Surface Modiﬁcation of Nanocrystalline TiO 2 Materials with Sulfonated Porphyrins for Visible Light Antimicrobial Therapy

: Highly-active, surface-modiﬁed anatase TiO 2 nanoparticles were successfully synthesized and characterized. The morphological and optical properties of the obtained (metallo) porphyrin@qTiO 2 materials were evaluated using absorption and ﬂuorescence spectroscopy, scanning electron microscopy (SEM) imaging, and dynamic light scattering (DLS). These hybrid nanoparticles e ﬃ ciently generated reactive oxygen species (ROS) under blue-light irradiation (420 ± 20 nm) and possessed a unimodal size distribution of 20–70 nm in diameter. The antimicrobial performance of the synthetized agents was examined against Gram-negative and Gram-positive bacteria. After a short-term incubation of microorganisms with nanomaterials (at 1 g / L) and irradiation with blue-light at a dose of 10 J / cm 2 , 2–3 logs of Escherichia coli , and 3–4 logs of Staphylococcus aureus were inactivated. A further decrease in bacteria viability was observed after potentiation photodynamic inactivation (PDI), either by H 2 O 2 or KI, resulting in complete microorganism eradication even when using low material concentration (from 0.1 g / L). SEM analysis of bacteria morphology after each mode of PDI suggested di ﬀ erent mechanisms of cellular disruption depending on the type of generated oxygen and / or iodide species. These data suggest that TiO 2 -based materials modiﬁed with sulfonated porphyrins are e ﬃ cient photocatalysts that could be successfully used in biomedical strategies, most notably, photodynamic inactivation of microorganisms.


Introduction
Application of inorganic semiconductors as heterogeneous photocatalysts, and in particular the role of TiO 2 in environmental and biomedical sciences and technologies, has already been extensively investigated. The most efficient methods in this respect are advanced oxidation processes (AOPs) including environmental [1][2][3][4] and biomedical photocatalysis [5][6][7][8][9]. Semiconductor-based photocatalysts are routinely used in the degradation of dyes present in waste-water for the following reasons: (i) they are inexpensive and can be obtained easily in a large-scale preparation; (ii) they are non-toxic; (iii) they exhibit tunable properties that can be modified by the size of the particles, doping, and/or sensitization; (iv) they facilitate electron transfer processes; and (v) they are capable of extending their use without substantial loss in the photocatalytic activity [10]. Furthermore, inorganic nanomaterials [35][36][37][38]. Unfortunately, titanium dioxide has a relatively wide bandgap and thus can absorb only light at λ < 400 nm, which may be intrinsically carcinogenic [39]. Various organic dyes can be attached to TiO 2 nanoparticle surfaces via functional groups (e.g., -OH, -SO 3 H, -OOCR) by direct linking between the dyes and the TiO 2 nanoparticle substrate [9,[40][41][42][43]. Alternative ways of encouraging interaction between the dye and semiconductor are electrostatic interaction through ion exchange, ion-pairing, donor-acceptor interactions, hydrogen bonding, and van der Waals forces. Among the various types of photosensitizers, transition metal complexes with low lying excited states (nitrogen heterocyclics with delocalized π electrons or aromatic ring system) such as (metallo)porphyrins and pthalocyanines [44] capable of the electron injection from their excited states to the conduction band of titania deserve particular attention [45][46][47][48]. Synergistic effects from the permeability by nanoparticles and ROS generation by conjugated photosensitizers facilitate photodynamic activity against bacteria, especially under solar irradiation [24,49].

Synthesis, Optical Properties, and Characterization of the Materials
Mixing of the colloidal form of titanium dioxide (qTiO 2 ) with porphyrin solutions in water at slightly acidic pH (pH = 6) resulted in colored colloidal solutions. After addition of qTiO 2 , the absorption spectrum of F 2 POH@qTiO 2 was broadened and red-shifted ( Figure 1).
These changes reflected a suggested electronic interaction between molecules and the support. As illustrated in Figure 1, TPPS attached to qTiO2 showed a blue shift of a Soret band. The spectral changes of TPPS in acidic pH have been previously reported and have been attributed to J-aggregates in which molecules are arranged in head-to-tail (Soret at: 413 nm pH > 7, 433 nm pH < 7) [50]. Thus, the presence of Soret band at 413 nm for TPPS@qTiO2 confirmed this interaction between the semiconductor and non-aggregated PS. The relevant spectral features of studied tetrapyrrolic compounds are reported in Table 1. Other modified porphyrins and their metal complexes have also been successfully used to sensitize TiO2 [9,51]. On the basis of the Tauc plots (Figure 1d,e,f; Figure S1) calculated from absorption spectra, the band gap energy for each modified porphyrin@TiO2 materials were estimated and reached ca. 3.37-3.47 eV. This value was higher than the typical band gap energy for anatase (3.2 eV). Considering the small size of TiO2 crystals (vide infra), such a large Eg value should be associated with the occurrence of the phenomenon of the quantum size effect (TiO2 abbreviated as qTiO2) [52,53]. Dynamic light scattering (DLS) was used to characterize the qTiO2 nanomaterials in suspension. The particle size of modified nanomaterials in aqueous solution had a homogeneous distribution with 13, 28, and 68 nm in diameter for TPPS@qTiO2, F2POH@qTiO2, and ZnF2POH@qTiO2, respectively ( Figure 2). The key parameter of the dispersion stability of nanoparticles is zeta potential (ζ), which is determined by degree of electrostatic repulsion of surface in an ionic environment at the boundary between the Stern layer and the diffuse layer. The obtained values of zeta potentials were in the range from −30 to −20 mV at pH = 6 ( Table 1), reflecting a strong binding of all organic modifiers to the titania surface and also a good stability (meant as a lack of aggregation or precipitation in the dark) of colloids. Table 1. Spectroscopic properties of sulfonyl porphyrin derivatives, mean hydrodynamic diameters, average size of aggregates from scanning electron microscopy (SEM) and zeta potential in water (pH = 6). qTiO2: colloidal form of titanium dioxide, DLS: dynamic light scattering.
These changes reflected a suggested electronic interaction between molecules and the support. As illustrated in Figure 1, TPPS attached to qTiO 2 showed a blue shift of a Soret band. The spectral changes of TPPS in acidic pH have been previously reported and have been attributed to J-aggregates in which molecules are arranged in head-to-tail (Soret at: 413 nm pH > 7, 433 nm pH < 7) [50]. Thus, the presence of Soret band at 413 nm for TPPS@qTiO 2 confirmed this interaction between the semiconductor and non-aggregated PS. The relevant spectral features of studied tetrapyrrolic compounds are reported in Table 1. Other modified porphyrins and their metal complexes have also been successfully used to sensitize TiO 2 [9,51]. On the basis of the Tauc plots (Figure 1d-f; Figure S1) calculated from absorption spectra, the band gap energy for each modified porphyrin@TiO 2 materials were estimated and reached ca. 3.37-3.47 eV. This value was higher than the typical band gap energy for anatase (3.2 eV). Considering the small size of TiO 2 crystals (vide infra), such a large E g value should be associated with the occurrence of the phenomenon of the quantum size effect (TiO 2 abbreviated as qTiO 2 ) [52,53]. Dynamic light scattering (DLS) was used to characterize the qTiO 2 nanomaterials in suspension. The particle size of modified nanomaterials in aqueous solution had a homogeneous distribution with 13, 28, and 68 nm in diameter for TPPS@qTiO 2 , F 2 POH@qTiO 2 , and ZnF 2 POH@qTiO 2 , respectively ( Figure 2). The key parameter of the dispersion stability of nanoparticles is zeta potential (ζ), which is determined by degree of electrostatic repulsion of surface in an ionic environment at the boundary between the Stern layer and the diffuse layer. The obtained values of zeta potentials were in the range from −30 to −20 mV at pH = 6 ( Table 1), reflecting a strong binding of all organic modifiers to the titania surface and also a good stability (meant as a lack of aggregation or precipitation in the dark) of colloids.

Scanning Electron Microscopy (SEM) Analysis of Prepared Nanomaterials
The morphological and structural development of the synthesized qTiO2-based nanomaterials was investigated by scanning electron microscopy (SEM). A high magnification SEM image of the nanoparticles shown in Figure 3 indicated that the diameter of the obtained nanomaterials was in the range of 30-50 nm in contrast to unmodified titania, which formed clusters and aggregates with a length of up to 100 nm (Table 1, Figures S2, S3). SEM analysis clearly demonstrated that after surface modification by porphyrins, the particles did not aggregate. In the field of environmental photocatalysis, nanoparticles with these particular dimensions generally show higher photocatalytic efficiency due to the large surface-to-volume ratio caused by the reduced particle size. Moreover,

Scanning Electron Microscopy (SEM) Analysis of Prepared Nanomaterials
The morphological and structural development of the synthesized qTiO 2 -based nanomaterials was investigated by scanning electron microscopy (SEM). A high magnification SEM image of the nanoparticles shown in Figure 3 indicated that the diameter of the obtained nanomaterials was in the range of 30-50 nm in contrast to unmodified titania, which formed clusters and aggregates with a length of up to 100 nm (Table 1, Figures S2 and S3). SEM analysis clearly demonstrated that after surface modification by porphyrins, the particles did not aggregate. In the field of environmental photocatalysis, nanoparticles with these particular dimensions generally show higher photocatalytic efficiency due to the large surface-to-volume ratio caused by the reduced particle size. Moreover, nanomaterials of these dimensions may be safely employed in biomedical research and for medicinal purposes.

Fluorescence Quenching by Colloidal qTiO2 Nanoparticles
The steady-state fluorescence spectra of sulfonyl porphyrins and their colloidal solution with qTiO2 were recorded in water (pH = 6). The maximum emission wavelengths are listed in Table 2. In all the studied scenarios, modification of the qTiO2 surface resulted in a decrease of fluorescence intensity and an increase of fluorescence lifetimes consistently ( Figure 4). For materials modified with free base porphyrins, almost complete quenching of the S1-S0 emission was observed. Furthermore, the formation of a new emission band derived from the electron transfer from the

Fluorescence Quenching by Colloidal qTiO 2 Nanoparticles
The steady-state fluorescence spectra of sulfonyl porphyrins and their colloidal solution with qTiO 2 were recorded in water (pH = 6). The maximum emission wavelengths are listed in Table 2. Table 2. Photophysical properties of sulfonyl porphyrin derivatives in the absence and presence of colloidal qTiO 2 (τ 0 is the lifetime in the absence of colloidal qTiO 2 , τ fl is the lifetime in the presence of colloidal qTiO 2 ). In all the studied scenarios, modification of the qTiO 2 surface resulted in a decrease of fluorescence intensity and an increase of fluorescence lifetimes consistently ( Figure 4). For materials modified with free base porphyrins, almost complete quenching of the S 1 -S 0 emission was observed. Furthermore, the formation of a new emission band derived from the electron transfer from the conduction band (CB) to the ground and oscillation states was observed (Figure 5a,b). The fluorescence intensity of ZnF 2 POH was strongly quenched after adsorption at the qTiO 2 surface (Figure 4c). The observed quenching of the excited state could be attributed to an energy or electron transfer from the excited dye to the semiconductor [54,55], or more precisely, to the electron injection from the excited state of the dye into the conduction band of titanium dioxide ( Figure 5). Although for F 2 POH@qTiO 2 and TPPS@qTiO 2 the quenching process in the presence of qTiO 2 was not so evident, the observed slight decrease in fluorescence intensity may also indicate less effective electron/energy transfer processes ( Figure 4). conduction band (CB) to the ground and oscillation states was observed (Figure 5a,b). The fluorescence intensity of ZnF2POH was strongly quenched after adsorption at the qTiO2 surface ( Figure 4c). The observed quenching of the excited state could be attributed to an energy or electron transfer from the excited dye to the semiconductor [54,55], or more precisely, to the electron injection from the excited state of the dye into the conduction band of titanium dioxide ( Figure 5). Although for F2POH@qTiO2 and TPPS@qTiO2 the quenching process in the presence of qTiO2 was not so evident, the observed slight decrease in fluorescence intensity may also indicate less effective electron/energy transfer processes ( Figure 4). The fluorescence quenching data clearly highlight the role of excited state of porphyrins in injecting their electron to the conduction band of qTiO2. Additionally, the fluorescence intensity of porphyrin ( Figure 4) decreased at a wavelength of ~600 nm and ~650 nm in the presence of qTiO2. Meanwhile, the fluorescence intensity attributed to the interaction between qTiO2 and porphyrin increased. An additional confirmation of this effect was the appearance of a new band at 700 nm ( Figure 4, red line). The increase in the fluorescence lifetime of porphyrin@qTiO2 compared to free porphyrin was the result of the emergence of a new relaxation path derived from the qTiO2 surface modification by porphyrins ( Figure 5). Increasing the lifetime of an excited state correlated with reducing the recombination effectiveness. This effect was achieved through the transfer of the electron from the LUMO level of the porphyrin to the qTiO2 conduction band. The presence of this mechanism was confirmed by the conduction band (CB) to the ground and oscillation states was observed (Figure 5a,b). The fluorescence intensity of ZnF2POH was strongly quenched after adsorption at the qTiO2 surface ( Figure 4c). The observed quenching of the excited state could be attributed to an energy or electron transfer from the excited dye to the semiconductor [54,55], or more precisely, to the electron injection from the excited state of the dye into the conduction band of titanium dioxide ( Figure 5). Although for F2POH@qTiO2 and TPPS@qTiO2 the quenching process in the presence of qTiO2 was not so evident, the observed slight decrease in fluorescence intensity may also indicate less effective electron/energy transfer processes ( Figure 4).  Increasing the lifetime of an excited state correlated with reducing the recombination effectiveness. This effect was achieved through the transfer of the electron from the LUMO level of the porphyrin to the qTiO2 conduction band. The presence of this mechanism was confirmed by the observed change in the fluorescence maxima for TPPS@qTiO2 and F2POH@TiO2. In the case of Increasing the lifetime of an excited state correlated with reducing the recombination effectiveness. This effect was achieved through the transfer of the electron from the LUMO level of the porphyrin to the qTiO 2 conduction band. The presence of this mechanism was confirmed by the observed change in the fluorescence maxima for TPPS@qTiO 2 and F 2 POH@TiO 2 . In the case of ZnF 2 POH@qTiO 2 , the changes in the maximum were not observed, so the difference in lifetimes (twice longer) can be related with a different mechanism of relaxation of photoexcited electrons (see Figure 5c) or the possible geometry distortion of the porphyrin ring due to adsorption on qTiO 2 . Moreover, the energy differences between fluorescence maxima for TPPS@qTiO 2 and F 2 POH@qTiO 2 were about 0.14 eV, which suggests that the S 1 state of these porphyrins (in contrary to ZnF 2 POH@qTiO 2 ) was located at higher energy than for the conduction band of qTiO 2 . Thus, the oxygen-centered radical formation may have been promoted and the reduction of molecular oxygen to O 2 •− and further HO • may have been preferred over that of ZnF 2 POH@qTiO 2 . Nevertheless, the mechanisms of ROS generation on the basis of the excitation of porphyrins themselves should also be considered. As indicated in Table 2, investigated porphyrins were characterized by different singlet oxygen quantum yield (Φ ∆ ). Φ ∆ values were strongly affected by the substitution pattern and show the following trend: TPPS ≈ F 2 POH < ZnF 2 POH, with respective values of 0.64, 0.65, and 0.85, respectively [9]. The increase in Φ ∆ may be related to fluorine atoms substitution, as well as to metal insertion. The improvement of Φ ∆ by about 25% for ZnF 2 POH suggests that this photosensitizer acted particularly through a type II photochemical mechanism. Consequently, after impregnation of studied porphyrins on the qTiO 2 surface, the prepared hybrid nanomaterials may generate both singlet oxygen as well as oxygen-centered radicals, which may improve the overall photochemical properties influencing the photodynamic activity of the photosensitizer [45].

Photoelectrochemical Properties of qTiO 2 Material
TiO 2 was characterized by the wide-band gap, hence the photoelectrochemical response upon irradiation was observed in the range of 300-400 nm (Figure 6), whereas for all porphyrin-based nanomaterials, the photocurrents appeared to be above 400 nm. These results confirm directly the photosensitization of TiO 2 with porphyrins within the visible light and are consistent with all spectroscopic data described above (including the mechanisms proposed in Figure 5). Moreover, the higher the photocurrent was, the more sufficient interfacial electron transfer (IFET) occurred. Therefore, the higher photocurrent value at 420 nm for F 2 POH@qTiO 2 , compared with others, clearly indicated that F 2 POH@qTiO 2 seemed to be the most photoactive material upon irradiation. Other materials, such as ZnF 2 POH@qTiO 2 and TPPS@qTiO 2 , should be slightly less active in the IFET process above 400 nm. It is worth noting that bare qTiO 2 showed no photocurrent in this range. In addition, especially for ZnF 2 POH@TiO 2 material, the activity of bare qTiO 2 in the UV range was not diminished [56,57], but increased significantly. Therefore, the higher photocurrent value at 420 nm for F2POH@qTiO2, compared with others, clearly indicated that F2POH@qTiO2 seemed to be the most photoactive material upon irradiation. Other materials, such as ZnF2POH@qTiO2 and TPPS@qTiO2, should be slightly less active in the IFET process above 400 nm. It is worth noting that bare qTiO2 showed no photocurrent in this range. In addition, especially for ZnF2POH@TiO2 material, the activity of bare qTiO2 in the UV range was not diminished [56,57], but increased significantly.

ROS Detection by Molecular Fluorescent Probes
To elucidate the activity of the porphyrin@qTiO 2 nanomaterials, several fluorescent probes for detection of various ROS were used. Photocurrents clearly confirmed electron transfer from the excited dye molecule to the conduction band of qTiO 2 . Titanium dioxide (especially anatase polymorphs) appeared to be a strong oxidant. Its photo-excitation in water resulted in efficient HO • generation as a result of water or surface hydroxyl group oxidation [58]. The 3-p-(hydroxyphenyl)fluorescein (HPF) probe, which possesses reasonable selectivity to hydroxyl radicals, shows that attaching (metallo)porphyrin to qTiO 2 led to an increased level of HO • generation (Figure 7). In the case of TPPS, which is a well-known type II PS, generation of HO • was also observed upon attachment to qTiO 2 . The experiment performed with 3-p-(aminophenyl)fluorescein (APF) also confirmed an increasing level of ROS generation after impregnation of qTiO 2 with studied porphyrins. As shown in Figure 5, photoexcited electrons were transferred to the CB of titania. Electrons from the conduction band of TiO 2 may reduce the molecular oxygen adsorbed at the semiconductor surface in one-or three-electron processes to superoxide anion (O 2 •− ) or hydroxyl radicals, respectively. Such a mechanism has been reported several times by other authors [59]. For superoxide ion generated upon irradiation of the studied colloids, the dihydroethidium (DHE) probe was used ( Figure 8). Moreover, to detect singlet oxygen in the studied systems, the Singlet Oxygen Sensor Green (SOSG) probe was also applied ( Figure 8). The observed increase in the specific fluorescence signal for each probe proved that both singlet oxygen and oxygen-centered radicals were generated, and that the changes indicate the relative contribution of these ROS. This is especially important because by combining multiple mechanisms of ROS generation into a single material, the possibility of overcoming multidrug resistance is more likely [20,60].
was used (Figure 8). Moreover, to detect singlet oxygen in the studied systems, the Singlet Oxygen Sensor Green (SOSG) probe was also applied (Figure 8). The observed increase in the specific fluorescence signal for each probe proved that both singlet oxygen and oxygen-centered radicals were generated, and that the changes indicate the relative contribution of these ROS. This is especially important because by combining multiple mechanisms of ROS generation into a single material, the possibility of overcoming multidrug resistance is more likely [20,60].

Antimicrobial Studies
Upon visible light irradiation, porphyrin-based nanomaterials seemed to exhibit antimicrobial activity at a very low concentration ( Figure 9). Unmodified qTiO2 at a concentration of 1 g/L exhibited a killing efficacy of one log reduction due to the low absorption in visible light about 400 nm and 2 logs after potentiation with H2O2 or KI addition. Materials modified with porphyrin derivatives showed improved phototoxicity against both studied bacterial strains in a broad spectrum of applied concentrations, short-term incubation (2 h), and irradiation with 10 J/cm 2 . Increased concentrations of materials caused further decrease in bacterial survival. In the case of photodynamic treatment without any potentiation (H2O2 or KI), the performed strategy reduced the colony forming unit (CFU) value at 1 g/L concentration of each material up to 3-4 logs. The most potent antibacterial effect was observed for F2POH@qTiO2. However, the complete bactericidal effect was not achieved in any case. Thus, for potentiation of PDI treatment, we performed the treatment with 100 mM H2O2 or 100 mM KI added to cells before irradiation. For all experimental groups we observed a huge potentiation of photodynamic effect with 2-4 additional logs in bacterial killing. A complete eradication of Escherichia coli was achieved for TPPS@qTiO2-PDI + H2O2 at a concentration of 0.5 g/L, for F2POH@qTiO2 PDI + H2O2 and PDI + KI at 1g/L, and PDI + H2O2 for ZnF2POH@qTiO2 at 0.5 g/L. This had a great advantage over traditional methods, which usually do not lead to the complete destruction of Escherichia coli because the outer membrane of Gram-negative bacteria inhibits the uptake of drugs and antibiotics. The huge potentiation of PDI combined with H2O2 may be related to

Antimicrobial Studies
Upon visible light irradiation, porphyrin-based nanomaterials seemed to exhibit antimicrobial activity at a very low concentration (Figure 9). Unmodified qTiO 2 at a concentration of 1 g/L exhibited a killing efficacy of one log reduction due to the low absorption in visible light about 400 nm and 2 logs after potentiation with H 2 O 2 or KI addition. Materials modified with porphyrin derivatives showed improved phototoxicity against both studied bacterial strains in a broad spectrum of applied concentrations, short-term incubation (2 h), and irradiation with 10 J/cm 2 . Increased concentrations of materials caused further decrease in bacterial survival. In the case of photodynamic treatment without any potentiation (H 2 O 2 or KI), the performed strategy reduced the colony forming unit (CFU) value at 1 g/L concentration of each material up to 3-4 logs. The most potent antibacterial effect was observed for F 2 POH@qTiO 2 . However, the complete bactericidal effect was not achieved in any case. Thus, for potentiation of PDI treatment, we performed the treatment with 100 mM H 2 O 2 or 100 mM KI added to cells before irradiation. For all experimental groups we observed a huge potentiation of photodynamic effect with 2-4 additional logs in bacterial killing. A complete eradication of Escherichia coli was achieved for TPPS@qTiO 2-PDI + H 2 O 2 at a concentration of 0.5 g/L, for F 2 POH@qTiO 2 PDI + H 2 O 2 and PDI + KI at 1g/L, and PDI + H 2 O 2 for ZnF 2 POH@qTiO 2 at 0.5 g/L. This had a great advantage over traditional methods, which usually do not lead to the complete destruction of Escherichi coli because the outer membrane of Gram-negative bacteria inhibits the uptake of drugs and antibiotics. The huge potentiation of PDI combined with H 2 O 2 may be related to the increased amount of ROS, which was generated during PDI. The Staphylococcus aureus eradication was obtained only after using materials based on halogenated porphyrins (F 2 POH and ZnF 2 POH) that used ten times lower material concentration equal to 0.1 g/L after PDI with both H 2 O 2 and KI potentiation. In TPPS@qTiO 2 -PDI combined with hydrogen peroxide or KI, the CFU notably decreased (1 log after H 2 O 2 addition and 2 additional logs after KI) compared to the TPPS alone. Nevertheless, the bactericidal effect was not observed. Thus, F 2 POH@qTiO 2 and ZnF 2 POH@qTiO 2 exhibited a higher antimicrobial activity than TPPS@qTiO 2 , which may have been related to fluorine atom substitution. Introduction of halogens into the macrocycle not only efficiently improved the photophysical properties of photosensitizers, but also their bioavailability biological activity [25,44].
Catalysts 2019, 9, x FOR PEER REVIEW 11 of 21 the increased amount of ROS, which was generated during PDI. The Staphylococcus aureus eradication was obtained only after using materials based on halogenated porphyrins (F2POH and ZnF2POH) that used ten times lower material concentration equal to 0.1 g/L after PDI with both H2O2 and KI potentiation. In TPPS@qTiO2-PDI combined with hydrogen peroxide or KI, the CFU notably decreased (1 log after H2O2 addition and 2 additional logs after KI) compared to the TPPS alone. Nevertheless, the bactericidal effect was not observed. Thus, F2POH@qTiO2 and ZnF2POH@qTiO2 exhibited a higher antimicrobial activity than TPPS@qTiO2, which may have been related to fluorine atom substitution. Introduction of halogens into the macrocycle not only efficiently improved the photophysical properties of photosensitizers, but also their bioavailability biological activity [25,44].

Bacteria Imaging by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM)
To visualize the photoinduced damages and the differences in the mechanisms of bacterial death, we performed SEM imaging immediately after each scheme of PDI treatment (PDI alone, PDI in combination with H2O2, and with KI, separately). The SEM images of Escherichia coli and Staphylococcus aureus are shown in Figures 10 and 11, and Figures S4 and S5. As may be observed, the PDI (incubation time: 2 h, material concentration of 0.5 g/L and light dose of 10 J/cm 2 ) against Escherichia coli indicated a bacteriostatic effect and may inhibit the bacteria division without visible membrane disruption. Nevertheless, it is believed that these bacteria were dying or losing cell functions with the intact cell walls. In contrast, after addition of 100 mM H2O2, the cells showed a substantially damaged cell membrane. In the case of PDI with addition of 100 mM KI, even more

Bacteria Imaging by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM)
To visualize the photoinduced damages and the differences in the mechanisms of bacterial death, we performed SEM imaging immediately after each scheme of PDI treatment (PDI alone, PDI in combination with H 2 O 2 , and with KI, separately). The SEM images of Escherichia coli and Staphylococcus aureus are shown in Figures 10 and 11, and Figures S4 and S5. As may be observed, the PDI (incubation time: 2 h, material concentration of 0.5 g/L and light dose of 10 J/cm 2 ) against Escherichia coli indicated a bacteriostatic effect and may inhibit the bacteria division without visible membrane disruption. Nevertheless, it is believed that these bacteria were dying or losing cell functions with the intact cell walls. In contrast, after addition of 100 mM H 2 O 2 , the cells showed a substantially damaged cell membrane. In the case of PDI with addition of 100 mM KI, even more severe damage can be observed ( Figure 10). The membrane appeared to be "shredded" and the cells showed leakage of cellular contents.
severe damage can be observed ( Figure 10). The membrane appeared to be "shredded" and the cells showed leakage of cellular contents.
Seemingly, a distant type of photodamage may have been detected for Staphylococcus aureus after PDI-treatment. Independently from the type of potentiation, the cells that received PDI with addition of 100 mM H2O2 or KI were killed completely ( Figure 11). The images show multiple wall debris or even the area of completely disrupted cells. After the PDI-treatment alone, only cell shrinkage and gradual shape change were observed, whereas the application of the PDI treatment with H2O2 and KI led to a more pronounced effect. Figure 10. SEM images of Escherichia coli before PDI treatment (control) and bacterial cell debris after different PDI procedures (2 h incubation, 10 J/cm 2 , 420 ± 20 nm LED light, material concentration of 0.5 g/L with addition of 100 mM H2O2 or 100 mM KI). However, because of the fact that the SEM images displayed similar structure of bacteria (in some cases only shapes were interrupted) we confirmed the presence of damaged cells by staining them with Hoechst33342 and propidium iodide (PI) (LIVE/DEAD staining) and imaging with CLSM. Figure 12 shows the images of untreated and photodynamically treated Escherichia coli and Staphylococcus aureus (incubation time: 2 h, material concentration of 0.5 g/L and light dose of 10 J/cm 2 ). The blue fluorescence signal is characteristic for intact living cells. In contrast, the red signal from PI clearly indicated that after PDI treatment, treated cells lost their integrity that allowed the dye (that is excluded by viable cells) to penetrate the cell membranes of disrupted or dead bacterial cells. Figure 11. SEM images of Staphylococcus aureus before PDI treatment (control) and bacterial cell debris after different PDI procedures (2 h incubation, 10 J/cm 2 , 420 ± 20 nm LED light, material concentration at 0.5 g/L with addition of 100 mM H 2 O 2 or 100 mM KI).
Seemingly, a distant type of photodamage may have been detected for Staphylococcus aureus after PDI-treatment. Independently from the type of potentiation, the cells that received PDI with addition of 100 mM H 2 O 2 or KI were killed completely ( Figure 11). The images show multiple wall debris or even the area of completely disrupted cells. After the PDI-treatment alone, only cell shrinkage and gradual shape change were observed, whereas the application of the PDI treatment with H 2 O 2 and KI led to a more pronounced effect.
However, because of the fact that the SEM images displayed similar structure of bacteria (in some cases only shapes were interrupted) we confirmed the presence of damaged cells by staining them with Hoechst33342 and propidium iodide (PI) (LIVE/DEAD staining) and imaging with CLSM. Figure 12 shows the images of untreated and photodynamically treated Escherichia coli and Staphylococcus aureus (incubation time: 2 h, material concentration of 0.5 g/L and light dose of 10 J/cm 2 ). The blue fluorescence signal is characteristic for intact living cells. In contrast, the red signal from PI clearly indicated that after PDI treatment, treated cells lost their integrity that allowed the dye (that is excluded by viable cells) to penetrate the cell membranes of disrupted or dead bacterial cells.

Discussion and Conclusions
Inorganic semiconductor photocatalysts offer a number of opportunities for environmental and biomedical applications. Particular attention has been given to titanium dioxide because of its unique properties, such as it being a wide-bandgap semiconductor, its non-toxicity to living organisms, its stability in water, and its strong photocatalytic activity when its crystal grain size is reduced to tenths of nanometers. Most importantly, it is also possible to modify its surface by many inorganic and organic molecules, and thus obtain hybrid materials that are able to extend the photoactivity of bare TiO 2 into the visible spectrum of light [9,10,42,45]. Our studies reveal that surface modification of qTiO 2 with commercially available TPPS and its fluorinated derivatives, F 2 POH and ZnF 2 POH, in aqueous medium at pH = 6 demonstrate efficient charge injection into the conduction band. This effect was confirmed by photocurrent measurements and steady-state fluorescence, as well as detection of a high amount of photogenerated ROS. Moreover, our photocatalysts based on highly-dispersed nanocolloidal TiO 2 were very efficient in the sensitization of semiconductor bandgap by interfacial charge-transfer transition (IFET). In contrast to TPPS in a homogeneous system, we demonstrated that prepared hybrid nanomaterials were able to generate not only singlet oxygen (as with free porphyrins in a homogeneous system), but also oxygen-centered radicals. Thus, their photoactivity was derived from multiple mechanisms of ROS generation. In fact, the ability of nanomaterials to facilitate electron transfer, and thereby promote ROS generation, is a fundamental property of these materials and guarantees their use as an efficient photocatalyst in antimicrobial treatment. Our data clearly show that the modification of the qTiO 2 surface led to the presence of partition of hydroxyl radicals' generation, which was associated with the physicochemical characteristics in terms of both mechanisms and activity.
Efficient ROS generation is considered a key factor in sufficient photocatalysis and photodynamic inactivation of bacteria [25]. We have demonstrated that the nanomaterials synthesized and characterized by us are promising antimicrobial agents. We have also studied the PDI potentiation with H 2 O 2 and KI to achieve complete eradication of pathogens. When KI was added, there was a huge increase in bacterial killing, ranging from 2 logs extra at 0.01 g/L, to >6 logs extra at 1 g/L in CFU reduction of Escherichia coli and Staphylococcus aureus after blue-light irradiation at a dose of 10 J/cm 2 . It can be hypothesized that hydrogen peroxide would improve the efficacy of the performed PDI-treatment against Escherichia coli and Staphylococcus aureus, as the antibacterial photodynamic effect was improved across a wide concentration range of prepared materials at a low concentration of H 2 O 2 . It can also be suggested that H 2 O 2 could increase the amount of ROS, which are generated during PDI [61]. However, some authors reported that the in vitro production of ROS did not increase in the presence of H 2 O 2 , meaning that the synergetic effect of H 2 O 2 in PDT has another mechanism [62]. McCullagh et al. suggested the quenching of triplet-excited methylene blue by H 2 O 2 in a type I process during PDT against Synechococcus leopoliensis [63]. In addition, hydrogen peroxide provides photodamage of bacterial membranes and increased drug accumulation in the cells [64]. The presence of H 2 O 2 outside the bacteria facilitates the diffusion of PS into the bacteria [61,62]. Accordingly, for PDI combined with KI, the mechanism of action has been recently described [65][66][67][68]. In this case, the potentiation is caused by the formation of long-lived antimicrobial species (probably hypoiodite and iodine) in the reaction mixture (detected by adding bacteria after light), as well as short-lived antibacterial reactive species (bacteria present during light) able to produce more killing [67]. Hamblin et al. also reported that the potentiation of TiO 2 -based materials in combination with UVA light, along with the presence of iodide, produces reactive iodine intermediates during illumination that kills microbial cells, and long-lived oxidized iodine products that kill after the application of light has ended [65].
Taken together, all of the performed studies indicate that TiO 2 nanoparticles modified with tetrapyrrolic compounds are an inorganic photoactive material with superior biological activity. Their antimicrobial properties in vitro in bacteria suspensions reveal that they are worth further investigation, especially in vivo in wound-healing processes and the elimination of localized infections.

Porphyrins@qTiO 2 Materials Preparation
In our experiments, 5% colloidal solution of TiO 2 nanoparticles in water was used and mixed with the water solution of a modifier (100 µM) in the ratio of 1:1 (v/v). In this way, color colloids of surface modified nanocrystalline TiO 2 were obtained. The pH of resulting colloid solution was adjusted with addition of 1% HCl solution to natural skin pH = 6. The solution was placed in a dialysis tube (Sigma, cut-off 14 kDa) and dialyzed two times against water in order to remove all low molecular-weight additives and the unbound modifier. Within this paper, the abbreviations TPPS@qTiO 2 , F 2 POH@qTiO 2 , and ZnF 2 POH@qTiO 2 have been assigned to modified colloids.

UV/VIS Electronic Absorption and Emission Spectra Measurements
UV/VIS absorption spectra were recorded with the Hewlett Packard HP8453 spectrophotometer in a 1 cm quartz cuvette. Fluorescence emission spectra were recorded from 550 to 750 nm with excitation at the Soret band (420 nm) using a Perkin Elmer Fluorescence Spectrometer LS 55.

Dynamic Light Scattering (DLS) and Zeta Potential Measurements
Zeta potential and hydrodynamic diameter measurements of nanomaterials in colloid materials were performed using Zetasizer Nano ZS (Malvern Instruments). The apparent diffusion coefficients of the nanoparticles were obtained from the normalized time correlation function of the scattered electric field, g(1)(τ), using the cumulants analysis. An average value was obtained from repeated measurements for each sample (N = 3).

Photoelectrochemical Measurements
Photocurrents of modified and unmodified qTiO 2 were measured using a three-electrode setup using 0.1 M solution of KNO 3 as electrolyte. The working electrode was prepared by spreading an aqueous suspension of studied material on ITO-coated transparent foil (60 Ω/sq resistance, Sigma-Aldrich) and dried afterwards in the stream of warm air (ca. 50 • C). A platinum wire and Ag/AgCl (3M) were used as the counter and reference electrodes, respectively. Photoelectrochemical measurements were performed using the photoelectric spectrometer (Instytut Fotonowy). The working electrodes were irradiated in the range of 330-500 nm from the backside through the ITO layer in order to minimize the influence of the film thickness on the measured photocurrents.

Detection of Reactive Oxygen Species Using Fluorescent Probes
3-p-(Aminophenyl)fluorescein (APF) and 3-p-(hydroxyphenyl)fluorescein (HPF) are selective probes for oxygen reduction products. Singlet Oxygen Sensor Green (SOSG) is a specific probe for singlet oxygen, whereas dihydroethidinum (DHE) traps superoxide ion. These probes were employed for the detection of ROS generated during illumination of the colloid materials with absorbance at the same level at about 1.0. Each fluorescent probe was added to a well at a final concentration of 10 µM. Freshly prepared solutions were then irradiated with LED (420 ± 20 nm) light for increasing time-intervals. A microplate reader (Tecan Infinite M200 Reader) was used for acquisition of fluorescence signal immediately before and after illumination. When APF and HPF were employed, fluorescence emission at 515 nm was acquired upon excitation at 490 nm. With SOSG, the corresponding values were 525 and 505 nm, and for DHE, 480 nm and 580 nm, respectively [25].

Photodynamic Inactivation (PDI) of Bacteria
The microorganisms used in PDI were Escherichia coli (K12) and Staphylococcus aureus (8325-4). The planctonic Escherichia coli were cultured in Luria Broth (LB) (BioShop Lab Science Products) and Staphylococcus aureus in brain heart infusion broth (BHI) (Sigma-Aldrich) in an orbital incubator (37 • C, 130 rpm) until the optical density reached OD = 0.5, which corresponds to approximately 10 7 CFU per mL. For PDI experiments, suspensions of bacteria were incubated with various concentrations of hybrid nanomaterials for 2 h in the dark at room temperature. For further potentiation effect, in other experimental groups, bacteria were also incubated with the addition of 100 mM H 2 O 2 or 100 mM KI. Then, aliquots (1 mL) were transferred to a 12-well plate and illuminated with a blue light. We used blue-LED array (420 ± 20 nm) to deliver a light dose of 10 J/cm 2 (measured with a radiometer).
After illumination (or dark incubation), samples were resuspended, diluted in water, mixed and plated (LB or BHI agars). Aliquots were taken from each well to determine the CFU value. Plates were segregated in triplicate and incubated for 24 h at 37 • C in the dark to allow colony formation. A control group of bacterial cells treated with light showed the same number of CFU as the absolute control (data not shown). The viability of bacteria was also monitored using LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen; monitors membrane integrity) according to the manufacturer's instructions. All values were expressed as average ± SD (standard deviation), which represented the standard deviation of the sample mean estimate of a population mean.

CLSM Fluorescence Imaging of Bacteria
Bacterial viability and photodynamically-induced damages were followed with fluorescence confocal imaging using a Zeiss LSM880 laser-scanning microscope equipped with an argon ion laser. The objective used was a water-immersion objective lens (63×, Carl Zeiss Ltd.) with a working distance of 1.46 mm. Selected bacteria were incubated with the photosensitizer and irradiated accordingly to the PDI protocol described above. Then, bacteria were stained with two nucleic acid dyes: propidium iodide (PI) to stain dead bacteria and Hoechst33342, which is able to stain only bacteria with an intact membrane (living). After washing, the microorganism cells were placed on the microscopic glass slides and imaged. Registered images were analyzed with the Zeiss ZEN software.

Statistics
All values were expressed as average ± SD (standard deviation), which represented the average squared deviation from the mean. All experiments were repeated at least three times with comparable results. The sample size in biological tests was N = 6-12 in each experimental group.
Supplementary Materials: The following are available online at Available online: www.mdpi.com/xxx/s1 (accessed on 8 May 2012).: Figure S1: Absorption spectra of qTiO 2 and the calculation of the band gaps based on the Tauc plot with baseline correction. Figure S2: SEM images of nanostructured qTiO 2 , TPPS@qTiO 2 , F 2 POH@qTiO 2 and ZnF 2 POH@TiO 2 recorded on carbon tape with size of particle highlighted. Figure S3: SEM images of nanostructured qTiO 2 , TPPS@qTiO 2 , F 2 POH@qTiO 2 , and ZnF 2 POH@TiO 2 recorded on carbon tape with size of particle highlighted. Figure S4: SEM images of Escherichia coli before PDI treatment (control) and bacterial cells debris after different PDI procedures. Figure S5: SEM images of Staphylococcus aureus before PDI treatment (control) and bacterial cells debris after different PDI procedures.