Mechanistic insight into the photodynamic effect mediated by porphyrin-fullerene C60 dyads in solution and in Staphylococcus aureus cells

The photodynamic action mechanism sensitized by a non-charged porphyrin-fullerene C60 dyad (TCP-C60) and its tetracationic analogue (TCP-C604+) was investigated in solution and in Staphylococcus aureus cells. The ability of both dyads to form a photoinduced charge-separated state was evidenced by the reduction of methyl viologen in N,N-dimethylformamide (DMF). Moreover, the formation of superoxide anion radicals induced by these dyads was detected by the reduction of nitro blue tetrazolium. Also, photosensitized decomposition of l-tryptophan (Trp) was investigated in the presence of reactive oxygen species (ROS) scavengers. The addition of β-carotene and sodium azide had a slight effect on reaction rate. However, photooxidation of Trp mediated by TCP-C60 was negligible in the presence of d-mannitol, while no protection was found using TCP-C604+. In a polar medium, these dyads mainly act by a contribution of type I pathway with low generation of singlet molecular oxygen, O2(1Δg). In S. aureus cell suspensions, an aerobic atmosphere was required for the photokilling of this bacterium. The photocytotoxicity induced by TCP-C60 was increased in D2O with respect to water, while a small effect was found using TCP-C604+. Furthermore, photoinactivation of microbial cells was negligible in the presence of sodium azide. The addition of d-mannitol did not affect the photoinactivation induced by TCP-C60. In contrast, S. aureus cells were protected by d-mannitol when TCP-C604+ was used as a photosensitizer. Also, generation of O2(1Δg) in the S. aureus cells was higher for TCP-C60 than TCP-C604+. Therefore, TCP-C60 appears to act in microbial cells mainly through the mediation of O2(1Δg). Although, a contribution of the type I mechanism was found for cell death induced by TCP-C604+. Therefore, these dyads with high capacity to produce photoinduced charge-separated state represent interesting photosensitizers to inactivate microorganisms by type I or type II mechanisms. In particular, TCP-C60 may be located in a non-polar microenvironment in the cells favoring a type II pathway, while a contribution of the type I mechanism was produced using the cationic TCP-C604+.


Introduction
Nowadays, infections caused by bacteria are becoming difficult to eradicate. This is mainly due to the continuous emergence of multidrug resistant strains. 1,2 Thus, infections that could be treated without problem in the past, today they have become a challenge to be eradicated in hospitals throughout the world. [3][4][5][6] Therefore, new treatments to deal with this situation are being sought. 7,8 A new alternative to controlling microorganism infections is photodynamic inactivation (PDI). 9 This therapy is based on the addition of a photosensitizer that rapidly binds to microbial cells. Excitation of the photosensitizer with light of an appropriate wavelength in the presence of molecular oxygen in the ground state, O 2 ( 3 S g ), produces reactive oxygen species (ROS). 10 In PDI, two mechanisms can be mainly involved aer activation of the photosensitizer. 11 In the type I pathway, the photosensitizer excited triplet state can react with different substrates by electron or proton transfer to form free radicals. These radicals can also interact with O 2 ( 3 S g ) producing ROS, such as superoxide anion radical (O 2 c À ), hydroxyl radical (HOc) and hydrogen peroxide (H 2 O 2 ). In the type II partway, the photosensitizer generates singlet molecular oxygen, O 2 ( 1 D g ), by energy transfer. Thus, generation of ROS can simultaneously occur through type I and type II mechanisms. The ROS generated can rapidly react with a variety of substrates in microorganism cells inducing damage in biomolecules. 12 Thus, these changes yield a loss of biological functionality leading to cell inactivation.
In recent years, varieties of molecules have been proposed as photosensitizers. The design and synthesis of new compounds is still developing in order to nd photosensitizers able to efficiently inactivate different bacterial strains in low concentration and in a shorter period of time. 13 In particular, porphyrin and fullerene C 60 derivatives were investigated as effective compounds to eradicate Gram-positive and Gram-negative bacteria aer illumination with visible light. 14,15 In a previous work, a novel porphyrin-fullerene C 60 dyad (TCP-C 60 ) was synthetized with three carbazoyl groups attached to the tetrapyrrolic macrocycle at the meso positions ( Fig. 1). 16 The three carbazoles and the pyrrolidinium ring, which links porphyrin covalently to fullerene C 60 , were positively charged by exhaustive methylation to produce a tetracationic photosensitizer (TCP-C 60 4+ , Fig. 1). The photoinactivation ability of these neutral and cationic dyads was for rst time investigated in Staphylococcus aureus. This microorganism was chosen because its ability to acquire resistance to antibiotics. 17,18 Antibiotic resistant S. aureus is endemic in hospitals worldwide and it causes substantial morbidity and mortality. 19 Ballatore et al. demonstrated for rst time that TCP-C 60 and TCP-C 60 4+ are efficient photosensitizers to inactivate S. aureus. 16 Therefore, in the present work we are interested in to obtain mechanistic insight about photodynamic processes involved in the inactivation of S. aureus cells mediated by TCP-C 60 and TCP-C 60 4+ .
First, photodynamic properties were investigated in solution to obtain evidence about the formation of charge separation state and production of ROS. Aer that, photoinactivation of S. aureus was studied under different conditions, varying the medium of cell suspensions and using specic scavengers of ROS. These experiments were used to increase the knowledge of the balance between type I and type II mechanisms sensitized by porphyrin-C 60 dyads in a Gram-positive bacterium.

General
Absorption spectra were performed on a Shimadzu UV-2401PC spectrometer (Shimadzu Corporation, Tokyo, Japan  substrates (NBT or NADH) and with both substrates without the dyad. The progress of the reaction was monitored by following the increase of the absorbance at l ¼ 560 nm. 21

Photooxidation and singlet excited state deactivation of tryptophan (Trp)
Solutions of dyad (absorbance 0.1 at Soret band) and Trp (20 mM) in DMF were irradiated with light at l irr ¼ 428 nm. Photooxidation of Trp was determined by the decrease of the uorescence intensity (I) at l ¼ 340 nm, exciting the samples at l exc ¼ 290 nm. Also, decomposition of Trp by dyads was investigated by adding b-carotene (5.6 mM) in DMF, sodium azide (50 mM) in DMF/2.5% water and D-mannitol (50 mM) in DMF/5% water. Solutions of Trp and dyad in the presence of different scavengers of ROS were irradiated with light at l irr ¼ 562 nm. Under these conditions, the uorescence intensity correlates linearly with Trp concentrations. The observed rate constants (k obs ) were calculated by a linear least-squares t of semilogarithmic plots of ln(I 0 /I) vs. time. 21 Singlet excited state deactivation of Trp by photosensitizer was investigated using the Stern-Volmer's eqn (1). 22 where I 0 and I are the uorescence intensity of Trp in the absence and in the presence of quencher, k q represents the biomolecule quenching rate constant, s 0 the excited state lifetime of Trp in the absence of photosensitizer, [Q] is the photosensitizer concentration and K SV is the Stern-Volmer quenching constants.

Bacterial strain and preparation of cultures
The microorganism used in this study was the reference strain S. aureus ATCC 25923. 16 This bacterium was grown in a rotator shaker (100 rpm) at 37 C in tryptic soy (TS, Britain, Buenos Aires, Argentina) broth overnight. Aliquots ($60 mL) of this culture were aseptically transferred to 4 mL of fresh medium and incubated with agitation at 37 C to middle of the logarithmic phase (absorbance $0.3 at 660 nm). Cells were centrifuged (3000 rpm for 15 min) and re-suspended in equal amount of 10 mM phosphate-buffered saline (PBS, pH 7.2) solution. Then the cells were diluted 1/1000 in PBS, corresponding to $10 6 colony forming units (CFU) mL À1 . In all the experiments, 2 mL of the cell suspensions in Pyrex brand culture tubes (13 Â 100 mm) were used. Cell suspensions were serially diluted with PBS and each solution was quantied by using the spread plate technique in triplicate. Viable bacteria were monitored and the number of CFU ml À1 was determined on TS agar plates aer $24 h incubation at 37 C.

Photoinactivation of bacterial cell suspensions
Cell suspensions of S. aureus (2 mL, $10 6 CFU mL À1 ) in PBS were incubated with 1 mM dyad in the dark at 37 C for 30 min with agitation (100 rpm). The dyad was added from a stock solution 0.5 mM in DMF. Aer that, the cultures were exposed to visible light for 15 min. Studies in anoxic conditions were performed by displacing the oxygen with argon in the cultures asks for 15 min before irradiation and keeping an argon atmosphere during the experiments. For photoinactivation assays in D 2 O, cells were centrifuged (3000 rpm for 15 min) and re-suspended in 2 mL PBS in D 2 O. Then the cell suspensions were incubated with 1 mM dyad as described above. Sodium azide or D-mannitol was added to bacterial suspensions from stock solutions in water (2 M and 1 M, respectively). Cell suspensions were incubated with 50 mM sodium azide or Dmannitol for 30 min at 37 C in dark before the treatment with 1 mM dyad, as previously described. 23

Steady state photolysis in bacterial cell suspensions
Cell suspensions of S. aureus (2 mL, $10 6 CFU mL À1 ) were treated with 10 mM 9,10-dimethylanthracene (DMA) for 30 min in dark at 37 C. Cells were harvested by centrifugation (3000 rpm for 15 min) and re-suspended in 2 mL PBS. Then, cells were incubated with 1 mM dyad for 30 min in dark at 37 C. Samples (2 mL) were irradiated in 1 cm path length quartz cells with visible light 455-800 nm. Photooxidation of DMA was determined by following the decrease in the uorescence intensity at l ¼ 427 nm, exciting the samples at l exc ¼ 378 nm. In these conditions, the uorescence intensity correlates linearly with DMA concentrations. The values of k obs were obtained by a linear least squares t of characteristics plots of ln(I 0 /I) vs. time. 23

Controls and statistical analysis
Control experiments of S. aureus cultures were carried out with and without dyad in the dark and without dyad with irradiated cells. The amount of DMF (<1% v/v) used in each experiment was not toxic to bacterial cells. Three values were obtained per each condition and each experiment was repeated separately three times. The signicance of the PDI effect of each dyad on microbial cells viability was assessed by one-way analysis of variance (ANOVA). A p-value below 0.05 was considered statistically signicant. Data were represented as the mean AE standard deviation of each group.

Photosensitized reduction of MV 2+
To test the electron transfer ability of TCP-C 60 and TCP-C 60 4+ , the photoreduction of MV 2+ in presence of TMN was studied in DMF/5% water. In these experiments, MV 2+ can act as an electron acceptor. 20 A comparison between the electron reduction potential of C 60 moiety (À0.66 V) and MV 2+ (À0.44 V) indicated that an electron transfer from C 60 to MV 2+ is exothermic by 0.22 eV. 20,24 Moreover, TMN can be used as an electron donor.
Taking into account the oxidation potential of TCP moiety (0.67 V) and the external electron donor TMN (0.43 V), electron transfer from TMN to TCP structure is expected to be exothermic by 0.24 eV. 20,24 Therefore, the formation of a photoinduced charge-separated state (TCPc + -C 60 c À ) can be accompanied by the reduction of MV 2+ and oxidation of TMN.
Under an argon atmosphere, the photoinduced chargeseparated state (TCPc + -C 60 c À ) can be formed by irradiation with light at 433 nm. This wavelength was used because the absorption spectra of TCP-C 60 and TCP-C 60 4+ showed a Soret band at 433 nm due to the free base porphyrin unit. 16 When dyad/MV 2+ /TMN system was irradiated, a progressive increase in the characteristic absorption bands of MVc + was noted at 398 and 606 nm (Fig. S1 †). The results aer different irradiation periods are shown in Fig. 2, monitoring the generation of MVc + at 606 nm. Thus, the reduction of MV 2+ and the oxidation of TMN were accomplished by the oxidation of TCPc + and reaction of C 60 c À in the dyad, respectively. By contrast, no reaction occurs in the dark or in the absence of the dyad under illumination. Moreover, it is important to note that no reaction occurs when irradiating the dyad with the electron acceptor or with the electron donor (results do not show). Thereby, both dyads are capable of forming a photoinduced charge-separated state in a polar medium as DMF/5% water. However, the MV 2+ reduction by TCP-C 60 was signicantly slower than that by TCP-C 60 4+ (Fig. 2). This difference may be due to the partial aggregation of non-charged TCP-C 60 by the addition of water to the DMF solution. On the other hand, in the presence of O 2 ( 3 S g ) the reduction of MV 2+ was not observed because the oxygen catalyzed the back electron transfer process between C 60 c À and TCPc + (Video S1 †). 25 It was previously demonstrated that TCP-C 60 and TCP-C 60 4+ have a very weak emission at $667 nm from the porphyrin moiety in DMF, with uorescence quantum yield of F F $ 4 Â 10 À3 . 16 The quenching efficiencies (ɳ q ) of the porphyrin excited singlet state by the attached fullerene moiety were estimated to be ɳ q > 0.96. These results are in accordance with the formation of a photoinduced charge-separated state (TCPc + -C 60 c À ). Also, a value of 0.62 eV was obtained for the driving force for the initial charge separation from 1 TCP* to C 60 . 16 Similar results were previously observed with a porphyrin-fullerene C 60 dyad metallated with Zn(II). 20 In this case, the efficiency of charge transfer was favoured by the presence of the metal. Fukuzumi et al. demonstrated that the generated C 60 c À moiety in ZnPc + -C 60 c À dyad and in ZnPc + -H 2 P-C 60 c À triad undergoes one electron oxidation by hexyl viologen (HV 2+ ), whereas the ZnPc + moiety was reduced by a NADH analogues, 1-benzyl-1,4dihydronicotinamide and 10-methyl-9,10-dihydroacridine. 26 Thus, ZnP-C 60 and ZnP-H 2 P-C 60 donor-acceptor ensembles act in benzonitrile as efficient photocatalysts for the uphill oxidation of NADH analogues by HV 2+ . On the other hand, Dallas et al. demonstrated the intramolecular charge transfer between pyrene donor and fullerene C 60 /C 70 acceptors, which showed the ability of these molecules to accept electrons. 27 Moreover, Wang et al. prepared a complex micelle from zinc tetrakis(4sulfonatophenyl)porphyrin (ZnTPPS), modied fullerene (mC 60 ) and poly(ethyleneglycol)-block-poly(L-lysine) (PEG-b-PLys) by electrostatic interactions. The complex micelle exhibited high electron transfer performance in the photocatalytic reduction of MV 2+ . In the micellar core, ZnTPPS and mC 60 molecules are surrounded by each other which ensured effective energy migration from the donor to the acceptor. 28 Therefore, porphyrin-fullerene C 60 dyads are effective molecular systems to form photoinduced charge-separated state.

Photodecomposition of NBT
Under aerobic conditions, the decomposition of NBT occurred predominantly through a type I process, indicating the formation of O 2 c À . 29,30 Thus, when solutions of dyads containing NBT and NADH were irradiated, the generation of diformazan was observed in DMF/10% water, as shown by the characteristic colour change of the reaction (Fig. S2 †). The reduction of NBT to diformazan was examined following the increase in absorption at 560 nm. Fig. 3 shows the changes in the absorption as a function of time aer irradiation of samples. For both dyads, reduction of NBT was negligible in the absence of NADH. Without the dyad in solution, a slight reduction of NBT was observed in presence of NADH (Fig. 3). In all cases, the presence of NADH was needed for the formation of diformazan.  Therefore, an increase in the photoreduction of NBT was observed in the presence of dyad and NADH with respect to solution without the photosensitizer. These results showed that TCP-C 60 or TCP-C 60 4+ in a polar homogeneous medium can form a photoinduced charge-separated state resulting in the production of O 2 c À . In a previous work, the generation of O 2 c À was found with a dicationic fullerene C 60 derivative in presence of NBT and NADH, in benzene/BHDC (0.1 M)/W 0 ¼ 10 reverse micelles. 31 Moreover, the decomposition of NBT sensitized by 5,10,15,20-tetrakis[3-(N-ethyl-N-methylcarbazoyl)]chlorin or its analogous porphyrin was found indicating that both photosensitizers considerably produced O 2 c À in presence of NADH. 32 Under similar conditions, the generation of O 2 c À was also observed with a complex micelle formed by zinc tetrakis(4sulfonatophenyl)porphyrin, modied fullerene and poly(ethylene glycol)-block-poly(L-lysine). 28 On the other hand, the production of O 2 ( 1 D g ) by these dyads was previously investigated in solvents of different polarities. 25 An efficiently produce O 2 ( 1 D g ) sensitized by TCP-C 60 (F D ¼ 0.56) was found in a non-polar solvent, such as toluene. However, the type II photoprocess mediated by both dyads considerably decrease (F D ¼ 0.01 and 0.02 for TCP-C 60 and TCP-C 60 4+ , respectively) in DMF. Therefore, a competitive process must be involved in this more polar solvent. This behaviour can be attributed to the formation of photoinduced charge-separated state, which can favour the generation of O 2 c À in a polar medium.

Photooxidation of Trp
Trp amino acid residues is one of the amino acids most susceptible to oxidation and can be a potential target of the ROS produced by photosensitizers in microbial cells. 33,34 Moreover, Trp can be photooxidized by both type I and type II mechanisms. 35 Photosensitized decomposition of Trp sensitized by TCP, TCP-C 60 and TCP-C 60 4+ was evaluated by the decrease of uorescence in DMF (Fig. S3 †). As shown in Fig. 4, the photodecomposition of Trp followed rst-order kinetics with respect to substrate concentration. The results of k Trp obs calculated for Trp decomposition are summarized in Table 1. A higher value was found for the reaction rate sensitized by TCP in comparison with dyads. It was previously demonstrated that the TCP presents considerable photodynamic activity in DMF because it has a signicant contribution of type II mechanism. 25 In contrast, a low O 2 ( 1 D g ) production by dyads was observed in a polar solvent. Therefore, the decomposition of Trp mediated by dyads may be mainly due to type I mechanism of action. Moreover, the results shown in Table 1 indicate a higher value of k Trp obs for the reaction photosensitized by TCP-C 60 4+ than TCP-C 60 . These results are not in agreement with the O 2 ( 1 D g ) production sensitized by these dyads. 25 A possible interaction between these dyads and Trp could be favouring an electron transfer process in the decomposition of the amino acid. Thus, the interaction of photosensitizers singlet excited state with Trp was studied by steady-state uorescence. Fig. S4 36 the values of k q ($1 Â 10 11 s À1 ) are over diffusion limit in DMF. The ability of the fullerene C 60 to interact with amino acids was previously calculated suggesting that the most favourable interactions of the fullerene are with arginine, leucine, and tryptophan, which is related to the backbone structure of the corresponding amino acids. 37 Therefore, to evaluate the main photodynamic mechanism involved in the Trp decomposition photosensitized by these dyads, experiments were carried out in presence of different ROS scavenges. The photodynamic effect on the Trp  decomposition was investigated in the presence of b-carotene, sodium azide or D-mannitol. Solutions of TCP, TCP-C 60 or TCP-C 60 4+ containing Trp and scavenger were irradiated with monochromatic light (l irr ¼ 562 nm). This wavelength was chosen to avoid b-carotene absorption and this light was mainly used to excite the porphyrin moiety in the dyads. A low amount of water addition was needed to solubilize sodium azide and Dmannitol. The values of k Trp obs in the presence and absence of scavengers were calculated from rst-order kinetic plots of the Trp (Fig. 5-7) sensitized by TCP, TCP-C 60 and TCP-C 60 4+ . The results of k Trp obs are shown in Table 1. First, the photodynamic effect was evaluated by decomposition of Trp in the presence of b-carotene (Fig. 5). Under these conditions, b-carotene can deactivate O 2 ( 1 D g ) through energy transfer or by chemical reaction. 38,39 The photodecomposition of Trp sensitized by TCP in the presence of b-carotene showed a lower value of k Trp obs than in absence of b-carotene (Table 1). Since the TCP mainly produces O 2 ( 1 D g ), it was expected a photoprotective effect by b-carotene. However, in the presence of TCP-C 60 or TCP-C 60 4+ the values of k Trp obs were slight affected by the addition of b-carotene. In a polar solvent, such as DMF, dyads can form a photoinduced charge-separated state diminishing its ability to generate O 2 ( 1 D g ). 23 Photodecomposition of Trp was studied in presence of sodium azide in DMF/2.5% water (Fig. 6). Azide ions can deactivate O 2 ( 1 D g ) and other compounds in its excited triplet state through an energy transfer. 40 A lower value of k Trp obs was obtained when for the reaction sensitized by TCP in the presence of azide ions (Table 1), according with the formation of O 2 ( 1 D g ). In contrast, for TCP-C 60 and TCP-C 60 4+ the values of k Trp obs were little affected by the addition of sodium azide, indicating a contribution of type I mechanism in Trp photooxidation process. Although the addition of b-carotene did not affect the rate of Trp photooxidation sensitized by TCP-C 60 4+ (Fig. 5C), the addition of sodium azide slowed it (Fig. 6C). This behavior may be due to the electrostatic interaction of the azide anions with the cationic TCP-C 60 4+ dyad, favoring the deactivation of the excited triplet state of the photosensitizer. The photosensitized decomposition of Trp was also analysed in the presence of D-mannitol in DMF/5% water (Fig. 7). This compound can act as a radical scavenger. 41 Therefore, it was used to verify the presence of type I mechanism. 23 The value of k Trp obs for TCP was not modied by the addition of D-mannitol (Table 1), which was due to TCP generates mainly O 2 ( 1 D g ). The photodecomposition of Trp sensitized by TCP-C 60 in the presence of D-mannitol was negligible, according with the formation a photoinduced charge-separated state diminishing its ability to generate O 2 ( 1 D g ). Therefore, the addition of D-mannitol proves evidence that its primary mechanism of action was type I in this polar medium. In the case of TCP-C 60 4+ , the presence of Dmannitol did not affect the value of k Trp obs . As indicated above, cationic photosensitizers may interact with Trp and produces the decomposition of the amino acid by a radical charge transfer process. Thus, the interaction between the cationic dyad and Trp avoid the effect D-mannitol on photooxidation rate. In presence of dyads, Trp can produce Trp radical, which   can react with O 2 c À to form Trp hydroperoxide that can then rearrange into N-formylkynurenine and kynurenine. 34

Photodynamic action in S. aureus cells
Different experimental conditions were used to detect ROS sensitized by TCP-C 60 or TCP-C 60 4+ in S. aureus cell suspensions.
The effect of medium on the photoinactivation of S. aureus was investigated in an atmosphere of argon and in cell suspensions in D 2 O. Moreover, the activity of scavengers of ROS on the photoinactivation of S. aureus was evaluated by the addition of sodium azide or D-mannitol. In a previous work, these dyads were effective photosensitizers to inactivate S. aureus aer irradiation with visible light. 16 The results showed that PDI mediated by TCP-C 60 4+ was slight more effective than by TCP-C 60 . In particular, the cationic dyad TCP-C 60 4+ exhibited a photosensitizing activity producing a 4.5 log decrease of cell survival, when the cells were treated with 5 mM dyad and irradiated for 30 min with visible light. In this work, studies of PDI were performed with this Gram-positive bacterium in order to obtain information about the main photoprocesses involved in the photokilling of cells. For cells treated with 1 mM dyad and 15 min irradiation, the decrease in cell survival was 1.8 log and 2.5 log for TCP-C 60 and TCP-C 60 4+ , respectively (Fig. 8, line 4).
Moreover, no toxicity was observed for S. aureus cells not treated with the dyads aer 15 min irradiation (Fig. 8, line 2) or treated with 1 mM dyad in dark (Fig. 8, line 3). Therefore, these conditions were used to determine the principal mechanism involved in PDI because it allowed seeing an increase or a decrease in the photoinactivation of bacteria. Photoinactivation of S. aureus cell by dyads was studied under anoxic condition. Cell viability was not modied aer irradiation without dyad in an atmosphere of argon (Fig. 8, line 5) or in the presence of dyad in the dark (result not shown). Furthermore, the photoinactivation of S. aureus was negligible for cells incubated with TCP-C 60 or TCP-C 60 4+ aer 15 min irradiation (Fig. 8, line 6). Although, the presence of oxygen is essential for the generation of O 2 ( 1 D g ) through the type II photosensitization mechanism, oxygen also plays a major role in the type I mechanism by adding to biochemical radicals. 12 In a type I process, the light excites the photosensitizers and it interacts with a substrate to yield radical ions by a hydrogen atom or electron transfer reaction. The majority of these radicals react with oxygen instantaneously and generate a complicated mixture of highly reactive oxygen intermediates, which can oxidize a wide variety of biomolecules. Therefore, these experiments are not decisive in establishing the predominant photoreaction process involved in the cytotoxicity. Nevertheless, this result shows that these dyads were not able to inactivate microorganisms under anoxic conditions. Similar results were found with Streptococcus mitis cell suspensions treated with the phthalocyanine ZnEPc 4+ under an argon atmosphere. 42 The loss of viability of S. mitis cells was highly oxygen dependent. Furthermore, other photosensitizers were reporter with the same behaviour. 23,40 To evaluate the O 2 ( 1 D g ) mediated photoinactivation of microorganisms, the PDI was performed in D 2 O because it is well known that D 2 O increases the lifetime of O 2 ( 1 D g ) (s 0 ¼ 68 ms in D 2 O and s 0 ¼ 4.2 ms in water). 43 No toxicity was detected in the presence of D 2 O under irradiation without dyad (Fig. 8, line 7) or cells treated with dyad in dark (result not shown). Irradiation of S. aureus in D 2 O with TCP-C 60 produced a more pronounced reduction in cell viability (1.5 log) than in PBS cell suspensions (Fig. 8A, line 8). The values found for the non-charged dyad showed that there was a contribution of O 2 ( 1 D g ) in cell inactivation. However, the photocytotoxic activity induced by TCP-C 60 4+ was similar in D 2 O respect to PBS cell suspensions ( Fig. 8B, line 8). This result with cationic dyad revealed a low contribution of type II mechanism. The different behaviours of dyads could be due to different intracellular localization of the photosensitizers in S. aureus cells. 23 Moreover, in order to assess the involvement of O 2 ( 1 D g ) in the photoinactivation of S. aureus cells, experiments were carried out in the presence of sodium azide. 44 S. aureus cells were treated with 50 mM sodium azide and 1 mM dyad. This amount of azide ions was not toxic aer 15 min with visible light (Fig. 8, line 9) or in the dark containing the dyad (result not shown). The presence of azide ions produced almost complete photoprotection of S. aureus cells treated with TCP-C 60 or TCP-C 60 4+ and irradiated for 15 min (Fig. 8, line 10). Sodium azide is known to inhibit the photodynamic damage of O 2 ( 1 D g ) in bacteria cells. 23 This is due to the fact that the azide ions can act as a quencher of O 2 ( 1 D g ) but also can deactivate compounds in their triplet excited state. 45 Thus, the presence of azide ions quenched the photocytotoxic species, producing a protective effect on S. aureus cells.
As well, the photoinactivation of S. aureus mediated by 1 mM dyad was examined aer incubation with 50 mM D-mannitol. This compound can be used as a scavenger of O 2 c À and HOc (type I reaction). 41 The addition of 50 mM D-mannitol was not toxic to irradiated cells without dyad (Fig. 8, line 11) or containing the D-mannitol and dyad in dark (result not shown). Aer 15 min irradiation of treated cells, the results indicated that phototoxicity efficacy of TCP-C 60 to inactivate S. aureus cells was not signicantly affected (Fig. 8A, line 12). Non-charged dyad may be located in a non-polar cellular microenvironment favouring the production of O 2 ( 1 D g ). On the other hand, $1 log photoprotective effect was found for bacterial cells treated with TCP-C 60 4+ in presence of D-mannitol (Fig. 8B, line   12). Therefore, a contribution of type I pathway was found for S. aureus sensitized by cationic dyad. Previous studies about the contribution of ROS in the photodynamic inactivation of a Gram-positive bacterium Enterococcus hirae by meso-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP) was investigated using several scavengers. 40 The results demonstrated that for Gram-positive bacteria, type I reactions appear to play a minor role compared to type II reactions. On the other hand, mechanistic studies on the photodynamic effect induced by fullerene C 60 derivative on S. aureus cells showed that under aerobic condition the photocytotoxicity activity induced by fullerene was mediated by mainly a contribution of type II process. 23 However, dyads TCP-C 60 and TCP-C 60 4+ have a porphyrin moiety covalently bound to fullerene C 60 . Therefore, the mechanism of photodynamic action involved will depend on the polarity of the medium in which the molecules were dissolved. In a polar microenvironment, a photoinduced charge separation state may be formed, favoring a type I photoprocesses. In a non-polar medium, an energy transfer mechanism can occurs, increasing the production of O 2 ( 1 D g ). 16 3.5. Photooxidation of DMA in S. aureus cells In order to conrm the production of O 2 ( 1 D g ) by TCP-C 60 and TCP-C 60 4+ dyads in S. aureus cells, the photooxidation of DMA was evaluated in bacterial suspensions monitoring uorescence emission decrease (Fig. S5 †). This anthracene derivative can act as photosensitizer itself producing O 2 ( 1 D g ) when it is irradiated. 46 Therefore, the system was illuminated in a range (l irr ¼ 455-800 nm) where DMA do not absorb. Hence, PDI of S. aureus was not affected by the addition of DMA. Furthermore, shorter irradiation times were used in kinetic studies, with a total time of 1 min. Considering that DMA quenches O 2 ( 1 D g ) by chemical reaction to form the corresponding endoperoxide, it is used as a method to evaluate the ability of dyads to produce O 2 ( 1 D g ) in S. aureus cells. 23 The values of the observed rate constant (k DMA obs ) were calculated from rst-order kinetic plots of the DMA emission at 428 nm with time ( Fig. 9). Values of k DMA obs ¼ (1.26 AE 0.06) Â 10 À2 s À1 and (4.18 AE 0.08) Â 10 À3 s À1 were obtained for TCP-C 60 and TCP-C 60 4+ , respectively. The decomposition rate induced by TCP-C 60 was three times faster than that of TCP-C 60

4+
. The results obtained in cell media showed that TCP-C 60 can be located in a less polar cellular environment than TCP-C 60 4+ . Therefore, TCP-C 60 can inactivate the microbe mainly by type II mechanism of photosensitization. Similar results were found using the photosensitizer zinc(II) 2,9,16,23-tetrakis[4-(Nmethylpyridyloxy)]phthalocyanine in Candida albicans cells. 47 On the other hand, the photooxidation of DMA mediated by N,N-dimethyl-2- [4-(3- production than TMAP 4+ in S. aureus cells. However, a different intracellular localization of the photosensitizers can also affect the decomposition rate of DMA due to the short lifetime of O 2 ( 1 D g ) inside the cells. Among other variables, the photodynamic mechanism of action depend on the medium. 48 Therefore, it is not possible to make direct correlations between the data obtained in solution and in microbial cells.

Conclusions
Type I and II mechanisms are present in the PDI induced by porphyrin-fullerene C 60 dyads both in solution and in S. aureus cells. Studies of steady-state photolysis in the presence of an electron acceptor and an electron donor conrm that TCP-C 60 Fig. 9 Photooxidation of DMA in S. aureus cells ($10 6 CFU mL À1 ) incubated with 10 mM DMA for 30 min followed by a washing step and treated with 1 mM TCP-C 60 (:) or TCP-C 60 4+ (;) for 30 min at 37 C in dark. Control: S. aureus cells incubated with 10 mM DMA (C); l irr ¼ 455-800 nm.
and TCP-C 60 4+ dyads are capable to form a photoinduced charge-separated state in a polar medium. Furthermore, both dyads produced O 2 c À in the presence of NADH, as a biological reducing agent. Although both dyads have a very low production of O 2 ( 1 D g ), they sensitized the photooxidation of Trp. A slight protection in the decomposition of Trp was found in presence of b-carotene and sodium azide, which indicates a low contribution of type II photoprocess. However, the oxidation of Trp sensitized by TCP-C 60 was negligible in solution containing Dmannitol, while the reaction rate was unaffected using TCP-C 60

4+
. This cationic dyad electrostatically interacts with Trp and electron transfer process can occur conducing to Trp decomposition. Therefore, both dyads oxidize Trp mainly by type I mechanism of action in DMF containing water. Moreover, the present study provides knowledge about the photodynamic mechanism that takes place in the PDI of S. aureus cells sensitized by TCP-C 60 and TCP-C 60 4+ . To elucidate the oxidative processes that occur during the killing of microbial cells, the effect of the media was analyzed on cell photoinactivation. It was observed that an oxygen atmosphere was necessary for an efficient photoinactivation. The photocytotoxicity induced by TCP-C 60 4+ dyad in D 2 O was similar than in PBS cell suspensions.
On the contrary, irradiation of bacteria treated with TCP-C 60 in D 2 O produced a greater reduction in cell viability than in PBS cell suspensions. Photoprotection was found using sodium azide as type II scavengers. The protective effect of D-mannitol was greater for the cells in the presence of TCP-C 60 4+ than TCP-C 60 . Additionally, the O 2 ( 1 D g ) production was higher for TCP-C 60 than TCP-C 60 4+ in the S. aureus cells. Due to the presence of positive charges on the periphery of TCP-C 60 4+ in comparison with non-charged TCP-C 60 , this dyad may be located in less polar cellular microenvironment. Therefore, TCP-C 60 inactivates the S. aureus cells mainly by type II mechanism of photosensitization, while a greater contribution of type I pathway is involved using TCP-C 60 4+ as photosensitizer.

Conflicts of interest
There are no conicts to declare.