Prof. Tebello Nyokong (PAS), Distinguished Professor of Chemistry and Director of Institute for Nanotechnology Innovation, Rhodes University, South Africa

Abstract: Modern medicine is exploring new approaches to combat infections associated with antimicrobial resistance. Among these, antimicrobial photodynamic therapy (aPDT) stands out for its ability to target pathogenic microbes effectively. This review focuses on methods to enhance aPDT on planktonic cells and biofilms. The combination of nanoparticles and antibiotics with the photosensitizers results in improved aPDT.

1. Introduction

Antimicrobial resistance (AMR) occurs when microorganisms no longer respond to antimicrobial medicines. Because of AMR, antimicrobial medicines become ineffective, increasing the risk of severe illness. Thus, preventing resistance is crucial in microbial treatment. AMR is a problem for all countries at all income levels. The World Health Organization (WHO) has listed the following as some of the high-priority pathogens in urgent need of drug development [1]: Methicillin-resistant Staphylococcus aureus [2], vancomycin-resistant Enterococcus faecium (VREF) [3], and multidrug resistant Klebsiella pneumoniae [4]. The emergence of multidrug resistant fungus Candida auris is of particular concern [5].

In addition, biofilms, linked to over 60% of antibiotic-resistant infections, present significant challenges in biomedicine [6]. Biofilms strongly resist antimicrobial agents [7]. The concentration of antimicrobials needed to eradicate biofilms is about 1,000 times higher than that for planktonic bacteria, partly due to the biofilm matrix acting as a physical barrier to antibiotics [8]. A biofilm is a well-organized community of bacteria embedded in an exopolymeric substance (EPS) that is attached to a biotic or abiotic surface [9]. EPS plays a key role in the development, maintenance as well as protection of biofilms against dehydration and the effects of antimicrobials [10]. Compared to planktonic cells, biofilm cells exhibit different physiological and metabolic states. They account for up to 80% of all bacterial chronic infections in humans and the formation of bacterial biofilms is one of the major causes of bacterial resistance [11]. Antimicrobial photodynamic therapy (aPDT) has been proposed for the elimination of drug resistance.

2. Antimicrobial photodynamic therapy (aPDT)

The concept of cell death induced by the interaction of light and chemicals was first reported by Osar Raab, a medical student working with Professor Herman von Tappeiner in Munich [12]. Subsequent work in the laboratory of von Tappeiner coined the term “Photodynamic action” and they showed that oxygen was essential for cell death.

aPDT is an antimicrobial treatment modality based on using light of a specific wavelength in combination with a photosensitizer, leading to a phototoxic reaction that induces micro-organism destruction, known as the photodynamic effect [13]. aPDT is expected to eradicate micro-organisms through the photosensitized production of toxic reactive oxygen species (ROS) including singlet oxygen in the presence of molecular oxygen [14], as shown in Fig. 1. This approach causes irreversible cell membrane damage preventing resistance against aPDT [15]. aPDT is particularly good for dental [16] and dermatological [17] applications, where there is light irradiation of a tissue containing microorganisms that were previously exposed to a photosensitizing (PS) dye.

In dentistry, aPDT can be considered as an adjunctive to conventional mechanical therapy. The liquid photosensitizer placed directly in the periodontal pocket can easily access the whole root surface before activation by the laser light through an optical fiber placed directly in the pocket [18].

In aPDT, the molecules absorb light to the first excited state (S₁) and can further undergo a spin conversion through intersystem crossing (ISC) from S₁ to long-lived triplet state (T₁), where they either emit photons through phosphorescence or can undertake other chemical reactions known as either type I or type II pathways. In type I, a photosensitizer reacts directly with the biological substrates or oxygen to generate ROS such as hydroxyl radical, peroxide and superoxide anion radicals through electron transfer. In the type II reaction, the activated PS transfers energy to the ³O₂ forming ¹O₂, Fig. 1. Both pathways can occur simultaneously and are responsible for the inactivation of multiple pathogens, with the most dominant pathway being largely dependent on the photosensitizer used [19].

An ideal photosensitizer should exhibit high photochemical reactivity, which is essential in producing ROS. The PS should have a high absorption coefficient, be photostable, have thermal and chemical stability. The PS should be able to produce high triplet quantum yields and have a long triplet lifetime to allow for efficient energy transfer to ground-state oxygen [20]. PSs can be classified into two categories, porphyrinoids and non-porphyriniods, Fig. 2. Porphyrinoids are aromatic tetrapyrrole macrocycles derived from natural pigments such as chlorophyll. Porphyrinoids include porphyrin, chlorin, phthalocyanine, bacteriochlorin, porphycene, texaphyrins, and sapphyrins [21,22]. The non-porphyriniods refer to aromatic dyes that can generate singlet oxygen, including methylene blue, rose bengal, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), eosin blue and erythrosine B [22,23], Fig. 2. aPDT can be used on various microorganisms such as fungi, parasites, bacteria, and viruses.

aPDT is effective against fungal infections since it can be combined with other therapies and is applicable to patients who are unresponsiveness to oral antifungal agents [24]. Currently, aPDT is widely used to treat many cutaneous fungal infections [24].

The emergence of drug resistance in parasites has been a major concern [25]. Human pathogenic parasites have been killed by combinations of PS and light, thus aPDT has successfully been employed against parasites [25]. In a similar fashion to viruses, there have been several reports on the use of aPDT to kill yeasts [26]. Membrane damage and the consequent increased permeability was reported to be the cause of cell death using aPDT for yeasts [26].

Antimicrobial PS should be able to kill multiple classes of microbial cells at relatively low concentrations and low fluences of light. The PS should also be reasonably nontoxic in the dark and should show selectivity for microbial cells over host mammalian cells.

2.1. Focus on Bacteria

The activity of photosensitizers on bacteria can been determined using Log reductions, equation 1

log reduction = log (A) - log (B)                                                                 (1)

A and B are the number of viable colonies (in colony forming units/mL) of bacteria for the untreated and treated samples. A log reduction > 3.0 and a 99.9% reduction in viability is classified as an efficient antimicrobial agent [27].

Gram-negative bacteria are responsible for many life-threatening infections, and they are often resistant to the most commonly used antibiotics, making the search for new antibacterial drugs and alternative therapies, such as aPDT, very important [28]. Neutral PSs have been efficiently applied in the aPDT of gram-positive bacteria while cationic PSs are effective for both gram-positive and gram-negative bacteria [29,30]. In aPDT, the PS employed needs to penetrate the cell wall. Cationic molecules can more easily bind to the cell wall of Gram-negative bacteria, which is negatively charged. Gram-negative bacteria have a double lipid bilayer sandwiching the peptidoglycan layer plus an outer layer of lipopolysaccharide, resulting in a low degree of permeability, while gram-positive bacteria possess a porous peptidoglycan layer and a single lipid bilayer [31]. The outer membrane of Gram-negative bacteria plays an important role that is related to resistance to many antibiotics that are highly effective against Gram-positive bacteria. This explains the higher prevalence of Gram-negative infections in the modern hospital environment [32]. Therefore, aPDT killing of Gram-positive bacteria is definitely much easier to accomplish than that of Gram-negative bacteria. Thus, it is more difficult to obtain highly potent PS to mediate PDT of Gram-negative bacteria since their membrane barrier prevents the uptake of anionic and neutral PS [33,34]. Nevertheless, different approaches have been documented aimed at efficiently killing Gram-negative bacteria via aPDT. Several of these approaches involve the optimization of the chemical structure of the PS.

2.2. The role of nanoparticles in aPDT

Nanoparticles (NPs) are particulates with one or more nanoscale dimensions (1–100 nm). Their superior physicochemical, optical, and thermal properties make them valuable functional materials for various technologies [35,36]. There are numerous varieties of NPs, including metallic and non-metallic NPs [37]. Nanoparticles based on metals and silica offer advantages over organic nanoparticles, such as having easy-to-control particle sizes, shape, porosity, and monodispersibility; however, these inorganic nanoparticles do not readily degrade in the biological system [38]. Some of the main advantages of using NPs include their ability to target drugs and enhance aqueous solubility of other materials; they have tuneable properties and large surface areas, which allow for multiple functional groups to be added to their surfaces [39]. In addition, NPs can influence the photophysicochemical properties of a photosensitizer and they tend to exhibit size-related properties that differ significantly from those observed in fine particles or bulk materials [40,41]. To enhance the efficacy of aPDT, nanoparticles can be conjugated with photosensitizers. NPs can act as a drug-delivery system and consequently facilitate the internalization of photosensitizers; this approach allows for the use of low concentrations of photosensitizers and the shortest light exposure time [42]. The loading of the PSs onto carbon nanomaterials can be achieved by π-π stacking noncovalent functionalization, since both have benzene rings in their structure. The covalent chemical functionalization with the PS molecule can be done through formation of amide or ester bonds. This surface functionalization helps to improve the photophysicochemical properties and the targeting and therapeutic efficiency of the PSs for aPDT. PSs are often ineffective when using lower concentrations and light doses, especially in the case of Gram-negative bacteria biofilms, due to the complexity of their envelope. aPDT activity improves (log reduction increase) in the presence of NPs.

2.3. aPDT-antibiotic dual therapy

aPDT-antibiotic dual therapy combines two therapies, photodynamic and chemotherapy, that act in two quite different therapeutic mechanisms resulting for reductions in treatment time, drug doses, side effects, and drug-resistance problem [43]. In dual photo-chemotherapy, three approaches can be considered: sequential administration of a PS and an antibiotic drug, the use of PS and antibiotic conjugates, and co-encapsulation of the two in a nanocarrier. As an example, ciprofloxacin (CIP), a quinolone-type antibiotic that is commonly used to treat infections in humans, [44] was employed together with a phthalocyanine for aPDT [45]. The data show that greater antibacterial activities are obtained in the dual therapy of aPDT and chemotherapy when the biofilms were sequentially photoinactivated with PSs, then incubated with ciprofloxacin. Coupling aPDT with CIP offers several advantages, preferably when aPDT precedes the antibiotic. This is because aPDT can disrupt the EPS layer, lower the expression of the antibiotic resistance-conferring genes and inactivate the drug modifying enzymes beforehand. Subsequently, this will result in an increased uptake of the antibiotic and potentiate a localized photo-destructive effect, making the cells inside the biofilm more susceptible to the antibiotic. It is worth emphasizing that the complete inhibition of biofilms only occurred under the influence of simultaneous treatment with aPDT and ciprofloxacin [45]. This combination totally eradicated the biofilm, regardless of the incubation time, Fig. 3. The presence of two modes of action enables the killing of bacteria in their stationary growth phase (inside biofilms) and to lower the toxic effects of antimicrobial chemotherapy on normal host tissues. The synergistic effect of aPDT combined with antibiotic treatment could be a promising strategy to overcome the multidrug-resistant bacterial infections caused by biofilms.

Other molecules such as ampicillin (AMP) and gallic acid have been used together with PS for aPDT [46,47]. AMP has antimicrobial activity [48]. According to reports, gallic acid (GA) has a potent antibacterial effect that induces cell death by disrupting the membrane integrity of some gram-negative and gram-positive bacteria [49-51]. Thus, combining AMP or GA with PS for aPDT resulted in enhanced antibacterial activity.

3. Conclusion

Antimicrobial photodynamic therapy (aPDT) of various microorganisms is effective against planktonic and biofilm forms, with cationic photosensitizers showing high log reductions values against bacteria. Combining photosensitizers with antibiotics or other antimicrobial agents enhances aPDT. Thus, aPDT is the solution to drug resistance. Conjugating photosensitizers to nanoparticles further improves aPDT.

 

Acknowledgments

This work was supported by the Department of Science and Innovation (DST), Innovation and National Research Foundation (NRF), South Africa through DSI/NRF South African Research Chairs Initiative for Professors of Medicinal Chemistry and Nanotechnology (UID 62620), Rhodes University, CSIR National Laser Centre Rental Pool Program and by DSI/Mintek Nanotechnology Innovation Centre-Sensors.

 

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