rapid eradication of antibiotic-resistant bacteria and biofilms by … · 2021. 2. 2. ·...

mater.scichina.com link.springer.com Published online 24 September 2020 | https://doi.org/10.1007/s40843-020-1451-7Sci China Mater 2021, 64(3): 748–758

Rapid eradication of antibiotic-resistant bacteria andbiofilms by MXene and near-infrared light throughphotothermal ablationFan Wu1†, Huiling Zheng2†, Wenzhao Wang1, Qiong Wu2, Qi Zhang3, Jiayu Guo1, Bangzheng Pu1,Xinyuan Shi1, Jiebo Li1*, Xiangmei Chen2* and Weili Hong1*

ABSTRACT With the development and rising of anti-microbial resistance, rapid and effective killings of bacteria areurgently needed, especially for antibiotic-resistant bacteriaand bacterial biofilms that are usually hard to be treated withconventional antibiotics. Here, a rapid and broad-spectrumantibacterial strategy is demonstrated through photothermalablation with MXene and light. Ti3C2 MXenes, when com-bined with 808 nm light, show significant antibacterial effectsin just 20 min. The antibacterial strategy is effective to 15bacterial species tested, including methicillin-resistant Sta-phylococcus aureus (MRSA) and vancomycin-resistant En-terococci (VRE). In addition, the rapid antibacterial strategyworks for MRSA biofilms, by damaging the structures as wellas killing bacteria in biofilms. Furthermore, the investigationof the antibacterial mechanisms shows that Ti3C2 with lightkills bacteria mainly physically through inserting/contact andphotothermal effect. This work broadens the potential appli-cations of MXene and provides a way to eradicate bacteria andbiofilms physically, without the likelihood of resistance de-velopment.

Keywords: MXenes, 2D materials, antibiotic-resistance, bacteria,photothermal

INTRODUCTIONDue to the overuse and misuse of antibiotics, the devel-opment of antimicrobial resistance has been exacerbated.Antibiotic-resistant bacteria (ARB), such as methicillin-

resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), are difficult or even im-possible to be treated with existing antibiotics [1–4],causing nearly 1 million related mortality each year in theworld [5–7]. It was estimated that this number will in-crease to 10 million by 2050 if no action is taken [8,9].Besides developing antibiotic resistance, bacteria can alsoform biofilms that are responsible for many persistentinfections. Due to the difficulties of antibiotics to pene-trate the dense matrix, bacterial biofilms are not easily tobe eradicated with antibiotics [10]. Thus, it is critical todevelop novel antibacterial approaches that can eradicateARB and biofilms effectively.In recent years, two-dimensional (2D) materials have

attracted extensive attentions in biomedicine [11–15].Some 2D materials, such as graphene, have shown anti-bacterial properties with greatly reduced bacterial re-sistance [16,17]. The antibacterial mechanisms of 2Dmaterials were believed to be both physical damagesthrough the sharp edges of the 2D material inserting orattaching on the surface of bacteria, causing the disrup-tion of bacterial membrane and the leakage of cell con-tents [18–23], and chemical damages through thegeneration of oxidative stress or charge transfer [17]. Theantibacterial mechanisms of both physical and chemicaldamages greatly reduced the ability of bacteria to developdrug resistance.MXenes, or 2D transition metal carbides or nitrides, are

1 Institute of Medical Photonics, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and MedicalEngineering, Beihang University, Beijing 100191, China

2 Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing100191, China

3 State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of the Chemical Physics, Chinese Academy of Sciences, Dalian 116023,China

† These authors contributed equally to this paper.* Corresponding authors (emails: [email protected] (Hong W); [email protected] (Chen X); [email protected] (Li J))

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a new and fast-growing family of 2D materials and havemany applications in energy storage, electromagneticapplication and biomedical field [13,24]. As a typicalMXene, Ti3C2Tx was also found to have antibacterialproperties toward Escherichia coli (E. coli) and Bacillussubtilis (B. subtilis), with a higher antibacterial efficiencythan the graphene-based material, graphene oxide (GO)[18]. Similar to graphene, Ti3C2Tx MXenes exhibit anti-bacterial activity through both physical effects by dis-rupting cellular membranes and chemical effects byoxidative stress through the generation of reactive oxi-dative species (ROS). However, the antibacterial proper-ties of MXenes have been demonstrated in limitedbacterial species only, whether they have broad applica-tion to other types of bacteria or ARB is unknown.Moreover, although the induction of oxidative stress is acommon antibacterial mechanism [25–27], some bacteria,such as MRSA, are capable of resisting the oxidant-basedclearance mechanism by producing antioxidants [28].Therefore, antibacterial mechanisms that involve oxida-tive stress may be less or even not effective to thesebacteria. In addition, it usually takes at least 4-h in-cubation for the 2D materials, including MXenes, to showeffective antibacterial activities. This long-time culturemay further induce the development of resistance.Therefore, novel antibacterial strategies that are not likelyfor bacteria to develop resistance are highly desirable.Photothermal therapy (PTT) is a promising new ap-

proach for bacterial disinfection [29]. Through the gen-eration of high local heat with light and photothermalagents, PTT eliminates bacteria physically and thereforedoes not raise the concern of drug-resistance develop-ment commonly faced by antibiotics [17]. MXenes exhibitexcellent hydrophilicity and high photothermal-conver-sion efficiency [30–34]. In particular, Ti3C2 MXenes havebeen demonstrated to be a highly effective PTT agent fortumor therapy [33,35]. However, MXenes through PTTfor antibacterial utilization have not yet been explored.Here, we investigated the antibacterial activity through

PTT with MXenes and demonstrated a rapid and effectivekilling of bacteria and biofilms with MXenes and near-infrared (NIR) light. We found that when combined with808 nm light, Ti3C2 MXenes showed a significant anti-bacterial activity toward both E. coli and S. aureus in just20 min. Antibacterial mechanisms of MXene with lightwere investigated and revealed that bacteria were killedmainly physically through photothermal effect. In addi-tion, we tested 15 bacterial species and showed that theantibacterial strategy was effective for all bacteria tested,including MRSA and VRE. Furthermore, we demon-

strated that PTT with MXene could eradicate bacterialbiofilms through damaging the structures as well as kill-ing bacteria in biofilms. Our work promises a great po-tential of MXene in biomedical field for rapid killings ofARB and biofilms.

EXPERIMENTAL SECTION

Preparation of MXenesMXenes were purchased from Shandong Xiyan NewMaterial Technology Co. Ltd, Shandong, China. MXenesolid particles or powders were dissolved in deionizedwater by ultrasound. Characterization of Ti3C2 MXene isshown in Figs S1–S4.

Bacterial speciesBacteria were cultivated in Luria Broth agar (LB Brothwith agar Lennox, Sigma-Aldrich) medium plates at 37°Cfor 24 h and then stored at 4°C for future use. In total, 15bacterial species, including 10 Gram-negative bacteria (E.coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,Acinetobacter baumannii, Salmonella typhi, Shigella spp,Burkholderia cepacia, Enterobacter cloacae, Enterobacteraerogenes, Proteus mirabilis) and 5 Gram-positive bacteria(S. aureus, VRE, Enterococcus faecalis, Streptococcus aga-lactiae, Bacillus subtilis), were tested for antibacterial ac-tivity.

In vitro antibacterial activityBacterial solutions were added to different MXene solu-tions (Ti3C2, V2C and Nb2C) and illuminated with/with-out 808 nm light. After treatment, bacterial suspension ofeach group was diluted 10, 102, 103, 104 and 105 folds,then 5 μL of the dilutions were dropped onto LB plates togrow for 12–24 h at 37°C. Colonies were calculated afterincubation, and each assay was carried out in quad-ruplicate.To examine the effect of MXene on bacterial regrowth,

the growth curve experiment was executed. After ex-posure of 808 nm light, 50 μL of bacterial solution wasthen transferred to 10-mL tubes, each containing 5 mL ofLB medium. The tubes were then put on a shaking in-cubator at 220 r min−1 and 37°C. Aliquots of the sampleswere withdrawn at specific time intervals and the opticaldensity (OD) value at 570 nm was measured with a UV-vis spectrometer.

Oxidation of GSHGlutathione (GSH) oxidation was measured with a UV-vis spectrometer. Before exposure to 808 nm light, bac-

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teria and MXene mixture was mixed with GSH(50 μmol L−1). After the exposure, GSH oxidation wasrecorded from the absorption of 412 nm by adding 5,5ʹ-dithiobis-(2-nitrobenzoic acid) reagent (DTNB, Invitro-gen). GSH oxidation by H2O2 (1 and 10 mmol L

−1) wasused as positive controls.

Superoxide radical assayXTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tet-razolium-5-carboxanilide) could be reduced by super-oxide radical anion to form water-soluble XTT-formazanwith maximum absorption at 470 nm. Phosphate bufferedsaline (PBS) was used to dissolve XTT. Then Ti3C2MXene (100 µg mL−1) was mixed with XTT (1 mmol L−1).The mixture was irradiated by 808 nm light (400 mW) for0–20 min. After irradiation, the mixture was filtered witha 0.22-µm filter to remove the Ti3C2 MXene. The changesin absorbance at 470 nm were monitored with a UV-visspectrometer.

Bacterial water bath assayTo examine the effect of temperature on bacterial growth,bacterial water bath experiment was executed on E. coli(ATCC 25922) and S. aureus (ATCC 29213). Bacteria(106–107 CFU mL−1, 1 mL) (colony forming unit (CFU))were added into a microcentrifuge tube and incubated inwater bath at 45, 50, 60, 70 and 80°C for 20 min. Afterincubation, all groups were conducted for plate CFUcounting and the survival rate of bacteria was determinedby counting the number of CFU.

Confocal laser scanning microscopy of live and deadbacteria in biofilmS. aureus (107 CFU mL−1) were diluted in a 1꞉50 ratio toLB containing 5% glucose and transferred to glass bottomdishes (35 mm, In Vitro Scientific). The dishes were in-cubated at 37°C for 24 to 48 h in order to form maturebiofilms. Thereafter, the media was removed, and thesurface of the dish was washed three times with sterilewater to remove planktonic bacteria. Then the biofilmswere incubated with 100 μg mL−1 Ti3C2 MXene and ex-posed to 400 mW 808 nm light for 20 min. After re-moving the reaction mixture, the surface of the dish waswashed twice gently with sterile water. Then the formedbiofilms were stained by a live/dead bacterial viability kit(Invitrogen) for 15 min, washed twice with sterile waterand then imaged by confocal laser scanning microscope.

Ultrafast transient absorption spectroscopy systemIn this work, we employed a homemade femtosecond

transient absorption system as reported before [36,37]. Inbrief, our experimental setup was as following: a broad-band oscillator (Coherent Vitesse, 1 W) generated seedpluses (20 fs pulse width, 80 MHz) with a center wave-length of 800 nm (80 nm bandwidth). The pulses weredelivered into the Coherent Legend Elite He + UPS-IIIregenerative amplifier. The output laser (35 fs pulsewidth, 1 kHz) was 7 mJ with a diameter of 10.5 mm, acenter wavelength of 800 nm with the bandwidth around40 nm. Then, the beam from amplifier was separated to apump beam and a probe beam for the pump-probe setup.The pump beam was put into optical parametric amplifier(OPA) (light conversion: Topas + UV/Vis) to generate alaser pulse of 240–2500 nm as the pump beam, with apulse width of about 70–150 fs and pulse energy around2 μJ (centered at 800 nm). Focused by a quartz lens withfocusing length of 750 nm, the diameter of the pumpbeam was changed to around 400 μm. The probe beamwith lower energy was focused on a sapphire plate toproduce the broadband white-light continuum pulses(450–1100 nm). Between the pump and probe beams, weemployed the motorized delay stage to adjust the timedelay. After passing through the sample, the pump beamwas blocked and probe beam was collected by a fiberspectrometer (AvaSpec-ULS2048CL-EVO, Avantes).For the study of MXene flakes’ thermal energy decay,

the final concentrations of bacteria and MXene wereadjusted to be 9×109 CFU mL−1 and 55 µg mL−1, respec-tively, so the ratio of the number of bacterial cells to thenumber of MXene flakes was 1꞉1.

RESULTS

Rapid eradication of bacteria with MXene and NIR lightTo investigate the effect of light exposure on MXenes’antibacterial efficiency, we mixed bacteria with MXenes,and exposed the mixture to NIR light (Fig. 1a). The an-tibacterial efficiency was examined by measuring the cellviability with CFU counts using the disc diffusion methodand bacterial growth curves with OD measurements.After mixing with Ti3C2 MXenes at a final concentrationof 100 µg mL−1 and exposure to 808 nm light at 400 mWfor 20 min, both S. aureus, the Gram-positive bacteria,and E. coli, the Gram-negative bacteria, showed a sig-nificantly reduced CFU as compared with the controlwithout treatment, treated with Ti3C2 alone, and treatedwith light alone (Fig. 1b). These results were furtherconfirmed by the growth curve measurements whichshowed that while both S. aureus and E. coli treated withlight or Ti3C2 alone grew with a similar growth curve to



Figure 1 Rapid eradication of bacteria with MXene and light combination. (a) Schematic diagram of bacterial treatment with MXene and light. (b)CFU images of S. aureus and E. coli without and with treatments. The red rectangles show the CFU counts of the same dilution after each treatment.(c) Growth curves of S. aureus and E. coli after no treatment, treated with light alone, MXene alone, and MXene with light for 20 min. (d, e) Statisticalanalysis of bacterial concentration after treatment with different types of MXenes (d) and different light wavelength (e). (f, g) Bacterial concentrationafter treatment as a function of light intensity (f) and MXene concentration (g). (h) Statistical analysis of bacterial concentration as a function ofexposure time with MXene and light combination. Error bars represent standard deviation. *p< 0.05, **p< 0.01, ***p< 0.001.



the control, bacteria treated with Ti3C2 and light si-multaneously did not grow after up to 10-h culture(Fig. 1c). These results collectively demonstrated that thecombination of Ti3C2 MXene and 808 nm light showed arapid and effective antibacterial activity.There are different types of MXenes. To explore whe-

ther the rapid antibacterial strategy of Ti3C2 with lightworks for other types of MXenes, we screened three dif-ferent MXenes, Ti3C2, V2C and Nb2C. Among them,Ti3C2 has been reported to have a good plasmonicproperty [38]. Again, the antibacterial efficiency was ex-amined against S. aureus and E. coli and evaluated bymeasuring the cell viability using CFU counts. ForMXenes’ concentration of 100 µg mL−1 and exposure timeof 20 min, Ti3C2 with light showed a significant anti-bacterial activity with mortality rates over 99% for S.aureus and 95% for E. coli. In contrast, Nb2C with lightshowed no antibacterial activity, and V2C with lightshowed an antibacterial activity to S. aureus only (Fig. 1dand Fig. S5). Interestingly, the mixture of Ti3C2 alonewith bacteria for 20 min showed an antibacterial activityto S. aureus but not to E. coli. Nevertheless, with si-multaneous exposure of Ti3C2 and light, the antibacterialefficiency to S. aureus was more than one order magni-tude significant than that with Ti3C2 alone. On the con-trary, the mixture of V2C or Nb2C alone with bacteria didnot show any antibacterial activity, indicating the uniqueproperty of Ti3C2 when combined with light for anti-bacterial treatment.Next, we used Ti3C2 MXenes and evaluated the de-

pendence of the antibacterial effects of Ti3C2 with light onlight wavelength. We tested 532, 808 and 980 nm lightand found that while the combination of Ti3C2 and808 nm light showed a significant antibacterial activity,532 and 980 nm light at the same intensity showed no ornegligible antibacterial activity (Fig. 1e and Fig. S6). Tounderstand the differences of antibacterial efficiency atdifferent light wavelength, we measured the absorption ofvarious MXenes in visible and NIR light region (Fig. S7).The absorption spectrum of Ti3C2 showed a peak from650 to 850 nm in the NIR region. Only the 808 nm light iswithin this peak, suggesting that light need to be absorbedby Ti3C2 to have a significant antibacterial effect.To investigate the dependence of antibacterial activity

of MXenes with light on light intensity, MXenes’ con-centration, and exposure time, the antibacterial efficiencywas further evaluated with Ti3C2 and 808 nm light. Weused 100 µg mL−1 Ti3C2 and evaluated the dependence ofthe antibacterial activity on light intensity first (Fig. 1fand Fig. S8). With an irradiation area of 0.8 cm2 and an

exposure time of 20 min, the combination of Ti3C2 and808 nm light started to show a significant antibacterialeffect at 400 mW. The antibacterial efficiency increasedwith elevating light intensity for both S. aureus and E.coli. Next, we used 400 mW light intensity and evaluatedthe dependence of antibacterial efficiency on Ti3C2 con-centrations (Fig. 1g and Fig. S9). With a light intensity of400 mW and an exposure time of 20 min, the combina-tion of Ti3C2 and 808 nm light started to show a sig-nificant antibacterial effect to S. aureus at 50 µg mL−1 andE. coli at 100 µg mL−1 Ti3C2. The antibacterial efficiencywas more significant at higher Ti3C2 concentrations forboth bacteria. These results further confirmed that theantibacterial strategy with Ti3C2 and 808 nm light is moreeffective to S. aureus than E. coli. Finally, we used400 mW light and 100 µg mL−1 Ti3C2 and evaluated thedependence of antibacterial efficiency on exposure time(Fig. 1h and Fig. S10). Our results showed that 5-minexposure started to show a significant antibacterial effectto S. aureus. This effect was more significant at longerexposure times. For E. coli, it took at least 15 min to showa significant antibacterial effect. With the consideration ofless photo-damage and MXenes’ doses, we then applied808 nm light at 400 mW, MXenes’ concentration of100 µg mL−1 and an exposure time of 20 min for thefollowing studies.

Antibacterial mechanisms of MXene with lightSince MXenes have a high photothermal-conversionefficiency under NIR light irradiation [31–33,39,40], andother nanomaterials that have a photothermal effect havebeen proven to be bactericidal [41], we inferred that theantibacterial strategy of MXenes with light involves thephotothermal effect. Besides, pioneer research has shownthat light illumination can also generate oxidative stressto damage cells [35,40]. In order to explore the anti-bacterial mechanisms of MXene with light, we conductedcomprehensive experiments of the pump-probe spectro-scopy, water bath temperature assay, GSH oxidation as-say, superoxide radical assay, and morphologymeasurements with scanning electron microscopy (SEM)and transmission electron microscopy (TEM), and pro-posed the antibacterial mechanisms of Ti3C2 with light(Fig. 2a): Ti3C2 flakes had the possibilities to attach ontoor insert into bacterial cells by their sharp edges, irra-diation energy of NIR light (808 nm) absorbed by Ti3C2flakes increased the temperature of Ti3C2 significantly,and the high temperature accelerated the destruction ofbacterial structures, leading to bacterial cell death.To verify these hypotheses, we conducted a series of



experiments. The pump-probe spectroscopy [42] wasemployed to study the thermal transfer from material tothe surrounding binding molecules in solutions. Ourprevious studies had demonstrated that bilayer moleculesfully covering MXenes significantly retarded MXenes’thermal relaxation dynamics from the flakes to water[43]. To understand the energy migration pathway fromTi3C2 flakes to bacteria, we measured the transient ab-sorption of Ti3C2 with bacteria (E. coli and S. aureus)mixture and Ti3C2 in water as contrast, and fitted thecurves of MXenes’ dynamics with a biexponential decay(Supplementary information (SI)). In our experiments,the pump light was set at 780 nm (Ti3C2 absorption peak)

to create a non-equilibrium MXenes’ excited state, andthen the probe light recorded the decay rates of the ex-cited state. Thus, the transient spectra dynamics (Fig. 2band Fig. S11a) could represent the MXene flakes’ thermalenergy decay [44]. The fitting results showed that pro-portion of the fast part of Ti3C2 and S. aureus mixture is9% less than that of Ti3C2 in water, while the proportionof the slow part of Ti3C2 and S. aureus mixture is 9%more than that of Ti3C2 in water, indicating that theproportion of the fast part decreased, while the propor-tion of the slow part increased after mixing with S. aur-eus. The time constants of the fast and slow parts in theTi3C2 and S. aureus mixture are 29% and 2% smaller than

Figure 2 Antibacterial mechanisms of MXene with light. (a) Schematic diagram of antibacterial mechanisms of MXene with light. (b) Pump-probespectroscopy of Ti3C2 and S. aureus mixture and Ti3C2 in water. (c) SEM images of S. aureus exposed to various treatment conditions. Green colorrepresents MXene and blue color represents S. aureus. Scale bar: 1 μm. (d) TEM image of S. aureus after exposure to Ti3C2 with light. The arrows showthe destroyed outer layer of bacterial cell (red arrow) and the area that Ti3C2 insert into or contact with the cell (blue arrow). (e) GSH loss assay atvarious conditions.



those in the control group (Table S1), respectively. Theseresults show that S. aureus retarded Ti3C2 MXenes’ ex-citation dynamics. In other words, some parts of MXenes’thermal energy were transferred to bacteria under lightirradiation. This could only occur when there were nowater molecules between MXene and bacteria in themixture. Therefore, our pump-probe results show thatbacteria could be in close contact with MXene flakes andtherefore could play the role as a direct energy acceptor todamp thermal energy migrating from MXene flakes un-der light irradiation.To further understand the antibacterial mechanisms of

MXene with light, we evaluated the morphologicalchanges of S. aureus (Fig. 2c and d) and E. coli (Fig. S11band c) after various treatments using SEM and TEM.SEM images of S. aureus showed that bacteria withouttreatment or with 20-min light exposure alone stillmaintained integrity with no bacterial membrane or cellwall destruction. After exposure to Ti3C2 alone for20 min, some bacterial cells contacted with Ti3C2 flakes(green) directly. Without light exposure, bacterial cellsremained intact after 20-min mixture with Ti3C2. How-ever, with the combination of Ti3C2 and light exposure,bacterial cell structures were destroyed (Fig. 2c andFig. S11b). Bacteria after exposure to Ti3C2 with lightwere further examined with TEM, which clearly showedthe contact of Ti3C2 with bacteria and the destruction ofbacterial outer layers (Fig. 2d and Fig. S11c). In contrast,TEM images of bacteria without treatment (Fig. S12)showed intact outer layers. We noticed that the TEMimages of E. coli showed that the outer layers of E. coliwere destroyed but there were no closely contacted Ti3C2flakes, probably because there were contacts betweenTi3C2 flakes and E. coli cells during the exposure, but E.coli cells detached from Ti3C2 flakes during the samplepreparation procedure.The antibacterial activity in MXene with light group

only showed that bacteria were not killed through phy-sical contact only with MXenes for 20 min. We inferredthat the antibacterial mechanisms of MXenes with lightcould be attributed to the generation of oxidative stressand/or photothermal effect. To rule out the mechanisms,we conducted GSH oxidation assay and superoxide ra-dical assay to detect the amount of oxidative stress gen-erated. Our GSH oxidation assay showed that there wereno significant GSH loss after 20-min exposure to Ti3C2and/or light, suggesting that there was no significantgeneration of O2

2− (Fig. 2e and Fig. S11d). CFU counts ofE. coli and S. aureus exposed to various concentrations ofH2O2 showed that exposure to H2O2 up to 8×10

−3 mol L−1

for 20 min could not kill bacteria efficiently, implyingthat even exposure to the maximum amount of O2

2−

produced by 100 μg mL−1 MXene had no antibacterialeffect (Fig. S13). Next, we detected the production ofsuperoxide anion using XTT assay. The results (Fig. S14)showed that no significant absorbance change was de-tected, revealing that no or negligible superoxide anionwas produced. Finally, we calculated the amounts ofperoxide radicals generated in Ti3C2 solution after lightirradiation. In principle, light interaction with MXenenanosheet would produce ROS at MXenes’ surface. Thischemical reaction could be accompanied by MXene’sdissociation, whose rate could be described by a singleexponential function [45]. According to our calculation,the reacted Ti3C2 was less than 0.4%, producing verysmall amounts of peroxide radical (less than 10 µmol L−1)in the initial 20 min (SI). Therefore, these results ex-cluded the mechanism that the bacteria were mainlykilled by oxidative stress in our antibacterial strategy.To examine whether the bacteria were killed by pho-

tothermal effect, we measured the temperature in Ti3C2solution and estimated the temperature of Ti3C2 flakesafter light irradiation. According to the temperature assay(Fig. S15), at Ti3C2 concentration of 100 µg mL

−1 andlight intensity of 400 mW, exposure of Ti3C2 solution to808 nm light resulted in the solution temperature in-creasing to 45°C from room temperature (25°C). As acontrol, the temperature in water without Ti3C2 did notincrease after light exposure. These results demonstratedthat Ti3C2 could convert 808 nm light efficiently into heatthrough the photothermal effect. However, this tem-perature could not kill bacteria effectively. As shown inthe water bath experiments (Fig. S16), when the entirebacterial solution was heated at 45°C water bath for20 min, the survival rate was about or higher than 50%,which was significantly higher than the 5% or less survivalrate in MXene and light treatment. To understand themechanisms, we inferred that the temperature in Ti3C2flakes should be higher than the solution since Ti3C2 wasthe only heating source that absorbs irradiation energyfrom light. We estimated roughly the temperature ofTi3C2 flakes in the solution (SI). According to our esti-mation, the temperature of Ti3C2 flakes could probablyincrease to ~75°C when the solution temperature was45°C. This temperature of Ti3C2 flakes was higher thanthe water bath temperature needed (between 50 and60°C) to totally kill bacteria, and was probably high en-ough to break down the structure of bacteria rapidly,resulting in cell death in those bacteria that Ti3C2 flakesinserted into or had direct contact with.



To build a correlation between the temperature of Ti3C2solution and cell viability of bacteria, we compared thetemperature of Ti3C2 solution with the corresponding cellviability (Fig. S17). As expected, cell viability of bacteriadecreased as the temperature of Ti3C2 solution increased.

Broad-spectrum antibacterial activity of MXene with lightTo test whether our antibacterial strategy works for otherbacterial species, we tested a total of 15 bacteria, with 10Gram-negative and 5 Gram-positive species. Accordingto the previously determined conditions, bacteria (106–107 CFU mL−1) were mixed with Ti3C2 (100 µg mL

−1) andexposed to 808 nm light (400 mW) for 20 min. CFUcounts showed that the combination of Ti3C2 and lighthad antibacterial effects to all bacteria tested, with mor-tality rates all over 94% (Fig. 3 and Fig. S18), amongwhich, the mortality rates of 13 bacterial species wereover 98%. These results showed the broad-spectrum an-tibacterial properties of Ti3C2 with 808 nm light.We compared the antibacterial efficiency between

Gram-positive and Gram-negative species and did notfind any significant difference. Two species that hadslightly lower mortality rates (between 94% and 98%)than the others are E. coli, which is Gram-negative, andVRE, which is Gram-positive bacteria.In addition to broad-spectrum antibacterial activity,

our antibacterial strategy with MXene in combinationwith light also has the advantage of not likely to developresistance since the antibacterial mechanisms are mainlyphysical damages through photothermal effects. Com-pared with the antibacterial strategy with MXene alone,which takes 4 h and works through both physical andchemical effects, MXene with light not only takes muchless time (20 min), but also is less likely to develop re-sistance through physical effects.

Rapid eradication of bacterial biofilms with MXene andlightBiofilms are hard to be treated with antibiotics due to thedifficulties in penetrating the matrix of biofilms, the ex-tracellular polymeric substances (EPS). To explore whe-ther MXene with light could eradicate bacteria inbiofilms, we grew MRSA biofilms on the bottom of a glassdish, and then exposed the biofilms to Ti3C2(100 μg mL−1) and 808 nm light (400 mW) simulta-neously for 20 min (Fig. 4a). A live/dead bacterial viabi-lity kit was used to quantify the survival of bacteria in thebiofilms. According to the laser confocal microscopyimages, the MRSA biofilms formed a dense matrix(Fig. 4b). However, after simultaneous exposure to Ti3C2

and light, connections between the matrixes in biofilmswere damaged. In addition, statistical analysis showedthat the survival rate after exposure to MXene with lightreduced by 95% compared with the control without ex-posure (Fig. 4c). In contrast, biofilms after exposure tolight or Ti3C2 alone still had intact structures with similar(light exposure alone) or slightly reduced (Ti3C2 exposurealone) survival rates compared with the control. Theseresults showed that the combination of Ti3C2 and lightcould not only kill bacteria in biofilms, but also damagethe structures of biofilms, and therefore, demonstrated arapid and effective eradication strategy of biofilms.To compare with the antibacterial strategy with MXene

alone for biofilm treatment, we grew MRSA biofilms andexposed them to various concentrations of Ti3C2 for 4 h(Fig. S19). According to the laser confocal microscopyimages, the structures of biofilms remained intact afterexposure to MXene for up to 400 µg mL−1. Statisticalanalysis of survival rates showed that Ti3C2 started toshow antibacterial activity to biofilms at the concentra-tion of 200 µg mL−1 and upregulated with increased Ti3C2concentration. The survival rate after exposure to Ti3C2 at400 µg mL−1 reduced by 87% compared with the control.At Ti3C2 concentration of 100 µg mL

−1, which was theconcentration we used for the combination of Ti3C2 andlight, 4-h exposure of Ti3C2 alone did not show any sig-nificant antibacterial effects to the biofilms. Therefore, thecombination of MXene and light showed a significantlyhigher antibacterial effect in significantly less time ascompared with MXene alone for eradication of biofilms.The mechanism of rapid eradication of bacterial bio-

films with MXene and light is proposed to be that thesharp structures of Ti3C2 MXene and the photothermaleffect through MXene and light could not only damagebacterial cells but also destroy the EPS structure of bio-

Figure 3 Bacterial mortality measurements of 15 bacterial species ex-posed to Ti3C2 with 808 nm light for 20 min. Error bars representstandard deviation.



films physically. After destruction of the surface EPS andbacterial cells, MXene flakes can flow into the inside ofbiofilms. Due to the deep penetration depth of NIR light,the eradication strategy works for structures inside thebiofilms, resulting in rapid and effective eradication ofbiofilms.

CONCLUSIONSIn conclusion, we demonstrated a rapid and broad-spec-trum antibacterial strategy through photothermal effectswith MXene and light. We showed that Ti3C2 MXenes,when combined with 808 nm light, exhibited a remarkableantibacterial effect to bacteria and biofilms in just 20 min.The combination of Ti3C2 and light showed a broad-spectrum antibacterial effect to 15 bacterial species tested,including both Gram-positive and Gram-negative species,and ARB. In addition, we showed that the combination ofTi3C2 and light could eradicate bacterial biofilms effi-ciently through destroying the intact structure as well askilling bacteria in biofilms. Finally, antibacterial mechan-isms of MXene with light were investigated and revealedthat bacteria were killed mainly by physical damagesthrough inserting/contact and photothermal effects.

Received 10 April 2020; accepted 2 July 2020;published online 24 September 2020

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Figure 4 Bacterial biofilm treated with MXene and light. (a) Schematic diagram showing the treatment of bacterial biofilms with MXene and light. (b)Fluorescence images from confocal laser scanning imaging of MRSA biofilms after no exposure (control), exposure to Ti3C2 (100 µg mL

−1) alone,exposure to light (400 mW) alone, and simultaneous exposure to Ti3C2 (100 µg mL

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Acknowledgements This work was supported by the National NaturalScience Foundation of China (81901790 and 21803006), the NaturalScience Foundation of Beijing (7204274), the Fundamental ResearchFunds for the Central Universities, and the Interdisciplinary MedicineSeed Fund of Peking University (BMU2017MX015).

Author contributions Wu F, Zheng H, Wang W and Wu Q per-formed the experiments; Zhang Q performed ultrafast transient ab-sorption spectroscopy experiment; Guo J, Pu B and Shi X contributed tothe establishment of the mathematical model; Wu F, Hong W, Li J andChen X wrote the paper with support from Zheng H. All authors con-tributed to the general discussion.

Conflict of interest The authors declare that they have no conflict of



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interest.

Supplementary information Experimental details are available in theonline version of the paper.

Fan Wu is currently a graduate student in theSchool of Biological Science and Medical En-gineering at Beihang University. He received hisbachelor’s degree from Tangshan College in2017. In 2018, he joined Prof. Weili Hong’s lab,focusing on the application of coherent Ramanmicroscope for antibiotic susceptibility testingand medical photonics.

Huiling Zheng is currently a graduate student inthe Department of Microbiology, Peking Uni-versity Health Science Center. She received herbachelor’s degree from XiangYa School of Med-icine, Central South University, in 2018. And inthe same year, she joined the research group ofassociate prof. Xiangmei Chen, focusing onstudying the pathogenesis of hepatitis B virus(HBV)-related hepatocellular carcinoma (HCC).

Weili Hong is an associate professor at BeijingAdvanced Innovation Center of Biomedical En-gineering at Beihang University, Beijing, China.He obtained his BSc degree from the Universityof Science and Technology of China, and PhDdegree from the University of Utah. After apostdoctoral training at Purdue University, hejoined Beihang University as an associate pro-fessor. His research focuses on the developmentand biomedical applications of optics and label-free imaging.Xiangmei Chen received her PhD degree ofmedical genetics from Harbin Medical Universityin 2006. She completed postdoctoral training atthe Department of Microbiology, Peking Uni-versity Health Science Center, in 2008. She be-came assistant professor of medical microbiologyat Peking University Health Science Center in2008 and was promoted to associate professor in2012. Her current research interest focuses onthe pathogenesis of infection-associated cancers,especially HBV-related HCC and helicobacterpylori-induced gastric cancers.

Jiebo Li gained his BSc and MSc from PekingUniversity, and PhD degree from Rice Uni-versity, Texas, USA. Now, he is an associateprofessor at Beijing Advanced Innovation Centerfor Biomedical Engineering, Beihang University.His research focuses on developing variousspectroscopy techniques to understand the in-terfacial problems in biomedical engineering re-lated areas.

MXene介导的近红外光热效应用于快速清除多种耐药菌以及菌膜吴凡1†, 郑惠玲2†, 王文钊1, 吴琼2, 张琦3, 郭佳钰1, 普邦政1,史芯源1, 李介博1*, 陈香梅2*, 洪维礼1*

摘要随着细菌耐药性的不断出现和加重, 针对耐药菌感染和菌膜这类难以用传统抗生素治疗的临床医疗问题, 需要发展新型高效快速的杀菌方法. 本文采用新型二维材料MXene与近红外激光相结合, 实现了在20 min内对细菌以及菌膜的快速高效杀除. 为了测试该方案的广谱抗菌性, 我们对包括耐药性的耐甲氧西林金黄色葡萄球菌(MRSA)和耐万古霉素肠球菌(VRE)等多种耐药菌进行了快速灭菌实验. 我们发现二碳化三钛类的MXene与808 nm激光的组合, 对所测试的15种细菌均展现出显著的抗菌特性. 并且该杀菌方案可通过破坏菌膜结构杀灭深层细菌, 快速清除由MRSA所形成的菌膜.此外,对抗菌机理的研究显示, MXene和近红外激光主要是通过物理性的MXene插入及接触和光热效应杀死细菌, 从而可以显著地降低细菌耐药性的产生. 本工作提出了一种物理性清除多种耐药菌和菌膜的方案, 并扩展了新型二维材料MXene在生物医学领域和临床上的潜在应用范围.



Rapid eradication of antibiotic-resistant bacteria and biofilms by MXene and near-infrared light through photothermal ablation INTRODUCTIONEXPERIMENTAL SECTIONPreparation of MXenesBacterial species antibacterial activity activityOxidation of GSHSuperoxide radical assayBacterial water bath assay Confocal laser scanning microscopy of live and dead bacteria in biofilmUltrafast transient absorption spectroscopy system

RESULTSRapid eradication of bacteria with MXene and NIR lightAntibacterial mechanisms of MXene with lightBroad-spectrum antibacterial activity of MXene with lightRapid eradication of bacterial biofilms with MXene and light

CONCLUSIONS

rapid eradication of antibiotic-resistant bacteria and biofilms by … · 2021. 2. 2. ·...

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