The effectiveness of photodynamic therapy on planktonic cells and biofilms and its role in wound healing.

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The effectiveness of photodynamic therapy on planktonic cells and biofilms and its role in wound healing Steven L Percival*,1,2, Louise Suleman2, Iolanda Francolini3 & Gianfranco Donelli4

ABSTRACT Photodynamic therapy (PDT) is the application of a photoactive dye followed by irradiation that leads to the death of microbial cells in the presence of oxygen. Its use for controlling biofilms has been documented in many areas, particularly oral care. However, the potential use of PDT in the treatment of chronic wound-associated microbial biofilms has sparked much interest in the field of wound care. The aim of this article is to provide an overview on the effectiveness of PDT on in vitro and in vivo biofilms, their potential application in both the prevention and management of wound biofilm infections and their prospective role in the enhancement of wound healing. The use of photodynamic agents and their application to health can be traced back to the 20th century [1] . In 1900, Raab first proposed that there was an interaction and chemical effect between a photodynamic agent, acridine orange, and visible light in the presence of oxygen [1] . However, the biological effects of this reaction were not evaluated. A later study by von Tappeiner and Jodlbauer (1904) reported on the light- and oxygen-dependent toxicity of fluorescent dyes against bacteria [2] . Later work in 1907 introduced the term ‘photodynamic action’ to describe these findings [3] . A more recent study coined the term ‘photoradiation therapy’ following the light activation of porphyrins. These porphyrins were being used to evaluate the effects of hematoporphyrin for the treatment of skin diseases [4] . The basic principles of this therapy consist of the binding of the photodynamic agent to a target cell or biological entity, followed by its activation by light of an appropriate wavelength in the presence of oxygen. The outcome of the reaction was the production of reactive oxygen species (ROS). Of course, the unbalanced release of ROS leading to oxidative stress is well known to have detrimental effects on both microorganisms and human cells [5,6] . However, the use of photosensitizers can be localized to specific areas of tissue, thus possibly lending themselves as promising treatment options in the destruction of malignant tissues and pathogens. More than 400 compounds are known to display photosensitizing properties [7] , most of which include dyes and also natural substances, such as δ-aminolevulinic acid (ALA), a natural precursor of protoporphyrin IX [8] . Photodynamic agents can be divided into azine dyes, which consist of acridine and phenothiazines, such as methylene blue (MB), toluidine blue O (TBO), macrocyclic photosensitizers (porphyrins, chlorins, phthalocyanines and a naphthodiathrone known as hypericin and others that are metallated derivatives, such as zinc porphyrins [9–12] . In the healthcare environment, photodynamic agents, such as MB and crystal violet, are commonly used photosynthesizing agents, as are erythrosine and phloxine B. A large portion of these photodynamic agents have been reported to have inherent antibacterial effects, which shall be discussed below. However, it is

KEYWORDS

• biofilms • chronic wounds • PDT • photodynamic

therapy

Surface Science Research Centre, University of Liverpool, Liverpool. UK Institute of Ageing & Chronic Disease, University of Liverpool, Liverpool. UK 3 Department of Chemistry, Sapienza University, Rome, Italy 4 Microbial Biofilm Laboratory, IRCCS Fondazione Santa Lucia, Rome, Italy *Author for correspondence: [email protected] 1 2

10.2217/FMB.14.59 © 2014 Future Medicine Ltd

Future Microbiol. (2014) 9(9), 1083–1094

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Review Percival, Suleman, Francolini & Donelli generally only during irradiation that the photodynamic agent demonstrates an antimicrobial, specifically bactericidal, effect [13] . Photodynamic therapy (PDT) has been used for the treatment of numerous diseases, mainly focusing on the treatment of tumors associated with the lung, esophagus, head and neck and skin [14,15] . The US FDA approved PDT in 1999 for the treatment of precancerous skin lesions on both the face and scalp. Research on the usage of PDT in precancerous cells has demonstrated that there is preferential uptake of photosensitizers by cancerous cells, with reports of relatively little damage occurring to the surrounding healthy cells. The reasons for this selective uptake are presently unknown, but the uptake is possibly due to the fact that the ROS and singlet oxygen (1O2) generated during PDT are only available for a short period of time [16] . In particular, erythrosine and a number of other photodynamic agents have been used for decades for diagnostic purposes, which have included disrupting dental plaque biofilms and their eradication in situ [17] . Maisch highlighted other medical conditions in which PDT has been useful, particularly those diseases and infections caused by microorganisms [18] . Maisch’s review in particular highlighted the fact that PDT can be used to kill the microbial biofilms that are responsible for oral and cutaneous diseases. Thus, the scope for utilizing PDT as an effective treatment in the eradication of biofilms is an area of much interest. Biofilms are known to be associated with nonhealing chronic wounds, and as such, these agents demonstrate interesting concepts [19] . Biofilms are complex communities of microorganisms that attach to both biotic and abiotic surfaces and are encased within a 3D structure of polysaccharides, proteins, glycoproteins, lipids and extracellular DNA called the extracellular polymeric substance (EPS) [20] . Biofilms are thought to be associated with 80% of all know infections and 65% of healthcare-associated infections, as reported by the NIH. This is principally due to biofilms being inherently tolerant and often completely recalcitrant to many different antimicrobial agents [21,22] . Consequently, it is important that old and new antimicrobials demonstrate efficacy on both planktonic, free-floating microorganisms and microorganisms that reside in the sessile/attached state of a biofilm, a requirement that has been previously overlooked in modern medicine [19] . A growing

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area of intensive research is the use of PDT in the treatment of biofilms and wounds specifically, as all chronic wounds should be considered to ‘house’ a biofilm [23] . The aim of this article is to provide an overview on the effectiveness of PDT on in vitro and in vivo biofilms, their potential application in both the prevention and management of wound biofilm infections and their prospective role in the enhancement of wound healing. PDT: mode of action PDT is based on the concept that a dye (photosensitizer) is applied and taken up by a cell. Following irradiation with the correct wavelength of light, the photosensitizer absorbs the light. This exposure causes the transition of the photosensitizer into an excited triplet state, whereby the photosensitizer undergoes a type I photodynamic reaction. This culminates in the release of ions that interact with surrounding molecules, leading to the liberation of free radicals, which react rapidly with molecules such as oxygen, culminating in the formation of ROS. The released ROS then causes cellular destruction. Examples of ROS include anionic molecules, such as peroxide or superoxides. In the triplet state, photosensitizers have been documented to also undergo a type II photodynamic reaction. Within this state, there is an energy transfer to molecular oxygen, which leads to the generation of an excited-state 1O2 [24] . Both ROS and 1O2 act as microbicidal species and are known to oxidize the cell wall constituents of both microbial and mammalian cells [25] . In addition, specific photodynamic agents have been shown to break down lipid membranes and nucleic acids, causing a reduction in ATP and enzyme levels, all ultimately leading to cellular death [26,27] . More specifically, ROS targets such cellular cytoplasmic membrane components as phospholipids, proteins, cytoplasmic enzymes and nucleic acids [10,28] . Ding and colleagues investigated the use of hematoporphyrin monomethyl ether (HMME) as a porphyrin-related photosensitizer for use in PDT [16] . The researchers demonstrated that HMME-PDT could induce cell death through both necrosis and apoptosis. Furthermore, the introduction of the oxygen quencher sodium azide or the hydroxyl radical scavenger d-mannitol protected HeLa cells from apoptosis and necrosis. The researchers demonstrated that ROS such as 1O2 and hydroxyl radicals

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The effectiveness of photodynamic therapy on planktonic cells & biofilms & its role in wound healing represented the method by which HMME-PDT induced HeLa cell death. It was hypothesized that the apoptosis caused by the generation of ROS may be due to Ca 2+ elevation, mediating cytochrome C release and caspase-3 activation, leading to the late stages of apoptosis [16] . Current photosensitizers & their effectiveness in antimicrobial PDT The main classes of photosensitizing agents that have been documented in the treatment of cancer and various tissue disorders include: ●● Porphyrin derivatives ●● Chlorins ●● Phthalocyanines ●● Porphycenes

Additional photosensitizing agents that display antimicrobial properties include phenothiazonium salts, such as cationic MB [29,30] and TBO [31] , merocyanines [32] and halogenated xanthenes, such as Rose Bengal (RB) [33] and erythrosine [33] . Nevertheless, the photosensitizing phthalocyanines and porphyrins have been most documented for their antimicrobial effects [34–36] . The cationic photosensitizers MB and TBO were investigated by Kashef et al. in order to establish their effect on Staphylococcus aureus and Escherichia coli. The photosensitizer concentrations utilized were 12.5, 25 and 50 μg/ml, with laser irradiation times of 10, 20 and 30 min. Results showed that TBO was more effective than MB, with optimum bacterial eradication at 50 μg/ml with 46.8 J/cm 2 laser light. Furthermore, the clinical multidrug-resistant (MDR) strains were found to be more resistant to PDT than laboratory reference strains [37] . Nevertheless, the effect of MB on E. coli regarding outer membrane, ribosome, nucleic acid and cell wall damage is well documented [38–41] . A study into the effect of first- and second-generation cationic photosensitizers in antibioticresistant strains of S. aureus and Pseudomonas aeruginosa showed that although both microorganisms were successfully eradicated, P. aeruginosa required higher doses of the photosensitizers with a longer irradiation period [42] . PDT has also been reported to be efficacious on anaerobic bacteria [43] . Other photodynamic agents, such as RB and poly-l-lysine chlorin(e6) conjugate, have been shown to mediate the killing of S. aureus, E. coli and Candida albicans in


Review

planktonic culture. The authors demonstrated that the poly-l-lysine chlorin(e6) conjugate photosensitizer was the most effective at low concentrations in the killing of these three different microbes [44] . Concerns regarding the development of resistance in microorganisms following the use of PDT were addressed by Giuliani and colleagues through the investigation of RLP068/Cl, a novel Zn(II) phthalocyanine, which is a photosensitizer that can be used for localized infections. The authors demonstrated that 20 consecutive antimicrobial PDT treatments with RLP068/Cl did not result in any resistant mutants and that, in dark conditions, only S. aureus strains had increased MICs of RLP068/Cl. However, even in this case, the susceptibility of the mutated bacteria to antimicrobial PDT was not affected by their MIC increase [35] . Effectiveness of PDT on clinically significant biofilms ●●In vitro biofilms

Despite there being an array of studies showing the effectiveness of antimicrobial PDT on various microorganisms, these studies focus on microorganisms in a free-floating, planktonic state. Many in vitro studies researching the effects of PDT on laboratory-grown biofilms have been reported. From an oral biofilm-related perspective, PDT has been advocated as an alternative to conventionally used antimicrobial agents, particularly for suppressing subgingival species in oral plaque biofilms and to treat periodontitis. Initial studies by Dobson and Wilson highlighted the effectiveness of PDT on oral biofilms using the photodynamic dyes crystal violet, MB and aluminium phthalocyanine [45] . Soukos and colleagues reported on the delivery of 50 mg/ml MB into in vitro biofilms of Actinomyces viscosus [46] . The study found that, following activation of the photodynamic agent, a 2 log10 killing of the biofilm bacteria was observed. The study also reported that the photomechanical pulses were able to reduce biofilm-derived cells of Actinomyces naeslundii by 2 log10 following the application of 5 mM of chlorin(e6) photosensitizer and irradiation [46] . Zanin et al. evaluated the antimicrobial effect of TBO in combination with a helium/neon light-emitting diode (LED) on S. mutans biofilms [47] . Within this study, biofilms were grown on hydroxyapatite discs in a constant film fermentor. It was found that the viability of S. mutans biofilms decreased

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Review Percival, Suleman, Francolini & Donelli when exposed to both TBO and light, resulting in a 99.99% reduction in bacterial viability. This effect was found to be light dose dependent [47] . Later studies by Soukos et al. found that 25 mg/ml of MB and red laser light was effective in killing 97% of Enterococcus faecalis within a biofilm in an experimentally infected root canal [48] . A later study by Wood and colleagues described how erythrosine in conjunction with white light resulted in several log10 reductions in constant depth film fermentergrown S. mutans biofilms. The concentration of the photodynamic agent used was 22 mM with an exposure time of 15 min [33] . Despite the success of research in antibiofilm PDT, Müller and colleagues used a commercially available oral PDT regime known as HELBO® Blue (Helbo Photodynamic Systems, Austria) coupled with 1 min of soft laser light; however, no significant effect on a multispecies biofilm of oral origin grown in vitro was found [49] . Nastri and colleagues evaluated the bactericidal effect of laser diodes (830 nm) after photosensitization with TBO on Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia grown within a biofilm [50] . The results suggested a very good association with TBO and laser light in killing bacteria in biofilms. The researchers concluded that PDT “can be considered a valid alternative approach to antimicrobial therapy of periodontitis.” Goulart et al. evaluated the inactivation of A. actinomycetemcomitans (an organism known to be responsible for periodontitis, both within the planktonic and biofilm states) by PDT [51] . The researchers discovered a dose-dependant effect of RB and irradiation on planktonic A. actinomycetemcomitans viability, with a 55% reduction in cell viability at a photoirradiation length of 1 min and a RB concentration of 0.1 mmol/l. In addition, the reduction of the biofilm using RB and irradiation was found to be dependent on the concentration of RB. Schneider et al. assessed the impact of laserinduced antimicrobial PDT on the viability of S. mutans cells, again utilizing an artificial biofilm model. The researchers concluded that laser irradiation was an essential component for reducing the bacteria within a biofilm at 10 mm [52] . Research beyond the field of dentistry has also demonstrated the effectiveness of PDT on biofilms. An early study by Wainwright and colleagues reported the effect of a new MB photosensitizer and white light against P. aeruginosa

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biofilms grown on silicone tubing, mimicking that of a biofilm on implanted medical devices. Results showed a photo-oxidative breakdown of biofilm EPS, suggesting a structural as well as microbicidal effect of PDT on biofilms [53] . In the context of cutaneous candidiasis, biofilms of the fungus Candida albicans grown in serumcoated six-well plates were shown to be susceptible to PDT using the photosensitizer Photofrin® (Pinnacle Biologics Inc., IL, USA) and visible light from a mercury arc lamp [54] . In this study, the use of 10 mg/mL of Photofrin followed by irradiation was shown to inactivate C. albicans. The authors also demonstrated that the mechanisms that Candida employs to circumvent antimicrobial oxidative defences or applied therapeutics are not operative during PDT [54] . The effects of PDT on biofilms of clinical isolates associated with cystic fibrosis has been demonstrated by Donnelly et al. using TBO or a porphyrin photosensitizer and red light from a Paterson lamp [55] . The researchers found that higher concentrations of photosensitizer were needed to achieve biofilm bacterial reductions compared with those achieved against their planktonic counterparts. This is contrary to other studies, which have shown that P. aeruginosa grown in planktonic and biofilm cultures demonstrate similar susceptibility to PDT – a discrepancy that the authors point out could be due to the variability of the strains and light doses used in the studies [56] . Biel et al. studied the effectiveness and safety of PDT for eradicating antibiotic-resistant biofilms grown in endotracheal tubes (ETTs) in vitro. After a single treatment with MB and 664-nm nonthermal activating light, the ETT polymicrobial biofilm was reduced by 99.9%, demonstrating the potential of MB-PDT in the treatment of ETT biofilms in the prevention of ventilator-associated pneumonia [57] . The same group also studied the effectiveness of PDT for eradicating antibiotic-resistant biofilms within an in vitro model in relation to chronic rhinosinusitis (CRS). Within this study, MRSA and P. aeruginosa were grown as biofilms on salastic sheets. These were treated with MB and a nonthermal activating light at 670 nm. The use of PDT reduced the polymicrobial biofilm by 99.9% after only a single treatment regime [58] . A more recent study by Biel and colleagues studied the effectiveness of PDT on biofilms known to cause CRS within a maxillary sinus in an in vitro model [59] . Biofilms of MRSA and


The effectiveness of photodynamic therapy on planktonic cells & biofilms & its role in wound healing P. aeruginosa were treated with a MB/ethylenediaminetetraacetic acid (EDTA) photosensitizer and a 670-nm activating light source. The study demonstrated that PDT reduced the CRS biofilm by 99.9% after only a single dose. While this collection of data demonstrates the potential of PDT in the treatment of biofilms, there still remains variability between the effectiveness of these treatments not only due to the different microorganisms used in these studies, but also the array of in vitro biofilm models used. Of course, laboratory-grown biofilm models will differ depending on the disease setting that the researchers wish to represent. However, concerns arise as to the physiological relevance of these models, raising questions regarding whether PDT will be as effective in vivo. ●●Ex vivo

The study of PDT effectiveness in vitro has provided the field with the necessary parameters for the potential use of PDT in the treatment of biofilm-associated infection. Nevertheless, commonly used laboratory reference strains that are grown on artificial surfaces does not account for the potential interactions between the microbial biofilm and the in vivo environment in which the biofilm resides. With this in mind, the use of ex vivo studies using either clinical microorganisms or explanted tissue to study the effect of photodynamic agents is a popular method, particularly in the field of dentistry. Wilson and colleagues studied bacteria in supragingival plaque scrapings from volunteers [60] . These plaques were exposed to TBO and laser light, resulting in a reduction of Streptococcus and Actinomyces. Further studies utilized the constant depth film fermenter to develop a 4-day-old biofilm of Streptococcus sanguis and showed that aluminium phthalocyanine and laser light delivered in a dose–response manner was very effective on biofilms, demonstrating a relationship between light dose and microbial survival [61] . Other studies on oral plaque biofilms have shown that biofilms treated ex vivo were susceptible to zinc phthalocyanine when exposed to white light [34] . Seal and colleagues studied ex vivo biofilms of Streptococcus intermedius, which were treated with TBO-PDT using low-power laser light. In this study, 100 mg/ml of TBO was used, which resulted in a 5 log10 reduction following 10 min of irradiation [62] . In addition, Zanin et al. studied Streptococcus spp. biofilms when grown on bovine enamel for


Review

5 days ex vivo. The biofilms were treated with 100 mg/ml of TBO and a LED [63] . Although there were no significant effects of the use of a photosensitizer or light alone, the PDT regime gave 1.5–4.0 log10 reductions in viable counts, which was found to be species dependent. Allan et al. described the use of TBO and laser light in the PDT of microcosm dental plaques. Within this study, 81.7 mM of TBO and laser light resulted in a 97% eradication of the plaque biofilm bacteria [64] . A commercial product based on 12.7 mg/ml of TBO and 150 s of red laser light has been shown to give 88–99% reductions in biofilms of oral pathogens grown in extracted root canals [65] . Electron microscopy work by this group showed that 1–2-day-old biofilms were destroyed efficiently, while 6-day-old biofilms showed evidence of retention of the 3D structure in deeper layers. Fontana and colleagues also investigated the ability of PDT to reduce the number of bacteria in biofilms from patients diagnosed with chronic periodontitis [66] . The researchers compared the photodynamic effects of MB on human dental plaque microorganisms both in the planktonic phase and in biofilms. It was found that PDT killed 63% of the bacteria in suspension and viability was reduced by only 32% in the biofilms. Overall, the authors concluded that PDT was more effective on planktonic than biofilm cells. Vilela et al. compared the action of malachite green with MB and TBO on clinical oral-derived strains of E. coli and S. aureus biofilms isolated from five human oral cavities [67] . The biofilms were grown on acrylic resin and exposed to PDT using a diode laser at 660 nm and concentrations of photodynamic agents of 37.5–3000 μM. A concentration of 300 μM of MB provided a microbial reduction of 0.8–1.0 log10, 150 μM toludine blue resulted in a log10 reduction of 0.9–1.0 and 3000 μM of malachite green caused a log10 reduction of 1.6–4.0. ●●Biofilms in an in vivo setting

In dentistry, numerous photodynamic agents have been employed, particularly erythrosine [68] . Numerous studies have shown the positive benefits of using a PDT protocol in reducing plaque biofilm bacteria in vivo [69–71] . Unfortunately, few in vivo studies on the effect of antimicrobial PDT on biofilm infection away from oral care have been documented. Nevertheless, a study by Bisland and colleagues demonstrated the effectiveness of both MB and 5-ALA against S. aureus


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Review Percival, Suleman, Francolini & Donelli biofilms associated with osteomyelitis. Within this study, bioluminescent strains of S. aureus biofilms were grown on Kirschner wires and then treated with photosensitizer either in vitro or after implantation into the tibial cavities of rats. Irradiation via an optical fiber prevented biofilm formation in vivo [72] . Although these results are promising for the potential treatment of osteomyelitis, it would be interesting to determine the effects of the same treatment regime on multispecies biofilms. Effectiveness on wound-related biofilms Pathogenic biofilms in chronic wounds, in conjunction with a patient’s pathophysiology, are underlying reasons as to why these wounds do not heal in a timely manner [19] . Interestingly, recent research and the lack of randomized controlled trials have highlighted that commonly used antimicrobials, including antibiotics and antiseptics used in the treatment of chronic wound infections, are ineffective as ‘antibiofilm’ agents. This is because these agents have not been specifically designed for the killing of microorganisms within a biofilm and the breaking down of the EPS, but have been designed to specifically focus on killing microorganisms in the planktonic state only [73] . ●●In vitro studies

Lin et al. conducted a study that compared the susceptibilities of planktonic and biofilm cells of S. aureus to PDT using a merocyanine photosensitizer and green light from a noncoherent lamp [32] . By using a 20-mg/ml photosensitizer and 450 J/cm2 light, no viable biofilm-derived cells cultured in a rotating disk reactor were detected. Planktonic S. aureus cells required a slightly lower concentration of photosensitizer (15 mg/ml) and only 210 J/cm2 to achieve the same level of viable count reduction. A similar comparative study was carried out by Lee et al. using the porphyrin precursor 5-ALA and a LED light source against planktonic and biofilm cells of P. aeruginosa [56] . The authors reported that 108 CFU/ml of planktonic cells were completely eradicated using 10 mM of 5-ALA and a 240 J/cm2 (40 min) LED light. Biofilms containing 109 CFU/cm 2 were also eradicated by 20 mM of 5-ALA and a 120 J/cm2 (20 min) light. These findings suggested a similar susceptibility of P. aeruginosa to PDT in both planktonic and biofilm forms. Sharma et al. reported that the chelating agent EDTA enhanced the photodynamic agents for

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the eradication of S. aureus and S. epidermis biofilms grown on plasma-coated 96-well plates by sequestering Mg 2+ and Ca 2+ from the EPS of the biofilm [31] . Initially, using 40 mM of TBO photosensitizer and a diode laser, a light dose-dependent reduction in biofilms cells was observed, so that at 100 J/cm2, 3–4 log10 reductions of S. aureus and S. epidermis were seen, increasing to >4 log10 reductions for S. aureus and >5 log10 for S. epidermis with 200 J/cm 2 light. Pretreatment of biofilms with 2% EDTA before the PDT regime resulted in an enhancement in cell killing. For example, using 40 mM of photosensitizer and 100 J/cm2 light, the normal PDT regime gave 3.8 log10 reductions, compared with 4.6 log10 reductions for S. epidermis with EDTA; against S. aureus, the PDT regime alone gave 3.4 log10 reductions, compared with 3.8 log10 reductions with EDTA [31] . Sbarra et al. observed a significant reduction in bacterial survival when biofilms were exposed to the cationic porphyrin and tetra-substituted N-methyl-pyridyl-porphine and simultaneously to visible light [74] . They also found that the extent of biofilm clearance was dependent on the biofilm’s state of maturity. As was predicted, mature biofilms were less susceptible to PDT than younger biofilms. In addition, PDT-treated biofilms exposed to vancomycin or subjected to the phagocytic action of whole blood were almost completely eradicated. The researchers suggested that the combination of PDT and antibiotics or host defenses could be used to inactivate S. epidermidis biofilms [74] . With this in mind, Di Poto and colleagues investigated the effects of photodynamic treatment combined with antibiotic action. The researchers examined the structure of polysaccharide intercellular adhesin-dependent or protein-based S. aureus biofilms [75] . They found that significant inactivation of bacteria was observed when exposed to a cationic porphyrin, tetra-substituted N-methyl-pyridyl-porphine and visible light. When PDT-treated biofilms were exposed to vancomycin or subjected to the phagocytic action of whole blood, this resulted in almost complete eradication. In conclusion, the authors suggested that “PDT combined with vancomycin and the host defenses may be a useful approach for the inactivation of staphylococcal biofilms adhering to medical implant surfaces” [75] . A later study by Nakonieczna and colleagues focused on testing the bactericidal efficacy of


The effectiveness of photodynamic therapy on planktonic cells & biofilms & its role in wound healing photoinactivation using a known photosensitizer – protoporphyrin IX – in sequential combination with silver nanoparticles on S. aureus [76] . A high bactericidal effect (7 log10 reduction) was observed. The authors also reported that the silver nanoparticles prevented bacterial regrowth. ●●In vivo studies

Although research into the effectiveness of PDT on bacteria-infected wounds in animals has been conducted, specifically in mice, researchers often fail to reference the biofilm phenotype. Within a study by Hamblin and colleagues, the researchers injected suspensions of bioluminescent bacteria into induced wound tissue in order to establish an infection [77] . After topical application of 100 mM of porphyrin photosensitizer (chlorin[e6]-poly-l-lysine) for 30 min, E. coli was reduced by 98%, as monitored by bioluminescence, upon exposure to 160 J/cm2 diode laser light. This was followed up with a report describing similar PDT (but using 1 mM of photosensitizer) of established S. aureus-infected mice leg wounds. The lack of bioluminescence and regrowth after 160 J/cm2 light was taken as complete bacterial eradication and was seen in three out of five animals [78] . A similar study looking at the use of PDT in infected burns in mice also used bioluminescence to monitor S. aureus reduction following PDT, but this time, only 98–99% eradication was observed, and there was bacterial regrowth after PDT, which demonstrated a need to optimize PDT regimes [79] . Further to this, Motta and Monti described two autoimmune skin ulcers in an Italian patient that showed clinical signs of infection and tested positive for S. aureus [80] . The wounds had previously been recalcitrant to systemic antibiotics and various topical wound dressings. The right ulcer (4.0-cm diameter) was treated with 10% 5-ALA in PEG ointment then irradiated for 8 min with red laser light; the left ulcer (3.8-cm diameter) was used as a dressing-only control. After 6 weeks, PDT resulted in a reduction of the wound from 4.0 to 1.8 cm in diameter and had no evidence of a sloughy biofilm. The authors suggested a number of possible mechanisms of action in order to explain these results, including: marginal keratinocyte activation; anti-inflammatory action; immunomodulatory activity; and antimicrobial activity [80] . A further case study by Clayton and Harrison involved a patient with a 19.6-cm 2 chronic


Review

venous leg ulcer colonized by MRSA that had not responded to treatment with potassium permanganate, silver nitrate, bacteriostatic dressings or larval therapy [81] . Twice-weekly PDT (5-ALA photosensitizer and a 633-nm red light source) was topically applied to the patient’s leg ulcer over 4 weeks. The patient tolerated the PDT well, reporting no discomfort, and the ulcer was shown to improve significantly. Interestingly, Tegos and Hamblin reported a potential drawback of using phenothia zonium photosensitizers, such as MB, TBO and 1,9-dimethylmethylene blue [82] . The authors reported that phenothiazoniums were being used as substrates for MDR pumps in common human pathogens. S. aureus, P. aeruginosa and E. coli were shown to be highly protected against PDT using phenothiazoniums in MDRoverexpressing strains and more susceptible in MDR-knockout strains. Conversely, porphyrin (poly-l-chlorin[e6]) and xanthenes such as RB photosensitizers were shown not to be substrates for MDR pumps. This was the first demonstration of a true bacterial resistance mechanism to PDT, but one that is particular to phenothiazoniums only. Further work c ontinues on this area. Maisch and colleagues have shown that when MRSA was grown on a porcine skin model ex vivo, susceptibility to PDT was reported when a porphyrin photosensitizer (XF73) was employed [36] . More recent work has demonstrated a significant reduction in bacterial load when treated with the cationic photosensitizer PPA904 (3,7-bis[N, N-dibutylamino] phenothiazin-5-ium bromide) in patients with chronic leg ulcers when compared with the placebo-treated controls. In this Phase IIa, randomized, blinded and single-treatment study, PPA904 was applied topically to the wound before applying 50 J/cm 2 of red light for 15 min. The results showed complete wound closure in 50% of patients that underwent PDT when compared with 12% of patients on placebo [83] . Potential benefits of PDT in wound healing In wounds, the photodynamic agent is applied topically to skin or a wound and then irradiated with laser light at an appropriate wavelength that causes the excitation of the photodynamic agent. Once excited, the agent is considered to be an agent that is able to modulate and potentially promote healing due to its antimicrobial action.


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Review Percival, Suleman, Francolini & Donelli An array of PDT studies concentrated on wounds has focused on the effects of PDT on stimulating wound healing. The beneficial effects of PDT on the healing of surrounding tissue post-therapy has been documented [84,85] . Although Parekh and colleagues reported that PDT (a porphyrin or phthalocyanine photosensitizer plus argon laser) had no effect on wound healing in mice [86] , there has been a succession of data to support the use of PDT in enhancing wound closure [87] . Work on both infected wounds and infected burns showed positive outcomes with increased wound closure following the reduction in S. aureus colonization [78,79] . In addition, the very low levels of ROS generated via endogenous chromophores are thought to be able to stimulate cellular activities involved in healing

[88] .

Furthermore, low-level laser light has been reported to increase cell energy via mitochondrial absorption. In particular, by reviewing the experimental and clinical use of lasers on 15 biological systems over a 20-year period, low-energy laser radiation was shown to have a stimulating effect on cells, while high-energy radiation had an inhibiting effect. Thus, the application of lasers to stimulate wound healing in cases of nonhealing ulcers has been recommended [89] . A study by Jayasree and colleagues discovered that PDT quickened the healing of rat wounds from 20 days in control subjects to 13 and 14 days [90] . Heckenkamp and colleagues used PDT (a phenothiazonium [i.e., MB] and laser light-modulated mRNA) to improve wound healing in rat models [91] . Further studies by Silva et al. used a phthalocyanine photosensitizer with

EXECUTIVE SUMMARY Photodynamic therapy: mode of action ●●

The concept behind photodynamic therapy (PDT) consists of the uptake of a photosensitizer (e.g., a dye) by a cell

followed by the subsequent irradiation of the photosensitizer using light. The excitement of the photosensitier leads to the production of reactive oxygen species, including hydroxyl radicals and singlet oxygen. Current photosensitizers & their effectiveness in antimicrobial PDT ●●

Photosensitizers can be categorized into porphyrin derivatives, chlorins, phthalocyanines and porphycenes. In

addition to the established use of PDT in the treatment of many cancers and skin disorders, PDT has also demonstrated antimicrobial properties in Gram-positive and Gram-negative bacteria and also yeast in planktonic form. Effectiveness of PDT on clinically significant biofilms ●●

The use of PDT in the eradication of clinically significant biofilms is an area of research that is steadily building, with

the majority of research focused in the field of dentistry. Despite the a plethora of studies showing positive results in the eradication of biofilms in vitro, the physiological relevance in vitro models is questionable in relation to how these treatment parameters would manifest in vivo. Effectiveness on wound-related biofilms ●●

Microbial biofilms have been strongly associated with the persistence of chronic wounds. Although there is research

into the effect of PDT on wound-associated pathogens in vitro, there is still a lack of appropriate in vivo studies in order to take biofilm eradication from the laboratory to providing a safe and effective clinical tool. Despite this, animal studies and randomized clinical trials are emerging. Potential benefits of PDT in wound healing ●●

PDT has been reported to enhance wound closure in chronic wounds. With many studies reporting the noncytotoxic effects of PDT, given at the correct dose, PDT presents itself as a potential and viable option in the treatment of nonhealing wounds.

Conclusion & future perspective ●●

This article has reported on several studies that have demonstrated the effectiveness of PDT on microorganisms in

both planktonic and biofilm forms, whether it is in the context of chronic wounds or oral healthcare, or in vitro, ex vivo or in vivo settings. Indeed, PDT is being considered in the treatment of many biofilm-associated conditions; however, it is clear that a great deal of work needs to be undertaken in order to establish the correct dosiometry for effective killing of microorganisms within a biofilm without effecting host tissue.

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The effectiveness of photodynamic therapy on planktonic cells & biofilms & its role in wound healing low-level laser light to show that collagen synthesis and re-epithelialization improved significantly when PDT was applied to rat wounds [92] . Human studies involving 160 patients treated with PDT and nitric oxide showed improvements in wound healing and reductions of wound healing periods. The researchers reported that this was attributed to the stimulation of phagocytosis, debridement, fibroblast proliferation and also epithelialization [93] . Paulino and colleagues reported that S. mutans was photoinactivated by up to 50 mM of RB. The study showed that the use of the photodynamic agent demonstrated no cytotoxic effects on fibroblasts [94] . A review was undertaken by Peplow and colleagues in 2012 concerning the use of PDT on wound healing and cells in vitro [95] . The authors reported from their research that PDT improved the healing of animal wounds and concluded that they “strongly support PDT for wound healing” [95] . Although this review did not document the use of PDT on biofilms, which is fundamentally significant considering their documented role in preventing a wound from healing [19] , PDT is becoming of interest as a potentially promising technology for use in wound care, particularly in the context of biofilm management. Conclusion & future perspective Numerous primary research papers are available that highlight the growing applications of PDT for both therapeutic and diagnostic purposes, as stated earlier, particularly the use of PDT for the treatment of oral conditions, where it has been used extensively to disrupt biofilms on and around the teeth and treat conditions such as dental caries and periodontitis. The effects of biofilms on disease and chronic conditions are well documented outside of the area of oral References

2

3

Raab O. Ueber die Wirkung fluorescirender Stoffe auf Infusorien. Zeitschrift fuer Biologie 39 524–546 (1900). von Tappeiner H, Jodlbauer A. Uber die wirkung der photodynamischen (fluorescierenden) stoffe auf protozoen und enzyme. Dtsch Arch. Klin. Med. 80, 427–487 (1904). von Tappeiner HE, Jodlbauer A. Die sinsibilisierende Wirkung fluorescierender


care, and as such, PDT is being utilized. In particular, the use of PDT as a topical treatment for skin and wound conditions has become an interesting area of research over the last decade. As there is selective uptake between microbial and human cells, the careful dosimetry of PDT could reduce ROS-induced cytotoxicity towards surrounding host cells, and as such, the appropriate application of antimicrobial PDT could concomitantly stimulate the woundhealing process. Not surprisingly, a number of companies are in clinical trials regarding the use of wound PDT. However, photosensitizers have to undergo lengthy legislation, and may be candidates for MDR pumps [82] . Although agents, such as erythrosine and RB in particular, are FDA approved for use in dental and ophthalmic applications, they may also be useful in wound therapeutics. Furthermore, there appears to be a low risk of microbial resistance to PDT (1O2 and ROS) being generated. Through the controlled delivery/availability of the photodynamic agent, the amount of photosensitizer and light energy applied can be used in such a way as to avoid toxicity towards host cells and eradicate resident microorganisms, particularly within a biofilm state, ultimately promoting wound closure. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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•

Interesting overview of PDT.

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Good review paper on the history of photodynamic therapy (PDT).

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Interesting research paper on the effects of PDT on biofilms.

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Photodynamic therapy of antibiotic‐ resistant biofilms in a maxillary sinus model. Int. Forum Allergy Rhinol. 3(6), 468–473 2013.

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δ-aminolaevulinic acid mediated


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Review

•

Interesting paper on persister cells.

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Update on the Role of Infection and Biofilms in Wound Healing: Pathophysiology and Treatment.

Evaluation of the Effects of Photodynamic Therapy Alone and Combined with Standard Antifungal Therapy on Planktonic Cells and Biofilms of Fusarium spp. and Exophiala spp.

Biofilms' role in planktonic cell proliferation.

Psd1 Effects on Candida albicans Planktonic Cells and Biofilms.

Effectiveness of five antidandruff cosmetic formulations against planktonic cells and biofilms of dermatophytes.

Photodynamic inactivation of Klebsiella pneumoniae biofilms and planktonic cells by 5-aminolevulinic acid and 5-aminolevulinic acid methyl ester.

Photodynamic Therapy and Its Role in Combined Modality Anticancer Treatment.

Photodynamic inactivation induced by carboxypterin: a novel non-toxic bactericidal strategy against planktonic cells and biofilms of Staphylococcus aureus.

Effect of negative pressure wound therapy on wound healing.

Effects of photodynamic therapy on Enterococcus faecalis biofilms.

The effects of pH on wound healing, biofilms, and antimicrobial efficacy.

Role of adipose-derived stem cells in wound healing.

Mast Cells and Wound Healing.

The role of myofibroblasts in wound healing, contraction and its clinical implications in cleft palate repair.

The role of lymphocytes in wound healing.

The Role of Nerve Growth Factor (NGF) and Its Precursor Forms in Oral Wound Healing.

Inactivation kinetics of various chemical disinfectants on Aeromonas hydrophila planktonic cells and biofilms.

Safety, effectiveness and economic aspects of maggot debridement therapy for wound healing.

Cell therapy for wound healing.

The Impact of Lipoproteins on Wound Healing: Topical HDL Therapy Corrects Delayed Wound Healing in Apolipoprotein E Deficient Mice.

The Role of Endothelial Progenitor Cells in Postnatal Vasculogenesis: Implications for Therapeutic Neovascularization and Wound Healing.

Effectiveness of a Short-Term Treatment of Oxygen-Ozone Therapy into Healing in a Posttraumatic Wound.

The role of mouse mast cell proteases in the proliferative phase of wound healing in microdeformational wound therapy.

Silver and Alginates: Role in Wound Healing and Biofilm Control.