Journal of Photochemistry and Photobiology B: Biology 148 (2015) 188–196

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Improved antibacterial phototoxicity of a neutral porphyrin in natural deep eutectic solvents Kristine Opsvik Wikene a,⇑, Ellen Bruzell b, Hanne Hjorth Tønnesen a a b

Department of Pharmacy, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway Nordic Institute of Dental Materials AS, Sognsveien 70A, 0855 Oslo, Norway

a r t i c l e

i n f o

Article history: Received 14 January 2015 Received in revised form 9 April 2015 Accepted 20 April 2015 Available online 2 May 2015

a b s t r a c t Neutral porphyrins for antibacterial photodynamic therapy (aPDT) have received little attention due to their tendency to aggregate in aqueous media and reports of low phototoxic effect. These compounds may be less toxic to cells than positively and negatively charged photosensitisers. The preparation of highly bacterial phototoxic formulations of neutral porphyrins remains an open field of research with great potential if achievable. The purpose of this study was to develop novel hydrophilic formulations of the neutral porphyrin 5,10,15,20-tetrakis(4-hydroxyphenyl)-porphyrin (THPP) by use of natural deep eutectic solvents (NADES) prepared by the solvent evaporation method. Physical and photochemical stability and in vitro photoinactivation of Enterococcus faecalis and Escherichia coli were investigated. Two of the 15 NADES investigated demonstrated superior solubilising properties of THPP. The photostability of THPP was higher in NADES than in methanol. A 100-fold dilution of the preparations with buffer to a final concentration of 0.5–5 nM THPP resulted in complete photoinactivation of E. faecalis and E. coli both in their exponential and stationary phase. THPP demonstrated significantly higher phototoxicity when formulated in NADES than in other aqueous preparations like phosphate buffered saline. NADES as a formulation concept for photosensitisers shows a great potential in aPDT. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Antibacterial photodynamic therapy (aPDT) is a potential treatment for bacterial infections. Systemic side effects may be greatly reduced and the bacteria specifically targeted possibly without the risk of inducing bacterial resistance [1,2]. aPDT utilises a combination of visible light, a photosensitiser (PS) and oxygen to produce cytotoxic species. Porphyrins are aromatic heterocyclic compounds that are ubiquitous in nature, and have been widely investigated in photodynamic therapy of tumours and microbial infections [3–10]. In cancer treatment neutral and negatively charged porphyrins (e.g. porfimer sodium) and the related chlorins (e.g. meso-tetrahydroxy phenylchlorin) have been studied as effective treatment modalities [4]. However, in aPDT there is a general agreement that positively charged groups on the porphyrin is fundamental for antibacterial photodynamic effect [9–12]. This understanding reasons in the outer wall and cytoplasmic membrane structure of Gram-positive and Gram-negative bacteria. The Gram-positive outer wall consists of a porous peptidoglycan layer with traversing lipoteichoic acids ⇑ Corresponding author. Tel.: +47 22856589. E-mail addresses: [email protected] (K.O. Wikene), ellen.bruzell@niom. no (E. Bruzell), [email protected] (H.H. Tønnesen). http://dx.doi.org/10.1016/j.jphotobiol.2015.04.022 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

where even 30,000–60,000 Da peptides may diffuse through [13]. The Gram-negative outer structure contains an additional highly organised outer lipid bilayer membrane composed of negatively charged lipopolysaccharides, polysaccharides, proteins and lipoproteins [14]. This structure usually renders the bacteria less vulnerable to negatively charged or neutral porphyrins which efficiently photoinactivate Gram-positive bacteria [1,7,14,15]. Previous formulations of porphyrins have mostly been based on simple preparations in water, liposomes and DMSO [10,12,16]. Particularly for the neutral porphyrins, these preparations have been inefficient in photoinactivation of Gram-negative bacteria. Water miscible formulations are advantageous in aPDT applications as the PS needs to penetrate aqueous exudate or other body fluids to reach the target bacteria. Also the bacterial outer cover is largely hydrophilic and will not allow close interactions with hydrophobic compounds [17]. A neutral, hydrophobic PS incorporated in liposomes will therefore not readily be released from the carrier and interact with the bacterial membrane [12,18]. Natural deep eutectic solvents (NADES) are a third type of liquid, separate from water and lipids, which is present in all living cells [19]. These solvents may also be prepared in an industrial scale. They were described for the first time by Choi et al. in 2011 [19]. NADES consist solely of natural compounds that are omnipresent in nature, such as amino acids, sugars and simple

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organic acids [19,20]. These liquids may be used e.g. for extractions, biopharmaceutical stabilisation and in chemical industry. The eutectic solvents may also be used for drug delivery, as they are non-toxic, environmentally friendly, sustainable, cheap, and have unique solubilising properties [19,20]. The aim of the present study was to create a hydrophilic eutectic solvent (i.e. NADES) that could dissolve the hydrophobic, neutral porphyrin 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (THPP, Fig. 1) to evaluate its potential as a PS and NADES as a solvent of PS in aPDT of Gram-positive and Gram-negative bacteria. 2. Materials and methods 2.1. NADES preparation The two components of each deep eutectic solvent investigated (Table 1) were dissolved in warm water (50 °C) and evaporated at 45 °C for 15 min with a rotatory evaporator (Büchi EL 131 Rotavapor, Flawil, Switzerland). The liquid obtained was transferred to polypropylene tubes with a tight cap. Water content was determined by Karl Fischer titration (C20 Coulometric KF Titrator, Mettler Toledo Inc., Schwerenbach, Switzerland). The pH was measured in undiluted NADES and after dilution 1:1, 1:100 and 1:200 with phosphate buffered saline without Ca2+ and Mg2+ (PBS, Lonza, Verviers, Belgium) using a pH 526 MultiCalÒ pH meter (WTW GmbH, Weilheim, Germany).

Table 1 Constituents of the natural deep eutectic solvents investigated and the apparent pH values. n.d. = Not measured due to instability; solid precipitate within 7 d. Component 1

Component 2

Molar ratio

pH

Acronym

Citric acid Citric acid Citric acid Citric acid Choline chloride Choline chloride Choline chloride Choline chloride Choline chloride D-(+)-glucose D-(+)-glucose D-(+)-glucose Sucrose Sucrose Sucrose

Sucrose D-(+)-trehalose Choline chloride Choline chloride DL-malic acid DL-malic acid Maleic acid Maleic acid Glycerol Sucrose D-( )-fructose DL-malic acid D-( )-fructose DL-malic acid Maleic acid

1:1 2:1 1:1 1:2 1:1 1:3 1:1 1:3 1:1 1:1 1:1 1:1 1:1 1:1 1:1

0.34 n.d. 0.30 0.47 0.76 0.69 0.30 0.32 4.75 6.03 5.33 0.32 6.17 0.36 0.23

CS

MC GC

MG

mobile phase was composed of 0.34% (w/v) sodium acetate and 0.48% (w/v) sodium chloride (pH 5.2 adjusted with acetic acid) and methanol (15:85). The retention time of THPP at flow 0.35 ml/min was equal in methanol and in NADES diluted more than 50 times in methanol: approximately 4.3 min at a column temperature of 30 °C. The selected NADES had to be diluted more than 50 times prior to HPLC analysis due to the high viscosity.

2.2. Solubility test 2.3. Photostability of THPP Solubility test was performed by saturating the NADES in polypropylene tubes with an excess amount of THPP (meso-tetra (p-hydroxyphenyl)porphine, Frontier Scientific Inc., UT, USA; Fig. 1). The dry powder was given 1 h to sink into the viscous liquid before the tubes were agitated horizontally on an Edmund Bühler shaker (at 250 rpm) protected from light at 22 °C for 16 h. The tubes were centrifuged (6918g, 60 min, 22 °C) before visual evaluation of solubility potential (i.e. colouration, particle distribution and dissolution). Four NADES were selected for further studies (i.e. CS, MC, GC and MG, cf. Table 1). After centrifugation triplicate samples of the solutions were filtered (0.45 lm, Spartan, 13/0.45 RC filter, Schleider & Schull, Dassel, Germany) and diluted 100 times with methanol to be analysed by HPLC at detection wavelengths 419 nm and 445 nm. The HPLC analysis was performed with isocratic elution on an Ultra Biphenyl 3 lm column (100  2.1 mm; Restek Corporation, Bellefonte, PA, USA). The

Investigation of the photostability of THPP in selected NADES and in methanol was performed by irradiation in a Suntest CPS+(Atlas, Linsengericht, Germany) equipped with a 1.8 kW xenon lamp and a glass filter (cut off 310 nm) according to Option 1 (ICH Guideline Q1B) [21]. The samples were exposed at 765 W/m2 (310–800 nm) to an endpoint corresponding to 1.2  106 lux
h (400–800 nm). The photostability of THPP was investigated in methanol and in undiluted CS and MG (cf. Table 1) containing 1  10 3 M THPP, in NADES containing THPP diluted 50 times (to 1  10 5 M THPP) in methanol or in MilliQ water, and of 1  10 5 M THPP in pure methanol. Three parallels of each sample for irradiation and as dark controls were prepared in small glass containers (light path 3 mm) covered with cling film. The dark controls were additionally covered with aluminium foil. The maximum temperature at the surface of the containers was measured with temperature recording strips (37–65 °C, VWR International, LLC, West Chester, PA, USA). A small volume (100 ll) for quantification by HPLC was withdrawn from each container before irradiation, hourly during the first 3 h, then after 5 h and approximately 8 h (corresponding to 1.2  106 lux
h). The undiluted samples containing 1  10 3 M THPP were diluted 100 times with methanol and the samples containing 1  10 5 M THPP were diluted 10 times before quantification with the previously described HPLC method (2.2 Solubility test).

2.4. Polarity measurements

Fig. 1. Molecular structure of THPP.

Polarity (ENR) testing of NADES was performed with Nile red (NR, Life Technologies, Eugene, OR, USA) as a solvatochromic probe. A small amount of NR was added to each deep eutectic solvent. The recorded absorption maximum (kmax) of NR was applied in the formula ENR (kcal/mol 1) = hckmaxNA where h is Planck’s constant, c is the velocity of light and NA is Avogadro’s constant [22,23].

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2.5. Absorption spectroscopy Absorption spectra were recorded between 190 and 700 nm on a Schimadzu UV-2101 PC UV–Vis scanning spectrophotometer using a quartz cuvette with 1 cm cell path. MC and GC containing 0.05 mg/ml THPP, CS and MG containing 0.1 mg/ml THPP, and samples diluted 0–200 times in MilliQ water, PBS and in 0.9% (w/v) NaCl in MilliQ water, were studied. Solutions of THPP were also prepared in methanol, in MilliQ water (by dilution of a stock solution of THPP in methanol to a residual of 1 % (v/v) methanol), formic acid (diluted 1:1 with MilliQ water or 1:1, 1:100 and 1:200 with PBS), citric acid (diluted 1:1, 1:100 and 1:200 with MilliQ water or PBS) and 5% (v/v) concentrated ammonia solution in MilliQ water. All samples were made in triplicate. 2.6. Fluorescence spectroscopy Fluorescence spectroscopy was performed on a Photon Technology International modular fluorescence system (London, Ontario, Canada) with Model 101 monochromator with f/4 0.2-m Czerny–Turner configuration. The instrument was equipped with a red-sensitive photomultiplier. An excitation and emission correction was automatically performed. The excitation and emission monochromator band passes were set at 2 nm and excitation wavelength at the absorption maximum of the solutions. Correction for the difference in absorbance at the excitation maximum was performed manually for all samples. All measurements were performed in quartz cuvettes with 1 cm cell path at 22 °C. 2.7. Stability of THPP in solution

1:100 with a citric acid solution. The citric acid solution was prepared by diluting a 2 M solution 1:100 with PBS. Bacterial suspensions in PBS were mixed (1:1) with PBS (controls), CS or MG with and without THPP diluted in PBS or with reference THPP solutions. Each assay contained one culture plate for irradiation and one for dark controls. The effect of incubation with the pH-neutral reference THPP solution before and after irradiation (10–60 min) and of different light doses (11–32 J/cm2) was investigated. The conditions that resulted in the highest log reduction of viable bacteria after treatment with neutral THPP were chosen as experimental conditions for the respective bacteria in the further studies. In agreement with previous studies, the Gram-negative bacterium E. coli required longer pre- and post-irradiation incubation and irradiation times than the Gram-positive bacterium E. faecalis for optimal phototoxicity [24–26]. E. faecalis samples were incubated (37 °C) for 10 min before irradiation, irradiated for 10 min (corresponding to 11 J/cm2 ± 10%) and incubated (37 °C) again for 10 min. Irradiation was performed in a previously described light-polymerisation unit containing three blue light fluorescent tubes emitting mainly blue light in the wavelength range 400–500 nm [27]. After irradiation the samples were diluted 40 times in PBS prior to incubation. E. coli samples were incubated (37 °C) for 30 min prior to irradiation, irradiated for 30 min (corresponding to 32 J/cm2 ± 10%) and subsequently incubated (37 °C) for 60 min. The samples were diluted 60 times in PBS after irradiation. After the final incubation the samples were plated onto TSB agar using an automatic spiral plater (Whitley, Don Whitley Scientific Ltd., Shirley, England, UK). Bacterial survival was estimated after 24 h incubation (37 °C) by counting CFUs using a colony counter (Acolyte, Symbiosis Europe, Cambridge, UK). Each treatment was performed with eight parallels.

Selected NADES containing 0.1 mg/ml THPP were diluted in MilliQ water 10–200 times (n = 2). Absorption and fluorescence maxima and intensity were measured after 0–6 weeks after dilution. As precipitation of sugar was observed in 100–200 times diluted CS solutions after 4 weeks and in all CS solutions after 6 weeks, these samples were filtered (0.45 lm, Spartan) before measurements. The same NADES containing 0.1 mg/ml THPP were also diluted 1:100 in PBS and the absorbance was measured 0–7 days after dilution (n = 3). The physical stability of THPP in 2 M citric acid solution (similar concentration of citric acid as in undiluted CS) diluted 1:100 in MilliQ water and in PBS was measured by absorption spectroscopy 0–7 days after dilution (n = 3). A concentration of 0.1 mg/ml THPP was prepared in ethanol followed by dilution 1:100 in each citric acid solution (i.e. 1% (v/v) residual ethanol).

Determination of statistically significant differences between treatments was performed using a two-sample t-test (Minitab 17) on the results from the study on photostability (2.3 Photostability of THPP) and phototoxicity (2.8 Bacterial phototoxicity of THPP). Evaluation of statistically significant differences in stability of solutions of THPP diluted in PBS (2.7 Stability of THPP in solution) was performed by one-way ANOVA using Fischer’s method for multiple comparisons (Minitab 17). A p-value of less than 0.01 was considered statistically significant.

2.8. Bacterial phototoxicity of THPP

3.1. Characterisation of selected NADES

Enterococcus faecalis (ATCC 19433) and Escherichia coli (ATCC 25922) were resuspended from glycerol at 20 °C in tryptone soy broth (TSB; Oxoid Ltd., Basingstoke, UK) and incubated (37 °C) for 24 h. Aliquots from the overnight cultures were diluted to OD600 0.03 in PBS and transferred to culture plate wells (24-well; Flat Bottom Cell+, Sarstedt, Inc., Newton, NC, USA) for studies of the bacteria in the stationary phase. For studies in the exponential phase aliquots from the overnight cultures were re-suspended in TSB and incubated (37 °C) to OD600 0.6. The bacterial suspensions were then centrifuged (4000g, 22 °C, 10 min), TSB was replaced with PBS and dilution to OD600 0.03 was performed. CS and MG were prepared with 50–500 nM THPP to give a final concentration of 0.5–5 nM after dilution 100 times in PBS. Reference supersaturated solutions of THPP were prepared by diluting a stock solution of THPP in ethanol with PBS; the final amount of ethanol was 1% (v/v). Acidic reference solutions of THPP were prepared by diluting a stock solution of THPP in ethanol

The prepared NADES had a water content of 20% (w/w) and appeared more polar than methanol (51.9 kcal/mol) and water (48.2 kcal/mol) (Table 2) [20]. The apparent pH values varied depending on the type of NADES (Table 1) and dilution factor (Table 2), but remained strongly acidic even upon 1:200 dilution

2.9. Statistical analysis

3. Results

Table 2 pH after dilution 1:1, 1:100 and 1:200 in PBS (pH 7.4), solubility of THPP, water content and polarity of selected deep eutectic solvents (n = 3). NADES

pH (1:1)

pH (1:100)

pH (1:200)

THPP (mg/ml)

Water content (% (w/w))

ENR (kcal/mol)a

CS MG MC GC

1.03 0.97 0.27 7.07

2.66 2.62 1.70 7.22

3.09 2.97 2.02 7.35

0.093 ± 0.003 1.036 ± 0.041 0.007 ± 0.004 n.d.

22.0 ± 1.5 22.1 ± 3.2 18.3 ± 2.8 n.i.

44.1 47.7 44.6 47.9

a ENR = hcNA/kmax. n.d. = below the quantification limit (2.17  10 HPLC method. n.i. = not investigated

8

M) of the

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with PBS. Addition of excess THPP to the NADES presented in Table 1 indicated the best solubilising properties of the NADES named CS, MG, MC and GC (cf. Table 1). These four NADES appeared green while the other solutions were either clear with blue particles or pale bluish green with THPP particles. CS, MG, MC and GC were therefore chosen to be further analysed by HPLC, UV–Vis and fluorescence. The amount of solubilised THPP in these selected NADES is listed in Table 2. CS and MG proposed the best solubilising properties and were chosen for further studies. Storage of the solutions for three days induced a colour change of THPP in GC from green to brown and an unpleasant smell. MC and GC were not studied beyond this point due to the inferior ability to solubilise THPP (Figs. S1 and S2, Supplementary data). 3.2. Absorption spectroscopy The eutectic solvents without THPP showed minimal absorbance above 350–370 nm (Fig. 2). Dilution of the pure NADES with PBS induced the largest changes in the absorption spectrum of CS (Fig. 2A) and MG (Fig. 2B), while GC (Fig. 2C) and MC (Fig. 2D) appeared to be less affected by the buffer. Dilution of the NADES with MilliQ water only resulted in reduced absorbance without any structural changes in the spectrum (results not shown). The absorption spectrum of THPP in methanol (a dark red solution) showed a maximum at 419 nm (Soret band) and four Q-bands (Table 3 and Fig. S3, Supplementary data). After a 100-fold dilution of THPP in methanol with purified water the solution turned yellow and the Soret band was slightly blue shifted (414 nm) and the Q-bands changed (Table 3 and Fig. S3, Supplementary data). The absorption maximum of THPP in ammonia was observed at

438 nm and two Q-bands appeared (Table 3 and Fig. S3, Supplementary data). In formic acid (diluted in water) the Soret band of THPP was red shifted to 445 nm, the four Q-bands disappeared and a new band appeared (Table 3 and Fig. S4, Supplementary data). The same pattern was observed in citric acid diluted in water (Table 3). Upon addition of THPP to MG and CS followed by dilution 0–100 times in water, the Soret band of THPP occurred at 445 nm and only one Q-band was visible (Table 3 and Figs. S4 and S5, Supplementary data). The absorbance decreased linearly as a function of the dilution factor. The absorbance spectra of THPP in GC and MC showed limited absorbance and broad Soret bands (Figs. S1 and S2, Supplementary data), indicating that only small amounts of THPP were fully dissolved in these NADES. THPP in CS and MG diluted with PBS had maximum absorption at 424 nm (Table 3 and Fig. S6, Supplementary data). The Soret band was broader and the absorbance was lower in samples diluted in PBS or in NaCl solution compared to similar samples diluted in MilliQ water. A similar blue-shift was observed upon dilution of the formic acid solution 1:100 in PBS or 0.9% (v/v) NaCl (Table 3 and Fig. S6, Supplementary data), in the citric acid solution diluted 1:100 in PBS or 0.9% (v/v) NaCl (Table 3), and after dilution of the NADES containing THPP 1:100 in 0.9% (w/v) NaCl aqueous solution (Table 3 and Fig. S7, Supplementary data). 3.3. Stability of THPP in NADES diluted in MilliQ water Samples of THPP in CS and MG diluted 10–200 times in water and stored for up to 6 weeks showed non-linear changes in fluorescence intensity, area under the emission curve (AUCem,

Fig. 2. Absorption spectra of undiluted (dash-dotted line), 1:1 diluted (dashed line), 1:100 diluted (dotted line) and 1:200 diluted (solid line) (A) CS, (B) MG, (C) GC and (D) MC in PBS in the absence of THPP.

Table 3 Soret band, Q-bands and emission maximum (nm) of THPP in different solutions.

a b

THPP solution

kSoret

kQ1

kQ2

kQ3

kQ4

kQ5

kem1

kem2

Methanolb Methanol: water (1:100)b Ammonia: water (5%)b Formic acid: water (1:1)b Formic acid: PBS (1:100)b Citric acid: water (1:100) Citric acid: PBS (1:100) MG/CSb MG/CS + water (1:100)b MG/CS + PBS (1:100)b MG/CS + NaCl (1:100)b MCb GCb

419 414 438 445 419 (453) 445 419 (453) 450 445 424 (454) 423 (453) 456 429a

517 529 – – – – – – – – – – –

555 578 – – – – – – – – – – –

593 – 594 – (657) – (658) – – (657) (657) – –

650 661 664 (623) 690 (623) 692 (623) – 693 692 (640) (645)

– – – 682 – 682 – 684 682 – – >700 >700a

658 – – – 732 – n.i. – – 736 732 – –

722 n.i. n.i. 736

Weak signal, broad peak. Absorption spectrum in Supplementary data. Brackets indicate shoulder. n.i. = not investigated.

n.i. 735 735

737 773a

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650–800 nm) and emission maximum. The absorbance wavelength maximum remained constant (445 nm) as the samples were diluted 10–200 times and during storage. The reduction in absorbance at 445 nm upon dilution was linear. The absorbance decreased in a linear manner in all samples during 6 weeks of storage (2–30%). An increase in fluorescence intensity that was inversely proportional to the decrease in absorbance was also observed for these samples during storage. Samples diluted 50 and 100 times resulted in the highest and most stable fluorescence intensity. The samples diluted 100 and 200 times, however, were the most stable with respect to the ratio AUCem/absorbance445nm (Table 4). The Stoke’s shift varied within 5% during storage of all samples (Table 4). A plot of AUCem/absorbance445nm was made to observe the concentration independent change in fluorescence (Fig. 3). The 10-fold dilution of CS showed a linear increase (r = 0.951) in AUCem/absorbance445nm with time, whereas the 50-fold dilution showed a non-linear increase in AUCem/absorbance445nm, and the 50-fold dilution of MG an overall decreasing trend in AUCem/absorbance445nm (Fig. 3). The other dilutions showed only minor changes in AUCem/absorbance445nm during storage (Table 4). Quantification of THPP in the diluted samples, performed by HPLC at t0 (freshly made dilutions of samples of THPP in CS and MG that had been stored for 42 d) and after 42 d (t42), revealed a 5–80% reduction in THPP concentration in the diluted samples after storage (results not shown). The highest reduction was seen in 200 times diluted CS (80% vs. 30% in MG) and the lowest reduction in MG diluted 50 and 100 times (10% and 5%, respectively). The THPP concentration decreased more in diluted CS than in diluted MG during storage. No new peaks were observed in the HPLC chromatograms after storage for 42 d (data not shown). Precipitation of sugars was observed in 100–200 times diluted CS solutions after 28 d and in all CS solutions after 42 d and may have entrapped some THPP. Otherwise no precipitation of THPP was observed. A supersaturated solution of THPP prepared from a stock solution in methanol by dilution with MilliQ water (1% residual Table 4 Emission (em) maxima of dilutions of NADES in water containing THPP (n = 2) after 0 (t0) and 42 d (t42) storage, the Stoke’s shifts and % change in AUCem/absorbance445nm. The Stoke’s shifts are based on the Q-band at 682 nm. kex t0–t42 was 445 nm. Preparation

Dilution factor

kem (nm)

Stoke’s shift (nm)

t0

t42

t0

t42

CS CS CS CS MG MG MG

10 50 100 200 50 100 200

735 ± 4 732 ± 2 731 ± 1 728 ± 0 735 ± 1 731 ± 0 732 ± 1

738 ± 1 733 ± 1 731 ± 1 730 ± 0 732 ± 1 732 ± 2 733 ± 3

53 ± 4 50 ± 2 49 ± 1 46 ± 0 53 ± 1 49 ± 0 50 ± 1

56 ± 1 51 ± 1 49 ± 1 48 ± 0 50 ± 1 50 ± 1 51 ± 3

Dt0?t42 AUCem/ abs445nm (%) +139 +16 7 +4 23 +5 3

methanol) was not physically stable, as demonstrated by a 25% decrease in absorbance at 418 nm after 3 h. In comparison, the absorbance of the 50 and 100 times diluted MG and CS with THPP decreased 1–3% after 7 d and totally 2–15% after 6 weeks (results not shown). The largest decrease was observed in the NADES diluted 200 times. 3.4. Dilution in MilliQ water vs. PBS The absorbance of THPP in NADES and in a citric acid aqueous solution diluted 1:100 in MilliQ water did not decrease significantly during storage for 7 d (p > 0.01, Tables 5 and 6). By dilution in PBS, however, the absorbance decreased markedly during storage (Tables 6 and 7). 3.5. Photostability of THPP THPP in undiluted NADES was more photostable than in methanol at equal concentration (Fig. 4a). The rate of THPP degradation was significantly higher in methanol than in the NADES, and in CS than in MG (p < 0.01). THPP appeared slightly more photostable in the NADES diluted in water compared to NADES diluted in methanol (p < 0.01, Fig. 4b). The degradation was more rapid in the diluted NADES samples than in pure methanol containing the same amount of THPP (p < 0.01). 3.6. Bacterial phototoxicity Both doses (11 and 32 J/cm2) of blue light in the presence of the NADES without PS induced approximately 0.4 log reductions in viable E. faecalis and E. coli upon irradiation (resulting pH 3.0; Fig. 5). A solution of citric acid (2 M) diluted 1:100 with PBS (i.e. 1:200 after mixing with bacterial suspension, resulting pH 3.1) resulted in a non-significant log reduction of viable E. coli (p > 0.01, results not shown). Exposure to the supersaturated solution of THPP in PBS (pH 7.4) combined with blue light resulted in >6 log reductions of E. faecalis in the stationary phase at 5 nM concentration (Fig. 5a; i.e. 2.5 nM after mixing with bacterial suspension). Supersaturated THPP solutions with the concentrations 1 lM and 10 lM induced a 0.4 and 2.3 log reduction in viable E. coli in the stationary phase, respectively. Increasing the concentration of THPP to 20 lM still resulted in 2.3 log reductions in viable E. coli, implying that increasing the concentration of THPP only leads to in aggregation, filter effect and little additional phototoxic effect (results not shown). The preparations of THPP in citric acid and PBS (1% (v/v) residual ethanol) induced 0.5–3.3 log reductions in viable E. coli after treatment with 1–10 nM THPP (Fig. 5b; i.e. 0.5–5 nM after mixing with bacterial suspension). Exposure to 5 nM THPP prepared in CS and MG (i.e. 2.5 nM) resulted in no viable E. faecalis and E. coli in the stationary phase (Fig. 5). E. faecalis

Fig. 3. Ratio of area under the curve (AUCem) of the emission band (650–800 nm) and the absorbance (a.u.) at the excitation wavelength (445 nm) after 0–42 d storage at 22 °C protected from light (average of n = 2 ± the highest and lowest value). THPP dissolved in (A) CS at 10 dilution, (B) CS at 50–200 dilution, and (C) MG at 50–200 dilution.

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THPP in MG + MilliQ water (1:100)

Time (h)

kmax (nm)

Absorbance ± SD

% Initial ± SD

kmax (nm)

Absorbance ± SD

% Initial ± SD

0 20 96 168

445 445 445 445

0.493 ± 0.018 0.489 ± 0.018 0.484 ± 0.016 0.483 ± 0.018

100 ± 4 99 ± 4 98 ± 3 98 ± 4

445 445 445 445

0.494 ± 0.014 0.490 ± 0.014 0.483 ± 0.009 0.478 ± 0.012

100 ± 3 99 ± 3 98 ± 2 97 ± 2

Table 6 Absorption maximum, absorbance and % of initial absorbance (±SD) of THPP in citric acid solutions diluted in MilliQ water or PBS after storage 0–168 h. Time (h)

0 20 96 168

THPP in citric acid + MilliQ water (1:100)

THPP in citric acid + PBS (1:100)

kmax (nm)

Absorbance ± SD

% Initial ± SD

kmax (nm)

Absorbance ± SD

% Initial ± SD

445 445 445 445

0.500 ± 0.015 0.493 ± 0.012 0.471 ± 0.015 0.468 ± 0.026

100 ± 3 99 ± 2 94 ± 3 94 ± 6

419 419 416 416

0.146 ± 0.003 0.109 ± 0.003 0.059 ± 0.005 0.029 ± 0.006

100 ± 2 75 ± 3 41 ± 9 20 ± 21

Table 7 Absorption maximum, absorbance and % of initial absorbance (± SD) of THPP in CS and MG diluted in PBS after storage 0–168 h. Time (h)

0 20 96 168

THPP in CS + PBS (1:100)

THPP in MG + PBS (1:100)

kmax (nm)

Absorbance ± SD

% Initial ± SD

kmax (nm)

Absorbance ± SD

% Initial ± SD

424 423 423 423

0.074 ± 0.005 0.053 ± 0.003 0.033 ± 0.004 0.014 ± 0.002

100 ± 6 72 ± 5 45 ± 11 19 ± 11

424 423 422 423

0.134 ± 0.007 0.092 ± 0.007 0.029 ± 0.004 0.013 ± 0.002

100 ± 5 69 ± 8 22 ± 14 9 ± 18

Fig. 4. (a) Photostability of 1  10 3 M THPP in methanol (diamond), in undiluted CS (square) and in undiluted MG (triangle) ±SD (n = 3). (b) Photostability of 1  10 5 M THPP in methanol (diamond), in MG diluted 50 times in water (triangle) or in methanol (), and in CS diluted 50 times in water (square) or in methanol (circles) ±SD (n = 3).

Fig. 5. Viable bacteria in the stationary phase after exposure to THPP with or without simultaneous irradiation expressed as mean colony forming units per ml (CFU/ml) + SD. THPP solutions in pure ethanol, CS or MG were diluted 100 times in PBS (or a combination of citric acid and PBS) to yield 1 nM, 5 nM, 10 nM or 1 lM THPP. Black columns = non-irradiated samples; white columns = irradiated samples; x = no viable bacteria. ⁄Significant log reduction in viable bacteria. (a) E. faecalis; light dose 11 J/cm2. (b) E. coli; light dose 32 J/cm2.

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Fig. 6. Overlap of the absorption spectra of 0.1 mg/ml THPP in citric acid (1% (v/v) residual ethanol), MG and CS diluted 1:100 in PBS (n = 3) with the emission spectrum of the blue light source.

was also completely photoinactivated by 1 nM THPP in the diluted MG preparation (Fig. 5a; i.e. 0.5 nM). In the exponential phase, only 0.5 nM and 1 nM THPP in CS and MG (0.25 nM and 0.5 nM after mixing with bacterial suspension) were needed for complete photoinactivation of E. faecalis and E. coli, respectively. The CS and MG dilutions containing 0.5 nM THPP (i.e. 0.25 nM) resulted in a 3.0 and 3.3 log reduction of viable E. coli in the exponential phase, respectively. The absorbance of the solution of 0.1 mg/ml THPP in citric acid diluted 1:100 in PBS had the most extensive overlap with the blue light source (Fig. 6). The area under the absorption curve (AUC) was 1:0.65:0.41 for THPP in citric acid:MG:CS diluted 1:100 in PBS (0.1 mg/ml THPP, 350–600 nm).

4. Discussion The neutral p-hydroxyphenyl porphyrin THPP was selected as PS to challenge the solubilising properties of the NADES and the current understanding of the lack of bacterial phototoxicity of neutral porphyrins. The selected four NADES appeared more polar than water, yet solubilised THPP well. For comparison, THPP in pure water adhered to the walls of the container above the liquid surface and showed no interaction with the solvent. The proposed structure of NADES resembles liquid crystals where the molecules are arranged through hydrogen bonding and other intermolecular interactions [20]. The water that remains after preparation also participates in the macromolecular structure [20]. A PS should exhibit both hydrophilic and hydrophobic properties to be able to interact with and penetrate the bacterial outer structure [3]. THPP is a relatively hydrophobic porphyrin with a log P value of 4.0 [28]. The hydrophobicity and lack of cationic charge could lower the interaction probability between the PS and the bacteria and result in formation of cytotoxic species at a non-lethal distance from the bacteria upon irradiation. To overcome this obstacle we designed hydrophilic preparations of THPP without encapsulating THPP in a carrier that would prolong its release time from the vehicle. The absorption spectra of THPP in undiluted CS and MG and after dilution in water were comparable to the spectrum of THPP in 50% (v/v) formic acid (Figs. S4 and S5, Supplementary data). These spectra are similar to those of THPP dissolved at pH below 4 [29,30]. Sobczyn´ski et al. (2013) asserted that the Q-band was characteristic for the protonated porphyrin core [29]. The similarity between the absorption spectra of THPP in NADES and in formic acid indicates the formation of the protonated form of THPP in NADES. It is not unlikely that the acids in selected NADES (e.g. citric acid in CS and malic acid in MG) can donate a hydrogen atom to a pyrrole in THPP, thus yielding a cationic porphyrin with one or

several positive charges. The assertion that NADES may act like a protic solvent and thus solubilise several poorly water-soluble compounds has been suggested [20,31]. The similarities between the absorption and fluorescence spectra of THPP in NADES and in formic acid confirm the presence of the protonated form of THPP in selected NADES (Figs. S4 and S8, Supplementary data). Guo et al. explained the bathochromic shift of the Soret band and the one Q-band of THPP as J-aggregate shapes due to formation of two-valence porphyrin cations [30]. In line with this suggestion the absorption spectra obtained for THPP in NADES indicate that J-aggregates are formed. The fluorescence pattern, however, does not show evidence of the formation of J-aggregates in NADES. THPP has two emission bands at approximately 658 nm and 722 nm in methanol (Table 3), ethanol, chloroform, DMF and in aqueous solutions of cyclodextrins [28,30,32]. J-aggregates are characterised inter alia by a small Stoke’s shift. THPP in NADES, including samples diluted in purified water, in PBS and in 0.9% (w/v) NaCl, has only one emission band at approximately 734 nm (Table 3 and Figs. S8 and S9, Supplementary data), which gives a fairly large Stoke’s shift (approximately 50 nm based on the Q-band). The emission peak is symmetrical and indicates only one emitting species. After dilution 1:100 with PBS or 0.9% (w/v) NaCl of CS and MG containing THPP the fluorescence intensity decreased. Fluorescence quenching by chloride is well established [33]. The change in absorption spectrum in the presence of chloride further indicates an interaction between the protonated THPP and chloride ions (possibly salt formation). The solubilising effect of 5% ammonia on THPP was hypothesised to be due to the high polarity and ionisation of the porphyrin as the resulting solution appeared green, similar to the formic acid solution. However, the two Q-bands were not consistent with the Q-bands of THPP in methanol, acids or NADES (Figs. S3 and S4, Supplementary data). The changes in absorbance of THPP in the presence of a strong base were therefore assumed to be due to deprotonation of the phenols [34]. The ionisation and change in molecule symmetry resulted in a bathochromic shift, a wide Soret band and two Q-bands due to the presence of different deprotonation states of THPP. The presence of coexisting absorbing species (i.e. monomers and aggregates) is probably also the reason for the wide Soret band (70 nm) of THPP in water (containing 1% (v/v) methanol; Fig. S3, Supplementary data). The wide, fairly symmetric Soret band of THPP in formic acid diluted with PBS and in NADES after dilution in PBS (Fig. S6, Supplementary data) or NaCl solution (Fig. S7, Supplementary data) was probably due to the equilibrium between protonated THPP and the THPP salt. The absorbance of THPP in these solutions was lower than in water, most likely due to aggregation and precipitation of the less soluble chloride salt. Dilution of a solution of THPP in pure methanol a 100-fold with water resulted in a slight blue shift compared to the pure methanol solution which can be attributed to H-aggregates and distortion of the porphyrin ring [32,35]. The increase in AUCem/absorbance445nm during storage of CS diluted 10 and 50 times in water indicates the formation of a more structured system. The THPP molecules seem to be rigidified in the NADES network resulting in a higher fluorescence quantum yield (without any changes in the absorption spectrum; Fig. 3). As the absorption spectra remained unchanged during storage, except for the small reduction of absorbance as THPP precipitated, there was no apparent change in monomer or aggregate state. The previously mentioned decrease in THPP concentration in MG and CS diluted 200 times in water (approximately 30% and 80%, respectively) during 6 weeks of storage did not coincide with the minimal change in AUCem/absorbance445nm of the same preparations (Table 4). This finding supports the theory that THPP is solubilised in the NADES network which becomes more rigid during storage. The extensive increase in AUCem/absorbance445nm during storage

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of the 10-fold dilution of CS indicates that this rigidification is more pronounced in these less diluted samples (Fig. 3a). Further dilution weakens the network which results in more molecular mobility and hence reduced fluorescence. The reduction in AUCem/absorbance445nm during storage of 50-fold diluted MG showed that the structure of the NADES affects the reaction pattern of THPP, i.e. which deactivation pathway that is given priority. The most pronounced decrease in absorbance was observed upon storage of the 200-fold diluted samples. This can probably be ascribed to a less structured NADES network (i.e. less solubilising effect) combined with a slight increase in pH moving the equilibrium towards the deprotonated and less soluble form of THPP. Formation of J-aggregates of THPP dissolved in this solvent is unlikely based on the combined results. The important observation from this stability study is that the solubilising interactions between NADES and THPP still remain intact even upon extensive dilution in water and storage for several weeks. Several factors may contribute to the photostabilisation of THPP in NADES such as higher viscosity leading to a slower reaction rate and the formation of a stabilising network between the NADES and THPP. In methanol THPP appeared more available for photochemical degradation due to lack of a protective surrounding network. THPP forms aggregates in methanol at this concentration (1  10 3 M). The photostabilising effect of the NADES was apparently higher than the shielding effect obtained by aggregate formation in methanol. The finding that the porphyrin was more photostable in NADES diluted in water than in methanol indicate that the addition of larger amounts of methanol (i.e. a 50-fold dilution) to the NADES disrupts the NADES network with the result that THPP was solubilised primarily by methanol. Dilution of the NADES 50 times in water appeared to maintain the solubilising NADES network. Upon further dilution with water the photostability of THPP decreased below the photostability in methanol in spite of a slightly higher viscosity in the former solution. Intermolecular interactions between THPP and the NADES and the unique NADES network appear to be the most prominent photostabilising factors in undiluted NADES. Many of these interactions are disturbed upon dilution. THPP is likely to be protonated and thereby dissolved in strongly acidic solutions (pKa of THPP central ring nitrogens is approximately 4.5 [29]). As the remaining water in the NADES was tightly bound and incorporated in the NADES network, the pH values obtained for the undiluted NADES (Table 1) are uncertain. Upon dilution, however, the pH values are more valid (Table 2). It was postulated that the NADES that appeared to be acidic would solubilise the porphyrin well. However, as seen from Table 1, only a few of the very acidic NADES could dissolve THPP. The solubilising effect of the NADES on THPP was probably due to intermolecular hydrogen bonding and proton donor–acceptor interactions. Different spacing and number of hydrogen donors and acceptors of the NADES are possibly some of the reasons why some NADES solubilised THPP well while others were poor solubilisers of this porphyrin. Upon dilution of THPP in NADES or a citric acid solution with PBS the absorbance of THPP decreased rapidly (Tables 6 and 7) compared to samples diluted in water (Tables 5 and 6). It seems plausible that the interaction between protonated THPP and chloride present in PBS leads to a gradual precipitation of the porphyrin. The presence of chloride ions thus disturbs the solubilising interactions between the NADES and THPP. There was apparently no significant difference in the physical stability of THPP in NADES and citric acid diluted in water or in PBS. The 1:100 and 1:200 diluted NADES appeared acidic. Samples of THPP in citric acid diluted in PBS were therefore prepared to investigate whether the enhanced phototoxicity was solely due to a pH effect or to the specific combination of THPP and NADES. The citric

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acid samples had a pH similar to the diluted NADES. The study was performed on E. coli as Gram-negative bacteria generally are considered more difficult to inactivate with aPDT [36]. Fig. 6 shows that THPP in citric acid solution had the most extensive overlap with the blue light source, yet THPP in NADES was more phototoxic to the bacteria (Fig. 5b). This means that even though the portion of excited THPP was lower in the NADES solutions than in the citric acid solution, the presence of NADES increased the phototoxic potential of the dissolved porphyrin in these preparations. After 200 times dilution with PBS, the obtained pH values suggest that the NADES itself could weaken the bacteria in the pre-irradiation incubation period. The citric acid solution (pH 3.1) did not affect the bacteria significantly (results not shown). The difference in toxicity was not significant between the diluted NADES and the citric acid solution. However, the amount of viable bacteria was slightly, and significantly, reduced after treatment with the diluted NADES without THPP (Fig. 5). This reduction was probably due to an acidic effect combined with the ‘‘NADES effect’’. The NADES might weaken the bacteria by extracting both water soluble and insoluble components from the bacterial membrane (NADES solubilise several small molecules and macromolecules, inter alia DNA [20]). The disruption of the cell wall of the bacteria due to the presence of delocalised charges in NADES has also been suggested [31]. The acid tolerance of bacteria differs between species and strains. Several E. coli strains tolerate exposure to pH 2.5 for several hours with only minimal reductions in viable CFU/ml, while other strains are more acid-sensitive [37]. E. faecalis has also been reported to tolerate low pH values, as expected from a bacterium associated with endodontic diseases, and only to be completely eradicated within 3 h of exposure to pH 2.5 [38]. The selected bacterial strains in the current study appeared to endure the acidic environment fairly well (only non-significant 0.2 log reductions in viable bacteria after exposure to a pH 3 citric acid solution). Nevertheless, an extensive study on the acid tolerance of these bacteria is beyond the scope of this article. THPP was more phototoxic to E. faecalis and E. coli in the exponential phase than in the stationary phase. This may be explained by the activation of the RpoS system in E. coli in the stationary phase resulting in increased defences against ROS and low pH [39,40]. In relation to the current study, E. faecalis has previously been reported to be less tolerant to general and photodynamic stress in the exponential phase than in the stationary phase [41,42]. There are, however, conflicting reports in the literature whether different bacteria are more susceptible to PDT in the exponential phase or the stationary phase [43–45]. The bacterial tolerance levels appear to depend on the bacterial strain, the PS and the excipients (and thus possibly also the light source used). 5. Conclusion Even though neutral porphyrins like THPP have been regarded as less phototoxic towards Gram-negative bacteria, only nanomolar amounts of THPP in NADES were needed for complete photoinactivation of E. coli. THPP became protonated in the NADES, but the phototoxic effect was higher in the NADES preparations than in an equally acidic citric acid solution. A synergistic antibacterial effect between THPP and NADES is evident. Preliminary results in our laboratory indicate that this effect is valid also for other photosensitisers. The physical- and photochemical stability of THPP in NADES in undiluted samples were superior to diluted samples indicating that these preparations should be stored undiluted. NADES as a delivery principle of photosensitisers shows potential for use in aPDT, especially in topical treatment of microbial infections on the skin and in the oral cavity. Further development might also reveal the potential of NADES in diagnosis were the product may be applied locally without extensive dilution with body fluids.

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Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This research received no grant from any funding agency in the public, commercial or not-for-profit sectors. Acknowledgements The authors thank Ivar Grove, MSc, for developing the method for HPLC analysis of the porphyrin. We are grateful for the E. faecalis strain FCC120 (ATCC 19433) received as a gift from Prof. Ingar Olsen at the Faculty of Dentistry, University of Oslo, Norway. We thank MSc. Inger Sofie Dragland (NIOM) for identity confirmation and gene sequencing of this strain in collaboration with the Faculty of Dentistry. We further wish to thank Dr. Håkon Valen Rukke (NIOM) for valuable feedback on the bacterial assay. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2015. 04.022. References [1] M.R. Hamblin, T. Hasan, Photodynamic therapy: a new antimicrobial approach to infectious disease?, Photochem Photobiol. Sci. 3 (2004) 436–450. [2] H. Ikai, Y. Odashima, T. Kanno, K. Nakamura, M. Shirato, K. Sasaki, Y. Niwano, In vitro evaluation of the risk of inducing bacterial resistance to disinfection treatment with photolysis of hydrogen peroxide, PLoS One 8 (2013) e81316. [3] A. Almeida, A. Cunha, M.A.F. Faustino, A.C. Tomé, M.G.P.M.S. Neves, Porphyrins as antimicrobial photosensitizing agents, Compr. Ser. Photoch. 11 (2011) 83– 160. [4] A.E. O’Connor, W.M. Gallagher, A.T. Byrne, Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy, Photochem. Photobiol. 85 (2009) 1053–1074. [5] T.S. Mang, D.P. Tayal, R. Baier, Photodynamic therapy as an alternative treatment for disinfection of bacteria in oral biofilms, Laser Surg. Med. 44 (2012) 588–596. [6] M. Berenbaum, S. Akande, R. Bonnett, H. Kaur, S. Ioannou, R. White, U. Winfield, Meso-tetra (hydroxyphenyl) porphyrins, a new class of potent tumour photosensitisers with favourable selectivity, Br. J. Cancer 54 (1986) 717. [7] Y. Nitzan, M. Gutterman, Z. Malik, B. Ehrenberg, Inactivation of gram-negative bacteria by photosensitized porphyrins, Photochem. Photobiol. 55 (1992) 89–96. [8] S.C. Karunakaran, P.S.S. Babu, B. Madhuri, B. Marydasan, A.K. Paul, A.S. Nair, K.S. Rao, A. Srinivasan, T.K. Chandrashekar, C.M. Rao, R. Pillai, D. Ramaiah, In vitro demonstration of apoptosis mediated photodynamic activity and NIR nucleus imaging through a novel porphyrin, ACS Chem. Biol. 8 (2013) 127–132. [9] C.S. Prasanth, S.C. Karunakaran, A.K. Paul, V. Kussovski, V. Mantareva, D. Ramaiah, L. Selvaraj, I. Angelov, L. Avramov, K. Nandakumar, N. Subhash, Antimicrobial photodynamic efficiency of novel cationic porphyrins towards periodontal gram-positive and gram-negative pathogenic bacteria, Photochem. Photobiol. 90 (2014) 628–640. [10] S. Banfi, E. Caruso, L. Buccafurni, V. Battini, S. Zazzaron, P. Barbieri, V. Orlandi, Antibacterial activity of tetraaryl-porphyrin photosensitizers: an in vitro study on Gram negative and Gram positive bacteria, J. Photochem. Photobiol. B: Biol. 85 (2006) 28–38. [11] I. Stojiljkovic, B.D. Evavold, V. Kumar, Antimicrobial properties of porphyrins, Exp. Opin. Invest. Drugs 10 (2001) 309–320. [12] M. Merchat, J.D. Spikes, G. Bertoloni, G. Jori, Studies on the mechanism of bacteria photosensitization by meso-substituted cationic porphyrins, J. Photochem. Photobiol. B: Biol. 35 (1996) 149–157. [13] C.L. Friedrich, D. Moyles, T.J. Beveridge, R.E.W. Hancock, Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria, Antimicrob. Agents Chemother. 44 (2000) 2086–2092. [14] T. Maisch, R.-M. Szeimies, G. Jori, C. Abels, Antibacterial photodynamic therapy in dermatology, Photochem. Photobiol. Sci. 3 (2004) 907. [15] Z. Malik, J. Hanania, Y. Nitzan, New trends in photobiology bactericidal effects of photoactivated porphyrins — an alternative approach to antimicrobial drugs, J. Photochem. Photobiol. B: Biol. 5 (1990) 281–293. [16] H.M. Tang, M.R. Hamblin, C.M.N. Yow, A comparative in vitro photoinactivation study of clinical isolates of multidrug-resistant pathogens, J. Inf. Chemother. 13 (2007) 87–91. [17] Z. Malik, H. Ladan, Y. Nitzan, Photodynamic inactivation of Gram-negative bacteria: problems and possible solutions, J. Photochem. Photobiol. B: Biol. 14 (1992) 262–266.

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