The MIC values of water-soluble PSs in the dark and under illumination. About 3 × 104 CFU mL−1 of
Increasing resistance of bacteria to antibiotics is a serious worldwide problem, and to combat resistant bacteria, new antibacterial approaches are to be developed. One alternative to traditional antibiotic therapy is photodynamic antimicrobial chemotherapy (PACT). PACT is based on excitation of photosensitizers (PS) capable of transferring the absorbed light energy to dissolved molecular oxygen causing generation of reactive oxygen species, which irreversibly damage bacterial cell components. The overall efficiency of PACT has been proven for Gram-positive and Gram-negative bacteria. The effectiveness of PACT can be increased by encapsulation of PS in liposomes providing more concentrated delivery of PS, enhanced cytotoxicity, improved pharmacokinetic properties, sustained release, and prolonged action of the PS. For continuous and reusable application, PS can be immobilized in polymers. Chemiluminescence, sonodynamic treatment, and radiofrequency irradiation allow to perform excitation of PS in the dark without external illumination, opening prospects for combating internal infections. Combination of PS with antibiotics can gain a synergistic effect, allowing in some cases to overcome the resistance of bacteria to antibiotics.
- photodynamic therapy (PDT)
- photodynamic antimicrobial chemotherapy (PACT)
- photosensitizer (PS)
- chemiluminescent antimicrobial chemotherapy (CPAT)
- sonodynamic antimicrobial chemotherapy (SACT)
- targeted drug delivery
1.1 History of photodynamic therapy
The therapeutic properties of light were observed already in ancient Greece, Egypt, and India. However, they were not widely used for many centuries . The history of modern photodynamic therapy (PDT) dates back to 1900, when Oscar Raab discovered the toxic properties of the dye acridine red on
1.2 Photosensitizers and their mechanism of action
PACT is based on the exposure of bacteria to photosensitive compounds—photosensitizers (PSs). When a PS located in the bacteria or on the bacterial surface is exposed to light (usually visible), it transfers from its low-energy ground state to an excited singlet state. Return of the PS to its ground state is accompanied by either emission of fluorescence or transition of the PS to a longer-living, higher-energy triplet state (PS*) via intersystem crossing. The PS* in turn reacts with surrounding molecules to form free radicals and hydrogen peroxide (Type I reaction) or transfers its energy to molecular oxygen to produce singlet oxygen and other highly reactive oxygen species (ROS; Type II reaction) [9, 10]. Type I and Type II reactions occur simultaneously, and the ratio at which they occur depends on both the PS type and the surrounding conditions. A detailed description of the photosensitization process can be found in the recent reviews of Castano et al.  and Cieplik . ROSs formed in this process oxidize biomolecules, damage the cell membrane, and ultimately lead to cell death . PACT usually proceeds predominantly through Type II processes. However, since Gram-negative bacteria are more susceptible to OH. radicals than to singlet oxygen, the Type I reaction may be more efficient against such microorganisms [13, 14].
1.3 Photosensitizers for PACT
Hundreds of compounds are currently available for mediating PDT in various areas of medicine, where some have been shown to be suitable for antimicrobial applications. PSs employed for medical uses should be a single pure compound, stable at room temperature and inexpensive. The PS must have a strong absorption peak in the visible spectrum between 600 and 900 nm and should possess a high-triplet quantum yield that will provide high production of ROS upon illumination. It should not be toxic in the dark (especially to mammalian cells), mutagenic or carcinogenic [15, 16, 17, 18]. In addition, when talking about PACT, it is very important that the PS will display preferential association with bacteria, accumulate within the cells, or bind to the bacterial cell envelope [14, 19].
PSs can generally be assigned to several chemical classes: tetrapyrroles (which include porphyrins, chlorins, bacteriochlorins, and phthalocyanines), synthetic dyes (phenothiazinium salts, Rose Bengal, squaraines, etc.), and naturally occurring compounds (such as riboflavin or curcumin). Cyclic tetrapyrroles present the most well-known class of clinically relevant PSs used mostly for anticancer applications . This structure can be found naturally in such important biomolecules such as haem, chlorophyll, and bacteriochlorophyll. Unlike other types of PSs, most tetrapyrroles (except for bacteriochlorins) are more likely to react by a Type II reaction with the creation of singlet oxygen , whereas bacteriochlorins act
2. Photosensitizer activation modes
2.1 Dark activity
The name photosensitizer implies the need for illumination in order to activate PS molecules and trigger their action. However, PSs possess some so-called “dark activity” even in the absence of illumination, leading to cell death in the dark [23, 24, 25, 26, 27, 28, 29]. This feature depends on the PS concentration and manifests itself in different ways for various PSs.
Shrestha demonstrated dark toxicity of RB against Gram-positive
In our studies, we also noted the dark toxicity of various PSs against different types of bacteria (Figures 1,2, Table 1). Figure 1 shows the effect of various RB concentrations on
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Although PSs are known to possess a certain dark activity, illumination noticeably increases their cytotoxic effect [6, 14]. An example of the difference in antibacterial activity of different PSs with and without illumination is shown in Table 1. In this experiment, the MIC of three PSs was determined for the bacterium
The main light sources used today for activation of PSs are lasers, light-emitting diodes (LED), and gas discharge lamps (GDL) [10, 31, 32]. There is no absolute advantage of one of these light sources over the others. The choice of light source depends on the specific application. Laser is a high-intensity monochromatic source. It can be easily coupled to a single optical fiber and installed on different lighting devices. LED lamps are cheaper and provide a wide emission spectrum. GDLs are also cheaper than lasers—both in acquisition and in maintenance and have a wide emission spectrum. However, GDLs transmit more heat to the illuminated area than lasers and LEDs, which can lead to tissue damage. In general, the emission spectrum and light intensity are more important for the excitation of a specific PS than the particular light source type [10, 31, 32].
2.3 Sonodynamic excitation of photosensitizers
Illumination is undoubtedly the easiest and most effective way to activate PSs. However, its use is restricted, due to limited penetration of visible light into tissues. There is an ongoing search for alternative methods of PS excitation in the dark in order to overcome this problem. Ultrasonic activation seems to be attractive as an alternative to illumination. As with light activation, ultrasound can be selectively focused on a specific area, thus activating only PS molecules located in the affected area. Ultrasound can also easily penetrate into tissues, which opens prospects for its application in treatment of internal lesions and infections, without the need for invasive devices [33, 34]. Ultrasonic irradiation of PSs initiates the formation of highly active cytotoxic species—ROS and free radicals—which lead to the death of pathogenic cells. It was found that some well-known PSs also have sonosensitizing properties. Among them are porphyrins , RB [36, 37], chlorin e6 derivative, photodithazine , and curcumin . Several studies found sonodynamic therapy (SDT) to be the promising treatment in various forms of cancerous tumors [39, 40, 41, 42, 43]. Sonodynamic therapy is also offered as treatment for atherosclerosis . The applicability of sonodynamic antimicrobial chemotherapy (SACT) for the treatment of infectious diseases has been confirmed by various research groups [33, 34]. We have previously demonstrated the effectiveness of RB activated by ultrasonication for eradication of Gram-positive
Figure 2 demonstrates the effect of ultrasonic activation that we showed on the antibacterial activity of two PSs—RB (Figure 2a) and MB (Figure 2b)—against
2.4 Activation of photosensitizers by radio waves
Another possible way for activating PSs in the dark is by using nonionizing radiofrequency electromagnetic waves. The ability of radiofrequency waves to heat human tissue has been known for a long time and has already been applied for local destruction of cancerous tumors [47, 48]. The effectiveness of this method can be significantly improved by using suitable sensitizers, which can be targeted to the affected area and activated by means of radiofrequency radiation for selective destruction of cells. Tamarov et al. proposed the use of crystalline silicon-based nanoparticles as sensitizers induced by 27 MHz radiofrequency waves for effective treatment of Lewis lung carcinoma
In our studies, we tested the possibility of using radiofrequency radiation to sensitize PSs in order to destroy microorganisms . For this purpose, we irradiated
To the best of our knowledge, our work was the first attempt to sensitize a PS by radio waves for destruction of bacteria. This topic naturally necessitates a broader and deeper study to understand the mechanisms of excitation and the possibilities of applying this method. The most likely mechanism of RB excitation by radio waves is conversion of electromagnetic energy into heat, which causes activation of RB, followed by energy transfer to dissolved oxygen and the formation of ROS, affecting the cells. We assume that when PSs are exposed to radiofrequency radiation, they actually behave like thermosensitizers excited by heat instead of light .
2.5 Chemiluminescent and bioluminescent excitation of photosensitizers
Another approach to overcoming the limitations of PACT in the treatment of deep infections is to replace the external light source by chemo- or bioluminescent light. Bioluminescence is a well-known phenomenon occurring in biological systems as a result of oxidation reactions of luciferins catalyzed by luciferases. This property is inherent in various microorganisms, worms, and insects, and the luciferins and luciferases of different organisms can be completely different. Bioluminescence is considered as a type of chemiluminescence, i.e., luminescence originating in the course of a chemical reaction. Bio- and chemiluminescence systems are used in various fields of medicine, pharmaceuticals, and bioanalytics [52, 53].
One of the well-studied and most effective chemical reactions involving light emission is oxidation of luminol [52, 54, 55]. Most applications of this reaction are associated with treatment of cancers [55, 56, 57]. Use of chemiluminescence as a light source for PACT has not been studied as extensively. Ferraz and colleagues evaluated the potential of chemiluminescent-excited photogem in killing
The dark effect of MB discussed in the above “Dark Activity” section can be seen in Figure 4, where the exposure of
3. Encapsulation of photosensitizers in liposomes
Since PSs are usually inactive in the absence of excitation, focusing the beam of light, ultrasound or radio wave radiation on the affected area is the easiest way to achieve selective action of a PS. However, surrounding healthy tissues may also be affected by the PS, even under such focused processing. It is therefore very important to target the treatment directly to the infected site. Highly biocompatible and low immunogenic liposomes can serve as carriers for targeted delivery of PSs encapsulated into liposomes to the infected site [61, 62, 63].
Liposomes are spherical multi- or unilamellar vesicles consisting of phospholipids (e.g., phosphatidylcholines) with an internal hydrophilic cavity. They vary in composition, size, charge, and number of layers and can encapsulate and deliver both hydrophilic and hydrophobic compounds, which can be retained in the water core of liposomes or be encapsulated in the phospholipid bilayer, respectively. A variety of methods have been developed for the production of liposomes with a controlled size and special properties. The most widely used method for producing liposomes is hydration of thin lipid films. In this case, lipids with or without active substances are dissolved in an organic solvent, which is evaporated on a rotary evaporator, producing a thin film on a flask wall. The lipid film is then rehydrated by an aqueous phase. Membrane extrusion and sonication methods are most commonly used for control of liposome size . Advanced strategies for liposome preparation include charging the liposomes, attaching the ligands such as antibodies or lectins to their surface, or altering the physiological conditions such as increasing the temperature or changing the pH in the target tissues to produce heat-sensitive or pH-sensitive liposomes . The works of Ghosh, Li, Bulbake, Abu Lila, and Alavi summarize the latest developments in the field of liposome design and optimization, including passive and active targeting, extended circulation, building multifunctional liposomes, and so on [62, 63, 64, 65, 66].
There exist several methods for PS encapsulation into liposomes (Figure 5). Hydrophilic PSs (e.g., MB, RB, or photofrin) are dissolved in aqueous buffer and are included into the internal cavity of liposomes. Hydrophobic compounds (such as temoporfin and bacteriochlorin a) are integrated in the phospholipid bilayer [62, 67]. Several groups have shown that encapsulation of PSs in liposomes improves their effectiveness against cancer
Liposomal PS preparations are suitable for antibacterial applications. This approach ensures the delivery of the compound at a higher concentration, thus increasing the cytotoxicity of the drug. In addition, the local use of liposomal preparations provides a slow release of active components, which helps prolong their effect in infected tissues. In Gram-negative bacteria, fusion between liposomes and the outer cell membranes leads to the delivery of concentrated liposome contents directly into the cytoplasm [70, 71, 72]. In Gram-positive bacteria, the PS is probably released when liposomes interact with the external peptidoglycan and diffuse through the cell wall [72, 73, 74]. Various researchers have demonstrated the effectiveness of liposomal formulations of various PSs against Gram-positive and Gram-negative microorganisms and also against fungal infections
In our studies, we tested the effect of different PSs in different liposome formulations on Gram-positive and Gram-negative bacteria. Figure 6 presents a comparison between the MICs of free and dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol liposome-encapsulated MB and NR against
As can be seen from the results, incorporation into liposomes significantly increased the antibacterial activity of MB and NR. Following encapsulation, the MIC of MB decreased by approximately 2-fold and that of NR by about 1.4-fold for both tested microorganisms (Figure 6). We tested the effect of liposome composition on the delivery of these PSs to cells and determined the conditions for efficient use of encapsulated PSs .
In addition, we tried to apply liposomal forms of PSs to CPAT by encapsulating not only PSs in liposomes but also luminol and introduced to activate PSs in sites inaccessible to external lighting . We monitored the survival of the cells following their exposure to either liposomal MB or luminol, as well as to liposomes containing both compounds together (Figure 7) when the experiments were carried out in the dark.
It can be seen (Figure 7) that luminol itself did not lead to cell damage. MB in the liposomal form exhibited certain dark activity, similar to that in a free form discussed in the “Dark Activity” section. The addition of luminol to MB liposomes markedly increased its antibacterial activity toward
New prospects of using PSs are opened by the immobilization of PSs onto a solid phase. This approach may allow repeated or continuous use of PSs. PSs can be immobilized by adsorption and covalent bonding onto solid supports and by ionic bonding to ion-exchange resins or incorporation into polymer films. The photodynamic properties of immobilized PSs are reported to be retained for a long time [79, 80, 81, 82, 83]. PSs studied in the immobilized form include RB, MB, and TBO; the porphyrin derivatives 5,10,15,20-tetrakis (p-hydroxy phenyl) porphyrin, 5,10,15,20-tetrakis (p-aminophenyl) porphyrin, and zinc (II) phthalocyanine tetrasulfonic acid; and the ruthenium salts tris (4,4′-diphenyl-2,2′-bipyridine) ruthenium (II), tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II), tris (1,10-phenanthrolinyl-4,7-bis (benzenesulfonate) ruthenate (II), and tris (4,40-dinonyl-1,10-phenan throline) ruthenium (II). Solid supports applied for immobilization of PSs include polyethylene, polypropylene, polystyrene, polycarbonate, polymethyl methacrylate, polyester isophthalic resin, silicone, cationic nylon, porous silicones, poly (vinylidene difluoride), cellulose membranes, and chitosan [82, 83, 84, 85, 86, 87, 88]. Immobilized PSs demonstrated antibacterial properties against Gram-negative and Gram-positive bacteria in batch and continuous regimes and under reuse. Immobilized PSs were found more stable and resistant to photobleaching than in a free form [82, 86, 88].
Our group immobilized PSs in polymers using several techniques. The first method included mixing solutions of PSs in chloroform with solutions of polymers in the same solvent, followed by evaporation of the solvent, which yielded thin polymeric films with homogeneously incorporated PSs. This technique was applied to RB and MB immobilized onto polystyrene, polycarbonate, and polymethyl methacrylate [88, 89, 90]. In all cases, the obtained polymer films showed high antibacterial activity against Gram-positive and Gram-negative bacteria when exposed to an external source of white light. However, since this method involves using an organic solvent, it cannot be considered environmentally friendly. The second method is based on dissolution of PSs in a melted polymer under extrusion and does not require any additional chemical reagents . The photosensitizers RB, Rose Bengal lactone, MB, and hematoporphyrin were immobilized in polyethylene and polypropylene using this method. The antibacterial efficiency of immobilized PSs obtained as polymeric strips and beads was tested against
Another immobilization technique was based on polymerization of silicon in the presence of RB as the photosensitizer. Silicon tablets produced by this method contained evenly distributed RB that was not bound to the support by covalent bonds . The antibacterial activity of the immobilized RB was tested under illumination and using ultrasonic activation in the dark (Figure 8). Figure 8 demonstrates the effect of immobilized RB on
Further development of immobilization methods and different PSs and polymers may expand the possibilities of this approach and yield the applications in various fields, such as the production of antibacterial surfaces and water disinfection.
Numerous studies show that photodynamic antibacterial chemotherapy is a powerful tool for killing microorganisms. Since this method requires external illumination, it can be successfully applied only to the treatment of local superficial skin and oral cavity infections. Development of new modes of PS excitation by ultrasound, radio waves, chemiluminescent, and bioluminescent light opens new prospects for their use in treating internal infections. Encapsulation of PSs in liposomes may solve the problem of using hydrophobic PSs with poor solubility in the aqueous phase. It can also provide delivery of a concentrated PS directly to the target site, thus increasing efficiency and reducing side effects of the treatment. Immobilization of PSs in a solid phase enables using them repeatedly or in a continuous mode. It can be assumed that PSs have a good potential for various clinical and nonclinical applications.
This work was supported by the Research Authority of the Ariel University, Ariel, Israel.
Conflict of interest
The authors declare no conflict of interest.