Visible light-induced biocidal activities and

Journal of Photochemistry & Photobiology, B: Biology 185 (2018) 199–205

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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Visible light-induced biocidal activities and mechanistic study of neutral porphyrin derivatives against S. aureus and E. coli

T

Jing Wanga,b, Xia Yanga,c,d, Hu Songa,b, Wei Liaoa, Liangang Zhuoa, Guanquan Wanga, ⁎ ⁎ Hongyuan Weia,c,d, Yuchuan Yanga,c,d, Shunzhong Luoa, , Zhijun Zhoua, a

Institute of Nuclear Physics and Chemistry, China Academy of Engineering Physics, 621900 Mianyang, PR China Key Laboratory of Nuclear Medicine and Molecular Imaging of Sichuan Province, 621999 Mianyang, PR China c Collaborative Innovation Center of Radiation Medicine of Jiangsu, Higher Education Institutions, 215123 Suzhou, PR China d Department of Nuclear Medicine, The Affiliated Hospital Southwest of Medical University, 646000 Luzhou, PR China. b

A R T I C LE I N FO

A B S T R A C T

Keywords: Biocidal activity Photoinactivation Porphyrin Electronic effect

Positive charged porphyrins have long been regarded as effective biocidal agents, however neutral porphyrins have rarely been studied in their ability photoinactivating microbials, and the structure-activity relationship such as correlation of electronic effect and biocidal activity of porphyrins still remains unclear. Herein, four neutral porphyrins with various electronic effects were selected to undergo light-induced biocidal processes. It turned out that the TPPOH and TPPNH2 with electron-donating groups eNH2 and eOH, respectively, exhibited much more powerful light-induced biocidal activities against E. coli and S. aureus than TPP and TPPNO2 with electron-withdrawing group eNO2. This phenomenon suggested that neutral porphyrins may be treated as a new class of biocidal agents and functional groups with various electronic effects on porphyrins can dramatically affect porphyrins' light-induced biocidal activities. Mechanistic studies demonstrate that despite a better lightinduced antibacterial ability of TPPOH, its singlet oxygen generation efficacy is a little lower than that of TPPNH2, together with charge characteristics and lipophilicity, it is clear that (1) the oxidative species singlet oxygen and ROS played the key role in the photo-activated antimicrobial processes of porphyrins, and (2) higher singlet oxygen or ROS yields of TPPOH and TPPNH2 may originate from their structural characteristics, namely electron-donating groups eOH or eNH2, and (3) a synergistic effect of all other factors including the electrostatic and hydrophobic effects must involve in the process and cooperatively determine their biocidal activities.

1. Introduction Every year millions of people died of bacterial infections. A bacterial infection caused great threat to the public health, also caused large social cost to society. Based on the data from acute care hospitals according to the U.S. centers for disease control and prevention, about 722,000 people infected in hospitals in 2011, and about 75,000 hospital patients died during their hospitalizations [1]. Despite antibiotics saving lives of many people, a realistic problem also should not allow to be ignored that a variety of bacteria have developed resistance to antibiotics owing to the abuse of antibiotics. Even more alarming is the appearance of multi- or pan-resistant Gram-negative strains with extended spectrum β-lactamase and the New Delhi metallo-β-lactamase resistance [2]. More recently, multidrug-resistant Acinetobacter baumann and Pseudomonas aeruginosa have become a major cause in controlling infectious diseases [3]. Against this threat, it is necessary to develop new antimicrobial strategies. For years, photodynamic ⁎

Corresponding authors. E-mail addresses: [email protected] (S. Luo), [email protected] (Z. Zhou).

https://doi.org/10.1016/j.jphotobiol.2018.06.003 Received 20 March 2018; Received in revised form 27 May 2018; Accepted 12 June 2018 1011-1344/ © 2018 Published by Elsevier B.V.

inactivation (PDI) of pathogens as an alternative strategy to deactivation of bacteria has attracted tremendous and intensive research interest due to its significant advantages in biocidal efficacy, no bacterial resistance developed, controllable, recyclable and green over traditional biocides [4–6]. PDI is dependent on light, oxygen, photosensitizer (PS) and with the following mechanism: upon irradiation of light, PS absorbs light and leads to its excitation and energy transfer, then ultimately produces strong oxidizing reactive oxygen species to inactivate pathogens [7]. Gram-positive and Gram-negative bacteria exhibit fundamentally different susceptibility to PS due to their distinct physiology [8]. A variety of PS has been developed over the past decades [9–11]. Among those promising PS, porphyrins, which have been found to be endogenous in cells, have emerged as a major type of photodynamic agents because porphyrins are able to be activated by visible light to perform cytotoxicity to certain bacterial species [12–15]. So far, a few photodynamic agents with porphyrin backbone have been approved for


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treating cancers and other diseases [16]. Porphyrin has several significant characteristics: (1) it owns the characteristics of both type I and type II photo processes [17] meaning that porphyrin can produce both reactive oxygen species (ROS) by electron transfer of the triplet state of PS followed by reacting with oxygen (type I), and high quantum yield of singlet oxygen (1O2) by energy transfer of the triplet state of PS to ground state oxygen (3O2) (type II) [18–20], (2) porphyrin is highly photostable, and is not prone to photobleaching, (3) the photophysical and biocidal properties of porphyrin is tunable by introducing side chains or metal ions into its structure [12]. These properties make porphyrin an attractive antimicrobial agent. Hence, intensive research efforts have been made to enhance porphyrin's biocidal ability. Up to date, positive charged porphyrins have long been regarded as effective biocidal agents, however neutral porphyrins have rarely been studied in their light-induced biocidal abilities, and the structure-activity relationship, such as correlation of electronic effect and biocidal activity of porphyrins, still remains unclear [21]. Additional reports observed that lipophilicity may also play an important role in cytotoxicity of porphyrins [22, 23]. Herein, four neutral porphyrins with various electronic effects were selected to undergo light-induced biocidal processes. It showed that TPPOH (4,4′,4″,4‴-(porphyrin-5,10,15,20-tetrayl)tetraphenol) and TPPNH2 (4,4′,4″,4‴-(porphyrin-5,10,15,20-tetrayl)tetraaniline) with electron-donating groups eNH2 and eOH, respectively, exhibited much more powerful light-induced biocidal activities against E. coli and S. aureus than TPP (5,10,15,20-Tetraphenylporphyrin) and TPPNO2 (5,10,15,20-tetrakis(4-nitrophenyl)porphyrin) with electron-withdrawing group eNO2. This phenomenon indicated that neutral porphyrins may function as a new class of biocidal agents and functional groups with various electronic effects on porphyrins can dramatically affect porphyrins' light-induced biocidal activities. In addition, in order to illuminate the biocidal mechanism of these porphyrins further, their ability of singlet oxygen generation, bacterial membrane perturbations, morphological damages, photostability and the lipophilicity were studied. To the best of our knowledge, there is no such comparison study made, nor study about the potential of these neutral porphyrins as biocide reported so far.

Scheme 1. Structure of selected porphyrins: TPP, TPPOH, TPPNO2, and TPPNH2.

[27] are performed based on corresponding references. 2.2. Biocidal Experiments Gram-negative bacterium E. coli and Gram-positive bacterium S. aureus were selected for this study. Bacterial samples were transferred from the frozen state onto agar plates 1.5% agar + standard Luria broth (LB) and incubated at 37 °C for 24 h, then stored at 4 °C for use in two weeks. A single colony from the slants was incubated in 5 mL of LB for 18 h with shaking at 30 °C. The bacterial culture was then centrifuged at 4000 r/min for 5 min and the pellet was suspended in 0.9% NaCl solution. This washing procedure was repeated in triplicate. The cell pellet was resuspended in 0.9% NaCl solution to OD600~1.0. The final concentration of bacteria was ~109 colony forming units (CFU)/mL. Porphyrin stock solutions were prepared by dissolving a porphyrin compound in dimethyl sulfoxide (DMSO) to form 1 mg/mL solution for biocidal evaluation. To exam the biocidal effect of porphyrins on Gram-negative and Gram-positive bacteria, E. coli and S. aureus were incubated with various concentrations of porphyrins (1.0, 3.0, and 9.0 μg/mL in 0.9% NaCl for E. coli and 0.01, 0.03, 0.09, and 0.3 μg/mL in 0.9% NaCl for S. aureus) at room temperature for 60 or 120 min in the dark or exposed to visible light (11 mw/cm2, Mejiro Genossen MVL-210 fiber light, wavelength: 400–800 nm). Then the biocidal solutions were taken out and diluted CFU of bacteria in the aforementioned samples were cultured on LB agar plates and incubated for 18–24 h at 30 °C. The ability of porphyrins to inactivate bacteria cells was determined by the plate counting method and was calculated as survival fractions (N/N0), where N is the number of CFU of the bacteria solution after exposed to porphyrins and N0 is the CFU of a control (bacteria alone in the dark or exposed to visible light).

2. Experimental 2.1. Material and Methods All porphyrin derivatives (Scheme 1), intermediates, 5(6)-Carboxyfluorescein (hereafter referred to as fluorescein), and culture media were purchased from Sigma Aldrich. 1,2-Dioleoyl-sn-glycero-3phospho-(1′-rac-glycerol) (sodium salt) (DOPG), E. coli total lipid, and cholesterol were purchased from Avanti Polar Lipids. Superfine Sephadex G-25 was purchased from GE Healthcare Bio-Science. All of the solvents were HPLC grade and purchased from Honeywell and used without further purification. E. coli strain ATCC25922 and S. aureus strain ATCC25923 were supplied by the Institute of Microbiology of Chinese Academy of Sciences. Ultrapure water was used throughout the study (Milli-Q, 18.2 MΩ/cm resistivity). Lipophilicity was obtained according to the shake-flask method by determining the distribution coefficient (logD7.4) of the porphyrin derivatives in n-octanol and PBS buffer (pH 7.4) as aqueous phase [24]. Care was taken to avoid cross-contamination between the phases. The partition coefficient was calculated as the average log ratio of the radioactivity in the organic fraction and the PBS fraction. The photostability of TPPNH2 and TPPOH under visible light irradiation was tested by comparing the absorbance of the porphyrins after light irradiation. The singlet oxygen generation was tested by comparing the absorbance of DPBF (1,3-Diphenylisobenzofuran) at 412 nm with and without porphyrins after light irradiation. The details of other experimental methods, including preparation of fluorescein-loaded vesicles and vesicle leakage assays [25, 26], and observation of cell morphology

2.3. Stability of TPPOH and TPPNH2 Under Light Irradiation Stock solutions (1 mg/mL) of TPPOH and TPPNH2 in DMSO were prepared, and 500 μL of each porphyrin stock solution was added in 3 mL H2O. Then the porphyrin solutions were irradiated under visible light (11 mw/cm2). Absorption spectra of porphyrin solutions were recorded every 20 min (a PerkinElmer Lambda 850 UV/Vis spectrometer) from 700 nm to 250 nm. The specific absorption peaks were observed. And their stability under light irradiation was evaluated by the change of the absorbance peak intensity. 200


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2.4. Singlet Oxygen Generation of TPPOH and TPPNH2 A fresh stock solution of DPBF (1 mg/mL) in CH3OH and porphyrin stock solutions (1 mg/mL) in DMSO were prepared and kept in dark. Into 3 mL CH3OH, 5 μL DPBF stock solution and 5 μL porphyrin stock solution were added. The sample was irradiated on visible light (11 mw/cm2) for 10s and the absorbance at 412 nm was collected, which was repeated till the sample had been irradiated for 60s in total. The ability to generate singlet oxygen was evaluated by comparing the UV absorbance decrease between DPBF solution itself and DPBF-porphyrin mixture solution. 3. Results and Discussion 3.1. Dark Biocidal Properties of Porphyrins The biocidal activities of TPP and porphyrin derivatives containing electron-withdrawing group eNO2 (TPPNO2), electron-donating groups eNH2 (TPPNH2) and eOH (TPPOH) against E. coli and S. aureus in the dark or exposed to visible light were evaluated at various concentrations. In the dark, four porphyrins showed no or very little biocidal activity against E. coli after 2 h incubation with each porphyrin at 9 μg/ mL, and a highest dark killing (10 ± 3.0)% was obtained from TPPNH2 (Fig. 1A). A lower survival rate was not observed with increasing concentrations of porphyrins due to their poor water solubility. In the case of S. aureus, porphyrins exhibited enhanced biocidal activities to a greater or lesser extent, and the most potent killing was observed with TPPNH2, which only showed a (30 ± 3.2)% killing (Fig. 1B). Below the concentration of 9 μg/mL, no evaluable killing was observed for all tested porphyrins against two bacterial strains.

Fig. 2. Survival rate (%) of E. coli (A) and S. aureus (B) after incubated with four porphyrins exposed to the light for 2 h with different optical density, where the concentration of each porphyrin is 9 μg/mL for E. coli and 0.3 μg/mL for S. aureus. Error bars represent the standard deviations of three parallel measurements. *Represents a statistically significant difference of p < 0.05 when compared to the control.

3.2. Visible Light-Induced Biocidal Activities of Porphyrins of four porphyrins were remarkably improved. At 9 μg/mL, in the E. coli suspension, the most potent porphyrin TPPOH showed a (95 ± 2.5)% killing, and the least potent porphyrin (TPP) showed a (20 ± 2.2)% killing (Fig. 2A). The photodynamic cytotoxicity of four porphyrins followed the sequence: TPPOH > TPPNH2 > TPPNO2 > TPP. For S. aureus, four porphyrins showed the same trend in their potency in killing bacteria, but S. aureus was much vulnerable to porphyrins, to obtain the same killing, the concentrations of porphyrins were much less than those against E. coli (Fig. 2B). Moreover, the biocidal activity was improved with increasing of optical density (Fig. 2). Based on the above research results, TPPNH2 and TPPOH have displayed more potent light-induced biocidal activities among the four porphyrins. Because their biocidal activities were induced by visible light, it became necessary to investigate their stability under visible light irradiation for their potential application. Herein, absorption spectra were applied to evaluate the photo-stability of both TPPOH and TPPNH2. As TPP is the most used reference in PDT, so we also provided the photo-stability of TPP and the comparison data. It can be seen from Fig. 3, TPPNH2, TPPOH and TPP displayed specific absorption peaks at 434 nm, 417 nm and 419 nm, respectively. Time-dependent absorption spectra indicated that the absorptions of TPPOH, TPPNH2 and TPP decreased under 11 mw/cm2 visible light irradiation. It can also be seen that the decomposition of TPPNH2 proceeded more rapidly than TPPOH. The absorbance decreases of TPPOH, TPPNH2 and TPP is 23%, 58% and 18%, respectively, which suggested that both TPPOH and TPPNH2 showed photo instability more or less. Together with the lightinduced biocidal ability, TPPOH kept more stable than TPPNH2 under light irradiation, serving as a solid explanation for the stronger biocidal activity of TPPOH than TPPNH2. Despite the partial decomposition of TPPOH and TPPNH2 during visible light irradiation, it can still consider both porphyrins to be reasonably stable and deserve excellent biocidal activities. Moreover, higher stability of TPPOH under light irradiation extends its potential antimicrobial application.

When exposed to the visible light irradiation, the biocidal activities

Fig. 1. Survival rate (%) of E. coli (A) and S. aureus (B) after incubated with four porphyrins in the dark for 2 h with various concentrations. Error bars represent the standard deviations of three parallel measurements. *Represents a statistically significant difference of p < 0.05 when compared to the control. 201


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Fig. 3. Time-dependent absorption spectra of TPPNH2 (A), TPPOH (B) and TPP (C) under 11 mw/cm2 visible light irradiation. Time-dependent relative absorption profile of TPPNH2, TPPOH and TPP at the maximum absorption wavelength (referring to Table 1) of each compound (D) under 11 mw/cm2 visible light irradiation.

or TPPNH2, the absorbance of DPBF decreased more evident than the solution containing only DPBF compound. To be noticed, the rate of absorbance decrease of TPPNH2 was faster than that of TPPOH, which indicated that the efficiency of TPPNH2 to generate singlet oxygen was higher than that of TPPOH. Considering that TPPOH is a little better than TPPNH2 in their biocidal activities, it is clear that despite the major role of singlet oxygen in antimicrobial activities of biocidal agents, a cooperated effect of other factors may not be neglected.

3.3. Singlet Oxygen Generation of TPPOH and TPPNH2 Antimicrobial ability of PS is dependent on a number of factors involving in biocidal process, however, it is believed that reactive oxygen species such as singlet oxygen may play the leading role. Among the tested porphyrins, TPPOH and TPPNH2 exhibited remarkable lightinduced biocidal activities, so it is important to determine their ability to generate singlet oxygen. Herein, the generation of the singlet oxygen was detected by the absorbance intensity decay of DPBF which was used as a singlet oxygen scavenger. The time-dependent photobleaching of DPBF with and without porphyrins under visible light irradiation were recorded. As shown in Fig. 4, DPBF is light-sensitive and fast photo-bleached, so the DPBF absorbance at 412 nm decreased by itself under visible light irradiation. However, upon addition of TPPOH

3.4. Lipophilicity of TPPOH and TPPNH2 As mentioned above, the efficacy of singlet oxygen generation of TPPNH2 and TPPOH is not consistent with their biocidal activities, and it was reported that lipophilicity may largely determine the membraneperturbation ability of a biocidal molecule, thus further affect the physiological function of bacteria and dramatically enhance the disruptive ability of singlet oxygen. It becomes very important to investigate lipophilicity of biocidal agents and reveal their functions in antibacterial activities. Porphyrin is generally regarded as a weak base originated from its pyrrole rings, thus four porphyrins are, more or less, positive charged in 0.9% NaCl solution. Furthermore, hydroxyl group and amino group as strong electron donors induce the increase of electron density on pyrrole rings, which makes TPPOH and TPPNH2 more basic and readily protonated than the porphyrin bearing electron-withdrawing group eNO2. So TPPOH and TPPNH2 present more positive charges than TPPNO2 and TPP in aqueous solution, thus, TPPOH and TPPNH2 are more in favor of absorption onto bacteria with negative charged surface and likely produce more powerful biocidal activities against E. coli and S. aureus. On the other hand, our previous studies [23] have proven that lipophilicity of biocides can also contribute to their activities against bacteria. We compared the lipophilicity of porphyrins by measuring their lipo-hydro partition coefficient (logD7.4) and the data was followed: TPPOH < TPPNO2 < TPPNH2 < TPP (Table 1). The above-

Fig. 4. Time-dependent photobleaching of DPBF absorbance at 412 nm, A412 (DPBF), upon visible light irradiation, in the absence and presence of porphyrins. 202


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Table 1 Lipophilicity, absorption, and emission data for selected porphyrins.

LogD7.4 Maximum Maximum Maximum Maximum

in H2O (nm) absorption in CH3OH (nm) emission in CH3OH (nm) emission in H2O (nm)

TPP

TPPNO2

TPPNH2

TPPOH

1.486 419 419 652 655

0.916 421 415 654 646

1.372 434 415 655 662

0.570 417 418 N.A. 655

mentioned values indicated that water-solubility of four porphyrins is as follows: TPPOH > TPPNO2 > TPPNH2 > TPP. Interestingly, the light-activated biocidal activity was mostly proportional to the hydrophilicity except for TPPNO2. It is generally believed that poor solubility will be beneficial for biocidal molecule internalization into bacterial membrane for the purpose of decreasing its Gibbs free energy, accordingly, with its proximity to bacterial living matter such as protein and DNA, the cytotoxicity upon light irradiation would become more significant. However, it must be noted that poor water-solubility might also lead to the significant reduction of effective concentrations of porphyrins. When the concentrations (for TPPNO2, TPPNH2, and TPP) were over 150 μg/mL, the absorbance didn't enhance significantly with higher concentrations of porphyrins in absorption spectra (data not shown).

3.5. Disruption of Bacterial Membrane Mimicking Liposomes Biological membranes, consisting largely of a lipid bilayer, are vital components of all living systems, forming the outer boundary of living cells or internal cell compartments and acting as important filters to regulate complex processes. For the antibacterial process of biocide, the interaction with the bacterium membrane is a committed step. Liposomes have been widely utilized as an experimental cell-surface model and allow us to gain insight into interaction between antimicrobial molecules and liposomes to mimic how biocides interact with biomembranes and to fundamentally understand the mechanism of their biocidal activities [28, 29]. In this study, on the basis of the differences in lipid composition between Gram-positive S. aureus and Gram-negative E. coli, two model anionic liposomes were utilized to simulate bacterial cell membranes (Table 2). The membrane perturbation activities of the porphyrins were evaluated by fluorescein release assays (Fig. 5). For the porphyrins dissolved in DMSO, the same amount of DMSO is added in the control group to eliminate the effects from DMSO in porphyrins groups. L-1, made from DOPG, was used as a model of S. aureus [30]. The dye released from L-1 as time went on even without porphyrins; however, adding porphyrins to the system accelerated the fluorescein release progress (Fig. 5A). The order of releasing effects of the porphyrins series against L-1 is as follows: TPPOH > TPPNH2 > TPPNO2 > TPP. Interestingly, this order is consistent with that of biocidal ability against S. aureus of porphyrins (Fig. 2B). The lipid composition of L-2 is E. coli total lipid, which was used as a model of E. coli. The order of release effects of the porphyrins series against L-2 (Fig. 5B) is also consistent with the order of porphyrins biocidal abilities against E. coli (Fig. 5). The porphyrins used in this study possess structural and singlet oxygen generating diversity, whereas the biocidal effects remain

Fig. 5. Fluorescein leakage profile from DOPG liposome (A) and E. coli total lipid liposome (B) with the addition of porphyrins in PBS buffer at room temperature under dark condition (Ex, 495 nm; Em, 510 nm). Fluorescence from liposomes incubated alone was subtracted.

consistent with the membrane perturbation abilities. The results from dye-release assays show that the membrane disruption ability is highly dependent on the groups that modify the electrostatic and hydrophobic interactions. 3.6. Visualization of Light-Enhanced Antimicrobial Actions Against GramNegative Bacteria Although interactions with the plasma membrane are necessary for the bactericidal actions of porphyrins compounds, interactions of these compounds with the bacterial cell envelope are also crucial because the cell envelope serves as the first point-of-contact for exogenous materials. The cell envelopes of Gram-positive and Gram-negative bacteria are compositionally and structurally different, and the resistance of E. coli to antibacterial agent is much stronger than that of S. aureus. Thus, we focus on the interactions of porphyrins with Gram-negative E. coli surface, which will provide a deep insight into the toxicity mechanism. The complexities of the cell envelopes make such biological entities difficult to mimic with model system. In this study, the interactions of porphyrins with Gram-negative Bacteria E. coli under visible light irradiation were studied by visualizing cell morphology using SEM (Fig. 6). SEM was used to image morphological damages of E. coli cells with porphyrins either under the visible light irradiation or in the dark. Cell morphological damage was neither observed in the dark with porphyrins (data not shown), nor was it observed under visible light irradiation without porphyrins (Fig. 6A) or with TPP (Fig. 6B) or with TPPNO2 (Fig. 6C). Meanwhile, some cell morphological damage was observed with TPPNH2 (Fig. 6D) and TPPOH (Fig. 6E). The cell morphology collapsed, which is probably induced by cytoplasm leakage. The morphological damage to E. coli was consistent with both the

Table 2 Liposome Abbreviations and Corresponding Compositions and Sizes. Liposome

Lipid composition

Net surface charge (mV)

Hydronamic diameter (nm)

L-1 L-2

DOPG E. coli total lipid

−31.3 ± 1.5 −19.4 ± 0.8

198.8 ± 4.2 152.8 ± 3.6

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Fig. 6. SEM images of E. coli cells (ATCC25922) (1 × 108 CFU/mL) alone (A) and incubated with 9 μg/mL TPP (B) or TPPNO2 (C) or TPPNH2 (D) or TPPOH (E) under visible light irradiation (11 mW/cm2).

Scheme 2. Presumed Mechanism of TPPNH2 and TPPOH against Bacteria.

adsorption of porphyrins to perform biocidal activity, while the latter is determined by a combination of hydrophobic and electrostatic interactions. Based on the light-induced biocidal activity and mechanistic study, the possible mechanism for TPPNH2 and TPPOH inactivating bacteria is proposed here (Scheme 2). TPPNH2 and TPPOH are protonated in aqueous solution (pH 7.4), present as positive charged state, then they may be adsorbed onto the bacterial surface by electrostatic interaction. And both of them perform better water-soluble, especially for TPPOH, which increase the effective concentration for antibacteria. Upon irradiation of visible light, the close contact generated singlet oxygen produce greater biocidal activity.

porphyrins-induced antibacterial activity (Fig. 2A) and the perturbation of bacterial membrane liposome (Fig. 5B). From the results mentioned above, the biocidal activity of porphyrins is related to their structure, concentrations, optical density, and bacteria strains closely. The various functional groups on porphyrins change the hydrophobicity and electronic effect significantly, which affect the interactions between porphyrins and bacterial membrane. TPPNH2 and TPPOH can be easily protonated and thus present as positively charged molecules in aqueous solution (pH 7.4), which promotes their rapid adsorption onto a negatively charged bacterial surface. Additionally, the results of interactions between porphyrins and liposome model have showed that TPPNH2 and TPPOH can much more easily induce fluorescein leakage. Hence, probably the enhanced interactions between TPPNH2 & TPPOH and bacterial membrane play a key role in their biocidal activities. Our results show that porphyrins cause great damage to both E. coli and S. aureus cells under visible light irradiation due to reactive singlet oxygen. Singlet oxygen has a relatively long lifetime (10−6–10−5 s) and diffusion range in pure water. However, for high reactivity of singlet oxygen to biomolecules, its diffusion distance and lifetime is remarkably reduced in cells [31, 32]. Due to the effective range of singlet oxygen within nanometers, the damage caused by singlet oxygen is likely related to its positions to the cells. In this study, porphyrins works as the generator of singlet oxygen, if they could absorb on the cell membrane, the distance between cells and singlet oxygen could be shorten, then the biocidal effect will be probably enhanced. There seems to be a synergic effect between singlet oxygen generation and

4. Conclusion The neutral porphyrin TPPOH and TPPNH2 showed much higher biocidal activities than TPP and TPPNO2 upon visible light irradiation against E. coli and S. aureus, however, all porphyrins in this manuscript exhibited no or little biocidal activity in the dark. Mechanistic studies demonstrate that despite a better light-induced antibacterial ability of TPPOH, its singlet oxygen generation efficacy is a little lower than that of TPPNH2, together with charge characteristics and lipophilicity, it is clear that (1) the oxidative species singlet oxygen and ROS played the key role in the photo-activated antimicrobial processes of porphyrins, and (2) higher singlet oxygen or ROS yields of TPPOH and TPPNH2 may originate from their structural characteristics, namely electron-donating groups eOH or -NH2, and (3) a synergistic effect of all other 204


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factors including the electrostatic and hydrophobic effects must involve in the process and cooperatively determine their biocidal activities. Together with our in vivo biodistribution study carried out before and their reasonable photostability [33], TPPOH and TPPNH2 showed a good potential as light-induced biocides.

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