Photodynamic Therapy Review: Past, Present, Future, Opportunities and Challenges

Abstract

Photodynamic therapy (PDT) is a medical treatment that utilizes photosensitizing agents, along with light, to produce reactive oxygen species that can kill nearby cells. When the photosensitizer is exposed to a specific wavelength of light, it becomes activated and generates reactive oxygen that can destroy cancer cells, bacteria, and other pathogenic micro-organisms. PDT is commonly used in dermatology for treating actinic keratosis, basal cell carcinoma, and other skin conditions. It is also being explored for applications in oncology, such as treating esophageal and lung cancers, as well as in ophthalmology for age-related macular degeneration. In this study, we provide a comprehensive review of PDT, covering its fundamental principles and mechanisms, as well as the critical components for its function. We examine key aspects of PDT, including its current clinical applications and potential future developments. Additionally, we discuss the advantages and disadvantages of PDT, addressing the various challenges associated with its implementation and optimization. This review aims to offer a thorough understanding of PDT, highlighting its transformative potential in medical treatments while acknowledging the areas requiring further research and development.

1. Introduction

Many of the novel treatment methods being developed today have origins rooted in ancient history and have been modified over multiple centuries to evolve into the modern medicine we know today. One such treatment that has progressed over time is photodynamic therapy (PDT) [1,2,3,4].

PDT is often used to treat cancerous tumors, bacterial infections, abnormal skin conditions, fungi, etc., as shown in Figure 1 [1,3,4,5,6,7,8]. Current investigations into PDT often revolve around overcoming current limitations such as a low concentration of produced ROS, tissue penetration, and PS localization [2,3,9,10,11,12,13]. While it has taken over a century to uncover the chemical mechanism behind PDT, the origins of light therapies can be traced back to ancient civilizations, with PDT first appearing in the literature around the mid-20th century [14,15].

2. The History of Light Therapies

The historical use of light therapies spans from ancient practices to modern advancements, reflecting a continuous evolution in understanding light’s therapeutic potential [14,15]. The Egyptians constructed temples that utilized sunlight and colored light for various healing purposes. Similarly, the Assyrians and Babylonians practiced therapeutic sun healing, known as heliotherapy. Herodotus, a renowned Greek physician often regarded as the father of heliotherapy, mentioned frequently the importance of sun exposure for health renewal. The Greek city of Heliopolis was famous for its healing temples and light rooms, which featured windows covered with different colored cloths believed to have unique healing properties. Hippocrates, the father of medicine, described how sunlight could be used to treat mood disorders. Many people believed that the red light of the sun played a significant role in these therapeutic effects.

One of the oldest medical documents of ancient Egypt, the Papyrus Ebers, dated to 1550 BC, mentioned the use of sunlight in combination with ingestion of the Ammi majus plant to treat vitiligo [16]. Similarly, the Atharvaveda, one of the oldest medicinal records of India dating back to 1200 BC, illustrated the use of sunlight and the seeds of the Psoralea corylifolia to treat leukoderma [17]. Additionally, sunlight and the oral extracts of Ammi majus were also mentioned in the book Mofradat El-Adwiya, written by Arab physician Ibn al-Bitar and dating back to 1100 AD [15]. These historic light therapies were often termed “heliotherapy”, meaning natural sunlight [18].

In the 19th century, additional discoveries were made that further advanced the field of phototherapy. For example, in 1877, Downes and Blunt published an article with the Royal Society examining the effect of light on bacteria [19]. They observed that “Light is inimical to the development of Bacteria and the microscopic fungi associated with putrefaction and decay...”. Similarly, in 1890, Palm examined the relationship between the prevalence of rickets and exposure to sunlight in various regions, deducing that higher exposure to sunlight prevented rickets [20]. One of the first documented treatments using phototherapy was conducted by Niels Ryberg Finsen in 1896, when he treated and resolved the lesions of an individual with lupus vulgaris [21]. After this treatment, Finsen treated an additional 800 individuals with lupus vulgaris. In 1903, Finsen received the Nobel Prize in 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”.

The early 20th century saw the refinement and expansion of light therapies. The development of lasers in the 1960s provided new tools for precise light application, enhancing the efficacy and safety of phototherapy. A pivotal moment occurred in 1978, when Thomas Dougherty demonstrated the effectiveness of photodynamic therapy (PDT) for treating cutaneous and subcutaneous malignant tumors, establishing PDT as a viable treatment modality. Following this, the discovery of new photosensitizers, such as 5-aminolevulinic acid (ALA), led to significant advancements in PDT, improving its effectiveness and safety for treating a range of conditions, including cancers and age-related macular degeneration. This progression from ancient light therapies to modern PDT reflects a rich history of innovation and expanding the understanding of the use of light for medical treatments.

3. Principles of PDT

PDT is a treatment that uses photosensitizing agents which when exposed to light become toxic to targeted bacteria, malignant cells, and other diseased cells. PDT has become a frequently used alternative method to antibiotics as it is capable of eliminating antibiotic-resistant pathogens [22,23,24,25,26,27]. PDT offers several advantages as compared to traditional antibiotics, including minimal side effects, shorter treatment times, minimal invasiveness, lower costs, precise targeting, repeated use, and suitability for outpatient use. There are three key components in PDT treatment, namely a photosensitizer, a light source, and tissue oxygen. The treatment of bacterial infections using PDT has been successfully applied in dermatology [28,29,30,31,32,33] and oncological diseases [34,35], where phenothiazine dyes are applied to the infected skin area and then illuminated with red light [36,37]. PDT is usually performed as an outpatient procedure; however, it can also be used in combination with surgery, chemotherapy, or other anti-cancer drugs, as well as radiation therapy.

PDT involves introducing a light-sensitive chemical called a photosensitizer (PS) administered either topically or intravenously at a specified treatment site. When the treatment area is irradiated with a certain wavelength of light (generally in the red and infrared spectral region), the photosensitizer compound undergoes a transition from a ground state into an excited singlet state, as shown in Figure 2. The excited compound can either relax back to the ground state or transition to an excited triplet state via intersystem crossing. Once in the excited triplet state, the activated PS reacts with molecular oxygen through one of two reactions to produce reactive oxygen species (ROS). Here, singlet and triplet states are derived using the equation for multiplicity, 2S + 1, where S is the total spin angular momentum, the sum of all electron spins. For the singlet state, two of the spins are oriented as in the ground state and are unpaired (+1/2, −1/2); therefore, the total spin would be 2(+1/2 + −1/2) + 1 = 1, while the multiplicity equation, 2(+1/2 + +1/2) + 1, would result in three for triplet states where spins are oriented in the same orientation (unpaired).

It should be noted that electrons are diamagnetic in the ground state and paramagnetic in the excited triplet state. This difference in spins makes the transition from singlet to triple, or the reverse, less likely than singlet-to-singlet transitions since they involve spin flips. Since singlet-to-triplet (or triplet-to-singlet) transitions involve a change in electronic state, the lifetime of the triplet state is significantly longer than the singlet state by 10⁴.

It should also be noted that a transition from the ground singlet electronic state to the triplet electronic state is because the electron spin is parallel to the spin in its ground state. This transition leads to a change in multiplicity and thus has a low probability of occurring. Thus, it is a partially forbidden transition.

4. Photosensitizers

An efficient photosensitizer should have high absorption at long wavelengths (the maximum transmittance of skin tissue is in the 700–800 nm region) for deeper penetration and better access to deep-seated tumors. Ideally, PSs should have a high quantum yield of bacteria-attacking singlet oxygen (¹O₂), be as selective as possible to the targeted tissue to avoid harming healthy cells, be eliminated from the body easily, be resistant to photobleaching, have minimal dark toxicity in the absence of light, have preferential localization in tumors, and have amphiphilicity [4,38,39].

Photosensitizers can be categorized into different types, with one significant category being organic photosensitizers. Organic photosensitizers are versatile and effective in various clinical scenarios. This section explores the different classes of organic photosensitizers with their unique structures, properties, and clinical applications.

Porphyrins and their derivatives: Porphyrins absorb light around 400–450 nm and play a key role in PDT [40,41]. Photofrin derived from the hematoporphyrin derivative (HPD) is widely used, while protoporphyrin IX (PpIX) from 5-aminolevulinic acid (ALA) is effective for skin cancers [42,43,44,45]. Their strong light absorption and versatility make them highly effective for PDT.

Chlorins: Chlorins with red-shifted absorption (650–700 nm) for deeper tissue penetration include Chlorin e6 (Ce6), known for high ROS generation, and talaporfin sodium (LS11), which has been approved for early-stage lung cancer [46,47,48,49]. Their absorption properties and stability make chlorins effective for deep tumors.

Phthalocyanines: Phthalocyanines, with large planar structures, absorb strongly in the near-infrared spectrum (600–700 nm) for deep tissue penetration [40,50,51]. Some examples are aluminum phthalocyanine (AlPc) and zinc phthalocyanine (ZnPc), known for high stability and ROS generation [52,53]. Silicon phthalocyanine (Pc4) shows promise in lung cancer treatment [54]. Despite aggregation issues, their NIR absorption is advantageous for deep PDT applications.

Bacteriochlorins: Bacteriochlorins, which absorb near-infrared light (700–800 nm), offer deep tissue penetration for PDT [55,56]. Bacteriochlorin a, derived from bacteriochlorophyll, and synthetic derivatives like 5,10,15,20-tetrakis(m-hydroxyphenyl) bacteriochlorin (m-THPBC) enhance stability and efficacy [57,58,59]. Their long-wavelength absorption makes them ideal for treating large or deep tumors.

Xanthene dyes: Xanthene dyes, like rose bengal and erythrosine B, are noted for their fluorescence and effective singlet oxygen generation in PDT [60,61,62,63,64,65]. Fluorescein, mainly a marker, also shows potential in PDT when modified [66,67]. Their versatility is enhanced by conjugation with targeting moieties.

Coumarins: Coumarins, such as COUPY derivatives and coumarin 6, are effective PDT photosensitizers with a strong absorption and high singlet oxygen yield [68,69].

Benzoporphyrins: Benzoporphyrins with extended conjugation include Verteporfin and BPD-MA, known for targeting specific tissues and effective ROS generation [70,71,72,73,74,75]. They are crucial in PDT for treating cancers and age-related macular degeneration [76,77].

Table 1. List of commercially available photosensitizers and their structure, wavelength and applications.

Name (Commercially Available)	Structure	Wavelength	Application
Allumera (Hexyl Aminolevulinate)	C11H21NO3	560 nm	Facial photodamage
Photofrin	C68H74N8O11 (for n = 0)	630 nm	Esophageal cancer, bladder cancer, non-small cell lung carcinoma
Visudyne	C41H42N4O8	689 nm	Retinopathy
Levulan (Aminolevulinic acid)	C5H9NO3	635 nm	Cancer diagnosis and experimental brain cancer treatment
Foscan (Temoporfin)	C44H32N4O4	652 nm	Squamous cell carcinoma of head and neck
Metvix (Methyl aminolevulinate)	C6H11NO3	630 nm	Non-melanoma skin cancer, basal cell carcinoma
Hevvix (Hexaminolevulinate)	C11H21NO3	360–450 nm	Bladder cancer
Laserphyrin	C38H41N5O9	665 nm	Lung cancer

below shows some of the commercially available products. PSs can also be categorized into different generations. The first-generation PSs consist of naturally occurring porphyrins and their derivatives such as hematoporphyrin derivatives (HPDs). Porphyrins are heteroaromatic compounds with a tetrapyrrolic structure that consists of four pentagonal pyrroles linked by four methylene bridges, also called the porphine structure, as shown Figure 3. It is characterized by an absorption spectrum with a specific band around 400 nm (Soret band) and usually four further absorption bands around 500–600 nm. These PSs have some drawbacks, such as a low absorption band, high hydrophobicity, cytotoxicity, poor clearance, and phototoxicity [78]. Those disadvantages promoted the development of the second-generation PSs.

Second-generation PSs were designed to improve some of these limitations and are synthetic compounds that include or originate from porphyrins, bacteriochlorin, benzoporphyrins, curcumin, and methylene. Some examples of the second-generation PSs include benzoporphyrin derivatives (BPDs), lutetium texaphyrin, tinethyletiopurpurin (SnET2), 5-aminolevulinic acid, talaporfin sodium, protoporphyrin IX, phthalocyanine, and chlorin such as Chlorin e6 and derivatives. For example, Ce6 is localized in lysosomes, where it induces damage after irradiation, and it strongly absorbs in the 670 nm region.

The third-generation PSs are the latest edition of PSs which incorporate innovative strategies of immunotechnology and nanotechnology to have cancer cell specificity and selectivity [79]. It is expected that third-generation PSs will improve targeting, absorption wavelength, and photostability. Cyanine dyes are considered a good candidate for PDT due to their wide absorption spectra. One of the cyanine dyes, a disulfonated heptamethine cyanine, absorbing light at 780 nm, has been used in cancer diagnostics in detecting abnormal pulmonary drainage since 2015 [80].

5. Light

Light is another critical component in PDT. There have been multiple different light sources used in PDT, such as lasers, LEDs, and incandescent light sources. Each has advantages and disadvantages, as shown in Figure 4.

For instance, lasers may provide high power and precise wavelengths, which can be not only used to synthesize multiple different nanoparticles and surface modifications, but it can also be used in medicine and to control a specific photosensitizer [81,82,83]. This is especially important because lasers minimize the thermal effects in PDT. However, they are usually large, expensive, and require additional optical equipment to guide and manipulate the beam. Both pulsed and continuous wave mode lasers have been used in PDT. Clinical diode lasers can provide irradiance up to 1 W cm⁻². Diode lasers with output wavelengths in the 415–690 nm range are typically used in PDT [84].

Light emitting diodes (LEDs) are semiconductor devices in which light is generated as a result of an electron–hole recombination from a P-type semiconductor (large hole concentration) and an N-type semiconductor (large electron concentration). LEDs are considered a low-cost alternative to lasers in addition to being less hazardous, small, and adaptable. Furthermore, they are available in flexible arrays. However, LEDs provide low power, a large beam divergence, a broad spectral width, and thermal effects.

Lamps are cost effective, with a simple design and broad field irradiation. The fluorescent, incandescent, metal halide, xenon arc, and sodium arc lamps are the types of lamps used in PDT. However, they also suffer from a very broad spectrum (300–1200 nm) and large beam divergence. Moreover, they require optical filtering due to the broad spectrum and high losses due to optical coupling present another challenge [85].

It should be noted that wavelengths beyond 800 nm (1.55 eV) do not have enough energy to promote oxygen from the triplet to singlet state. The penetration depth of the light is limited by the absorption of light by blood and scattering. Therefore, the selection of an appropriate wavelength for PDT should be based on the depth of the lesion, the optical properties of the tissue, and the absorption spectrum of the PS. Each of these characteristics should all be taken into consideration when choosing the appropriate wavelength and light source.

6. Oxygen

Molecular oxygen is the third key element in photodynamic therapy. Oxygen is required for reactive oxygen species generation. The amount of ROS generation depends on the concentration of oxygen which varies with the types of tumors, different regions of the same tumor, and the density of the vasculature. Within any tumor, oxygen levels are extremely heterogeneous and can include mild hypoxia (≤2% O₂) and severe levels of hypoxia (<0.1% O₂) [86,87].

Tumor hypoxia is a major driver for cancer aggressiveness and is associated with the stabilization of hypoxia inducible factor-1 (HIF-1), which regulates the expression of genes that are involved in tumor progression, aggressiveness, and metastasis [88]. Tumor hypoxia presents a challenge for the deeper part of the tumor, where the environment usually lacks oxygen. Furthermore, PDT itself may cause a further increase in hypoxia as a result of the decreased blood flow to the tumor tissue [89]. Multiple strategies have been proposed to alleviate the challenges of hypoxia. These strategies include the direct transport of oxygen by O₂ carriers (hemoglobin, perfluorocarbon, and the metal–organic framework), in situ O₂ generation by catalase or O₂ self-supply materials, a decrease in oxygen consumption in mitochondrial respiration, and other strategies, such as increasing blood flow and the downregulation of HIF-1 [87,90].

Given the importance of the concentration of oxygen in tumors, determining the real-time concentration of tissue oxygen levels in PDT will be one of the main challenges in the future. Once the oxygen levels are determined, fluence and fraction optimization can then be adjusted.

Singlet oxygen has been used in many areas such as PDT, blood sterilization, wastewater treatment, polymer science, and photo-oxidation [91]. Singlet oxygen can be detected using various methods, including chemically, using the disodium salt of 9, 10-anthracene diproprionic acid (ADPA) as a singlet oxygen sensor.

Reactive oxygen species (ROS) and singlet oxygen cause lethal oxidative stress and membrane damage in bacteria and viruses. In the case of a tumor, reactive oxygen species and singlet oxygen lead to cell death by direct cytotoxicity and have a dramatic antivascular action that impairs the blood supply to the area of light exposure [92]. The excited singlet state decays within 1 ns to the long-lived MB triplet state [93,94]. The triplet state in turn transfers electrons (reaction type I) or energy (reaction type II) to the ground state atmospheric molecular triplet oxygen, ³O₂, to form OH radicals and singlet oxygen. Both of these species are very reactive, as shown by their reactive mean free paths (~10 Å for OH radicals and ~100 Å for singlet oxygen) [95,96]. Therefore, to be effective, the reactive species must be generated very close to the wall of the bacteria.

7. Mechanism of PDT

As discussed previously, when a photosensitizer absorbs light, the atom goes from a ground state to an excited singlet state, as depicted in the Jablonski diagram in Figure 2. This excited singlet state where all the electron spins are paired can either return to the ground state, releasing energy as fluorescence, or undergo intersystem crossing to a long-lived triplet state where electron spins are unpaired and parallel.

In the triplet state, the photosensitizer can interact with molecular oxygen, leading to the formation of ROS, such as singlet oxygen. These ROS are highly reactive and can induce cellular damage by oxidizing cellular components, including lipids, proteins, and nucleic acids, ultimately leading to cell death. The Jablonski diagram effectively illustrates these energy transitions and the pathways through which the photosensitizer mediates the production of cytotoxic species, highlighting the critical role of light and oxygen in the PDT mechanism.

Type 1 reactions involve the activated PS interacting with surrounding biological substrates to form free radicals and anion radicals [1,2,3,4]. These radicals can then go on to react with molecular oxygen to form a super oxide anion (O₂^•−) and hydroxyl radicals (OH•), a type of reactive oxygen species. Type 2 reactions, however, occur when the activated PS interacts directly with molecular oxygen to form singlet oxygen (¹O₂), which has strong oxidizing properties that facilitate its interactions with cellular material. It has been demonstrated that type 2 reactions occure more frequently, indicating that the production of singlet oxygen is the primary mechanism of action for PDT. Singlet oxygen and ROS present in the system can interact with proteins, mitochondria, or lysosomes, resulting in apoptosis, necrosis, or autophagy, respectively [1,2,3,4].

8. Limitations of PDT

PDT’s efficacy is restricted to areas accessible to light, usually no more than few tens of millimeters [97,98]. This limitation confines the use of PDT to surface or near-surface lesions, such as skin cancers, and makes it less effective for treating deeper-seated tumors. The challenge of reaching and effectively treating deeper tissues remains a significant hurdle for PDT’s broader application.

The effectiveness of PDT largely depends on the selective accumulation of photosensitizers in the tumor tissue while sparing the surrounding healthy tissue. However, this selective targeting is not always achieved with high precision. Photosensitizers can accumulate in healthy tissues, leading to unintended damage when exposed to light. Moreover, the heterogeneous nature of tumors means that the photosensitizer may not distribute evenly within the tumor, resulting in incomplete treatment and possible recurrence of the disease.

In addition, PDT relies on the presence of molecular oxygen to produce reactive oxygen species (ROS), which are responsible for inducing cell death. However, as mentioned previously, many tumors are hypoxic, meaning they have low oxygen levels, which can significantly reduce the efficacy of PDT. In hypoxic environments, the production of ROS is limited, leading to suboptimal treatment outcomes [87]. Addressing the challenge of hypoxia in tumors is crucial for improving the effectiveness of PDT, but it remains a difficult problem to solve.

There is also a concern about the potential for cancer cells to develop resistance to PDT. Although PDT targets cells through a different mechanism than traditional therapies like chemotherapy, there is evidence that some cancer cells can adapt to the oxidative stress induced by PDT, leading to resistance [99,100,101]. Additionally, incomplete treatment or inadequate targeting of the tumor can result in recurrence, which poses a significant challenge for long-term cancer management.

Additionally, practical challenges include the difficulty in determining optimal variables for specific treatments, resistance from healthcare staff and institutions to adopting new therapeutic approaches, and the high capital costs of establishing PDT centers and acquiring light sources [102]. Post-treatment photosensitivity due to the drugs used presents another challenge. Photosensitizing agents can render the skin and eyes extremely sensitive to light for several weeks following treatment. This necessitates the strict avoidance of sunlight and other bright lights to prevent severe phototoxic reactions, such as burns or blisters. This prolonged photosensitivity can significantly affect a patient’s lifestyle and limit their daily activities.

Furthermore, PDT is not applicable for treating metastatic cancer. Metastatic cancer involves cancer cells spreading through the bloodstream or lymphatic system to distant organs. Since PDT requires direct light exposure to activate the photosensitizer, it is ineffective against cancer cells that are dispersed throughout the body. Systemic cancers cannot be targeted by PDT because it is impossible to illuminate all metastatic sites simultaneously [3,103,104]. Metastatic tumors often vary in size, location, and number, making it difficult to achieve a uniform treatment response with PDT. The variability in how well the photosensitizer accumulates and the inconsistent light exposure across multiple metastatic sites further complicate treatment.

9. Adjuvants in Photodynamic Therapy

The effectiveness of PDT can be significantly enhanced by incorporating nanoparticles as adjuvants. Nanoparticles offer unique advantages due to their small size, large surface area, and ability to be functionalized with various chemical groups. This section explores the different types of nanoparticles used as adjuvants in PDT and their contributions to enhancing the therapy’s efficacy.

10. Gold Nanoparticles

Gold nanoparticles (AuNPs) are among the most widely studied nanoparticles in aPDT. Their surface can be easily functionalized with photosensitizers, enhancing the local concentration of these molecules at the infection site. Additionally, AuNPs can enhance the optical properties of photosensitizers through surface plasmon resonance, which increases light absorption and subsequently the production of ROS. Furthermore, gold nanoparticles can be designed to target specific microbial cells, improving selectivity and reducing damage to surrounding healthy tissues [105,106]. Akhtar et al. (2021) present a phototheranostic approach using toluidine blue-conjugated chitosan-coated gold–silver core–shell nanoparticles (TBO–chit–Au–AgNPs) to enhance PDT for treating diabetic foot ulcers (DFUs) caused by multidrug-resistant bacteria [107]. The study demonstrates that TBO–chit–Au–AgNP-mediated PDT achieved a 6.86 log10 CFU/mL reduction in S. aureus, a 5.4 log10 CFU/mL reduction in P. aeruginosa, and a 5.31 log10 CFU/mL reduction in polymicrobial biofilms. In vivo experiments on diabetic rats show that daily treatment with TBO–chit–Au–AgNPs followed by laser irradiation effectively heals DFUs, reduces inflammation, and promotes tissue regeneration.

Chi et al. demonstrated that gold nanoparticles (GNPs) conjugated with 5-aminolevulinic acid (5-ALA) significantly enhanced the efficacy of PDT in cutaneous squamous cell carcinoma cells [108]. Results showed that 5-ALA–GNP treatment increased cell apoptosis and singlet oxygen generation by up to 60% compared to 5-ALA alone, particularly in A431 carcinoma cells, suggesting that GNPs potentiate PDT’s anti-tumor effects.

Mokoena et al. demonstrated that conjugating hypericin to gold nanoparticles (AuNPs) significantly enhanced the PDT effect in MCF-7 breast cancer cells [109]. Quantitative results showed that at a PDT fluence of 10 J/cm², 49.1% of cells underwent early apoptosis and 18.1% experienced late apoptosis compared to the untreated control, emphasizing the compound’s potential to improve cancer cell death via apoptosis.

11. Silver Nanoparticles

Silver nanoparticles (AgNPs) are another type of nanoparticle frequently used in aPDT [110,111,112]. Silver has well-known antimicrobial properties and when combined with photodynamic therapy, it can significantly enhance the antimicrobial effect. AgNPs can generate ROS independently and in combination with the ROS produced during aPDT, leading to a synergistic effect that results in higher microbial kill rates [110,113]. Additionally, silver nanoparticles can disrupt microbial cell membranes, making the cells more susceptible to the effects of aPDT.

Cao et al. (2022) introduce a silver/bismuth molybdate (Ag/BMO) nanozyme optimized using charge separation engineering for enhanced antibacterial PDT and catalytic therapy against methicillin-resistant S. aureus (MRSA) [113]. The Ag/BMO nanozyme shows superior NIR-II light absorption and peroxidase-mimicking activity, generating significant amounts of singlet oxygen and hydroxyl radicals under 1064 nm laser irradiation. In vivo studies using a MRSA-infected mouse model demonstrate effective wound healing and bacterial eradication with minimal toxicity. The Ag/BMO nanozyme achieved approximately 99.9% bactericidal performance against methicillin-resistant S. aureus (MRSA) under 1064 nm laser irradiation.

Hakimov et al. (2022) explore the antimicrobial properties and cytotoxicity of silver nanoparticles (AgNPs) combined with methylene blue (MB) for PDT [110,111]. AgNPs were synthesized using pulsed laser ablation in different mediums and then coupled with MB. The MB/AgNP mixture completely deactivated 10⁸ CFU/mL of E. coli within 3 to 8 min and S. aureus within 6 to 8 min, depending on the medium used (citrate, PVP, or PVA). Cytotoxicity assays on HEK 293T cells reveal that AgNPs are not significantly toxic up to 50 μg/mL, suggesting that the MB/AgNP combination has potential as a safe and effective PDT agent for treating bacterial infections.

Habiba et al. explored the potential of silver nanoparticles decorated with graphene quantum dots (Ag–GQDs) for combined chemo–photodynamic cancer therapy [114]. Quantitative results demonstrated that Ag–GQDs loaded with doxorubicin reduced the viability of HeLa and DU145 cancer cells by approximately 75% under irradiation, highlighting the synergy between chemotherapy and photodynamic therapy.

Liu et al. developed a near-infrared (NIR) dye-coated silver nanoparticle/carbon dot nanocomposite (CyOH–AgNP/CD) for targeted tumor imaging and enhanced PDT [115]. Quantitative results showed that CyOH–AgNP/CD significantly improved singlet oxygen generation, achieving approximately 90% cell death in 4T1 tumor cells post-irradiation, highlighting its potential as an effective photosensitizer for cancer treatment.

12. Silica Nanoparticles

Silica nanoparticles are often used as carriers for photosensitizers [116,117]. These nanoparticles can encapsulate photosensitizers within their porous structure, protecting them from degradation and ensuring a controlled release at the target site. This encapsulation also allows for the simultaneous delivery of multiple therapeutic agents, further enhancing the effectiveness of aPDT. Moreover, silica nanoparticles can be easily functionalized to improve their biocompatibility and targeting capabilities. Tang et al. (2019) developed multifunctional nanoagents using fluorescent silicon nanoparticles (SiNPs) functionalized with a glucose polymer and loaded with Chlorin e6 (Ce6) for ultrasensitive imaging and photodynamic treatment of Gram-negative and Gram-positive bacteria [118]. The study shows these nanoagents are internalized by bacteria via the ATP-binding cassette transporter pathway, allowing dual-emission imaging and effective photodynamic antibacterial activity with efficiencies of 98% against S. aureus and 96% against P. aeruginosa.

Prieto-Montero et al. developed mesoporous silica nanoparticles (MSNs) functionalized with various photosensitizers (PSs) for enhanced PDT in cancer cells [119]. Quantitative results showed that MSNs loaded with rose bengal decreased cell viability by 80% under light exposure compared to 50% with free rose bengal, highlighting their improved phototoxic effect and efficient internalization in HeLa cells.

13. Magnetic Nanoparticles

Magnetic nanoparticles, such as iron oxide nanoparticles, have unique properties that make them useful in aPDT [120]. These nanoparticles can be directed to specific sites within the body using an external magnetic field, ensuring a high concentration of therapeutic agents at the infection site [121]. Magnetic nanoparticles can also enhance the penetration of light into deeper tissues, improving the overall effectiveness of aPDT. The study found that using a magnetic-based photosensitizer (MagTBO) significantly enhanced the effectiveness of aPDT against mature S. mutans biofilms. While conventional aPDT using toluidine blue ortho (TBO) showed no significant impact, the MagTBO microemulsion without a magnetic field achieved a 4 log reduction in biofilm viability. With the application of a magnetic field, this reduction increased to 5 log. However, the treatment did not improve the microhardness of the underlying dentin [121]. Additionally, they can generate heat when exposed to an alternating magnetic field, providing a combined photothermal and photodynamic effect for enhanced antimicrobial action.

14. Graphene Derivatives

Although they are quite effective, current PDT agents still have drawbacks, including poor water dispersibility, low photostability, and low absorption at high wavelengths [22,23]. Semiconductor quantum dots (QDs), especially those that are cadmium-based, have been introduced as an alternative to standard photosensitizers [22]. One of the biggest advantages of semiconductor QDs over organic photosensitizers is their tunable optical and chemical properties. Also, semiconductor quantum dots have been shown to have better photostability, water solubility, and large dipole moments [22]. Despite the fact that QDs have superior properties over standard photosensitizers, their practical applications are impeded by low singlet oxygen quantum yields and cytotoxicity [22,24]. Therefore, a biocompatible PDT agent that has a high singlet oxygen yield, high water dispersibility, and excellent photostability is needed.

Carbon products have a variety of applications in environmental, energy, and biomedical fields due to their superior chemical inertness and biocompatibility [28,30,122,123]. One of these products is a graphene quantum dot (GQD), a single-atom thick sheet of sp² hybridized carbon atoms with nanoscale lateral dimensions less than 100 nm. Among the most important properties of a GQD are its low toxicity, high solubility in various solvents, and its ability to equip with functional groups at its edges [34,36]. In addition, GQDs were recently reported to be rather effective photodynamic agents, producing high amounts of singlet oxygen when irradiated by visible light [30,34,106,124,125]. Graphene has a broad absorption spectrum between 400 and 700 nm and GQDs have a tunable bandgap which varies with size and surface chemistry [34,126].

Importantly, GQDs were recently shown to exhibit a high singlet oxygen (¹O₂) generation yield and hence and improved antibacterial effect [106,124,127]. The presence of defects and free radicals at the surface of GQDs and the direct energy transfer from GQDs to oxygen are believed to be responsible for the high singlet oxygen generation with a new multistage sensitization process, resulting in a yield more than 1.3 compared to less than 1.0 for conventional photosensitizers. During GQD excitation, in addition to the conventional energy transfer from the triplet state, the energy transfer during intersystem crossing from the singlet to the triplet state (³O₂) may also lead to singlet oxygen generation [30,106,128]. GQDs killed U251 human glioma cells when irradiated with 470 nm blue light by causing oxidative stress [30], and it has been shown that the irradiation of GQDs caused a morphology change, including shrinkage and the formation of blebs in HeLa cells [29]. In addition, an antibacterial system combining GQDs with low doses of H₂O₂ was proven to be an effective antibacterial agent by improving OH generation, and the designed system showed effectiveness against both Gram-positive and Gram-negative bacteria [129]. We have recently shown that graphene quantum dots can be used to deactivate E. coli under light irradiation [130].

Graphene oxide nanoparticles have recently gained attention for their potential in aPDT. Graphene oxide has a high surface area and excellent photothermal properties, which can be exploited to enhance the effects of aPDT. When combined with photosensitizers, graphene oxide nanoparticles can improve light absorption and ROS production. Additionally, these nanoparticles can act as carriers for multiple therapeutic agents, allowing for a combination of antimicrobial strategies.

Pourhajibagher et al. (2022) developed a targeted bio-theragnostic system using DNA–aptamer–nanographene oxide (NGO) for aPDT against Porphyromonas gingivalis [131]. Their study demonstrates the high binding specificity of DNA–aptamer–NGO to P. gingivalis, confirmed by flow cytometry. Upon irradiation, the system generates ROS, significantly reducing P. gingivalis viability, biofilm formation, and metabolic activity. It showed a decrease in the cell viability of P. gingivalis with a reduction in Log10 CFU/mL by 4.33 and 3.42 when using 1/2× and 1/4× MIC of DNA–aptamer–NGO, respectively (p < 0.05). The treatment also alters the expression of genes related to biofilm formation and oxidative stress responses.

Ge et al. (2014) present a PDT agent based on graphene quantum dots (GQDs), which shows remarkable singlet oxygen generation with a quantum yield of 1.3, the highest reported for PDT agents [29]. The GQDs exhibit broad absorption across the UV and visible spectrum and a strong deep-red emission, making them suitable for imaging and highly efficient cancer therapy. In vitro and in vivo studies confirm their potential for simultaneous imaging and treatment, offering superior photostability, water dispersibility, pH stability, and biocompatibility compared to conventional agents.

Kholikov et al. (2018) used sulfur-doped graphene quantum dots (SGQDs) combined with methylene blue (MB) for enhanced singlet oxygen generation and antimicrobial activity in PDT [130]. The study demonstrates that the SGQDs significantly improve the singlet oxygen yield compared to conventional GQDs, resulting in a more effective bacterial eradication of both Gram-negative E. coli and Gram-positive Micrococcus luteus under light irradiation. The combination of SGQDs with MB not only enhances the antimicrobial efficacy but also shows minimal cytotoxicity to human cells in the dark, making it a promising candidate for advanced PDT applications. Similarly, the exploration of different quantum dot materials continues to reveal new possibilities for improving photodynamic therapy.

Mir et al. (2018) report on the synthesis and characterization of cadmium-free water-soluble silver indium sulfide (AgInS) and AgInS@ZnS core–shell quantum dots (QDs) with tunable bandgaps and high quantum yields [132]. These QDs exhibit excellent luminescence properties and enhanced photodynamic and antifungal activities, particularly against Candida albicans. The study demonstrates that these QDs generate significant reactive oxygen species (ROS) under illumination, which contributes to their antifungal efficacy. Additionally, the core–shell QDs show superior biocompatibility and photostability, making them promising candidates for bioimaging and PDT applications.

Thakur et al. synthesized GQDs from Ficus racemosa leaves for combined photothermal and photodynamic cancer therapy. Quantitative results showed that GQDs, when irradiated with a near-infrared laser (808 nm), induced a temperature rise of up to 49 °C in MDA-MB-231 cells, resulting in less than 10% cell survival at a concentration of 1000 μg/mL, showcasing their efficacy in cancer treatment [124].

15. Organic Adjuvants

Organic adjuvants play a crucial role in enhancing the efficacy of PDT. Among these, polymers such as polyethylene glycol (PEG), chitosan, and poly(lactic-co-glycolic acid) (PLGA) are frequently utilized due to their biocompatibility and their ability to improve the solubility and stability of photosensitizers. For instance, PEGylation—the process of attaching PEG chains to photosensitizers—can prolong their circulation time in the bloodstream, enhancing the targeting of microbial cells [133]. Chitosan, a naturally occurring polymer, enhances the penetration of photosensitizers into microbial cells by interacting with their negatively charged membranes [134,135]. PLGA is often employed to create nanoparticles that encapsulate photosensitizers, protecting them from degradation and ensuring controlled release at the target site [136,137].

Perni et al. (2021) demonstrated the use of poly-beta-amino esters (PBAEs) to boost the efficacy of aPDT with toluidine blue O (TBO) [138]. Leveraging the cell-penetrating properties of PBAEs, they significantly reduced the required exposure time for effective aPDT. Their study identified specific PBAE–TBO complexes achieving inactivation rates up to 30 times faster than pure TBO. Moreover, PBAEs not only enhanced TBO uptake but also increased reactive oxygen species (ROS) production.

In another study, De Freitas et al. (2018) investigated the synergistic effects of aPDT enhanced by the peptide aurein 1.2 against E. faecalis and other clinically relevant pathogens [139]. Combining aPDT with aurein 1.2 led to a significantly improved bacterial reduction, particularly when using methylene blue or Chlorin e6 as photosensitizers. Aurein 1.2 enhanced photosensitizer uptake and ROS generation, leading to the complete elimination of E. faecalis and vancomycin-resistant E. faecium.

Lipid-based carriers such as liposomes and micelles also play a significant role in aPDT [140]. Liposomes, spherical vesicles composed of lipid bilayers, can encapsulate both hydrophobic and hydrophilic photosensitizers, enhancing their solubility and stability. The lipid bilayer structure of liposomes can fuse with microbial cell membranes, facilitating the direct delivery of photosensitizers into the cells. Similarly, micelles formed by the self-assembly of amphiphilic molecules can encapsulate hydrophobic photosensitizers, increasing their bioavailability and ensuring effective delivery to the target site. Functionalizing liposomes and micelles with targeting ligands can further improve the specificity of aPDT.

Rout et al. (2017) explored using lipid nanoparticles for encapsulating toluidine blue O (TBO) to enhance photosensitization-based antimicrobial therapy (PAT) [141]. The lipid nanoparticle delivery system improved stability, reduced aggregation, and increased the cellular uptake of the photosensitizer, leading to more effective antimicrobial action against both Gram-positive and Gram-negative bacteria. Encapsulated TBO generated reactive oxygen species more efficiently, resulting in greater bacterial inactivation and DNA damage compared to TBO in an aqueous solution. This highlights the potential of lipid nanoparticles to overcome conventional photosensitizer delivery limitations in PAT.

Papain gel, derived from the papaya plant, is another notable organic adjuvant in aPDT. Papain, a proteolytic enzyme, can break down proteins, making it effective for disrupting microbial biofilms and enhancing photosensitizer penetration [142,143]. When used with aPDT, papain gel can degrade the extracellular matrix of biofilms, exposing the embedded micro-organisms to photosensitizers and ROS. This results in a more effective treatment of biofilm-associated infections, often resistant to conventional antimicrobial therapies. Additionally, papain gel has anti-inflammatory properties that can help reduce inflammation and promote healing in infected tissues.

Silva Jr. et al. (2016) investigated the efficacy of a papain gel containing methylene blue (PapaMBlue) for removing dental caries and antimicrobial photoinactivation against S. mutans biofilms [142]. The study showed that PapaMBlue produced significantly more reactive oxygen species than free methylene blue, enhancing its antimicrobial effects. The combination of papain and methylene blue in the gel effectively reduced S. mutans biofilms without damaging fibroblasts or the collagen structure, making it a promising tool for minimally invasive dental treatments.

Cyclodextrins are cyclic oligosaccharides with a hydrophobic cavity and a hydrophilic exterior, suitable for enhancing the solubility and stability of photosensitizers [144]. By forming inclusion complexes with photosensitizers, cyclodextrins protect them from degradation and improve their delivery to microbial cells. Their unique structure allows for the controlled release of photosensitizers, ensuring sufficient concentrations at the site of infection during light exposure.

Dendrimers, which are highly branched tree-like polymers, can encapsulate or conjugate with photosensitizers, offering a high loading capacity and precise control over size and surface functionality [145]. Functionalized dendrimers can enhance the selective uptake of photosensitizers by microbial cells, increasing the efficacy of aPDT while minimizing damage to surrounding healthy tissues.

Natural organic compounds such as essential oils and plant extracts have also been explored as adjuvants in aPDT [146,147]. These compounds can exhibit intrinsic antimicrobial properties, which when combined with aPDT result in synergistic effects that enhance microbial kill rates. Essential oils can disrupt microbial cell membranes, increasing permeability and facilitating photosensitizer entry. Additionally, certain plant extracts contain antioxidants that can modulate ROS production during aPDT, optimizing the therapeutic effect.

5-aminolevulinic acid (5-ALA) is an endogenous compound that is a precursor in the biosynthesis of heme. In PDT, including aPDT, 5-ALA is used as a prodrug leading to the accumulation of protoporphyrin IX (PpIX), a potent photosensitizer within cells [148,149]. Tan et al. (2018) investigated the effects of 5-aminolevulinic acid photodynamic therapy (ALA–PDT) on the P. aeruginosa biofilm structure, virulence factor secretion, and quorum sensing (QS) [150]. Their study revealed that ALA–PDT significantly inhibits planktonic P. aeruginosa growth, destroys biofilm structures, and reduces the secretion of virulence factors like pyocyanin and elastase. The treatment also downregulated QS-related gene expression, crucial for biofilm formation and virulence.

Organic adjuvants in PDT are showing great promise in improving the effectiveness of cancer treatments. Polymeric nanoparticles and micelles enhance the delivery of photosensitizers (PSs) due to their small size and biocompatibility. These nanoparticles allow for controlled drug release, increasing the therapeutic impact. For example, methylene blue-conjugated nanoparticles and Chlorin e6-loaded particles combined with doxorubicin have proven highly effective in targeting resistant cancer cells [151,152,153]. Boron dipyrromethene (BODIPY) derivatives functionalized with mannose have also shown strong tumor inhibition in both lab and animal studies [154,155].

Block copolymer micelles, which offer a high drug-loading capacity, have demonstrated significant tumor inhibition when functionalized with peptides like Lyp-1, showing promise in both PDT and chemotherapy [156,157,158]. Liposomes, another type of lipid-based nanoparticle, improve PS delivery to tumors and have been shown to increase ROS production when exposed to light, effectively reducing tumor size [159,160].

Biological nanoparticles, such as virus-like particles (VLPs) and albumin-based nanoparticles, provide added benefits due to their biocompatibility and low toxicity [161,162]. Functionalized organic nanoparticles, such as HER2-targeting and mannose-functionalized nanoparticles, enhance PDT precision by targeting specific cancer cell receptors. These systems improve PS accumulation in tumor sites, reducing side effects and improving treatment outcomes [163,164]. In summary, these advancements in organic nanoparticle technology are overcoming key challenges in PDT, making it a more effective and safer option for breast cancer therapy.

16. Inorganic Adjuvants

Inorganic adjuvants can significantly enhance the effectiveness of PDT. Metal ions and their complexes are a prominent class of inorganic adjuvants in aPDT [165,166]. Metal ions like zinc, iron, and copper can form complexes with photosensitizers, enhancing their photophysical properties. For example, zinc phthalocyanine, a widely used metal complex in aPDT, exhibits high ROS generation efficiency upon light activation [167]. These metal complexes often have improved photostability and enhanced light absorption, leading to more efficient ROS production and greater microbial killing. Additionally, metal ions possess intrinsic antimicrobial properties, contributing synergistically to the overall antimicrobial effect of aPDT.

Porphyrins and their metal complexes are another important group of inorganic adjuvants in aPDT [168,169]. Porphyrins can form stable complexes with various metal ions, resulting in metalloporphyrins such as hematoporphyrin and protoporphyrin IX. These metalloporphyrins have been extensively studied for their ability to generate ROS upon light activation. The incorporation of metal ions into porphyrins enhances their photodynamic activity and improves their solubility and stability in biological environments. Metalloporphyrins can be designed to target specific microbial cells, increasing the selectivity and effectiveness of aPDT.

Phosphate-based compounds like hydroxyapatite are also used as adjuvants in aPDT [170,171,172]. Hydroxyapatite, a naturally occurring mineral form of calcium phosphate, can serve as a carrier for photosensitizers, improving their stability and delivery to the target site. When combined with aPDT, hydroxyapatite enhances photosensitizer penetration into biofilms, structured communities of micro-organisms resistant to conventional antimicrobial treatments. By facilitating the delivery of photosensitizers into biofilms, hydroxyapatite ensures that the ROS generated during aPDT can effectively reach and kill the embedded micro-organisms.

Titanium dioxide (TiO₂) is another inorganic compound explored as an adjuvant in aPDT [172,173]. TiO₂ is known for its photocatalytic properties, generating ROS when exposed to UV light [174]. Combining TiO₂ with aPDT significantly increases ROS production, leading to more effective microbial killing. Additionally, TiO₂ can coat surfaces, providing a self-cleaning and antimicrobial effect activated by light.

Potassium iodide (KI) acts as an adjuvant by reacting with ROS produced during photodynamic therapy, particularly singlet oxygen, to form reactive iodine species [175,176,177,178,179]. These iodine species have potent antimicrobial properties, adding an additional mechanism of microbial killing to the aPDT process. The dual action of ROS and reactive iodine species results in a synergistic effect, leading to higher kill rates of bacteria, fungi, and other pathogens. Furthermore, KI is relatively non-toxic and can be used safely in various clinical applications, making it a valuable addition to aPDT protocols. Huang et al. (2018) reported the enhancement of aPDT for treating urinary tract infections (UTIs) using a combination of methylene blue (MB) and potassium iodide (KI) [175]. In a female rat model, they demonstrated that the combination of MB and KI significantly potentiated the antibacterial effects of aPDT against uropathogenic E. coli (UPEC). Bioluminescent imaging confirmed the light dose-dependent reduction of bacterial burden in the bladder, with no detectable damage to the bladder lining. This study highlights the potential clinical application of MB and KI in aPDT as a non-antibiotic approach to combat drug-resistant UTIs.

Xuan et al. (2018) examined the potentiation of aPDT by potassium iodide (KI) when used with tetracyclines such as demeclocycline (DMCT) and doxycycline (DOTC) [180]. The study showed that KI significantly enhances the antibacterial effects of these tetracyclines under blue or UVA light by up to 5 logs of extra killing, primarily through the production of reactive iodine species and hydrogen peroxide. They also demonstrated that the tetracycline–KI combination is effective in both aerobic and anaerobic conditions, providing a robust mechanism for bacterial inactivation. This research highlights the potential of combining KI with tetracyclines for effective aPDT, especially in treating infections resistant to traditional antibiotics.

Another inorganic compound with similar potential is sodium azide (NaN3) [181,182]. Sodium azide can enhance aPDT by generating nitrogen-centered radicals when exposed to ROS. These radicals are highly reactive and can further damage microbial cells, contributing to the overall antimicrobial effect. However, it is essential to note that sodium azide is toxic and must be used with caution, particularly in clinical settings.

Hydrogen peroxide (H₂O₂) is an inorganic compound commonly used as an adjuvant in aPDT [183,184,185]. The additional ROS generated from the decomposition of H₂O₂ can increase the oxidative stress on microbial cells, leading to more effective killing. The use of hydrogen peroxide in aPDT can be particularly advantageous in treating biofilms, as the increased ROS production can penetrate and disrupt these structured microbial communities more effectively than aPDT alone.

Sodium nitrite (NaNO₂) is another inorganic compound that can act as an adjuvant in aPDT [186]. Sodium nitrite can react with ROS to form reactive nitrogen species, which have potent antimicrobial effects. Additionally, sodium nitrite has been shown to disrupt biofilms, making it a valuable tool in treating chronic and resistant infections [187].

Sarda et al. (2019) evaluated the antimicrobial efficacy of PDT, diode lasers, and sodium hypochlorite (NaOCl), along with their combinations, against endodontic pathogens E. faecalis and S. mutans [188]. The study demonstrated that combining NaOCl with either a diode laser or PDT significantly enhances bacterial reduction, achieving a 98% reduction in E. faecalis and a 96–97% reduction in S. mutans compared to individual treatments. The research suggests that combining NaOCl with PDT offers a superior disinfection method for root canal treatments, leveraging the advantages of lower wavelength lasers to minimize the damage to surrounding tissues while maximizing antimicrobial efficacy.

17. Antibiotics

Integrating antibiotics as adjuvants in aPDT can significantly enhance its efficacy, providing a dual attack on microbial infections. Combining antibiotics with aPDT can lead to synergistic effects where the combined action is greater than the sum of individual effects. One example is the use of tetracyclines, a class of broad-spectrum antibiotics that inhibit protein synthesis in bacteria [189]. When used in conjunction with aPDT, tetracyclines can increase the permeability of bacterial cell membranes, making the cells more susceptible to the ROS generated during photodynamic therapy [180,190,191]. This enhanced permeability allows photosensitizers to enter bacterial cells more effectively, leading to increased microbial kill rates.

Quinolones, another class of antibiotics, are known for their ability to inhibit DNA gyrase and topoisomerase IV, enzymes crucial for bacterial DNA replication. When combined with aPDT, quinolones can potentiate the damage caused by ROS to bacterial DNA [192,193,194]. This dual action of DNA damage from both the antibiotic and the ROS results in a higher probability of bacterial cell death. Additionally, the oxidative stress induced by aPDT can interfere with bacterial repair mechanisms, making it harder for bacteria to survive the combined treatment.

Beta-lactam antibiotics, such as penicillins and cephalosporins, disrupt the synthesis of bacterial cell walls by inhibiting penicillin-binding proteins. This disruption weakens the bacterial cell wall, making it more vulnerable to the oxidative damage induced by aPDT [190]. The ROS generated during photodynamic therapy can further compromise the integrity of the bacterial cell wall, leading to cell lysis and death.

Macrolides, such as erythromycin and azithromycin, inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit. These antibiotics can also exhibit anti-inflammatory properties, which can be beneficial in treating infections accompanied by inflammation. When used as adjuvants in aPDT, macrolides can enhance the uptake of photosensitizers into bacterial cells, facilitating ROS generation within the cells [195]. The anti-inflammatory effects of macrolides can also help reduce tissue damage and inflammation associated with infections, contributing to a more effective overall treatment.

Aminoglycosides, including gentamicin and tobramycin, bind to the bacterial 30S ribosomal subunit, causing the misreading of mRNA and inhibiting protein synthesis. These antibiotics can also increase the permeability of bacterial cell membranes. When combined with aPDT, aminoglycosides can facilitate the entry of photosensitizers into bacterial cells, enhancing ROS production and leading to increased bacterial cell death [196,197]. The combination of aminoglycosides with aPDT can be particularly effective against Gram-negative bacteria, which often exhibit resistance to other types of antibiotics.

Rifamycins, such as rifampin, inhibit bacterial RNA synthesis by binding to the RNA polymerase enzyme. This inhibition can interfere with the bacterial response to oxidative stress [198,199]. When used in combination with aPDT, rifamycins can enhance the susceptibility of bacteria to ROS by impairing their ability to produce protective stress response proteins. This combined action can lead to more efficient bacterial killing, especially in biofilms or dormant states that are typically harder to eradicate with conventional treatments.

18. PDT in Fungi Treatment

Photodynamic therapy (PDT) has also shown promise as an innovative treatment for fungal infections, particularly those caused by dermatophytes and yeasts [200,201,202]. This approach is beneficial for its ability to target fungal cells selectively while minimizing damage to surrounding healthy tissues. The effectiveness of PDT against various fungal pathogens, including Candida albicans, Aspergillus niger, and Trichophyton rubrum, were shown [203,204,205]. Studies have demonstrated that PDT can reduce fungal viability and biofilm formation, which are critical factors in the treatment of chronic and resistant fungal infections. Moreover, PDT’s ability to penetrate and treat superficial fungal infections makes it a valuable tool in dermatology, offering a non-invasive alternative to conventional antifungal therapies. However, challenges remain, such as optimizing light parameters and photosensitizer formulations to enhance efficacy and reduce treatment time. Continued research is essential to fully establish PDT as a standard treatment modality for fungal infections [206].

19. Novel Strategies

One innovative approach to overcoming this issue involves the synergistic use of PDT and efflux pump inhibitors (EPIs). This strategy aims to enhance the antimicrobial efficacy of PDT agents, such as silver nanoparticles (AgNPs) and methylene blue (MB), by inhibiting the action of bacterial efflux pumps, which are known to expel antibiotics and other antimicrobial agents from the cell.

Efflux pumps, particularly the AcrAB-TolC pump in E. coli, play a critical role in antibiotic resistance by extruding a wide range of toxic substances, including photosensitizers like MB. The use of EPIs, such as reserpine, have shown promise in blocking these pumps, thereby increasing the intracellular concentration of PDT agents and enhancing their antimicrobial effects. In a recent study by Allamyradov et al., the combination of reserpine with AgNPs and MB resulted in a significantly higher bacterial deactivation rate compared to the use of AgNPs and MB alone [112]. The research demonstrated that the enhanced efficacy was not due to increased singlet oxygen production but rather the inhibition of the AcrAB-TolC efflux pump, which prevented the removal of MB from bacterial cells.

20. Modulation of Environment

Recent advancements in the treatment of bacterial biofilm infections have highlighted the potential of combining PDT with strategies that modulate the infection microenvironment. One such innovative approach involves potentiating the hypoxic microenvironment within biofilms to enhance antibiotic activation. In the study by Xiu et al., PDT is employed not only to directly eradicate methicillin-resistant S. aureus (MRSA) biofilms under normoxic conditions but also to create a hypoxic environment that triggers the anaerobic metabolism of MRSA, thereby activating the antibacterial properties of metronidazole (MNZ) [207].

Biofilms present a significant challenge for traditional antibiotics due to the varied metabolic states of bacteria within the biofilm microenvironment (BIM). By inducing hypoxia through PDT, MRSA switches to anaerobic metabolism, which is conducive to the activation of MNZ. This dual approach not only enhances the direct antimicrobial action of PDT but also leverages the hypoxia-induced activation of MNZ to target metabolically less active bacteria that are typically resistant to antibiotics. Furthermore, this combined therapy polarizes macrophages to an M2-like phenotype, promoting the healing of biofilm-infected wounds in murine models.

21. Macrophages in PDT

The innovative use of adoptive macrophage-directed PDT presents a promising approach to enhancing innate immunity and effectively targeting and eliminating bacteria. This strategy, as explored by Wang et al., involves the transfer of macrophages loaded with a near-infrared photosensitizer (Lyso700D) into lysosomes [208]. These engineered macrophages, referred to as D-RAWs, utilize their innate immune capabilities to locate and engulf MDR bacteria, delivering the photosensitizer directly to the bacterial cells within the lysosomes. Upon activation by near-infrared light, the photosensitizer generates reactive oxygen species (ROS), which exhibit potent antibacterial effects, efficiently killing the bacteria while minimizing damage to surrounding healthy tissues.

The study demonstrated that this method could successfully eradicate MDR S. aureus (MRSA) and Acinetobacter baumannii (AB) infections in murine models, achieving a 100% survival rate compared to the 10% survival rate in control groups. Additionally, in a rat model of central nervous system bacterial infection, the therapy proved highly effective, with all treated rats surviving the infection. This therapeutic approach capitalizes on the precise targeting ability of macrophages and the broad-spectrum efficacy of PDT, offering a potential clinical treatment for severe bacterial infections, particularly in immunocompromised patients.

22. PDT Tolerance Development

In a study by Rapacka-Zdonczyk et al., S. aureus was subjected to repeated sub-lethal exposures of aPDI and aBL to assess the potential development of tolerance [209]. The study utilized the photosensitizer rose bengal (RB) for aPDT and endogenous photosensitizers such as porphyrins for aBL.

The findings revealed that S. aureus developed substantial tolerance to both aPDT and aBL after 15 cycles of sub-lethal treatment. This tolerance was stable even after five cycles of subculturing without exposure to the phototreatments. The study also indicated an increased mutation rate in S. aureus subjected to sub-lethal phototreatments, likely due to the activation of the stress-responsive, error-prone DNA polymerase V, encoded by the umuC gene. This was further supported by the increased expression of umuC and the dependence on the SOS response for tolerance development, as demonstrated by the lack of tolerance in recA and umuC transposon mutants of S. aureus.

Interestingly, the aPDI/aBL-tolerant S. aureus strains exhibited increased susceptibility to antibiotics such as gentamicin (GEN) and doxycycline (DOX), suggesting that the genetic alterations induced by the phototreatments might also affect other resistance mechanisms. This study underscores the importance of considering the risk of tolerance development when designing aPDI/aBL protocols and highlights the complex interplay between phototreatments and bacterial genetic adaptation.

23. Endogenous Photosensitizers

The development of antimicrobial blue light bathing therapy offers a novel, non-pharmacological approach to wound infection management, addressing the critical challenge of antibiotic resistance. In a study conducted by Jie Hui et al., a prolonged, continuous exposure to blue light at low irradiance levels (5 mW/cm²) was employed to maintain bacteriostatic pressure on wound infections, thereby reducing bioburden and promoting healing without the photothermal risks associated with higher irradiance levels [210]. This innovative light bathing strategy utilizes wearable, light-emitting patches, enabling the extended and safe application of blue light in preclinical trials on rat models.

The study demonstrated the efficacy of this approach in suppressing infections caused by methicillin-resistant S. aureus (MRSA) and multidrug-resistant P. aeruginosa. Continuous exposure to 410 nm blue light successfully inhibited bacterial growth, kept the wound bioburden low, and facilitated healing, as evidenced by clinical wound signs, immunohistochemical analysis, and endpoint colony-forming unit enumeration from wound biopsies. This method overcomes the limitations of traditional blue light therapy, which requires high optical intensities and poses significant photothermal risks, by maintaining effective antimicrobial pressure through sustained, low-level illumination.

The results of this study highlight the potential of blue light bathing as a groundbreaking strategy in wound care, offering a safe and effective alternative to conventional antibiotic treatments. The ability to use low irradiance levels significantly enhances the practicality of blue light therapy, paving the way for its clinical application in managing chronic and acute wound infections.

24. The Future, Opportunities, and Challenges of Photodynamic Therapy

The future of PDT promises significant advancements in several key areas. There is tremendous effort on the development of novel photosensitizers with enhanced efficacy, selectivity, and reduced side effects. Those efforts include new nanoparticles and organic molecules that can be specifically tailored for various therapeutic applications. In addition, combining photosensitizers with other treatments, such as chemotherapy or immunotherapy, is being investigated to improve outcomes for complex cancer types. Enhanced targeting and delivery systems are also of utmost importance, with efforts directed toward developing advanced delivery mechanisms like nanoparticles and conjugates to ensure the precise targeting of tumor sites while minimizing off-target effects. Improved imaging technologies are being utilized to monitor photosensitizer distribution and activation in real time, which could significantly enhance treatment accuracy and effectiveness. Moreover, personalized treatment approaches are on the horizon, focusing on creating tailored PDT protocols based on individual patient profiles and employing dynamic treatment monitoring to adjust parameters based on immediate feedback.

There are promising opportunities for PDT to expand its clinical applications and innovations in its technology. PDT has been investigated for a wider range of cancers, including those that are currently difficult to treat with conventional methods, such as metastatic or deep-seated tumors. In addition, PDT is being studied for treating other medical conditions, including infections, inflammatory diseases, and dermatological disorders. Technological innovations, such as advancements in light delivery systems and the integration of PDT with digital health technologies, like wearable devices and AI, offer exciting possibilities for optimizing treatment planning and patient management.

As mentioned above, there are challenges for PDT. Despite its numerous advantages, PDT faces notable limitations, such as issues with selective targeting, light penetration, and resistance development. However, with the advent of new developments in photosensitizer design, advanced delivery systems, and improved imaging technologies, many of these challenges are expected to be addressed. These advancements promise to enhance the effectiveness of PDT and broaden its applicability, ultimately overcoming some of the current limitations and paving the way for more effective treatments.

25. Conclusions

PDT is a versatile and effective treatment modality with a broad range of clinical applications. PDT can selectively destroy its target by utilizing photosensitizers and light to generate reactive oxygen species. Unlike some treatments, PDT can be repeated multiple times if necessary, which is crucial for managing recurrent conditions. PDT has proven successful in treating dermatological conditions, such as actinic keratosis and basal cell carcinoma, and its potential is being explored in oncology for cancers such as those of the esophagus and lungs, as well as in ophthalmology for conditions like age-related macular degeneration. Currently, PDT is being investigated for a wider range of applications in cancer, infectious diseases, and even neurological conditions.

The development of new photosensitizers with improved specificity, deeper tissue penetration, and lower toxicities presents new challenges and opportunities. In addition, Integrating PDT with other treatments such as immunotherapy, chemotherapy, or radiotherapy to enhance overall efficacy is another key area for future research. Furthermore, PDT will also be explored for a personalized treatment based on genetic, phenotype, and environmental factors to maximize its effect and minimize side effects.

Nanoparticles are also being studied to improve photosensitizer delivery, targeting, and activation, thereby increasing the precision and effectiveness of PDT.

To fully utilize the potential of PDT, photosensitizers should be improved for better absorption, distribution, metabolism, and excretion. In addition, light delivery systems should be further enhanced for the precise delivery of light in deeper and less accessible tumors. Strategies to mitigate side effects, such as skin sensitivity and inflammation, and standardized protocols for different clinical treatments and regulatory and approval processes must be developed.

PDT has tremendous potential in various medical fields, promising advancements in treatment efficacy and patient outcomes. By addressing the challenges above and seizing future opportunities, the ongoing research and development of new photosensitizers and light delivery systems will likely expand the applications and effectiveness of PDT, making it an increasingly integral part of modern therapeutic strategies.