Photo-catalytic Staphylococcus aureus inactivation and biofilm destruction with novel bis-tridentate iridium(III) photocatalyst

Shiho Chen,Zhishng Zhng,Li Wei,Zhongxin Fn,Yue Li,Xuezhi Wng,Tongming Feng,?,Huiyi Hung,?

a The Fourth People’s Hospital of Foshan,Foshan 528000,China

b School of Pharmaceutical Science (Shenzhen),Shenzhen Campus of Sun Yat-sen University,Shenzhen 518107,China

c College of Chemistry and Environmental Engineering,Shenzhen University,Shenzhen 518060,China

Keywords:Ir(III) photocatalyst NADH depletion Reactive oxygen species Antimicrobial photocatalytic therapy

ABSTRACT Bacterial infection is currently a serious challenge globally,causing death of thousands of human beings.New antimicrobial agents with novel mechanism of action are urgently needed.Transition metal complexes have shown great potentials in photodynamic and photocatalytic therapy.Herein,we take full advantage of metal photocatalyst and successfully developed a novel cyclometalated iridium(III) complex (Ir1) with higher biofilm damage efficiency than the clinical antibiotics.Ir1 synergistically generates reactive oxygen species and coenzyme photocatalytic activity with high efficiency under white light irradiation.Combined with these properties,Ir1 exhibited excellent photoinactivation of S.aureus and effectively damaged the biofilm.This work provides a new approach for the development of antibacterial photodynamic therapy.

The discovery of organic antibiotics several decades ago has dramatically reduced the death caused by bacterial infections [1].However,due to the abuse of antibiotics,multidrug-resistant bacteria have emerged and threatened thousands of human’s health around the world nowadays [2].For example,the methicillinresistantStaphylococcusaureus(S.aureus) is a common type of hospitalization infectious bacteria that shows resistance to several antibiotics [3].Thus,it is important to investigate antimicrobial agents with novel mechanism of action (MoA).

All the time,transition metal complexes are widely used in cancer diagnosis and treatment [4–7],which arises people to explore their effectiveness to antibacterial.Therefore,in addition to the traditional organic antibiotic,transition metal complexes which exhibit inherent phosphorescent and readily tunable photophysicochemical properties have been reported to combat the severe drug-resistant bacterial.Collins’s group discovered several series of polynuclear complexes Ru(II) complexes for antimicrobial applicationviaintercalating with nucleic acids [8,9].On the other hand,iridium complexes have also been discovered as potential antibacterial agents.In 2018,Sadleretal.synthesized Ir(III) complexes with metformin which showed antimicrobial activity [10].In addition,Sasmaletal.reported a series of cyclometalated Ir(III)complexes with simultaneous ultrasensitive detection properties to eliminate drug-resistant bacteria [11].However,the above metal complexes function as antimicrobial chemotherapeutic drugs lacking spat-temporal selectivity and thus may induce side effects.

Antimicrobial photodynamic therapy (APDT) has been recognized as a promising therapeutic,providing patio-temporal control over drug activation to inactive bacteria.Upon light activation,APDT uses photosensitizers (PSs) to generate diverse reactive oxygen species (ROS) to induced intracellular oxidative damageviatwo main pathways.The type I pathway includes electron transfer between the excited state PS with substrate and oxygen at the ground states,generating diverse radicals and superoxide anion.Type II pathway involves direct energy transfer from excited state PS to oxygen,producing the highly reactive singlet oxygen [12].Currently,photosensitizers applied for antibacterial mainly include type II organic photosensitizers,such as porphyrin [13],4,4-difluoro-boradiazaindacene (BODIPY) [14] and aggregation-induced emission PSs analogues [15].However,the poor water solubility and high oxygen dependent MoA limited their applications.It is important to mention that metal PSs are rarely investigated in antimicrobial photo-therapy.Only until recently,Sadler and co-workers reported a photoactivatable Ru(II)-isoniazid complex consist of the labile coordinated antimicrobial drug isoniazid for mycobacteria [16].Chen and coworkers reported Ir(III) complexes targeting negatively charged bacterial membrane,despite extremely high light dose required (white light,484.2 J/cm2) [17].It is necessary to mention that the above mentioned metal PSs did not or can only slightly inhibit the capability of antibiofilm.Therefore,developing highly efficient metal complexes with the ability to destruct biofilms are urgently needed.

Our group previously reported tri-dentate Ir(III) photocatalysts exhibited high photocatalytic activity for coenzyme I,the reduced nicotinamide adenine di-nucleotide (NADH),as novel photocatalytic mechanism for phototherapeutics.NADH participates in the maintenance of intracellular redox balance,and as a coenzyme for cellular oxidoreductases [18].However,the APDT application of bis-tridentate Ir(III) photocatalysts were seldom reported.Herein,we reported a novel Ir(III) photocatalyst (Ir1) (Scheme 1) for synergistically photodynamic and photocatalytic antimicrobial therapy.Ir1effectively inactivated Gram-positiveS.aureusviagenerating diverse ROS and photocatalytic oxidation of NADH under low light dose (21.1 J/cm2).Importantly,Ir1damaged the cell membrane ofS.aureusleading to protein leakage and showed stronger ability to damage the biofilm capability than the clinical used antibiotic.

Scheme 1.The chemical structure of antibiotics,NADH,NAD+ and the bistridentate Ir(III) photocatalyst used in this study.

The synthetic routes for the tridentate ligands andIr1were shown in Scheme S1 (Supporting information) with slightly modification from literature [19].Ir1was characterized by1H NMR,13C NMR and HRMS (Figs.S1 and S2 in Supporting information).Notably,the positive-chargedIr1may improve the potent interaction with the negatively charged bacterial membrane [11].The absorption and photoluminescent spectra ofIr1were depicted in Figs.1a and b.Differ from the widely studied tri-bidentate Ir(III) complexes,Ir1exhibited extended absorption bands tailing to 600 nm and emit intense deep-red phosphorescence (λmax=685 nm) [20].Importantly,the phosphorescent intensity enhanced about 3-fold in the absence of oxygen,indicating strong interaction between the excited state ofIr1and the molecular oxygen (Fig.1b).In view of the absorbance spectrum ofIr1,the white light source was employed to trigger the photo-induced activity in phosphate buffer solution (PBS) buffer solution.

Fig.1.Photophysical and photochemical properties of Ir1.(a) Absorption spectra of Ir1 in CH2Cl2.(b) Phosphorescence spectra of Ir1 in CH2Cl2 at 298 K,λex=525 nm.(c) Photo-stability of Ir1 and Ce6.(d) ABDA photodegradation by Ir1.(e) Change of fluorescent intensity of DHR123 by Ir1.(f) Photocatalytic oxidation of NADH by Ir1.Insert: H2O2 production after white light exposure.

The dark-and photo-stability are two important factors for a potent photosensitizer candidate to avoid off-target dark toxicity and photo-degradation.Ir1exhibited higher photostability than the widely used organic PS chlorin e6 (Ce6),as evaluated by the time-dependent UV–vis spectra under continuous light irradiation(Fig.1c).The singlet oxygen generation ability (type II energy transfer pathway) ofIr1was detected by 9,10-anthracenediylbis(methylene) dimalonic acid (ABDA) [21].Upon white light irradiation,the absorbance of ABDA decreased readily (Fig.1d),compared with the standard metal PS [Ru(bpy)3]2+(Fig.S7 in Supporting information).Since superoxide anion can inactive bacteriaviatype I electron transfer pathway [22].Thus we detected the superoxide anion generation efficiency with a fluorescent sensor dihydrorhodamine 123 (DHR123) [23].As shown in Fig.1e,after extremely short white light irradiation (4 s),DHR123 was rapidly oxidized and generated the highly fluorescent rhodamine 123.Especially,when irradiated for 12 s,the fluorescent intensity enhanced 3-fold,indicating high superoxide anion generation ability ofIr1.

We previously reported that Ir(III) PSs can catalyze cellular NADH oxidation upon light irradiation and induce intracellular redox imbalance [24,25].Thus,we explored the NADH catalytic activity ofIr1.In the absence of light,the absorbance of NADH remained unchanged withIr1(Fig.S6 in Supporting information).After 5 min white light irradiation,significant decrease of absorbance at 339 nm was observed,indicating effective depletion of NADH(TOF=219.9 h-1,Fig.1f).This property might be a critical factor for antibacterial activity,since NADH plays an important role in cell metabolism of living bacteria [18].Moreover,H2O2generation was detected by H2O2test paper during NADH photocatalysis (Fig.1f,inserted photograph).The above results indicated thatIr1can generate diverse ROS and oxidized NADH upon white light activation.

The excellent photosensitization and photocatalytic activity ofIr1promoted us to investigate the antimicrobial activity toward typical bacteria such as the Gram-positive bacteria (S.aureus),the Gram-negative bacteriaE.coli(E.coli) andMycolicibacteriumsmegmatis(M.smegmetis).The antibiotic used in clinic such as ampicillin,methicillin and streptomycin (Scheme 1) were tested as well.The minimum inhibitory concentration (MIC90) was defined.After incubating withIr1for 4 h,the bacteria were either kept in the dark or received white light irradiation (21.1 J/cm2),following by 14 h incubation in the dark.The survival rates of bacteria after dark or light treatment were shown in Figs.2a and b and Fig.S8 (Supporting information).The light MIC90ofIr1againstS.aureuswas 3.13 μmol/L while there was no obvious inactivation ability toward Gram-negative bacteria.Notably,Ir1also exhibited obvious dark toxicity againstS.aureus.These results illustrated thatIr1exhibited synergistic effects againstS.aureus,functioning as chemotherapeutic agent in the dark and phototherapeutic agent upon light activation.The dark and light MIC90values ofIr1and several clinical antibiotics were summarized in Fig.S8 and Table 1.Moreover,light MIC90ofIr1againstS.aureusreaches to 1.56 μmol/L when administrated with 3-fold intensity of light power (63.3 J/cm2,Fig.S10 in Supporting informaton),indicating excellent photo-stability ofIr1and the antimicrobial activity can enhanceviaincreasing the light dose.Since the fact that low light dose can decrease side effects,further experiments were performed with white light power of 21.1 J/cm2.

Table 1MIC90 values of complexes towards S.aureus and E.coli.

Bacteria can generate biofilm,a stronger barrier,to protect themselves from antibiotics.As a result,though the clinical used antibiotics exhibit strong antimicrobial activity toward bacteria,the therapeutic effect will be significantly inhibited treating with biofilm.Given the excellent activity ofIr1againstS.aureus,the capability of biofilm destruction was further investigated.We employed the crystal violet staining to explore the efficacy of biofilm inhibition and biofilm destruction,respectively [26].The inhibition of biofilm formation was detected 44 h after dark or light treatment withS.aureusat 37 °C.As shown in Fig.2c,the inhibition efficiency of biofilm formation strongly depends on the density of bacteria (106–108CFU/mL) and the dose ofIr1.For 106CFU/mLS.aureus,4× MIC90concentration ofIr1can inhibit ～90%of biofilm biomass upon light irradiation.When the bacterial density increased,Ir1can still inhibit biofilm formation but was less effective than ampicillin (Fig.S11 in Supporting informaton).It is important to mention that ampicillin exhibited inconspicuous mature biofilm elimination capability (Fig.2d),which promoted us to investigate the matureS.aureusbiofilm destruction ability ofIr1.As shown in Fig.2d,Ir1can effectively damage the matureS.aureusbiofilm (destruction rate,～35%-40%,over 4× MIC90concentration) after white light treatment.

Fig.2.Photo-inactivation of S.aureus (a) and E.coli (b) with Ir1 under white light(21.1 J/cm2).(c) Biofilm formation inhibition experiments,and S.aureus are incubated with Ir1 for 44 h after 21.1 J/cm2 white light treatment.(d) Mature biofilm destruction experiments,and mature S.aureus biofilm are incubated with Ir1 for 3 h after 21.1 J/cm2 white light treatment.(e) The first two pictures are ROS and superoxide anion produced in bacteria of biofilm treated with various conditions,detected by DCFH-DA and DHE.On the right is S.aureus biofilm staining with SYTO 9 and PI.

The excellent ability ofIr1towards both inhibition of biofilm formation as well as the mature biofilm destruction promoted us to further investigate the potent mechanism of action towardsS.aureus.Firstly,the positive-charged nature ofIr1may enhance the drug interaction with the negatively charged bacterial membrane.In order to figure out the interaction mode,we measured the change of zeta potential ofS.aureussurface.However,the zeta potential ofS.aureusjust changed slightly in the presence or absence ofIr1(Fig.3a).Thus,Ir1did not locate at the surface of bacteria due to negligible impact on zeta potentials change of the bacteria[27,28].Considering the inconspicuous change of zeta potential ofS.aureusand strong inactivation effect ofIr1,it is expected thatIr1was able to insert into the poriferous cell wall ofS.aureus.Besides,ROS produced in biofilm is able to destroy component of biofilm containing DNA,proteins,lipids,polysaccharide,etc.As expected,Ir1can provide ROS upon biofilm,causing significant death ofS.aureusbiofilm (Fig.2e).Herein,ROS is very important for biofilm destruction,which may explain why ampicillin exhibits low effect on biofilm.

Fig.3.(a) Zeta potentials of S.aureus in H2O pretreated with or without Ir1 for 60 min.(b) BCA released from bacteria following receiving various treatments.The amount of BCA released from Ir1-treated bacteria versus from that of other formulations-treated bacteria.(c) NADH deletion within S.aureus following receiving various treatments.(d) Hemolysis test results of Ir1.

Subsequently,the enhanced bicinchoninic acid (BCA) protein assay kit was employed to evaluate the bacterial cell membrane integrity after treatment [26].As shown in Fig.3b,the BCA protein released fromIr1and light treated group was higher than the other groups,indicating that a certain degree of bacterial cell membrane was ruptured after treatment.Additionally,NADH inS.aureuswas also eliminated effectively when bacteria incubated withIr1was irradiated with white light (21.1 J/cm2) (Fig.3c).

Hemolysis,which involves the disruption of erythrocyte membranes,is an obstacle for potent pre-clinical compounds.We explored the hemolysis ofIr1preliminary by treating 10% erythrocyte stock solution with diverse concentrations ofIr1(0–6.25 μmol/L).As shown in Fig.3d,Ir1caused less than 10% hemolysis after 1 h of incubation at 37 °C,suggesting thatIr1exhibited high biocompatibility.

Compare with Ir(III) photocatalysts published previously[20,21,24,25,29],we may conclude the structure activity relationship that Ir(III) photocatalysts with bis-tridentate system can significantly extend the light absorption wavelength as well as enhance the molar extinction coefficient.Thus,bis-tridentate Ir(III) photocatalysts may be a suitable platform to develop long wavelength light excited Ir(III) photocatalyst for the treatment of bacterial infection in deep tissue.However,it should also be concerned that multiple side product may occur during synthesis which results in low synthetic yield due to the high reaction temperature (200 °C).

In summary,we designed and synthesized a novel bistridentate Ir(III) photocatalyst and evaluated the antimicrobial photodynamic therapy activity.Ir1generated diverse ROS and induced photocatalytic oxidation of NADH upon low-dose white light irradiation.Ir1exhibited excellentS.aureusinactivation activityviasynergistic type I and type II mechanisms of action.Additionally,Ir1showed promising biofilm formation inhibition and mature biofilm destruction activity,indicating relatively low antimicrobial drug resistance.The antibacterial and anti-biofilm mechanisms ofIr1may be attributed to high lipophilicity,excellent photocatalytic ROS production and NADH oxidation activity.These results suggest thatIr1may be a promising antimicrobial PDT candidate for the treatment of Gram-positive bacterial infection.As one of the few studies about iridium complexes for antimicrobial PDT,this work would further facilitate the exploration of transition metal antibacterial materials to combat the severe bacterial infection.

Declaration of competing interest

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

Acknowledgments

This work was financially supported by the “Summit Plan”High-Level Hospital Construction Project of Foshan (No.FSSYKF-2020002),the Medical Scientific Research Projects of Foshan Health Bureau (No.20210358),the National Natural Science Foundation of China (NSFC,Nos.22277153,22007104),Guangdong Basic and Applied Basic Research Foundation (No.2021B1515020050),Science,Technology and Innovation Commission of Shenzhen Municipality Project (No.JCYJ20190807152616996) and the Fundamental Research Funds for the Central Universities (No.22lgqb37).

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2023.108412.