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. Author manuscript; available in PMC: 2019 Jan 1.
Published in final edited form as: J Photochem Photobiol B. 2017 Oct 31;178:277–286. doi: 10.1016/j.jphotobiol.2017.10.036

Potentiation by potassium iodide reveals that the anionic porphyrin TPPS4 is a surprisingly effective photosensitizer for antimicrobial photodynamic inactivation

Liyi Huang 1,2,3, Ahmed El-Hussein 2,4, Weijun Xuan 2,5, Michael R Hamblin 2,3,6,*
PMCID: PMC5771845  NIHMSID: NIHMS922267  PMID: 29172135

Abstract

We recently reported that addition of the non-toxic salt, potassium iodide can potentiate antimicrobial photodynamic inactivation of a broad-spectrum of microorganisms, producing many extra logs of killing. If the photosensitizer (PS) can bind to the microbial cells, then delivering light in the presence of KI produces short-lived reactive iodine species, while if the cells are added after light the killing is caused by molecular iodine produced as a result of singlet oxygen-mediated oxidation of iodide. In an attempt to show the importance of PS-bacterial binding, we compared two charged porphyrins, TPPS4 (thought to be anionic and not able to bind to Gram-negative bacteria) and TMPyP4 (considered cationic and well able to bind to bacteria). As expected TPPS4 + light did not kill Gram-negative Escherichia coli, but surprisingly when 100 mM KI was added, it was highly effective (eradication at 200 nM + 10 J/cm2 of 415 nm light). TPPS4 was more effective than TMPyP4 in eradicating the Gram-positive bacteria, methicillin-resistant Staphylococcus aureus and the fungal yeast Candida albicans (regardless of KI). TPPS4 was also highly active against E. coli after a centrifugation step when KI was added, suggesting that the supposedly anionic porphyrin bound to bacteria and Candida. This was confirmed by uptake experiments. We compared the phthalocyanine tetrasulfonate derivative (ClAlPCS4), which did not bind to bacteria or allow KI-mediated killing of E. coli after a spin, suggesting it was truly anionic. We conclude that TPPS4 behaves as if it has some cationic character in the presence of bacteria, which may be related to its delivery from suppliers in the form of a dihydrochloride salt.

Keywords: antimicrobial photodynamic inactivation, TPPS4, TMPyP4, ClAlPCS4, potassium iodide, cationic porphyrin, anionic porphyrin

Introduction

Antimicrobial photodynamic inactivation (aPDI) is rapidly emerging as a viable alternative strategy to kill pathogenic microorganisms in this modern age of antibiotic resistance [1]. aPDI is based on the use of non-toxic dye molecules called photosensitzers (PS) [2] or “photoantimicrobials” [3] which generate toxic levels of reactive oxygen species only when excited by visible light of the correct wavelength. Selectivity for killing microbial cells while preserving host mammalian cells is provided by the following: (i) choosing a PS that specifically binds to bacteria or fungi etc. and not to mammalian cells; (ii) selecting a short drug-light interval as PS bind rapidly to bacteria but only slowly to host cells; (iii) employing topical or local delivery of the PS; and (iv) confining the illumination spot to the area where the infection is located. The therapeutic use of aPDI to treat infections is called antimicrobial photodynamic therapy (aPDT) and has photochemical mechanisms in common with the better known PDT used to treat cancer [4] or retinal diseases [5].

It has been shown in numerous studies that aPDI is largely independent of the antibiotic resistance status of the microbial cells [6], and moreover, cannot itself induce the development of resistance even after 20 cycles of partial killing and regrowth [7]. It was discovered about 25 years ago that Gram-negative bacteria are relatively resistant to the photodynamic action of many PS (especially those used in cancer therapy) while Gram-positive bacteria and fungi are efficiently killed [8]. It was found that PS with a pronounced cationic charge can be very efficient at killing Gram-negative species [9], and that this preferential effect is partly due to the fact that cationic PS bind well to the anionic Gram-negative bacterial cells, and partly due to the so-called “self-promoted uptake pathway” described by RW Hancock [10] by which cationic (but not anionic) PS penetrate to the interior of the bacterial cells [11].

We have recently discovered that aPDI can be potentiated by addition of several different inorganic salts. We first showed this potentiation using sodium azide combined with phenothiazinium salt PS [12, 13]. Because azide is well-known to quench singlet oxygen, we showed that this paradoxical potentiation was a type I photochemical process, probably involving a photoinduced 1-electron transfer reaction from azide anion to the triplet PS form azide radicals and PS radical anions. We then showed that sodium thiocyanate could potentiate aPDI mediated by methylene blue (MB), and in this case the mechanism was shown to be mediated by singlet oxygen with formation of the sulfur trioxide radical anion [14]. We then went on to show that potassium iodide (KI) could also potentiate aPDI using MB, and originally we assumed that the mechanism involved an electron-transfer reaction (similar to that found with azide) to form iodine radicals and molecular iodine [15]. This hypothesis was corroborated by the findings that KI could potentiate aPDI mediated by functionalized fullerenes [16] and the photocatalytic nanoparticles made from titanium dioxide [17] (both of these compounds are known to be good agents for photoinduced electron transfer). However we then discovered that KI could also potentiate aPDI carried out by PS that are well-known to produce singlet oxygen, such as Photofrin [18] and Rose Bengal [19]. The reaction appears to involve addition of singlet oxygen to form peroxyiodide, which subsequently decomposes into molecular iodine and hydrogen peroxide [18]. The killing is caused by a mixture of extracellular free iodine (I2/I3−), and reactive iodine radicals (I•/I•2) depending on the degree of binding of the PS to the microbial cells. This is presumably because the diffusion distance of radicals is very short and they are much more toxic if generated very close to the target cells. On the other hand free iodine is stable and can kill microbial cells when generated in solution. In the case of titanium dioxide there is evidence that hypoiodite (semi-stable) was also formed and could kill microbial cells for some time before it decomposed.

In order to gain information about the importance of binding of the PS to the microbial cells, and the subsequent interaction with iodide anion, we decided to investigate a pair of porphyrins. These were the tetracationic 5,10,15,20-tetrakis(N-methyl-4-pyridinium)porphyrin tetratosylate (TMPyP4) and the tetraanionic 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin dihydrochloride (TPPS4) (see Figure 1). We supposed that TMPyP4 would bind to all classes of microbial cells (particularly bacteria), while TPPS4 would not bind well to any classes of microbial cells (with the possible exception of Gram-positive bacteria).

Figure 1.

Figure 1

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Structures of the compounds used as photosensitizers

Materials and Methods

Compounds

Meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TPPS4), meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate, (TMPyP4), chloroaluminum phthalocyanine tetrasulfonate (ClAlPCS4) were purchased from Frontier Scientific (Logan, UT)and potassium iodide (KI) were purchased from Sigma-Aldrich (St. Louis, MO). All PS stock solution (2.5 mM) were prepared in distilled H2O (dH2O) and were stored at 4°C in the dark for no more than 2 weeks prior to use. KI solution was prepared in dH2O as required immediately before experimentation.

Cells and culture conditions

The following microbial strains were used: Gram-positive bacterium: methicillin-resistant Staphylococcus aureus (MRSA) USA 300; Gram-negative bacteria: Escherichia coli (E.coli) K-12 (ATCC 33780), Pseudomonas aeruginosa (P. aeruginosa) ATCC 19660 (Xen 5P), Proteus mirabilis (P. mirabilis) ATCC 51393 (Xen 44), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii) ATCC BAA 747 and E. coli UTI 89, UTI89 was a generous gift from Dr. Patrick Seed’s laboratory, which is a clinical cystitis isolate strain from humans previously described by Mulvey et al [20]. For a fungal yeast: strain CEC 749 of the luciferase-expressing fungal yeast Candida albicans (C. albicans). A colony of bacteria was suspended in 5 mL of brain heart infusion (BHI) broth and grown overnight in a shaker incubator (New Brunswick Scientific, Edison, NJ) at 120 rpm under aerobic conditions at 37 °C. An aliquot of 1 mL from an overnight bacterial suspension was refreshed in fresh BHI for 2–3 h at 37 °C to mid-log phase. Cell concentration was estimated by measuring the optical density (OD) at 600 nm (OD of 0.6 = 108 cells/mL). A colony of C. albicans was suspended in 10 mL of yeast extract-peptone-dextrose (YPD) and grown overnight in a shaker incubator at 30 °C. The C. albicans cell number was assessed with a hemocytometer and was generally between 107 and 108 cells/mL.

aPDI experiments

Three types of aPDI experiments were carried out. The first used cells with a fixed light dose and varied the all PS concentration and with or without a spin. The second type of experiment used cells with a fixed PS concentration and a fixed light dose and varied the KI concentration, and with or without a spin, and when the PS and KI were exposed to 10 J/cm2 of light, then cells were added. The third type of experiment used cells with a fixed PS concentration and KI concentration and varied the light dose.

The initial studies used PDI with suspensions of bacteria (108 cells/mL) or C. albicans (107 cells/mL) incubated in the dark at room temperature with different concentration of TPPS4 or TMPyP4 for 30 min, cells with or without spin (5 min, 4,000 rpm) irradiating with 10J/cm2 of blue light. The light source we used was an Omnilux Clear-U light-emitting diode (LED) array (Photo Therapeutics, Inc., Carlsbad, CA) that emitted blue light at a center wavelength of 415 nm to deliver 10 J/cm2 at an irradiance of 50 mW/cm2 as measured with a power meter (Coherent, Santa Clara, California). The aliquots were serially diluted tenfold in PBS to give dilutions of 10−1 to 10−5 times in addition to the original concentration and 10 μL aliquots of each of the dilutions were streaked horizontally on square BHI agar plates for bacteria or YPD agar plates for Candida. Plates were streaked in triplicate and incubated for 12–18 h at 37 °C (bacteria) or for 24–36 h at 30 °C (Candida) in the dark to allow colony formation. Each experiment was performed at least three times.

Suspensions of bacteria (108 cells/mL) or Candida (107 cells/mL) were incubated in the dark at room temperature for 30 min with 200 nM TPPS4 (for MRSA and C. albicans) or 10 μM TPPS4 (for E. coli), 200 nM TMPyP4 (for MRSA and E. coli) or 2 μM TMPyP4 (for C albicans) and added a range of KI concentrations between 0 and 100 mM in pH 7.4 PBS. For the spin experiment, centrifugation (5 min, 4,000 rpm) of 1mL aliquots was used to remove the excess of TPPS4 or TMPyP4 that was not taken up by the microbial cells, after that we added different concentration of KI. When experiments required it, 200 nM TPPS4 or TMPyP4, 10 μM TPPS4 or 2 μM TMPyP4 plus a range of KI concentrations between 0 and 100 mM in pH 7.4 PBS were exposed to 10J/cm2 blue light (cell free), then was added MRSA and C albicans cells to the light activated 200 nM TPPS4 concentration, while E. coli cells were added to the light activated 10 μM TPPS4 concentration, MRSA and E. coli cells were added to the 200 nM TMPyP4 group, C albican cells were added to the 2 μM TMPyP4 group. After 30 min incubation, the aliquots were serially diluted as before. Each experiment was performed at least three times.

Suspensions of bacteria (108 cells/mL) or C. albicans (107 cells/mL) were irradiated with different fluences of blue light (0, 0.2, 0.4, 0.8, 1J/cm2 for bacteria; 0, 2, 4, 8, 12, 16, 20J/cm2 for Candida) with different concentration of TPPS4 (200 nM for all cells), TMPyP4 (200 nM for MRSA and E. coli cells, 2 μM for C. albicans), together or without 100 mM KI. The aliquots were serially diluted tenfold in PBS as before. Each experiment was performed at least three times.

For the ClAlPCS4 compound, cells were incubated with varying concentrations of ClAlPCS4 and then centrifuged or not before being exposed to 10 J/cm2 of 670 nm light. A red-light source consisting of a white lamp with a band-pass filter probe (660 ±15 nm, Lumacare, Newport Beach, CA, USA) was used with a spot size of 2.5 cm diameter a power density of 100 mW/cm2. The aliquots were serially diluted tenfold in PBS as before. Each experiment was performed at least three times.

When experiments required it, cells were incubated with stated concentrations of ClAlPCS4 and varying concentrations of KI and exposed to 10 J/cm2 of 670 nm light using “In”, “Spin” and “After” formats as described before, the aliquots were serially diluted as before. Each experiment was performed at least three times.

A control group of cells treated with light alone (no TPPS4, TMPyP4 or ClAlPCS4 added) showed the same number of CFU as absolute control (data not shown). Survival fractions were routinely expressed as ratios of CFU of microbial cells treated with light and TPPS4, TMPyP4 or ClAlPCS4 (or TPPS4, TMPyP4 or ClAlPCS4 in the absence of light) to CFUs of microbes treated with neither.

Uptake experiments

Suspensions of microbial cells (108/ml) were incubated in the dark at room temperature with concentrations in the range 0.5–2.5 μM of all compounds for 30 minutes. The cell suspensions were centrifuged, the supernatant was aspirated and bacteria were washed with PBS and centrifuged. Finally, the cell pellet was dissolved in 200 μl of SOLVABLE solution (PerkinElmer Inc., Waltham, MA) at 37°C in an incubator for 2 hours to give a homogeneous solution. The fluorescence of the cell lysates was measured using appropriate excitation and emission wavelength for each compound with a fluorescence plate reader (Molecular Devices, Sunnyvale CA). Following the fluorescence measurement, the total protein concentration was determined with a bicinchoninic acid protein assay kit (Sigma) using bovine serum albumin to prepare protein calibration curves. Individual fluorescence calibration curves were prepared for known concentrations of each of all compounds.

Confocal microscopy

C. albicans cells (107 cells/ml) were incubated with 10 μM TPPS4 or 30 μM TMPyP4 in PBS (pH 7.4) at room temperature, after 30 min cells were washed in PBS. Fluorescent probes were added to stain the cellular organelles, which were MitoTracker Green to stain mitochondria (excitation 490-nm and emission 516-nm, 50 nM), and Hoechst trihydrochloride trihydrate to stain nuclei (excitation 350-nm, emission 510-nm, 4 ug/ml, 40 μL). All probes were incubated for 45 minutes at room temperature. Cells were washed in PBS, pelleted, and resuspended in 200 μl PBS, and 10 μL was placed on a microscope slide and covered with a coverslip. An Olympus Fluoview 1000-MPE multiphoton confocal microscope (Olympus Corporation, Tokyo, Japan) was used to image the cells at a resolution of 1,024 × 1,024 pixels with a 100 × 1.4-numerical aperture (NA) oil immersion lens. Images were acquired using Fluoview 10-ASW software (version 2.0; Olympus Corporation, Tokyo, Japan).

Results

aPDI with TMPyP4 and TPPS4

We initially compared the microbial killing mediated by these two porphyrins both in the dark and when excited by 10 J/cm2 of 405 nm light. The data are shown in Figure 2 for the three representative microorganisms: Figure 2A for Gram-positive bacteria MRSA; Figure 2B for Gram-negative bacteria E. coli; Figure 2C for fungus C. albicans. For MRSA we see that TPPS4 is significantly more powerful than TMPyP4, giving 1–2 logs more killing at concentrations of 100–200 nM, until both compounds produced eradication at 1 μM. There was no detectable dark toxicity. For E. coli we see that there was no effect of TPPS4 either in the light (only about 30% killing) or in the dark at concentrations up to 10μM. Even at 100 μM there was less than 1 log of aPDI killing (data not shown). For TMPyP4 and E. coli we see that eradication was achieved at 1μM and there was a distinct degree of dark toxicity (about 1 log) at concentrations up to 10μM. For C. albicans both porphyrins were effective in producing eradication. For TPPS4 this occurred at 1μM while for TMPyP4 eradication did not occur until 10 μM was reached.

Figure 2. Effect of porphyrin concentration.

Figure 2

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Concentration dependent killing of microbial cells incubated for 30 min with TPPS4 or TMPyP4 and exposed (light) or not exposed (dark) to 10 J/cm2 of 405 nm light. (A) Gram-positive MRSA; (B) Gram-negative E. coli; (C) Fungal yeast C. albicans.

To gain information on the binding of the porphyrins to the microbial cells we compared aPDI killing with (spin) and without (no spin) an intermediate centrifugation step after the iuncubation and before the light. The data are shown in Figure 3. For MRSA (Fig 3A) we can see that centrifugation makes a significant and roughly equivalent difference to the aPDI killing mediated by each porphyrin, but that even with a centrifugation step eradication was still achieved if about 3–5 times the concentration of porphyrin was used. For E. coli (Fig 3B) we see that TMPyP4 was less effective (needing ten times higher concentration to produce eradication) after a spin. As expected there was no effect with TPPS4. For C. albicans (Fig 3C) both porphyrins were significantly less effective after a spin, needing about ten times higher concentrations to produce eradication.

Figure 3. Comparison of spin and no spin.

Figure 3

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Concentration dependent killing of microbial cells incubated for 30 min with TPPS4 or TMPyP4 and centrifuged (spin) or not (no spin) before being exposed to 10 J/cm2 of 405 nm light. (A) Gram-positive MRSA; (B) Gram-negative E. coli; (C) Fungal yeast C. albicans.

The fact that TPPS4 was significantly more effective than TMPyP4 in killing both MRSA and C. albicans was surprising, as we had initially expected exactly the opposite result. On the other hand, the ineffectiveness of TPPS4 in killing E. coli was exactly what we expected.

Potentiation of aPDI with KI

In Figure 4 we show the effect of adding increasing concentrations of KI to the different aPDI experiments. We used three different experimental set-ups. Firstly the set-up with all three ingredients (porphyrin, KI, light) present with the cells at the same time. This was called “in” in the graphs and was designed to measure killing produced both by short-lived reactive iodine species, and by stable free iodine, (in addition to the killing produced by ROS alone shown at KI = zero mM). Secondly we have “after “where the cells were not added to the illuminated mixture of porphyrin and KI until after the light had been switched off. This was designed to measure killing by the stable free iodine species (and conceivably the semi-stable hypoiodite). Thirdly we have the situation called “spin” where the cells are incubated with the porphyrin, centrifuged, resuspended and then KI is added, followed immediately afterwards by light delivery. This was designed to maximize the chance of seeing killing by reactive iodine species that are only effective when produced very close to the microbial cells such as when the PS is bound to the outside of the cells. We varied the KI concentration while keeping the porphyrin concentration constant (specifically chosen for each type of cell) and the light fluence constant at 10J/cm2, since we have previously shown that production of reasonable (bactericidal) quantities of free iodine needs a fairly high KI concentration (10, 50 or even 100 mM). The data are shown in Figure 4.

Figure 4. Effect of KI concentration.

Figure 4

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Microbial cells incubated for 30 min with stated concentration of TPPS4 or TMPyP4. “In” means cells, porphyrin and KI were added together followed by exposure to 10 J/cm2 of 405 nm light. “Spin” means cells and porphyrin were incubated, then centrifuged, KI was added, followed by exposure to 10 J/cm2 of 405 nm light. “After’ means pophyrin and KI were exposed to 10 J/cm2 of 405 nm light, then cells were added. (A) Gram-positive MRSA with 200 nM TPPS4; (B) Gram-negative E. coli with 10μM TPPS4; (C) Fungal yeast C. albicans with 200 nM TPPS4; (D) Gram-positive MRSA with 200 nM TMPyP4; (B) Gram-negative E. coli with 200 nM TMPyP4; (C) Fungal yeast C. albicans with 2 μM TMPyP4.

For MRSA with TPPS4 (Fig 4A) there was eradication with 10mM KI (but no effect with lower concentrations) in both “in” and “after” set-ups. For “spin” there was eradication with 50mM KI showing that TPPS4 must have bound to the cells. These data are consistent with the extra killing being caused by free iodine. For MRSA with TMpyP4 (Fig 4D) we see a slightly different picture. Although the basic relationship of the potentiation is similar, there does appear to be more effect of the KI at lower concentrations (0.1 and 1 mM). This data is consistent with some contribution to killing mediated by short-lived reactive iodine species, as they appear to be active at lower concentrations (formed from KI at lower concentrations), while free iodine needs to reach a threshold concentration (only possible at higher KI concentrations) in order to kill microbial cells. In the case of E. coli with TPPS4 (Fig 4B) the three curves are super-imposable. These data are consistent with all the killing being caused by free iodine that reached a microbicidal concentration at 10 mM KI. However what we found surprising was the fact that this killing was also seen after a spin, as we did not expect that TPPS4 would bind to Gram-negative bacteria such as E. coli, which was consistent with its well-known lack of bacterial killing in Gram-negatives. For E. coli and TMPyP4 (Fig 4E) we see potentiation between 10 mM and 100 mM KI (but not at lower KI concentrations), which is consistent with killing being due to free iodine. For C. albicans and TPPS4 (Fig 4C) we see a more gradual effect of increasing the KI concentration especially in the “in” set-up. This is consistent with some of the killing being due to short-lived iodine species. The killing seen with “after” confirms that TPPS4 binds to the fungal cells. For C. albicans and TMPyP4 (Fig 4F) again there was a more gradual increase in killing with increasing KI concentration consistent with the involvement of short-lived species.

Figure 5 shows five different Gram-negative bacterial (E. coli UTI, K pneumoniae, P. mirabils, A. baumannii and P. aeruginosa) species to explore the generality of the surprising observation with TPPS4 and E. coli. In every case the addition of 100 mM KI led to eradication of the bacteria incubated with 10 μM TPPS4 after delivery of 10 J/cm2 405 nm light. In the case of A. baumannii and P. aeruginosa 1 and 2 logs of killing was obtained without any KI, but with the other 3 species no killing at all was seen. After a spin, no killing was obtained for any species without KI, but when 100 mM KI was added eradication was obtained in every case (6 logs of additional killing).

Figure 5. Effect of TPPS4 on five Gram-negative species.

Figure 5

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Cells were incubated with 10 μM TPPS4 for 30 min in presence or not of 100 mM KI, and exposed to 10 J/cm2 405 nm light (no spin). Cells were incubated with 10 μM TPPS4 for 30 min, centrifuges, 100 mM KI was added or not added, and exposed to 10 J/cm2 405 nm light (spin).

One of the parameters it is necessary to vary in aPDI experiments is the light dose (or fluence measured in J/cm2). Hitherto we had used a fixed dose of 10 J/cm2 of 415 nm light, but we wanted to show that delivering more light killed more microbial cells. In Fig 6 we show that increasing the number of J/cm2 of light produces correspondingly more killing. This was true of MRSA with 200 nM porphyrins (TPPS4 and TMPyP4) + 100 mM KI (Figure 6A) and for E. coli again with 200 nM porphyrins and 100 mM KI (Figure 6B).

Figure 6. Effect of light dose (J/cm2).

Figure 6

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Microbial cells were incubated for 30 min with TPPS4 or TMPyP4 at stated concentrations + 100 m M KI and exposed to increasing fluences of 405 nm light. (A) Gram-positive MRSA; (B) Gram-negative E. coli; (C) Fungal yeast C. albicans.

Effects of ClAlPCS4

Since we found unexpected results with TPPS4 and Gram-negative cells, we asked whether there was something special about this particular anionic porphyrin tetrasulfonate, which was different from other tetrapyrrole tetrasulfonates. To explore this question we obtained some ClAlPCS4, that has been previously investigated as a PDT agent [21]. In Figure 7 we show the aPDI experiments repeated with ClAlPCS4. In the aPDI experiments with ClAlPCS4 alone (Fig 7A–C) we see that only MRSA is killed by aPDI using 10J/cm2 of 660 nm light. This provided equal killing regardless of spin or no spin showing that ClAlPCS4 did in fact bind to MRSA. However ClAlPCS4 (eradication at 10 μM) was substantially less effective than TPPS4 (eradication at 1μM). There was no detectable killing with either E. coli or C. albicans. In Figs 7D–F we show the effects of adding KI. For MRSA (Fig 7D) we used a low concentration (200nM) to compare with TPPS4 (Fig 3D). There was eradication with 50 and 100mM KI in “in” and “spin” but not in “after”. As mentioned above we did see eradication with 200 nM TPPS4 in “after”, suggesting that ClAlPCS4 is somewhat less efficient in producing iodine in solution than TPPS4. However the lack of killing seen with “after” suggests that there was a contribution from short-lived iodine species as killing was seen with “in”. For E. coli with ClAlPCS4 at 10 μM there was killing at 10 mM KI in both “in” and “after” but no killing with “spin” even at 100 mM KI (a stark difference between ClAlPCS4 and TPPS4 where we got eradication at 10 mM KI in “spin”). Fig 7F shows the results with C. albicans and 10μM ClAlPCS4. There was eradication with 50 and 100 mM KI in both “in” and “after” but again no killing with “spin”. There was a slight hint that there was more killing with “after” than “in” at 10 mM KI. The data suggest that ClAlPCS4 does not bind to either E. coli or C. albicans, and that for these species the potentiation by KI was solely due to production of free iodine.

Figure 7. aPDI with ClAlPCS4 with and without KI.

Figure 7

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(A–C) Cells were incubated with varying concentrations of ClAlPCS4 and then centrifuged or not before being exposed to 10 J/cm2 of 670 nm light. (A) Gram-positive MRSA; (B) Gram-negative E. coli; (C) Fungal yeast C. albicans. (D–F) Cells were incubated with stated concentrations of ClAlPCS4 and varying concentrations of KI and exposed to 10 J/cm2 of 670 nm light using “In”, “Spin” and “After” formats as described in Figure 4. (D) Gram-positive MRSA; (E) Gram-negative E. coli; (F) Fungal yeast C. albicans.

Uptake

Since one of the remarkable findings of this study was that TPPS4 appeared to bind to Gram-negative bacteria we decided to confirm this by measuring uptake, in other words porphyrin fluorescence converted to nmol of porphyrin remaining bound to the cell pellet (measured as mg protein) after a spin. The data is shown for the 3 different compounds and the 3 different species in Fig 8. In Fig 8A the uptake for MRSA is in the following order TMPyP4 > TPPS4 > ClAlPCS4. For E. coli (Fig 8B) the uptake is lower than for MRSA and is in the order TPPS4 ≈ TMPyP4 > ClAlPCS4. For C. albicans (Fig 8C) the uptake is about the same magnitude as E. coli and is in the order TPPS4 > TMPyP4 > ClAlPCS4.

Figure 8. Cell uptake of the three tetrapyrrole PS.

Figure 8

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Cells were incubated with increasing concentrations of PS, centrifuged, the pellet was dissolved in Solvable and fluorescence and protein measured. Calibration curves were prepared for each PS and each cell type. (A) Gram-positive MRSA; (B) Gram-negative E. coli; (C) Fungal yeast C. albicans.

Confocal Imaging in C. albicans

Since we unexpectedly found that TPPS4 was taken up well into Candida cells, we used confocal microscopy to determine the intracellular localization after co-incubation with fluorescent organelle-specific probes for the nucleus (Hoechst) and the mitochondria (Mitotracker green). Figure 9 shows that TPPS4 localizes in the nucleus with almost perfect overlap with Hoechst (Fig 9E) and no overlap with mitochondria (Fig 9F). Figure 10 shows the experiment repeated with TMPyP4, with almost identical results. TMPyP4 is well known to accumulate in the nucleus of mammalian cells [22], and has been studied as a specific binding agent for G-quadruplex DNA [23]. Therefore it was not surprising to see accumulation in the nucleus of Candida.

Figure 9. Confocal micrographs of C. albicans incubated with TPPS4.

Figure 9

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(A) Bright field; (B) Hoechst; (C) TPPS4; (D) Mitotracker green; (E) Overlap of Hoechst (blue) and TPPS4 (red); (F) Overlap of TPPS4 (red) and Mitotracker (green). Scale bar = 5 μm.

Figure 10. Confocal micrographs of C. albicans incubated with TMPyP4.

Figure 10

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(A) Bright field; (B) Hoechst; (C) TMPyP4; (D) Mitotracker green; (E) Overlap of Hoechst (blue) and TMPyP4 (red); (F) Overlap of TMPyP4 (red) and Mitotracker (green). Scale bar = 5 μm.

Discussion

The present study contains several novel and surprising findings. Perhaps the most surprising finding is that the well-known anionic porphyrin, TPPS4, is actually a surprisingly effective anti-microbial PS. In some cases (MRSA and C. albicans), TPPS4 was substantially better than the equally well-known cationic porphyrin, TMPyP4, and was active at 200nM concentration. There are dozens of papers where TMPyP4 is used as a PS to mediate aPDI [24–28], but virtually none looking at TPPS4 in this role (except perhaps a few where it was used as a negative control [29, 30]). Another surprising finding is that TPPS4 and ClAlPCS4 are actually very different from each other regarding their relative activity as PS for aPDI; in our opinion most workers would have thought they would be similar. TPPS4 binds to all three classes of microbial cells (although it does not produce photokilling of Gram-negative cells, with the possible exception of some killing of P. aeruginosa and A. baumannii). The third interesting aspect is that the potentiation of aPDI using addition of KI revealed the binding of TPPS4 to Gram-negative cells, which would have been unlikely to be discovered otherwise. The uptake of TMPyP4 by MRSA is much higher than that of TPPS4 (Figure 8A), however, the PDI activity of the former is much lower than that of the later (Figure 2A). We believe that this is due to the fact that some PS with a large number of cationic charges (such as the four cationic charges present on TMPyP4) encourage a high level of binding to Gram-positive species, but do not allow the PS to penetrate inside as well as those PS with a lesser number of cationic charges. There was experimental evidence of this hypothesis in a study we published a few years ago [31]. There we had four stable synthetic bacteriochlorins that had different numbers of cationic groups: either zero (2 basic amino groups), 2 quaternary, 4 quaternary or 6 quaternary ammonium groups. The most active PS against Gram-positive bacteria was the compound with zero (2 basic amino groups) cationic groups, while the most active compound against Gram-negative bacteria was the compound with 6 quaternary cationic groups.

Since we have recently published several papers [18, 19] showing that addition of KI can turn neutral/anionic PS such as Rose Bengal and Photofrin into broad-spectrum antimicrobial PS, it might have been expected that the same phenomenon would have occurred with TPPS4, which is known to be highly effective in generating singlet oxygen in solution, especially when excited by blue light that hits the Soret band (ε = 250,000 M−1cm−1). However the discovery that we could eradicate E. coli after a spin (but only when KI was added otherwise there was no killing) was surprising. The logical conclusion was that TPPS4 was bound to the outside of E. coli cells, but since it does not kill the bacteria when excited, the explanation for this must be that the singlet oxygen does not penetrate through the bacterial outer membrane. The literature contains reports that the permeability barrier of Gram-negative bacteria is sufficient to protect them from non-cationic PS [8], but also that the same permeability barrier protects them from extracellular generated singlet oxygen [32]. In the case of TMPyP4 which does kill E. coli without a spin, and to lesser extent with a spin, the explanation must be that the more pronounced cationic character of the tetra-pyridinium porphyrin allows it to penetrate through the outer membrane, so that the singlet oxygen is generated in a location where it can do some damage.

Our previous reports on KI potentiation of aPDI attempted to distinguish between microbial killing by extracellular generated free iodine (I2/I3−) and short-lived reactive iodine species, which are hypothesized to be iodine radicals (I•/I•2). We propose that free iodine needs to reach a sufficient threshold concentration to be microbicidal. The amount of free iodine produced depends on the amount of singlet oxygen produced, but also on the concentration of iodide anion present in solution. The reason for the big effect of iodide concentration is hypothesized to be due to the very short lifetime of singlet oxygen, so the likelihood of singlet oxygen being quenched by iodide is much higher when the iodide concentration is relatively high. We originally used a concentration of 10mM KI combined with MB as the PS [15], but subsequently found that concentrations up to 100mM KI were necessary when the PS did not bind to the bacteria, as demonstrated for Photofrin [18] and for Rose Bengal [19]. In the paper with MB and Ki we concluded that short-lived reactive iodine species were principally involved [15] rather than long-lived molecular iodine. It is inherently difficult to tease apart the microbial killing produced by these two species (I2/I3− and I•/I•2), but we believe that by looking at the shape of the dose response curve with increasing KI concentration, some information can be gleaned. If the dose response curve shows an abrupt threshold value (e.g. Figs 3A, B, E) then free iodine is the main killing species, but if there is amore gradual increase in killing (e.g. Figs 3C, D, F), then there is a contribution from short-lived reactive iodine species.

The mechanisms of oxidation of iodide by singlet oxygen is hypothesized to involve an initial addition reaction to give peroxyiodide (eq 1)

 1O2+2I−+H2O→HOOI−2+HO− Eq 1

The peroxyiodide can decompose by two different mechanisms. The first involves the production of iodine and hydrogen peroxide (eq 2).

HOOI−2+H2O→I2+H2O2+HO− Eq 2

It Is at present uncertain if the H2O2 contributes any additional microbial killing, but it is certainly possible, because a mixture of iodine and H2O2 has been shown to possess greater microbicidal activity compared to either compound used alone (especially for E. coli and yeast) [33].

The second mechanism involves a homolytic fission process to give a pair of free radicals, which are expected to be very reactive and to have short lifetimes (eq 3)

HOOI−2→I2•−+HOO• Eq 3

The surprising finding that TPPS4 can bind to Gram-negative bacteria, and be taken up by C. albicans, while ClAlPCS4 cannot, is presumably explained by the fact that TPPS4 has a more cationic character than ClAlPCS4, as suggested by the fact that TPPS4 is supplied from the manufacturer as a dihydrochloride salt. Although it should be possible to measure the pKa values of these tetrapyrrole compounds [34–36], we were unable to find this data in the literature. D’Urso et al discuss the zwitterionic character of porphyrin tetrasulfonates due to the higher pKa values of the two central pyrrole nitrogen atoms [37].

There are a large number of papers looking at TPPS4 and TMPyP4, in which the former is used as an example of an anionic porphyrin, while TMPyP4 is used as an example of a cationic porphyrin. These papers are looking at different kinds of porphyrin aggregation (J-aggregates and H-aggregates). J-aggregates consist of “side-by-side” porphyrin arrays with characteristic red-shifted absorption, whereas H-aggregates are composed of “face-to-face” porphyrin monomers that show blue-shifted absorption For instance Wu et al reported that diprotonated TPPS4 produced J-aggregates in the presence of the ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate [38]

TMPyP4 is used as a typical porphyrin ligand that stabilizes the non-canonical DNA structure called G-quadruplex (GQ) DNA [39]. GQs are nucleic acid structures formed by guanine tetrads that π-stack on top of each other, forming a right-handed helix. Since these GQs occur more often in telomeres, stabilization by TMPyP4 could disrupt the telomere structure and could therefore be used as an anticancer strategy [40].

Both TPPS4 and TMPyP4 were localized in the nucleus of C. albicans cells. There have been a few papers where the aPDI effects of TPPS4 have been studied (mostly against bacteria) [30, 41, 42] but the surprising effectiveness found in our study was not reported. There is a paper comparing the localization of TPPS4 and TMPyP4 in CT26 mouse cancer cells. Both compounds were initially taken up into lysosomes but relocalized to the nucleus under the influence of light or after induction of oxidative stress (750 μM H2O2 for 1 h [43]

TPPS4 was seriously investigated as an anti-cancer PS some years ago [44–46]. However its exploration was abandoned when there were reports it could exert neurotoxic effects in experimental animals even in the dark [47]. Other researchers later questioned this conclusion and suggested the clinical use of TPPS4 could be reconsidered [48]. TPPS4 was clinically tested in a trial of 9 patients with cutaneous metastases of breast cancer, who received TPPS4 injected locally into the tumor at a dose of 0.15–0.3 mg followed by 150 J/cm2 red laser. Complete destruction of the tumor was observed in 3 patients, reduction by > 50% in 2 patients, reduction by < 50% in 2 patients and no regression in 2 patients [49].

Although most of the studies investigating PDT with “chloroaluminum sulfonated phthalocyanine” have used a preparation that contained a mixture of mono-, di-, tri- and tetrasulfonates [50, 51], there is one paper that studied the pure ClAlPCS4 for PDT of tumors [52].

The ability of KI to transform almost any PS that produces reasonable amounts of singlet oxygen upon photoexcitation, into a broad-spectrum antimicrobial PS is remarkable. The approach has been shown to work in vivo in several models of localized infections [15, 16, 19, 53], and deserves consideration for early translation into clinical trials.

Highlights.

  • Cationic porphyrins are accepted as broad-spectrum antimicrobial photosensit1zers

  • Anionic porphyrins (TPPS4) were considered inactive against Gram-negative bacteria

  • TPPS4 localizes in the nucleus of Candida cells

  • Potentiation by KI revealed TPPS4 bound to Gram-negative bacteria

  • Anionic ClAlPCS4 behaves differently from TPPS4

Acknowledgments

This work was supported by US NIH grants R01AI050875 and R21AI121700. Liyi Huang was supported by National Natural Science Foundation of China (81260239, 81472002), Guangxi Scientific and Technological Project (1355005-1-2), Guangxi Natural Science Foundation (2016GXNSFAA380312)

Footnotes

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