**3. PDT fundamentals**

PDT consists of a photochemical reaction between a photosensitizing agent and the oxygen that selectively destroys the target tissue, constituting an alternative modality clinically approved by several health agencies in many countries [25–29]. The photodynamic effect consists of causing a powerful and sustained photochemical reaction between light at a given wavelength, the photosensitizer (PS) and oxygen in the target tissue. Consequently, after the irradiation of light, PS converts O2 into cytotoxic reactive oxygen species (ROS), where cell death can occur through mechanisms such as apoptosis, necrosis, or autophagy (**Figure 2**). However, recent studies have demonstrated the existence of other mechanisms with characteristics of necrosis and apoptosis. These new pathways of cell death, collectively called regulated necrosis, include a variety of processes triggered by different stimuli [14, 30].

### **Figure 2.**

*Basic scheme of the photodynamic reaction. (A) Formation of ROS. (B) Porphyrin group of photosensitizer absorbs a photon that excites it to the short-lived singlet state and may decay by non-radioactive relaxation with heat emission or fluorescence emission to the long-lived triple state. In this triplet state, PS can interact with molecular oxygen in two ways, type-1 and type-2, leading to the formation of oxygen radicals and singlet oxygen [31].*

### *Light and Phages on Tackle of Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.96425*

The light is formed by subatomic particles given off by atoms and are endowed with high luminous energy, and energy differences result in different colors called photons. The laser (Light Amplification by Stimulated Emission of Radiation) consists of a monochromatic, non-ionizing and highly concentrated beam of light. Each wave has identical coherence in size and physical shape along its axis, producing a specific form of electromagnetic energy. This wave is characterized by spatial coherence, that is, the beam can be well defined. The intensity and amplitude of the beam follow the curve of the Gaussian beam bell as most of the energy is in the center, with a rapid drop at the edges. There is also a temporal coherence, which means that the emission of the single wavelength has identical oscillations over a period. The final laser beam starts in a collimated form and can be emitted over a long distance in this way. However, bundles emanating from optical fibers generally diverge at the tip. When using lenses, all the beams can be precisely focused, and this monochromatic and coherent beam of light energy can achieve the treatment goal [16, 32, 33].

Photosensitizing agents (PS) consist of molecules in the singlet state in their fundamental state because they have two electrons with opposite spins that allow the transport and transfer of light energy for a chemical reaction, where each PS has unique characteristics for successful activation such as wavelength and creep intensity [34–36].

Most of them are derived from endogenous dyes and are characterized by not being toxic to cells. The molecular structure of most PSs used in PDT is based on a tetrapyrrol skeleton. This type of structure occurs naturally in several important biomolecules, such as heme, chlorophyll, and bacteriochlorophyll, being called "pigments of life" [36]. Therefore, PSs based on porphyrin structures satisfy most of the desirable properties of PSs, such as the high efficiency of singlet generation ( 1 O2), absorption of the higher wavelengths of the electromagnetic spectrum and a relatively greater affinity for malignant cells, in addition, due to the internal dimensions of the macrocycle cavity and the chelate effect, the porphyrin macrocycle can coordinate transition metals in various oxidation states [36].

### **4. Bacteriophages and PDT**

Resistance to antibiotics spreads rapidly in relation to the discovery of new compounds and their introduction into clinical practice. In addition, the increase in bacterial adaptation can be directly correlated to the scarcity of new classes of antimicrobial agents. In the last decades, synthetic tailoring has been the main strategy to improve the nuclear scaffolding established through analog generation. Although this approach has been beneficial, this research has faced a 'Discovery Void' for 30 years, of which no new class of drugs effective against problematic ESKAPE pathogens. In addition, pathogenic microorganisms generally have the ability to form biofilms. This cellular superstructure may exhibit greater resistance to antibiotics and cause serious and persistent health problems in humans [37–39].

Bacteriophages (phages) are ubiquitous viruses which cause no harm to human or animal cells but are capable to specifically infect, replicate, and kill bacteria [40, 41]. Bacteriophages have been described for delivering successfully antimicrobial agents into bacteria, which consist in a potential alternative for the treatment of infectious diseases [42, 43] caused by bacteria.

The first *in vivo* evidence of effective phage therapy against *Klebsiella pneumoniae*, one of Gram-negative bacterium of ESKAPE group listed in the critical priority tier [44, 45] as serious opportunist in nosocomial infections in the respiratory and urinary tracts, wound sites and blood [46], was demonstrated by Anand et al. [47]. The authors observed significant reduction in the lung lesion severity in the mouse model, suggesting the efficacy of a novel lytic phage VTCFPA43 therapy against virulent *K. pneumoniae* infection by the intranasal route.

According to [48, 49], the delivery systems based on a phage-carrying PS exhibit increased effective killing by the concentrated fluence at the bacterial cell wall, and consequently, reduced side damage to the indigenous microbiota by the site singlet oxygen. Moreover, they investigated the photodynamic effects of the photosensitizer tin (IV) chlorin e6 (SnCe6) (**Figure 3A**) covalently linked to phage 75 on several strains of *S. aureus*, including methicillin- and vancomycinintermediate strains. Pathogens such as Methicillin-resistant *Staphylococcus aureus* (MRSa) show that antibiotic resistance rates are surpassing 50% in 5 out of 6 world regions of the World Health Organization (WHO) [37, 50]. Results showed that the phage 75 conjugated with SnCe6 was not capable to damage human epithelial cells whereas potently showed bactericide effect against vancomycin-intermediate and MRSa. Additionally, other exogenous photosensitizers (protoporphyrin IX and protoporphyrin diarginate) have been successfully *in vitro* evaluated against clinical strains of MRSa [51].

*Acinetobacter baumannii* is other important Gram-negative bacterium multidrug-resistant involved in nosocomial infections [51]. Due its capacity to form biofilms, they have the capacity to survive and persist in intensive care unit environment and medical devices [52], what also make of *A. baumannii* one of critical-priority pathogens encompassing the ESKAPE group, for which new antibiotics and combating strategies are urgently needed [11, 12]. In this way, [53] used for the first time the strategy of combining of cationic photosensitizer (NB), structurally modified to produces ROS, and bacteriophages (APB)-based photodynamic antimicrobial agent (APNB) for eradication biofilm formed by multi-drug resistant *A. baumannii*. Both *in vitro* and *in vivo* assays demonstrated that APBN was efficient to treat *A. baumannii* infection, including being more efficient than some antibiotics when evaluated *in vivo*. These results demonstrated the potential of APNB in combating multidrug-resistant bacteria and biofilm ablation.

*Candida albicans* and more recently *C. auris* are opportunistic polymorphic fungal pathogens, which exhibits almost 40% mortality rates for superficial and systemic infections in humans [54–57]. Likewise, the increasing occurrence of antibiotic-resistant among *C. albicans* strains, demands new approaches to control this life-threatening pathogen [58]. In [59], it reported the photodynamic inactivation of *C. albicans* by the Pheophorbide A (PPA) (**Figure 3B**), a chlorophyll-based

**Figure 3.** *Chemical structure of (A) SnCe6 and (B) PPA.*

### *Light and Phages on Tackle of Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.96425*

photosensitizer crosslinked associated with single-chain variable-fragment phage (JM), which possesses high affinity to β-glucanase mannoprotein (MP65), an essential cell-wall mannoprotein of *C. albicans*. The complex PPA-JM-phage was capable to induce a caspase-dependent apoptosis pathway in *C. albicans.*

The second-generation of PSs exhibits improved photophysical properties in relation to first-generation PSs, which halogens or other substituents are added in the meso- positions of the porphyrin macrocycle. These porphyrins present a better activation of the ring, since the halogens act as removers of electronic density of the ring [18]. Chlorins, which are essentially reduced porphyrins derived from chlorophyll bacteria fetophorides, stable derivatives of chlorophyll varieties that are found in bacteria, are related to porphyrins and are simple to produce. Phthalocyanines and naphthalocyanines, which are derived from azaporphyrin, have high stability, and selectivity [36].

On the other hand, light absorption capacity is an important factor, since most tissues present a comparatively low absorption in the spectral range that extends from 500 nm to about 1500 nm (**Table 1**). This wavelength range is popularly known as the therapeutic window or the diagnostic window (**Figure 4**) [26, 61].


### **Table 1.**

*Capacity penetration (mm) of light in different tissues. Adapted from [60].*

#### **Figure 4.**

*Electromagnetic spectrum and their respective wavelengths in the region of visible light as a function of the different types of LASERs.*
