Selective Photodynamic Inactivation of Bacterial Cells over

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Selective Photodynamic Inactivation of Bacterial Cells over Mammalian Cells by New Triarylmethanes Ke Li,†,‡ Wanhua Lei,† Guoyu Jiang,† Yuanjun Hou,† Baowen Zhang,† Qianxiong Zhou,*,† and Xuesong Wang*,† †

Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡ University of the Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: Three new triarylmethane dyes (TAMs), MPCV, DPCV, and AEV, were synthesized and their photodynamic inactivation abilities against E. coli and human pulmonary carcinoma A549 cells were compared to two commercial TAMs, CV and EV. The enhanced hydrophilicity of MPCV and AEV decreases their cellular uptake to A549 cells dramatically. However, their binding affinity toward E. coli cells are comparable to that of CV and EV by virtue of the improved electrostatic attraction with highly negatively charged E. coli outer membranes. MPCV and AEV were also found to generate hydroxyl radicals more efficiently upon irradiation than CV and EV. Consequently, MPCV and AEV exhibited markedly improved photodynamic inactivation of E. coli cells but remarkably diminished photodynamic inactivation of A549 cells than CV and EV. The photodynamic inactivation ability of DPCV was much lower than that of CV due to its high propensity for bleaching in neutral aqueous solution. Our work demonstrates that the introduction of protonatable groups in a proper manner into the structures of TAMs may lead to selective binding and photodynamic inactivation toward bacterial cells over mammalian cells. This strategy may be extended to other types of photodynamic antimicrobial chemotherapy (PACT) agents to improve their clinical potential.

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INTRODUCTION The rapidly increasing emergence of antibiotic resistance among pathogenic bacteria urges the development of alternative antibacterial therapeutics that may not elicit resistance. In this regard, photodynamic therapy (PDT), a minimally invasive tumor treatment modality that has been successfully applied in clinics, is drawing more and more attention as a promising solution to this issue.1−3 In PDT, the interplay of a photosensitizer, oxygen, light of appropriate wavelength, and bioactive molecules generates reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide anion radical (O2•−), and hydroxyl radical (•OH), which in turn damages bioactive components of tumor cells, such as cytoplasmic membranes, intracellular proteins, and DNA, and lead to apoptosis and/or necrosis of the cells.4,5 The multipletargeting character of ROS makes tumor cells difficult to develop resistance to PDT. Similar benefits are expected in the antimicrobial application of PDT, which is usually referred to as photodynamic antimicrobial chemotherapy (PACT).1−3 In fact, PACT has long been applied in blood disinfection and was recently used to treat cutaneous leishmaniasis.6 Over the past several years, the burgeoning PACT studies witnessed © XXXX American Chemical Society

numerous new types of PACT agents, including transition metal complexes,7−9 conjugated polymers,10−12 and nanoparticles,13−16 along with porphyrin derivatives17−20 and novel organic chromophores.21−24 The high reactivity of ROS makes themselves very shortlived,25,26 which may confine the photodynamic effect within the irradiated area, accounting for the high spatial selectivity of PACT/PDT. On the other hand, the short-lived nature of ROS makes the association of photosensitizers to their biotargets extremely important. Accumulated experimental evidence has demonstrated that Gram-negative bacteria are generally more difficult to inactivate by PACT,1−3 particularly when neutral and anionic photosensitizers were used. The reason mainly stems from the different membrane structures of both types of bacteria.1−3 For Gram-positive species, their cytoplasmic membrane is surrounded by a thick but porous layer of peptidoglycan and lipoteichoic acid.1−3 In contrast, despite the layer of peptidoglycan being much thinner in Gram-negative Received: July 22, 2014 Revised: November 12, 2014

A

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Scheme 1. Chemical Structures of the Examined TAMs; the Counter Anions Are All Chloride

extending into the phototherapeutic window (600−900 nm). Crystal Violet (CV), a typical representative of TAMs, can be irreversibly fixed by Gram-positive bacteria and constructs the foundation of the Gram stain. Besides, CV was found to be trypanocidal in the dark and the trypanocidal activity may be enhanced greatly by irradiation.35,36 The fascinating biological properties of CV encouraged extensive and intensive studies of TAMs, including their basic photophysical properties,37−40 aggregation behavior,41−43 PDT activity and mechanisms toward tumor cells,44−46 PACT ability against bacteria, virus, and parasites,6,47−49 as well as their versatile applications such as chemosensors and smart materials.50−52 Despite TAMs being regarded as promising PACT agents, no efforts were paid to tune their selective binding/uptake to bacterial cells over mammalian cells via delicate chemical structure modification. To examine our aforementioned conjecture, we synthesized monopiperazine modified CV (MPCV), dipiperazine modified CV (DPCV), and aliphatic amine-modified ethyl violet (AEV) as shown in Scheme 1, and studied their binding/uptake and photodynamic inactivation efficiencies toward mammalian cells and bacteria, using human pulmonary adenocarcinoma A549 cells, rat erythrocyte, and E. coli as biotargets, respectively, and compared their properties with that of two commercial TAMs (CV and EV (ethyl violet), Scheme 1). Experimental results demonstrate that introducing protonatable groups into the structures of TAMs may promote the photodynamic inactivation capability toward E. coli, but at the same time restrict that toward mammalian cells. Our strategy may be extended to other types of PACT agents to improve their antibacterial selectivity over human cells.

species, there is a densely organized outer membrane of lipopolysaccharides (LPS) and lipo-proteins, which provides a robust physical barrier between the cell and its environment.1−3 Though membranes of both types of bacteria are highly negatively charged, the porous membranes of Gram-positive species allow neutral and even anionic photosensitizers to cross. In the cases of Gram-negative species, only cationic photosensitizers may strongly bind to the outer membrane and interrupt its integrity.1−3 Thus, cationic photosensitizers usually present a broader antibacterial spectrum than neutral and anionic ones and therefore are drawing more attention in developing new PACT agents. The high reactivity of ROS also confronts PACT with a great challenge in clinical application, i.e., how to find a therapeutic window in vivo where bacteria but not human cells are killed, an issue that must be addressed but is less explored so far.3,10,27,28 Most of the above-mentioned PACT agents exhibited strong PDT activity toward mammalian cells, e.g., many kinds of tumor cell lines. To improve the selectivity of PACT, a viable strategy is the pursuit of new PACT agents that can preferentially associate with bacterial cells over mammalian cells. Though both mammalian and bacterial cells have insidenegative transmembrane potentials, the magnitude of transmembrane potential is much smaller in mammalian cells than in bacteria. Moreover, unlike bacteria, acidic phospholipids are sequestered in the inner leaflets of the mammalian cell membranes, leading to much lower negative charge density on the cell surface.10,29 These differences may be utilized to develop PACT agents of high selectivity. Generally, a photosensitizer with higher lipophilicity can be taken up by mammalian cells (including tumor cells) more easily, provided it is water-soluble enough to prevent aggregation.30−33 It is anticipated that a photosensitizer bearing more positive charges may be more hydrophilic and thus more difficult to be taken up by mammalian cells, but still has strong binding affinity toward bacteria by virtue of electrostatic attraction.34 In this work, several new triarylmethanes (TAMs) were designed and studied to test this hypothesis. Triarylmethanes (TAMs) are a type of unique cationic photosensitizers that display long wavelength absorption

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EXPERIMENTAL SECTION

Oil/Water Partition Coefficients (log PO/W). The oil/water partition coefficients were determined at room temperature following a reported method.53 Typically, the stock aqueous solutions (1 mM) of the examined dyes were prepared at first. One milliliter of diluted solutions with the concentration of 30 μM were then mixed with 1 mL of n-octanol and sonicated for 30 min. After separation by centrifugation, the amounts of dye in each phase were quantified by the absorbance at absorption maximum of the examined dye. The results were the average of three independent measurements. B

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Figure 1. Normalized absorption spectra of CV, MPCV, DPCV (a) and EV and AEV (b) in 5 mM PBS (pH = 7.4). Cellular Binding and Uptake. The apparent binding capacities of the examined dyes to E. coli cells were estimated as follows. Suspensions of E. coli (∼108 cells/mL) in PBS (5 mM, pH = 7.4) were incubated with a dye (0.5, 1, 3, and 5 μM) at 37 °C for 30 min in the dark, then the suspensions were centrifuged at 8000 rpm for 10 min. The absorbance values of the filtrates were recorded and contrasted with the dye samples without E. coli treatment. Cellular uptake of the examined dyes by human pulmonary adenocarcinoma A549 cells was estimated as follows. A549 cells were grown in DMEM containing 10% fetal bovine serum by maintaining at 37 °C under an appropriate atmosphere with 5% CO2. Five milliliter aliquots of A549 cells were seeded into flasks with bottom area of 25 cm2 at a cell density of 8 × 104 cells mL−1 in DMEM medium, supplemented with 10% (v/v) fetal calf serum and penicillin/streptomycin. After the cells were grown to confluence, the medium was replaced by a dye-containing medium and the cells were incubated further for 1 h. Due to the high dark cytotoxicity of the examined TAMs, lower concentrations (0.5, 1, and 3 μM) were used to evaluate their cellular binding and uptake toward A549 cells. Then, the cells were washed with dye-free medium, and extracted with 1 mL methanol. The dye content of each extract was determined spectrophotometrically using calibration curves which were obtained by recording the absorption spectra of the methanol extractions of the untreated A549 cells containing known concentrations of the corresponding dye. The cellular uptake was also monitored by fluorescence microscopy on a Nikon C1Si inverted fluorescent microscope and the magnification employed was 10 × 60. MTT Assay. MTT assay was utilized to analyze cell viability of A549 cells in varied conditions. A549 cells were plated at 2 × 105 per well in a Nunc 96 well plate and allowed to grow for 24 h. The cells were exposed to increasing concentrations (0.1−10 μM) of the examined dyes and incubated for 4 h at 37 °C, and then activated for 1 h with light ≥550 nm (using an Oriel 91192 Solar stimulator as the light source and a 550 nm cutoff filter to remove the short wavelength light. The irradiation intensity was about 14 mW/cm2 and the total light dose was approximately 50 J/cm2) at 25 °C. After 20 h of cell incubation, the loading medium was removed and the cells were fed with medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide). Dark controls were run in parallel. The cell viability data were obtained by analysis of the absorbance at 490 nm of each sample using a Thermo MK3Multiscan microplate reader. The signal was normalized to 100% viable (untreated) cells. Hemolytic Activity Assay. In microcentrifuge tubes, 500 μL aliquots of varied concentrations of dye solution were mixed with 480 μL PBS and 20 μL washed rat erythrocytes so that the final concentration of erythrocytes was 2% and those of dye were 1, 5, 10, and 50 μM, respectively. A positive control (100% hemolysis) was prepared with 980 μL water and 20 μL erythrocytes. A negative control was prepared with 980 μL PBS and 20 μL erythrocytes. Samples were incubated at 37 °C for 2 h on a rocker, then centrifuged for 5 min. The optical density of the supernatant was determined by transferring 200 μL to a 96-well microtiter plate and reading at 405

nm. The absorbance of CV, EV, and AEV at all examined concentrations of 10−50 μM and MPCV at the concentrations of 10−40 μM was negligible. The absorbance of MPCV at 405 nm at 50 μM is much smaller than that of the hemolytic sample and the OD value of the hemolytic sample in this case can be corrected by deducting the absorbance of MPCV. The percentage hemolysis was determined by blanking the OD against that of the negative control and presenting the resulting OD as a proportion of the OD of the positive control (blanked with water). Antimicrobial Experiments. The photodynamic antimicrobial properties of the examined dyes were determined by incubation with E. coli cell suspensions (∼108 cells/mL) for 20 min in the dark at 37 °C and then exposed to an irradiation of ≥550 nm for 20 min (the total light dose was ca. 16.8 J/cm2). For dark toxicity, the examined dyes were incubated with E. coli cell suspensions for 40 min in the dark at 37 °C. The treated bacterial samples were diluted in PBS and were spread on 3 M Petrifilm E. coli Count Plate and incubated at 37 °C for 48 h. The number of colony-forming units (CFU) was counted by a Shineso G6 Colony Counter.

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RESULTS AND DISCUSSION Design and Synthesis of TAMs. MPCV, AEV, and DPCV were synthesized in good yields by lithiation of the corresponding 4-bromoaniline derivatives with butyllithium and then nucleophilic addition to Michler’s ketone, bis(4(diethylamino)phenyl)methanone, or 4-(dimethylamino)benzoate and finally ionization after treatment with concentrated hydrochloric acid, as shown in Supporting Information Scheme S1. Both MPCV and DPCV are designed as derivatives of CV, where the trimethyl amine group is fused with a N,Ndimethyl group of CV to form a piperazine group. The peripheral nitrogen atom of the piperazine group is expected to be protonated in neutral aqueous solution (pKa of N,N′dimethylpiperazine is 8.81, data from ChemSpider, RSC), giving the divalent cation of MPCV and trivalent cation of DPCV, respectively. Similarly, AEV may be regarded as a derivative of EV. The aliphatic tertiary amine in AEV has more flexibility with respect to that in MPCV and DPCV, and the protonation of which is also anticipated in neutral aqueous solution. Physicochemical Properties of TAMs. Figure 1 shows the absorption spectra of MPCV, DPCV, and AEV in PBS buffer along with those of CV and EV for comparison. The introduction of one or two aliphatic tertiary amine group(s) into CV and EV leads to a bathochromic shift of the absorption maximum by 4−10 nm. Interestingly, the red shift effect is more significant in MPCV (10 nm vs CV) than in AEV (3 nm vs EV), implying that the rigidity of the tertiary amine group may play a role. Additionally, the molar extinction coefficients C

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formation of carbinol. Luckily, this effect is far less important in MPCV and AEV, favorable for their application in PACT. Cellular Binding/Uptake of TAMs. Selective binding and uptake of photosensitizer molecules by bacteria over mammalian cells seems an effective strategy to achieve selective photodynamic antibacterial effect. In this work, we chose human pulmonary carcinoma A549 cells and Gram-negative E. coli cells as mammalian and bacterial cell models, respectively, to examine the cellular binding/uptake of the five TAMs. In bacteria binding measurement, an E. coli suspension was incubated in the dark in the presence of a kind of TAM for 30 min. After centrifugation to remove E. coli cells, the absorbance of filtrate was measured and correlated to the percentage of free TAM (see details in the Experimental Section). As shown in Figure 3a, the bound TAMs were 0.62, 0.53, and 0.85 nmol/106 cells for CV, MPCV, and DPCV, respectively, at the concentration of 5 μM. At other examined concentrations, the binding affinity toward E. coli cells also follows the order DPCV > CV > MPCV. The results may be reasoned as follows. On one hand, the improved hydrophilicity of both MPCV and DPCV causes their preferential partition in water phase over E. coli outer membranes. On the other hand, the additional positive charges in MPCV and DPCV enhance their electrostatic attraction with the highly negatively charged E. coli outer membranes. Consequently, the balance of both opposite effects makes the binding ability of MPCV toward E. coli cells a little bit lower than that of CV, but that of DPCV is much stronger than that of CV. Similarly, the binding ability of AEV toward E. coli cells is lower than that of EV. The uptake of the TAMs by A549 cells was also measured by monitoring their absorbance. Differently, the absorbance of the bound instead of the free dye molecules was recorded in this case (see details in Experimental Section). As shown in Figure 3b, the uptake capacities of the TAMs are highly dependent on their hydrophilicity, in the order of EV > CV > AEV > MPCV > DPCV. With 1 μM of the TAMs as an example, the uptake decreased 50% from CV to MPCV and another 55% from MPCV to DPCV. The fluorescencent feature of the examined TAMs allowed the cellular uptake visualized by confocal fluorescence imaging (Supporting Information Figure S3). After incubation for 30 min of A549 cells and the TAMs (1 μM), the red fluorescence was observed throughout the interior of the cells except nucleus, indicating the internalization of the TAMs. The sharp contrast between the binding/uptake behaviors of the TAMs toward A549 and E. coli cells may mainly result from the different membrane structures of both types of cells. Despite both types of cellular membranes being negatively charged, the inside-negative transmembrane potential is much smaller in mammalian cells than in bacteria. More importantly, a large quantity of negative charges are on the outer surface of bacterial membranes, while in mammalian cells the negative charges mainly locate in the inner leaflets of the cell membranes. As a result, the electrostatic attraction between positively charged photosensitizer molecules and negatively charged cellular membranes is much stronger for bacterial cells than for mammalian cells, which leads to a high correlation between hydrophilicity and celluar uptake in the case of mammalian cell lines but not in the case of bacteria. Hemolysis is not only an important toxicity index for a PACT agent, but also a good measure of the binding and interaction strength of a PACT agent with mammalian cell membranes. The hemolysis results of the TAMs are presented

follow the order CV > MPCV > DPCV and EV > AEV (Table 1). Table 1. Physicochemical Properties of the Examined TAMs compound

CV

MPCV

DPCV

EV

AEV

ε × 104a (L·mol−1·cm−1) λb (nm) log PO/Wc

6.66 590 1.2

4.10 600 −0.2

3.34 596 −1.4

6.74 595 2.2

3.26 598 0.4

a Molar extinction coefficient in 5 mM PBS (pH = 7.4). bAbsorption maximum in 5 mM PBS (pH = 7.4). cOil/water partition coefficient.

As expected, the introduced tertiary amine group has a large effect on the hydrophilicity of the molecules. The n-octanol/ water partition coefficients (log PO/W) were measured to follow the order of EV > CV > AEV > MPCV > DPCV (Table 1), indicating that the introduction of aliphatic tertiary amine group and its protonation may improve the hydrophilicity of the corresponding photosensitizer markedly. Consequently, lower aggregation tendencies are anticipated for MPCV, DPCV, and AEV. In fact, no aggregation behaviors were observed for the five TAMs at 10 μM in PBS buffer (Supporting Information Figure S1). The aggregation of TAMs was found to impair their photodynamic damage abilities toward biomolecules and tumor cells.41 The low aggregation tendency of MPCV, DPCV, and AEV is beneficial for their application in PACT or PDT. The enhanced electrostatic repulsion interaction between the photosensitizer molecules that bear more positive charges may also contribute to the low aggregation tendency of these TAMs. In aqueous solutions, TAMs exist in an equilibrium with their colorless carbinol forms (Supporting Information Scheme S2, taking CV as an example).54 Changing the structures might have some influence on this equilibrium. We found that the PBS solution of DPCV underwent gradual bleaching in the dark as shown in Supporting Information Figure S2b. The acid titration reversed the process, suggesting that the bleaching originated from the transformation from DPCV to its carbinol form. Figure 2 presents the comparison of the bleaching rates of the examined TAMs. Clearly, DPCV bleached far efficiently than the other TAMs, indicating that the additional two positive charges located on both peripheral nitrogen atoms of the piperazine groups may greatly improve the electrophilic character of the central carbon cation and therefore favor the

Figure 2. Bleaching curves of the examined TAMs (5 μM) in 5 mM PBS (pH = 7.4). D

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Figure 3. Binding/uptake isotherms of the examined TAMs toward E. coli cells (a) and human pulmonary carcinoma A549 cells (b).

in Figure 4 and Supporting Information Table S1. Due to the poor PACT activity of DPCV (see below), its hemolysis

Figure 5. Photodynamic inactivation of E. coli cells by the TAMs.

cells (Figure 3a) undoubtedly make their intrinsic PACT power, i.e., ROS generation ability, decisively important (see below). Among the five TAMs, DPCV is the least active one in photoinactivation of E. coli, presumably due to its high bleaching propensity in aqueous solution (Figure 2). The PDT activities of the TAMs toward A549 cells were also investigated (Supporting Information Figure S4) and IC50 values are shown in Table 2. DPCV was not involved in the

Figure 4. Hemolytic activities of the examined TAMs at varied concentrations.

behavior was not studied. CV showed negligible hemolysis at concentrations lower than 10 μM. At 50 μM level, 58.1% rat erythrocyte cells were lysed by CV. In contrast, the hemolytic activity of MPCV was still negligible even at 50 μM. Due to the higher lipophilic nature, EV can damage rat erythrocyte cells effectively at 5 μM. However, no hemolysis was observed for AEV even at 10 μM. The hemolysis capabilities of the TAMs are in good agreement with their cellular uptake capacities, demonstrating that our structure modification is effective in promoting the binding/uptake selectivity by bacterial cells over mammalian cells. Phototoxicity. The PACT and PDT activities of the five TAMs toward E. coli and A549 cells were compared upon irradiation with light ≥550 nm. As shown in Figure 5, the PACT activities of all TAMs showed strong concentration dependence. At 1 μM, the colony-forming unit (CFU) reduction by AEV was as high as 5.2 log units, whereas EV decreased log CFU by only 2.6 units, suggesting that the PACT activity of AEV is two orders of magnitude higher than that of EV. Similarly, MPCV photoinactivated E. coli cells far more efficiently than CV. All four TAMs showed negligible inactivation activity to E. coli in the dark even at 5 μM (Supporting Information Table S2).The binding affinities of MPCV and AEV to E. coli cells cannot explain their much higher PACT activities than that of CV and EV. However, the comparable binding affinities of the four TAMs toward E. coli

Table 2. Phototoxicity against A549 Cells and Reduction Potentials of the TAMs

a

compound

CV

MPCV

EV

AEV

IC50 (μM) Ereda (V, vs SCE)

1.39 −0.71

2.38 −0.67

0.24 −0.79

1.54 −0.76

Reduction peak potential in DMSO.

measurements owing to its poor PACT activity. The IC50 value of MPCV is about twice that of CV, and that of AEV is 6.4-fold that of EV. The greatly diminished PDT activities of MPCV and AEV should mainly originate from their markedly decreased cellular uptake, which in turn is the result of the much enhanced hydrophilicity of both MPCV and AEV. When PACT and PDT results are compared, one may immediately realize that improving the hydrophilic property by way of introducing protonatable groups into a TAM skeleton may restrict its PDT activity but at the same time improve its PACT activity. It is worth noting that MPCV and AEV are still very active in photodynamic killing of the mammalian cells. Particularly, both E

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a better •OH generator than EV (Figure 6b). In these cases, TAM itself acted as electron donor, because no EPR signals were observed at lower concentration (0.1 mM). However, in the presence of 1 mM DTT (DL-Dithiothreitol), DMPO−OH signals may be recorded even at low concentration of the TAMs, as shown in Supporting Information Figure S5. The enhanced •OH generation abilities of MPCV and AEV are in line with their much improved PACT activities than CV and EV. No DMPO-OOH adduct signals were observed, mainly because that the reaction rate constant of DMPO with •OH is higher than O2•− by nearly 8 orders of magnitude.56 We also measured T−T absorption spectra of CV, MPCV, EV, and AEV in PBS; however, no signals were observed in the time domain of ns and μs, indicative of their low quantum yields of triplet excited state. Using TEMP (2,2,6,6-tetramethyl4-piperidone) as spin trapping agent, we did not obtain TEMPO signals upon irradiation the PBS solutions of CV, MPCV, EV, and AEV, indicating their disability in 1O2 generation, which is in good agreement with their low quantum yields of triplet excited state. TAMs are generally short-lived (order of ps) in their singlet excited states in low-viscosity solvents due to efficient internal conversion via a synchronous rotation of the phenyl rings.60 As a result, low intersystem crossing efficiency and poor 1O2 quantum yield are common features for TAMs.60

MPCV and AEV are highly toxic against A549 cell line in the dark (Supporting Information Figure S4), despite the dark toxicity being lower than their counterparts of CV and EV. Introducing more protonatable groups into MPCV and AEV might decrease dark/phototoxicity toward mammalian cells. In light of the bleaching propensity of DPCV, additional protonatable groups should be spatially more isolated from the central carbon cation of the corresponding TAM. Moreover, our strategy may be applied to other types of PACT/PDT agents to achieve higher selectivity of PACT over PDT. PACT/PDT Mechanism of TAMs. Though the binding affinities of MPCV and AEV are a little bit lower than those of CV and EV, the PACT activities of MPCV and AEV toward E. coli cells are much higher than those of CV and EV, implying that MPCV and AEV may possess higher ROS generation abilities. Different from porphyrin-based photosensitizers which generally exhibit PDT activities by 1O2 generation (Type II mechanism), TAMs have been proved to show PDT activities via radical generation (Type I mechanism), e.g., carboncentered TAM radicals, O2•− and •OH.31,55 Generally, the radical generation is initiated by photoinduced electron transfer from an electron donor to a TAM, as shown in Scheme 2.35,42,55−57 Many biological species, such as DNA and

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Scheme 2. Type I Mechanism of TAMs

CONCLUSIONS Three new TAMs, MPCV, DPCV, and AEV, were designed and synthesized, and their PACT activities against E. coli cells and PDT activities against human pulmonary carcinoma cells A549 were compared to two commercial TAMs, CV and EV. Both MPCV and AEV exhibited much higher PACT activity toward E. coli cells but far lower PDT activity toward A549 cells than CV and EV. It was found that MPCV and AEV possess higher capability to generate •OH, and enhanced hydrophilicity than CV and EV. The improved PACT selectivity of MPCV and AEV mainly results from the enhanced hydrophilicity by way of the introduction of a protonatable aliphatic tertiary amine group into the structures. Because E. coli cells have highly negatively charged outer membrane and lots of negative charges located on the surface of the outer membranes, the strong electrostatic interaction makes the binding affinity of MPCV or AEV to E. coli cells comparable to that of CV or EV, though MPCV and AEV are more hydrophilic than CV or EV and more prone to stay in the water phase. In sharp contrast, the A549 cells have much smaller inside-negative transmembrane potential and the negative charges are buried in the inner leaflets of the membranes. The electrostatic attraction interaction is no longer important in this case and the cellular uptake of MPCV and AEV by A549 cells decrease greatly with respect to CV and EV. For DPCV, the bleaching tendency impairs its PACT activity. Our work demonstrates that the introduction of protonatable groups in a proper manner into the structures of TAMs may promote their PACT selectivity. This strategy may be extended to other types of PACT agents to improve their antibacterial selectivity over mammalian cells.

protein, may serve as electron donors. In the case of high concentration of TAM, TAM itself can also act as electron donor.57 We measured the reduction potentials of the TAMs in DMSO. The anodic shift of the reduction peak potentials of MPCV and AEV (vs CV and EV, respectively, Table 2) suggests they are stronger electron acceptors than CV and EV, favorable for ROS generation. EPR was used to compare the radical generation abilities of MPCV, CV, AEV, and EV in PBS (5 mM, pH = 7.4), using DMPO as spin trapping agent. As shown in Figure 6a, the

Figure 6. EPR spectra of CV, MPCV (a), EV and AEV (b) (1 mM) upon irradiation by a 100 W high pressure mercury lamp in the presence of DMPO (0.1 M) in PBS (5 mM, pH = 7.4).

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irradiation of MPCV in the presence of DMPO led to a fourline EPR signal with intensity ratio of 1:2:2:1 and hyperfine coupling constant of aH = aN = 15.1 G, assignable to DMPO− OH adduct.58,59 In the case of CV, the signal of DMPO−OH adduct was also observed, but the signal intensity was much lower than that of MPCV, vindicating that MPCV can generate •OH more efficiently than CV. Similarly, AEV was found to be

ASSOCIATED CONTENT

* Supporting Information S

Experimental details, normalized absorption spectra of TAMs, absorption spectrum changes of TAMs in PBS upon standing in the dark, cytotoxicity against A549 cells of dyes in the dark or upon irradiation, EPR spectra of TAMs in the presence of F

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(15) Huang, C.; Xing, C.; Wang, S.; Li, Y.; Liu, H.; Lai, S.-W.; Che, C. M.; Li, Y. Fabrication of a Well Ordered Microspheres Film for Efficient Antibacterial Activity. Chem. Commun. 2011, 47, 7644−7646. (16) Hayden, S. C.; Allam, N. K.; El-Sayed, M. A. TiO2 Nanotube/ CdS Hybrid Electrodes: Extraordinary Enhancement in the Inactivation of Escherichia coli. J. Am. Chem. Soc. 2010, 132, 14406−14408. (17) Dosselli, R.; Tampieri, C.; Ruiz-Gonzalez, R.; De Munari, S.; Ragas, X.; Sanchez-Garcia, D.; Agut, M.; Nonell, S.; Reddi, E.; Gobbo, M. Synthesis, Characterization, and Photoinduced Antibacterial Activity of Porphyrin-Type Photosensitizers Conjugated to the Antimicrobial Peptide Apidaecin 1b. J. Med. Chem. 2013, 56, 1052− 1063. (18) Liu, K.; Liu, Y.; Yao, Y.; Yuan, H.; Wang, S.; Wang, Z.; Zhang, X. Supramolecular Photosensitizers with Enhanced Antibacterial Efficiency. Angew. Chem., Int. Ed. 2013, 52, 8285−8289. (19) Jiang, G.; Lei, W.; Hou, Y.; Wang, X. Photodynamic Inactivation of Escherichia coli by Porphyrin Cytochrome c. New J. Chem. 2012, 36, 2180−2183. (20) Hager, B.; Strauss, W. S. L.; Falk, H. Cationic Hypericin Derivatives as Novel Agents with Photobactericidal Activity: Synthesis and Photodynamic Inactivation of Propionibacterium acnes. Photochem. Photobiol. 2009, 85, 1201−1206. (21) Johnson, G. A.; Muthukrishnan, N.; Pellois, J.-P. Photoinactivation of Gram Positive and Gram Negative Bacteria with the Antimicrobial Peptide (Klaklak)(2) Conjugated to the Hydrophilic Photosensitizer Eosin Y. Bioconjugate Chem. 2013, 24, 114−123. (22) Velema, W. A.; van der Berg, J. P.; Hansen, M. J.; Szymanski, W.; Driessen, A. J. M.; Feringa, B. L. Optical Control of Antibacterial Activity. Nat. Chem. 2013, 5, 924−928. (23) Takahashi, D.; Miura, T.; Toshima, K. Photodegradation of Lipopolysaccharides and the Inhibition of Macrophage Activation by Anthraquinone-Boronic Acid Hybrids. Chem. Commun. 2012, 48, 7595−7597. (24) Frimannsson, D. O.; Grossi, M.; Murtagh, J.; Paradisi, F.; O’Shea, D. F. Light Induced Antimicrobial Properties of a Brominated Boron Difluoride (BF2) Chelated Tetraarylazadipyrromethene Photosensitizer. J. Med. Chem. 2010, 53, 7337−7343. (25) Jiang, G. Y.; Lei, W. H.; Zhou, Q. X.; Hou, Y. J.; Wang, X. S.; Zhang, B. W. A New Phenol Red-Modified Porphyrin as Efficient Protein Photocleaving Agent. Phys. Chem. Chem. Phys. 2010, 12, 12229−12236. (26) Ogilby, P. R. Singlet Oxygen: There Is Indeed Something New under the Sun. Chem. Soc. Rev. 2010, 39, 3181−3209. (27) Soncin, M.; Fabris, C.; Busetti, A.; Dei, D.; Nistri, D.; Roncucci, G.; Jori, G. Approaches to Selectivity in the Zn(II)-PhthalocyaninePhotosensitized Inactivation of Wild-Type and Antibiotic-Resistant Staphylococcus aureus. Photochem. Photobiol. Sci. 2002, 1, 815−819. (28) Soukos, N. S.; Wilson, M.; Burns, T.; Speight, P. M. Photodynamic Effects of Toluidine Blue on Human Oral Keratinocytes and Fibroblasts and Streptococcus Sanguis Evaluated in vitro. Lasers Surg. Med. 1996, 18, 253−259. (29) Matsuzaki, K.; Sugishita, K.; Fujii, N.; Miyajima, K. MolecularBasis for Membrane Selectivity of an Antimicrobial Peptide, Magainin2. Biochemistry 1995, 34, 3423−3429. (30) Pavani, C.; Uchoa, A. F.; Oliveira, C. S.; Iamamoto, Y.; Baptista, M. S. Effect of Zinc Insertion and Hydrophobicity on the Membrane Interactions and PDT Activity of Porphyrin Photosensitizers. Photochem. Photobiol. Sci. 2009, 8, 233−240. (31) Sholto, A.; Lee, S.; Hoffman, B. M.; Barrett, A. G. M.; Ehrenberg, B. Spectroscopy, Binding to Liposomes and Production of Singlet Oxygen by Porphyrazines with Modularly Variable Water Solubility. Photochem. Photobiol. 2008, 84, 764−773. (32) Sugita, N.; Kawabata, K.-i.; Sasaki, K.; Sakata, I.; Umemura, S.-i. Synthesis of Amphiphilic Derivatives of Rose Bengal and their Tumor Accumulation. Bioconjugate Chem. 2007, 18, 866−873. (33) Cauchon, N.; Tian, H. J.; Langlois, J.; La Madeleine, C.; Martin, S.; All, H.; Hunting, D.; van Lier, J. E. Structure-Photodynamic

DTT, uptake of TAMs by A549 cells and Log CFU reduction of E. coli cells by TAMs in the dark. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology (2013CB933801, 2012AA062903) and NNSFC (21390400, 21172228, 21101163, 21273259, 21301182, 81171633).

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REFERENCES

(1) Jori, G.; Brown, S. B. Photosensitized Inactivation of Microorganisms. Photochem. Photobiol. Sci. 2004, 3, 403−405. (2) Hamblin, M. R.; Hasan, T. Photodynamic Therapy: a New Antimicrobial Approach to Infectious disease? Photochem. Photobiol. Sci. 2004, 3, 436−450. (3) Maisch, T.; Szeimies, R. M.; Jori, G.; Abels, C. Antibacterial Photodynamic Therapy in Dermatology. Photochem. Photobiol. Sci. 2004, 3, 907−917. (4) O’Connor, A. E.; Gallagher, W. M.; Byrne, A. T. Porphyrin and Nonporphyrin Photosensitizers in Oncology: Preclinical and Clinical Advances in Photodynamic Therapy. Photochem. Photobiol. 2009, 85, 1053−1074. (5) Detty, M. R.; Gibson, S. L.; Wagner, S. J. Current Clinical and Preclinical Photosensitizers for Use in Photodynamic Therapy. J. Med. Chem. 2004, 47, 3897−3915. (6) Baptista, M. S.; Wainwright, M. Photodynamic Antimicrobial Chemotherapy (PACT) for the Treatment of Malaria, Leishmaniasis and Trypanosomiasis. Braz. J. Med. Biol. Res. 2011, 44, 1−10. (7) Shao, Q.; Xing, B. Enzyme Responsive Luminescent Ruthenium(II) Cephalosporin Probe for Intracellular Imaging and Photoinactivation of Antibiotics Resistant Bacteria. Chem. Commun. 2012, 48, 1739−1741. (8) Heilman, B. J.; John, J., St; Oliver, S. R. J.; Mascharak, P. K. LightTriggered Eradication of Acinetobacter baumannii by Means of NO Delivery from a Porous Material with an Entrapped Metal Nitrosyl. J. Am. Chem. Soc. 2012, 134, 11573−11582. (9) Lei, W.; Zhou, Q.; Jiang, G.; Zhang, B.; Wang, X. Photodynamic Inactivation of Escherichia coli by Ru(II) Complexes. Photochem. Photobiol. Sci. 2011, 10, 887−890. (10) Zhu, C.; Yang, Q.; Liu, L.; Lv, F.; Li, S.; Yang, G.; Wang, S. Multifunctional Cationic Poly(p-phenylene vinylene) Polyelectrolytes for Selective Recognition, Imaging, and Killing of Bacteria Over Mammalian Cells. Adv. Mater. 2011, 23, 4805−4810. (11) Xing, C.; Xu, Q.; Tang, H.; Liu, L.; Wang, S. Conjugated Polymer/Porphyrin Complexes for Efficient Energy Transfer and Improving Light-Activated Antibacterial Activity. J. Am. Chem. Soc. 2009, 131, 13117−13124. (12) Corbitt, T. S.; Sommer, J. R.; Chemburu, S.; Ogawa, K.; Ista, L. K.; Lopez, G. P.; Whitten, D. G.; Schanze, K. S. Conjugated Polyelectrolyte Capsules: Light-Activated Antimicrobial Micro ″Roach Motels″. ACS Appl. Mater. Interfaces 2009, 1, 48−52. (13) Huang, J.; Ho, W.; Wang, X. Metal-Free Disinfection Effects Induced by Graphitic Carbon Nitride Polymers under Visible Light Illumination. Chem. Commun. 2014, 50, 4338−4340. (14) Sun, L.; Qin, Y.; Cao, Q.; Hu, B.; Huang, Z.; Ye, L.; Tang, X. Novel Photocatalytic Antibacterial Activity of TiO2 Microspheres Exposing 100% Reactive {111} Facets. Chem. Commun. 2011, 47, 12628−12630. G

dx.doi.org/10.1021/la5028724 | Langmuir XXXX, XXX, XXX−XXX

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Article

Activity Relationships of Substituted Zinc Trisulfophthalocyanines. Bioconjugate Chem. 2005, 16, 80−89. (34) Engelmann, F. M.; Mayer, I.; Gabrielli, D. S.; Toma, H. E.; Kowaltowski, A. J.; Araki, K.; Baptista, M. S. Interaction of Cationic meso-Porphyrins with Liposomes, Mitochondria and Erythrocytes. J. Bioenerg. Biomembr. 2007, 39, 175−185. (35) Docampo, R.; Moreno, S. N. J.; Cruz, F. S. Enhancement of the Cytotoxicity of Crystal Violet against Trypanosoma-Cruzi in the Blood by Ascorbate. Mol. Biochem. Parasitol. 1988, 27, 241−248. (36) Docampo, R.; Moreno, S. N. J.; Muniz, R. P. A.; Cruz, F. S.; Mason, R. P. Light-Enhanced Free-Radical Formation and Trypanocidal Action of Gentian-Violet (Crystal Violet). Science 1983, 220, 1292−1295. (37) Aiassa Martínez, I. M.; Montes de Oca, M. N.; Iriarte, A. G.; Ortiz, C. S.; Argüello, G. A. Study on the Interaction of Basic Violet 2 with Hydroxypropyl-β-Cyclodextrin. Dyes Pigm. 2012, 92, 758−765. (38) Punzi, A.; Martin-Gassin, G.; Grilj, J.; Vauthey, E. Effect of Salt on the Excited-State Dynamics of Malachite Green in Bulk Aqueous Solutions and at Air/Water Interfaces: a Femtosecond Transient Absorption and Surface Second Harmonic Generation Study. J. Phys. Chem. C 2009, 113, 11822−11829. (39) Bhasikuttan, A. C.; Mohanty, J.; Nau, W. M.; Pal, H. Efficient Fluorescence Enhancement and Cooperative Binding of an Organic Dye in a Supra-Biomolecular Host-Protein Assembly. Angew. Chem., Int. Ed. 2007, 46, 4120−4122. (40) Sanguinet, L.; Twieg, R. J.; Wiggers, G.; Mao, G.; Singer, K. D.; Petschek, R. G. Synthesis and Spectral Characterization of Bisnaphthylmethyl and Trinaphthylmethyl Cations. Tetrahedron Lett. 2005, 46, 5121−5125. (41) Kandela, I. K.; Bartlett, J. A.; Indig, G. L. Effect of Molecular Structure on the Selective Phototoxicity of Triarylmethane Dyes towards Tumor Cells. Photochem. Photobiol. Sci. 2002, 1, 309−314. (42) Bartlett, J. A.; Indig, G. L. Effect of Self-Association and Protein Binding on the Photochemical Reactivity of Triarylmethanes. Implications of Noncovalent Interactions on the Competition between Photosensitization Mechanisms Type I and Type II. Photochem. Photobiol. 1999, 70, 490−498. (43) Lueck, H. B.; Rice, B. L.; McHale, J. L. Aggregation of Triphenylmethane Dyes in Aqueous Solution Dimerization and Trimerization of Crystal Violet and Ethyl Violet. Spectrochim. Acta, Part A 1992, 48, 819−828. (44) Montes de Oca, M. N.; Vara, J.; Milla, L.; Rivarola, V.; Ortiz, C. S. Physicochemical Properties and Photodynamic Activity of Novel Derivatives of Triarylmethane and Thiazine. Arch. Pharm. 2013, 346, 255−265. (45) Indig, G. L.; Anderson, G. S.; Nichols, M. G.; Bartlett, J. A.; Mellon, W. S.; Sieber, F. Effect of Molecular Structure on the Performance of Triarylmethane Dyes as Therapeutic Agents for Photochemical Purging of Autologous Bone Marrow Grafts from Residual Tumor Cells. J. Pharm. Sci. 2000, 89, 88−99. (46) Wainwright, M.; Burrow, S. M.; Guinot, S. G. R.; Phoenix, D. A.; Waring, J. Uptake and Cell-Killing Activities of a Series of Victoria Blue Derivatives in a Mouse Mammary Tumour Cell Line. Cytotechnology 1999, 29, 35−43. (47) Budin, G.; Chung, H. J.; Lee, H.; Weissleder, R. A Magnetic Gram Stain for Bacterial Detection. Angew. Chem., Int. Ed. 2012, 51, 7752−7755. (48) Wainwright, M. ’Safe’ Photoantimicrobials for Skin and SoftTissue Infections. Int. J. Antimicrob. Agents 2010, 36, 14−18. (49) Saji, M.; Taguchi, S.; Uchiyama, K.; Osono, E.; Hayama, N.; Ohkuni, H. Efficacy of Gentian-Violet in the Eradication of Methicillin-Resistant STaphylococcus-Aureus from Skin-Lesions. J. Hosp. Infect. 1995, 31, 225−228. (50) Li, Z.; Smeu, M.; Afsari, S.; Xing, Y.; Ratner, M. A.; Borguet, E. Single-Molecule Sensing of Environmental pH-an STM Break Junction and NEGF-DFT Approach. Angew. Chem., Int. Ed. 2014, 53, 1098−1102.

(51) Zhang, Q. M.; Xu, W.; Serpe, M. J. Optical Devices Constructed from Multiresponsive Microgels. Angew. Chem., Int. Ed. 2014, 53, 4827−4831. (52) Gotor, R.; Costero, A. M.; Gil, S.; Parra, M.; Martinez-Manez, R.; Sancenon, F.; Gavina, P. Selective and Sensitive Chromogenic Detection of Cyanide and HCN in Solution and in Gas Phase. Chem. Commun. 2013, 49, 5669−5671. (53) Kepczynski, M.; Pandian, R. P.; Smith, K. M.; Ehrenberg, B. Do Liposome-Binding Constants of Porphyrins Correlate with their Measured and Predicted Partitioning between Octanol and Water? Photochem. Photobiol. 2002, 76, 127−134. (54) Goldacre, R. J.; Phillips, J. N. The Ionization of Basic Triphenylmethane Dyes. J. Chem. Soc. 1949, No. JUL, 1724−1732. (55) Harrelson, W. G.; Mason, R. P. Microsomal Reduction of Gentian-VIolet - Evidence for Cytochrome P-450-Catalyzed FreeRadical Formation. Mol. Pharmacol. 1982, 22, 239−242. (56) Reszka, K.; Cruz, F. S.; Docampo, R. Photosensitization by the Trypanocidal Agent Crystal Violet - Type-I versus Type-II Reactions. Chem. Biol. Interact. 1986, 58, 161−172. (57) Viola, A.; Hadjur, C.; Jeunet, A.; Julliard, M. Electron Paramagnetic Resonance Evidence of the Generation of Superoxide (O2•−)) and Hydroxyl (•OH) Radicals by Irradiation of a New Photodynamic Therapy Photosensitizer, Victoria Blue BO. J. Photochem. Photobiol., B 1996, 32, 49−58. (58) Wang, J.; Hou, Y.; Lei, W.; Zhou, Q.; Li, C.; Zhang, B.; Wang, X. DNA Photocleavage by a Cationic BODIPY Dye through Both Singlet Oxygen and Hydroxyl Radical: New Insight into the Photodynamic Mechanism of BODIPYs. ChemPhysChem 2012, 13, 2739−2747. (59) Sun, Y.; Hou, Y.-J.; Zhou, Q.-X.; Lei, W.-H.; Chen, J.-R.; Wang, X.-S.; Zhang, B.-W. Dinuclear Cu(II) Hypocrellin B Complexes with Enhanced Photonuclease Activity. Inorg. Chem. 2010, 49, 10108− 10116. (60) Duxbury, D. F. The Photochemistry and Photophysics of Triphenylmethane Dyes in Solid and Liquid-Media. Chem. Rev. 1993, 93, 381−433.

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