Journal of Photochemistry and Photobiology B: Biology xxx (2015) xxx–xxx

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Strategies to optimize photosensitizers for photodynamic inactivation of bacteria Maisch Tim Department of Dermatology, Antimicrobial Photodynamic and Cold Plasma Research Unit, University Hospital, Regensburg, Germany

a r t i c l e

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Article history: Received 27 November 2014 Received in revised form 13 May 2015 Accepted 15 May 2015 Available online xxxx Keywords: Phenothiazinium Porphyrines Fullerenes Perinaphthenone Vitamin B2 Susceptibility Wounds Bacteria

a b s t r a c t The Infectious Diseases Society of America (IDSA) highlights that over the past several years, the number of new antibacterial drugs approved continues to decrease (Boucher et al., 2009) [1]. Bacteria are very good in developing resistance against antibiotics in a short time. Therefore new approaches like antibacterial photodynamic inactivation of bacteria (aPDI) will become more important in the future as antimicrobial resistance is expected to continue to increase. This review summarises the potential of the susceptibility of bacteria to aPDI and the strategies to optimize leading photosensitizers which are useful for aPDI. The most appropriate photosensitizers belonging to the chemical classes of phenothiazinium, porphyrine, fullerene and perinaphthenone. They all share the following characteristics: positively-charged, water-soluble and photostable. Taken together the most promising clinical applications of aPDI are (i) decolonization of pathogens on skin, (ii) treatments of the oral cavity like periodontitis and root canal infection and (iii) superinfected burn wounds, because these are relatively accessible for photosensitizer application and illumination. Ó 2015 Published by Elsevier B.V.

1. Introduction Today the increasing resistance of bacteria against antibiotics is one of the most important clinical challenges; the so-called ‘‘ESKAPE’’-pathogens (Enterococcus faecium, Staphylococcus aureus,

extracellular or intracellular localization of the photosensitizer is needed and no specific targets are in the focus for the oxidative burst mediated by aPDI [4]. Therefore aPDI will become more important in the future as antimicrobial resistance is expected to continue to increase.

Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter strains) are the superbugs of the 21th century, because they can ‘‘escape’’ more or less any single kind of antibiotic treatment. The actual WHO’s 2014 report on global surveillance of antimicrobial resistance reveals that ‘‘antibiotic resistance is no longer a prediction for the future; it is happening right now, across the world, and is putting at risk the ability to treat common infections in the community and hospitals [2]. Without urgent and coordinated action, the world is heading towards a post-antibiotic era, in which common infections and minor injuries, which have been treatable for decades, can once again kill’’ [2]. Both selection and evolution of antibiotic-resistant bacteria is a non-stoppable process due to the random rate of mutation in the bacteria that can provoke antibiotics ineffective within short time [3]. Therefore new approaches are needed acting as a multi-target process to avoid the development of resistances in bacteria. The antibacterial photodynamic inactivation of bacteria (aPDI) represents such a multi-target damaging process. No specific E-mail address: [email protected]

2. General aspects of photodynamic inactivation of bacteria At the beginning of the 20th century Proteus vulgaris was inactivated by the combination of a fluorescent dye, light and oxygen for the first time [5,6]. Tappeiner termed this interaction of light, oxygen and a dye as ‘‘photodynamic reaction’’. Nowadays the lethal effect of aPDI is based on the principle that visible light activates a photosensitizer to lead the formation of reactive oxygen species, which induces a phototoxic damage immediately during illumination. The absorption of light (visible wavelength range 400– 700 nm) by the ground state of a PS leads to a transition to its singlet state and via intersystem crossing to its excited triplet state, than two mechanisms of action take place [7]. Type I photosensitization processes can produce different kinds of reactive intermediates. In the present of oxygen, type I processes can induce the formation of species like hydrogen peroxide (H2O2), superoxide radical anion (O2–) and hydroxyl radical (OH) via Fenton reaction. These ROS are known to effectively oxidize a wide variety of biomolecules and ultimately cause substantial biological damage.

http://dx.doi.org/10.1016/j.jphotobiol.2015.05.010 1011-1344/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: M. Tim, Strategies to optimize photosensitizers for photodynamic inactivation of bacteria, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.05.010

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In type II reactions the excited PS transfers energy to molecular oxygen and generates highly reactive singlet oxygen (1O2) which photo-oxidizes biomolecules like lipids and proteins leading to lysis of cell membranes. The singlet oxygen quantum yield UD describes the ratio of the type II mechanism of a given photosensitizer [8]. In addition both type I and type II reactions can occur simultaneously. However several photosensitizers have shown different quantum yields of singlet oxygen. The antimicrobial photodynamic process exhibits several positive aspects for the treatment of microbial infections, including a broad spectrum of action, the efficient inactivation of antibiotic-resistant strains, the low mutagenic potential, and the lack of selection of photoresistant microbial cells [4]. Therefore an appropriate photosensitizer for aPDI should fulfill the following criteria in order to have a pronounced antimicrobial efficacy (minimum > 3log10 reduction of CFU) and low toxicity towards mammalian cells (‘‘therapeutic window’’):  High 1O2 quantum yield.  Photostable.  Broad spectrum of antimicrobial action (bacteria, fungi, parasites).  High binding affinity for microorganisms (positively charged PS for good adherence to negatively charged bacterial cell walls).  Low binding affinity and low toxicity for mammalian cells.  No mutagenicity (DNA damage must be avoided).  No dark toxicity.  Therapeutic window (photodynamic inactivation parameters necessary where bacteria are killed efficiently without damage of eukaryotic cells).

3. Susceptibility of bacteria to aPDI In general bacteria have developed several mechanisms to elude oxidative stress from the environment. This protective system consists of an enzyme network of proteins like catalase, peroxidase or superoxide dismutase detoxifying reactive oxygen species. Furthermore anti-oxidative molecules like carotenoids quenching the singlet oxygen as well as the triplet state of chlorophyll in photosynthetic active organisms like cyanobacteria [9]. In Rhodobacter sphaeroides a model bacteria to study bacteria photosynthesis singlet oxygen was found as a direct inducer of an alternative RpoHII-type sigma factor which is required for the expression of defense factors and that deletion of RpoHII leads to increased sensitivity against exposure to singlet oxygen originated by methylene blue and light [10]. Upon activation of the RpoHII genecluster an oxidative-stress defense system is expressed where proteins are involved for quenching of ROS, detoxification of peroxides and regulate redox and iron reactions. So far in human pathogenic bacteria defense systems against singlet oxygen itself are not present. In case of type-I induced ROS by a given photosensitizer, like methylene blue, TBO or curcumin, bacteria can produce oxidative-stress defense system to avoid an oxidative damage caused by aPDI. One major oxidative stress defense system is called the two stage soxR and soxS oxidative stress regulon [11]. Here the superoxide dismutase is one of the key player enzymes which metabolize super oxide anions to hydrogen peroxide and oxygen (Eq. (1)) [12,13]. þ ð2O 2 þ 2H ! H2 O2 þ O2 Þ

ð1Þ

Hydrogen peroxide itself can be scavenged by both alkyl hydroperoxide reductase (ahpCF) and catalase (katEG) to water and ground state oxygen [14,15]. Thereby Hydrogen peroxide serves as a sensor molecule for the transcription factor OxyR which

regulates the oxyR gene regulon (catalases and peroxidases) [14,15]. Furthermore H2O2 oxidizes Fe2+ via Fenton reaction whereby hydroxyl radicals are generated [16] (Eq. (2))

Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH

ð2Þ

The hydroxyl radical reacts with many organic compounds (e.g. fatty acids) by removal of a hydrogen atom, forming water and an alkyl radical (Eq. (3)). Than the alkyl radical reacts rapidly with oxygen forming a peroxy radical (Eq. (4))

OH þ R-H ! H2 O þ R

ð3Þ

R þ O2 ! R-O2

ð4Þ

Besides singlet oxygen, hydroxyl radicals, super oxide anions and reactive oxygen intermediates derived from singlet oxygen are the meaningful molecules that induce oxidative stress in bacteria. In aPDI the photosensitizers produce different amounts of singlet oxygen depending on the chemical structure (e.g. methylene blue: UD 0.52 [17], TMPyP: UD 0.77 [18] and SAPyR: UD 0.99 [19]). Depending on the localization of the photosensitizer singlet oxygen rapidly oxidizes all double bonds of fatty acids and proteins in the direct surrounding due to its high reactivity (+0.98 eV energy), short lifetime (5log10 steps could be achieved within a total treatment time of a few seconds. Another porphyrin derivative XF73 demonstrated a high antibacterial photodynamic efficacy (Fig. 2). Here the two positive charges (tri-methyl-ammonium) were arranged symmetrically and separated by a propyl-spacer (–C3H7) from the tetrapyrrol-ringsystem [22]. Such a spacer might provide a higher flexibility of the positive charge and therefore again a better interaction with the outer cell wall areas of bacteria. Next to the porphyrines cationic water-soluble Zn-phthalocyanines have also shown photodynamic inactivation of both Gram-negative and Gram-positive bacteria [48,49].

Please cite this article in press as: M. Tim, Strategies to optimize photosensitizers for photodynamic inactivation of bacteria, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.05.010

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Fig. 1. Chemical structures of new developed phenothiazinium dyes. Selection of new developed methylene blue derivatives (DMB [39], NMB [39], MB(3) [41] and MB(4) [42]) compared to MB or TBO.

Furthermore a multiple positive charged tetracationic Zn(II)phthalocyanine was able to kill bacteria but not TBO which was used as a reference photosensitizer [50]. 4.3. Fullerenes

Fig. 2. Chemical structures of TMPyP and XF73 [22].

Another class of interesting dyes is named fullerenes which are configured in a soccer ball like structure consisting of 60–70 carbon atoms. Tegos et al. functionalized such a soccer ball with hydrophilic and cationic groups to act as a photosensitizer for aPDI [51]. Fullerenes with polar diserinol and quarternary pyrrolidinium groups were reported to kill effectively microorganisms upon light activation (Fig. 3) [51]. Again increasing the number of positive charges was done to improve aPDI efficacy. Huang et al. showed that water-soluble deca-cationic fullerenes (containing one penta-cationic malonate ester moiety on each side of the dye) were able to eradicate both Gram-positive and Gram-negative bacteria [52]. Furthermore the authors concluded from this study, that singlet oxygen generated by these deca-cationic fullerenes is the relevant reactive oxygen specie to kill more Gram-positive bacteria, whereas the less permeable outer membrane area of Gram-negative bacteria needs the more reactive hydroxyl-radical to cause aPDI induced damage [52]. However both fullerenes generate the same amount of singlet oxygen, but in the presence of an electrolyte salt buffer system (PBS) the fullerene produces HO⁄ via a photoninduced electron-transfer (type-I mechanism) from the electron-donating counterion (I) and the excited triplet state of the fullerene. Such a reaction is driven by the electrolyte salts of the buffer system which facilitates the ion exchange and the iodide-dissociation. Thus a fullerene-radical is generated which in turn reacts with oxygen to produce a

Please cite this article in press as: M. Tim, Strategies to optimize photosensitizers for photodynamic inactivation of bacteria, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.05.010

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M. Tim / Journal of Photochemistry and Photobiology B: Biology xxx (2015) xxx–xxx

(A)

(B)

(C)

Fig. 3. Chemical structures of cationic fullerenes [51]. (A) Fullerene with quarternary pyrrolidinium groups (n = 1–3). (B) Fullerene with diserinol groups (n = 1– 4).

superoxide-radical that is the precursor molecule to the formation of hydroxyl-radicals [52–54]. 4.4. Perinaphthenone In order to optimize the efficacy of aPDI with respect to a type II mechanism of action only, the aromatic ketone 1H-phenalen-1-one structure was considered [55]. This perinaphthenone is a non-water soluble molecule and has shown a singlet quantum yield of UD 97%. In order to make this perinaphthenone water-soluble, Nonell et al. developed the 1-H-phenalen-1-one-2-sulfonic acid (PNS) molecule which is generating singlet oxygen almost quantitatively according to type-II mechanism (singlet quantum yield UD 1.03 ± 0.10) in aqueous media (Fig. 4) [56]. The negative charge of the sulfonic acid substituent of PNS prohibits the application as an antibacterial photosensitizer, but stabilizes the aromatic ringsystem against photobleaching [57]. Based on this PNS a new (2-((4-pyridinyl)methyl)-1Hphenalen-1-one (SAPYR) was developed which contains a positive charge (Fig. 4) [19]. For SAPYR, a 1 O2 quantum yield of UD 0.99 ± 0.05 could be determined. Cieplik et al. concluded that SAPYR acts via a dual mechanism of action [19]. Firstly SAPYR can disrupt the 3D-structure of a biofilm even without illumination, because it can act as a surfactant. This is attributed to the chemical structure of SAPYR which consists of the combination of a hydrophilic pyridinium group (positive charge group) and a hydrophobic tail. This similarity is known for surfactants such as cetylpyridinium chloride which is used as an antiseptic compound in mouthwashes, toothpastes and nasal sprays [58]. Busscher et al. demonstrated that cetylpyridinium-containing formulations can stimulate bacterial detachment [58]. Secondly light-activated SAPYR demonstrated efficient photodynamic

Fig. 4. Chemical structures of perinaphthenone molecules. (A) 1H-phenalen-1-one (PN) [56]; (B) 1-H-phenalen-1-one-2-sulfonic acid (PNS) [56]; and (C) (2-((4pyridinyl)methyl)-1Hphenalen-1-one (SAPyR) [19].

inactivation of bacteria in a polymicrobial biofilm consisting of E. faecalis, Actinomyces naeslundii and Fusobacterium nucleatum after one single treatment. 4.5. Vitamin B2 derivatives Vitamin B2 (riboflavin) is a naturally occurring compound which can be activated by UV to generate ROS [59]. Such a combination is used for photochemical inactivation of pathogens present in blood components like platelets concentrates [60]. To avoid the application of harzard UV new Vitamin B2 derivatives were synthesized to design safe (no broad UV irradiation) and effective photosensitizer to kill superbugs [61] (Fig. 5A). Therefore positive charges (NH+4) were added to the chromophore using a short alkyl chain linker. These new synthesized flavin derivatives showed a high quantum yield of singlet oxygen of approximately 75%. Light-activation of these riboflavin-derivatives decreased the viable number of bacteria up to 6 log10 steps of multi-resistant Superbugs (MRSA, EHEC, P. aeruginosa and A. baumanii) [61]. This new class of photosensitizer is considered safe in humans showing a great potential of bacterial killing without harming the adjacent tissue (‘therapeutic window’), because decomposition of flavin

Please cite this article in press as: M. Tim, Strategies to optimize photosensitizers for photodynamic inactivation of bacteria, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.05.010

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Fig. 5. Chemical structures of Vitamin B2 (riboflavin) derivatives and curcumin. (A) Riboflavin derivative FLASH-01a [61]. (B) Curcumin (1,7-bis-(4-hydroxy-3methoxyphenyl)-hepta-1,6-dien-3,5-dione) existing in tautomeric forms, including a 1,3-diketo form (left) and two equivalent enol form (right, only one enol form is pointed) [66,68].

molecules is ubiquitous (vitamin decomposition in milk products). The toxicity of products of decomposition of flavins by oxidation and hydrolysis processes is imperceptible because this is a daily process in foodstuff manufacture and inside the human body [62,63]. 4.6. Curcumin Another natural occurring photosensitizer is named curcumin which belongs to the group of diphenylheptanoids. These molecules are acting as a small group of plant secondary metabolites. Curcumin has shown antimicrobial photodynamic efficacy against several microorganisms (Fig. 5B) [64,65]. Due to the poor water solubility further optimization processes were done to improve the water-solubility. Winter et al. examined the applicability of polyvinylpyrrolidone (PVP)-curcumin mediated photodynamic inactivation of bacteria [66]. PVP does have the advantage to provide an advanced solubility of curcumin in aqueous solution without the need of organic solvents as penetration enhancers [67]. A reduction of viable bacteria of 99.9999% was achieved after incubation of 5 lM PVP-curcumin for 5 min and a light dose of 33.8 J/cm2 (435 ± 10 nm). Furthermore curcumin is known as a food additive (E100). Therefore the combination of food coloring by curcumin and acting as antimicrobial photodynamic active agents is of interest in foodstuff manufacture. Tortik et al. investigated the photodynamic decontamination of foodstuff (chicken meat and peppers) from S. aureus based on polyvinylpyrrolidone formulations of curcumin [68]. A Photodynamic inactivation of S. aureus on the food-surfaces was achieved. Bacteria applied on cucumber, pepper and chicken meat surfaces were reduced by 99.8%, 99.7% and 98%, respectively [68]. This study shows that decontamination of bacteria by aPDI might have a potential to improve food safety for the consumer. 4.7. Antibody-photosensitizer conjugates In order to enhance specify of a given photosensitizer to kill selectively a certain bacteria specie antibody-photosensitizer conjugates were developed to recognize bacterial antigens. Such a combination of a photosensitizer coupled to the Fc-part of an antibody would not only kill bacteria selectively in vivo but also prevent non-specific damage to normal tissues at the infection site.

An overview about the current research activities into targeting of photosensitizer to enhance the selectivity is given elsewhere [69]. Already in 1994 Berthiaume et al. demonstrated that antibody-targeted photolysis of bacteria works in vivo [70]. The conjugate was prepared by site-selective covalent coupling of chlorin e6 as the given photosensitizer to the oligosaccharide moiety (Fc-part) of anti-Pseudomonas monoclonal antibody (clone PO37) [71]. In vivo killing of P. aeruginosa by local injection of antibody-photosensitizer conjugates in a mice infection model showed that 75% decrease in the number of viable bacteria treated with a specific antibody-conjugate, whereas normal bacteria growth was detected after treatment with a non-specific conjugate or untreated [70]. Furthermore a murine monoclonal antibody against Pseudomonas gingivalis LPS were linked with TBO for lethal photo-sensitization of P. gingivalis in the presence of Streptococcus sanguis or human gingival fibroblasts [72]. In this study a selective killing of P. gingivalis was demonstrated by the reductions in viable counts of 5 log10 vs 0.1 log10 for S. sanguis, when the antibody-TBO conjugate was used. However the higher molecular weight of such antibody-conjugates compared to the TBO (270 g/M) alone would inhibit penetration into intact skin for selective decolonization of bacteria (see 500 Dalton rule for the skin penetration of chemical agents [73]). Larger molecules cannot simply pass the corneum layer. Furthermore the better selectivity of an antibody the major feasibility of resistance is given (‘‘lock-key’’ principle) by the bacteria to overcome the photodynamic inactivation susceptibility by changing the bacterial receptor quality to avoid antibody binding.

4.8. Cationic BODIPY derivatives A new class of photosensitizer suitable for aPDI are the distyryl boron dipyrromethene (BODIPY) dyes containing one pyridinium cationic group and two iodine atoms at the dipyrrolylmethene structure [74]. Such cationic BODIPY dyes (0.5 lM or 5 lM) were able to eradicate Staphylococcus xylosus and E. coli upon light activation by up to 6 log10 using a green LED (5 min, 1.38 J/cm2). No dark toxicity was observed. Overall Caruso et al. remarked that BODIPY dyes in general feature some advantages (high extinction coefficient of absorbance in the visible spectrum; strong fluorescence for diagnostic imaging) and disadvantages (lipophilicity; formation of aggregates) relating aPDI. Therefore BODIPY needs to be optimized in terms of efficient photo toxicity. However the

Please cite this article in press as: M. Tim, Strategies to optimize photosensitizers for photodynamic inactivation of bacteria, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.05.010

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combination of high fluorescence intensity of BODIPY for diagnostic imaging in combination with efficient antimicrobial activity would be of interest in the future. 5. Conclusion and outlook Recently a first phase IIa randomized, placebo-controlled study of super infected chronic leg ulcers and chronic diabetic foot ulcers demonstrated that aPDI was able to reduce the bacterial load immediately post-treatment and an apparent trend toward wound healing was observed [75]. The phenothiazinium photosensitizer PPA904 (3,7-bis(di-n-butylamino)phenothiazin-5-ium bromide) or placebo was applied topically as a cream to wounds for 15 min, followed by a single treatment of red light illumination with 50 J/cm2. After a following up to 3 month, 50% (four of eight) of patients with actively treated chronic leg ulcer showed complete healing, compared with 12% (one of eight) of patients on placebo. The authors stated that the result of this study is slightly encouraging that a trend towards accelerated wound healing was observed because unlikely that only a single photodynamic treatment would be optimal. Nevertheless this first clinical study emphasizes the potential of aPDI to validate this approach for the topical treatment bacterial infections in the future. Furthermore new supporting strategies for effective MRSA decolonization of patients is of interest in respect to control and prevent bacteria spread in hospital daily routine. Bryce et al. could demonstrate that the combination of a newly developed nasal photodisinfection device and chlorhexidine decrease surgical site infections [76]. 3068 patients were treated with chlorhexidine 24 h preceding surgeries and additional received an intranasal photodynamic treatment in the pre-operative area [76]. The authors concluded that the combination chlorhexidine and the photodynamic approach immediately before surgery reduced surgical site infections, achieved excellent compliance, and was easily integrated into the pre-operative routine [76]. These results emphasize the potential of aPDI to use it as an add-on approach for the eradication of bacteria, because the clinical outcome of standard disinfectants for MRSA eradication varied between 6% and 75% [77]. A successful decolonization depends on how many body regions are colonized by MRSA and on the compliance of the decolonization regime by the patients and the health care workers. Furthermore self-disinfecting surfaces may play an important role as an additional preventive step hampering nosocomial infections caused in the clinic in the future. An overview about such surfaces is given elsewhere [78–81]. Such self-disinfecting surfaces can maintain general disinfection approaches; thereby surfaces of catheters, medical devices, door handle and intensive care units are coated with an antibacterial layer. The reliability of such photodynamic active surfaces has been reported by several authors [82– 85]. For self-disinfecting photodynamic active surfaces PS are needed with a high singlet oxygen quantum yield, not acting with the coating material. Furthermore the photodynamic active polymer coated on the bulk material must provide good diffusion properties for oxygen and should not act as a quencher for the generated ROS. Overall a series of new photosensitizers were developed in the past ten years which have demonstrated a great photodynamic potential to kill the superbugs of the 21th century. The non-specific killing of microorganisms at multiple sites is one of the advantages of aPDI compared to the ‘‘lock-key’’ principle of antibiotics. So far resistance against aPDI seems to be unlikely, whether bacteria develops resistance to ROS, especially singlet oxygen, is questionable and currently under investigation. However aPDI is usually used to treat superficial infections, but may prevent multi-resistant bacteria reaching the bloodstream which would lead to the worsening of the infection. In such a

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scenario aPDI would help to reduce the bacterial load in immunocompromised patients to gain in time for an optimized and adapted antimicrobial treatment strategy. When bad bugs have already affected the bloodstream neither aPDI nor disinfectants helps, so you have fall back on appropriate working antibiotics again. Recently Tanaka et al. demonstrated that aPDI with PhotofrinÒ in MRSA arthritis in the mouse knee could treat the infection, not only by directly killing of bacteria but also by stimulating the host immune defense [86]. Photodynamic treatment with an appropriate light dose that causes both maximal bacterial killing and neutrophil cell accumulation into the infected wound area may results in a new mechanism of action. Here in this study Tanaka et al. showed a correlation between accumulated neutrophils at the infections site and bactericidal effect after aPDI [86]. Such a new mechanism is of interest, because neutrophils are the key players for host defense against bacterial infections. In this study increased levels of macrophage-inflammatory protein 2 were detected which facilitates neutrophils accumulation to the infected wound which results in neutrophil-mediated antibacterial effect of aPDI on MRSA arthritis. Considering that the development of new antibiotics is a downward trend aPDI would help to slow down or even stop the severe and fatal consequences of resistant strains. So far aPDI mediated by a light-activated photosensitizer has a great potential for the treatment of infectious diseases by directly killing of bacteria as well as by stimulation of the host immune defense to fight against bacteria in the future.

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Please cite this article in press as: M. Tim, Strategies to optimize photosensitizers for photodynamic inactivation of bacteria, J. Photochem. Photobiol. B: Biol. (2015), http://dx.doi.org/10.1016/j.jphotobiol.2015.05.010