Rapid and highly effective bacteria-killing by polydopamine/IR780@MnO2–Ti using near-infrared light

Xingfu Teng ,Xingmei Liu,** ,Zhenduo Cui ,Yufeng Zheng ,D-fu Chen ,Zhoyng Li ,Ynqin Ling,Shengli Zhu,Shuilin Wu,*

a Hubei Key Laboratory of Polymer Materials,Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials,School of Materials Science &Engineering,Hubei University,Wuhan,430062,China

b The Key Laboratory of Advanced Ceramics and Machining Technology by the Ministry of Education of China,School of Materials Science &Engineering,Tianjin University,Tianjin,300072,China

c State Key Laboratory for Turbulence and Complex System and Department of Materials Science and Engineering,College of Engineering,Peking University,Beijing,100871,China

d Beijing JiShuiTan Hospital,Beijing Research Institute Orthopaedics &Traumatology,Lab Bone Tissue Engineering,Beijing,100035,China

Keywords:Antibacterial Photothermal Photocatalytic MnO2 Implant

ABSTRACT

1.Introduction

Medical titanium(Ti)and its alloys are widely used in blood vessels[1],artificial joints[2],dental implants[3,4]and bone screws[5],due to their excellent mechanical properties,good corrosion resistance and good biocompatibility.Because these materials do not have antibacterial capabilities,the surrounding tissues still easily suffer from the invasion of bacteria to induce infections along the surface of implants,resulting in delayed healing,implantation failure and repeated surgery in spite of the strict sterilization and systemic antibiotic prevention before the implantation surgery [6].At present,the commonly used method to resolve bacterial infections is to use antibiotics.However,the excessive use of antibiotics not only weakens the biological functions of immune system,but also makes bacteria resistant[7],which in turn can produce super bacteria [8].Therefore,there is a need to develop artificial implant materials with excellent self-antibacterial properties.

In order to solve bacterial infections on implants,various strategies have been proposed,including antibacterial substrates and antibacterial surfaces.The former is to add antibacterial metal elements to the matrix during the melting process to form alloys with self-antibacterial ability by leaching metallic ions [9–11].The latter is to build antibacterial coatings on the surface of these implants to release bactericidal agents [12,13].The commonly used strategies to endow these coatings with antibacterial activity include loading antibiotics [14],doping inorganic bactericides [15,16],and mixing other organic antibacterial agents with adhesion-resistant materials [17,18].These endogenous antibacterial strategies usually require more time to kill bacteria,which can make bacteria resist these agents,even nano silver[19].In addition,once these coated implants are implanted in the body,it is difficult to control their performance before the secondary operation,and the released antibacterial agents often induced serous sideeffects during long-lasting contact with surrounding tissues [20].Therefore,there is an urgent need to develop a fast and controllable surface strategy to kill bacteria without the use of antibiotics while improving or maintaining the original biological properties of such coatings simultaneously.

Light radiation excited photothermal therapy (PTT) and photodynamic therapy (PDT) have unique advantages,such as non-drug resistance [21–23],fewer side effects,and low cytotoxicity [24],which are promising strategies for the treatment of bacterial infections.The underlying mechanism of the former is that the materials are excited to generate heat on the surface under light irradiation in a short time.Consequently,those pathogens attached on the surface are killed by local hyperthermia.The shortcoming for pure PTT is that a long exposure time and higher temperature over 60 °C are required for killing bacteria completely through destructing bacterial proteins [7,8,25],which cause burns and thermal damage to normal tissues nearby.The latter refers to the process in which photosensitizers are excited to produce a large amount of electrons under light irradiation.These photo-electrons are captured by the surrounding oxygen atoms or H2O to generate radical oxygen species (ROS),thereby killing bacteria rapidly through oxidizing the proteins inside bacteria and destroying bacterial membrane[26].However,there are still some limitations.For instance,the low ROS yields of photosensitizers inevitably lead to insufficient bacteria-killing ability while large amount of ROS is harmful for the normal cells and tissue by destructing the metabolism of cells[27,28].

In this work,a composite coating was constructed on Ti plates through a simple hydrothermal treatment and the following chemical grafting methods,as illustrated in Scheme 1.This surface system was composed of MnO2,IR780 and polydopamine (PDA).Among them,MnO2,as a biocompatible metal oxide with high photothermal performance,was hydrothermally grown on the surface of Ti plate.The positively charged IR780,a photosensitizer with strong NIR absorbance ability,was adsorbed on the negatively charged MnO2layer by electrostatic binding and then the PDA was grafted on the surface.These components possessed the functions of PTT and PDT under the irradiation of 808 nm NIR light,which endowed the implants with highly effective bactericidal ability within a short time through the synergistic effects of both ROS and hyperthermia.The detail mechanism will be discussed in the main text.

Scheme 1.The schema illustrating the fabrication process of the PDA/IR780@MnO2 coating on the surface of Ti plates.

2.Experimental

2.1.Preparation of samples

Ti plate pretreatment The biomedical Ti plates with a diameter of 6 mm and a thickness of 2.5 mm were polished step-by-step with sandpapers of different mesh sizes of 240,400,and 800,and then ultrasonically cleaned with acetone,ethanol,and deionized (DI) water and finally dried in an oven at 37 °C for further use.

Preparation of MnO2-Ti 0.225 g of potassium permanganate was dissolved in 40 mL of DI water,then 0.5 mL of 37% concentrated hydrochloric acid was added into the above solution with continuously stirring for 30 min.The mixed solution was transferred into a 100 mL high-temperature resistant polytetrafluoroethylene reactor kettle with the fixed Ti plates obliquely in it.Those Ti plates were placed with the smooth side downward.Then,the sealed reactor was placed in an oven at 100°C for 12 h,and naturally cooled to room temperature.The plates were taken out,and washed with DI water and ethanol to remove impurities,and then dried at 60 °C for 24 h.The obtained Ti plates modified with manganese dioxide nanosheets were labeled as MnO2–Ti.

Preparation of PDA/IR780@MnO2-Ti A 0.02 mg/mL IR780 solution was prepared in the solvent of dichloromethane.20 mL of the solution was dropped on the surface of the above MnO2–Ti.Then these plates were dried under vacuum to obtain IR780@MnO2–Ti.In the followed process,2 mg/mL dopamine(DA)hydrochloride solution was prepared and utilized to soak the above IR780@MnO2–Ti for 12 h in the dark to complete the polymerization process of DA on the surface of IR780@MnO2–Ti.Finally,these PDA coated samples were rinsed with DI water and dried in vacuum for further use,which were named as PDA/IR780@MnO2–Ti.

2.2.Characterization of prepared coatings

A field emission scanning electron microscope(FE-SEM,JSM7100F,JEOL,JP),a transmission electron microscope (TEM,Tecnai G20,FEI,USA)and a scanning electron microscopy(SEM,JSM6510LV,JEOL,JP)were utilized to observe the morphologies of specimens.The X-ray diffractometer (XRD,D8A25,Bruker,GER) was used to determine the phase structure of the samples.Confocal Raman microspectroscopy(Renishaw,UK) was employed to obtain the Raman spectra.The X-ray photoelectron spectroscopy (XPS,Escalab 250Xi,Thermofisher,USA)was used to determine the surface chemical compositions.Ultraviolet–visible near-infrared spectrometer (UV-3600,Shimadu,Japan) was used to study the ultraviolet–visible near-infrared absorption spectra.The light source (808 nm,LOS-BLD-0808) was used to excite the materials to produce photothermal and photodynamic effects.A FLIR E50 instrument (FLIR Systems,USA) was used to perform photothermal imaging measurements on the samples.The fluorescence images were obtained using an inverted fluorescence microscope(Olympus,IX73,Japan).

2.3.Photothermal

The photothermal effect of samples under continuous 808 nm NIR light was measured,the light source power density was constant(0.5 W/cm2) with the spot size of 1.6 cm diameter.Ti,MnO2–Ti,IR780@MnO2–Ti and PDA/IR780@MnO2–Ti samples were immersed in 200 μL of phosphate buffer solution (PBS).During the light irradiation for 15 min,the surface temperature was recorded every 1 min using a thermal imager.In order to evaluate the photothermal stability,the real-time heating/cooling curves of the PDA/IR780@MnO2–Ti were recorded for 3 cycles.

2.4.Detection of ROS

The 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) assay(Reactive Oxygen Species Assay Kit,cat#S0033;Beyotime)was used to determine the ROS production of the samples under NIR irradiation because DCFH can capture ROS as a probe.All tests were performed in a dark environment to eliminate the effects of light.Firstly,the value of 200 μL DCFH was measured in a dark environment using a microplate reader (SpectraMaxi3,Molecular Devices).Then,all samples (Pure Ti,MnO2–Ti,and PDA/IR780@MnO2–Ti)were placed into a 96-well plate,and then 200 μL of DCFH was added into each well.Each sample was irradiated with 808 nm NIR light (power density:0.5 W/cm2) for 15 min.The liquid was measured every 2 min with a microplate reader(wavelengths 488 nm and 525 nm).The ROS yields of the samples were obtained by comparing the values recorded by the microplate reader.

2.5.Antimicrobial tests

Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) were utilized as two kinds of typical bacteria to evaluate the antibacterial ability of the prepared samples.The antibacterial activity of different samples against S.aureus and E.coli can be determined by the plate coating method and SEM morphology observation,which can be referred to our previous publication in detail [29].

Briefly,all samples and equipment used were sterilized by UV light for 30 min,then the dried samples (control group:Pure Ti,experimental group:MnO2–Ti,IR780@MnO2–Ti,PDA/IR780@MnO2–Ti)were placed in a 96-well plate with and 200 μL of bacterial suspension(S.aureus:1 × 107colony-forming units per milliliter (CFU/mL),E.coli:1 × 107CFU/mL).Each sample was divided into two groups,the dark group and the light group.The dark group samples were covered with tin foil and left for 15 min to exclude the effect of light.The light group was exposed to 808 nm near infrared(NIR)light(0.5 W/cm2)for 15 min.The E.coli and S.aureus suspensions in each well were then diluted 100-fold and 200-fold,respectively,and 10 μL of the diluted bacterial suspension was evenly spread on the agar medium.Finally,the medium was incubated in a constant temperature incubator at 37°C for 24 h,the number of colonies was calculated,and the antibacterial efficiency was calculated according to the following formula [30].

Where Nc was the number of colonies in pure Ti dark group,and Ne was the number of colonies in other groups.

The bacterial morphological changes were examined by SEM after antibacterial experiment.The details were described in our previous work [31].

2.6.Cytocompatibility evaluation

Cell incubation and viability assayThe methyl thiazolyl tetrazolium(MTT)assay was used to evaluate the cytotoxicity of the samples in vitro.The cells used in the experiment were MC3T3-E1 cells.As with in vitro antibacterial experiments,all samples and equipments involved in cell experiments were sterilized under the same condition.200 μL of diluted MC3T3-E1 cell suspensions(1×105cells/mL)were added into a 96-well plate with samples at the bottom,and incubated in a constant temperature carbon dioxide incubator at 37°C for different times(1,3,and 7 days).After removing the culture solution,200 μL of 0.5 mg/mL MTT solution(solvent:PBS)was added and further incubated in a 37°C incubator for 4 h and then removed.Next,200 μL of DMSO was added into the well plate with continuous shaking for 10 min to complete the dissolution of the purple precipitate on the surface.Finally,the OD value at 490 nm was recorded on the microplate reader.Except for light irradiation,the MTT measurement of the light treatment groups was the same as described above.Briefly,at the beginning of each culture period (1,3 and 7 days),after the cells have completely attached onto the surface of samples,each sample was irradiated with 808 nm NIR light with a power of 0.5 W/cm2for 15 min,and then cultured for the same MTT treatment at a later stage.Each set of experiments consisted of three parallel groups.

Cell fluorescenceCells were examined using an inverted fluorescence microscope to evaluate their adhesion,spread,growth and proliferation behaviors.The diluted MC3T3-E1 cell suspensions were cocultured with different samples in a 96-well plate for 24 h (37 °C constant temperature carbon dioxide incubator)to ensure that the cells were evenly attached onto the sample surface.The cell-attached samples were rinsed with sterilized PBS,and then fixed with 4% formaldehyde for 10 min.The samples were then washed with PBS,and the MC3T3-E1 cells were stained with FITC (YiSen,Shanghai) for 30 min in the dark,and then stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) in the dark.After 30 s,the sample with cells growth on the surface was rinsed with PBS solution and dried for further use.Finally,an inverted fluorescence microscope was employed to observe the cell morphology on the surface of the sample.When the light source of the microscope illuminated the cells on the surface of the sample,the stained cytoplasm and nucleus emitted fluorescence,thus the image of cell fluorescence staining was captured.

3.Results and discussion

3.1.Characteristic of morphology and structure

As shown in Fig.1A,pure Ti exhibited smooth surface (insert highresolution image) with mechanical scratches on the surface.After hydrothermal treatment in an acidic KMnO4solution,a regular dense film composed of many nanosheets with a thickness of about 50 nm appeared on the surface (insert high-resolution image in Fig.1B).The coating thickness of the prepared MnO2nanosheets was about 466 nm(Fig.1C).The HR-TEM images (Fig.1D and E) showed obvious crystal lattice with a spacing of 0.71 nm,corresponding to the (001) crystal plane of MnO2[32],indicating that MnO2nanosheets were successfully prepared on the surface of Ti plates.As shown in Fig.1F,after covering IR780 solution on the surface,the surface morphology remained almost the same compared with MnO2–Ti.After immersion in DA solution,the surface morphology of the sample changed slightly due to the polymerization of DA on the surface (Fig.1G),and the thickness of the MnO2nanosheets increased to about 90 nm(insert chart in Fig.1G).As shown in Fig.1H,the thickness of the PDA/IR780@MnO2coating was approximately 648 nm.

Fig.1.(A)FE-SEM images of Pure Ti;Characterization of MnO2–Ti:(B)surface morphology and(C)cross-sectional images of MnO2–Ti and(D–E)HR-TEM image of scratched MnO2 nanosheets;FE-SEM image of (F) IR780@MnO2–Ti and (G) PDA/IR780@MnO2–Ti;(H) cross-sectional image of PDA/IR780@MnO2–Ti.

Fig.2.(A) XRD patterns of Pure Ti and MnO2–Ti;(B) Raman spectrum of MnO2–Ti;(C) XPS survey spectra of samples,(D) high-resolution spectra of Mn 2p from PDA/IR780@MnO2–Ti;high-resolution spectra of C 1s obtained from (E) IR780@MnO2–Ti,and (F) PDA/IR780@MnO2–Ti.

Fig.3.(A)UV–Vis–NIR absorbance spectra of Pure Ti,PDA-Ti,MnO2–Ti,IR780@MnO2–Ti and PDA/IR780@MnO2–Ti,(B)heating experiments of Pure Ti,MnO2–Ti,IR780@MnO2–Ti and PDA/IR780@MnO2–Ti in 200 μL of PBS aqueous solution under NIR light irradiation (15 min,0.5 W/cm2),(C) the corresponding real-time infrared thermal images of different samples immersed into PBS aqueous solution under continuous 808 nm NIR light irradiation,(D)temperature rising and cooling profiles of PDA/IR780@MnO2–Ti

XRD,Raman and XPS were used to further determine the phase components and chemical compositions of the prepared coatings.As shown in Fig.2A,three peaks were observed at 12.3°,36.7° and 66.2°,corresponding to the typical (001),(111) and (311) crystal planes of MnO2[32].The typical Raman peak of MnO2–Ti was located at 633.8 cm-1,further proving the successful preparation of MnO2on Ti plates(Fig.2B)[33].As shown in Fig.2C,the XPS survey scan disclosed the existence of the elements of C,N,O,and Mn in the prepared coatings.And with the introduction of IR780 and PDA,the signals of C,N and O elements were significantly enhanced while the peak intensity of Mn element was significantly reduced,suggesting the increase of the coating's thickness after PDA covering.According to Mn 2p high-resolution spectrum obtained from PDA/IR780@MnO2–Ti (Fig.2D),there were two typical characteristic peaks at the binding energy of 640.4 eV and 652.5 eV,corresponding to Mn (IV) 2p1/2and Mn (IV)2p3/2spin orbits.In the C 1s high-resolution spectrum of IR780@MnO2–Ti(Fig.2E),the peaks of C=C,C–C,C–N and C–Cl were located at 284.6 eV,285.0 eV,285.4 eV and 286.5 eV [34],respectively,suggesting that IR780 successfully bound to MnO2–Ti surface.For the C 1s high-resolution spectrum of PDA/IR780@MnO2–Ti (Fig.2F),it was fitted into four sub-peaks of 284.1 eV,284.6 eV,285.4 eV and 286.4 eV,which were the typical characteristic peaks of PDA,corresponding to C–C,C=C,C–N and C–O [35],respectively,indicating the successful covering of PDA on IR780@MnO2–Ti.

3.2.In vitro photothermal effects and reactive oxygen species detection

The light absorption ability of materials plays the key role for their photothermal the photocatalytic properties.As shown in Fig.3A,compared with Ti,the absorption of MnO2–Ti from visible light to the NIR wavelength was significantly enhanced and the further covering of IR 780 could not improve this ability.Pure PDA coating improved the light absorption of Ti,but did not achieve maximum absorbance.In contrast,after PDA covering the surface of IR780@MnO2–Ti,PDA/IR780@MnO2–Ti exhibited the best light absorption among these samples,especially in the range of 700–1000 nm,due to the higher absorption of PDA in NIR region.The temperature rise curves of the samples under 808 nm NIR light irradiation is shown in Fig.3B.After 15 min of 808 nm NIR light irradiation,the temperature of pure Ti hardly increased to 43.8 °C,and the temperature of MnO2–Ti and IR780@MnO2–Ti was similar,reaching up to 52.5 °C and 53.5 °C,respectively.PDA/IR780@MnO2–Ti showed the highest temperature of 56.4 °C,indicating that the photothermal effect of the composite coating was originated from MnO2,not IR780 photosensitizer or Pure Ti.PDA also played an important role in strengthening the photothermal capacity.These results are consistent with light absorption results.The corresponding real-time photothermal images(Fig.3C)also visually proved that PDA/IR780@MnO2–Ti had the best photothermal conversion efficiency among these samples.The laser on-offcycle curves shown in Fig.3D disclosed that repeated irradiation did not affect the photothermal performance of PDA/IR780@MnO2–Ti,suggesting that the prepared composite coating had a stable photo-stability.

The photocatalytic properties of the coatings are shown in Fig.4.After 15 min of 808 nm NIR irradiation,both pure Ti and MnO2–Ti groups showed almost no production of ROS.In contrast,PDA/IR780@MnO2–Ti showed a large amount of ROS production,resulting from NIR excited photosensitizer IR780 [34].The results revealed that the prepared PDA/IR780@MnO2–Ti had good photodynamic properties under 808 nm NIR light irradiation.

Fig.4.ROS production of Pure Ti,MnO2–Ti and PDA/IR780@MnO2–Ti under 808 nm NIR light irradiation as determined by DCFH.

3.3.In vitro antibacterial activity

Under the condition of 808 nm NIR light irradiation for 15 min,the antibacterial rate of the samples towards S.aureus and E.coli was measured by the plate coating method.As shown in Fig.5A,after the samples and the bacterial solution were co-cultured in the dark environment for 15 min,the total number of S.aureus colonies on all plates did not change significantly,indicating that these samples have almost no effects on the viability of the bacteria in the dark.In contrast,after 15 min of 808 nm NIR light irradiation,except for pure Ti,the number of S.aureus colonies on MnO2–Ti,IR780@MnO2–Ti and PDA/IR780@MnO2–Ti decreased in different degrees,and the group of PDA/IR780@MnO2–Ti showed the minimum number.The corresponding antibacterial efficacy is shown in Fig.5B.The antibacterial efficacy of MnO2–Ti and IR780@MnO2–Ti against S.aureus was 35.12% and 72.56%,respectively,which was ascribed to the photothermal effects of MnO2in the coating.In contrast,PDA/IR780@MnO2–Ti showed the best antibacterial efficacy of 99.94% toward S.aureus,which was attributed to the synergistic of photothermal effects of MnO2and photocatalytic performance of IR780 in the coating.Similarly,all the samples exhibited almost the same trend against E.coli,and the spread plate results and calculated antibacterial efficacy are shown in Fig.5C and D,respectively.PDA/IR780@MnO2–Ti also showed the best antibacterial efficacy of 99.89% against E.coli.

The above results were further confirmed by SEM observation of bacterial morphological changes before and after the antibacterial experiment.As shown in Fig.6A and B,in the dark condition,both S.aureus and E.coli attached to the surface of pure Ti,MnO2–Ti,IR780@MnO2–Ti and PDA/IR780@MnO2–Ti show distinct edges and intact cell walls.In contrast,after 808 nm NIR light irradiation for 15 min,those bacteria adhering to IR780@MnO2–Ti and PDA/IR780@MnO2–Ti(black arrow) showed obvious depressions,cracks,deformations and other damages.

The underlying antibacterial mechanism is schematically illustrated in Scheme 2.When irradiated by 808 nm NIR light,the photosensitizer of IR780 in the coatings can convert the ground state oxygen of3O2to the singlet oxygen of1O2,one kind of ROS.The interaction between ROS and bacteria causes oxidative damage to the bacterial cell membranes,and even ruptures the cell membrane,making the bacteria more susceptible to heat [36].Combined with the use of PTT,the local high temperature generated by the MnO2nanosheets will increase the permeability of the bacterial membrane,which makes them more vulnerable to ROS attack,which eventually leads to the damage to the bacterial membrane and the subsequent protein leakage [37].In addition,the temperature increase can significantly accelerate the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) [38],thereby weakening the antioxidant capacity of bacteria,thereby resulting in the enhancement of the antibacterial efficiency of the prepared coatings against S.aureus and E.coli.

Scheme 2.The schema illustrating the bacteria-killing mechanism of PDA/IR780@MnO2–Ti under near-infrared light.

Fig.5.(A) photographs of colonies of S.aureus,(B) antibacterial ratio histogram of S.aureus on different samples,(C) photographs of colonies of E.coli,(D)antibacterial ratio histogram of E.coli on different samples (mean ± SD,n=3,＊P ＜0.05,＊＊P ＜0.01,＊＊＊P ＜0.001).

Fig.6.Morphologies of (A) S.aureus,and (B) E.coli incubated with samples under the irradiation of 808 nm NIR light for 15 min or in darkness (scale bar=500 nm).

Fig.7.MTT assay of cells incubated on the Pure Ti,MnO2–Ti,IR780@MnO2–Ti and PDA/IR780@MnO2–Ti for 1,3,and 7 day:(A) under 808 nm NIR light for 15 min,and(B)no light(n=3,mean ± SD:＊p ＜0.05,＊＊p ＜0.01,＊＊＊p ＜0.001),(C)fluorescence photos(scale bar=50 μm)of cells after being seeded on different samples for 24 h under dark conditions.

3.4.In vitro cytocompatibility

The cytotoxicity of different samples evaluated by MTT tests is shown in Fig.7A and B.As shown in Fig.7A,after 808 nm NIR light irradiation for 15 min,the viability of cells growth on the samples exhibits more or less reduction compared with the one on pure Ti group,which is possibly ascribed to the damage from hyperthermia and ROS during irradiation.After one day of culture,the viability value of the cells dropped below 70%.However,with the increase of culture time,the cell viability value recovers,and the cell viability value of the PDA/IR780@MnO2–Ti recovers faster than the one of R780@MnO2–Ti.The cells viability of PDA/IR780@MnO2–Ti eventually recovers 100%after 7 days of culture,indicating that the covered PDA on IR780@MnO2–Ti has great biocompatibility and can reduce the toxicity caused by hyperthermia and ROS.In order to evaluate whether the prepared coating has effects on cell viability alone,the MTT results under nonlight conditions are shown in Fig.7B.Obviously,the viability of PDA/IR780@MnO2changes little,suggesting that this composite coating has excellent cytocompatibility.

It is well accepted that the initial adhesion and spreading behaviors of cells on the sample surface play important roles for the subsequent proliferation and differentiation of these cells.Therefore,it is of great significance to characterize the variation of cell morphologies on the samples.In this work,F-actin and nucleus of cells on the samples were observed by FITC and DAPI staining,respectively.As shown in Fig.7C,after culturing for 24 h in the dark,the density of cells on pure Ti and MnO2–Ti is relatively higher with scattered distribution homogeneously,and the cell diffusion area on MnO2–Ti is better than that of pure Ti because MnO2nanosheets(insert image in Fig.1B)can provide more active sites for the anchoring of filopodia of cells.After the introduction of IR780,the number of cells on IR780@MnO2–Ti decreased due to the following two factors.On the one hand,the covering of IR780 reduces the anchoring cites (Fig.2F).On the other hand,the photosensitizer of IR780 has some toxicity.However,after further introduction of PDA,the number of cells increases again and the cells spread well,showing obvious flocculent and unidirectional stripe lipid membrane extension because of the excellent biocompatibility of PDA.This result is well consistent with the result of Fig.7B,showing that the prepared PDA/IR780@MnO2composite coating is beneficial for the initial adhesion,spread,and growth of cells.

4.Conclusions

A photoresponsive composite coating of PDA/IR780@MnO2on Ti implant materials has been successfully prepared,in which the NIR responsive photosensitizer of IR780 provides a photocatalytic activity while MnO2nanosheets shows strengthened photothermal performance because of the enhanced light absorption ability with the assistance of PDA covering.Under 808 nm NIR light irradiation for 15 min,the former can produce sufficient ROS while the latter can generate hyperthermia on the surface locally.The synergistic action of both ROS and hyperthermia endows the composite coating on Ti with highly effective bacteria-killing efficiency of 99.94% and 99.89% against S.aureus and E.coli respectively under 15 min 808 nm NIR light irradiation.In the addition,this composite system exhibits good cytocompatibility due to the excellent of biocompatibility of PDA.In addition,the nanosheet structure of MnO2provides more active sites for anchoring of the filopodia of cells on the surface.Therefore,this composite coating modified Ti can be used as a potential biomaterials for manufacturing biomedical equipment or surgical instruments with rapid photoresponsive bacteria-killing ability.

Declaration of competing interest

The authors declare that they do not have any financial relation with the commercial identities or associative interests that represents a conflict of interest in connection with the work submitted.

Acknowledgements

This work is jointly supported by the Natural Science Fund of Hubei Province,2018CFA064,Beijing Municipal Health Commission (Grant No.BMHC-2019-9,BMHC-2018-4,PXM2020_026275_000002),National Natural Science Foundation of China nos.51871162,51671081,and the National Science Fund for Distinguished Young Scholars 51925104.