Science against microbial pathogens: photodynamic therapy approaches

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1 Science against microbial pathogens: photodynamic therapy approaches Constance L.L. Saw1 1 Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey, USA. constancesaw@gmail.com There is an emerging area of research to identify the application of photodynamic therapy (PDT) as a means to kill microbial pathogens. In fact, the first recorded observation in more than 100 years ago of photodynamic processes was inactivation of microorganism. In this volume of the Formatex Microbiology book titled: Science against microbial pathogens: communicating current research and technological advances, this chapter will focus on the use of photosensitizer and light as anti-microbial agent against various microbes in different settings. The mechanism of action of PDT inactivating microorganisms, anti-microbial photosensitizing agents and light sources used for eliminating microorganisms will be covered. The success and challenges of using PDT to eradicate bacteria including antibiotic resistant bacteria will be discussed. Keywords PDT; against microbial; antimicrobial; photosensitizers; light 1. Introduction There is an emerging area of research to identify the application of photodynamic therapy (PDT) as a means to kill microbial pathogens. In fact, the first recorded observation in more than 100 years ago of photodynamic processes was inactivation of microorganism, paramecia by Oscar Raab [1]. It was an incidental finding that in the presence of acridine and illumination from a thunderstorm, resulted in the death of paramecia. He demonstrated that the death of paramecia was possible only when light and acridine were present. Additionally, it was found that the toxic effect was not due to heat [2] and the term photodynamic reaction was coined in 1904 [3]. For detailed history of PDT, please refer to existing literature [4]. PDT is based on the dual selectivity: (i) selective localization of photosensitizer targeting at tumor or other lesion of interest and (ii) specific delivery of light eliciting the PDT at the target sites. Although PDT was originally developed as a cancer therapy approach and it is still being developed [5], furthermore it has already been developed as a treatment for age-related macular degeneration [6, 7], psoriasis [8, 9], barrett s oesophagus [10, 11] etc. Taking PDT in cancer as a classic example, after photosensitizers have accumulated in pre-cancerous and cancerous tissues, then appropriate light of specific wavelength will be applied, causing the tumor undergo photo-induced chemical reactions that cause apoptosis or necrosis [12]. Similarly, the uptake of photosensitizers and upon activation by light, PDT induced destruction to pathological or infectious tissues/regions is the common working mechanism among various diseases, including infection. 2. Mechanism of photodynamic reaction There are two mechanisms involved in the photodynamic reaction / PDT. Upon irradiation with an appropriate wavelength of light, a photosensitizer will be activated from its lowest energy ground state to a higher energy triplet stage, which will further react directly with biomolecules to produce free radicals (Type I mechanism) or react with oxygen to form reactive singlet oxygen, 1O2 (Type II mechanism) [13]. 1O2 is very reactive and has strong oxidizing power that can produce potent cytotoxic effects [4]. In cells, it has been reported that 1O2 has a lifetime of less than 0.05 s and a maximal diffusion distance of 0.02 m from the site of its production [14], such characteristics explain in part the specificity of photodynamic reaction for PDT in cancers or photodynamic inactivation (PDI) for killing of antibiotic resistant bacteria. Therefore, conventionally it is thought that the targets of PDT are places where the photosensitizer is localized [15]. However, recent findings suggest that even if some photosensitizers do not bind to the bacteria, yet can cause PDI of bacteria if the distance between the singlet oxygen source and bacteria is close [16]. Please see further discussion in section 5, mechanism of PDI damage to bacteria. 3. The need to search for new antibacterial therapeutics Due to the emergence of antibiotic resistance bacteria, particularly with Staphylococcus aureus after the introduction of methicillin [17], there is an urgent need to find alternative antibacterial therapeutics. While hospital-associated Methicillin-resistant Staphylococcus aureus (HA-MRSA) was once observed in immunocompromised hosts, the rapid emergence of community-associated MRSA (CA-MRSA) has caused a concern and the global epidemiology of CAMRSA appears to be heterogenous [18]; moreover, both HA-MRSA and CA-MRSA have been found to circulate in community due to loose used of terms [19]. Since the fist report of vancomycin-intermediate Staphylococcus aureus 668

2 (VISA) in 1996 in Japan [20], the appearance of MRSA gaining new resistance against vancomycin has been observed repeatedly in other countries including USA. Thus, there is an urgent need to develop new anti-microbial strategies in addition to the implementation of some precautions such as educating the health-care providers, reduction of unnecessary use of antibiotics and local disinfectants. 4. PDT against microbial pathogens Various studies have shown that there is a fundamental difference in susceptibility to PDT between Gram (+) and Gram (-) bacteria. In general, anionic and neutral photosensitizers are efficiently bound to Gram (+) bacteria, and they photodynamically inactivate these Gram (+) bacteria effectively after illuminating with appropriate light, which is not the case for Gram (-) bacteria [21]. The higher susceptibility of Gram (+) species is explained by their physiology where their cytoplasmic membrane is surrounded by a relatively porous layer of peptidoglycan and lipoteichoic acid that allows photosensitizers to cross [21]. For Gram (-) bacteria, they have an additional negatively charged outer membrane that forms a protective barrier between the cells and the environment. Several approaches have been adopted to kill Gram (-) bacteria using PDT. 4.1 Pre-treatment with ethylenediaminetetraacetic-acid (EDTA) Treatment with EDTA will made Gram (-) cells lose up to 50% of their lipopolysaccharide (LPS) because of the increased electrostatic repulsion between LPS caused by removal of divalent cations upon EDTA treatment. Bertoloni et al. tested the pre-treatment with tris-edta could induce photosensitivity and the analysis on irradiated cells suggest that the cytoplasmic membrane was an important target of the photoprocess [22] Pre-treatment with polycation Pre-treatment of E. coli and P. aeruginosa with polycation polymyxin B nonapeptide (PMBN) would assist the uptake of deuteroporphyrin and with the exposure of light, inhibited the cell growth, which was not seen when PMBN was used alone [23]. The main advantage of using PMBN is that this is an agent acting on the growing bacteria in their neutral environment in the culture medium, it is known as a membrane disorganizing agent working on the structure but does not cause metabolic leakage from the cells [24]. Unlike EDTA, PMBN does not cause the release of LPS, but expands the outer leaflet of the outer membrane such that the expanded membrane will become more permeable and facilitate the partition of the hydrophobic molecules from the external medium [21]. It was also shown that there was an interaction between PMBN and deuteroporphyrin which was thought to be assisting the penetration of deuteroporphyrin [23]. This method has been used to kill a multi-antibiotic resistant strain of a pathogenic aerobic Gram (-) bacteria, Acinetobacter baumannii, significantly by deuteroporphyrin after pre-treatment with PMBN [25]. Importantly, it is worth taking note that this particular strain of A. baumannii was resistant to the following antibiotics: ampicillin, mezlocillin, piperacillin, cefoxitin, ceftriaxone, aztreonam, tetracycline, chloramphenicol, gentamycin, tobramycin, amikacin, cirprofloxacin and morfloxacin Using photosensitizers with intrinsic positive charge Because Gram (-) bacteria are resistant to PDI that are commonly lead to PDI of Gram (+) bacteria [21], and photosensitizers bearing cationic charge or the use of agents such as PMBN that increase the permeability of the outer membrane of Gram (-) are reported to be effectively increase killing Gram (-) bacteria [23], therefore, photosensitizers with positive charge are preferred to combat Gram (-) bacteria [26-28]. Studies have shown that photosensitizers that have more cation charges are better in killing the bacteria [29], the cationic porphyrins having three and four charges were highly effective in PDI of both Gram (+) and Gram (-) bacteria, and the mono-cationic photosensitizer was the least effective [30]. Though some studies show the contradicting findings that some di- and tricationic porphyrins were more effective than tetracationic photosensitizers [31], other factors to be considered are substitution groups on the ring [30]. charge distribution and nature of 4.4 Using cell-penetrating polymer to improve delivery of photosensitizers This approach is similar to the treatment with EDTA, however, the photosensitizers are delivered together with the cationic and often amphipathic, cell-penetrating polymers such as Tat peptide was used to conjugate tetrakis(phenyl)porphyrin [32]. Though the Tat peptide is a positively-charged mammalian cell-penetrating peptide with potent antimicrobial activity, the Tat-porphyrin conjugate was found to have the bactericidal effect for both Gram (+) and Gram (-) bacteria, and dependent on the concentration and light dose [32]. The PDI of the bacteria was attributed to the membrane destabilization synergistic action of the Tat peptide and PDT. Polycationic conjugates of chlorin e6 and poly-l-lysine was made and successfully penetrate otherwise the impermeable outer membrane of Gram (-) [33]. 669

3 Cationic fullerenes were also used to deliver photosensitisers and after a short incubation followed by white light illumination, they have a broad-spectrum antimicrobial activity and can rapidly kill more than 99.99% of bacterial and fungal cells [34]. This approach of using cationic fullerenes was also successfully to rescue mice from lethal wound infection with Proteus mirabilis and Pseudomonas aeruginosa [35], which has great clinical potential. Using this delivery approach to deliver cationic photosensitizers appears to be the most popular and best approach towards PDI of bacteria research recently. 5. Mechanism of PDI damage to bacteria It has long been regarded that porphyrin binding to the cytoplasmic membrane is a prerequisite for the photsensitization of Gram (+) [36] and Gram (-) bacteria [23], regardless whether they are aerobic or anaerobic [37]. However, there are reports of PDI of Gram (-) bacteria in which that the photosensitizers did not even come into contact with the bacterium or penetrate the bacterium to be effective. These reports suggest that if singlet oxygen can be generated in sufficient quantities near the outer membranes of the cells, it can diffuse into the cells to cause damage to the vital structures [16]. Though it was found that Gram (+) bacteria were more sensitive than Gram (-) bacteria to the killing of singlet oxygen [38], the role of singlet oxygen was confirmed by measuring the decrease in cytotoxicity as the distance between the singlet oxygen source and the bacteria was increased [16]. It was also concluded that the structure of the cell wall thus plays an important role in susceptibility to singlet oxygen [38].When using photosensitizer Rose Bengal for PDI of different bacteria, it was again found that several Gram (+) species were inactivated about 200 times more quickly (99% inactivation) than a Gram (-) Salmonella typhimurium strain [39]. As it was estimated that the diffusion distance of singlet oxygen is approximately 0.02 m [14], the failure of some photosensitizers bind to Gram (-) species to produce any killing, would mean that the reactive species produced on photoactivation were unable to diffuse towards the critical target sites of the bacteria. Taking these observations into account, through generation of singlet oxygen [16, 38] there are two basic mechanisms that have been proposed for the lethal damage caused to bacteria by PDI as reviewed by Hamblin and Hasan [40]: (i) damage to the cytoplasmic membrane, allowing leakage of the cellular contents or inactivation of membrane transport systems and enzymes and (ii) DNA damage. 6. Preclinical studies focused on animal models Table 1 lists preclinical studies (animal models) of PDI to eradicate various bacteria, various photosensitizers tested by photosensitizer alone, combination or conjugated forms and the outcomes are promising. Nowadays, methylene blue seems to be the popular photosensitizer investigated which could be an extension of the previous success from in vitro findings [41, 42]. However, other newer photosensitizers and delivered by carriers such as fullerenes are also being developed. Table 1 is not meant to be extensive, but those relevant to the development of using PDT to kill bacteria and significant findings are listed here. Readers are encouraged to always refer to the PubMed for latest findings. Table 1 Preclinical studies of PDI to eradicate bacteria Bacteria and testing models Pseudomonas aeruginosa infected the dorsal skin in mice Photosensitizer MRSA infected the burn wounds of guinea pigs and biopsy after treatment to see culture growth Deuteroporphyrin IX dihydrochloride (deuteroporphyrin) and hemin used separately or in mixture 670 Specific and nonspecific tin (IV) chlorin e6-monoclonal antibody conjugates Light source and treatment regimen 630 nm light with a power density of 100 mw cm-2 for 1600 seconds In vitro photosensitization was performed on bacterial cultures in liquid medium by illumination with two 100 W incandescent lamps that provided a light intensity of 37 Em-2 s-1, equalling 1100 lux. Outcomes Reference Greater than 75% decrease in the number of viable bacteria at sites treated with a specific conjugate, whereas normal bacterial growth was observed in animals that were untreated or treated with a nonspecific conjugate. Deuteroporphyrin alone was strongly bactericidal only after photosensitization, hemin alone was moderately bactericidal but light independent, a combination of both deuteroporphyrin and hemin was extremely potent even in the dark and did not require illumination to eradicate the bacteria. [43] [44]

4 A real-time monitoring of infection in excisional wound of the back of mice, infected with non-pathogenic strain of E. coli that expressed the lux operon from Photorhabdus luminescens; these cells emitted a bioluminescent signal that allowed the infection to be rapidly quantified, using a low-light imaging system An excisional wound on the mouse back was contaminated with one of two bioluminescent Gram-negative species, Proteus mirabilis and Pseudomonas aeruginosa Oral candidiasis in an immunosuppressed murine model, mimicking what is found in human patients; SCID mice were inoculated orally with Candida albicans by swab 3 times a week for a 4-week period Epidemic methicillinresistant Staphylococcus aureus (EMRSA- 16) infected in excisional and Poly-L-lysine chlorine 6 conjugate C60 fullerene functionalized with three dimethylpyrrolidinium groups (BF6) Methylene blue Methylene blue Mice were illuminated with 665 nm light delivered by a 1 W diode laser coupled with a 200 mm fiber that gave a circular spot of 3 cm diameter on the mice and equally illuminated wounds with an irradiance of 100 mw cm -2. Mice were given a total fluence of 160 J cm -2 in four 40 J cm -2 aliquots, with imaging taking place after each aliquot of light. The total illumination time was 27 min. 180 J cm -2 of broadband white light ( nm) Mice received a topical oral cavity administration of 0.05 ml methylene blue at g ml -1, 10 min later, they were irradiated with 664 nm light from a diode laser light with a cylindrical diffuser; swabs were taken to determine colony forming unit of bacteria. 360 J cm -2 of laser light (670 nm) in the presence of 100 g ml - 1 of methylene blue was given. Reduction of more than 99% of the viable bacteria was noted after the mixture, an effect that lasted for up to 24 h. Light-dose dependent loss of luminescence in the wound treated with conjugate and light, which was not seen in untreated wounds. Treated wounds healed as well as control wounds, showing that the PDT did not damage the host tissue. Fullerene-mediated PDT of mice infected with P. mirabilis led to 82% survival compared with 8% survival without treatment (p < 0.001). PDT of mice infected with P. aeruginosa did not lead to survival, but when PDT was combined with a suboptimal dose of the antibiotic tobramycin (6 mg kg -1 for 1 day) there was a synergistic therapeutic effect with a survival of 60% compared with a survival of 20% with tobramycin alone (p < 0.01). Methylene blue dosedependent PDI of Candida albicans was found: g ml -1 reduced fungal growth but did not eliminate Candida albicans; g ml -1 totally eradicated Candida albicans from the oral cavity. Compared to the control, significant 25-fold and 14-fold reduction in the number of viable EMRSA was seen in excision wounds and superficial wounds respectively. [45] [35] [46] [47] 671

5 superficial wounded mice Oral candidiasis in rats infected with Candida albicans Methylene blue The light source used was a galliumaluminum-arsenide (GaAlAs) laser with a wavelength of 660 nm, output power of 100 mw, energy density of 245 J cm-2, and time of 69 seconds. The irradiation laser was applied to the tongue dorsum by contact. After 5 days, different treatments were administered: laser and photosynthesizer methylene blue 0.1 mg ml-1 (L+P+); laser only (L+P-); photosensitizer only (L-P+); and physiologic solution only (L-P-). Samples of the oral cavity were collected for a count of colony-forming units per ml. The number of C. albicans recovered from the oral cavity of the rats was similar between the groups (P = 0.106). The L+P+ group showed fewer microscopic lesions of candidiasis than the L-P- group (P = 0.001), suggesting that PDT showed some effect on experimental candidiasis in rats. The L+P+ group presented lower proteinase activity compared with the other groups, with significant difference between the groups L+P+ and L-P+ (P = 0.018). [48] 7. Clinical applications Hamblin s group has published an extensive review on the clinical applications of PDT for infectious diseases such as acne, human papillomatosis virus, dental infection and gastric Helicobacter pylori infection [49]. A clinical report published later shows the promising antifungal PDT effects of using methylene blue in 10 patients infected with chromoblastomycosis [50]. The patients selected did not receive antifungal treatment in the past 6 months before the treatment and did not present any additional disease or predisposing condition for other infections. A 20% methylene blue preparation in Eucerin cream was applied to the skin lesions for 4 h under a gauze occlusive dressing, which was then removed prior to the irradiation with red light emitting diode (LED, GaAlAs, wavelength absorption 660 nm, energy dose of 28 J cm-2) for 15 min. All of the ten patients treated presented reduction in the compromised area after six PDT applications, considering clinical and microscopical aspects. Though the complete healing was not achieved in any patient, and the mycological tests were still positive, except for one patient; these findings show the potential of coadjuvant therapy with conventional antifungal chemotherapy which can improve patient quality of life by reducing the infection area and degree as well as the length of time for antifugal therapy. 8. Conclusion and future perspectives Though there are obvious advantages of using PDT to combat microbial pathogens such as killing drug resistant bacteria, lack of induction of PDT resistance, the approach is not without challenges [49]. Some of the challenges are cessation of PDI on bacteria when the light is turned off, and the selectivity for microbial cells over host. Several means are being developed in this field, such as when using PDT to kill bacteria, in vivo bioluminescence imaging has been developed to allow real-time monitoring of PDI of bacteria [45, 51], new animal models are being developed to test against the standard antibiotics and new photosensitizers [52]. In order to make PDI treatment to be of clinical use, light and photosensitizer must be able to be delivered successfully to the target tissue, therefore attempts of using fiber optics to deliver light to activate nebulized methylene blue to lung have been made [53]. Antimicrobial PDT will become more important in the future as antibiotic resistance is expected to continue to increase. Therefore, we expect the solution in part to come from PDT using photosensitizers with suitable light source and appropriate delivery means. 672

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