Biosafety Evaluation And Filtration Performance Of Novel Self-assem-bled Electrospun Composite Films

A new respiratory virus,the novel coronavirus（severe acute respiratory syndrome coronavirus-2,SARS-Co V-2）,is ravaging the world.Respiratory infectious diseases are mainly transmitted by contact,respiratory droplets and pathogen aerosols.Wearing suitable masks is the most cost-effective way to control the spread of respiratory infections.At present,there are three main kinds of masks on the market,namely ordinary disposable masks,disposable medical masks and disposable medical protective masks and there are various brands,and the quality is also good and bad.Most of the commonly used dis-posable masks achieve the protective effect through physical barrier and elec-trostatic adsorption,and they do not have the antibacterial and antivirus effects.Based on this,our group prepared 13 kinds of cellulose acetate spun films con-taining different concentrations of nano silver and cetylpyridine bromide anti-bacterial agent and 12 kinds of polyepoxy-polyethyl lactone films containing different concentrations of chitosan and cetyltrimethyl ammonium bromide by electrostatic spinning technology.After screening 25 kinds of new self-assem-bled materials,biosafety evaluation,filtration performance evaluation and anti-bacterial performance evaluation were carried out to explore the application prospect of new electrostatic spinning composite films with antibacterial func-tion,and to provide reference for the research and development of new antibac-terial mask materials.The experiment is divided into three parts.PartⅠ Safety evaluation and screening of 25 self-assembled nano-electro-spinning antibacterial materials containing different antibacterial agentsObjective:Screening and evaluating the safety of cellulose acetate composite nano-a ntibacterial mask materials containing different concentrations of nano-silver a nd cetylpyridine bromide and epoxy ethane-polyethylene lactone-antibacterial materials containing different concentrations of chitosan and cetyltrimethylam monium bromide self-assembled by electrospinning technology,discuss the bi ological safety and application prospects of newly developed antibacterial mas k materials and provide reference for the research and development of antibact erial mask materials.Methods:The cellulose acetate composite nano antibacterial mask materials LL1-LL13 containing different concentrations of silver nanoparticles and cetylpyri-dinium bromide and the epoxy ethane-polyethylene lactone antibacterial mate-rials T1-T12 containing different concentrations of chitosan and cetyltrime-thylammonium bromide were used to prepare sterile extracts using MEM cell complete medium as extraction medium and filtrating through 0.22μm filter membrane.1.NCTC clone 929 were cultured in 100%sterile extracts of the 25 mate-rials as the experimental group.L929 cells were cultured in MEM as the nega-tive control group.MTT colorimetric method was used to detect the cytotoxicity of different composite films,and the antibacterial materials that met the require-ments of GB/T 16886.5-2017 with a relative cell proliferation rate of not less than 70%within 24 h were screened out.The extracts of the selected antibacte-rial materials were diluted,and the cytotoxicity of the diluent was detected again by MTT colorimetry.2.Three kinds of 100%extracts of self-assembled nano-electrospinning antibacterial materials screened out in 1 were selected as the experimental group,and the cell culture medium of MEM was used as the negative control group.The effect of the antibacterial agent in the selected antibacterial materials on the morphology of L929 cells was observed by optical microscope.3.Self-assembled nano-electrospinning antibacterial materials that meet the requirements screened in 1 were selected as the experimental group,defatted cotton immersed in normal saline was used as negative control,and 4%formal-dehyde solution was used as positive control.The erythema and edema at each contact site were observed and recorded at 24 h,48 h and 72 h after the materials were in contact with the skin of New Zealand rabbits.The skin irritation of the three composite film materials to New Zealand rabbits was determined,and the primary irritation index of each group was determined.HE staining was used to observe the infiltration of inflammatory cells in the dermis of skin tissue at the stimulated site.4.Thirty guinea pigs were randomly divided into 5 groups.Three kinds of100%extracts of self-assembled nano-electrospinning antibacterial materials screened out in 1.were selected as the experimental group,normal saline as the negative control group,and 4%formaldehyde solution as the positive control group.After intradermally induced,locally induced and challenged,the skin er-ythema and edema of guinea pigs in each group were observed to determine the sensitization of the material.HE staining was used to observe the infiltration of inflammatory cells in the dermis of skin tissue at the stimulated site.Results:1.In vitro cytotoxicity test results showed that only LL11,LL12 and LL13met the requirements of GB/T 16886.5-2017 that the cell survival rate of 100%extracts decreased within 24 h,but not less than 70%.The cytotoxicity results of the extracts of CA/Ag NPs,CA/CPB,CA/Ag NPs/CPB at four dilution levels of 25%,50%,75%and 100%showed that there was no statistical significance between the CA/Ag NPs groups and the control group at 24 h（F=0.9862,P>0.05）.There were significant differences between CA/CPB group and con-trol group in dilution of 25%,50%,75%and 100%（F=23.52,P<0.05）.The dilution of 50%,75%and 100%in CA/Ag NPs/CPB group was statistically dif-ferent from that in control group（F=18.26,P<0.05）.2.Under the light microscope,the cells in the negative control group were normal spindle-shaped or irregular triangular,while the cells in each experi-mental group had a certain degree of cell body thinning and flat,and the cyto-plasm increased,and the effect of the extract of each group on the cell morphol-ogy became more obvious with the extension of contact time.3.Results of skin stimulation experiment:the primary stimulation index of CA/Ag NPs,CA/CPB and CA/Ag NPs/CPB groups was 0,0 and 0.1,while that negative control group was 0,showing no irritation.Primary irritation index of positive control group:5.0,indicating strong stimulation.The primary irritation index of the three experimental groups was significantly decreased,and the dif-ference was statistically significant compared with the positive control group（F=25.44,P<0.05）.HE staining results showed that a large number of inflam-matory cells infiltrated in the dermis of the positive control group,while the inflammatory cells in the dermis of the negative control group were signifi-cantly reduced.The experimental group was similar to the negative control group,but significantly different from the positive control group.4.Results of skin sensitization tests were as follows:there was no obvious inflammatory reaction such as erythema and edema in the upper abdominal skin of the experimental group and the negative control group compared with the positive control group within 24 h,48 h and 72 h after allergen removal.The results of HE staining showed that a large number of eosinophils infiltration in the dermis of the positive control group was significantly different from that of the negative control group and the experimental group.Conclusions:LL11,LL12,LL13,namely CA/Ag NPs,CA/CPB,and CA/Ag NPs/CPB,meet the requirements of GB/T 16886.5-2017 medical device biosafety evalua-tion,and have no skin irritation and sensitization.It is expected to be a material for the preparation of new antibacterial masks.PartⅡExperimental study on bacterial filtration efficiency and bacterio-static performance of different types of new antibacterial mask materialsObjective:The bacterial filtration efficiency of medical surgical masks,disposable protective masks,N95 respirator and three electrospun composite films（CA/Ag NPs,CA/CPB,CA/Ag NPs/CPB）against Escherichia coli and Staphy-lococcus epidermosus and the antibacterial effect of three antibacterial compo-site films were tested.To evaluate the barrier properties and antibacterial prop-erties of CA/Ag NPs,CA/CPB,and CA/Ag NPs/CPB films,and explore the ap-plication prospect of antibacterial mask materials and provide reference for the development of new antibacterial mask materials.Methods:1.The filtration efficiency of three different disposable masks and three electrospun composite film antibacterial mask materials（CA/Ag NPs,CA/CPB,CA/Ag NPs/CPB）against Escherichia coli ATCC 25922 and Staphylococcus epidermosus ATCC 12228 was detected by object surface sampling,bacterial culture and colony counting using TK-3 aerosol generator and FA-1H microbial sampler at a resting gas flow of 5 L/min for 30 min,60 min,120 min,and 240min.2.Escherichia coli ATCC 25922 was represented.The changes of filtration efficiency of different disposable masks and three kinds of electrospun compo-site film antibacterial mask materials（CA/Ag NPs,CA/CPB,CA/Ag NPs/CPB）were detected aspirated for 30 min at different inspiratory flow rates of 10 L/min,20 L/min,and 30 L/min.3.Refer to FZ/T 73023-2006,antibacterial knitwear.The inhibition zone diameters of CA/Ag NPs,CA/CPB,CA/Ag NPs/CPB against Staphylococcus epidermidis,Staphylococcus aureus,Escherichia coli,Enterobacter cloacae,Serratia marcescens and Klebsiella pneumoniae were qualitatively detected by AGAR diffusion method to evaluate the antibacterial effects of the three anti-bacterial film materials.Results:1.At 30 min,there was no difference in the filtration effect of medical surgical masks,disposable protective masks and N95 masks on Escherichia coli and Staphylococcus epidermidis,with BFE above 98%（P>0.05）.At 60 min and120 min,the filtration effect of N95 protective masks on Escherichia coli was higher than that of medical surgical masks and disposable protective masks（P<0.05）,but there was no statistical difference between medical surgical masks and disposable protective masks（P>0.05）.There was no statistical difference in the filtering effect of the three masks on Staphylococcus epidermidis（P>0.05）.At 240 min,the filtration efficiency of N95 protective masks against Esche-richia coli and Staphylococcus epidermidis was higher than that of surgical masks（P<0.05）,and there was no statistical difference between N95 masks and disposable protective masks（P>0.05）.Overall protective effect:N95 mask is higher than disposable protective mask and medical surgical mask.The filtration efficiency of the three antibacterial materials on Escherichia coli for 30 min～240 min was as follows:LL11-CA/Ag NPs was 89.78%,87.28%,75.23%,69.41%,LL12-CA/CPB was 84.52%,83.38%,77.47%,68.79%,LL13-CA/Ag NPs/CPB was 84.39%,72.49%,68.23%,49.88%;Filtra-tion efficiency of Staphylococcus epidermidis:LL11-CA/Ag NPs was 99.61%,99.54%,97.19%,93.93%,LL12-CA/CPB was 99.61%,98.05%,95.73%,86.91%,LL13-CA/Ag NPs/CPB was 99.45%,98.37%,97.44%,83.67%.LL11was higher than LL12 and LL13 on the whole,and the filtration efficiency of the three materials on Staphylococcus epidermidis was higher than that of Esch-erichia coli（P<0.05）.2.The filtration effect of disposable masks and three antibacterial materials on Escherichia coli aerosol decreased with the increase of ventilation flow.3.The antibacterial zones of CA/Ag NPs,CA/CPB and CA/Ag NPs/CPB were all≥0.5 mm,and there was no bacterial reproduction under the samples.According to GB/T 20944.1-2007 and FZ/T 73023-2006,it can be considered to have a good antibacterial effect.Conclusions:After 240 min of continuous use in resting state,the filtration effect of N95mask on bacterial aerosol was higher than that of disposable protective mask and medical surgical mask.The self-assembled nano-electrospinning antibacte-rial materials CA/Ag NPs and CA/CPB have the same filtering effect on bacte-rial aerosol as surgical masks（H=2.000,P>0.05）,and are expected to be used as antibacterial mask filters after improvement.PartⅢ Establishment of visual LAMP system for Staphylococcus epider-midis and application to the rapid detection of filtration properties of new materialsObjective:To establish a LAMP system for visual and rapid detection of Staphylococ-cus epidermidis,and apply it to the detection of filtered mask samples,to ex-plore the feasibility of using LAMP to test the filtering effect of materials,so as to provide ideas for the immediate detection of the filtering effect of materials and guide the use and replacement of masks.Methods:1.The genomic DNA of Staphylococcus epidermidis was extracted.Ses B gene of Staphylococcus epidermidis strain ATCC 12228 was used as the re-search object to establish LAMP reaction system.The Mg²⁺,betaine,reaction temperature and reaction time in the reaction system were optimized,and then the reaction system was compared with Salmonella paratyphi A,Klebsiella pneumoniae,Mycobacterium smegmatis,Streptococcus pneumoniae and Staph-ylococcus aureus stored in the laboratory to determine the specificity of the re-action system.Finally,the sensitivity of the gradient dilution DNA template de-tection system was compared with that of PCR.2.The visualization methods were screened by comparing the turbidity of LAMP reaction system,or adding calcein or neutral red indicator to the LAMP reaction system before the reaction,and observing the reaction results with na-ked eyes.After screening,it was applied to the optimization of LAMP reaction system,and its accuracy was verified by comparing with 2%agarose gel elec-trophoresis.3.The filtered samples collected from CA/Ag NPs/CPB at different time points in part II were used as samples to extract DNA.The optimized LAMP system was used to visually and rapidly detect mask samples,and the results were compared with PCR.Results:1.The color changes of the system were obvious before and after the reac-tion of calcein and neutral red.Calcein negative is light orange,positive is green;Neutral red negative is orange yellow,positive is orange red.There was little difference between the naked eye observation before and after the turbidity comparison reaction.Calcein,as a visual dye,is relatively stable in the reaction system,so it is selected as a visual dye.2.A visualization system for detecting Staphylococcus epidermidis reac-tion by LAMP was established and optimized.The optimal concentration of Mg SO₄ in LAMP reaction system was 4 m M,betaine was 0.4 M,temperature was 63℃and the reaction time was 60 min.The specificity of the reaction sys-tem was good,and the minimum detection limit was 10 copies/μL.The mini-mum detection limit of PCR was 10²copies/μL,and the sensitivity of LAMP reaction was 10 times higher than that of PCR.Visualization results of calcein in LAMP system were consistent with those of agarose gel electrophoresis.3.Sample detection showed that both LAMP and PCR could detect Staph-ylococcus epidermidis from the outer side of the mask from 30 min,and a band began to appear on the inner side of LAMP mask at 60 min,and a band began to appear on the inner side of PCR at 120 min.The Kappa value of LAMP and PCR was 0.832,which was in good agreement.Conclusions:In this study,the visualization ring mediated isothermal amplification sys-tem was successfully established for the detection of Staphylococcus epider-midis,and was applied to the bacterial filtration detection of novel antibacterial materials.There was no difference in detection effect between LAMP and PCR（Kappa=0.832）.This method can be applied to detect the surface contamination of masks and guide users to change masks in time.