Enhanced photodynamic therapy: a review of combined energy sources

Citation: Rodrigues, J.A.; Correia, J.H. Enhanced Photodynamic Therapy: A Review of Combined Energy Sources. Cells 2022,11, 3995. https://doi.org/10.3390/cells11243995 Academic Editors: Fabio Mammano and Greta Varchi Received: 11 November 2022 Accepted: 6 December 2022 Published: 10 December 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Review Enhanced Photodynamic Therapy: A Review of Combined Energy Sources JoséA. Rodrigues 1,2,* and JoséH. Correia 1,2 1CMEMS-UMinho, University of Minho, 4800-058 Guimarães, Portugal 2LABBELS-Associate Laboratory, 4800-122 Braga, Portugal *Correspondence: jr[email protected] Abstract: Photodynamic therapy (PDT) has been used in recent years as a non-invasive treatment for cancer, due to the side effects of traditional treatments such as surgery, radiotherapy, and chemotherapy. This therapeutic technique requires a photosensitizer, light energy, and oxygen to produce reactive oxygen species (ROS) which mediate cellular toxicity. PDT is a useful non-invasive therapy for cancer treatment, but it has some limitations that need to be overcome, such as low-light-penetration depths, non-targeting photosensitizers, and tumor hypoxia. This review focuses on the latest innovative strategies based on the synergistic use of other energy sources, such as non-visible radiation of the electromagnetic spectrum (microwaves, infrared, and X-rays), ultrasound, and electric/magnetic fields, to overcome PDT limitations and enhance the therapeutic effect of PDT. The main principles, mechanisms, and crucial elements of PDT are also addressed. Keywords: photodynamic therapy; non-visible radiation; ultrasound; electric field; magnetic field; synergistic strategies 1. Introduction 1.1. Principles of Photodynamic Therapy and Photodynamic Reaction Photodynamic therapy (PDT) is a therapeutic modality that is based on the combination of three factors to promote the selective destruction of a target tissue: photosensitizer (PS), light with a specific wavelength, and the presence of molecular oxygen [ 1 – 4 ]. None of them are toxic individually, but together they initiate a photochemical reaction that culminates in the generation of reactive oxygen species (ROS) responsible for oxidative cell damage that can lead to the destruction of the target tissue [ 5 – 8 ]. The typical PDT procedure consists of two sequential steps: administration (intravenous or topically) of a PS and subsequent irradiation using light of a specific wavelength (usually a red lamp or laser beam) at the tumor’s location. Between PS administration and light irradiation, an appropriate time interval (drug–light interval) is required for the photosensitizer to accumulate in the tumor [ 5 , 9 ]. The drug–light interval depends on the route of administration, the type of PS, and its pharmacokinetic and biodistribution properties [10]. The photodynamic reaction starts with the absorption of light by the PS in the target tissue, which triggers a series of photochemical reactions that lead to the generation of ROS [ 7 , 9 – 12 ]. The PS in its ground state (singlet state, 1 PS) has a stable electronic configuration, i.e., it has two electrons with opposite spins. The absorption of a photon of light of a specific wavelength leads the PS to a short-lived (a few nanoseconds) electronically excited singlet state ( 1 PS*). This excited state is very unstable, losing the excess of energy by emitting light (fluorescence) or producing heat (internal conversion). However, the singlet state can undergo a process known as intersystem crossing to form a more stable, longlived (10 −6 to 10 −3 s), electronically excited state (triplet state, 3 PS*). The PS in the triplet state can decay back to the ground state through the emission of light (phosphorescence) or undergo two types of reactions. The long lifetime of the triplet state is sufficient to Cells 2022,11, 3995. https://doi.org/10.3390/cells11243995 https://www.mdpi.com/journal/cells Cells 2022,11, 3995 2 of 18 transfer its energy directly to the molecular oxygen (O 2 ). This energy transfer step leads to the formation of singlet oxygen ( 1 O 2 ) and the ground state of a PS, called a type II reaction [ 7 , 9 , 11 , 13 , 14 ]. The type I reaction can also occur if the PS in the excited state reacts directly with a cellular substrate, such as the cell membrane or a molecule, and undergoes electron transfer reactions, forming ROS. This mechanism may involve the acquisition or donation of an electron to form cationic or anionic radicals, respectively. These radicals react with molecular oxygen, producing ROS, such as superoxide anion radical (O 2−• ), hydroxyl radical (HO • ), and hydrogen peroxide (H 2 O 2 ) [ 7 , 9 , 11 , 13 ]. Figure 1shows the modified Jablonski diagram of the PDT action mechanism. Cells 2022, 11, x 2 of 18 or undergo two types of reactions. The long lifetime of the triplet state is sufficient to transfer its energy directly to the molecular oxygen (O2). This energy transfer step leads to the formation of singlet oxygen (1O2) and the ground state of a PS, called a type II reaction [7,9,11,13,14]. The type I reaction can also occur if the PS in the excited state reacts directly with a cellular substrate, such as the cell membrane or a molecule, and undergoes electron transfer reactions, forming ROS. This mechanism may involve the acquisition or donation of an electron to form cationic or anionic radicals, respectively. These radicals react with molecular oxygen, producing ROS, such as superoxide anion radical (O2−•), hydroxyl radical (HO•), and hydrogen peroxide (H2O2) [7,9,11,13]. Figure 1 shows the modified Jablonski diagram of the PDT action mechanism. Figure 1. Modified Jablonski diagram of the PDT action mechanism. Adapted from [7]. The products resulting from type I and II reactions lead to tumor ablation by three interrelated mechanisms: direct cytotoxic effects on tumor cells (apoptosis and necrosis), indirect damage to the tumor-associated vasculature, and induction of an inflammatory response that can activate an immune response against the tumor cells [8,9,11–13]. Both reactions can occur simultaneously, however the ratio between these two processes depends on the type of PS used, concentrations of substrate and molecular oxygen, as well as the binding affinity of PS to the substrate. Due to the high reactivity and short half-life of the ROS, only cells that are close to the ROS production area (PS location area) are directly affected by PDT [7,14]. The extent of damage and cytotoxicity resulting from PDT is multifactorial, depending on the type of PS, its extracellular and intracellular location and the total dose administered, light dose (light fluence) and light fluence rate, availability of oxygen, and drug–light interval [5,7,10,12]. 1.2. Light, Photosensitizers and Oxygen Light is an essential component of PDT. The clinical efficacy of PDT is highly dependent on the accuracy of light delivery to the target tissue and its dose, which translates into light fluence rate, light fluence, light exposure time, and light delivery mode (single or fractionated) [7,15]. Light-fluence rate is the incident energy per second across a sectional area of the irradiated spot and is expressed as W/cm2. Light fluence is the total energy of exposed light across a sectional area of an irradiated spot and is expressed in J/cm2 [7]. These two parameters define the light exposure time, which is obtained by dividing the light fluence by the light fluence rate. High light doses in a short period of time, i.e., high light fluence rates, have been reported as a non-ideal practice in PDT, reducing its efficiency. The main reason for this is the rapid tissue oxygen depletion, limiting the generation of reactive oxygen species and thus the potential for tumor destruction. Furthermore, low light-fluence rates increase the selective apoptosis of tumor cells, which is more desirable than the inflammation and edema that usually occurs with the uncontrolled rupturing of cellular contents during necrosis [7,12,15]. Molecular oxygen is a fundamental element for PDT-induced cytotoxicity. So, tissue oxygenation is extremely important for the efficacy of PDT. The oxygen concentration can Figure 1. Modified Jablonski diagram of the PDT action mechanism. Adapted from [7]. The products resulting from type I and II reactions lead to tumor ablation by three interrelated mechanisms: direct cytotoxic effects on tumor cells (apoptosis and necrosis), indirect damage to the tumor-associated vasculature, and induction of an inflammatory response that can activate an immune response against the tumor cells [ 8 , 9 , 11 – 13 ]. Both reactions can occur simultaneously, however the ratio between these two processes depends on the type of PS used, concentrations of substrate and molecular oxygen, as well as the binding affinity of PS to the substrate. Due to the high reactivity and short half-life of the ROS, only cells that are close to the ROS production area (PS location area) are directly affected by PDT [ 7 , 14 ]. The extent of damage and cytotoxicity resulting from PDT is multifactorial, depending on the type of PS, its extracellular and intracellular location and the total dose administered, light dose (light fluence) and light fluence rate, availability of oxygen, and drug–light interval [5,7,10,12]. 1.2. Light, Photosensitizers and Oxygen Light is an essential component of PDT. The clinical efficacy of PDT is highly dependent on the accuracy of light delivery to the target tissue and its dose, which translates into light fluence rate, light fluence, light exposure time, and light delivery mode (single or fractionated) [ 7 , 15 ]. Light-fluence rate is the incident energy per second across a sectional area of the irradiated spot and is expressed as W/cm 2 . Light fluence is the total energy of exposed light across a sectional area of an irradiated spot and is expressed in J/cm 2 [ 7 ]. These two parameters define the light exposure time, which is obtained by dividing the light fluence by the light fluence rate. High light doses in a short period of time, i.e., high light fluence rates, have been reported as a non-ideal practice in PDT, reducing its efficiency. The main reason for this is the rapid tissue oxygen depletion, limiting the generation of reactive oxygen species and thus the potential for tumor destruction. Furthermore, low light-fluence rates increase the selective apoptosis of tumor cells, which is more desirable than the inflammation and edema that usually occurs with the uncontrolled rupturing of cellular contents during necrosis [7,12,15]. Molecular oxygen is a fundamental element for PDT-induced cytotoxicity. So, tissue oxygenation is extremely important for the efficacy of PDT. The oxygen concentration can vary significantly between different tumors and even between different regions of the same tumor, depending on the density of the vasculature. Especially in deeper solid tumors, Cells 2022,11, 3995 3 of 18 often characterized by their anoxic microenvironment, lack of oxygen can be a limiting factor. As mentioned above, the light fluence rate is related to photochemical oxygen depletion during the generation of cytotoxic singlet oxygen by PDT. Oxygen depletion occurs when the oxygen consumption rate by the photodynamic reaction is greater than the rate of oxygen diffusion in the irradiated area. Continuous adjustment of the light fluence rate (maintaining the total light dose) during PDT allows the oxygen consumption rate to not exceed the rate of oxygen diffusion into the target tissue. This balance can also be achieved by using fractionated light [7,10]. Another essential component of PDT, besides light and oxygen, is the presence of PSs. These substances are defined as substances capable of absorbing light with a specific wavelength and triggering photochemical or photophysical reactions [ 1 , 16 ]. Some of the features that should be found in an ideal PS are [5,7,10,12,16–19]: •High purity and chemical stability; • Strong absorption with a high molar extinction coefficient ( ε ) for higher light wavelengths (600 to 800 nm); •High singlet oxygen quantum yield (Φ∆); •Low dark toxicity; • Accumulation and retention, preferentially in the target tissues and rapid clearance from healthy tissues; •Amphiphilicity; •Inexpensive, simple synthesis and easy availability. Most of the PSs used in PDT are porphyrins or their reduced derivatives, such as chlorins or bacteriochlorins, which have in common the tetrapyrrole macrocycle, similar to that of the protoporphyrin contained in hemoglobin [ 5 , 10 ]. The first compounds to demonstrate therapeutic potential for PDT of cancer were hematoporphyrin derivatives (HpD), of which the purified version and commercially approved porfimer sodium (Photofrin ® ) represents the first-generation of PS [ 1 , 5 , 10 , 16 ]. PDT for cutaneous indications commonly utilizes a topical photosensitizer, such as 5-aminolevulinic acid or methyl aminolevulinate, which are precursors of protoporphyrin IX. Treatment of visceral tumors requires an intravenous or oral photosensitizer, and the most commonly used photosensitizing agent for this indication is porfimer sodium [ 15 ]. The second-generation PSs arose to overcome some drawbacks of the first-generation ones. The second-generation PSs are characterized by a higher chemical purity, higher yield of singlet oxygen formation, and better light penetration to deeply located tissues, due to their maximum absorption in the wavelength range 650–800 nm. The third-generation PSs are molecules with improved selectivity for tumor regions, due to the conjunction of the PS with targeting molecules (antibody conjugates) or its encapsulation into carriers (e.g., liposomes micelles, nanoparticles) [ 12 , 15 , 16 ]. Table 1shows some PS used in the PDT. Table 1. Examples of PS used in the PDT [1,5,7,9,10,16,20–24]. Photosensitizer (Generation) λof Max. Absorption (nm) Main Applications Porfirmer sodium or Photofrin (1st generation) 630 FDA approved: treatment of carcinomas Phase I clinical trials: ovarian, breast, skin metastases Phase II clinical trials: lung, head and neck, bladder, brain Phase III clinical trials: esophagus, bile duct Fimaporfin or Amphinex (2nd generation) 633 Phase I clinical trials: Superficial cancers, colon Phase II clinical trials: bile duct 5-aminolevulinic acid or Levulan (2nd generation) 635 FDA approved: skin Phase I/II clinical trials: bladder, brain, esophagus Cells 2022,11, 3995 4 of 18 Table 1. Cont. Photosensitizer (Generation) λof Max. Absorption (nm) Main Applications Methyl-aminolevulinate or Metvix (2nd generation) 635 FDA approved: skin Hexyl-aminolevulinate or Hexvix (2nd generation) 635 FDA approved: bladder Phase I/II clinical trials: prostate, colon Temoporfin or Foscan (2nd generation) 652 EU approved: advanced head and neck squamous cell carcinoma Phase II clinical trials: lung, brain, bile duct, pancreas, skin, breast Talaporfin (2nd generation) 660 Japan approved: early-stage endobronchial carcinoma Phase II clinical trials: brain, liver, colon, breast, skin metastases Rostaporfin or Purlytin (2nd generation) 660 Phase II/III clinical trials: breast, bile duct, ovarian Bremachlorin (2nd generation) 662 Phase II clinical trials: skin, lung HPPH 1or Photochlor (2nd generation) 665 Phase II clinical trials: head and neck, esophagus, lung Ce6-PVP 2or Photolon (2nd generation) 665 Phase II clinical trials: skin, lung, brain Verteporfin or Visudyne (2nd generation) 690 FDA approved: choroidal neovascularization in wet age-related macular degeneration (AMD) Phase I/II clinical trials: pancreas, breast Motexafin lutetium or Lutrin (2nd generation) 732 Phase I clinical trials: prostate, breast, ovarian, colon, stomach, skin metastases Redaporfin or LUZ11 (2nd generation) 749 Phase II clinical trials: head and neck, biliary tract Padeliporfin or Tookad (2nd generation) 762 EU approved: prostate Phase I/II clinical trials: esophagus 12-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a; 2chlorin e6-polyvinylpyrrolidone. 1.3. Limitations of Photodynamic Therapy Like other therapies, the breadth and depth of PDT’s effectiveness have not been fully realized due to limitations, such as depth of light penetration, inefficient PSs, targeted delivery, and tumor hypoxia [ 2 , 11 , 17 ]. When considering PDT as a treatment option, the accuracy of target tissue irradiation is the most important point because PDT is effective only when light hits the target area. So, deep tumors (not easily accessible without surgical intervention) are difficult to treat due to the low penetration of visible light into the tissue. Disseminated metastases are also very difficult to treat with the currently available technology [ 2 , 7 , 17 ]. The difficulty in systemic administration of this form of therapy is because PSs are generally easy to aggregate and lack targeting, limiting the clinical efficacy of PDT [ 2 , 7 , 11 , 17 ]. Furthermore, due to the excessive proliferation of cancer cells and insufficient blood supply in the tumors, the O 2 content in the tumors is severely insufficient, resulting in a significant reduction in the effectiveness of PDT [7,8,11,17]. In recent years, great efforts have been devoted to overcoming the limitations of PDT and a number of strategies have been proposed to increase its efficiency [ 11 ]. In this review, we focus on the latest innovative strategies based on the use of non-visible radiation of the electromagnetic spectrum (microwaves, radio waves, infrared, and X-rays), ultrasound, and electric/magnetic fields to overcome PDT limitations and enhance its therapeutic effect (Figure 2). Cells 2022,11, 3995 5 of 18 Cells 2022, 11, x 5 of 18 Figure 2. Schematic illustration of other energy sources (in addition to the visible light source) that can be used in PDT to overcome its limitations and enhance its therapeutic effect. 2. Illumination of the Tissue Different types of illumination sources have been proposed for photodynamic treatment. The choice of the light source should be based on the PS absorption spectrum, pathology characteristics (location, lesion size, accessibility, and tissue characteristics), and cost. Typically, tissue illumination in PDT can be performed by four different types of light sources: lamps, light-emitting diodes (LEDs), lasers, and daylight [5,7,10,19,25]. 2.1. Lamp Light Lamps have the advantage of being affordable, portable, easy to use, requiring low maintenance, having the ability to cover a large area, and providing a wide spectral output. This type of light source includes halogen, xenon, and metal-halide lamps. The use of narrowband filters allows the selection of a range of wavelengths that correspond to the maximum absorption of the PS. Optical filtering is also required to ensure unnecessary ultraviolet and infrared wavelengths are removed from the output light. Loss of energy in the form of heat, low-light intensity, and restriction of irradiation to easily accessible places (e.g., skin) are some disadvantages of this type of light source [2,7,10,26]. 2.2. Light-Emitting Diodes LEDs are characterized by fixed narrowband emission compared with lamps, eliminating the need for optical filters. They can be assembled to cover large areas of irradiation or complex anatomic shapes. LEDs have advantages over other PDT light sources, such as low cost and low hazard. In addition, LEDs have other advantages: they are compact, lightweight, thermally non-destructive, easily available in flexible arrays, and require low amounts of energy when producing desired wavelengths [2,5,10,19,27]. 2.3. Laser Light The development of the laser was a major milestone in PDT and is currently the most widely used light source. There are four types of laser light sources that have been used in PDT: argon-pumped lasers, metal-vapor-pumped lasers (Auor Cu-vapor lasers), solid-state lasers (Nd:YAG lasers, Ho:YAG lasers, KTP:YAG/dye lasers), and diode lasers. Lasers produce high-intensity coherent monochromatic light. They can be coupled to optical fibers to reach inaccessible locations (e.g., lung and bladder) and decrease light loss due to scattering. For the treatment of superficial lesions, in order to cover a relatively Figure 2. Schematic illustration of other energy sources (in addition to the visible light source) that can be used in PDT to overcome its limitations and enhance its therapeutic effect. 2. Illumination of the Tissue Different types of illumination sources have been proposed for photodynamic treatment. The choice of the light source should be based on the PS absorption spectrum, pathology characteristics (location, lesion size, accessibility, and tissue characteristics), and cost. Typically, tissue illumination in PDT can be performed by four different types of light sources: lamps, light-emitting diodes (LEDs), lasers, and daylight [5,7,10,19,25]. 2.1. Lamp Light Lamps have the advantage of being affordable, portable, easy to use, requiring low maintenance, having the ability to cover a large area, and providing a wide spectral output. This type of light source includes halogen, xenon, and metal-halide lamps. The use of narrowband filters allows the selection of a range of wavelengths that correspond to the maximum absorption of the PS. Optical filtering is also required to ensure unnecessary ultraviolet and infrared wavelengths are removed from the output light. Loss of energy in the form of heat, low-light intensity, and restriction of irradiation to easily accessible places (e.g., skin) are some disadvantages of this type of light source [2,7,10,26]. 2.2. Light-Emitting Diodes LEDs are characterized by fixed narrowband emission compared with lamps, eliminating the need for optical filters. They can be assembled to cover large areas of irradiation or complex anatomic shapes. LEDs have advantages over other PDT light sources, such as low cost and low hazard. In addition, LEDs have other advantages: they are compact, lightweight, thermally non-destructive, easily available in flexible arrays, and require low amounts of energy when producing desired wavelengths [2,5,10,19,27]. 2.3. Laser Light The development of the laser was a major milestone in PDT and is currently the most widely used light source. There are four types of laser light sources that have been used in PDT: argon-pumped lasers, metal-vapor-pumped lasers (Auor Cu-vapor lasers), solid-state lasers (Nd:YAG lasers, Ho:YAG lasers, KTP:YAG/dye lasers), and diode lasers. Lasers produce high-intensity coherent monochromatic light. They can be coupled to optical fibers to reach inaccessible locations (e.g., lung and bladder) and decrease light loss due to scattering. For the treatment of superficial lesions, in order to cover a relatively large Cells 2022,11, 3995 6 of 18 target tissue with uniform irradiance, the laser can be coupled with beam-expanding lenses. Irradiation through lasers with a defined wavelength facilitates the accurate calculation of the light dose. The complex, bulky, and expensive laser systems used in the past have been replaced by easy-to-use, reliable, and cost-effective laser diodes [5,10,19,25,26,28]. 2.4. Daylight Another relevant light source for PDT is natural light. The concept of daylight PDT is based on the use of natural light instead of an artificial light source to treat skin lesions, such as actinic keratosis. Daylight PDT has made PDT more widespread, cheaper, less painful, and with shorter clinic visits (patients can complete their therapy at home) [ 2 , 7 ]. However, important drawbacks include the difficulty of scheduling due to daylight dependence on weather and times, as well as the inconvenience of controlling daylight exposure [2]. 2.5. Light Absorption in Biological Tissues Light penetration into biological tissue is very complex, as it can be reflected, scattered, or absorbed. The extent of these processes depends on the type of tissue and light wavelength (Figure 3) [ 7 , 10 , 18 , 29 ]. Light absorption is mainly due to endogenous chromophores existing in tissues, such as hemoglobin, myoglobin, melanin, and cytochromes, which can decrease the photodynamic process by competing with PS in the absorption process. The region between 600 and 1200 nm is often called the “tissue optical window”, due to the absorption of lower wavelength light by the endogenous chromophores, combined with reduced light scattering at longer wavelengths and the occurrence of water absorption at wavelengths greater than 1200 nm [ 5 , 7 , 18 , 30 ]. Shorter wavelengths (<600 nm) have less tissue penetration and are more absorbed, resulting in high skin photosensitivity. On the other hand, longer wavelengths (>850 nm) do not have enough energy to generate triplet states of PS that can efficiently transfer their energy to molecular oxygen. Therefore, the highest tissue permeability occurs between 600 and 850 nm. This band, called the “phototherapeutic window”, is predominantly used in PDT [7,10,18]. Cells 2022, 11, x 6 of 18 large target tissue with uniform irradiance, the laser can be coupled with beam-expanding lenses. Irradiation through lasers with a defined wavelength facilitates the accurate calculation of the light dose. The complex, bulky, and expensive laser systems used in the past have been replaced by easy-to-use, reliable, and cost-effective laser diodes [5,10,19,25,26,28]. 2.4. Daylight Another relevant light source for PDT is natural light. The concept of daylight PDT is based on the use of natural light instead of an artificial light source to treat skin lesions, such as actinic keratosis. Daylight PDT has made PDT more widespread, cheaper, less painful, and with shorter clinic visits (patients can complete their therapy at home) [2,7]. However, important drawbacks include the difficulty of scheduling due to daylight dependence on weather and times, as well as the inconvenience of controlling daylight exposure [2]. 2.5. Light Absorption in Biological Tissues Light penetration into biological tissue is very complex, as it can be reflected, scattered, or absorbed. The extent of these processes depends on the type of tissue and light wavelength (Figure 3) [7,10,18,29]. Light absorption is mainly due to endogenous chromophores existing in tissues, such as hemoglobin, myoglobin, melanin, and cytochromes, which can decrease the photodynamic process by competing with PS in the absorption process. The region between 600 and 1200 nm is often called the “tissue optical window”, due to the absorption of lower wavelength light by the endogenous chromophores, combined with reduced light scattering at longer wavelengths and the occurrence of water absorption at wavelengths greater than 1200 nm [5,7,18,30]. Shorter wavelengths (<600 nm) have less tissue penetration and are more absorbed, resulting in high skin photosensitivity. On the other hand, longer wavelengths (> 850 nm) do not have enough energy to generate triplet states of PS that can efficiently transfer their energy to molecular oxygen. Therefore, the highest tissue permeability occurs between 600 and 850 nm. This band, called the “phototherapeutic window”, is predominantly used in PDT [7,10,18]. Figure 3. Schematic representation of wavelength-dependent light penetration into biological tissue. The schematic shows a section of the skin layers (epidermis, dermis, and subcutaneous layer). The arrows represent the penetration power of light with different wavelengths in the tissues. Blue light penetrates less efficiently into the tissue, while red light penetrates more deeply. 3. Enhanced PDT Figure 3. Schematic representation of wavelength-dependent light penetration into biological tissue. The schematic shows a section of the skin layers (epidermis, dermis, and subcutaneous layer). The arrows represent the penetration power of light with different wavelengths in the tissues. Blue light penetrates less efficiently into the tissue, while red light penetrates more deeply. 3. Enhanced PDT The typical PDT procedure is based on the irradiation of tumor tissue with visible light. In recent years, new strategies have been used to overcome the limitations of PDT and enhance its therapeutic effect. These strategies rely on the use of non-visible radiation of the electromagnetic spectrum, ultrasound, and electric/magnetic fields. Cells 2022,11, 3995 7 of 18 3.1. Non-Visible Radiation Visible light corresponds to a narrow slice of the electromagnetic spectrum that is visible to the human eye. However, electromagnetic radiation also includes non-visible radiation, such as radio waves, microwaves, infrared, ultraviolet, X-ray, and gamma radiation (Figure 4) [6]. Cells 2022, 11, x 7 of 18 The typical PDT procedure is based on the irradiation of tumor tissue with visible light. In recent years, new strategies have been used to overcome the limitations of PDT and enhance its therapeutic effect. These strategies rely on the use of non-visible radiation of the electromagnetic spectrum, ultrasound, and electric/magnetic fields. 3.1. Non-Visible Radiation Visible light corresponds to a narrow slice of the electromagnetic spectrum that is visible to the human eye. However, electromagnetic radiation also includes non-visible radiation, such as radio waves, microwaves, infrared, ultraviolet, X-ray, and gamma radiation (Figure 4) [6]. Figure 4. Electromagnetic spectrum. 3.1.1. Microwaves Microwaves have been widely explored for tumor ablation in clinical settings due to their depth of penetration into tissues, high heating efficiency, and negligible side effects [31,32]. Microwave ablation uses electromagnetic waves in the microwave energy spectrum to produce tissue heating effects that ultimately generate tissue necrosis within solid tumors. Microwave energy is capable of propagating through all types of tissue and nonmetallic materials, including water vapor and dehydrated, charred, and desiccated tissue created during the ablative process [33,34]. The combination of microwaves and PDT has been reported as a new therapy for deep cancer treatment. Microwave irradiation can cause local hyperthermia. When tissues are heated, blood vessels dilate and blood flow increases, thereby enhancing the effectiveness of PDT treatment [2,34]. Several PSs for microwave-induced PDT have been used to produce ROS under microwave irradiation and destroy tumor cells, such as copper-cysteamine (Cu–Cy) nanoparticles [33], g-C3N4 quantum dots [35], TiO2 nanoparticles [36], Fe-metal organic framework nanoparticles [37], liquid metal supernanoparticles [31], Cu2ZnSnS4 nanocrystals [38], Mn-doped zirconium metal-organic framework nanocubes [39], and gold nanoparticles [40]. 3.1.2. Infrared Light The most direct approach to excite PS for PDT in deeper tissues is to use radiation that lies within the NIR (near-infrared) optical window (700 to 1100 nm) [41]. Compared to visible light, NIR light minimizes the degree of tissue scattering with a penetration depth greater than 1 cm. The use of NIR light not only allows deeper penetration of the tumor but also reduces phototoxicity in healthy tissues [11]. There are three main approaches for using NIR radiation to perform PDT in deeper tissues: two-photon absorption, nonlinear optical photon conversion techniques, and the use of upconversion materials and nanoparticles [29]. Figure 4. Electromagnetic spectrum. 3.1.1. Microwaves Microwaves have been widely explored for tumor ablation in clinical settings due to their depth of penetration into tissues, high heating efficiency, and negligible side effects [ 31 , 32 ]. Microwave ablation uses electromagnetic waves in the microwave energy spectrum to produce tissue heating effects that ultimately generate tissue necrosis within solid tumors. Microwave energy is capable of propagating through all types of tissue and non-metallic materials, including water vapor and dehydrated, charred, and desiccated tissue created during the ablative process [ 33 , 34 ]. The combination of microwaves and PDT has been reported as a new therapy for deep cancer treatment. Microwave irradiation can cause local hyperthermia. When tissues are heated, blood vessels dilate and blood flow increases, thereby enhancing the effectiveness of PDT treatment [ 2 , 34 ]. Several PSs for microwave-induced PDT have been used to produce ROS under microwave irradiation and destroy tumor cells, such as copper-cysteamine (Cu–Cy) nanoparticles [ 33 ], g-C 3 N 4 quantum dots [ 35 ], TiO 2 nanoparticles [ 36 ], Fe-metal organic framework nanoparticles [ 37 ], liquid metal supernanoparticles [ 31 ], Cu 2 ZnSnS 4 nanocrystals [ 38 ], Mn-doped zirconium metal-organic framework nanocubes [39], and gold nanoparticles [40]. 3.1.2. Infrared Light The most direct approach to excite PS for PDT in deeper tissues is to use radiation that lies within the NIR (near-infrared) optical window (700 to 1100 nm) [ 41 ]. Compared to visible light, NIR light minimizes the degree of tissue scattering with a penetration depth greater than 1 cm. The use of NIR light not only allows deeper penetration of the tumor but also reduces phototoxicity in healthy tissues [ 11 ]. There are three main approaches for using NIR radiation to perform PDT in deeper tissues: two-photon absorption, nonlinear optical photon conversion techniques, and the use of upconversion materials and nanoparticles [29]. A single NIR photon does not have enough excitation energy to produce singlet oxygen molecules and induce photodynamic tissue damage [ 41 ]. Two-photon absorption is a nonlinear optical process involving the simultaneous absorption of two infrared photons that combined, promote an electron to a higher energy level than a single photon [ 29 , 42 – 44 ]. A key feature of two-photon excitation is the nonlinearity of photon absorption which makes it possible to activate PSs at the focal point of the laser beam. This allows for better spatial Cells 2022,11, 3995 8 of 18 control of PS activation in three dimensions during PDT, reducing off-target damage to surrounding healthy tissues [ 29 , 41 , 44 ]. Several studies revealed that two-photon excitation of a commonly used PS was insufficient to induce PDT phototoxicity in vivo , so PSs specific for direct two-photon excitation were designed [ 29 , 41 ]. The first proof-of-concept in vivo study for two-photon PDT was reported in 2008 by H. A. Collins et al., who developed a new family of porphyrin-based PSs with high two-photon cross-sections [ 42 , 45 ]. Since then, other PSs were developed, as can be seen in [ 46 – 51 ]. An effective PS for two-photon PDT requires both the maximal two-photon absorption cross-section (e.g., gold nanorods) and a sufficient singlet oxygen quantum yield (e.g., porphyrin derivatives) [ 41 ]. To enable direct excitation of the PS through two-photon absorption, light sources with ultra-fast pulses of high photon density (femtosecond laser) are typically required due to the low absorption probability of the two-photon absorption process in most PSs [29,42]. PSs can also be excited indirectly (also using ultra-fast high-intensity lasers) by exploiting nonlinear optical photon conversion mechanisms occurring in many biological tissue constituents. Second-harmonic generation is a second-order nonlinear optical process that occurs in collagen (abundant in tumors). Four-wave mixing, including coherent anti-Stokes Raman scattering, is a third-order nonlinear optical process produced by the natural intracellular macromolecules (proteins and lipids). Kachynski et al. demonstrated deeper light penetration and phototoxicity effects with lower radiation thresholds by using a combination of these novel techniques as compared to two-photon absorption alone. With the same irradiation dose, 4500 J/cm 2 , the two-photon excitation was at the threshold level of phototoxicity, while these new techniques showed that 70% of the cells were necrotic or detached [29,52]. Another indirect excitation of the PSs is based on the upconversion of NIR photons into visible photons using dedicated upconversion materials, such as nano-transducers, or upconverting nanoparticles (UCNPs) [ 29 ]. There has been an increased interest in using energy nano-transducers to locally absorb incident NIR radiation to subsequently activate the PS. These nano-transducers can have various origins: chromophores [ 53 , 54 ], plasmonic gold nanorods [ 55 , 56 ], semiconductor quantum dots [ 57 ], and carbon quantum dots [ 58 ]. Regarding UCNPs, they are usually made of a ceramic lattice doped with rare-earth ions (lanthanides) that allow the sequential absorption of two photons through a metastable energy level [ 41 , 59 ]. The metastable state lifetime is typically on the order of a microsecond, which makes it possible to use continuous wave lasers and, more importantly, lower energy densities for UCNP excitation (1–10 3 W/cm 2 for UCNP excitation vs. 10 6 –10 9 W/cm 2 for two-photon activation) [ 41 ]. UCNPs have the ability to convert NIR light to visible light, which can then activate PS through the transfer of electronic excitation energy, either radiative (i.e., absorption of upconverting luminescence photons by PS) or non-radiative (i.e., via Förster resonance energy transfer or Dexter mechanisms of the electronic excitation energy transfer) [ 41 , 44 , 60 ]. Several strategies for conjugating UCNPs and PSs have been developed: PSs can be covalently conjugated to UCNPs through surface functionalization and chemical binding procedures, PSs can be non-covalently attached to the surface of UCNPs through either hydrophobic–hydrophobic interactions or electrostatic interactions, PSs can be embedded in a mesoporous silica matrix to coat the UCNPs, and a core-shell architecture comprising the UCNPs as the core and PS (TiO 2 , ZnO) as the shell [ 42 , 60 , 61 ]. There are a large number of in vitro and in vivo studies reporting an efficient effect of UCNPs-induced PDT, however their in vivo bioclearance and toxicity still need thorough investigation to reinforce clinical applicability [41]. 3.1.3. X-ray X-ray has been widely applied in clinical tumor imaging and therapy because X-ray photons have an unlimited penetration depth in the human body compared to visible or near-infrared light [ 2 , 11 , 32 ]. Recently, PDT and X-ray have been merged to establish a new mode of PDT to treat deep tumors, X-ray-induced PDT (X-PDT) [ 26 , 62 ]. Figure 5shows the schematic illustration of the classic X-PDT. PS cannot be directly excited by X-rays because Cells 2022,11, 3995 9 of 18 of the significant energy mismatch between therapeutic X-rays and PS [ 26 ]. So, the PS must be activated using radioluminescence of nanoparticles: scintillation nanoparticles (SCNPs) or persistent luminescence nanoparticles (PLNPs) [2,26,29,63]. Cells 2022, 11, x 9 of 18 3.1.3. X-Ray X-ray has been widely applied in clinical tumor imaging and therapy because X-Ray photons have an unlimited penetration depth in the human body compared to visible or near-infrared light [2,11,32]. Recently, PDT and X-ray have been merged to establish a new mode of PDT to treat deep tumors, X-ray-induced PDT (X-PDT) [26,62]. Figure 5 shows the schematic illustration of the classic X-PDT. PS cannot be directly excited by Xrays because of the significant energy mismatch between therapeutic X-rays and PS [26]. So, the PS must be activated using radioluminescence of nanoparticles: scintillation nanoparticles (SCNPs) or persistent luminescence nanoparticles (PLNPs) [2,26,29,63]. Figure 5. Schematic illustration of the classic X-PDT. X-rays excite a nanoscintillator to generate X-ray luminescence, which in turn activates a PS to produce cytotoxic ROS. SCNPs downconvert X-ray energy into visible light through a scintillation process and then transfer the energy to nearby PSs to initiate PDT [26,29,64]. SCNPs can be classified into two major groups: doped scintillators and semiconductors [29,64]. Some important characteristics are a high material density (for a good interaction with ionizing radiation), high scintillation quantum yield and efficient energy transfer, biocompatibility, and adapted in vivo biodistribution [29]. The doped scintillators based on lanthanides elements have been the most widely explored, due to their high material density, high atomic number, and strong luminescence intensity [29,64]. Table 2 shows some SCNPs used in X-PDT and the energy required to activate them. Table 2. Some SCNPs (nanoscintillators and photosensitizers) used in X-PDT. The energy required to activate them is also presented. Adapted from [29,64]. X-Ray Scintillator (Emission) Photosensitizer (Absorption) X-Ray Energetics Ref CeF3 (340 nm) Verteporfin (370, 420 nm) 6 MeV, 30 keV, 1–6 Gy [65] SrAl2O4:Eu2+ (520 nm) Merocyanine (540 nm) 50 keV, 1–10 Gy [66] LaF3:Ce3+ (520 nm) PpIX 1 (409 nm) 90 keV, 3 Gy [67] LaF3:Tb (544 nm) Rose Bengal (560 nm) 75 keV [61] LaF3:Tb silica coated (540 nm) Rose Bengal (560 nm) 75 keV [68] LaF3:Tb (540 nm) MTPC 2 (516 nm) 80 keV [69] GdEuC12 (595 nm) Hypericin (590 nm) 15 keV [70] Hf-nMOL (500 nm) Ir[bpy(ppy)2]+ (355 nm) or [Ru(bpy)3] 2+ (450 nm) 225 keV, 2 Gy [71] ZnS:Cu,Co (510 nm) TBrRh123 (518 nm) 120 keV, 2 Gy [72] LiYF4:Ce (305 nm) ZnO (290 nm) 220 keV, 8 Gy [73] LiGa5O8:Cr (720 nm) NC 3 (775 nm) 50 keV, 5 Gy [74] NaLuF4:Gd,Eu (543 nm) Rose Bengal (560 nm) 160 keV, 5 Gy [75] Y2.99Pr0.01Al5O12@SiO2 (300-450 nm) PpIX 1 (408 nm) 1.48 keV [76] 1 Protoporphyrin, 2 Meso-tetra(4-carboxyphenyl)porphyrin, 3 2,3-naphthalocyanine. Figure 5. Schematic illustration of the classic X-PDT. X-rays excite a nanoscintillator to generate X-ray luminescence, which in turn activates a PS to produce cytotoxic ROS. SCNPs downconvert X-ray energy into visible light through a scintillation process and then transfer the energy to nearby PSs to initiate PDT [ 26 , 29 , 64 ]. SCNPs can be classified into two major groups: doped scintillators and semiconductors [ 29 , 64 ]. Some important characteristics are a high material density (for a good interaction with ionizing radiation), high scintillation quantum yield and efficient energy transfer, biocompatibility, and adapted in vivo biodistribution [ 29 ]. The doped scintillators based on lanthanides elements have been the most widely explored, due to their high material density, high atomic number, and strong luminescence intensity [ 29 , 64 ]. Table 2shows some SCNPs used in X-PDT and the energy required to activate them. Table 2. Some SCNPs (nanoscintillators and photosensitizers) used in X-PDT. The energy required to activate them is also presented. Adapted from [29,64]. X-ray Scintillator (Emission) Photosensitizer (Absorption) X-ray Energetics Ref CeF3(340 nm) Verteporfin (370, 420 nm) 6 MeV, 30 keV, 1–6 Gy [65] SrAl2O4:Eu2+ (520 nm) Merocyanine (540 nm) 50 keV, 1–10 Gy [66] LaF3:Ce3+ (520 nm) PpIX 1(409 nm) 90 keV, 3 Gy [67] LaF3:Tb (544 nm) Rose Bengal (560 nm) 75 keV [61] LaF 3 :Tb silica coated (540 nm) Rose Bengal (560 nm) 75 keV [68] LaF3:Tb (540 nm) MTPC 2(516 nm) 80 keV [69] GdEuC12 (595 nm) Hypericin (590 nm) 15 keV [70] Hf-nMOL (500 nm) Ir[bpy(ppy)2]+(355 nm) or [Ru(bpy)3]2+ (450 nm) 225 keV, 2 Gy [71] ZnS:Cu,Co (510 nm) TBrRh123 (518 nm) 120 keV, 2 Gy [72] LiYF4:Ce (305 nm) ZnO (290 nm) 220 keV, 8 Gy [73] LiGa5O8:Cr (720 nm) NC 3(775 nm) 50 keV, 5 Gy [74] NaLuF4:Gd,Eu (543 nm) Rose Bengal (560 nm) 160 keV, 5 Gy [75] Y2.99Pr0.01Al5O12@SiO2 (300–450 nm) PpIX 1(408 nm) 1.48 keV [76] 1Protoporphyrin, 2Meso-tetra(4-carboxyphenyl)porphyrin, 32,3-naphthalocyanine. 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