Mechanisms of PDT action
The distance of photodynamic damage is limited to the immediate intracellular localization of the PS (Moan et al., 1989) due to the short lifetime 1O2 (approximately 10-320 ns) and limited diffusion in biological media (up to 100 nm) (Dysart and Patterson, 2005). Singlet oxygen is a very reactive species, much more electrophilic than its ground state, and very rapidly oxidizes closest biomolecules such as proteins and lipids. Hundred nanometers range of its oxidation determined the subcellular level of PDT damage. Thus, intracellular localization is crucial for effective PDT and PSs displaying a higher cellular uptake are usually more cytotoxic (Jensen et al., 2010).
Direct tumor cell death effects
PS accumulates in various intracellular compartments and can induce multiple cell death subroutines at the cellular level (Figure 1.3). There are two main types of cell death subroutines: accidental (necrosis) and regulated (apoptosis, autophagy, etc). Necrosis is an uncontrollable form of cell death characterized by the disruption of the plasma membrane, cytoplasm swelling, and destruction of organelles. Necrosis has been associated with incidental cell death, caused by extreme physical, chemical, or mechanical cues.
Figure 1.3 The main cell death subroutines induced by PDT. Reprinted from (Agostinis et al., 2011)
In turn, regulated cell death subroutines could be pharmacologically or genetically modulated through the one or more signal transduction modules. Regulated cell death subroutines, which could be induced upon PDT mainly include apoptosis and autophagy. Apoptosis or programmed cell death is identified in single cells, and morphologically characterized by cell shrinkage, chromatin condensation, cleavage of chromosomal DNA into internucleosomal fragments, blebbing of the plasma membrane, and the formation of apoptotic bodies without plasma membrane breakdown (Agostinis et al., 2004; Almeida et al., 2004). It was identified that among all subcellular structures mitochondria plays the most critical role in the initiation of PDT-mediated apoptosis (Castano et al., 2005; Kessel and Luo, 1999). PDT-induced damage of mitochondria results in a cascade of reactions including the rapid release of mitochondrial cytochrome c into the cytosol followed by activation of the apoptosome and procaspase-3 that eventually cause the apoptosis (Oleinick et al., 2002).
Autophagy, as one of the regulated cell death subroutines, is a specific type of cell damage which relies on degradation redundant or damaged cell’s elements. This subroutine is characterized by a massive vacuolization of the cytoplasm and can be stimulated by various stress signals, including oxidative stress (Dewaele et al., 2010). During autophagy, normal cell functions used to degrade components of the cytoplasm are involved and characterized by appearance autophagosomes, autolysosomes, electron-dense membranous autophagic vacuoles, myelin whorls, multivesicular bodies, as well as engulfment of entire organelles (Castano et al., 2005). Autophagy is upregulated in response to extra– or intracellular stress and signals such as starvation, growth factor deprivation, ER stress and pathogen infection (Kessel and Oleinick, 2009). Along with the mentioned cell death mechanisms, the subroutines such as mitotic cell death (Castedo et al., 2004), cathepsin-mediated lysosomal death pathway (Leist and Jäättelä, 2001), programmed necrosis (Bizik et al., 2004; Vanlangenakker et al., 2008) were also reported for PDT.
Mechanism of cell death depends on cell and PS type, treatment protocol, and photodynamic dose (Agostinis et al., 2011). PS localization within or on the cell surface is critical to determine the mode of cell death induction and thus the cellular response to photodamage. Apoptosis is preferable for PDT treatment because the cells undergoing apoptosis release the “find me” and “eat me” signals in the interstitial medium which are required for the clearance of the remaining apoptotic bodies by phagocytes that usually prevents inflammation response. On the other hand, during necrosis, the release of cellular contents and pro-inflammatory molecules into the intracellular media also caused an intense inflammatory reaction. Nevertheless, non-homogenous distribution of PS within the tumor, insufficient depth of light penetration into the tumor or distance of tumor cells from the vessels (Korbelik and Krosl, 1994) as well as availability of oxygen (Tromberg et al., 1990) make difficulties to achieve the tumoricidal effect in vivo only through direct damage of tumor cells. Along with direct tumor cell damage, PDT could also damage the tumor-associated vasculature and could induce robust inflammatory reactions activating an immune response against tumor (Figure 1.4).
Antivascular effects of PDT
According to the literature, tumor blood vessels are fundamentally different from normal vasculature (Eberhard et al., 2000; Siemann, 2011). Tumor vasculature is characterized by the vessels that are immature in nature with poorly developed, chaotic growing architecture and discontinuous endothelial linings (Eberhard et al., 2000). Photodamage of endothelium cells lining tumor vessels could incite the modifications in cytoskeleton proteins organization with consecutive induction of a signal to the platelets and neutrophils activation which adhere on the vessel wall (Senge and Radomski, 2013). It was demonstrated, that endothelial cells accumulate more PS and possess higher sensitivity to PDT compared to muscle cells (West et al., 1990). Finally, the combination of vessel constriction, thrombus formation, and increased interstitial pressure leads to tumor tissue ischemia (Fingar et al., 2000; Henderson et al., 1985) and restricts nutrient supply to the proliferating tumor cells inhibiting tumor growth, making the tumor vasculature a promising target for anticancer treatment. Moreover, PDT vascular destruction may be accompanied by an inflammatory response (Korbelik, 1996).
PDT and the immune response
PDT-response is closely connected with the immune system. It could be said that photosensitizers, vascular and immune system together create a group to fight with tumor cells (Oniszczuk et al., 2016). To date, it was established that PDT could stimulate both innate immunity and adaptive immunity (Castano et al., 2006). Preclinical studies have shown that low-dose PDT regimens can induce anti-tumor immunity (Shams et al., 2015). The oxidative stress induced by PDT could incite a strong acute inflammatory response observed as localized edema at the target site (Dougherty, 2002). Inflammatory response promotes local healing with the restoration of normal tissue function after the disruption of homeostasis and the removal of damaged cells. PDT treatment stimulates the regulation of the release of pro- or anti-inflammatory cytokines by macrophages (Korbelik and Krosl, 1994; Lynch et al., 1989). Furthermore, the lysates generated after PDT can activate dendritic cells and increase the ability to stimulate T-cells, which play an essential role in the development of a cellular immune response (Gollnick et al., 2002). Finally, removal of inflammatory cells or inhibition of their activity demonstrated significant reducing of the total therapeutic effect of PDT (Agostinis et al., 2011).
However, PDT-mediated immune response depends on the many factors, such as the type PS, features of pharmacokinetic and location within tumor and surrounding cells (especially immune cells), the area treated and treatment regimen (e.g., drug-light interval (DLI) and settings of irradiation) (Kousis et al., 2007; Reinhard et al., 2015). So, the regimens that result in rapid cell death (within 1h of treatment) and maximal tissue damage were demonstrated to cause minimal acute inflammation, presumably because of the vascular shutdown, which would prevent neutrophil infiltration and systemic release of cytokines. In contrast, regimens that cause diffuse tumor damage should allow neutrophil infiltration followed by induction of expression and release of inflammatory mediators critical for enhancement of anti-tumor immunity (Henderson et al., 2004). In summary, the cytotoxic effects mediated by PDT are strongly depended on the localization of PSs. Thus, the distribution processes of PSs could be considered as key factors of successful PDT alongside the PS photophysical properties and treatment regimen.
As it was mentioned above, the contribution of tumor eradication mechanisms as well as PDT efficiency strongly depends on the settings of irradiation and light source used. To date, with various light sources including lasers, incandescent light, laser emitting diodes, and daylight were applied for PDT (Agostinis et al., 2011; Fitzmaurice and Eisen, 2016). Nowadays, the diode lasers are widely used due to their compact size, cost-effectiveness, automated dosimetry and calibration, simple installation and long operational life. Light-emitting diodes are alternative light sources with relatively narrow spectral bandwidths and high fluence rates (Juzeniene et al., 2006). To deliver the light to the hard-to-reach places (e.g., urinary bladder and the digestive tract) lasers beams are coupled into fibers with diffusing tips. Moreover, there are already in the market inflatable balloons, covered on the inside with a strongly scattering material and formed to fit an organ (Beyer, 1996).
No single light source is ideal for all PDT indications, even with the same PS. The choice of the light source should, therefore, be based on PS absorption (fluorescence excitation and action spectra), disease (location, size of lesions, accessibility, and tissue characteristics), cost and size. Preferably, the light should efficiently penetrate the tissue to be delivered across the whole tumor mass. Due to light scattering and absorption of tissue chromophores, shorter wavelengths (< 600 nm) have less tissue penetration and are mostly absorbed. Blue light penetrates only several hundred micrometers, whereas red and infrared radiations penetrate more deeply (up to 1 cm) (Figure 1.5). The most effective for the human organism is wavelength above 630 nm (Castano et al., 2004). The light in this range can penetrate human tissue on the appropriate required level, which provides the success of the therapy. More precise tuning of laser excitation wavelength is performed for certain PSs according to its action spectra. However, the longer the excitation wavelength, the less energy the PS will absorb, and the longer the exposure time will be (Henderson et al., 2004). The clinical efficacy of PDT is dependent on complex dosimetry: total light dose, light exposure time, and light delivery mode (single vs fractionated or even metronomic).
PS is the most significant component having an influential impact on the PDT process. Appropriate choice of the compound is a guarantee of success. The main characteristics of PS are selectivity for tumor cells, formation a long-lived triplet excited state in reaction, activation with wavelength appropriate for tissue and high chemical purity (Robertson et al., 2009; Zhang et al., 2018). To date, more than 400 compounds are known as PSs, which are used in various fields such as medicine, cosmetics, chemicals, etc. (Robertson et al., 2009). The most significant groups of the substances are tetrapyrrolic compounds (porphyrins, chlorins, phthalocyanines, etc) (Josefsen and Boyle, 2012; O’Connor et al., 2009), non-porphyrin PS (5-aminolevulinic acid, xanthenes, phenothiazines, triarylmethanes, curcumin, hypericin) (Craig et al., 2015; Kübler, 2005), and other classes of compounds such as synthetic dyes (e.g., phenothiazinium, squaraine), transition metal complexes, and natural products (e.g., hypericin, riboflavin, curcumin) (Abrahamse and Hamblin, 2016).
To be considered as an efficient PS, the substance concurrently should match the following criteria (Pushpan et al., 2002):
• Strong absorption (high extinction coefficient, ε) of light in the red or far-red region;
• The high QY of ROS generation;
• Low photobleaching (self-oxidation) rate;
• Low dark toxicity for both photosensitizer and its metabolites;
• Selectively accumulates in tumor tissues and cellular compartments;
• Rapid clearance from healthy tissues upon completion of treatment.
Additionally, PS should be in a pure form (known chemical composition) and stable compound with relatively easy and low coast synthesis. From a clinical point of view, the substance should be commercially available, reliable, and pain-free upon activation, acceptable for outpatient treatment with versatile and easy administration.
PDT drugs are generally classified as first, second or third generation PSs. Сurrently, only a few PSs, have official approval worldwide and are being used clinically for cancer treatments (Table 1.1). Photofrin® and Photogem®, the first-generation PDT sensitizers, are porphyrin-based PS which have been for a very long time the only PSs used in clinical PDT. They consist of a mixture of monomers, dimers, and oligomers of hematoporphyrin derivatives. However, due to the complex composition, non-optimal photophysical properties, lack of tumor selectivity, poor bioavailability and prolong patient photosensitivity (poor clearance) their application in PDT was limited (Macdonald and Dougherty, 2001).
Table of contents :
CHAPTER I. INTRODUCTION
1. Photodynamic therapy
1.1. History and basic principles
1.2. Basis of PDT therapy
1.3. Mechanisms of PDT action
1.4. Light sources
2.1. General properties and usage
2.2. Uptake and localization
2.3. Biodistribution and pharmacokinetic properties
2.4. Nanoscale delivery systems
3. Drug-in-cyclodextrin-in-liposome (DCL) nanoplatform
3.2. Cyclodextrin-based inclusion complexes
CHAPTER II. CD INCLUSION COMPLEXES
4. Characterization of inclusion complex formation
5. Development of spectral techniques for monitoring the distribution of inclusion complexes
6. Nanoshuttle mechanism of mTHPC delivery
CHAPTER III. MTHPC-IN-CYCLODEXTRINS-IN-LIPOSOME
7. Development and optimization of mTHPC-DCL
8. Double loaded mTHPC-DCL