treatment times are very short and the lighting is very painful for this reason the treatments are recommended under anesthesia [18].

Due to their submicron size, nanoparticles as PS delivery system have numerous advantages such as the protection against enzymatic PS degradation, the control of PS release allowing a constant and uniform concentration into target cells, and the ability to penetrate target cells [19].

into cells. Also, due to their submicron size, nanoparticles as a PS delivery system have other advantages such as protection against PS enzymatic degradation, the control of PS release that allows a constant and uniform concentration in target cells, and the ability to penetrate target cells [19].

PS incorporation of nanoparticles opens new perspectives and challenges to this eld.

Third Generation

of Photosensitizers

The low speci city of current photosensitizers to localize and reach tumor tissue is one of the most important problems of PDT to achieve greater ef cacy.

Second-generation photosensitizers bound to carriers such as antibodies and liposomes for selective accumulation within tumor tissue are referred to as third-generation photosensitizers and currently represent an active research area in the eld [20].

A third generation of PSs results by modi cation of the second generation with biologic conjugates such as carriers, antibodies, or liposomes to improve their physical, chemical, and therapeutic properties. Recently, signi cant efforts have employed in the synthesis of pure chemical derivatives in order to create new sensitizers with improved activity and minimal side effects.

In this sense, the transport of photosensitizers by means of nanoparticles is increasingly being investigated to improve permeability and retention [21]. The incorporation of PS into nanostructured drug delivery systems, such as polymeric nanoparticles (PNPs), solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), gold nanoparticles (AuNPs), hydrogels, liposomes, liquid crystals, dendrimers, and Cyclodextrin is a potential strategy to overcome this dif culty [22].

This type of nanoparticles is synthesized biomolecules for drug delivery, which are used in PDT to enhance transcytosis across epithelial and endothelial barriers and optimize delivery of poorly water-soluble PS and co-delivery of PS

PDT Reaction

Figure 14.1 involves administration of a photosensitizing agent followed by light irradiation of the previously sensitized tissue, at a wavelength corresponding to an absorbance band of the PS. In the presence of oxygen, a series of events lead to direct tumoral cell death, damage to the microvasculature, and induction of a local infammatory reaction [23].

The photochemical reaction requires three fundamental components: a sensitizer, photons, and oxygen. The mechanism of action of PDT is determined by the uptake of a photosensitizer (PS) molecule which, upon being excited by light in a determined wavelength, reacts with oxygen and generates oxidant species: singlet oxygen, radicals that produce peroxidative reactions in cell membranes, cytoplasm, or organelles that cause cell damage and death by apoptosis or necrosis of tumor cells, closes the vasculature of the tumor, and further affects the immune system [23, 24].

This photodynamic reaction is activated when an appropriate wavelength of light is emitted on a previously sensitized tissue. When the light arrives at the target tissue, it is absorbed by the photosensitizer (Ps) who has two electrons with opposite spin in their ground state that makes that one of them jumps to an excited energy singlet state, generating an unstable excited photosensitizer. This unstable excited photosensitizer can emit excess energy as heat and/or fuorescence (a feature which can be used for the purposes of diagnostics and optical monitoring).Alternatively, the electron may have its spin inverted (parallel to its counterpart) creating a new excited triplet

PS*excited singlet state

Light

Ground state triplet O2

3PS*

1PS*

Excited singlet state O2

e e.g.NADH

Ground state triplet O2

1PS*

Fig. 14.1  Type I and Type II reactions in PDT (“photodynamic reaction”) Schematic Jablonski’s diagram showing PDT’s mechanism of action. Following light absorption, the PS reaches an excited singlet state (PS*). After an intersystem crossing, photosensitizer in a triplet excited state (3PS*), can react in two ways: (1) it reacts with biomolecules

ROS e.g.O2

through a hydrogen atom transfer to form radicals, which react with molecular oxygen to generate ROS (Type I reaction); (2) 3PS* can react directly with oxygen through energy transfer, generating singlet oxygen (1O2) (Type II reaction). PS photosensitizer, PS* excited singlet, 3PS* excited triplet singlet, ROS reactive oxygen species, 1O2 singlet oxygen

state in a process called intersystem crossing. From here, one of two things can happen: The particle reacts directly with the surrounding tissue (substrate) forming a radical anion or cation which then reacts with oxygen in the air to produce reactive oxygen species (ROS) (Type 1 reaction); or all of the electron’s energy is transferred to oxygen from the air forming singlet oxygen (Type II reaction) [2].

The balance between these two processes (type I and II reaction) depends on the nature of PS being used, the concentrations of oxygen and substrate, and af nity of the PS with the substrate. Both types of reactions reach cell death but in general, under hypoxic conditions primarily occurs a type I photodynamic reaction, while in oxygenated conditions prevail the generation of type II photochemical reactions which is the most important pathway for PDT clinical use, because the PS interacts with oxygen to generate singlet oxygen, which is considered to be the basis of PDT’s tumor and vascular ablation ability. This oxygen-dependent type II PDR is essential for PDT [25–28].

Tumor Damage Process

Tumor destruction is based on the following facts: After the administration, the photosensitizer is distributed in all cells and can be found in the liver, spleen, kidney, bone marrow, and tumor tissue. Normal organs quickly clear this substance, but in tumor cells it remains inside for more than 48 h. Due to differences in the vasculature and lymphatic drainage of tumors and its uptake, the photosensitizer is selectively retained by the tumor, its cells, and its interstitial tissue, so that after 2 days its concentration will be higher in the tumor than in the rest of the tissues. Finally, the photosensitizer is activated at the target tissue by an appropriate wavelength light. The photosensitizing substance will absorb the light energy and oxygen derivatives (singlet oxygen) will be produced, with the consequent destruction acting on the different cell structures from the membrane to the nucleus. PDT also causes a cessation of vascular tumor circulation with tissue hypoxia and secondary tumor necrosis, while nearby tissues are not affected. This reaction is the main cause for tumor destruction.

Events related to the reaction have drawn attention lately, such as the antitumor activity of infammatory cells and the triggered immune reaction by the tumor sensitized with a photodynamic substance. These two reactions are initiated by photodynamic damage and contribute to a more complete tumor destruction.

Some factors can limit tumor cell death, such as an inhomogeneous distribution of the photosensitizing substance within the tumor, and also the availability of oxygen. These factors have been largely avoided with the appearance of new third-generation photosensitizers, as we will see later.

PDT reaches the cytotoxic effects on tumor cells by indirect and direct mechanisms. Indirect mechanisms lead to changes in the tumor microenvironment as anti-vascular effect (vasoconstriction, thrombosis or vessel leakage) and anti-tumor immune response (release pro-­ infammatory cytokines and tumor associated antigens or xation of complement). Direct mechanisms produce cell killing due to macromolecule damage with apoptosis and necrosis process. The reactive oxygen species (ROS) that are produced during PDT have been shown to destroy tumors by multifactorial mechanisms [29, 30].

ROS (reactive oxygen species) and singlet oxygen have a high reactivity but a short half-life (40 ns). Due to this, PDT directly affects only those biological substrates that are close to the region where these species are generated, usually within a 20 nm radius [25].

Apoptotic cell death tends to predominate in the most PDT sensitive cell lines at lower light/ photosensitizer doses [31, 32], while the necrotic mechanism tends to predominate at higher light/ photosensitizer doses [33]. Activation of an autophagic mode of cell death following irradiation of certain photosensitizers have been also described by changes in the cellular morphology, chromatin condensation, loss of mitochondrial membrane potential and formation of vacuoles containing cytosolic components [34].

The damage of a speci c subcellular target depends on the location of the photosensitizer, due to the reduced capacity of migration of oxy-

gen. Photofrin®, one of the most used photosensitizers, is accumulated in the mitochondria and once activated causes apoptosis. Other PSs have empathy for determinate organelles, like lysyl chlorin p6 for lysosomes. The initial damages that PDT produces in cells membranes can be observed after light exposure: edema, blistering, ruptured vesicles containing enzymes, reduction of active cell transport, plasmatic membrane depolarization producing more photosensitizer income, increased chromate permeability and ATPase inhibition [35].

The administration of some medications will also affect the nal result of the photodynamic effects. The two best known are Adriamycin and glucocorticoids. Both improve the effects of therapy increasing the area of tumor necrosis when administered 24 h after photoradiation [36–39].

Additionally, other animal studies have shown that the photodynamic reaction is time-­dependent, even when it starts almost immediately after exposure to light, the process of tumor cell destruction continues to act slowly over a rather long time. “In vivo” models showed that tumor cells transplanted immediately after treatment were able to be implanted and to reproduce, while those transplanted 24 h after treatment were not [40].

Procedure

For PDT treatment of malignant tumors in the tracheobronchial tree, bronchoscopy is performed under topical anesthesia or conscious sedation 48 h. After a slow intravenous injection of the photosensitizer, doses and window period until bronchoscopy are variable depending on the chosen photosensitizer. Photofrin® is used at a dose of 2 mg/kg 48 h before bronchoscopy while Npe6 is administered at doses of 40 mg/m2 4 h before. The PS should be preferably excited by light of a wavelength included in the therapeutic range between 600 and 800 nm, which has greater capacity for tissue penetration. The period of time between the drug is given and the light is applied, called the drug-to-light interval, can be anywhere from a couple of hours to a couple of days, depending on the chosen agent [41].

Tumors should be irradiated (630 nm light) through a fexible beroptic bronchoscope 40 ± 50 h after oral or intravenous administration of PS. The tumor area is illuminated for 500 s with a 630 nm wavelength of a with non-thermal effect laser light, such as Argon-Dye laser or Diode laser (Fig. 14.2). In case of using Photofrin® as a photosensitizer, the residual tumor could receive additional illumination within 6–7 days since the photosensitizer concentration is still in the therapeutic range.

Two different radiation bers are used depending on the type and the size of the tumor: front light microlens bers (Fig. 14.3) or 360° diffusing light cylindrical bers (Fig. 14.4). Microlens bers are used for small and super cial tumors such as “in situ” lesions. When a radical treatment is intended, it is necessary to carry out an exhaustive inspection of the entire bronchial tree, especially at the peripheral areas. The extent of the tumor should be mapped and any synchronous airway lesion should be con rmed or excluded before the procedure.

Fig. 14.2  Diodes laser

Fig. 14.3  Microlens ber

The use of the ultra ne berscope and EBUS can be very useful for diagnosing small peripheral lesions inaccessible to the conventional berscope. The procedure has to be performed in an appropriate suite following the laser safety rules (Fig. 14.5).

For exophytic tumors more than 0.5 cm and for parallel bronchial lumen tumors or those that involve small branches of the bronchial tree, the cylindrical ber or 360° diffusing light cylindrical ber inserted directly inside the tumor is appropriate.

Between 2 and 5 days after treatment, a new bronchoscopy is performed to clean out debris. A second illumination is advisable if parts of the tumor show no signs of necrosis 96 ± 120 h after the rst illumination.

PDT clinical ef cacy is dependent on complex dosimetry of total light dose, light exposure time, and light delivery mode [42]. Current protocols use a power of 200–400 mW/cm2 to apply a total light dose of 100–200 J/cm2 in a treatment time of 500 s [43].

In addition to Argon-Dye and Diode lasers, other laser types have been used, such as gold vapor laser, copper-dye laser, laser-dye excimer, yttrium laser, aluminum and garnet (YAG) with a crystal of potassium titanyl phosphate laser and an optical parametric oscillator. The therapeutic window for the majority of PDT applications lies in the red region of the spectrum between 620 and 850 nm achieving optimal tissue penetration and PS activation. For the delivery of light, both lasers and incandescent light have proven to be effective [43, 44].

Fig. 14.5  PDT room

Photosensitivity is the most common secondary effect of PDT. It usually lasts for 4–6 weeks. During all this time patients are

advised to cover their skin completely when exposed to direct or indirect sunlight, and to avoid bright indoor light. The patient must also wear sunglasses and should not use hair dryers or other devices that give direct heat to the skin. The new generations of photosensitizers have less photosensitivity, reducing this period from weeks to days.

As we mentioned, one or several clean-up bronchoscopy should be performed2 or 3 days after tumor illumination, in order to remove viscous debris and detritus from the tumor process destruction to avoid complications such as atelectasia, infections, respiratory distress, or respiratory failure. A new follow-up bronchoscopy should be done if the patient persists or develops bronchial obstruction symptoms.