Photodynamic therapy (PDT) is a therapeutic modality which uses visible light wavelengths, mainly in the red and near-infrared (NIR) regions, for the activation of photosensitizing molecules (PSs). In the presence of molecular oxygen, light energy absorption by PSs triggers a sequence of events, ultimately leading to the formation of singlet oxygen (¹O₂) and other cytotoxic reactive oxygen species (ROS) [1]. Thus, any kind of cells or tissues in which a PS accumulates and that has been exposed to appropriate light wavelengths can be killed by irreversible damages caused by oxidative stress. This phototherapeutic approach has been largely investigated and proposed for the treatment of several pathological conditions, even if PDT is mainly applied in the oncology field for the treatment of selected types of solid tumors [2]. The first clinical studies of PDT in humans date back to the 1970s, while regulatory approval of PDT for the treatment of certain types of cancer dates back to the 1990s ^[3][4][5][3,4,5]. Many clinical trials have been completed and others are still ongoing, but PDT remains an underused modality for treating solid tumors, compared to conventional radiotherapy, surgery, or chemotherapy ^[6][7][6,7]. However, PDT is largely used in dermatology for the removal of non-melanoma skin cancers and the treatment of precancerous conditions such as actinic keratosis, being minimally invasive and with therapeutic efficacy comparable to surgery ^[8][9][8,9].

Limited light penetration into deep-sited tumor masses and poor oxygen availability can impair the efficacy of PDT. Even under this circumstance, its selectivity and absence of important side effects, if compared to chemotherapy, continues to stimulate the research of innovative approaches to overcome limitations, while fully exploiting PDT advantages. Despite recent successes in the clinical application of PDT, chemotherapy continues to remain the therapy of choice for the treatment of most cancers due to its high potential to eradicate cancer cells. However, chemotherapy is a very aggressive treatment with severe side effects. For this reason, current research in the oncology field is focusing on developing combinations of PDT and chemotherapy, with the aim to achieve a synergistic effect between the two therapies. These multi-therapeutic strategies could provide a way to reduce the recommended doses of chemotherapeutics, while at the same time overcoming the limitations of PDT. The advantages derived from this combinatorial approach can be better exploited by adopting strategies for delivering combinations of drugs selectively into tumor, concomitantly minimizing undesired side effects in normal tissues. Recent studies are mainly directed toward the design and realization of smart nanocomplexes and nanoplatforms to deliver PSs in combination with additional anticancer agents to optimize the efficiency and selectivity of drugs uptake into malignancies and to potentiate the overall anticancer responses.

As anticipated, PDT is a strategy for treating tumors based on three components (light, PS, and molecular oxygen), neither of which is per se harmful to cells, but whose interplay triggers the formation of ROS that, above a given threshold, causes severe/lethal cell damages. The PDT starts with a systemic administration of the PS (or topical in the case of skin cancers); after a selected time, which allows the PS accumulation in the neoplastic lesion, the tumor mass is irradiated with light wavelengths absorbed by the PS, leading to the production of ROS. Commonly used PSs produce mainly ¹O₂, whose lifetime in biological environments is ~4 µs, with a corresponding diffusion radius in the tissue of no more than ~220 nm ^[10][11][10,11]. This implies that the photooxidative stress induced by PDT remains confined to the cells and tissue in which the PS has been accumulated and activated, thus conferring intrinsic selectivity to the therapy. Most of the PSs are macrocyclic molecules such as porphyrins or their derivatives, such as chlorins, bacteriochlorins, and phthalocyanines. All these molecules absorb light in the red and far-red regions of the visible spectrum, the so-called “PDT therapeutic window” (660–800 nm). In this spectral range, tissues are more transparent due to the limited light absorption of endogenous chromophores, thus allowing deeper light penetration [1].

ROS produced during PDT treatment are harmful to cells and organisms, because they oxidize a variety of biological components, with proteins and membrane lipids being the major targets [12]. Because of the short diffusion capacity of ROS, the primary damaged cellular sites are determined by the intracellular localization of the PS during light irradiation, which in turn depends on the physico-chemical features of the PS. In general, hydrophobic PSs mainly localize in the intracellular membranes of endoplasmic reticulum (ER), Golgi apparatus and mitochondria, while the hydrophilic ones are mainly found in endosomes and lysosomes ^[13][14][13,14]. However, the intracellular localization of a PS is determined also by additional parameters, such as incubation time and cell type ^[15][16][15,16]. Depending on the PS localization and on the primary sites of photodamage, different cellular responses can be triggered by PDT. In particular, if the oxidative insult exceeds the cellular capacity to restore homeostasis, cell death can occur mainly via necrosis, apoptosis, or autophagy ^[17][18][17,18] or via less known PDT-induced cell death pathways, such as necroptosis ^[19][20][19,20], ferroptosis [21], and parthanatos ^[22][23][22,23]. It has been well characterized that high PSs and light doses cause heavy cellular damages and induce accidental necrosis, similar to PSs that are associated with the plasma membrane, which cause oxidative stress to proteins and phospholipids. This results in rapid loss of membrane integrity and subsequent cell lysis ^[15][24][15,24]. On the contrary, low PDT doses and PSs localized in mitochondria, ER, or lysosomes lead to apoptosis, which is induced by direct damages to the anti-apoptotic proteins Bcl2 and Bcl-xL, without affecting the pro-apoptotic protein Bax [25]. Damage-associated molecular patterns (DAMPs or alarmins) are released by or exposed on the surface of dying cancer cells. DAMPs are perceived as “alarm signals” and stimulate a specific anti-tumor response to the PDT-treated tumor as well as distal untreated metastasis ^[26][27][26,27]. Thus, PDT can inhibit tumor growth by the direct killing of cancer cells through one of the mechanisms outlined above and subsequent activation of innate and adaptative immunity ^[28][29][30][28,29,30].

PDT can also damage and affect the functionality of components of the tumor microenvironment (TME), which often plays a fundamental role in favoring cancer cell proliferation, tumor progression, and development of drug resistance [31]. As an example, disorganized and hyperpermeable tumor vasculature sustains rapid cancer cell proliferation with oxygen and nutrients supply. Tumor vessels are important targets of PDT, as they are easily affected by oxidative stress, thus resulting in vessel constriction, damages to endothelial cells, thrombi formation, and ultimately complete stasis of the blood flow, leading to severe hypoxia and tumor necrosis. In general, with PDT protocols based on short drug-light intervals, in which light is delivered to tumor when high PS levels are still circulating in the bloodstream, the vasculature represents the main target of the treatment, while cancer cells are killed indirectly through oxygen and nutrient deprivation [32].