Photoinactivation of Escherichia coli using porphyrin derivatives with different number of cationic charges

FEMS Immunology and Medical Microbiology 44 (2005) 289–295 www.fems-microbiology.org

Photoinactivation of Escherichia coli using porphyrin derivatives with different number of cationic charges Mariana B. Spesia a, De´bora Lazzeri a, Liliana Pascual b, Marisa Rovera b, Edgardo N. Durantini a,* a

Departamento de Quı´mica, Universidad Nacional de Rı´o Cuarto, Rı´o Cuarto, Agencia Postal Nro 3, X5804BYA Rı´o Cuarto, Co´rdoba, Argentina b Departamento de Microbiologı´ae Inmunologı´a, Universidad Nacional de Rı´o Cuarto, Rı´o Cuarto, Agencia Postal Nro 3, X5804BYA Rı´o Cuarto, Co´rdoba, Argentina Received 9 August 2004; received in revised form 16 September 2004; accepted 21 December 2004 First published online 7 January 2005

Abstract The photodynamic effect of meso-substituted cationic porphyrins, 5-[4-(trimethylammonium)phenyl]-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin iodide 1, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 2 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were compared in N,N-dimethylformamide. Fluorescence quantum yields (/F) of 0.10, 0.06 and 0.08 were calculated for porphyrins 1, 2 and 3, respectively. The singlet molecular oxygen, O2(1Dg), production was evaluated using 9,10-dimethylanthracene yielding values of 0.66, 0.36 and 0.42 for porphyrins 1, 2 and 3, respectively. Guanosine 5 0 -monophosphate was used as biological substrate model. Similar decomposition of guanosine 5 0 -monophosphate was obtained using these cationic porphyrins as sensitizer. In biological medium, photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. E. coli cultures were treated with sensitizer at 37
C for 30 min in dark. In both procedures, a higher photoinactivation of cells (>99.999%) was found for cells treated with 10 lM of tricationic porphyrin 3 and irradiated for 5 min with visible light. Porphyrins 1 and 2 only show an important photodamage when the cells are irradiated without washing step. These results indicated that the tetracationic porphyrin 3 could be a promising sensitizer with potential applications in the photoinactivation of bacterial cells by photodynamic therapy.  2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Cationic porphyrin; Photoinactivation; Bacteria; Escherichia coli; Singlet oxygen; Photodynamic therapy

1. Introduction Tetrapyrrolic macrocycle ring systems have showed great potential as phototherapeutic agents for the treatment of a variety of oncological and non-oncological diseases [1–3]. Photodynamic therapy (PDT) of cancer

*

Corresponding author. Fax: +54 358 467 6233. E-mail address: [email protected] (E.N. Durantini).

involves the administration of a photosensitizer, which is selectively incorporated in tumor cells. The subsequent exposure to visible light specifically inactivates neoplastic cells [4,5]. Two oxidative mechanisms are considered to be principally implicated in the photodamage of cells. In the type I photochemical reaction, the photosensitizer interacts with a biomolecule to produce free radicals, while in the type II mechanism, singlet molecular oxygen, O2(1Dg), is produced as the main species responsible for cell inactivation [5]. Both mechanisms can occur simultaneously and the ratio

0928-8244/$22.00  2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2004.12.007

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better accumulation in cells [21,22]. Thus, the main purpose of this study was to compare the photoinactivation efficiencies of these cationic photosensitizers 1–3 with different number of positive charges in a gram-negative bacterium E. coli.

between two processes depends mainly of the sensitizer, substrate and the nature of the medium [6,7]. PDT has been suggested as an alternative approach for treating local infections [3]. Thus, pathogenic microorganisms growing in vivo as localized foci of infection, on skin or on accessible mucous membrane, would be candidates for photodynamic destruction [8,9]. In this way, photodynamic treatment by porphyrins was established for photoinactivation of bacteria, in an attempt to overcome the problem of bacterial multidrug resistance [8–16]. Gram-positive bacteria are susceptible to the photosensitizing action of a variety of porphyrins [10,11]. However, gram-negative bacteria exhibit a remarkable resistance to negatively charged or neutral porphyrins, unless the permeability of the outer membrane is artificially increased by treatment with chemical or biological agents, which stimulates the membrane translocation of the sensitizers [17]. The resistance of gram-negative bacteria to the action of photoactivated sensitizers has been ascribed to the presence of highly organized outer membrane, which hinders the interaction of the photosensitizer with the cytoplasmic membrane and intercepts the photogenerated reactive species [14]. Studies with cationic porphyrin derivatives have shown that these compounds can cause direct photoinactivation of gram-negative bacteria even in the absence of additives [10–13]. In previous studies, we have investigated the photodynamic activity of amphiphilic porphyrin in different media and in vitro on the Hep-2 human larynx carcinoma cell line [18–20]. An increase in the uptake into Hep-2 cells was observed for a porphyrin substituted by a cationic trimethylammonium group in the periphery of the structure [20]. In this case, a higher cellular incorporation was also accompanied by a higher photocytotoxic activity on Hep-2 cells. In this study, the photodynamic activity of cationic porphyrins 1–3 (Scheme 1) was compared in homogeneous medium bearing biologically related substrates and in a typical gram-negative bacterium Escherichia coli. The asymmetric charge distribution at the peripheral position of the porphyrin produces an increase in the amphiphilic character of the structure, which can help a

2. Materials and methods 2.1. General UV–vis and fluorescence spectra were recorded on a Shimadzu UV-2401PC spectrometer and on a Spex FluoroMax fluorometer, respectively. All the chemicals from Aldrich (Milwaukee, WI, USA) were used without further purification. Guanosine 5 0 -monophosphate (GMP) from Sigma (St. Louis, MO, USA) was used as received. Solvents (GR grade) from Merck were distilled. Ultrapure water was obtained from Labonco equipment model 90901-01. 2.2. Porphyrins 5-[4-(Trimethylammonium)phenyl]-10,15,20-tris(2,4,6trimethoxyphenyl) porphyrin iodide 1, 5,10-bis(4-methylphenyl)-15,20-bis(4-trimethylammoniumphenyl)porphyrin iodide 2 and 5-(4-trifluoromethylphenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 3 were synthesized as previously described [18,21]. Pure porphyrins were used as evaluated using thin layer chromatography (TLC) analysis (silica gel, dichloromethane/ methanol) and 1H NMR spectroscopy. 2.3. Spectroscopic studies Absorption spectra were recorded at 25.0 ± 0.5
C using 1-cm path length cells. The fluorescence quantum yield (/F) of porphyrins were calculated by comparison of the area below the corrected emission spectrum with that of tetraphenylporphyrin (TPP) as a fluorescence standard, exciting at kex = 515 nm [23]. Values of /F = 0.12 was used for TPP in N,N-dimethylformamide (DMF) [18,24].

H3CO

R1 1 R1:

+

-

N (CH3)3I

R2=R3=R4:

N

NH

H3CO

R2

R4 N

HN R3

OCH3

2 R1=R2: 3 R1=R2=R3:

+

-

N (CH3)3I

N+(CH3)3I-

R3=R4: R4:

Scheme 1. Molecular structures of porphyrins 1–3.

CH3 CF3

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2.4. Steady-state photolysis Solutions of 9,10-dimethylanthracene (DMA, 30 lM in DMF, 2 ml) and photosensitizer were irradiated in quartz cuvettes with monochromatic light at k = 418 nm (porphyrin absorbance 0.15,
1 lM) from a 75 W high-pressure Xe lamp through a high intensity grating monochromator, Photon Technology Instrument [24]. The light intensity was determined as 0.68 mW/cm2 (Radiometer Laser Mate-Q, Coherent). The kinetics of photooxidation were studied by following the decrease of the absorbance (A) at kmax = 376 nm for DMA. The observed rate constants (kobs) were obtained by a linear least-squares fit of the semilogarithmic plot of Ln A0/A vs time. Photooxidation of DMA was used to determine singlet molecular oxygen, O2(1Dg), production by the photosensitizers. Measurements of the sample and reference under the same conditions afforded UD for porphyrins by direct comparison of the slopes in the linear region of the plots [18,25]. The studies in presence of GMP (110 lM) were performed using light of 418 nm (porphyrin absorbance 1.0,
7 lM) and 2.60 mW/cm2 in DMF–water 10% (v/v), 2 ml [18,26]. The disappearance of GMP was monitored by decrease in the absorption peak at 272 nm. All experiments were performed at 25.0 ± 0.5
C. The pooled standard deviation of the kinetic data, using different prepared samples, was less than 5%. 2.5. Bacterial strain and preparation of cultures Escherichia coli strain EC 7 (gram-negative) recovered from clinical urogenital material was used. The bacterial strain was identified according to conventional procedures [27]. The antibiotic resistance profiles are: ceftazidime (sensitive), levofloxacin (sensitive), ampicillin (resistant), aminosulbactam (resistant), cephalotin (limit) gentamycin (sensitive) and sulfamethoxazol-trimethoprim (resistant). E. coli strain was grown aerobically at 37
C in 30% (w/v) tryptic soy (TS) broth overnight. Aliquots (
40 ll) of the culture was aseptically transferred to 4 ml of fresh medium (30%, w/v, TS broth) and incubated at 37
C to mid logarithmic phase (O.D.
0.6 at 660 nm). Cells in the logarithmic phase of growth were harvested by centrifugation of broth cultures (3000g for 15 min), washed once with 10 mM phosphate-buffered saline (PBS, pH 7.0) and re-suspended in 4 ml of PBS. Then the cells were diluted 1/1000 in PBS, corresponding to
106 colony forming units (CFU)/ml. In all experiments, 2 ml of the cell suspensions in Pyrex brand culture tubes (13 · 100 mm) were used and the sensitizer was added from a stock solution of porphyrin (3.0 · 103 M) in DMF. Viable bacteria were monitored and their number calculated by counting the number of colony forming units after appropriate dilution on agar plates [18,26]. Bacterial

291

cultures grown under the same conditions and light exposures, but without addition of any photosensitizer served as controls. 2.6. Photosensitized inactivation of bacteria cells Cell suspensions of E. coli (2 ml,
106 CFU/ml) in PBS were incubated with porphyrin at 37
C for 30 min in the dark. After that, two protocols were followed: (a) irradiation of cultures without the cell washing step, which means the porphyrin remained in the bacterial suspension and (b) washing once the cells with PBS after irradiation. In both cases, the cultures were exposed for different time intervals to visible light. The light source used was a Novamat 130 AF slide projector equipped with a 150 W lamp. The light was filtered through a 2.5-cm glass cuvette filled with water to absorb heat. A wavelength range between 350–800 nm was selected by optical filters [28]. The light intensity at the treatment site was 90 mW/cm2 (Radiometer Laser Mate-Q, Coherent). Control experiments were carried out without illumination in the absence and presence of sensitizer, and with addition of DMF excluding the porphyrins. Control and irradiated cell suspensions were serially diluted with PBS, each solution was plated in triplicate on TS agar and the number of colonies formed after 18–24 h incubation at 37
C was counted. Dark experiments involved incubation for 30 min and for the period of irradiation of each sample after plating. Each experiment was repeated separately three times. Minimal bactericidal concentration (MBC) of each porphyrin was taken as the lowest concentration that showed no growth on agar plate. To determine MBC, the cultures prepared as described above were treated with different concentrations of porphyrin (1–45 lM). The MBC values were evaluated after irradiation with visible light for 10 and 30 min.

3. Results and discussion 3.1. Spectroscopic studies The absorption spectra of porphyrins 1–3 in DMF show the typical Soret and Q-bands, characteristics of free-base (Fig. 1A) [29]. As can be observed, a broadening of Soret band was observed for porphyrin 2 and 3 in DMF, indicating that aggregation occurs, as is typical for many porphyrin derivatives [30–32]. This effect was not observed for porphyrin 1 (10 lM), which remains mainly unaggregated as shown by sharp absorption bands. The steady-state fluorescence emission spectra of these porphyrins 1–3 show two bands in the red spectral region (Fig. 1B), which are characteristic for similar free-base porphyrins and they have been assigned to

normalized intensity (AU)

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normalized absorbance

292

1.0 0.8 0.6 0.4 0.2 0.0 400

(A)

500 600 wavelength (nm)

700

1.0 0.8 0.6 0.4 0.2 0.0 550

(B)

600 650 700 750 wavelength (nm)

800

Fig. 1. (A) Absorption and (B) fluorescence emission spectra of porphyrins 1 (solid line), 2 (dashed line) and 3 (dotted line) in DMF.

Q(0–0) and Q(0–1) transitions [33]. By comparison with TPP as a reference, values of fluorescence quantum yield (/F) of 0.10 ± 0.01, 0.06 ± 0.01 and 0.08 ± 0.01 were calculated for porphyrin 1, 2 and 3, respectively, in DMF. The lower values of /F for porphyrins 2 and 3 can be associated with the partial aggregation of the sensitizer in this medium [1,24].

Table 1 Kinetic parameters for the photooxidation of DMA sensitized by porphyrins 1–3 and singlet oxygen quantum yield (UD) in DMF Porphyrin

k DMA (s1) obs

UDa

1 2 3

(9.4 ± 0.5) · 104 (5.0 ± 0.3) · 104 (5.9 ± 0.4) · 104

0.66 ± 0.05 0.36 ± 0.05 0.42 ± 0.05

a

Using porphyrin 1 as Ref. [18].

3.2. Photosensitized decomposition of substrates

Ln (A0 / A) (376 nm)

3.2.1. Photooxidation of 9,10-dimethylanthracene The aerobic irradiation of photosensitizer 1–3 was performed in the presence of DMA. Fig. 2 shows the semilogarithmic plots describing the progress of the reaction for DMA. From these plots, the values of the observed rate constant ðk DMA obs Þ were obtained (Table 1). The quantum yield of O2(1Dg) production (UD) were calculated comparing the slopes for the porphyrins in Fig. 2. As shown in Table 1, a higher efficiency in the O2(1Dg) production was found for porphyrin 1 than for 2 and 3, in DMF. The values of UD can significantly change in a different medium, diminishing when the sensitizer is partially aggregated [1,11]. This could be the case of porphyrin 2 and 3 in this homogeneous medium.

3.2.2. Decomposition of guanosine 5 0 -monophosphate The nucleotide GMP was used as substrate model for the compounds of biological interest that would represent potential targets of porphyrin photosensitization [24,26,34,35]. Continuous irradiation of porphyrins 1–3 leads to GMP decomposition as evidenced by the formation of a broad absorption band above 300 nm (Fig. 3A). The results indicate a similar behavior of GMP reaction using porphyrins 1–3 as sensitizers (Fig. 3B). Under aerobic conditions the decomposition of GMP occurs predominantly through a type II photoreaction process [26,36]. However, since cationic porphyrins bind to GMP by electrostatic attraction, an electron transfer pathway may also be contributing to its decomposition under these conditions [26,37].

0.25

3.3. Photosensitized inactivation of Escherichia coli

0.20

After the treatment of E. coli cells with sensitizers 1–3 of different concentrations (1–10 lM) at 37
C for 30 min in the dark, the cultures were irradiated with visible light without a washing step (Fig. 4A). As can be observed, a decrease in the cell survival was obtained when increasing the concentration of porphyrin in the treatment. Therefore, 10 lM of sensitizer was used to evaluate the efficacy of these agents on E. coli cells either without washing step or after one washing step. Under these conditions, control experiments showed that the viability of E. coli was unaffected by illumination alone or by dark incubation with 10 lM of the photosensitizer for 30 min, indicating that the cell mortality obtained after irradiation of the cultures treated with the

0.15 0.10 0.05 0.00

0

100 200 irradiation time (s)

Fig. 2. First-order plots for the photooxidation of DMA (30 lM) photosensitized by porphyrin 1 ( ), 2 (n) and 3 (m) in DMF; kirr = 418 nm. Values represent means ± standard deviation of three separate experiments.



M.B. Spesia et al. / FEMS Immunology and Medical Microbiology 44 (2005) 289–295

0.5 ∆ absorbance (272 nm)

∆ absorbance

0.2

0.0

-0.2

-0.4 300 (A)

293

400 500 600 wavelength (nm)

0.4 0.3 0.2 0.1 0.0

700 (B)

0

20 40 60 80 irradiation time (min)

100

7

7

6

6

5

5 Log UFC/mL

Log UFC/mL

Fig. 3. (A) Difference absorption spectra of porphyrin 2 (absorbance = 1.0 at 418 nm) in the presence of GMP (110 lM) and the same solution after 10 min intervals of irradiation (kirr = 418 nm) in DMF–water 10%; (B) photosensitized decomposition of GMF by porphyrins 1 (d), 2 (j) and 3 (m) monitored from decrease of GMP absorption peak at 272 nm.

4 3 2

(A)

3 2 1

1 0

4

0

0

5 [porphyrin] (M)

10

(B)

0

10 15 5 irradiation time (min)

20

Fig. 4. (A) Survival curves of E. coli incubated with different porphyrin concentration at 37
C for 30 min in the dark and exposed to visible light for 5 min; (B) photoinactivation of E. coli incubated with 10 lM of porphyrin 1 (d), 2 (j) and 3 (m) at 37
C for 30 min in the dark and exposed to visible light for different irradiation times and washing once the cells before illumination 1 (s), 2 (h) and 3 (n). Control culture untreated e. Values represent means ± standard deviation of three separate experiments.

porphyrin is due to the photosensitization effect of the agent produced by visible light. As observed in Fig. 4B, the E. coli cells are rapidly photoinactivated when the unwashed cultures treated with cationic porphyrin 1–3 are exposed to different doses of visible light. In particular, the tricationic porphyrin 3 exhibits a higher photosensitizing activity causing a
5.5 log decrease of cell survival. These results represent a value greater than 99.999% of cellular inactivation. Moreover, after 20 min irradiation of porphyrins 1 and 2, a high diminishment in the cellular viability (
4.5 log) was observed, which represents a 99.9% of E. coli inactivation. These photoinactivation activities are similar to that reported before for 5,10,15,20-tetrakis-(4-N-alkylpyridil)porphyrin derivative bearing chains of 10 (96%) and 14 (100%) carbons on E. coli cells treated under similar conditions [14]. However, these values are considerably higher than that for 5,10,15,20-tetrakis-(4-N-methylpyridil)porphyrin (29%) using 8.3 lM. Under the above conditions, MBC were estimated for these porphyrins. The susceptibility of E. coli organism

to the photodynamic activity of these cationic porphyrins depends of the light dose. Thus, after 10 min of irradiation, the values of MBC were 36.0 ± 0.5 lM for porphyrins 1, >20 lM for porphyrin 2 and 8.0 ± 0.5 lM for porphyrin 3. Likewise, MBC were evaluated after 30 min of irradiation, giving 2.0 ± 0.5 lM for porphyrin 3, 20.0 ± 0.5 lM for porphyrin 1 and >20.0 ± 0.5 lM for porphyrin 2. The MBC values for porphyrin 2 were not possible to determine, because precipitation of the sensitizer occurs at concentrations above 20 lM in the PBS buffer. Therefore, these results also give evidence of the highest efficiency of porphyrin 3 in comparison with porphyrins 1 and 2. On the other hand, after one washing step, the photoinactivation of E. coli cells follows the tendency showed in Fig. 4B. Under such conditions, the photodynamic effect is mainly associated with porphyrins that are more effectively bound to cells. As can be observed, the photoinactivation activities of tricationic porphyrin 3 remain similar than those found for unwashed cells. However, for cultures treated with porphyrin 2, the photocytotoxic effect diminishes
1.5 log after 20 min

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of irradiation (Fig. 4B). Similar behavior was previously observed for 5,10-di(4-N-methylpyridil)-15,20-di(phenyl)porphyrin on the survival of E. coli cells [11]. This decrease in the photoinactivation of cells treated with porphyrins 1 and 2 can be associated with a loss of the amount of cell-bound sensitizer after one washing step. Thus, these results indicate that tricationic porphyrin 3 produces an efficient photodamage still after one washing step of E. coli cells. These results may indicate that this porphyrin has a more effective binding site to E. coli cells and therefore differs in its target from the other compounds or it may be that there are differences in levels of uptake or binding to the external peptidoglycan layers [11]. In conclusion, these studies provide information on the photodynamic activity of cationic porphyrin derivatives 1–3 with different number of positive charges on the periphery of the tetrapyrrolic macrocycle. In homogeneous medium, similar photodynamic effect was observed for porphyrins 1–3 with biological substrates. However, this behavior is not always expected in cellular media [38]. The biological microenvironment where the sensitizer is localized can induce significant changes in the photophysical properties of the porphyrin established in solution. Thus, although porphyrin 3 presents a lower value of UD in DMF than 1, probably due to aggregation of 3 in this media, the MBC values indicate that 3 is an efficient sensitizers to photoinactivate E. coli cells. Therefore, the structure–function relationship for E. coli photoinactivation follows the order 3 > 2 > 1, which is also the arrangement of increasing cationic charges on the molecules. These studies show that the tricationic porphyrin 3 is a promising photosensitizer agent with potential applications in bacteria inactivation by photodynamic treatment.

Acknowledgments Authors are grateful to Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET) of Argentina and Fundacio´n Antorchas for financial support. E.N.D. is a Scientific Member of CONICET. D.L. thanks Fundacio´n Antorchas for a research fellowship.

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