New techniques in antimicrobial photodynamic therapy: scope of application and overcoming drug

New techniques in antimicrobial photodynamic therapy: scope of application and overcoming drug resistance in nosocomial infections

Faina Nakonechny1,2, Marina Nisnevitch1, Yeshayahu Nitzan2 & Michael A. Firer1,* 1Department of Chemical Engineering and Biotechnology, Ariel University Center of Samaria, Ariel 40700, Israel 2The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel *Correspondent author

Given the ever increasing problem of antibiotic resistance in nosocomial pathogens it is important to promote alternate technologies that may be more affective than current antibiotics. This article reviews Photodynamic Antimicrobial Chemotherapy (PACT), a technology based on the use of a photosensitizer activated by visible light illumination and found to be effective against most types of microbial pathogens, including those resistant to antibiotics. PACT nonetheless has certain limitations, particularly against internal and blood-borne infections. To this end, we are developing Chemiluminescent Photodynamic Antimicrobial Therapy (CPAT). This review also summarizes our recent data on CPAT. The practical advantages of CPAT emphasize that this novel technique could expand efforts to control nosocomial pathogens, including those responsible for systemic infections.

Keywords Photodynamic therapy; chemiluminescence; targeted drug delivery; Photodynamic Anti-Microbial Chemotherapy; PACT; Chemiluminescent Anti-Microbial Chemotherapy; CPAT; liposomes.

1. The problem of hospital-borne infections

The need to develop novel technologies to combat the evolution of bacterial drug resistance is clearly a matter of public concern and urgency. The main reasons for this situation include the widespread use of antibiotics over a period of decades both in the clinic and in animal husbandry and the subsequent mutation-derived adaptation of bacteria to antibiotic challenge. The prospect of microbial development of antibiotic resistance is not new; indeed the discoverer of penicillin, Sir Alexander Fleming, warned of this possibility. Antimicrobial resistance is a growing and worldwide problem that impinges on the treatment of both nosocomial (hospital-borne) and community-acquired infections and encompasses the complete range of human pathogens, including bacteria, fungi, and viruses. Studies that track the development of important bacterial pathogens such as Methicillin-Resistant Staphylococcus aureus (MRSA), Klebsiella pneumonia, multidrug-resistant strains of Acinetobacter, gonococci, cholera and Salmonella all point towards an underlying theme - the development of resistance to currently available antibiotics, in developed as well as developing countries at a time when the pipeline for new antimicrobials is drying up [1]. The possibility that we may soon return to a “pre-antibiotic” era must stimulate the development of new technologies to correct the current situation [2]. Antibiotic resistance of nosocomial pathogens in particular, is resulting in increased human morbidity and mortality and is escalating health costs [3-5]. A 2009 report from the US Centers for Disease Control estimated the annual direct medical costs of healthcare-associated infections to range between $28-45 billion [6]. One quarter of all nosocomial infections involve patients in intensive care units, and most patients who die in these wards succumb to infection(s) [7]. Gram-positive bacteria such as S. aureus and Enterobacter species account for about 60% of nosocomial systemic infections in US hospitals and the incidence of resistance to important antimicrobials such as methicillin and vancomycin is increasing in these strains [8]. A similar trend is seen for Gram-negative infections with K. pneumonia, Pseudomonas aeroginosa and Stenotrophomonas maltophilia [9] and the incidence of resistance to cephalosporins, quinolones and carbapenems. Unfortunately, while the alarm bells raised by this precarious situation is now appreciated by both scientists and government [10] and has provided impetus for increased academic and pharmaceutical research (a search of the PubMed database using the terms “antibiotic resistance and hospital infections” returned 87 hits for 1980 and 1287 for 2010), few new antimicrobial compounds have so far made a practical impact in the clinic [11]. It therefore seems prudent to look for additional therapeutic strategies.

2. Photodynamic Therapy (PDT)

One attractive approach is the use of photodynamic therapy (PDT). PDT is a two-stage procedure based on two nontoxic components that combine to induce oxidation of membrane phospholipids and proteins, leading to membrane leakage and cytolysis [12]. The first component is a photosensitizer (PS) molecule, such as porphyrin, phenothiazinium,

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Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)______________________________________________________________________________

phthalocyanine or chlorin derivatives. When activated by visible light of a particular wavelength (for example from a laser or LED), the PS transfers energy to molecular oxygen, resulting in production of reactive oxygen species (ROS) that lead to direct and indirect damage of cellular and membrane components and consequently to cell death. The structures of several representative PS molecules mentioned in this review are shown in Figure 1 and a list of PSs approved for clinical use can be found in Ref [13]. The basic principles of PDT are outlined in Figure 2. PDT has been used in biomedical research as well as in the clinic for over 100 years [14], not only against microbial infections but also for the treatment of several types of cancer and skin diseases [15, 16]. The history, mechanism of action and biomedical applications of PDT have been extensively reviewed [17-22].

Figure. 1 Chemical structures of some photosensitizers used in PDT. Aminolevulinic acid, Hematoporphyrin and Photofrin are approved for clinical use.

Figure. 2 A schematic outline of PDT action. Type I and Type II pathways are explained in the text that follows.

As shown in Figure 2, PDT can induce bacterial cell death through two pathways. In Type I PDT, external light activation of endogenous photosensitive molecules such as porphyrins and flavins results in the transfer of electrons to molecular oxygen generating the superoxide radical anion O2

- which in turn converts to the hydroxyl free radical and singlet oxygen in the presence of H2O2. In Type II PDT, exogenous photosensitizers that have been taken up by the bacterium are activated by external light of appropriate wavelength to excite molecular oxygen into its singlet state 1O2. The highly reactive ROS produced by both pathways oxidize various important cellular and membrane components leading to cell disruption (reviewed in [23]).

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Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)_______________________________________________________________________________

3. Photodynamic Antimicrobial Chemotherapy (PACT)

PDT technology has been extensively studied for antimicrobial therapy and has been termed PACT or photodynamic inactivation (PDI). Indeed the sensitivity of microorganisms to PACT has been tested against a range of Gram-positive and Gram-negative bacteria [23], fungi [24], enveloped and non-enveloped viruses [25]. Importantly for nosocomial infections, the efficacy of PACT towards Gram-positive MRSA and Gram-negative antibiotic-resistant P. aeruginosa has been demonstrated in a number of studies [26-28]. However this seems to depend on the type of PDT used. Sharma and colleagues succeeded in substantial eradication of antibiotic resistant P.aeruginosa by first exposing cells to -aminolaevulinic acid (ALA) and glutathione, which caused increased synthesis of endogeneous protoporphyrins, followed by light irradiation [29]. Moreover, it was shown previously [30], that methicillin-sensitive S. aureus (MSSA) are almost twice as sensitive to endogenous PDT as are MRSA. However in our more recent experiments, MRSA are more sensitive to Type II PDT in the presence of methylene blue (MB) (Figure 3). Illumination caused a 1.7-2.6 log10 reduction in CFU of the methicillin-sensitive cells but a 3.2-3.6 log10 reduction in the resistant cells.

3.1. Resistance to PACT.

It is particularly encouraging to note that despite the large number of studies on the effect of PACT against different microorganisms, the development of resistance to PDT has not been reported [22, 31]. This important phenomenon seems not be confined to microorganisms either as studies, including our own, show that aside from rare situations [32] cancer cells do not develop resistance to PDT either [15, 33]. It is not yet clear why PDT is different in this regard from other cytotoxic strategies such as antibiotic and anti-cancer chemotherapy where the development of multi-drug resistance is the norm following repeated exposure to free drug [34, 35]. This subject deserves further investigation as understanding the mechanisms involved may help in developing improved strategies for other forms of drug therapy.

3.2. Sensitivity of Gram-positive versus Gram-negative bacteria to PACT

Gram-positive and Gram-negative bacteria react differently to PACT. Gram-negative bacteria were initially found to be resistant to PDT until it was appreciated that phospholipids, complex lipoproteins and polysaccharides present in the additional outer envelope of E.coli, P.aeruginosa, K.pneunomia and H.influenza, inhibit the binding of anionic PS molecules [36], unless additional manipulations are used that facilitate membrane transport [37]. Fortunately, a number of alternate strategies have been developed to overcome this barrier. These include the use of uncharged (e.g. deuteroporphyrin, prochlorphyllide) or positively charged (e.g. tetra-cationic porphyrin substitutes, cationic phthalocyanines, toluidine blue O, methylene blue) PS, particularly when coupled with membrane penetrating peptides (reviewed in [18, 22]). Interestingly, cationic PS demonstrate a level of intrinsic advantage from a clinical perspective in that their rate of uptake into bacterial cells is far greater than for mammalian cells [38]. Learning to manipulate this phenomenon may have become important in controlling any side effects to PACT therapy (see Section 4 below).

3.3. PACT effectiveness against microbial biofilms

Of particular concern in the treatment of bacterial infections is that over 60% are the result of bacterial growth in biofilms [39]. Biofilms are communities of cells supported by an extracellular polymeric network; alternatively cells can grow as small colonies or as single (planktonic) organisms as is the case in bacteraemia. Biofilms are extremely

Figure 3. PACT effect on the viability of ofMSSA (ATCC 25923) and MRSA (ATCC 43300).Cells at initial concentration of 107 cells/ml wereincubated with 25 µM of MB for 20 min in thedark and then illuminated with a whiteluminescent lamp with a fluence rate of 1.6mW/cm2 for 30 min at 25oC under temperaturecontrol. After treatment, cells were diluted in 10-fold dilutions and evenly spread over BH-agarplate. Plates were incubated at 37oC overnight andCFU were counted. b/t – before treatment

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important in infectious disease. They are probably produced by all bacterial pathogens and form not only on the skin and internal organs but also on medical devices in direct contact with patients, such as drips and catheters. Cells in different sections of the biofilm are in different phases of growth, including the stationary phase, a factor that contributes to differential susceptibility to most antibiotics. Interestingly, when extracted in the laboratory almost all cells in a biofilm are susceptible to antibiotics [40], demonstrating that there is no intrinsic resistance of biofilm residents to these drugs. In practice however, it is thought that antibiotic treatment eliminates most biofilm bacteria but that the presence of the extracellular polysaccharide polymer network severely inhibits the elimination of remaining bacteria by the immune response [40]. Thus “persistent” bacteria survive, particularly when the levels of antibiotic wane, allowing their growth and repopulation within the polymer network. The effect of PACT on biofilms has received considerable attention, particularly in relation to skin and oral infections where direct access to the site of infection is available [13, 41, 42]. Most in vitro studies demonstrate that while PACT is indeed more effective than conventional antibiotic treatment in reducing biofilm populations [43], the effect is not complete. One limitation may be the penetration of PSs through the complex biofilm matrix which, depending on the chemistry of the latter, may bind to and therefore inhibit the PS from actually reaching its target. This might be overcome by using alternative strategies of PS delivery such as nanoparticle packaging (see below) or conjugation to protein carriers. Another alternative is the concurrent use of chelating agents such as EDTA [44] that may assist in PS diffusion through the matrix.

3.4. Demonstration of PACT efficacy in animal models of infection

While an in depth review of the literature is outside the scope of this chapter, it is worthwhile noting that investigators have gone to considerable effort to devise experimental set-ups that recapitulate as much as possible conditions that result in clinical infection. These include infections resulting from burns or surgical wounds [45, 46] which account for about one-third of all nosocomial infections. Other studies have used models of various soft-tissue infections, oral and dental infections, osteomyelitis and localized mycobacterial infection. The results of these and other animal studies clearly validate two points highlighted throughout the in vitro studies on PDT. First, PACT is a safe therapeutic strategy that induces minimal collateral damage to normal tissue and cells. Second, PACT is effective against a variety of infectious microbes in vivo [47, 48]. A comprehensive overview of this field can be found in a recent review [22].

4. Antimicrobial properties of liposome encapsulated photosensitizers

There are several issues which should be addressed if PACT technology is to find expanded use in the clinic. One of these is the accumulation of PS into cells. While cationic PS may accrue faster in microbial than normal mammalian cells as mentioned above [38], the non-specific accumulation of PS in normal cells of the body may still result in side effects such as cutaneous photosensitivity [17]. One way to overcome this problem might be to package the PS into nanoparticles such as liposomes labelled with a carrier molecule specific for the target cell. This approach not only localizes the PDT effect to the bacteria but also results in a more concentrated compound delivery and enhanced cytotoxicity. This principle has already been demonstrated with Scanning Electron Microscopy which showed that fusion between antibiotic-containing liposomes and Gram-negative bacteria outer membranes results in the delivery of the liposomal contents into the cytoplasm [49-51]. For Gram-positive bacteria, interaction of the liposome with the external peptidoglycan probably enables release of PS and its diffusion through the cell wall [52]. Moreover, local application of liposomal entrapped drugs helps prolong their action in infected tissues and provides for sustained release of active components [53]. Even without the added effect of targeting, encapsulation can result in enhanced localization of active drug into the target tissue compartment. For example, early studies by Beaulac and colleagues [54] showed that liposome-encapsulated tobramycin administered to rats with chronic pulmonary P. aeruginosa infection maintained a high level of activity in the lungs while only low quantities were found in the kidney. Administration of free drug resulted in the complete opposite effect. Similarly Drummond [55] reported a 3- 15-fold greater accumulation of doxorubicin in tumour cells when the drug was delivered via liposomes. Tsai studied the bactericidal efficacy of liposome or micelle entrapped hematoporphyrin and chlorin e6 against a number of Gram-positive bacteria, including MRSA, and showed that liposomal drug forms exhibited 0.4 to 2 log10 reduction of bacteria survival compared to free drug forms and PS entrapped into micelles exerted complete bactericidal effect [56]. Entrapment of PS into nanoparticles does not always result in enhanced cytotoxic activity. Ferro [57, 58] reported that chlorophyll a was pronouncedly more efficient in a free form than in any liposomal form, whereas hematoporphyrin as well as a positively charged PS 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphyrin were less effective in free form than when enclosed into a cationic lipid or incorporated into liposomes made of phosphatidylcholine derivatives. The results were explained by differences in PS chemistry which would influence their association with liposomal components, lipid fluidity and localization in liposome vesicles.

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We have previously shown that MB entrapped into liposomes composed of the neutral dipalmitoyl phosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (EPC) with or without of additions of dimyristoyl phosphatidylglycerol (DMPG) or octadecylamin (OA), was effectively delivered to a number of Gram-positive and Gram-negative bacteria among them: S. lutea, S. aureus, S. epidermidis, E. coli and S. flexneri [59]. The lipid composition of liposomes indeed affected PS delivery to cells. The best results were obtained for DPPC/DMPG and EPC liposomes. DPPC/DMPG and EPC liposomes were the most effective with MB, due to the positive charge of the PS, which was helpful for its incorporation into the negative charged liposomes; indeed MB was less efficiently entrapped into the cationic vesicles. We tested the influence of PS encapsulation on PACT and found that free and liposomal forms of PS were similarly able to sensitize S.aureas under external illumination. The light-dose response curves for the free and liposome-encapsulated MB were very close, although there was a 2-fold improvement in bacterial growth inhibition with liposome-enclosed MB. These results suggest that at least in vitro, PS incorporation into Gram-positive bacteria is only moderately enhanced by liposome encapsulation.

5. Chemiluminescent Photodynamic Antimicrobial Therapy (CPAT)

An serious limitation of PACT is the requirement for an external light source, which may be from a diode, laser beam or LED. So in its current configuration, PACT is not applicable for systemic or blood-borne infections and despite advances in phototherapy [16], PACT is also limited in the treatment of deep infections due to the limited tissue penetration of external light sources. To overcome this limitation we [15, 60] and others [61] developed a new approach in which the external light source was replaced by chemiluminescent light emitted in a course of a chemical reaction. We used chemiluminescent (CL) oxidation of luminol (LM), in which the in situ conversion of molecular oxygen to superoxide ions and the subsequent release of light energy are achieved without electrical or thermal input. The mechanism of this CL reaction has been known for some time [62] and it is commonly used in a variety of CL-based bioassays. Initially we demonstrated that LM induced intracellular CL in murine myeloma cells and effectively lead to their eradication [15]. More recently we reported that this technology, which we call Chemiluminescent Photodynamic Antimicrobial Therapy, CPAT, was effective against both Gram-positive and Gram-negative bacteria [60]. In those experiments, our data showed that CPAT was almost as effective as PACT in reducing the viability of S.aureus and E.coli. Experiments were performed with both free or DPPC-liposome entrapped MB (lip-MB) and in CPAT experiments we used both free or DPPC-liposomes encapsulated LM (lip-LM). CPAT treatment of the cells with free MB in the presence of free LM, as well as by a mixture of lip-MB and lip-LM resulted in a significant reduction (2-3 log10) in bacterial viability [60]. By comparing PACT to CPAT we calculated that the chemiluminescent light intensity produced by CPAT had a fluence rate of 1.6-12.1 mW/cm2. The mechanism of activity of CPAT has not yet been fully delineated although control experiments in the absence of H2O2 only gave cytotoxicity equal to that of the control dark effect (without LM). In mammalian tumour cells, the presence of ROS and H2O2 are necessary for PDT induced cell killing and preliminary experiments indicate that like PDT, chemiluminescence-activated PDT induces apoptosis in mammalian cells (M. Firer, unpublished). Interestingly, work by Chang and colleagues [63] showed that in response to H2O2 exposure, S.aureas upgrade expression of genes involved in a variety of defence mechanisms. On the other hand, S.aureas is known to be PACT sensitive [29, 57, 59, 64], so presumably the cytotoxic effects of PDT such as oxidation of membrane lipids and proteins by oxygen radicals and other ROS can overcome these defensive strategies. By extension, we assume that CPAT is inducing similar biochemical effects in the cell, although this awaits experimental substantiation. It was of a special interest to compare between a CPAT effect on MRSA and MSSA strains. Both were incubated in the presence of free MB and LM or lip-MB and lip-LM in the dark. As can be seen in Figure 4, CPAT in both free and encapsulated forms was effective in eradication of MRSA and MSSA – the CFU was reduced by two orders of magnitude. Separate incubation of the cells with free or encapsulated MB, or free or encapsulated LM with H2O2 together with a catalyst, did not affect cell viability, demonstrating that as in PACT, CPAT requires the presence of both a light source and a PS.

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Figure 4. CPAT effect on the viability of MSSA (ATCC 25923) and MRSA (ATCC 43300). 107 cells/ml were incubated with 25 µM of free or encapsulated MB (lip-MB) for 1 or 2 h at 25oC together with 0.15 mM of free or encapsulated luminol (lip-LM) in the presence of 4 M FeSO4 and 3M H2O2. In control experiments cells were incubated separately with each of the conponents (free or encapsulated MB and free or encapsulated LM together with FeSO4 and H2O2). Strict precautions were taken to avoid external illumination of the system during addition of components and further incubation. After the treatment aliquots of mixtures were diluted in 10-fold dilutions and evenly spread over BH-agar plate. Plates were then incubated at 37oC overnight and CFU were counted taking dilutions into account.

These results demonstrate that CPAT can become a practical and effective alternative to traditional PACT in killing and inhibiting the growth of both Gram-positive and Gram-negative bacteria, including antibiotic-resistant strains. The important advantage of bypassing the need for an external light source to activate the PS suggests that CPAT might be effective for internal infections that are difficult to locate or target using traditional PDT, which encourages further assessment of CPAT as a novel antimicrobial therapeutic strategy.

Conclusion

The continued development of antibiotic resistant bacterial strains in hospitals has generated a serious public health care issue. Physicians in both primary health care and specialist wards such as Intensive Care Units already face situations where certain infections are untreatable. To overcome this crisis, it is imperative to look for novel anti-microbial strategies. PACT has been extensively studied and has already demonstrated efficacy in the laboratory, in various animal models and in the treatment of periodontal disease and additional clinical trials should be initiated, particularly for topical infections where it should be most effective. In addition, PACT should be further developed for improved sterilization of medical devices used on, in, or in the vicinity of patients. CPAT, our novel improvement of PACT that alleviates the need external activation of PS, may further extend the application of PDT to internal and blood borne infections. One bottleneck in the wider application of PDT-based technologies for clinical infections is the lack of highly effective antimicrobial PS. Currently, clinically approved PSs include earlier generation molecules such as phenothiazinium dyes (MB and TBO), ALA, porphyrin derivatives (Photofrin, Visudyn) and meta-tetra-hydroxyphenyl chlorin (Foscan). While these have efficacy to some pathogens, their photodynamic potency is much weaker than later-generation PS. Unfortunately, the latter have yet to be subjected to the rigorous and costly toxicological and safety studies necessary for approval for human use. PACT and CPAT appear to represent realistic technologies that may well aid in the fight to control nosocomial antibiotic resistant bacteria. Efforts should be made to encourage the pharmaceutical and biotechnology industries to develop these strategies into clinical products.

Acknowledgements This work was supported by the Research Authority of the Ariel University Center of Samaria and the Rappaport Foundation for Medical Microbiology, Bar Ilan University, Israel (to Y.N.).

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New techniques in antimicrobial photodynamic therapy: scope of application and overcoming drug