 cm-1was designated C=N stretching vibration of PHMG;1 285 cm-1belonged to C-N of the newly created amide bond III belt.The XPS spectra also successfully proved the introduction of PHMG (Fig.2(b)).

Fig.2 Fabrication and structure characterization of the PET@PHMG: (a) FT-IR;(b) XPS spectra of PET@PHMG;(c,d) the TG curves of PET@PHMG,the insert in (c) was DSC curves;(e) Tensile strength and strain of PET with different PHMG content;(f)dynamic contact angle of single fiber of PET@PHMG

The weight loss ratio was detected by thermogravimetric analysis of PET@PHMG,the initial thermal decomposition temperature of PET gradually decreased from 387.7 to 348.5 ℃ with the increase of PHMG content (Figs.2(c),2(d)).And,the melting temperature of PET@PHMG also decreased from 252 to 246 ℃.The reason was that the composition of guanidine salt and PET destroyed the regularity and molecular skeleton structure of PET.And,mechanical properties have also declined because the deterioration of the PET molecular backbone (Fig.2(e)).The tensile strength of PET decreased significantly when PET and PHGB was mixed by melt coextrusion.And,the more added amount,the lower tensile strength.The tensile strength decreased about 50% when 0.5% PHGB was mixed with PET,and the strength decreased about 90% when 5% PHGB was mixed with PET.So,PET@PHMG alone,as antibacterial material,is not suitable for the fields with high mechanical strength requirements.It is difficult to achieve the balance between mechanical properties and antibacterial properties by adjusting the proportion of PET@PHMG.However,using PET@PHMG as antibacterial composite material could solve the defect in mechanical properties;for example,PET@PHMG could be used as the sheath layer of sheath-core fiber or the antibacterial functional layer of the multilayer composite membrane.PHMG not only endowed PET antibacterial ability,but also improved its hydrophilicity.The dynamic contact angle of single fiber of PET@PHMG was measured to evaluate its hydrophilicity.With the increasing of PHMG content,the contact angle decreased from 81.3oto 48.2o(Fig.2(f)).As the result showed,its hydrophilicity is significantly improved after compositing with PHMG.This is beneficial for improving its comfort as clothing textile and its biological safety as medical and health supplies.

3.2 Antibacterial activity and mechanism

As a hydrophobic commercial polymer,bacteria tend to adhere onto the surface of polyester through hydrophobic interactions[24].The contaminated PET was the pathogen sources,which could spread diseases in multiple application areas.Excellent antibacterial ability help PET maintaining “clean” state.The antibacterial ability of composite PET showed obvious PHMG concentration dependence,and which have higher antibacterial activity against Gram-positive bacteria (S.aureus) than Gram-negative (E.coli).As shown in Fig.3,the antibacterial activity of composite PET had no obvious difference againstS.aureusandE.coliwhen the PHMG content was more than 2.5%,and it was over 99%.At low PHMG content (0.1%-1%),there is significant difference in antibacterial properties.The inhibitory rate againstS.aureuswere 48.0%,82.7% and 88.7%,when the PHMG contents were 0.1%,0.5% and 1% respectively;however,the inhibitory rate againstE.coliwere 5.4%,25.2% and 41.6%.At low PHMG content,PHMG composite PET had more effective antibacterial ability to Grampositive bacteria than Gram-negative bacteria.

Fig.3 The antibacterial mechanism and ability of PET@PHMG: (A) Schematic illustration showing the antibacterial mechanism;(B-C) the antibacterial ability of composite polyester against E. coli (a-f) and S. aureus (g-i) (the PHMG contents was decreased from 5 wt% to 0.1 wt%,a and g were negative control)

In order to further verify the antibacterial ability of composite polyester,E.coliandS.aureuswere stained with acridine orange and ethidium bromide mixed fluorescent dyes for LIVE/DEAD bacterial survival test.Bacterial cells are observed to be green/red using a fluorescence microscope,which indicates that the cell membrane was intact/damaged,and the bacteria are live/dead.Fig.3 showed the fluorescence images ofE.coliandS.aureusbefore and after treatment with PET@PHMG 5 and 30 min.The bacterial cells are green before treatment with composite polyester,indicating that the bacteria are alive.After treatment with PET@PHMG for 5 min,some bacteria are green and some bacteria are red,that indicated that many bacterial was killed.All bacterial cells were red when treat with composite polyester 30 min,indicating that all the bacterial was dead.LIVE/DEAD fluorescence test further confirmed that the composite polyester could kill bacteria quickly and effectively.

In order to clarify the antibacterial mechanism of composite PET,SEM were used to observe the surface morphology changes ofS.aureusandE.colibefore and after with composite polyester for 5 and 30 min.The untreatedS.aureusandE.colihas a smooth surface,showing a complete surface membrane structure (insert images in Fig.4).In contrast,the surface morphology changed forS.aureusafter treatment with composite polyester for 5 min.Surface morphology forS.aureustreated with composite polyester for 30 min exhibited a distinct and stark change,compared with the negative control.Similar morphological changes ofE.coliwere also observed from the SEM images.PHMG,as antibacterial agents,had positively charged,which could pierce the negatively charged bacterial cells membrane through electrostatic attraction.The damaged cytoplasmic membrane causes the destruction of membrane integrity,leading to the death of bacteria.Due to different membrane structures of gram-positive bacteria and negative bacteria,gram-negative bacteria have outer membrane,while gram-positive bacteria have not.This is the reason why the composite polyester showed different antibacterial properties toE.coliandS.aureus.

Fig.4 Fluorescence micrograph and SEM images of E. coli and S. aureus after being treated with composite polyester for 0,5 and 30 min

3.3 Cytocompatibility

Good cytocompatibility is the prerequisite for promising application of antibacterial composite PET.There is quantitative correlation between the absorbance of sample and the activation of the cells.The cell viability is revealed by the ratio of the absorbance of the cells incubated with the composite PET to the cells incubated with culture medium.The results reveal that the composite polyester had low cytotoxicity towards HSF and HUVEC cells(Fig.5).＞85% cells survived after 24 when the PHMG contents was equal or lower than 2.5%,even if the concentration was 100 mg/mL.The composite PET had weak cytotoxicity,when PHMG contents increased up to 5.0%.The cell viability was 70.9% and 55.4% against HSF and HUVEC cells respectively,when the concentration was 100 mg/mL.The cell viability was also above 80% when the concentration was lower than 50 mg/mL;at the same time,the antibacterial ability was over 99% against bothE.coliandS.aureus.All the results showed that composite PET had excellent cytocompatibility.The good cytocompatibility lays the foundation of application in multiple fields to combat pathogenic.

Fig.5 In vitro toxicity test of composite polyester.Cell viability after incubation with composite polyester extracts by CCK-8 assay for 24 h: (a)HSF cells;(b) HUVEC cells

4 Conclusions

A one-step eco-friendly method was proposed to prepare antibacterial composite PET,and the method was suitable for large-scale production.Beside evenly mixing of PHMG and PET in the melt coextrusion procedure,PET also undergone amidation reaction with PHMG under high temperature (230-270 ℃).The antibacterial ability of composite PET showed obvious PHMG concentration dependence,and antibacterial activity reached more than 99% when PHMG content was 2.5 wt%.Moreover,LIVE/DEAD fluorescence test further confirmed that composite PET could kill bacteria quickly and effectively (within 30 min);while negligible cytotoxicity was observed to HSF and HUVEC cells.One-step eco-friendly fabrication of composite antibacterial PET was accomplished by on-line melt coextrusion.The composite antibacterial PET had potential use in multiple fields to combat pathogenic including textiles,packaging materials,decoration materials and biomedical devices,etc.

Conflict of interest

All authors declare that there are no competing interests.