Photophysical, photobiological, and mycobacteria photo-inactivation properties of new meso-tetra-cationic platinum(II) metalloderivatives at meta position

Abstract

In this manuscript, we report the photo-inactivation evaluation of new tetra-cationic porphyrins with peripheral Pt(II) complexes ate meta N-pyridyl positions in the antimicrobial photodynamic therapy (aPDT) of rapidly growing mycobacterial strains (RGM). Four different metalloderivatives were synthetized and applied. aPDT experiments in the strains of Mycobacteroides abscessus subsp. Abscessus (ATCC 19977), Mycolicibacterium fortuitum (ATCC 6841), Mycobacteroides abscessus subsp. Massiliense (ATCC 48898), and Mycolicibacterium smegmatis (ATCC 700084) conducted with adequate concentration of photosensitizers (PS) under white-light conditions at 90 min (irradiance of 50 mW cm⁻² and a total light dosage of 270 J cm⁻²) showed that the Zn(II) derivative is the most effective PS significantly reduced the concentration of viable mycobacteria. The effectiveness of the molecule as PS for PDI studies is also clear with mycobacteria, which is strongly related with the porphyrin peripheral charge and coordination platinum(II) compounds and consequently about the presence of metal center ion. This class of PS may be promising antimycobacterial aPDT agents with potential applications in medical clinical cases and bioremediation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01201-0.

Introduction

Porphyrins are organic compounds present in essential metabolic processes for life [1–4]. These biologically important roles stem from the functional versatility of these molecules whose main characteristic is the presence of the macrocyclic heterocyclic ring. This ring is composed of four pyrrole units that are interconnected at their α-carbon atoms through methine bond bridge. This highly π-conjugated structure grants porphyrins a very extensive absorption range, which extends from the ultraviolet–visible (UV–Vis) to the near infrared (NIR) region [2, 5].

Due to their unique photochemical properties and excellent prospects for development in pharmaceutical synthesis, porphyrins have gained prominence in medical applications, mainly through photodynamic therapy (PDT) applications [6]. Porphyrin derivatives are widely used as photosensitizers (PS) in PDT due to their absorption bands being observed in the visible light region, their high molar absorption coefficient, biocompatibility, and good stability against light radiation (UV–Vis) [6, 7]. However, despite the widespread use of this molecule and its derivatives as conventional PS, they easily form aggregates in aqueous media due to π-π interactions and their hydrophobic nature, thus causing chromophoric changes and decreased production of reactive oxygen species (ROS), such as singlet oxygen species (¹O₂) [2, 8–10].

To overcome these disadvantages, modifications in the porphyrin structure have been carried out in order to improve characteristics such as aggregation, biodistribution, stability and toxicology of the molecule [6, 11, 12]. The addition of peripheral complexes in the porphyrin structure, for example platinum(II) and palladium(II) derivatives, has been described as potentiating the photodynamic activity of these compounds [13–16]. Its aromatic nature provides a higher electron density located in the four meso positions of the ring, making them more reactive. Therefore, meso-tetra-substituted porphyrins are largely synthesized and studied as potential antimicrobial agents [17–22].

Likewise, studies report that the addition of transition metal ions coordinates to the macrocyclic ring results in changes in the photophysical and photobiological properties of these molecules, presenting a better efficiency in the photo-oxidation process through a greater ROS generation [23–26]. Thus, tetra-cationic porphyrins contain Zn(II), Cu(II), Ni(II), Fe(III), Co(III), or Mn(III) metal ions were, in some cases, more soluble in water and may be excellent candidates for application in photo-oxidative processes, since these derivatives produced good amounts of reactive oxygen species in solution, with high stability and low tendency to aggregation [27].

Based on the above considerations, we designed a series of novel meta-substituted meso-(pyridyl)metalloporphyrins, which are more soluble and less prone to aggregate formation in solution, with photodynamic properties to act as antibacterial agents in the control of mycobacterial infections. For this study, we consider the excellent efficacy already shown, in previous studies by our research group, by meso-substituted porphyrins with peripheral platinum(II) complex, more specifically free-base 3-PtTPyP against gram-negative and gram-positive bacteria, mycobacteria, and fungi and in the Aedes larvae control [14, 28, 29]. Moreover, photophysical and photobiological assays will also be discussed throughout the manuscript, in order to better understand the behavior and influence of these metalloderivatives in the presence of microorganisms, in this case, rapid growing mycobacteria (RGM).

Experimental section

Tetra-cationic porphyrin photosensitizers Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP)

Meta-substituted metalloporphyrins were prepared slightly by methodology modification by da Silveira and co-workers [30] reacting free-base tetra-cationic 3-PtTPyP (0.015 g, 0.052 mmol; 1.0 equiv.) with 5.0 equivalents (0.262 mmol) of metal(II) acetate (zinc, copper, cobalt and manganese), in DMF reflux system (10 mL), for 24-h period time. The solvent was removed in an evaporator and the solid washed with cold distilled water and diethyl ether, recrystallized, filtered and dried in a vacuum system. Elemental analysis (CHN%) and molar conductimetry analysis are presented in the Supplementary information section (see Table S1). Also, all metalloporphyrins tested in this study (Fig. 1) are soluble in DMSO and stable in this solution (see results and discussion section).

Fig. 1.

Representative structures of the meta-substituted porphyrins used in this work. AcO = axial acetate ligand

Photophysical studies

Electronic UV–Vis absorption spectroscopy analysis was recorded using a Shimadzu UV-2600 spectrophotometer (data interval, 1.0 nm) in dimethyl sulfoxide (DMSO) and DMSO(5%)/Tris–HCl pH 7.4 buffer mixture solution as solvents at 250–800-nm range. Steady-state fluorescence emission spectra for the porphyrins in the same solutions were measured in a Horiba Yvon-Jobin Fluoromax Plus (Em/Exc; slit 5.0 mm) at 500–800-nm range. Fluorescence quantum yield (Φ_F) values of the derivatives Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP) were determined by comparing the corrected fluorescence spectra with those obtained by meso-tetra(3-pyridyl)porphyrin (3-H₂TPP) in DMSO solution (Φ_F = 2.4%, λ_exc = 419 nm) [31] as the Φ_F standard as the fluorescence yield and Eq. 1 was used to determine the Φ_F values:

$${\mathrm{\Phi }}_F={\mathrm{\Phi }}_{F_{\mathrm{std}}}\frac{I}{I_{\mathrm{std}}}\frac{{(1-{10}^{-A})}_{\mathrm{std}}}{\left(1,-,{10}^{-A}\right)}\frac{\eta 2}{{\eta 2}_{\mathrm{std}}}$$ 1

where Φ_F, I, A, and η are the fluorescence quantum yield, integral area of fluorescence, absorbance in λ_exc, and refractive index of selected solvents (DMSO = 1.479). The subscript “std” refers to the standard molecule.

Lifetime fluorescence decays (τ_f) in the singlet-excited state were recorded using Time-Correlated Single Photon Counting (TCSPC) method with a DeltaHub controller in conjunction with a Horiba spectrofluorometer. Data was processed with DAS6 and Origin® 8.5 software using exponential (mono-exponential) fitting of raw data. NanoLED (Horiba) source (1.0 MHz, pulse width < 1.3 ns at 441-nm excitation wavelength) was used as an excitation source. In this way, radiative (k_r) and non-radiative (k_nr) constants can be determined by knowing the fluorescence quantum yield and fluorescence lifetime, as following Eqs. 2 and 3 [32, 33]:

$$k_r={\varphi }_f/{\tau }_f$$ 2

$$k_{\mathit{nr}}=\left(1,-,{\varphi }_f\right)/{\tau }_f$$ 3

Electrochemical analysis

Cyclic voltammetry measurements were carried out using an Metrohm Autolab Eco Chemie PGSTAT 128N potentiostat/galvanostat using a conventional three-electrode system constituted by a glassy carbon working electrode, a platinum wire as auxiliary, and a platinum wire as pseudo-reference electrode (using ferrocene in DMF as internal standard; E_1/2 =  + 0.40 V) [34]. The experiments were carried out using 0.1 M of tetrabutylammonium hexafluorophosphate salt (TBAPF₆) in dry DMF as a support electrolyte.

Aggregation and stability study by UV–Vis analysis

UV–Vis experiments were conducted as a function of successive increase of porphyrin concentration (1.0 to 30 μM) in DMSO and DMSO(5%)/Tris–HCl pH 7.4 buffer solutions and changes in the λ_max in the 250–800 nm range were monitored, according to the related literature [30]. The stability experiments in pure dimethyl sulfoxide (DMSO) and in DMSO(5%)/Tris–HCl pH 7.4 buffer solution of related porphyrin were also monitored by absorption UV–Vis measurements at several days (seven days). In order to investigate the possibility of exchanging chloride ligands (Cl⁻) for water molecules (H₂O) in the coordination sphere of peripheral Pt(II) complexes or in the metal center coordination sphere, an assay via UV–Vis spectroscopy of metalloporphyrins in the presence of amount of DMSO (2.0 mL) was conducted by the time of acquisition of the absorption spectra (t = 48 h) at 25ºC. All experiments were performed in duplicate and independently.

Photostability experiments

The photostability experiments in dimethyl sulfoxide (DMSO) and in DMSO(5%)/Tris–HCl pH 7.4 buffer solution of related compound was also monitored by absorption electronic UV–Vis measurements at different exposure times (0 to 30 min) under white-light LED lamp (400 at 800 nm) at irradiance of 50 mW cm⁻² and a total light dosage of 90 J cm⁻² and calculated according the Eq. 4. All experiments were performed in duplicate and independently.

$$\mathrm{Photostability}\left(\%\right)=\frac{\mathrm{Abs}\mathrm{at}a\mathrm{given}\mathrm{time}\mathrm{of}\mathrm{irradiation}}{\mathrm{Abs}\mathrm{before}\mathrm{irradiation}}$$ 4

Water/n-octanol partition coefficients (log POW)

The partition coefficient of porphyrins was determined using octanol (3.0 mL) and water (3.0 mL), according to the literature [32]. The log P_OW value was calculated from Eq. 5, with A_org and A_aq being the maximum absorbances of the Soret band in the organic and aqueous phases and V_org and V_aq being the final volumes of the organic and aqueous phases, respectively.

$$log{P}_{\mathit{OW}}=log\left[(A_{\mathit{org}}/A_{\mathit{aq}}),(V_{\mathit{aq}}/V_{\mathit{org}})\right]$$ 5

Singlet oxygen (¹O₂) generation and superoxide assays

For ¹O₂ determination, typical 1,3-diphenylisobenzofuran (DPBF) singlet oxygen quencher using in photodegradation experiments, the maximum volume of 1.0 mL which contained 10 μM DPBF in DMSO was mixed with 0.5 mL (50 μM) of porphyrins Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP). The flask was then filled with 2.0 mL of DMSO to a final volume of 3.5 mL. In order to measure singlet oxygen generation, absorption UV–Vis spectra of each solution were recorded at different exposure times (0 to 600 s, using red-light diode laser (Thera Laser DMC®, São Paulo; potency of 100 mW at 660 nm) according to the literature [35]. The singlet oxygen production quantum yield (Φ_Δ) was calculated applying Eq. 6:

$${\mathrm{\Phi }}_{\mathrm{\Delta }}={\mathrm{\Phi }}_{{\mathrm{\Delta }}_{\mathrm{std}}}\frac{k}{k_{\mathrm{std}}}\frac{{(1-{10}^{-A})}_{\mathrm{std}}}{(1-{10}^{-A})}$$ 6

where (1 − 10^A)std/(1 − 10^A), Φ_Δstd is the singlet oxygen quantum yield of standard compound (meso-tetra(3-pyridyl)porphyrin platinum(II) 3-PtTPyP in DMSO, Φ_Δstd = 0.50) [14], k and k_std are the photodegradation kinetic constants for porphyrins and 3-PtTPyP (standard), respectively, and A_std and A are the absorbances for 3-PtTPyP and studied porphyrin, respectively.

The NBT assays were used to detect the formation of superoxide radical species (O₂^·−). This approach was carried out using at the same conditions in the literature, using NBT and NADH in DMSO(5%)/Tris–HCl buffer (pH 7.4) mixture solution [36–38]. Control experiments were performed in the absence of porphyrins. Samples were irradiated under aerobic conditions with white-light conditions (irradiance at 50 mW cm⁻² total light dosage at 90 J cm⁻² for 30 min). The progress of the reaction was monitored by following the increase of the absorbance at 560 nm (color change from yellow to violet). The superoxide generation constant (k_SO) can be obtained from the slope.

Irradiation source

For the PS photoinactivation studies, 96-well plates were submitted to irradiation with white-light LED array system (400–800 nm) at irradiance of 50 mW cm⁻² and a total light dosage of 270 J cm⁻² (total time = 90 min). The plates remained closed according to biosafety standards, and the required distance between the plate and white-light source was 8–10 cm. A light control (LC) was performed in each lighting experiment under the same lighting conditions as the samples, but without PS; a dark control (DC) containing the PS at the same concentrations was also carried out but kept in the dark.

Mycobacteria strains

In this study, mycobacterial strains growing for less than seven days were used. The rapid growth mycobacterium (RGM) standard strains Mycobacteroides abscessus subsp. Abscessus (ATCC 19977), Mycobacteroides abscessus subsp. Massiliense (ATCC 48898), Mycolicibacterium fortuitum (ATCC 6841), and Mycolicibacterium smegmatis (ATCC 700084) were kept frozen at − 80 °C and for their use were cultivated on Löwenstein-Jensen agar (HiMedia Laboratories Pvt. Ltd, India).

Susceptibility test

The photoactive action of the metalloporphyrins under study was evaluated by the susceptibility assay according to the standard protocol of the CLSI M24-A2 [39]. Different concentrations of PS were obtained by means of serial dilutions in Mueller Hinton Broth medium (Sigma-Aldrich® Pvt. Ltd, India). The inoculum density was standardized according to the 0.5 MacFarland scale and subsequently diluted to a concentration of 5.0 × 10⁵ CFU mL⁻¹ and transferred to 96-well microplates. The antimycobacterial activity of each PS was verified in the presence and absence of white-light irradiation. In this way, a group of plates was kept incubated in the dark, at 30 °C, for 72 h. The second group was kept under the same conditions, but with lighting for a period of 90 min at 0, 24, and 48 h of incubation. The minimum inhibitory concentration (MIC) was considered the lowest concentration capable of inhibiting visible mycobacterial growth.

ROS scavenger assays

The scavenger assay identified the investigative possible ROS mechanism resulting from the metalloporphyrins photo-inactivation process under study. Ascorbic acid (AA; Sigma-Aldrich®), dimethyl sulfoxide (DMSO; Sigma-Aldrich®), tert-butanol (t-BuOH; Merck®), and N-acetylcysteine (NAC; Sigma-Aldrich®) were used for detecting respective species: singlet oxygen (¹O₂), superoxide radical (O₂^·‒), hydroxyl radical (·OH), and hydroperoxyl radical (·OOH), respectively. The PS dilution followed the MIC determination assay conditions and, in the 96-well microplates, the sub-inhibitory concentrations of each sequestrant and the standardized inoculum were added. The plates were kept at 30ºC for 72 h with 90-min irradiation at times 0, 24, and 48 h. The higher MIC concentration was used to determine the type of ROS produced by the tetra-cationic porphyrins at meta position [28].

Atomic force microscopy (AFM) analysis

The photo-damage promoted by metalloporphyrins in RGM strains was evaluated by the AFM microscopy analysis. In this assay, the topographical, nanomechanical, and electrical properties were measured after 24 h of incubation of the Mycolicibacterium smegmatis strain (ATCC 700084) under different conditions of exposure to white-light conditions and PS. After treatment, the samples were diluted in water and deposited on small square pieces of freshly cleaved mica. AFM characterization was performed using a Park NX10 microscope (Park Systems, Suwon, Korea) equipped with SmartScan software (version 1.0.RTM13c2020-1). Topographic and bond strength images were taken simultaneously using the PintPoint Nanomechanical mode. Measurements were carried out using a silicone tip (TAP300-G Budget Sensors, Sofia, Bulgaria) with a nominal frequency of 300 kHz and a force constant of 40 N m⁻¹. Electrical force maps were obtained using a PtIr-coated silicone tip (PPP-EFM, Nanosensors, Neuchâtel, Switzerland) with a nominal frequency of 75 kHz and a force constant of 2.8 N m⁻¹. All measurements were made under ambient conditions of temperature of 21 ± 5ºC and relative humidity of 55 ± 10%. The images obtained were later treated using the XEI software version 4.3.4Build22.RTM1.

Statistical analysis

All tests were performed in triplicate and the results expressed as the mean ± standard derivation. The time-kill curve was performed using GraphPad Prism version 5.01 (GraphPad Software®, La Jolla, CA, USA), and statistical differences were considered when p < 0.05.

Results and discussions

General characterization

The free-base meso-tetra(3-pyridyl)porphyrin platinum(II) (3-PtTPyP) was previously described and fully characterized by Naue and co-workers [40]. The tetra-platinated metalloporphyrin synthesis was prepared slightly methodology modification by da Silveira and co-workers [30], and all compounds were confirmed by elemental analysis (CHN%), and molar conductimetry (see Supplementary information section, Table S1). Also, the choice of porphyrins at the meta positions is due to the greater solubility of this isomer (compared to the para position) and less tendency to aggregate in solution.

Photophysical analysis

The electronic absorption UV–Vis spectra of the metalloporphyrins in DMSO and DMSO(5%)/Tris–HCl pH 7.4 buffer mixture solution consist of an envelope of superimposed absorption bands in the range of 400–700 nm, arising from the characteristic absorption properties of meso-tetra-substituted porphyrin derivatives (Fig. 2a, b).

Fig. 2.

Normalized UV–Vis absorption spectra of Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP) metalloporphyrins, in a DMSO and b DMSO(5%)/Tris–HCl pH 7.4 buffer mixture solutions

The corresponding absorption Soret and Q-bands for metalloderivatives with Zn(II), Cu(II), Co(III) and Mn(III) complexes are observed between 420 and 470-nm range for Soret transition and at 525–660 nm for Q bands, respectively (Table 1). As usual, Mn(III) derivative Mn(3-PtTPyP) exhibits electronic transitions at 375–425-nm region. These transitions can be attributed to MLCT type, as reported in the literature [30, 41]. In spite of their similarities, it should be noted that the Soret band is quite narrow for the Zn(II) and Cu(II) complexes, when compared with that of Co(III) and Mn(III) metalloporphyrins. This observation is consistent with a tetra-coordinated ambient of Zn(II) and Cu(II) ions in the distorted square-planar and penta-coordinated coordination sphere of Co(III) and Mn(III) in a distorted pyramidal square-base environment into the N₄-macrocycle (Fig. 2a).

Table 1.

Photophysical data of studied porphyrin Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP)

	DMSO
Parameters	Zn(3-PtTPyP)	Cu(3-PtTPyP)	Co(3-PtTPyP)	Mn(3-PtTPyP)
λabs, nm (log ε)a	318 (4.38); 432 (5.40); 561 (3.99); 600 (3.46)	320 (4.61); 422 (5.40); 545 (4.15); 584 (3.35)	317 (4.51); 420 (5.15); 537 (4.09)	325 (4.77); 382 (4.69); 463 (5.25); 565 (4.14); 600 (3.90)
λem, nm (ΦF, %)b	606, 660 (9.0%)	648, 714 (1.0%)	651, 715 (3.0%)	n.d.*
τf, ns (χ2)c	1.30 (1.06668)	n.d.*	2.15 (1.11378)	n.d.*
kr, s−1 (× 108)d	0.69	n.d.*	0.14	n.d.*
knr, s−1 (× 108)d	7.00	n.d.*	4.50	n.d.*
	DMSO(5%)/Tris–HCl pH 7.4 buffer
Parameters	Zn(3-PtTPyP)	Cu(3-PtTPyP)	Co(3-PtTPyP)	Mn(3-PtTPyP)
λabs, nm (log ε)a	315 (4.72); 428 (5.22); 564 (4.22); 605 (3.64)	320 (4.88); 415 (5.22); 547 (4.22)	315 (4.66); 413 (5.10); 543 (4.12)	320 (4.87); 387 (4.83); 463 (5.19); 562 (4.22); 595 (3.93)
λem, nm (ΦF, %)b	665 (4.0%)	n.d.*	n.d.*	n.d.*
τf, ns (χ2)c	1.50 (0.91552)	n.d.*	n.d.*	n.d.*
kr, s−1 (× 108)d	0.26	n.d.*	n.d.*	n.d.*
knr, s−1 (× 108)d	6.40	n.d.*	n.d.*	n.d.*

*n.d. not determined (without emission)

^aSolution at 5.0 µM

^bSolution at 2.0 µM, using meso-tetra(3-pyridyl)porphyrin (3-H₂TPP) in DMSO solution (Φ_F = 2.4%) with an error around 5%

^cUsing NanoLED at 441 nm and values with an error around 5%

^dCalculated by Eqs. 2 and 3

Porphyrin derivatives generally present two fluorescence emission transitions in the excited state (Fig. 3a). The different values for the fluorescence quantum yield (Φ_f) values may be a clue concerning the electronic nature of metal ion in the porphyrin ring (Table 1), and the decrease in the fluorescence can be associated with an increase of non-radiative processes [31, 42]. Only the Zn(II) derivative Zn(3-PtTPyP) showed fluorescence emission peak in DMSO and in DMSO(5%)/Tris–HCl pH 7.4 buffer mixture solution. The Φ_f values of metalloporphyrin derivatives are smaller than the emission quantum yield of the standard TPP (Table 1), which can be attributed to an increase unpaired d electron in metallocompounds and the presence of Pt(II) complexes (heavy atom effect). In the case of the Zn(II) metal ion, the macrocycle resulted in an increase of the emission intensity and quantum yield, because of the full-shell d¹⁰ electron configuration. The different values for the quantum yields may be a clue concerning the electronic coupling between the [Pt(bpy)Cl]⁺ units and the porphyrin ring related to the presence metal ion center.

Fig. 3.

Steady-state fluorescence emission spectra of Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP) metalloporphyrins, in a DMSO and b DMSO(5%)/Tris–HCl pH 7.4 buffer mixture solutions

Time-resolved fluorescence was measured by exciting the porphyrins at excitation in 441 nm (NanoLED source). The fluorescence lifetimes (τ_f; Table 1) were obtained through a mono-exponential method, and the best fits are presented in the Supplementary information section (Figures S1–S2). In this way, it was observed that both metalloderivatives, in both solutions, presented the lower fluorescence quantum yield and the shorter fluorescence lifetime, in agreement to the literature [31]. These results can be probably attributed to the lower fluorescence emission values, associated with an increase of non-radiative processes (Table 1).

Aggregation and solution stability studies

The evaluation of the aggregation behavior of metalloporphyrins in solution was studied by UV–Vis analysis in DMSO and DMSO(5%)/Tris–HCl pH 7.4 buffer mixture. In all cases, no significant shift at the maximum absorbance wavelength was observed. A linear increase was observed in the UV–Vis absorption spectrum as a function of the variation of the concentration from 1.0 to 30 μM (see Supplementary information section; Figures S3–S10). The low tendency of aggregation can be attributed to the positive charges in the porphyrin periphery, following the stereochemistry of the molecule (complexes coordinated to the 3-N positions of pyridine).

In terms of stability in solution, the derivatives studied here were monitored by absorption spectroscopy for a period of 7 days in DMSO solution. The UV–Vis spectra of metalloporphyrins are listed in the Supplementary information section (Figures S11–S14). Analyzing the data obtained, it is possible to say that these compounds are stable for a long period of time and can be easily used in photobiology assays.

Finally, the metalloderivatives were analyzed for a period of 48 h for the presence of water in the DMSO solution, to see the possibility of changes in the UV–Vis electronic spectra. Thus, it is possible to notice that there was no significant change in the absorption spectra, leading us to believe that there was no possible Cl-ligand exchange in the platinum(II) coordination sphere and in the metal center ion in solution. The compiled UV–Vis spectra are listed in the Supplementary information section (Figures S15–S18).

Electrochemical properties

The cyclic voltammograms of porphyrins Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP) in anhydrous DMF are shown in Table 2, and CV analyses are listed in the Supplementary information section (Figure S19). For metalloporphyrins in oxidation state 2 + , in this case, Zn(3-PtTPyP) and Cu(3-PtTPyP), two oxidation and reduction processes (both irreversible) were observed (Table 2). In the case of derivative Co(3-PtTPyP), one oxidation/reduction processes (irreversible) were observed (Table 2). In the positive region, the two redox processes observed in all metalloporphyrins are probably assigned to the monoelectronic oxidations of the porphyrin ring, generating species of a π-cation radical and a di-cation, respectively (Table 2). Moving to the negative region, the observed reduction processes can be assigned to the monoelectronic reductions of the macrocycle ring, by formation of a π-radical anion and a di-anionic porphyrin species (Table 2). In none of the cases, it was possible to determine the oxidation or reduction metal ion porphyrin center, probably due to the restricted window of the solvent used.

Table 2.

Electrochemical data of metalloporphyrins in dry DMF solution (E versus SHE)

	Redox potentials				Energy
Porphyrin	Ered1 (V)	Ered2 (V)	Eox1 (V)	Eox2 (V)	EHOMO (eV)d	ELUMO (eV)e	ΔE (eV)f
Zn(3-PtTPyP)	− 1.12a	− 0.71c	+ 0.80b	+ 1.10b	− 5.20	− 3.69	1.51
Cu(3-PtTPyP)	− 1.02c	− 0.67c	+ 0.76b	+ 0.90b	− 5.16	− 3.38	1.78
Co(3-PtTPyP)	− 1.38a	–	+ 0.90b	–	− 5.30	− 3.02	2.28
Mn(3-PtTPyP)	− 1.32a	− 0.71a	+ 0.81b	+ 1.13b	− 5.21	− 3.08	2.13

^aE_pc = cathodic peak

^bE_pa = anodic peak

^cE_1/2 = E_pa + E_pc/2

^dE_HOMO =  − [4.4 + E_ox1 (vs. SHE)]

^eE_LUMO =  − [4.4 + E_red2 (vs. SHE)]

^fΔE = E_LUMO − E_HOMO

Photobiological parameters

In the photostability experiments, metalloderivatives remained photo-stable at least over a period of 30 min under white-light LED irradiation (irradiance of 50 mW cm⁻² and a total light dosage of 90 J cm⁻²), and the results are presented in the Supplementary information section (Figures S20–S21).

The log P_OW was measured, and the values found for the tetra-cationic metalloderivatives are in accordance with the literature. When compared to the free-base 3-PtTPyP porphyrin [14, 29], metalloporphyrins Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP) showing a more hydrophilic character (Table 3). These results can be attributed to the presence of coordinated ions in the porphyrin ring and a possible interaction of water molecules in the coordination sphere of the metallic center.

Table 3.

Photobiological data of studied metalloporphyrins

	3-PtTPyP*	Zn(3-PtTPyP)	Cu(3-PtTPyP)	Co(3-PtTPyP)	Mn(3-PtTPyP)
log POWa	+ 0.244	− 0.380	+ 0.104	− 0.420	− 1.50
kpo (s−1)b	1.14 × 10−3	1.23 × 10−3	0.26 × 10−3	0.37 × 10−3	0.17 × 10−3
ΦΔ (%)b	63.0	68.0	14.0	20.0	9.0
kSO (M−1 s−1)c	3.12 × 10−2	3.48 × 10−3	1.71 × 10−3	3.21 × 10−3	3.68 × 10−3

*By references [14, 28, 29]

^aIn octanol/water mixture solution

^bIn DMSO solution

^cSuperoxide formation constant, by NBT reduction assay

For singlet oxygen quantum yields (Φ_Δ), the metalloporphyrins Zn(3-PtTPyP), Cu(3-PtTPyP), Co(3-PtTPyP), and Mn(3-PtTPyP) are capable of producing ¹O₂ species, mainly the Zn(II) derivative (Table 3). Compared to the standard free-base 3-PtTPyP, the presence of metal center ion can interfere in the ¹O₂ generation, probably by differences in the intersystem crossing taxes (spin–orbit coupling) and the half-filled d orbitals of the Cu(II), Co(III) and Mn(III) ions. All UV–Vis spectra are presented in the Supplementary information section (Figures S22–S25).

The capacity of studied to generate superoxide species (O₂^·−) by type I pathway was investigated in DMSO solution. For this application, solutions of metalloderivatives containing NBT and the reducing agent NADH were irradiated with white-light source under aerobic conditions. The reaction of NBT with O₂^·− species produced diformazan molecule that can be monitored following the absorption band of this product, which is centered around 560 nm (Supplementary information section, Figure S26). The superoxide generation constant (k_SO) by the NBT reduction assays is shown in Table 3. These results indicate that tetra-cationic meta-isomer of peripheral platinum(II) porphyrins, after white-light irradiation conditions, can form O₂^·− in the presence of an electron donor agent (NADH), in comparison, for example, to the free-base tetra-platinated porphyrin (Table 3). These results lead us to believe that these metalloderivatives can, in addition to generating ¹O₂, produce radical species via a Type I mechanism.

Mycobacteria photoinactivation assays

Antimicrobial treatment triggered by light sources, the so-called antimicrobial photodynamic therapy (aPDT), presents itself as a powerful and reversible tool to eradicate microorganisms directly by remote control of light irradiation [7, 43]. Porphyrins and their derivatives are among the most exploited photosensitizers in aPDT [1, 43], and their excellent prospects for development in pharmaceutical synthesis favor modifications in their structure, thus allowing the control of their physicochemical and pharmacological properties of this class of molecules [1, 3]. In this way, these molecules are at the center of medical applications, with an increasing focus on their ability to generate ROS for PDT, along with bioimaging and biosensing properties [44].

In this context, metalloporphyrin derivatives have emerged as a new class with significant properties when applied to aPDT. Considering the liposolubility of these molecules, the insertion of a central metallic ion in the porphyrin nucleus can alter its structure, absorption, fluorescence, and even solubility properties [45–47]. Its ability to coordinate with the most varied transition metal ions is due to the fact that the nitrogen atoms of the pyrrole rings can generate complexes with more or less rigid square-quadratic geometry [1, 48]. However, the choice of metal ion for structure coordination is extremely important for biological applications [49, 50]. Our study, for example, shows greater antimycobacterial activity when complexes are formed with Zn(II) ions, which are full d-orbitals, which is one of the crucial factors to favor the ROS formation. However, metalloporphyrins containing Cu(II), Co(III), or Mn(III) ions did not show significant activity.

Susceptibility test

The photoactive action shown by the metalloporphyrins against RGM strains is described in Table 4. It was possible to observe that in the absence of light (dark conditions), the metalloporphyrins showed similar activity against the microorganisms tested. However, in the irradiation conditions with white-light LED system (irradiance at 50 mW cm⁻² and a total light dosage of 270 J cm⁻² at 90 min), the metalloporphyrins had better activity in relation to the dark assays, with emphasis on porphyrin contain Zn(II) ions Zn(3-PtTPyP), which was more active when compared to the other metallocompounds tested.

Table 4.

MIC values (μg/mL; μM) of metalloporphyrins against RGM strains under dark and white-light conditions (irradiance at 50 mW cm⁻² and a total light dosage of 270 J cm⁻² at 90 min)

	Dark conditions
	MIC values (µg/mL; µM)
Microrganism	3-PtTPyP*	Zn(3-PtTPyP)	Cu(3-PtTPyP)	Co(3-PtTPyP)	Mn(3-PtTPyP)
M. abscessus	93.75; 38.8	93.75; 37.8	93.75; 37.9	93.75; 37.0	46.87; 18.5
M. massiliense	93.75; 38.8	93.75; 37.8	93.75; 37.9	93.75; 37.0	46.87; 18.5
M. fortuitum	93.75; 38.8	93.75; 37.8	93.75; 37.9	93.75; 37.0	93.75; 37.1
M. smegmatis	93.75; 38.8	46.87; 18.9	93.75; 37.9	93.75; 37.0	23.43; 9.27
	White-light conditions
	MIC values (µg/mL; µM)
Microrganism	3-PtTPyP*	Zn(3-PtTPyP)	Cu(3-PtTPyP)	Co(3-PtTPyP)	Mn(3-PtTPyP)
M. abscessus	0.73; 0.30	46.87; 18.9	93.75; 37.9	93.75; 37.0	46.87; 18.5
M. massiliense	1.46; 0.60	23.43; 9.45	93.75; 37.9	93.75; 37.0	46.87; 18.5
M. fortuitum	0.36; 0.15	23.43; 9.45	46.87; 18.9	46.87; 18.5	46.87; 18.5
M. smegmatis	0.73; 0.30	1.46; 0.59	46.87; 18.9	46.87; 18.5	11.71; 4.63

*Free-base derivative, in reference [28]

The photodynamic efficiency resulting from the coordination of porphyrin derivatives with Zn(II) ions has already been reported against several types of cells, including microorganisms [18, 51–54]. Skwor et al. compare a sequence of metalloporphyrins and demonstrate pronounced bactericidal activity when the compound is a Zn(II) metalloporphyrin. However, as in our study, the results with Cu(II) metalloporphyrins were not significant. This study also associates the weak photobiological activity of the Cu(II) derivative with its low capacity to produce singlet oxygen [26]. Socoteanu et al. also point out that tetrapyrrole derivatives of Zn(II) are more efficient and safe candidates as PS for PDT in tumors. In this study, compounds containing Zn(II) proved to be more suitable for potential biomedical use than, for example, Cu(II) porphyrins [53].

Likewise, N-alkylpyridylporphyrin derivatives when coordinated with Mn(III) are widely known as superoxide dismutase mimetics [55]; however, the same group of porphyrins, after shifting the metal center to Zn(II) ions, shows potential as PS, now being extensively studied in aPDT [18]. The literature also reports a study involving RGM strains and water-soluble metalloporphyrins [27]. The effect of the metal atom on the photophysical properties of these porphyrins was compared with the freebase porphyrin. In this study, Zn(II) metalloporphyrin was able to eradicate the growth of most strains with only one white light irradiation session; however, metalloporphyrins containing Cu(II), Ni(II), Mn(III), and Fe(III) did not show positive results for aPDT, which was attributed to its low capacity to generate ROS.

ROS scavenger assays

Tests were performed with ROS scavengers to determine which reactive oxygen species participated in the photo-inactivation process, and Zn(3-PtTPyP) porphyrin was used with the best result. The MIC values in the absence and presence of the selected ROS scavengers are shown in Table 5.

Table 5.

MIC values (in µg/mL; µM) for Zn(3-PtTPyP) porphyrin in the presence of ROS scavengers (ascorbic acid (AA); dimethyl sulfoxide (DMSO); terc-butanol (t-BuOH); and N-acetylcysteine (NAC)), under white-light conditions at 90 min (50 mW cm⁻² and 270 J cm⁻²)

		ROS scavengers
Microorganism	Absence	AA	DMSO	t-BuOH	NAC
M. fortuitum	46.87; 18.9	23.43; 9.45	23.43; 9.45	23.43; 9.45	23.43; 9.45
M. abscessus	23.43; 9.45	46.87; 18.9	46.87; 18.9	46.87; 18.9	46.87; 18.9
M. massiliense	23.43; 9.45	23.43; 9.45	23.43; 9.45	23.43; 9.45	23.43; 9.45
M. smegmatis	1.46; 0.59	1.46; 0.59	1.46; 0.59	1.46; 0.59	1.46; 0.59

In general, in the presence of the ascorbic acid (¹O₂ scavenger), DMSO (O₂^·− scavenger), terc-BuOH (·OH scavenger), and NAC (·OOH scavenger), the values found for MIC were close or similar to those presented for the photo-inactivation of all tested microorganisms in the absence of the scavenger species (Table 5). Many of the expected results of this work can be attributed to the potential of ROS generation, which is possible in a simultaneous oxidation mechanism (Type I and II), but we do not rule out any hydrolytic process that may be occurring. It is known that porphyrins absorb a significant portion of the visible spectrum and, in the presence of oxygen molecule, produce ROS that may contribute to photodynamic action.

Although Zn(II) porphyrin Zn(3-PtTPyP) is a good singlet oxygen generator, the observed results are less encouraging when compared with free-base porphyrin 3-PtTPyP, related by Rossi and co-workers [28]. In this way, we can say that there is influence of the metallic center in the photodynamic activity.

Atomic force microscopy (AFM) analysis

The effect of Zn(II) metalloporphyrin Zn(3-PtTPyP) treatment on the morphology, adhesive forces, and electrical properties of the mycobacterium M. smegmatis was evaluated using AFM analyses. Figure 4 shows representative topographic maps displaying the morphology of mycobacteria exposed to different conditions: dark, light, and treatment with metalloporphyrin under both dark and white-light exposure. As seen in Fig. 4a, AFM topography of untreated mycobacterium under dark condition displays the common rod shape morphology with the outer membrane well preserved. Again, mycobacterium maintains its common morphology even after exposure to white-light irradiation (Fig. 4b). Subtle topographical modifications can be seen after the treatment with Zn(II) metalloporphyrin under dark conditions, with the bacillus undergoing wilt at its center and small protrusions at the ends (Fig. 4c). On the other hand, the treatment with the porphyrin under white-light irradiation is capable provoke extensive damages to the mycobacterium, wrinkling the whole surface while increasing its roughness (Fig. 4d).

Fig. 4.

Representative topographic images of M. smegmatis when submitted to four processing: a dark condition, b white-light condition at 90 min (50 mW cm⁻² and 270 J cm⁻²), c Zn(3-PtTPyP) treatment in dark condition, and d Zn(3-PtTPyP) treatment in white-light condition 90 min (50 mW cm⁻² and 270 J cm⁻²), respectively

Table 6 presents the AFM adhesion force values for the mycobacteria subjected to same conditions mentioned above. Adhesion data were obtained in real time from force-distance curves in nanomechanical mode (PinPoint). In dark conditions and without the porphyrin treatment, the mycobacteria have adhesive forces of 0.83 μN, but decreasing to 0.41 μN under white-light irradiation or to 0.38 µN after the porphyrin treatment (under dark conditions). The treatment with porphyrin under white-light irradiation (90 min; 50 mW cm⁻² and 270 J cm⁻²) is capable to reduce adhesive forces down to 0.29 μN, indicating that both bacterial growth and anchoring is reduced after the treatment. Table 6 also presents the average values of width and length of the bacteria submitted to the different conditions studied. A significant decrease in both the length and width values of the mycobacteria is observed after treatment with Zn(II) metalloporphyrin under white-light conditions.

Table 6.

Adhesion force, length and width of the M. smegmatis before and after the Zn(3-PtTPyP) treatment in dark and white-light condition (90 min; 50 mW cm⁻² and 270 J cm⁻²). Standard deviations are results from five measurements

Condition	Adhesion force (μN)	Length (μm)	Width (μ m)
Dark condition	0.83 ± 0.04	2.45 ± 0.23	0.96 ± 0.12
Light condition	0.41 ± 0.02	1.57 ± 0.15	0.78 ± 0.01
Dark condition + porphyrin	0.38 ± 0.01	2.26 ± 0.15	1.00 ± 0.03
Light condition + porphyrin	0.29 ± 0.01	1.47 ± 0.14	0.5 ± 0.03

Figure 5 presents another set of topography maps with the correspondent electrical images for the mycobacterium Mycolicibacterium smegmatis submitted to different conditions. 2D AFM topography images have the same features that were observed in Fig. 4, but the electric force maps present important additional information. For both dark and light conditions, the mycobacterium strain that was not submitted to the porphyrin treatment has positive charges surrounding the membrane-substrate interface. Electrostatic charges play an important role in the electrostatic adhesion of bacteria to the substrate surface, but the mycobacteria under the Zn(3-PtTPyP) treatment in both dark and white-light conditions loses most of these charges. In fact, for the porphyrin treatment, the electrostatic potential at the membrane-substrate interface has very low contrast. Additionally, the electrostatic map reveals the cavities formed in the mycobacterial membrane under porphyrin and white-light irradiation, evidenced by the darker points in the EFM images.

Fig. 5.

Topographic (top) and electric force (bottom) maps of Mycolicibacterium smegmatis when submitted to dark condition, light condition, and porphyrin treatment in dark and white-light conditions (90 min; 50 mW cm⁻² and 270 J cm⁻²)

It is known that the molecular mechanisms involved in bacterial death induced by aPDT are not fully elucidated. Since photogenerated reactive species have short lifetimes, damage is limited to proximity to the PS. However, studies describe that oxidative damage to DNA and plasma membrane components are the main targets responsible for the inactivation of bacteria. [11, 56]. This damage promoted to the bacterial cell structure can be better understood by our study through the data obtained by the AFM analysis. The damage caused by metalloporphyrins to the bacterial structure is evidenced by changes in morphological, adhesion force and electrical properties of mycobacteria. The morphological images indicate the formation of ruptures caused by the treatment, mainly in light conditions. These ruptures show the inactivation of the mycobacteria after the photodynamic treatment.

The nanomechanical properties of the mycobacteria that were analyzed using the AFM technique reveal significant changes after the photodynamic treatment. Mycobacterial membrane adhesion force decreased after Zn(II) porphyrin application, mainly under white light irradiation conditions. In fact, several microbial pathogens may depend on adhesion to biomaterials and host factors to initiate an infection, given that such microbes have the ability to adhere to biotic and abiotic media, such as catheters and host cells [57]. In addition, several microbes prefer to associate in communities on surfaces, forming biofilms, which gives them greater protection from various environmental stresses, such as desiccation or the application of antibiotics. Thus, the decrease in the bacteria’s adhesion force, and consequent inactivation, after photo-treatment with porphyrin may be important in different environments, such as hospitals and industries [58, 59].

The attachment of bacteria to different surfaces also depends on other contributing factors, such as van der Waals forces, hydrophilicity, and electrostatic interactions [60]. The electric force maps of the bacteria under the tested conditions provide valuable information about the electrical adhesion before and after the photodynamic treatment. The electrical force maps show that the mycobacterial membrane has positive charges responsible for the adhesion on the substrate. In dark conditions and without the addition of porphyrin, the interface between the bacteria and the substrate presents a positive electrical potential, but after the aPDT, the electrostatic component decreases considerably. The treatment also leads to the formation of cavities in the membrane that were not apparent to the AFM topography images but are easily observed in the EFM maps.

The presented results demonstrate that the treatment with Zn(II) metalloporphyrin in inactive light conditions prevents the mycobacteria from anchoring and growing on surfaces, preventing the formation of biofilms, for example. Other works have already been published by this research group [27, 28, 61] using the AFM technique to evaluate the changes in the bacterial membrane caused by treatment with porphyrins. Accordingly, such works also revealed the formation of cavities in the membrane of microorganisms and changes in the width and length of bacteria after treatment. In fact, the AFM technique has been very versatile to evaluate the physicochemical properties of microorganisms, in order to obtain useful information about photo-inactivation and prevention of biofilm formation on the most diverse surfaces.

Conclusions

This work demonstrates that tetra-cationic porphyrins with peripheral [Pt(bpy)Cl]⁺ complexes at meta N-pyridyl positions may be considered interesting PS for the photo-inactivation of mycobacteria. In the case of zinc(II) porphyrin, the ROS production (mainly singlet oxygen), photostability, lower aggregation in solution, and higher white-light dosage effects on the mycobacteria are most likely the factors underlying the satisfactory performance of tetra-cationic porphyrins against mycobacteria. By AFM analysis, we detected again that, due to the presence of ROS, damage to the membrane of these strains may occur. The results of this study confirm the potential of tetra-cationic porphyrin derivatives with coordination chemistry for antimicrobial photodynamic applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

MMAC and BAI idealized the work. IT conducted the porphyrin synthesis. GGR conducted the microbiological assays. KSM and TALB conducted the AFM experiments. GGR, BAI, MMAC, and TALB wrote, revised, and corrected the manuscript.

Funding

This study was financed by CNPq, CAPES, and FAPERGS. Bernardo A. Iglesias thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq–Brazil; Universal process 409150/2018–5 and PG-2021 grant process 305458/2021–3) and FAPERGS (PQ Gaúcho 21/2551–0002114-4), and Marli Matiko Anraku de Campos thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq–Brazil; Grants 404541/2016–0). Thiago A. L. Burgo thanks MCTIC/CNPq (465452/2014–0), FAPESP (2014/50906–9), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES)-Finance Code 001 through INCT/INOMAT (National Institute for Complex Functional Materials) and MCT/Finep/CT-Infra 02/2010.

Declarations

Ethics approval

This article does not contain any studies with human participants or animals performed by any authors.

Conflict of interest

The authors declare no competing interests.