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Photodynamic Inactivation of Microbial Pathogens: Medical and Environmental Applications
By Michael R. Hamblin, Giulio Jori The Royal Society of Chemistry
Copyright © 2011 European Society for Photobiology
All rights reserved.
ISBN: 978-1-84973-308-3
CHAPTER 1
Antimicrobial Photodynamic Therapy: Basic Principles
Giulio Jori, Monica Camerin, Marina Soncin, Laura Guidolin and Olimpia Coppellotti
Department of Biology, University of Padova, Via Ugo Bassi 58B, 35131 Padova, Italy
Table of Contents
1.1. Introduction
1.2. Photosensitized Inactivation of Microbial Cells: General Aspects
1.2.1. The Target
1.2.2. Photobiological Processes in the Photosensitized Inactivation of Microbial Cells
1.2.3. The Photosensitizer
1.3. Kinetics and Mechanism of the Photosensitized Inactivation of Microbial Cells
1.4. Conclusions
References
1.1. Introduction
Photodynamic therapy (PDT) represents a well-established modality for the treatment of a variety of malignant tumours; on the other hand, this technique is presently being extended to several non-cancerous conditions, especially those which are characterized by overgrowth of unwanted or abnormal cells, such as inter alia several dermatological diseases, benign prostatic hyperplasia and age-related macular degeneration. An increasing amount of attention is also being focused on the application of PDT for the treatment of infectious diseases. In actual fact, the possibility to kill microorganisms by the combined action of visible light and a photosensitizing dye was discovered at the very beginning of the twentieth century, when Raab observed that the presence of some exogenously added visible light-absorbing compounds, such as acridine orange, was necessary for sunlight or artificially produced visible light to promote the death of paramecia. Raab's observations were repeated with a variety of multi- and unicellular organisms, while Jodlbauer and von Tappainer demonstrated that the presence of oxygen is an essential requisite for photosensitization to occur. The combined effect of the three elements, namely light, photosensitizer and oxygen, has been termed photodynamic action. However, the potential of PDT against diseases of microbial origin was not exploited until the beginning of the present century, since it was repeatedly observed that some well-known pathogens, especially Gram-negative bacteria and protozoa in the cystic stage, are poorly responsive to PDT with the most traditional photosensitizing agents, including xanthene or acridine derivatives and those negatively charged porphyrins that have been frequently used in the PDT of tumours (e.g. Photofrin, benzoporphyrin derivative, tetrasulfonated porphyrins or phthalocyanines); furthermore, the discovery of antibiotics raised the belief that microbiologically based diseases would have been gradually reduced to a level that no longer had a serious impact on human health. On the contrary, at present the therapy of infectious diseases is increasingly challenged by the rapid evolutionary changes and large number of pathogens encountered, as well as by the continuous onset of multi-drug-resistant pathogens; as a consequence, quite a few types of infections, which, in the past, could be readily treated, now often lead to increased morbidity and may become life-threatening even in a nosocomial environment. The problem is further exacerbated by the truly large variety of mechanisms adopted by microbial cells to increase their resistance to external insults. These include a thickening of their outer wall, encoding of new proteins which prevent the penetration of drugs, onset of mutants deficient in those porin channels allowing the influx of externally added chemicals, etc. As a consequence, it has so far proven to be very difficult to identify a comprehensive strategy for overcoming this problem.
A significant step forward in the overall scenario of antimicrobial PDT occurred during the 1990s owing to the discoveries independently made by three research groups showing that visible light-activated cationic photosensitizers belonging to the family of phenothiazines, porphyrins and phthalocyanines induce a fast and extensive killing of typical Gramnegative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, in addition to the inactivation of fungi and Gram-positive bacteria.
Several lines of evidence indicate that PDT is particularly suitable for the treatment of localized microbial infections, including those which have become chronic after prolonged chemotherapy. Favourable features of PDT include:
– the possibility to develop treatment regimes efficiently acting on most classes of microbial pathogens, including Gram-positive and Gram-negative bacteria, yeasts, fungi and parasitic protozoa;
– the high susceptibility to PDT exhibited by antibiotic-resistant microbial strains with no parallel selection of photoresistant species even after multiple treatments;
– the selectivity of microbial cell killing in comparison with the constituents of host tissues;
– the low risk of inducing mutagenic effects;
– the possibility to activate the photodynamic sensitizers by means of inexpensive and safe visible light sources. In particular, while PDT can be easily applied for the therapy of infections in external readily accessible organs, the continuous progresses in optical fibre technology makes even deep-seated infections amenable to the photodynamic approach.
Thus, even though PDT is still in its infancy as regards microbiological applications, it is likely to become a mainstream therapeutic option in the near future, at least for specific indications.
1.2. Photosensitized Inactivation of Microbial Cells: General Aspects
1.2.1. The Target
Microbial cells display a truly large variety of size, sub-cellular architecture and biochemical composition; as a consequence, the susceptibility to photosensitized processes can be significantly different for the various microorganisms.
Thus, Gram-positive bacteria are surrounded by an outer wall, which is separated from the plasma membrane by a periplasmic space. The 20–80 nm thick wall represents a protective mesh mainly constituted by peptidoglycan layers, which are traversed by negatively charged lipoteichoic and teichuronic acids anchored in the membrane. This kind of spatial arrangement does not act as a strict permeability barrier, since several macromolecules with molecular weight up to about 60 000 Da were found to readily diffuse through the wall to reach the inner membrane. Therefore, the most common photosensitizing agents, whose molecular weight is generally not greater than 1500 Da, can more or less promptly cross the outer wall and localize in the immediate surroundings of the photosensitive endocellular sites. On the other hand, Gram-negative bacteria are characterized by the presence of an additional 10–15 nm thick structural element, external to the peptidoglycan network, whose constituents (e.g. lipoproteins, lipopolysaccharides, teichoic and lipoteichoic acids) provide the outer surface with a quasi-continuum of densely packed negative charges: this highly organized system inhibits the penetration of compounds with molecular weight larger than 600–700 Da. A thick external wall, composed of a mixture of lipoproteins, chitin, glucan and mannan, is also present in yeasts, a group of rather heterogeneous eukaryotic organisms, which are the causative factors of various infectious diseases. The wall is again separated from the cytoplasmic membrane by a periplasmic space. The permeability properties of such a wall are fairly similar to those typical of Gram-positive bacteria.
Pathogenic protozoa also represent a group of structurally rather disparate organisms, which raise a complicated problem for any therapeutic approach. Protozoa include parasitic and opportunistic pathogenic forms, which are characterized by a variety of complex life cycles and are often incapable of living outside the host except as resistant stages. As an example, the general pattern of life cycle of amoebic protozoa, such as Entamoeba histolytica and Acanthamoeba spp., is a trophic or feeding stage, alternating with a resting cystic stage. The cysts, which develop as environmental conditions become unfavourable (lack of food, waste product accumulation, desiccation), survive in nature until growth conditions improve and can remain viable for as long as 25 years. Indeed, the cysts are enclosed by a wall that may consist of two or three layers, each up to 300 nm thick, which forms a physical barrier. In Acanthamoeba castellanii the wall is constituted by fibrous material in which cellulose and an acid-insoluble protein have been identified as the principal components. On the other hand, the trophozoite is poorly equipped to survive for prolonged periods of time and is generally responsible for the lesions in infections of different tissues where it rapidly propagates.
On the basis of these considerations, it appears that Gram-negative bacteria and cysts from protozoa represent the most challenging targets for any type of antimicrobial treatment. It is worth emphasizing that in several cases the infections are caused by a heterogeneous microbial flora, hence the protocol adopted for the treatment of such infections cannot be focused on just one type of pathogens, rather it must be characterized by the possibility to efficaciously act on microbial pathogens with very different biological, morphological and physiological characteristics. As discussed below, PDT shows peculiar features which allow this problem to be addressed in an adequate manner.
1.2.2. Photobiological Processes in the Photosensitized Inactivation of Microbial Cells
As it is well known, photodynamic processes proceed by two competitive mechanisms, both of which require the participation of the long-lived photoexcited triplet state of the photosensitizer (3Sens) as the reactive intermediate. The type I pathway involves an electron transfer step between the triplet photosensitizer and a nearby substrate (Sub) with generation of radical species; the latter are then intercepted by ground state molecular oxygen yielding a variety of oxidized products. A typical simplified scheme for a type I photosensitization mechanism can be outlined as follows:
3Sens + Sub [right arrow] Sens(-1) + Sub(+) (1.1)
Sub(+) + O2 [right arrow] Subox (1.2)
The direction of the electron transfer event between the photosensitizer and the substrate is controlled by the relative redox potentials of the two species.
Alternatively, the type II pathway involves an electronic energy transfer process from the triplet photosensitizer to a suitable acceptor, most frequently oxygen, which is exceptionally a triplet in its ground state. The latter compound is converted to a highly reactive derivative, named singlet oxygen (1O2), which in turns attacks photosensitive targets in its surroundings:
3Sens + 3O2 [right arrow] Sens + 1O2 (1.3)
1O2 + Sub [right arrow] Subox (1.4)
In both cases, the oxidative attack is of electrophilic nature, hence it is preferentially directed toward specific biomolecules, including
(a) amino acids possessing an aromatic or heterocyclic side chain (e.g. tryptophan, tyrosine, histidine), as well as amino acids containing sulfur atoms (e.g. cysteine, methionine);
(b) pyrimidine and purine bases of DNA/RNA, with emphasis on guanosine;
(c) unsaturated lipids, such as oleic, linoleic and arachidonic acids, and steroids (especially cholesterol).
It is generally assumed that porphyrin- and phenothiazinephotosensitized processes proceed via formation of singlet oxygen, which thus represents the main cytotoxic agent responsible for the photodynamic inactivation of microbial cells. Indeed, it has been observed that the cultivation of Staphylococci in the presence of singlet oxygen scavengers, such as methionine or histidine, provides a high level of protection for the photosensitized cultures. At the same time, some authors demonstrated that singlet oxygen, generated by a photosensitizer deposited on an inert matrix and physically separated from the microbial cell culture, can diffuse through an air-equilibrated medium and cause an irreversible damage of Streptococcus faecium and E. coli. The decrease in the overall efficiency of the cell photoinactivation process upon increasing the distance between the immobilized photosensitizer and the bacterial cells corresponds very well with the measured lifetime of 1O2 in the reaction medium. However, the possibility of an at least partial contribution to the overall photoprocess from type I (radical-involving) reaction pathways cannot be ruled out, since a close spatial relationship between a cell-associated photosensitizer and photosensitive cell constituents could favour a direct interaction. In actual fact, the photoinduced formation of a radical anion from an E. coli-bound phthalocyanine has been detected.
In general, all the photosensitizers which have been found to be very active as photoantimicrobial agents preferentially localize at the level of the cytoplasmic membrane. One important exception is represented by acridines, such as proflavine or acridine orange, which largely intercalate with DNA bases. As a consequence, the main alterations of cell functions and morphology, caused by photodynamic inactivation, are typical of damaged membranous domains (see Table 1.1). No involvement of the genetic material is generally observed until the late stages of the overall photoprocess, which indicates that such photodamage is not correlated with cell death. This pattern of photoinduced sub-cellular damage is in agreement with the lack of mutagenic effects, as it has been repeatedly reported by different investigators, as well as with the lack of selection of photoresistant microbial strains even after several photosensitization treatments.
1.2.3. The Photosensitizer
Several photosensitizing agents of photodynamic nature proved to induce an efficient inactivation of at least some classes of microbial pathogens. On the basis of the experience developed so far and the considerations on the biochemical and morphological features of microbial cells (see paragraph 1.2.1), it is possible to outline which are the optimally desirable properties of an antimicrobial photosensitizer:
(a) Suitable photophysical characteristics, including a high extinction coefficient in the deeply tissue-penetrating wavelength regions (mainly the red and far-red spectral interval) even though for the treatment of superficial infections also the intensely absorbed blue light (400–420 nm) can be very useful; a long-lived electronically excited triplet state; and a high quantum yield for the generation of reactive oxygen species (in primis singlet oxygen) when exposed to excitation with visible light.
(b) A large affinity for the broadest possible classes of microbial cells, taking advantage of either the relatively high permeability of the outer wall (e.g. Gram-positive bacteria, trophozoites) or the presence of specific biochemical properties in this structural element (e.g. the presence of a vast array of negatively charged carboxylate functions in Gram-negative bacteria).
(c) The preferential binding of the photosensitizer with the cytoplasmic membrane, whose photosensitive constituents (e.g. unsaturated lipids, proteins) will consequently represent the main targets of the photoinactivation process; as a consequence, cell death will be predominantly due to membrane damage rather than involving the genetic material.
(d) The mechanisms involved in cell photoinactivation should have a minimal risk, if any, to induce the onset of mutagenic processes and to lead to the selection of microbial strains which are resistant to photodynamic action. The multi-target nature of the photodynamic killing of microbial cells can be considered as a strong warranty for reaching this objective.
(e) A broad spectrum of action on bacteria, fungi, yeasts and parasitic protozoa, in order to facilitate the achievement of an efficacious therapeutic effect also in the treatment of those infectious diseases which are characterized by the presence of a heterogeneous flora of pathogens.
(f) The possibility to identify a therapeutic window allowing an extensive mortality of the microbial pathogens with minimal simultaneous damage to the constituents of the host tissue, as well as the re-growth of the pathogens after the treatment. The achievement of the latter goal can especially rely on the lack of mutagenic effects or increased photoresistance in the photodynamically inactivated microbial cells; this would allow the repetition of the PDT approach in case of insufficient response of the infective agents to the primary treatment or the recurrence of the infection after a given time interval from the primary treatment.
While those photosensitizers which fulfil the criteria (a) and (b) are readily inducing an extensive killing of Gram-positive bacteria, fungi and trophozoites from parasitic protozoa, the goals mentioned in point (e) are generally much more difficult to achieve, largely owing to the tight organization of the outer wall and, respectively, the exocyst in Gram-negative bacteria and cysts derived from protozoa. Only photosensitizers which are characterized by the presence of positively charged functional groups in either the axial ligands to the centrally coordinated metal ion or the peripheral substituents located in the pyrrole rings or the meso positions of the macrocycle exhibit a sufficiently high photoactivity against these two classes of microorganisms. The substituents imparting a broad and markedly efficient antimicrobial photosensitizing activity are generally represented by moieties, such as pyridine, piperidine, morpholine, etc., whose nitrogen atoms can be quaternarized by alkylation reactions. In addition, even tertiary amino groups can represent an acceptable alternative provided their basic strength is sufficiently high to allow for protonation of the lone electron pair in the nitrogen atom at neutral pH values.
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Excerpted from Photodynamic Inactivation of Microbial Pathogens: Medical and Environmental Applications by Michael R. Hamblin, Giulio Jori. Copyright © 2011 European Society for Photobiology. Excerpted by permission of The Royal Society of Chemistry.
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