Antimicrobial photodynamic therapy (aPDT), also referred to as photodynamic inactivation (PDI), photodisinfection (PD), or photodynamic antimicrobial chemotherapy (PACT), is a photochemical antimicrobial method that has been studied for over a century. Supported by in vitro, in vivo and clinical studies, aPDT offers a treatment option for broad-spectrum infections, particularly in the context of rising antimicrobial resistance. Its multi-target mode of action allows aPDT to be a viable therapeutic strategy against drug-resistant microorganisms. The procedure involves the application of photosensitizing compounds, also called photoantimicrobials, which, upon activation by light, generate reactive oxygen species (ROS). These ROS lead to the oxidation of cellular components of a wide array of microbes, including pathogenic bacteria, fungi, protozoa, algae, and viruses.
Historical perspective In the early 20th century, decades before the first chemical antibiotics were developed, Dr. Niels Finsen discovered that blue light could be used to treat skin infections. In the following years, Finsen's phototherapy was used in many European medical institutions as a topical antimicrobial. In 1903, the Nobel Prize committee awarded him for his work in Physiology/Medicine, "in recognition of his contribution to the treatment of diseases, especially lupus vulgaris, with concentrated light radiation, whereby he has opened a new avenue for medical science".Nobel Prize Website
Similarly, in the beginning of the 20th century, Oscar Raab, a German medical student supervised by Professor Herman Von Tappeiner, accidentally made a scientific observation of the antimicrobial effects of light-activated dyes. While conducting experiments on the viability of motile protozoa, Raab noticed that fluorescent dyes, like some acridine and xanthene dyes, could kill stained microbes when sunlight was directed onto the stained samples. These effects were particularly pronounced during the summer, when sunlight is brightest. This chance observation highlighted the ability of certain fluorescent compounds, now termed "photosensitizers" (PS), to artificially induce light sensitivity in microorganisms and enhance the known antimicrobial effects of sunlight. Shortly thereafter, Von Tappeiner and Jodlbauer discovered that oxygen was crucial for light-mediated reactions, leading to the creation of the term "photodynamische wirkung" (photodynamic effect).
However, it wasn't until the 1970s that researchers began to systematically explore the potential of photodynamic therapy for medical applications. Since then, significant progress has been made in understanding the underlying mechanisms and optimizing the efficacy of photodynamic therapy (PDT) for treatment of cancers and age-related macular degeneration. Today, the branch of PDT focused on killing microbial cells is considered as an option to prevent and treat infectious diseases in a manner that avoids the emergence of antimicrobial drug-resistance.
Mechanism of action
The photochemical principle underlying antimicrobial photodynamic therapy involves the activation of a photosensitizer, a light-sensitive compound that can locally generate reactive products, such as radicals and reactive oxygen species (ROS), upon exposure to specific wavelengths of light. An ideal photosensitizer selectively accumulates in the target microbial cells, where it remains inactive and non-toxic until it is activated by irradiation with light of a specific wavelength. This activation promotes the photosensitizer molecules to a short-lived excited state that possesses different chemical reactivity relative to its ground-state counterpart. When the photosensitizer molecule is in an excited triplet state, it can induce local Type 1 photodynamic reactions by direct contact with molecular oxygen, inorganic ions or biological targets. These redox reactions (Type 1) involve charge transfers, by donation of electron (e–) or Hydrogen ion (H+), to form radicals and ROS, such as anion radical superoxide, hydrogen peroxide and hydroxyl radicals. The excited triplet-state photosensitizer can also transfer energy to molecular triplet-state oxygen producing singlet oxygen via Type 2 photodynamic reactions. The photoinduced burst of active reactants affect cellular redox regulations and can cause oxidative damage to vital structures made of proteins, lipids, carbohydrates and nucleic acids, leading to localized cellular death.
Efficacy against drug-resistant pathogens The efficacy of antimicrobial photodynamic therapy, using various distinct photosensitizers, has been studied since the 1990s. Most studies have yielded positive outcomes, often achieving disinfection levels, as defined by infection control guidelines, exceeding 5 log10 (99.999%) of microbial inactivation. Over the past decade, a collection of novel photoantimicrobials has been developed, exhibiting improved efficiencies in antimicrobial photodynamic action against various bacterial species. These studies have primarily focused on the inactivation of planktonic cultures, which are free-floating bacterial cells. This method serves as a convenient approach for high-throughput antimicrobial screening of multiple compounds, such as evaluating whether minor chemical modifications to a given photosensitizer can enhance antimicrobial efficacy. However, when present in biofilms, microbial populations can exhibit distinct characteristics compared to their planktonic counterparts, including significantly higher tolerance towards antimicrobials (up to 1,000-fold). Among the various factors contributing to this enhanced tolerance is the biofilm matrix composed extracellular polymeric substance (EPS). The EPS can shield constituent bacteria from antimicrobials through dual mechanisms: 1) by impeding the penetration of antimicrobial substances throughout the biofilm due to interactions between positively charged agents and negatively charged EPS residues, and also by 2) redox processes and π-π interactions involving aromatic surfaces generally acting to dismute the incoming active substance. EPS must be considered in the rational design of antimicrobial photosensitizers, because the densely cross linked matrix may also obstruct diffusion of photosensitizer into deeper biofilm layers.
The multi-target mechanisms of aPDT avoid antimicrobial resistance, which continues to be a major global health concern. The likelihood of developing resistance in pathogens is higher for antimicrobial strategies that have a specific target structure, following the key-lock principle, embodied in many antibiotics or antiseptics. In such cases, pathogens can evade the antimicrobial challenge through specific mutations, upregulation of efflux pumps, or production of enzymes that deactivate antimicrobials. In contrast, aPDT acts through a variety of non-specific oxidative mechanisms targeting multiple structures and pathways simultaneously, making the technique far less prone to resistance. The possibility of bacteria developing tolerance to aPDT has therefore been deemed highly unlikely. Several studies have demonstrated the efficacy of aPDT against various drug-resistant pathogens, including the World Health Organization (WHO) priority pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, Candida auris, Escherichia coli and many others.
Light sources Light is required to excite the photosensitizer, which leads to the photochemical production of ROS. To efficiently transfer photon energy to the electron structure of the photosensitizer, the wavelength of the light source must be matched to the absorption spectrum of the photosensitizer. Different light sources have been used in aPDT, such as lamps (e.g. tungsten filament, Xenon arc and fluorescent lamps), lasers and light emitting diodes (LEDs). Lamps typically emit white light, but a filter can be used to select the appropriate wavelength to be absorbed by the photosensitizer and to avoid undesired thermal effects. In contrast, lasers are monochromatic light sources that can be easily coupled to optical fibers to access non-surface regions. LEDs are also monochromatic light sources, although their spectral emission bands are wider than those of lasers. However, the coupling of LEDs and optical fibers is not efficient, resulting in significant loss of light. More recently, organic LEDs (OLEDs) have been used in aPDT as wearable light sources because they can be made to be more flexible, thinner, and lighter than conventional LEDs. Sunlight can also serve as a source of light for aPDT; however, exact illumination parameters may be difficult to precisely reproduce.
Light dosimetry aPDT results depend on the interplay of three physical quantities: irradiance, radiant exposure and exposure time. Irradiance is defined as the optical power of the light source in Watts, divided by the area of tissue illumination conventionally described in square meters or centimeters (W/m2 or W/cm2). The irradiance, as a photodynamic parameter, is limited by the onset of adverse thermal factors in exposed tissue, or by degradative consequences to the sensitizer itself (commonly referred to as "photobleaching"). Radiant exposure is given by the product of irradiance and exposure time in seconds, divided by the illuminated area (J/cm2), and is commonly termed the light dose. This parameter is often limited by acceptable treatment times because lengthy treatment times can be unacceptable in a point-of-care setting. Fluence is a different physical quantity often used by aPDT practitioners, which considers the backscattering flux of light-tissue interaction causing re-entry of photons back into the treated area.
Photosensitizers Photodynamic action relies on absorption of electromagnetic radiation by the photosensitizing compound and conversion of this energy into redox chemical reactions or transfer to ground-state oxygen, producing the highly oxidizing species, singlet oxygen. Consequently, the photosensitizer can be considered a photocatalyst, but it is also true that the sensitizer directly interacts with target moieties such as microbes to establish, for example, molecular targeting. This explains why not all photosensitizers are useful as photoantimicrobials.
The most effective photosensitizer molecules carry a positive charge (cationic). This promotes electrostatic attraction with negatively charged groups found on microbial cell surfaces (e.g. phosphate, carboxylate, sulfate), thus ensuring that during illumination, production of reactive oxygen species occurs in close contact with the targeted cellular population. Consequently, negatively charged photosensitizers are less effective, particularly against gram-negative bacterial cells that carry a strongly negative zeta potential.
The most widely employed photosensitizer in clinical practice is the phenothiazine derivative, methylene blue, which carries a +1 charge. Methylene blue is also favored due to its long record of safe use in patients, both in surgical staining and the systemic treatment of methemoglobinemia. Many other photosensitizers have been suggested, from various chemical classes, such as porphyrins, phthalocyanines and xanthenes, but the requirement for cationic nature and proven safety for human/animal use represents a high barrier to new chemical entity development.
aPDT Enhancement by inorganic salts and gold nanoparticles