me as a researcher.

My sincere thanks also goes to my advisor Dr. Lina Bolotine, for giving me the opportunity to join their team as intern, for her time and for long discussions of the research problems as well as of the research articles in the process of writing, and for directing me to the exciting world of the European science. I could not have imagined having a better advisor and mentor for my Ph.D study.

My research benefited significantly from the help of Dr. Henri-Pierre Lassalle, for the continuous support of my Ph.D study and related research. His guidance helped me continuously during my research and writing of this thesis.

I am indebted to Prof. Sandy MacRobert for accepting to review my thesis manuscript. His big experience in bio-and photophysics, formidable commentaries and questions has greatly contributed to the finalization of this work. I thank you for your keen interest in this thesis, and for giving me a wonderful opportunity to discuss it with you. I am very grateful to Dr. Andrey Klymchenko for reviewing my thesis manuscript. It was an honor for me to have my work evaluated by this distinguished scientist, and I have profited greatly from his insightful and profound questions on the work. I would like to thank the rest of my thesis committee: Prof. Geneviève Ubeaud-Sequier, Prof. Vladimir Kostyuk, and Dr. Ariane Boudier, for their kind and gracious acceptance to take part in the thesis defense as a member of the jury, for their insightful comments and fundamental discussion that we have had during the defense. phase evaporation and (D) dehydration-rehydration methods [START_REF] Gharib | Liposomes incorporating cyclodextrin-drug inclusion complexes: Current state of knowledge[END_REF] ..............58

GENERAL INTRODUCTION

Photodynamic therapy (PDT) is an alternative cancer treatment which offers a more targeted and less invasive treatment regimen compared to traditional modalities. Over the last decades, it has been demonstrated that the combination of PDT drugs (photosensitizers, PSs) with nanomaterial platforms enable precise delivery of PS to the target tissues improving the PDT effectiveness. Since the most effective PSs tend to be insoluble hydrophobic molecules with a high tendency to aggregate, their encapsulation into nanocarriers could improve their pharmacokinetic properties. Additionally, nanoparticles provide the opportunity for active targeting, control release and multimodal combined therapies.

Temoporfin (mTHPC, medicinal product name: Foscan ® ), is one of the most potent clinically approved PS. However, its poor solubility in aqueous medium caused several complications of its administration. Thus, many attempts were done in the application of nanoscale delivery systems for temoporfin delivery. Over the last 25 years, many nanoplatforms such as host-guest inclusion complexes, lipid-and polymer-based nanoparticles and carbon nanotubes were loaded with temoporfin. In particular, liposomal formulation offers an excellent opportunity to achieve selective drug targeting which is expected to optimize the pharmacokinetic parameters, prevent local irritation, and reduce drug toxicity. While, cyclodextrin-based inclusion complexes provide alteration of temoporfin biodistribution.

To improve the transport of mTHPC to target tissue and to strengthen its intra-tissue accumulation, the coupling of two these independent delivery systems was proposed. Thus, the main objective of present work was to develop and optimize temoporfin-loaded drug-incyclodextrin-in-liposomes (DCL) nanoconstruct with the aim to achieve synergetic combination of both delivery systems for efficient delivery of mTHPC in the target tumor tissue. With this purpose, the complexation of mTHPC with CDs and behavior of these complexes in 3D tumor tissue in vitro model have been studied. In addition, new approaches for analyzing mTHPC release from CDs have been developed to characterize the structure of mTHPC-DCL. In fine, the composition of developed mTHPC-DCL have been optimized

according to the studies on 2D and 3D in vitro tumor models.

CHAPTER I. INTRODUCTION 1. Photodynamic therapy 1.1. History and basic principles

Photodynamic therapy (PDT) employs a combination of photosensitizer, light, and molecular oxygen to selectively target cells like tumor cells via cytotoxic activity [START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF]. Since the Ancient, Indian and Chinese civilizations, PDT has now been in application for the treatment of various diseases including vitiligo, rickets, psoriasis, and skin cancer [START_REF] Daniell | A history of photodynamic therapy[END_REF]. Nowadays, PDT has been clinically utilized for more than 25 years as a cancer treatment. In 1972 Diamond et al. published the first medical study on PDT for cancer and introduced hematoporphyrin as a powerful agent for the selective lightmediated killing of glioma cells in vitro and tumors in vivo [START_REF] Diamond | PHOTODYNAMIC THERAPY OF MALIGNANT TUMOURS[END_REF]. In 1960 when new generations of less toxic drugs were developed for diagnosis and treatment of solid tumors, PDT became a hot scientific area attracting many researchers to the field.

Already in 1999, PDT was the first drug-device combination approved by the US Food and Drug Administration (FDA) to treat pre-cancerous skin lesions of the face or scalp.

Nowadays, numerous clinical studies show potential of PDT, especially in multimodal approach, while preclinical studies indicate there still is a room for improvement on multiple fronts [START_REF] Van Straten | Oncologic Photodynamic Therapy: Basic Principles, Current Clinical Status and Future Directions[END_REF].

The action of PDT consists in the interaction of three principal components: (i) a lightabsorbing compound that initiates the photochemical reaction, namely photosensitizer (PS),

(ii) the light of a specific wavelength and (iii) tissue/molecular oxygen [START_REF] Dolmans | Photodynamic therapy for cancer[END_REF].

All together they initiate a photochemical reaction that culminates in the generation of toxic reactive oxygen species (ROS), while individually none of these components is toxic. The ROS can rapidly cause significant toxicity leading to cell death via apoptosis or necrosis [START_REF] Castano | Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death[END_REF]. PDT is a complex procedure which includes the following steps (Figure 1.1). Firstly, PS is administrated topically, locally or systemically (e.g., intravenously, IV). Then, PS redistributes into the body and selectively accumulates into the target tissue.

Afterward, the localized illumination of the tumor region is carried out with a light source at emission wavelength corresponding to PS absorbance spectrum. Absorption of this light by tumor-localized sensitizer leads to ROS generation, which finally results in the destruction of malignant tissue [START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF]. There are three interconnected mechanisms of PDT-mediated tissue destruction: the direct killing of target cells, damage to the vasculature, and induction of an inflammatory reaction that can lead to the immune response [START_REF] Castano | Photodynamic therapy and antitumour immunity[END_REF]. It worth noting that collateral PDT-damage to healthy tissue can be minimized by the selective PS accumulation in the target tissue, as well as by delivering the light in a spatially confined and focused manner. This concept of dual selectivity is one of the chief benefits of PDT compared to other treatment modalities.

Basis of PDT therapy

The biological effects of PDT are a consequence of basic photophysical and photochemical reactions involving PS, light, and oxygen, which can be illustrated using a modified Jablonski diagram (Figure 1.2). Most PSs initially are in the ground state (usually singlet) that corresponds to two electrons with opposite spins in the low energy molecular orbital. The initial step of all photoreactions is the absorption of a photon with appropriate wavelength by PS molecule. Following the absorption of the photons, one of the electrons is boosted into a high-energy orbital (Sn, where n depending on the PS and excitation wavelength used) but keeps its spin (excited singlet state). Excited PS molecule will finally fall from a higher excited state Sn to the first excited singlet state S1 by internal conversion.

This process is very fast and takes a fraction of a nanosecond between 10 -14 and 10 -11 s, therefore we consider only S1 on the diagram. The upper excited states may be filled only under the special conditions of multiphoton absorption (short pulse, high intensities of irradiation) leading to the complex photophysical and photochemical processes [START_REF] Smith | An Efficient Oxygen Independent Two-Photon Photosensitization Mechanism[END_REF]. In turn, this singlet state is very unstable and can rapidly return to the ground state level S0 emitting this excess energy as light (fluorescence) and/or heat (internal conversion).

The probability of emitting the energy through the fluorescence pathway is described by the value of quantum yield (QY) of fluorescence as a ratio between emitted photons to the total absorbed photons. It worth noting that fluorescence is widely used for PS detection in photodiagnosis applications [START_REF] Matoba | Clinical application of photodynamic diagnosis and photodynamic therapy for gynecologic malignant diseases: A review[END_REF]. Reprinted from [START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF].

The excited singlet PS' state may also undergo as intersystem crossing whereby the spin of the excited electron inverts forming an excited triplet state (T1). This triplet state has electron spins parallel and is more stable than singlet one: the lifetime of T1 is τ > 10 -4 s, while τ ~ 10 -9 s for the singlet S1 state. As well as for excited singlet state, PS molecules tend to return to the ground state level S0 from triplet state T1 by emitting a photon (phosphorescence) or undergoing non-radiative intersystem crossing followed by vibrational relaxation. The relatively long lifetime of excited triplet state (T1) increases the probability of a reaction with neighboring substrate molecules making possible the collisional transfer of energy to surrounding oxygen molecules.

There are two various types of photochemical reactions which simultaneously occur during PDT (Figure 1.2). Type I reaction involves the direct reaction of PS with an organic molecule resulting in forming a radical by a hydrogen atom or electron transfer [START_REF] Henderson | How does photodynamic therapy work?[END_REF]. Type I reactions result in the generation of superoxide anion radical (O2 •-), hydrogen peroxide (H2O2), oxidant hydroxyl-radical (HO • ), etc., which could subsequently participate in different kinds of oxidation reactions.

Type II photochemistry is much simpler. In Type II process, the reaction proceeds via energy transfer from the excited triplet-state PS to the molecular oxygen in its triplet state ( 3 O2). As a result, highly reactive singlet oxygen ( 1 O2) is generated. Most PSs are believed to operate via a Type II rather than Type I mechanism. However, only PSs which possessed an energy gap between the ground state and the excited triplet state higher than the energy needed to excite oxygen into its excited singlet state (94.29 kJ/mol according to van Lier & Spikes, 1989) can generate singlet oxygen. The energy limit of 94.29 kJ/mol corresponds to the photon wavelength of 1270 nm, however only PSs which absorb photons with wavelengths longer than 800 nm could generate 1 O2 [START_REF] Juzeniene | Biophysical aspects of photodynamic therapy[END_REF].

Mechanisms of PDT action

The distance of photodynamic damage is limited to the immediate intracellular localization of the PS [START_REF] Moan | Intracellular localization of photosensitizers[END_REF] due to the short lifetime 1 O2 (approximately 10-320 ns) and limited diffusion in biological media (up to 100 nm) [START_REF] Dysart | Characterization of Photofrin photobleaching for singlet oxygen dose estimation during photodynamic therapy of MLL cellsin vitro[END_REF].

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 [START_REF] Jensen | Effect of overall charge and charge distribution on cellular uptake, distribution and phototoxicity of cationic porphyrins in HEp2 cells[END_REF].

Direct tumor cell death effects

PS accumulates in various intracellular compartments and can induce multiple cell death subroutines at the cellular level (Figure 1. 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 [START_REF] Agostinis | Regulatory pathways in photodynamic therapy induced apoptosis[END_REF][START_REF] Almeida | Intracellular signaling mechanisms in photodynamic therapy[END_REF]. It was identified that among all subcellular structures mitochondria plays the most critical role in the initiation of PDT-mediated apoptosis [START_REF] Castano | Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death[END_REF][START_REF] Kessel | Photodynamic therapy: a mitochondrial inducer of apoptosis[END_REF]. 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 [START_REF] Oleinick | The role of apoptosis in response to photodynamic therapy: what, where, why, and how[END_REF].

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 [START_REF] Dewaele | ROS-mediated mechanisms of autophagy stimulation and their relevance in cancer therapy[END_REF]. 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 [START_REF] Castano | Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death[END_REF]. Autophagy is upregulated in response to extra-or intracellular stress and signals such as starvation, growth factor deprivation, ER stress and pathogen infection [START_REF] Kessel | Chapter 1 Initiation of Autophagy by Photodynamic Therapy[END_REF]. Along with the mentioned cell death mechanisms, the subroutines such as mitotic cell death [START_REF] Castedo | Cell death by mitotic catastrophe: a molecular definition[END_REF], cathepsin-mediated lysosomal death pathway [START_REF] Leist | Triggering of apoptosis by cathepsins[END_REF], programmed necrosis [START_REF] Bizik | Cell-cell contacts trigger programmed necrosis and induce cyclooxygenase-2 expression[END_REF][START_REF] Vanlangenakker | Molecular mechanisms and pathophysiology of necrotic cell death[END_REF] were also reported for PDT.

Mechanism of cell death depends on cell and PS type, treatment protocol, and photodynamic dose [START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF]. 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 [START_REF] Korbelik | Cellular levels of photosensitisers in tumours: the role of proximity to the blood supply[END_REF] as well as availability of oxygen [START_REF] Tromberg | In vivo tumor oxygen tension measurements for the evaluation of the efficiency of photodynamic therapy[END_REF] 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 [START_REF] Eberhard | Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies[END_REF][START_REF] Siemann | The Unique Characteristics of Tumor Vasculature and Preclinical Evidence for its Selective Disruption by Tumor-Vascular Disrupting Agents[END_REF]. Tumor vasculature is characterized by the vessels that are immature in nature with poorly developed, chaotic growing architecture and discontinuous endothelial linings [START_REF] Eberhard | Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies[END_REF]. 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 [START_REF] Senge | Platelets, photosensitizers, and PDT[END_REF]. It was demonstrated, that endothelial cells accumulate more PS and possess higher sensitivity to PDT compared to muscle cells [START_REF] West | A Comparison of the Sensitivity to Photodynamic Treatment of Endothelial and Tumour Cells in Different Proliferative States[END_REF]. Finally, the combination of vessel constriction, thrombus formation, and increased interstitial pressure leads to tumor tissue ischemia [START_REF] Fingar | Vascular damage after photodynamic therapy of solid tumors: a view and comparison of effect in pre-clinical and clinical models at the University of Louisville[END_REF][START_REF] Henderson | Tumor destruction and kinetics of tumor cell death in two experimental mouse tumors following photodynamic therapy[END_REF] 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 [START_REF] Korbelik | Induction of tumor immunity by photodynamic therapy[END_REF].

Photodynamic therapy

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 [START_REF] Oniszczuk | The potential of photodynamic therapy (PDT)-Experimental investigations and clinical use[END_REF]. To date, it was established that PDT could stimulate both innate immunity and adaptive immunity [START_REF] Castano | Photodynamic therapy and antitumour immunity[END_REF]. Preclinical studies have

shown that low-dose PDT regimens can induce anti-tumor immunity [START_REF] Shams | Development of photodynamic therapy regimens that control primary tumor growth and inhibit secondary disease[END_REF].

The oxidative stress induced by PDT could incite a strong acute inflammatory response observed as localized edema at the target site [START_REF] Dougherty | An update on photodynamic therapy applications[END_REF]. 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 [START_REF] Korbelik | Cellular levels of photosensitisers in tumours: the role of proximity to the blood supply[END_REF][START_REF] Lynch | Systemic immunosuppression induced by photodynamic therapy (PDT) is adoptively transferred by macrophages[END_REF]. 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 [START_REF] Gollnick | Generation of effective antitumor vaccines using photodynamic therapy[END_REF]. Finally, removal of inflammatory cells or inhibition of their activity demonstrated significant reducing of the total therapeutic effect of PDT [START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF].

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) [START_REF] Kousis | Photodynamic therapy enhancement of antitumor immunity is regulated by neutrophils[END_REF][START_REF] Reinhard | Photodynamic therapy as a new treatment modality for inflammatory and infectious conditions[END_REF]. 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 [START_REF] Henderson | Choice of oxygen-conserving treatment regimen determines the inflammatory response and outcome of photodynamic therapy of tumors[END_REF]. 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.

Light sources

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 [START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF][START_REF] Fitzmaurice | Daylight Photodynamic Therapy: What is Known and What is Yet to be Determined[END_REF].

Nowadays, the diode lasers are widely used due to their compact size, cost-effectiveness, automated dosimetry and calibration, simple installation and long operational life. Lightemitting diodes are alternative light sources with relatively narrow spectral bandwidths and high fluence rates [START_REF] Juzeniene | Biophysical aspects of photodynamic therapy[END_REF]. 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 [START_REF] Beyer | Systems for light application and dosimetry in photodynamic therapy[END_REF].

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 [START_REF] Castano | Mechanisms in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization[END_REF]. 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 [START_REF] Henderson | Choice of oxygen-conserving treatment regimen determines the inflammatory response and outcome of photodynamic therapy of tumors[END_REF].

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).

Photosensitizers

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 [START_REF] Robertson | Photodynamic therapy (PDT): A short review on cellular mechanisms and cancer research applications for PDT[END_REF][START_REF] Zhang | An updated overview on the development of new photosensitizers for anticancer photodynamic therapy[END_REF]. To date, more than 400 compounds are known as PSs, which are used in various fields such as medicine, cosmetics, chemicals, etc. [START_REF] Robertson | Photodynamic therapy (PDT): A short review on cellular mechanisms and cancer research applications for PDT[END_REF].

The most significant groups of the substances are tetrapyrrolic compounds (porphyrins, chlorins, phthalocyanines, etc) [START_REF] Josefsen | Unique diagnostic and therapeutic roles of porphyrins and phthalocyanines in photodynamic therapy, imaging and theranostics[END_REF][START_REF] O'connor | Porphyrin and Nonporphyrin Photosensitizers in Oncology: Preclinical and Clinical Advances in Photodynamic Therapy[END_REF], nonporphyrin PS (5-aminolevulinic acid, xanthenes, phenothiazines, triarylmethanes, curcumin, hypericin) [START_REF] Craig | Photosensitisersthe progression from photodynamic therapy to anti-infective surfaces[END_REF][START_REF] Kübler | Photodynamic therapy[END_REF], and other classes of compounds such as synthetic dyes (e.g., phenothiazinium, squaraine), transition metal complexes, and natural products (e.g., hypericin, riboflavin, curcumin) [START_REF] Abrahamse | New photosensitizers for photodynamic therapy[END_REF].

To be considered as an efficient PS, the substance concurrently should match the following criteria [START_REF] Pushpan | Porphyrins in photodynamic therapy -a search for ideal photosensitizers[END_REF]:

• 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 [START_REF] Macdonald | Basic principles of photodynamic therapy[END_REF]. Compared with the first generation, the composition and structure of the secondgeneration PSs are definite; the photosensitivity, absorption spectrum and tissue selectivity have significantly been improved. As a result, the variety of new PS molecules such as metalloporphyrins or texaphyrins (Lutrin ® and Lutex ® ) [START_REF] Magda | Motexafin gadolinium: A novel redox active drug for cancer therapy[END_REF][START_REF] Young | Lutetium Texaphyrin (PCI-0123): A Near-Infrared, Water-Soluble Photosensitizer[END_REF]; benzoporphyrins (Visudyne ® ) [START_REF] Battaglia Parodi | Photosensitizers and Photodynamic Therapy: Verteporfin[END_REF]; chlorins (Fotolon ® , Radachlorin ® , Photodithazine ® , Aptocine ® , Foscan ® ) [START_REF] Kochneva | Photosensitizer Radachlorin®: Skin cancer PDT phase II clinical trials[END_REF][START_REF] Miki | Effect of talaporfin sodium-mediated photodynamic therapy on cell death modalities in human glioblastoma T98G cells[END_REF][START_REF] Senge | Temoporfin (Foscan®, 5,10,15,20-tetra(mhydroxyphenyl)chlorin)--a second-generation photosensitizer[END_REF][START_REF] Shliakhtsin | Pharmacokinetics and biodistribution of Photolon® (Fotolon®) in intact and tumor-bearing rats[END_REF][START_REF] Wen | The effects of photodynamic therapy with Photodithazine on HPV 16 E6/E7 associated cervical cancer model[END_REF]; porphycenes [START_REF] Guardiano | Tetra-n-propylporphycene as a tumour localizer: pharmacokinetic and phototherapeutic studies in mice[END_REF][START_REF] Szeimies | 9-Acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)porphycene (ATMPn), a novel photosensitizer for photodynamic therapy: uptake kinetics and intracellular localization[END_REF]; pheophorbides (Tookad ® ) [START_REF] Taneja | Final Results of a Phase I/II Multicenter Trial of WST11 Vascular Targeted Photodynamic Therapy for Hemi-Ablation of the Prostate in Men with Unilateral Low Risk Prostate Cancer Performed in the United States[END_REF][START_REF] Trachtenberg | Vascular-targeted photodynamic therapy (padoporfin, WST09) for recurrent prostate cancer after failure of external beam radiotherapy: a study of escalating light doses[END_REF]; purpurins (Purlytin ® ) [START_REF] Forsyth | Synthesis and structure of a 5,15-bis(4-pyridyl)purpurin[END_REF]; phthalocyanines (Photosens ® ) [START_REF] Miller | Photodynamic therapy with the phthalocyanine photosensitizer Pc 4: The case experience with preclinical mechanistic and early clinical-translational studies[END_REF][START_REF] Muehlmann | Aluminiumphthalocyanine chloride nanoemulsions for anticancer photodynamic therapy: Development and in vitro activity against monolayers and spheroids of human mammary adenocarcinoma MCF-7 cells[END_REF][START_REF] Trushina | Photosens PDT at the treatment of virus-associated precancer and non-invasive cervical cancer[END_REF], protoporphyrin-IX precursors (Hexvix ® , Metvix ® and Levulan ® ) [START_REF] Kennedy | Photodynamic therapy with endogenous protoporphyrin: IX: Basic principles and present clinical experience[END_REF][START_REF] Sachar | Protoporphyrin IX: the Good, the Bad, and the Ugly[END_REF] have been developed and approved for clinical applications (Figure 1.6). Moreover, along with clinically approved PSs, phenothiazines (methylene blue and toluidine blue) [START_REF] Tardivo | Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications[END_REF]; cyanines (merocyanine540, indocyanine green) [START_REF] Delaey | A comparative study of the photosensitizing characteristics of some cyanine dyes[END_REF][START_REF] Shirata | Near-infrared photothermal/photodynamic therapy with indocyanine green induces apoptosis of hepatocellular carcinoma cells through oxidative stress[END_REF]; dipyrromethenes (BODIPYs) [START_REF] Awuah | Boron dipyrromethene (BODIPY)-based photosensitizers for photodynamic therapy[END_REF]; hypericin [START_REF] Garg | Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin[END_REF]; xanthenes (RoseBengal) [START_REF] Panzarini | Rose Bengal Acetate PhotoDynamic Therapy (RBAc-PDT) Induces Exposure and Release of Damage-Associated Molecular Patterns (DAMPs) in Human HeLa Cells[END_REF] and curcumin [START_REF] Ellerkamp | Photodynamic Therapy Potentiates the Effects of Curcumin on Pediatric Epithelial Liver Tumor Cells[END_REF] have been also considered as good second-generation PSs for experimental PDT.

However, the second generations PSs are still poorly selective for tumors and require the improvements in their delivery to the target site due to the hydrophobic nature of many of them. Third-generation PS are now being developed to improve the PDT outcomes using nanotechnology approaches. Considerable efforts have been devoted to developing specific carriers for delivery of PSs in order to improve the selectivity of PS uptake in the target tissue and to avoid phototoxicity to healthy tissues, such as skin [START_REF] Marchal | Anticancer Drug Delivery: An Update on Clinically Applied Nanotherapeutics[END_REF]. The first and second-generation PSs, covalently attached to various biological modifiers like oligosaccharides, lipoproteins, antibodies and amino acids, or formulated into drug delivery systems like liposomes, polymeric nanoparticles, cyclodextrins, and emulsions are classified as third-generation PSs.

Of particular interest for this study is temoporfin (Foscan ® ), which is an extremely potent PS (approximately 100 times more photoactive than Photofrin ® ) (Mitra and Foster, 2005;[START_REF] Senge | Temoporfin (Foscan®, 5,10,15,20-tetra(mhydroxyphenyl)chlorin)--a second-generation photosensitizer[END_REF]. Senge MO raised the question on the ways of development of third-generation PS on the example of temoporfin [START_REF] Senge | mTHPC -A drug on its way from second to third generation photosensitizer? Photodiagnosis Photodyn[END_REF], the properties of which will be described in detail in the next chapter.

Meta-tetra(hydroxyphenyl)chlorin

General properties and usage

5,10,15,20-Tetra(m-hydroxyphenyl)chlorin (mTHPC, Figure 2.1) was discovered and synthesized by the group of professor Raymond Bonnett in 1989 as a second-generation PS [START_REF] Berenbaum | meso-Tetra(hydroxyphenyl)porphyrins, a new class of potent tumour photosensitisers with favourable selectivity[END_REF][START_REF] Bonnett | Hydroporphyrins of the meso-tetra(hydroxyphenyl)porphyrin series as tumour photosensitizers[END_REF]. mTHPC (generic name temoporfin, proprietary name Foscan ® ) possesses pure-compound preparation, hence efficient light tissue penetration, certain selectivity of tumor uptake, a low administration dose and attractive photophysical characteristics [START_REF] Senge | Temoporfin (Foscan®, 5,10,15,20-tetra(mhydroxyphenyl)chlorin)--a second-generation photosensitizer[END_REF]. , 2005;[START_REF] Bonnett | Hydroporphyrins of the meso-tetra(hydroxyphenyl)porphyrin series as tumour photosensitizers[END_REF][START_REF] Bonnett | Photophysical properties of 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (m-THPP), 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (m-THPC) and 5,10,15,20-tetrakis(m-References hydroxyphenyl)bacteriochlorin (m-THPBC): a comparative study[END_REF] and the main characteristics are summarized in Table 2.1.

Temoporfin possesses an extinction coefficient of 29,600 M -1 cm -1 in the red region of spectra (650 nm). Furthermore, triplet state QY formation is 0.89, hence QY singlet oxygen is 0.43 in aerated methanol [START_REF] Bonnett | Photophysical properties of 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (m-THPP), 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (m-THPC) and 5,10,15,20-tetrakis(m-References hydroxyphenyl)bacteriochlorin (m-THPBC): a comparative study[END_REF]. There are however compounds superior to it in terms of in certain photophysical properties. For example, bacteriochlorin-type TOOKAD ® exhibits extinction coefficient 90,000 M -1 cm -1 at 763 nm [START_REF] Koudinova | Photodynamic therapy with Pdbacteriopheophorbide (TOOKAD): Successful in vivo treatment of human prostatic small cell carcinoma xenografts[END_REF], while tin ethyl etiopurpurin (Purlytin TM ), possess the highest quantum yield of singlet oxygen ΦΔ= 0.7 [START_REF] Pogue | Photophysical Properties of Tin Ethyl Etiopurpurin I (SnET2) and Tin Octaethylbenzochlorin (SnOEBC) in Solution and Bound to Albumin[END_REF]. Nevertheless, mTHPC is considered as one of the most potent PSs due to both a very low drug dose (0.15 mg kg -1 ) and light intensity (order of 10 J cm -2 ) applied in clinical PDT.

So, the resulting total PDT dose (light dose × PS dose) is more than 100 times lower compared to other clinically approved first-generation PSs, such as haematoporphyrin derivatives [START_REF] Savary | Photodynamic therapy for early squamous cell carcinomas of the esophagus, bronchi, and mouth with m-tetra (hydroxyphenyl) chlorin[END_REF][START_REF] Savary | Photodynamic therapy for early squamous cell carcinomas of the esophagus, bronchi, and mouth with m-tetra (hydroxyphenyl) chlorin[END_REF] and Photofrin [START_REF] Ball | The high photoactivity of m-THPC in photodynamic therapy. Unusually strong retention of m-THPC by RIF-1 cells in culture[END_REF]. In 2001, mTHPC was approved in the EU and used as a solvent-based formulation (Foscan ® ; Biolitec Research GmbH, Jena, Germany) for the palliative treatment of head and neck cancers [START_REF] Senge | Temoporfin (Foscan®, 5,10,15,20-tetra(mhydroxyphenyl)chlorin)--a second-generation photosensitizer[END_REF]. In addition, mTHPC has been successfully utilized for the clinical treatment of other types of cancers such as early squamous cell carcinoma [START_REF] Jerjes | Photodynamic therapy outcome for T1/T2 N0 oral squamous cell carcinoma[END_REF][START_REF] De Visscher | mTHPC-mediated photodynamic therapy of early stage oral squamous cell carcinoma: a comparison to surgical treatment[END_REF], basal cell carcinoma [START_REF] Betz | Long-term outcomes following Foscan®-PDT of basal cell carcinomas[END_REF], biliary tract carcinoma [START_REF] Wagner | Temoporfin improves efficacy of photodynamic therapy in advanced biliary tract carcinoma: A multicenter prospective phase II study[END_REF], prostate [START_REF] Swartling | System for interstitial photodynamic therapy with online dosimetry: first clinical experiences of prostate cancer[END_REF][START_REF] Swartling | Online dosimetry for temoporfin-mediated interstitial photodynamic therapy using the canine prostate as model[END_REF], pancreatic cancer [START_REF] Bown | Photodynamic therapy for cancer of the pancreas[END_REF] and nonmelanoma skin cancers [START_REF] Horlings | mTHPC mediated, systemic photodynamic therapy (PDT) for nonmelanoma skin cancers: Case and literature review[END_REF]. Furthermore, there are reports in preclinical models on mTHPC-PDT application in antibacterial therapy [START_REF] Ossmann | Photodynamic killing of Enterococcus faecalis in dentinal tubules using mTHPC incorporated in liposomes and invasomes[END_REF], rheumatoid arthritis [START_REF] Hansch | Photodynamic treatment as a novel approach in the therapy of arthritic joints[END_REF], ulcerate colitis and prevention of colitisassociated carcinogenesis [START_REF] Reinhard | Photodynamic therapy as a new treatment modality for inflammatory and infectious conditions[END_REF]. Taking into account moderate photophysical characteristics, it was supposed that the potency of mTHPC-PDT should mainly rely on the features of mTHPC behavior during its distribution in biological systems.

Uptake and localization

First of all, the biological behavior of drugs is determined by the physical-chemical properties of PS. Temoporfin molecules have a highly hydrophobic nature characterized by octanol-water partition coefficient (logP) of 8.8, according to Pubchem database (PubChem), thus forming large aggregates in an aqueous environment [START_REF] Kruijt | Monitoring interstitial m-THPC-PDT in vivo using fluorescence and reflectance spectroscopy[END_REF][START_REF] Tikhomirov | Investigation of aggregates of dyes by the method of resonance light scattering: correction of spectra[END_REF]. As a rule, mTHPC is administrated intravenously and formation of non-photoactive aggregates in blood flow after its injection distorts the pharmacokinetics and reduces drug bioavailability. Molecular aggregates of mTHPC slowly dissociates in the presence of serum proteins redistributing PS to the lipoproteins or cellular membranes due to the high lipophilicity of mTHPC [START_REF] Melnikova | Subcellular localization of meta-tetra (hydroxyphenyl) chlorin in human tumor cells subjected to photodynamic treatment[END_REF][START_REF] Sasnouski | Investigation of Foscan interactions with plasma proteins[END_REF][START_REF] Triesscheijn | The pharmacokinetic behavior of the photosensitizer meso-tetrahydroxyphenyl-chlorin in mice and men[END_REF]. According to the literature data, about 95% of mTHPC in plasma is associated with high-density lipoproteins (HDL) and low-density lipoproteins (LDL), and the binding with HDL is twice higher than that with LDL [START_REF] Reshetov | Interaction of liposomal formulations of meta-tetra(hydroxyphenyl)chlorin (temoporfin) with serum proteins: protein binding and liposome destruction[END_REF][START_REF] Triesscheijn | The pharmacokinetic behavior of the photosensitizer meso-tetrahydroxyphenyl-chlorin in mice and men[END_REF].

However, as compared with other hydrophobic drugs, the kinetics of mTHPC redistribution between serum proteins and/or other biomembrane is very slow [START_REF] Sasnouski | Investigation of Foscan interactions with plasma proteins[END_REF][START_REF] Sasnouski | Redistribution of Foscan® from plasma proteins to model membranes[END_REF]. The redistribution process takes hours, thus making intracellular uptake of mTHPC very sluggish. For instance, time-dependent mTHPC accumulation in A431 human epidermoid carcinoma cells reaches a plateau only at 24 h [START_REF] Berlanda | Comparative in vitro study on the characteristics of different photosensitizers employed in PDT[END_REF]. High lipophilicity of mTHPC also determines a high affinity to lipid microenvironment of cellular membranes. It worth noting that temoporfin is strongly confined to model biomembranes [START_REF] Kachatkou | Unusual photoinduced response of mTHPC liposomal formulation (Foslip)[END_REF], demonstrating tight binding in the cells without remarkable release [START_REF] Ball | The high photoactivity of m-THPC in photodynamic therapy. Unusually strong retention of m-THPC by RIF-1 cells in culture[END_REF][START_REF] Berlanda | Comparative in vitro study on the characteristics of different photosensitizers employed in PDT[END_REF]Mitra and Foster, 2005). Overall, the cellular uptake of mTHPC could be affected by various factors. As discussed above, due to the high affinity of mTHPC to lipoproteins, HDL-mediated endocytosis was proposed as the main mode of temoporfin transport into the cells [START_REF] Sasnouski | Redistribution of Foscan® from plasma proteins to model membranes[END_REF]. At the same time, tumor cells could internalize mTHPC in its aggregated form, followed by slow monomerization [START_REF] Rezzoug | Parameters Affecting Photodynamic Activity of Foscan® or Meta-tetra(hydroxyphenyl)chlorin (mTHPC) In Vitro and In Vivo[END_REF], attested by fluorescence lifetime imaging microscopy data [START_REF] Lassalle | Fluorescence imaging of Foscan® and Foslip in the plasma membrane and in whole cells[END_REF]. In addition, photosensitivity of cells increased with decreasing pH, while uptake of mTHPC in tumor cells has been identified as pH-independent in the range of pH 6.8-7.8 [START_REF] Ma | Photochemotherapy of tumours with mesotetrahydroxyphenyl chlorin is pH dependent[END_REF]. Overall, all these factors lead to the heterogeneity of mTHPC distribution in the tumor tissue. Indeed, in vitro studies in multicellular tumor spheroid model demonstrated that mTHPC did not penetrate deeper than 50 µm and was localized only in the peripherical cell layers of spheroids [START_REF] Marchal | Necrotic and apoptotic features of cell death in response to Foscan photosensitization of HT29 monolayer and multicell spheroids[END_REF]Millard et al., 2018;Mitra and Foster, 2005;Mitra et al., 2005).

As have been described above, the mechanism of PDT cell death strongly depend on the PS intracellular localization [START_REF] Castano | Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death[END_REF]. The analysis of preferential sites of mTHPC localization in cells demonstrated the presence of PS in cellular organelles, except perinuclear space [START_REF] Melnikova | Subcellular localization of meta-tetra (hydroxyphenyl) chlorin in human tumor cells subjected to photodynamic treatment[END_REF]. [START_REF] Teiten | Endoplasmic reticulum and Golgi apparatus are the preferential sites of Foscan localisation in cultured tumour cells[END_REF] reported preferential mTHPC accumulation in the Golgi apparatus and endoplasmic reticulum in MCF-7 human adenocarcinoma cells. On the other hand, mitochondrial damage and cytochrome c release after mTHPC-PDT have been described for various cancer cells [START_REF] Marchal | Modality of cell death induced by Foscan-based photodynamic treatment in human colon adenocarcinoma cell line HT29[END_REF][START_REF] Yow | Cellular uptake, subcellular localization and photodamaging effect of temoporfin (mTHPC) in nasopharyngeal carcinoma cells: comparison with hematoporphyrin derivative[END_REF]. An apoptotic response after mTHPC-PDT in vitro has been observed by Kessel due to the mitochondrial damage and subsequent activation of caspase cascade caused by the release of cytochrome c [START_REF] Kessel | Transport and localisation of m-THPC in vitro[END_REF]. Although mitochondria are not affected directly, apoptotic signals originating in the endoplasmic reticulum and/or Golgi apparatus converge on mitochondria triggering afterward apoptotic cell death [START_REF] Marchal | Relationship between subcellular localisation of Foscan and caspase activation in photosensitised MCF-7 cells[END_REF].

Photoinduced mode of cell death varies widely depending on the cell type. Indeed, mTHPC-PDT in human colon cancer cells was shown to be a strong inducer of necrosis with a negligible percentage of apoptotic cells [START_REF] Marchal | Necrotic and apoptotic features of cell death in response to Foscan photosensitization of HT29 monolayer and multicell spheroids[END_REF]. Alternatively, the recent study of mTHPC-PDT on other human cell lines (lung, oral cavity, esophagus, bladder and cervix) reported mainly apoptotic cellular death and autophagy after mTHPC-PDT with a minor role of necrosis [START_REF] Lange | Comparison of Cellular Death Pathways after mTHPC-mediated Photodynamic Therapy (PDT) in Five Human Cancer Cell Lines[END_REF].

Biodistribution and pharmacokinetic properties

The issues of mTHPC pharmacokinetics were studied in detail in various preclinical models. It was reported that temoporfin's half-life in plasma was estimated in a range of 26-45 h depending on the animal model [START_REF] Campbell | Tissue levels, histologic changes and plasma pharmacokinetics of meta-Tetra (hydroxyphenyl) chlorin (mTHPC) in the cat[END_REF][START_REF] Glanzmann | Pharmacokinetics of tetra(m-hydroxyphenyl)chlorin in human plasma and individualized light dosimetry in photodynamic therapy[END_REF][START_REF] Ronn | Human tissue levels and plasma pharmacokinetics of temoporfin (Foscan®, mTHPC)[END_REF][START_REF] Triesscheijn | The pharmacokinetic behavior of the photosensitizer meso-tetrahydroxyphenyl-chlorin in mice and men[END_REF]. It was demonstrated that the mean values for plasma drug concentrations were in good agreement with the injected dose of mTHPC.

Pharmacokinetics of radiolabeled mTHPC in tumor-bearing rats was described using a triexponential model with a half-time of 0.46, 6.91 and 82.5 h, respectively [START_REF] Jones | Photodynamic therapy effect of m-THPC (Foscan) in vivo: correlation with pharmacokinetics[END_REF].

At the same time, the pharmacokinetics in mice and humans is quite different [START_REF] Campbell | Tissue levels, histologic changes and plasma pharmacokinetics of meta-Tetra (hydroxyphenyl) chlorin (mTHPC) in the cat[END_REF][START_REF] Triesscheijn | The pharmacokinetic behavior of the photosensitizer meso-tetrahydroxyphenyl-chlorin in mice and men[END_REF][START_REF] Whelpton | Distribution of temoporfin, a new photosensitizer for the photodynamic therapy of cancer, in a murine tumor model[END_REF]. In humans the mean plasma concentration of mTHPC was constant at high level for several hours after IV administration [START_REF] Triesscheijn | The pharmacokinetic behavior of the photosensitizer meso-tetrahydroxyphenyl-chlorin in mice and men[END_REF]. The authors suggested that the prolonged plateau in drug concentrations in human plasma is the result of the formation of drug depot, probably in the vascular compartment immediately after IV injection. In addition, it was shown that neither drug distribution over the lipoproteins nor the metabolism of lipoproteins influenced plasma pharmacokinetics of mTHPC [START_REF] Triesscheijn | The pharmacokinetic behavior of the photosensitizer meso-tetrahydroxyphenyl-chlorin in mice and men[END_REF]. Recently, Jablonka and co-workers confirmed these suggestions simulating in silico pharmacokinetics and biodistribution of Foscan ® in humans assuming precipitated mTHPC in the injection site (drug depot) [START_REF] Jablonka | Advanced in silico modeling explains pharmacokinetics and biodistribution of temoporfin nanocrystals in humans[END_REF].

The biodistribution studies demonstrated that the uptake of mTHPC depends on the perfusion degree of the organ. So, highly perfused organs (liver, spleen, kidney and lung) contained higher amount of the drug compared with less perfused organs [START_REF] Campbell | Tissue levels, histologic changes and plasma pharmacokinetics of meta-Tetra (hydroxyphenyl) chlorin (mTHPC) in the cat[END_REF][START_REF] Whelpton | Distribution of temoporfin, a new photosensitizer for the photodynamic therapy of cancer, in a murine tumor model[END_REF], while the maximum level of mTHPC varies in different animal models and depends on the administration doses [START_REF] Ronn | Human tissue levels and plasma pharmacokinetics of temoporfin (Foscan®, mTHPC)[END_REF][START_REF] Ronn | Comparative biodistribution of meta-Tetra(Hydroxyphenyl) chlorin in multiple species: clinical implications for photodynamic therapy[END_REF]. mTHPC is not metabolized and is excreted unchanged via the biliary route in the feces in a murine model (Cai et al., 1999a(Cai et al., , 1999b)). Nearly 40% of the drug was excreted by the feces during the first day, and less than 0.2% of the administrated dose was observed in the urine [START_REF] Whelpton | Distribution of temoporfin, a new photosensitizer for the photodynamic therapy of cancer, in a murine tumor model[END_REF].

The intratumoral distribution of mTHPC was studied in detail by Mitra and co-workers using high-resolution confocal fluorescence imaging (Mitra et al., 2005). The authors simultaneously mapped microscopic intratumoral mTHPC localization with respect to perfused vasculature in the function of time after injection and demonstrated the maximal mTHPC fluorescence in the perivascular region 3h after injection. Localization of mTHPC in tumor parenchyma was observed only at 24h post-administration. It corresponds to the results of Jones with co-workers [START_REF] Jones | Photodynamic therapy effect of m-THPC (Foscan) in vivo: correlation with pharmacokinetics[END_REF], where mTHPC had a small initial volume of distribution with strong retention in the vasculature together with two peaks of PDT efficacy (2 and 24h) in rats. As it is known for for the majority of PSs, PDT at the early time of irradiation could result mainly in the destruction of the microvasculature of the tumor, while at DLI of 24h it affects the tumor tissue. Similarly, mTHPC exhibits two peaks of PDT efficacy

(2 and 24h) in rats [START_REF] Jones | Photodynamic therapy effect of m-THPC (Foscan) in vivo: correlation with pharmacokinetics[END_REF]. In fact, a fractionated double injection (3 and 24h prior to PDT) should be superior to cumulative single administration, the fact was confirmed in mice bearing EMT6 tumors [START_REF] Garrier | Compartmental targeting for mTHPC-based photodynamic treatment in vivo: Correlation of efficiency, pharmacokinetics, and regional distribution of apoptosis[END_REF]. In fine, although direct cells damage is predominant in tumor destruction by mTHPC-PDT, other mechanisms play an important role in the complete tumor destruction and prolonged PDT-effect. Early pre-clinical studies have demonstrated the depth of necrosis of 3.79 ± 0.28 mm for mTHPC-PDT in PC6 tumorbearing mice [START_REF] Bonnett | Hydroporphyrins of the meso-tetra(hydroxyphenyl)porphyrin series as tumour photosensitizers[END_REF]. Also, it was shown that mTHPC mediates cell photodamage, principally through singlet oxygen formation [START_REF] Melnikova | Subcellular localization of meta-tetra (hydroxyphenyl) chlorin in human tumor cells subjected to photodynamic treatment[END_REF] and its efficacy is sensitive to oxygenation conditions [START_REF] Coutier | Effect of irradiation fluence rate on the efficacy of photodynamic therapy and tumor oxygenation in meta-tetra (hydroxyphenyl) chlorin (mTHPC)-sensitized HT29 xenografts in nude mice[END_REF]. However, several authors observed the absence of correlation between mTHPC concentration in tumor and PDT efficiency [START_REF] Garrier | Compartmental targeting for mTHPC-based photodynamic treatment in vivo: Correlation of efficiency, pharmacokinetics, and regional distribution of apoptosis[END_REF][START_REF] Ris | Photosensitizing effects of m-tetrahydroxyphenylchlorin on human tumor xenografts: correlation with sensitizer uptake, tumor doubling time and tumor histology[END_REF][START_REF] Veenhuizen | Does tumour uptake of Foscan determine PDT efficacy?[END_REF].

Nevertheless, mTHPC exhibits several side effects. The major issue in the clinical management of mTHPC is poor-selectivity and prolong post-treatment skin photosensitivity [START_REF] Senge | mTHPC -A drug on its way from second to third generation photosensitizer? Photodiagnosis Photodyn[END_REF]. Skin and eye photosensitivity of mTHPC is less than that of Photofrin ® (van Geel et al., 1995; Wagnieres et al., 1998). However, it persists for up to 6 weeks (usually 2-3 weeks) after mTHPC administration [START_REF] Allison | Oncologic photodynamic therapy photosensitizers: a clinical review[END_REF]. Usually, Foscan-based PDT resulted in mild to moderate skin photosensitivity and caused neither functional nor cosmetic impairments after treatment of early oral squamous cell carcinoma [START_REF] O'connor | Porphyrin and Nonporphyrin Photosensitizers in Oncology: Preclinical and Clinical Advances in Photodynamic Therapy[END_REF].

Most common side effects of Foscan ® include headache, hemorrhage, dysphagia and edema [START_REF] Baskaran | Clinical development of photodynamic agents and therapeutic applications[END_REF]. Moreover, there is evidence indicating a mild to moderate pain in the treated area [START_REF] Allison | Oncologic photodynamic therapy photosensitizers: a clinical review[END_REF]. Nevertheless, temoporfin remains one of the most potent PSs on the market, while most side effects are caused by non-optimal mTHPC distribution in the organism and could be significantly reduced using nanoparticle-based delivery platforms.

Nanoscale delivery systems

The era of nanoparticle-based mTHPC-PDT started in 1995 [START_REF] Westermann | Comparison of the influence of a water-soluble polymer carrier on the tumor localization and biodistribution of mesotetrametahydroxyphenylchlorin (mTHPC) in two animal models[END_REF] when mTHPC was covalently linked with polyethylene glycol (PEG) to increase its solubility and improve its bioavailability. Currently, there are numerous nano-platforms developed for efficient and targeted mTHPC delivery. The information on the features of each nanoplatform for mTHPC was summarized in the scientific review below.

Drug-in-cyclodextrin-in-liposome (DCL) nanoplatform

Due to the high hydrophobicity of many clinically effective drugs, the entrapment of hydrophobic drugs in the form of water-soluble drug-cyclodextrin (CD) complexes in liposomes has been investigated as a new strategy to combine the relative advantages of

CDs and liposomes into one system, namely drug-in-CD-in-liposome (DCL) (Figure 3.1). This concept was firstly proposed by [START_REF] Mccormack | In fine, the application of DCLs provides the benefits in the delivery of anticancer drugs resulting in (i) better antiproliferative activity than free anticancer drugs (EF24 and LPSF/AC04)[END_REF][START_REF] Mccormack | Entrapment of cyclodextrin-drug complexes into liposomes: potential advantages in drug delivery[END_REF][START_REF] Mccormack | Entrapment of cyclodextrin-drug complexes into liposomes: potential advantages in drug delivery[END_REF] in an effort to combine the advantages offered by both CDs and liposomes in one nano-platform. DCL approach can be useful in increasing drug solubility and vesicles stability, in controlling the in vivo fate of hydrophobic drugs and in avoiding burst release of drug from the vesicles. Relevant characteristics of liposomes and CDs will be briefly described in the next paragraphs.

Liposomes

The pioneering observation of spherical self-closed bilayered structures formed by phospholipids in aqueous systems was done by Alec Bangham about 55 years ago [START_REF] Bangham | Negative staining of phospholipids and their structural modification by surface-active agents as observed in the electron microscope[END_REF]. Ten years later, liposomes were already suggested as drug carriers in cancer chemotherapy by [START_REF] Gregoriadis | Drug-carrier potential of liposomes in cancer chemotherapy[END_REF]. Since then, the interest in lipid vesicles has widely grown and liposomal systems are currently being extensively studied as drug carriers [START_REF] Olusanya | Liposomal Drug Delivery Systems and Anticancer Drugs[END_REF][START_REF] Torchilin | Recent advances with liposomes as pharmaceutical carriers[END_REF]. Interestingly, a liposomal formulation of doxorubicin (PEGylated liposomal-based doxorubicin, Doxil ® ) became the first nano-drug approved by the FDA in 1995 [START_REF] Barenholz | Doxil®--the first FDA-approved nano-drug: lessons learned[END_REF], demonstrating a significant role of liposomes in pharmaceutics and particular in nanomedicine.

Composition and classification

Briefly, liposomes are spherical vesicles formed of one or more concentric phospholipid bilayers, separated by aqueous compartments [START_REF] Olusanya | Liposomal Drug Delivery Systems and Anticancer Drugs[END_REF][START_REF] Torchilin | Recent advances with liposomes as pharmaceutical carriers[END_REF]. Lipid vesicle can encapsulate both hydrophilic and hydrophobic agents, protecting the cargo during the circulation in the body. The properties of lipid vesicles strongly depend on the lipid bilayers composition and a wide range of phospholipids with varying physical-chemical properties provide high versatility of liposomes. Liposomes can be made from different natural and/or synthetic phospholipids (Phosphatidylethanolamine, Phosphatidylglycerol, Phosphatidylcholine (DPPC), Phosphatidylserine, Phosphatidylinositol). Phosphatidylcholine (also known as lecithin) and phosphatidylethanolamine constitute two major structural components of most biological membranes.

Varying the composition of lipid mixture, the physical properties of the lipid bilayer, such as permeability and fluidity, which greatly influence the performance of liposomes in vitro and in vivo, could be specifically tailored [START_REF] Slingerland | Liposomal drug formulations in cancer therapy: 15 years along the road[END_REF]. Different natural and synthetic phospholipids have different phase transition temperature (T M), which is a very important parameter influencing drug encapsulation efficiency and both storage and in vivo stability. With increasing temperature, lipids pass from the ordered gel to a disordered liquidcrystalline phase, making easier the permeation of encapsulated drug through the bilayer.

Therefore, phospholipids whose TM is higher than the body temperature are often used to prepare more stable liposomes as drug delivery carriers for in vivo use. Stability against leakage may be additionally promoted by adding cholesterol [START_REF] Samad | Liposomal drug delivery systems: an update review[END_REF][START_REF] Vemuri | Preparation and characterization of liposomes as therapeutic delivery systems: a review[END_REF]. However, while cholesterol may increase the lipid membrane rigidity and stabilize the liposomes, it may also increase the particle size of liposomes in a dosedependent manner [START_REF] López-Pinto | Effect of cholesterol and ethanol on dermal delivery from DPPC liposomes[END_REF]. The application of physiological lipids, which decrease the danger of acute and chronic toxicity, is a clear advantage of liposomes application.

Liposomes can be categorized in different ways. Generally, liposomes are classified according to their size and number of bilayers (also referred to as lamellae) [START_REF] Samad | Liposomal drug delivery systems: an update review[END_REF]. Both the size and the number of lamellae in the liposomal structure are considered to be the most crucial factors affecting the vesicles biological behavior and drug encapsulation efficiency [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF]. Liposomes with a single bilayer are called unilamellar vesicles, which are subdivided into the small (SUV, 20-200 nm), large (LUV, 200-1000 nm)

and giant (GUV, >1000 nm) ones. The vesicles consisting of one to five bilayers are called oligolamellar vesicles and are characterized by the size of 100-1000 nm. Finally, multilamellar vesicles composed of multiple bilayers (MLV, 5-25 lamella) usually possess the diameter from 500 nm up to 5 μm. Liposomes can be also categorized based on the lipid surface properties such as the charge, PEGylation, active targeting, etc [START_REF] Torchilin | Recent advances with liposomes as pharmaceutical carriers[END_REF] and preparation method [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF]. Ideally, the liposome formulations should be optimized in terms of the species and contents of lipids as well as preparation parameters in order to achieve desired properties, such as particle size, surface charge, encapsulation efficiency, and stability.

Methods of liposome preparation

The methods of preparing the liposomes involve the general stages such as (i) drying down the lipids from an organic solvent, (ii) dispersing the lipid suspension in aqueous media and (iii) purification of the resultant liposomes [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF]. Drug loading can be attained either passively (i.e., during liposome formation) or actively (i.e., after liposome formation). There are several passive loading techniques, which are used for liposomes preparation: mechanical dispersion (sonication, extrusion, micro-emulsification, etc), solvent dispersion (ether or ethanol injection, reverse phase evaporation) and detergent removal methods (dialysis, chromatography, chemical adsorbers). The preparation of liposomes will be further described on the example of small unilamellar vesicles, as most widely used for the delivery of macromolecules.

Among the methods available for liposome preparation, the most commonly employed one is thin-layer hydration proposed by Bangham et al. [START_REF] Bangham | Diffusion of univalent ions across the lamellae of swollen phospholipids[END_REF]. According to this method, a suspension of multilamellar lipid vesicles is readily obtained by co-dissolving the desired lipids (including any hydrophobic cargo) in an organic solvent, subsequent drying using rotatory evaporator and finally hydrating under agitation above the lipid phase transition temperature. In order to decrease the lamellarity and heterogeneity of resulting suspension, mechanical dispersion methods such as sonication or membrane extrusion could be applied. The resulting liposomes are characterized by narrow size distribution and are ready to use. Hydrophilic drugs could also be encapsulated within an aqueous compartment of liposomes. In this case, the addition of the active compound is carried out at the step of thin lipid hydration when the drug is dissolved in the hydration buffer. As a result, the drug is encapsulated in the inner aqueous core of liposomes. In fine, the resultant suspension is purified by means of dialysis, chromatography or ultracentrifugation techniques.

It worth noting, that the trapping efficiency strongly depends on the solubility and partitioning characteristics of encapsulated drug [START_REF] Gulati | Lipophilic drug derivatives in liposomes[END_REF]. Hence, highly lipophilic drugs (logP > 5) are entrapped almost completely in the lipid bilayer of the liposomes (trapping efficiency ≈100%), e.g. mTHPC [START_REF] Kuntsche | Temoporfin-loaded liposomes: Physicochemical characterization[END_REF]. While highly hydrophilic drugs with logP < (-0.3), are located exclusively in the aqueous compartments of the liposomes. In that case, the efficiency of passive encapsulation of hydrophylic drug is Drug-in-cyclodextrin-in-liposome (DCL) nanoplatform depends on the preparation technique and lipid concentration (ratio between aqueous volume of liposomes and unentrapped volume left in the void space due to the curvature of liposomes) and generally do not exceed 30% [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF].

The behavior of liposomes in vivo

Liposomal formulations have shown the ability to enhance the pharmacokinetics and pharmacodynamics of encapsulated drugs since these formulations can induce rapid uptake and long retention by the target tissues [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF]. The advantages of liposome-mediated drugs also include greater solubility, longer circulation times, greater exposure and targeted delivery of the enclosed drug [START_REF] Torchilin | Passive and active drug targeting: drug delivery to tumors as an example[END_REF]. Concerning anticancer therapy, numerous different liposome-based formulations demonstrated lower toxicity than free drugs [START_REF] Olusanya | Liposomal Drug Delivery Systems and Anticancer Drugs[END_REF]. Liposomes selectively deliver anticancer drugs to the tumor tissue by means of a passive targeting via the enhanced permeability and retention (EPR) effect of the tumor vasculature. Tumor vessel units are leaky and are characterized by the absence of lymph drainage [START_REF] Maeda | The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting[END_REF]. Thus, due to the size of hundreds of nanometers, liposomes could permeate into the tumor interstitium (typically over 24-48 h) via leaky tumor vasculature through molecular movement within fluids. However, the EPR effect is relatively modest, offering less than a 2-fold increase in delivery compared with critical normal organs [START_REF] Matsumura | A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs[END_REF][START_REF] Nakamura | Nanodrug Delivery: Is the Enhanced Permeability and Retention Effect Sufficient for Curing Cancer?[END_REF]. Advancements in liposomal vesicle development have achieved both controlled drug release and targeted drug delivery (disease-specific localization) by means of the modification of the liposomal surface by active targeting moieties [START_REF] Torchilin | Recent advances with liposomes as pharmaceutical carriers[END_REF]. Additionally, stimuli-responsive moieties could be embedded in order to control the release of the drug at the preferable sites.

The pharmacokinetics and biodistribution of liposome-encapsulated drugs are controlled by the interplay of two factors: the rate of plasma clearance of the liposome carrier and the stability of the liposome-drug association in the circulation. The clearance of intravenously-injected liposomes is governed by the mononuclear phagocyte system (MPS), or the reticuloendothelial system (RES) [START_REF] Abra | Liposome disposition in vivo. III. Dose and vesiclesize effects[END_REF]. MPS includes monocytes, macrophages and dendritic cells located primarily in the liver and spleen determining the preferable biodistribution of liposomal cargo. To be recognized by MPS, specific proteins (opsonins) adsorb immediately after the liposomes contact with plasma in vivo. Moreover, along with opsonins, serum proteins (i.e., lipoproteins) interact with liposomes causing intensive lipid mass transfer between them. This, in turn, leads to the release of the encapsulated drug from the liposomes to the serum proteins and also could result in liposome disintegration. To avoid the interaction of liposomal vesicles with serum components and RES, the liposomal surface could be modified by the addition of charged (either positive or negative) lipids [START_REF] Jain | Gold nanoparticles as novel agents for cancer therapy[END_REF] and/or surface-grafted hydrophilic polymers (i.e., PEG) [START_REF] Ishida | Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes[END_REF]. This unique and flexible variety in the liposomal structure and composition distinguishes liposomes as the preferred nanocarriers for a broad spectrum of therapeutic agents, including anticancer drugs [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF].

In conclusion, liposomes are non-toxic, flexible, biocompatible, completely biodegradable and non-immunogenic drug nanodelivery systems for systemic and nonsystemic drugs administrations. Lipid vesicles increase stability and solubility of the hydrophobic drug via encapsulation resulting in the reduced drug toxicity and side effects such as the exposure of sensitive tissues to toxic drugs [START_REF] Akbarzadeh | Liposome: classification, preparation, and applications[END_REF]. The main drawback of liposomes is its hindered penetration into the tissues. Thus, liposomes act well as containers for the hydrophobic drug delivery o the tumor but are not an ultimate nanoplatform. On the other hand, inner aqueous core allows encapsulating of soluble drugs or its formulations (i.e., micelles, CD-based inclusion complexes) forming hybrid nanoplatforms (i.e., drug-in-micelles-in-liposomes, drug-in-cyclodextrin-in-liposomes) [START_REF] Chen | Drug-in-cyclodextrin-in-liposomes: a promising delivery system for hydrophobic drugs[END_REF][START_REF] Franzè | Drug-in-micelles-inliposomes (DiMiL) systems as a novel approach to prevent drug leakage from deformable liposomes[END_REF]. Recently, a similar strategy was also adopted to encapsulate drug-loaded micelles into liposomes making drug-in-micelle-in-liposome [START_REF] Franzè | Drug-in-micelles-inliposomes (DiMiL) systems as a novel approach to prevent drug leakage from deformable liposomes[END_REF].

Cyclodextrin-based inclusion complexes

The inclusion complexes of CDs with a wide variety of organic and non-organic compounds refer to the class of the simplest supramolecular systems extensively studied for the last decades [START_REF] Carneiro | Cyclodextrin-Drug Inclusion Complexes: In Vivo and In Vitro Approaches[END_REF]. CDs were discovered by French scientist Villiers in 1891 when he noticed that Bacillus amylobacter produces a crystalline substance from starch after the digestion [START_REF] Crini | Review: A History of Cyclodextrins[END_REF]. However, industrial-scale production of CD in pure form was performed only in 1984, attracting the attention of pharmaceutical and food industries [START_REF] Szejtli | Introduction and General Overview of Cyclodextrin Chemistry[END_REF]. CDs belong to the family of cage molecules; that is, the core of their structure is composed of a dimensionally stable hydrophobic cavity that can trap or encapsulate other molecules. To date, CDs are widely used in different areas such as pharmaceuticals, drug delivery systems, cosmetics, food and chemical industries [START_REF] Carneiro | Cyclodextrin-Drug Inclusion Complexes: In Vivo and In Vitro Approaches[END_REF]. They can be found in commercially available medications, including tablets, eye drops and ointments [START_REF] Loftsson | Pharmaceutical applications of cyclodextrins: basic science and product development[END_REF]. There are above 40 products or formulations containing various CDs, especially β-CD and its derivatives such as sulfobutyl ether of β-cyclodextrin or 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), which are currently used in at least six FDA-approved products (four for sulfobutyl ether of β-cyclodextrin and two for HP-β-CD) [START_REF] Gidwani | A Comprehensive Review on Cyclodextrin-Based Carriers for Delivery of Chemotherapeutic Cytotoxic Anticancer Drugs[END_REF]. Nowadays, a broad spectrum of CDs containing materials with versatile supramolecular architectures (nanoparticles, nanosponges, nanomicelles, nanovesicles, etc.) has been synthesized to assemble functional platforms for drug delivery [START_REF] Duchêne | Cyclodextrins and Their Inclusion Complexes[END_REF]. Also, CDs have been recently proposed as a perspective nanoplatform for the delivery of PSs [START_REF] Ben Mihoub | Use of Cyclodextrins in Anticancer Photodynamic Therapy Treatment[END_REF][START_REF] Kryjewski | Functionality stored in the structures of cyclodextrin-porphyrinoid systems[END_REF].

Structure and derivatives

CDs are cyclic oligosaccharides, formed by six α-cyclodextrin (α-CD), seven β-cyclodextrin (β-CD), eight γ-cyclodextrin (γ-CD) D-glucopyranose units linked by α- [START_REF]Drug solubilization and stabilization[END_REF]4) bonds (Table 3.1). It worth noting that the formation of CDs with fewer than 6 units cannot be performed due to steric hindrances while the higher CDs homologs with 9 or more glucose units are very difficult to purify [START_REF] Challa | Cyclodextrins in drug delivery: An updated review[END_REF]. Generally, the cavity size of α-CD is insufficient for many drugs and γ-CD is expensive, while β-CD is affordable and has cavity size suitable for the widest range of drugs. Thus, β-CD has been more widely used in pharmaceutical applications than other native CDs [START_REF] Rajewski | Pharmaceutical applications of cyclodextrins. 2. In vivo drug delivery[END_REF]. hydrogens are located in the cavity of CDs [START_REF] Valle | Cyclodextrins and their uses: a review[END_REF]. As result CD molecules have a hydrophilic outside (logP is between (-13) and (-17)), and apolar cavity, which provides a hydrophobic matrix comparable to 60-70% of aqueous ethanol solution [START_REF] Connors | The Stability of Cyclodextrin Complexes in Solution[END_REF][START_REF] Loftsson | Drug Solubilization and Stabilization by Cyclodextrin Drug Carriers. In Drug Delivery Strategies for Poorly Water-Soluble Drugs[END_REF]. The increase in the number of glucopyranose units results in the enlargement of the hydrophobic cavity (Table 3.1). Despite the high hydrophilicity of CD's outer surface, the solubility in water could vary from 18 mg/ml for β-CD to 250 mg/ml for γ-CD [START_REF] Duchêne | Cyclodextrins and Their Inclusion Complexes[END_REF]. The relatively low water solubility of CDs, especially of β-CD, significantly reduces their applicability. CDs are chemically stable under neutral and basic conditions in aqueous solutions [START_REF] Frömming | Cyclodextrins in Pharmacy[END_REF] and are quite resistant to light within the ultraviolet-visible spectrum and infrared ranges [START_REF] Loftsson | Pharmaceutical applications of cyclodextrins[END_REF].

To overcome these problems and extend the physicochemical properties and inclusion capacity of parent CDs, chemically modified CD derivatives were produced by aminations, esterifications or etherifications of primary and secondary hydroxyl groups of the CDs [START_REF] Szente | Highly soluble cyclodextrin derivatives: chemistry, properties, and trends in development[END_REF]. It should be however noted, that the location of substituted groups could vary at different (2, 3 or 6) positions on the parent CD molecule [START_REF] Blanchard | Some important considerations in the use of cyclodextrins[END_REF].

The degree of substitution (DS) is defined as the average number of substituents per CD molecule (Table 3.1). Thus, the methylated β-CDs (Me-β-CD, TM-β-CD) possess the water solubility of > 500 mg/ml and hydroxypropyl derivative more than 600 mg/ml. The modification of substitutions changes the hydrophobic cavity volume and can improve solubility, physical and microbiological stability. Such high versatility of CD structure helps to select an optimal CD for each specific drug depending on the size of the guest molecules or its side substituents [START_REF] Szente | Cyclodextrin-Lipid Complexes: Cavity Size Matters[END_REF]. In fine, it reduces parenteral toxicity and may help to control the chemical activity of guest molecules [START_REF] Challa | Cyclodextrins in drug delivery: An updated review[END_REF][START_REF] Davis | Cyclodextrin-based pharmaceutics: past, present and future[END_REF].

Complex formation

The presence of a hydrophobic interior cavity allows the formation of inclusion hostguest complexes with an extensive range of solid, liquid and gaseous compounds [START_REF] Valle | Cyclodextrins and their uses: a review[END_REF][START_REF] Laza-Knoerr | Cyclodextrins for drug delivery[END_REF][START_REF] Vyas | Cyclodextrin based novel drug delivery systems[END_REF]. There are two key factors influencing the ability of CDs to form an inclusion complex with a guest molecule, namely steric and thermodynamic. Thus, the main quantitative parameters describing the mechanism of formation of the inclusion complexes are the stoichiometry and binding constants. From the steric point of view, the formation of the complex is a dimensional fit between the host cavity and guest molecule (Muñoz-Botella et al., 1995). The stoichiometry displays the number of molecules involved in the formation of the complex and determines the binding model, while the value of the binding constant reflects the degree of affinity between the complexants [START_REF] Thordarson | Determining association constants from titration experiments in supramolecular chemistry[END_REF]. The stoichiometry of the complex varies depending on the combination of CD-drug. Depending on their respective size, one CD molecule can bind more than one drug molecule, as a drug molecule may form complexes with several CD molecules (Figure 3.3). The most common stoichiometry among them is 1:1, but others like 1:2, 2:1, 2:2 can also be encountered [START_REF] Szejtli | Introduction and General Overview of Cyclodextrin Chemistry[END_REF]. It worth noting that inclusion complexation with CDs is a reversible process (dynamic equilibrium), i.e., each molecule can dissociate from the complex. Thus, often, the system with the high stoichiometric ratio is the mixture of the complexes with simple stoichiometry [START_REF] Thordarson | Determining association constants from titration experiments in supramolecular chemistry[END_REF]. Concerning the thermodynamic of complexation, the formation of inclusion complex is accomplished by the intermolecular interaction between CDs (host molecule) and guest molecule, which is driven by non-covalent interactions, including van der Waals, hydrophobic, electrostatic and charge transfer interactions, metal coordination, hydrogen bonding and steric effects [START_REF] Rekharsky | Complexation Thermodynamics of Cyclodextrins[END_REF]. It is crucial that no covalent bonds are broken or formed during the formation of the inclusion complex [START_REF] Schneiderman | Cyclodextrins: a versatile tool in separation science[END_REF]. In aqueous solutions, the inclusion of the (dehydrated) guest into the nonpolar cavity of the CD is accompanied by the water release from the CD cavity and subsequent penetration of the guest molecule wholly or partly into the central cavity of the CDs [START_REF] Song | Inclusion complexation, encapsulation interaction and inclusion number in cyclodextrin chemistry[END_REF]. Moreover, the conformational changes or strain release of the cyclodextrin molecule upon complexation also contributes in the formation of inclusion complex [START_REF] Saenger | Cyclodextrin Inclusion Compounds in Research and Industry[END_REF]. It is worth noting that the efficiency of the formation of inclusion complexes is determined by many factors, including temperature, solvents composition, amount of water (the degree of complexes dilution), complexation techniques. Ionization of the guest, salt formation, formation of metal complexes, application of supercritical fluids and addition of organic co-solvents to the drug/CDs aqueous solution will, if the conditions are correct, lead to enhanced complexation efficiency (e.g., enhanced solubility) [START_REF] Arun | Cyclodextrins as Drug Carrier Molecule: A Review[END_REF][START_REF] Loftsson | Cyclodextrins and their pharmaceutical applications[END_REF][START_REF] Van Hees | Application of supercritical carbon dioxide for the preparation of a piroxicam-betacyclodextrin inclusion compound[END_REF]. In addition, CDs are able to form non-inclusion complexes and complex aggregates [START_REF] Loftsson | Self-association of cyclodextrins and cyclodextrin complexes[END_REF] In fine, the quantitative analysis of the intermolecular interactions of interest is one of the fundamental issues in supramolecular chemistry. To measure the efficiency of binding, supramolecular titration experiments were performed, providing the data for the estimation of the binding (association) constant values [START_REF] Thordarson | Determining association constants from titration experiments in supramolecular chemistry[END_REF]. The thermodynamic association (binding) constant (K) is related to the kinetics of the particular system, hence K = k1/k-1, with the k1 and k-1 as forward (on) and backward (off) rate constants for the complex equilibria.

Traditionally, there are different methods of drug/CD complex preparation such as coprecipitation, kneading, spray drying, freeze-drying, solvent evaporation, or saturated aqueous solution methods [START_REF] Gharib | Liposomes incorporating cyclodextrin-drug inclusion complexes: Current state of knowledge[END_REF]:

• Complex preparation in the aqueous phase (co-precipitation): CD is dissolved in an aqueous phase and the drug is then added. Water-soluble inclusion complexes are formed after stirring the mixture for a specific period of time at a specific temperature.

• Kneading: CD and aqueous solution are mixed in a mortar to make a homogenous paste; the drug is then added slowly, while grinding, to dissolve the drug. Then, the mixture was ground with an appropriate quantity of water added to maintain a suitable consistency.

• Co-evaporation (solvent evaporation method): mixing of the organic phase containing drug and an aqueous solution of CD by stirring to achieve molecular dispersion. The resulting suspension is followed by evaporation of the solvent under vacuum until dried mass obtained.

• Spray drying: mixing of the drug in an organic solution (ethanol) with an aqueous solution of CD at 1:1 molar ratio. The resulting mixture is then stirred at room temperature to attain equilibrium followed by removing the solvent using spray drying.

• Freeze-drying (lyophilization): the drug and CD at 1:1 molar ratio are either dissolved in ethanol or water respectively or both in the aqueous phase. The resulted solution is stirred for a specific time at a certain temperature and then filtered, frozen and lyophilized under reduced pressure.

The fate of inclusion complexes in vivo

The inclusion complexes can be administered through different routes such as transdermal, ophthalmic, nasal, oral, intravenous, etc. [START_REF] Shimpi | Cyclodextrins: application in different routes of drug administration[END_REF].

Pharmacokinetics of CDs and their excretion from the body will be defined inter alia by route of administration. Concerning parenteral route of CD's administration, CDs are quickly eliminated from systemic circulation and more than 90% of the administrated CD are renally excreted intact (Stella and He, 2008). In addition, CDs exhibit a small volume of distribution in animal models or humans, indicating the preferable CD distribution in the extracellular compartments without involving deep compartments or storage pools (Stella and He, 2008).

Although cyclodextrin complexes enable lipophilic drugs to be administered in much greater concentrations than in their free form, on injection, these complexes will rapidly dissociate depending to some extent on their affinity constants [START_REF] Mccormack | Entrapment of cyclodextrin-drug complexes into liposomes: potential advantages in drug delivery[END_REF].

Dissociation occurs on dilution by the plasma and extracellular fluids [START_REF] Stella | Mechanisms of drug release from cyclodextrin complexes[END_REF]. In addition to dilution, drug release mechanisms involve: (i) drug-protein binding causing a decrease in free drug concentration, (i) competitive displacement by endogenous and exogenous molecules (i.e. cholesterol), (iii) and accumulation of the drug into tissues, which are not accessible by CDs [START_REF] Stella | Mechanisms of drug release from cyclodextrin complexes[END_REF]. According to that, the behavior of the drug coadministrated with CDs is determined by the CD affinity to the drug molecule. Thus, the pharmacokinetics of the drugs, which exhibit binding constant values K > 10 5 M -1 , can be altered by CDs (Stella and He, 2008). Thus, the dissociated drug will be eliminated from blood and tissues irrespective of its carrier and any remaining complexed drug will reach the kidneys only. Nevertheless, the methylation of the β-CD core increases CD's affinity for certain drugs as well as for the components of cellular membranes, resulting to a larger volume of distribution (VD ≈ 2.5 L/kg) and longer plasma half-life (t1/2 ≈7h) in animal models compared to native β-CD (VD ≈ 0.2 L/kg; t1/2 ≈0.3h) [START_REF] Thompson | Cyclodextrins--enabling excipients: their present and future use in pharmaceuticals[END_REF]. Moreover, the enhanced "CD-to-drug" affinity decreases the probability of the drug release upon the dilution providing stronger alteration of drug biodistribution and pharmacokinetics.

Biodistribution of CDs includes the following tissues: bladder, adrenal glands and liver with the highest concentration in the kidneys, while CD concentration in lungs and muscles is almost undetectable [START_REF] Antlsperger | Toxicological Comparison of Cyclodextrins[END_REF][START_REF] De Bie | Disposition of [14C]gammacyclodextrin in germ-free and conventional rats[END_REF][START_REF] Gerloczy | Absorption, distribution and excretion of 14C-labelled hydroxypropyl βcyclodextrin in rats following oral administration[END_REF][START_REF] Kubota | Absorption, distribution and excretion of beta-cyclodextrin and glucosyl-beta-cyclodextrin in rats[END_REF][START_REF] Van Ommen | Disposition of 14C-alphacyclodextrin in germ-free and conventional rats[END_REF]. Concerning tumor-bearing animal models, the accumulation of CD in tumor tissue was observed in primary effusion non-Hodgkin lymphoma [START_REF] Gotoh | The antitumor effects of methyl-β-cyclodextrin against primary effusion lymphoma via the depletion of cholesterol from lipid rafts[END_REF] and various carcinoma in xenograft tumor-bearing mice [START_REF] Grosse | Antiproliferative effect of methylbeta-cyclodextrin in vitro and in human tumour xenografted athymic nude mice[END_REF]Onodera et al., 2013a).

To date, there are a considerable number of publications, including a detailed review of the toxicological status of CDs and their biological activity [START_REF] Antlsperger | Toxicological Comparison of Cyclodextrins[END_REF][START_REF] Arima | Recent Findings on Safety Profiles of Cyclodextrins, Cyclodextrin Conjugates, and Polypseudorotaxanes[END_REF][START_REF] Gould | 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD): A toxicology review[END_REF][START_REF] Irie | Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation[END_REF][START_REF] Loftsson | Pharmaceutical applications of cyclodextrins: basic science and product development[END_REF][START_REF] Mosher | Complexation: Cyclodextrins[END_REF][START_REF] Thompson | Cyclodextrins--enabling excipients: their present and future use in pharmaceuticals[END_REF][START_REF] Zidovetzki | Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies[END_REF].

Generally, CD toxicity is related to the ability of CD to interact with membrane components such as cholesterol and lipids [START_REF] Kilsdonk | Cellular cholesterol efflux mediated by cyclodextrins[END_REF][START_REF] Mohammad | Cholesterol depletion by methyl-β-cyclodextrin augments tamoxifen induced cell death by enhancing its uptake in melanoma[END_REF][START_REF] Zidovetzki | Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies[END_REF], resulting in the increase of membrane fluidity and induction of membrane invagination through a loss of bending resistance and as such causing cell lysis [START_REF] Challa | Cyclodextrins in drug delivery: An updated review[END_REF]. Since kidneys are the main absorption site of CDs in the body, the renal toxicity was demonstrated, especially for β-CD and its methylated derivatives [START_REF] Frank | Cyclodextrin nephrosis in the rat[END_REF][START_REF] Irie | Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation[END_REF][START_REF] Thompson | Cyclodextrins--enabling excipients: their present and future use in pharmaceuticals[END_REF]. Nevertheless, it worth noting that the coadministration of CDs with drugs decreases toxicological effects of both substances [START_REF] Arima | Recent Findings on Safety Profiles of Cyclodextrins, Cyclodextrin Conjugates, and Polypseudorotaxanes[END_REF][START_REF] Mosher | Complexation: Cyclodextrins[END_REF].

In conclusion, CDs represent the example of nanocages imparting useful features to particles drug delivery system. Due to the ability to encapsulate hydrophobic drugs, i.e., the formation of CD-drug inclusion complexes, CDs are widely used in the medicinal and pharmaceutical applications, including the delivery of PSs in PDT [START_REF] Ben Mihoub | Use of Cyclodextrins in Anticancer Photodynamic Therapy Treatment[END_REF].

Generally, complexation with CDs leads to an improvement of the water solubility, stability, bioavailability, release and under certain conditions, the permeability of the encapsulated drug. In the case of PS, CDs could particularly improve the photophysical characteristics of PSs [START_REF] Lang | Photophysical properties of porphyrinoid sensitizers non-covalently bound to host molecules; models for photodynamic therapy[END_REF], including the generation of ROS and even altering PS delivery to targets cells/tissues (Yankovsky et al., 2016). However, the application of CDs in vivo is limited due to the dilution effects and rapid renal excretion of CD from the blood. To overcome these limitations, drug/CD inclusion complexes could be encapsulated in liposomal vesicles in the DCL system.

DCL

DCL is an example of hybrid nanoplatform combining the advantages offered by CDs and liposomes [START_REF] Chen | Drug-in-cyclodextrin-in-liposomes: a promising delivery system for hydrophobic drugs[END_REF]. Traditionally, pre-formed drug/CD complexes are encapsulated into liposomal inner aqueous phase by different methods to obtain DCLs.

DCLs are commonly prepared by means of thin-film hydration [START_REF] Maestrelli | Preparation and characterisation of liposomes encapsulating ketoprofen-cyclodextrin complexes for transdermal drug delivery[END_REF][START_REF] Piel | Ca2+channel blocker drugs as nifedipine (Škalko et al[END_REF], reverse-phase evaporation [START_REF] Maestrelli | Effect of preparation technique on the properties of liposomes encapsulating ketoprofencyclodextrin complexes aimed for transdermal delivery[END_REF], freeze and thaw [START_REF] Ascenso | Novel tretinoin formulations: a drug-in-cyclodextrin-in-liposome approach[END_REF], ethanol injection [START_REF] Chen | Preparation, Characterization and Pharmacokinetics of Liposomes-Encapsulated Cyclodextrins Inclusion Complexes for Hydrophobic Drugs[END_REF][START_REF] Fatouros | Liposomes encapsulating prednisolone and prednisolone-cyclodextrin complexes: comparison of membrane integrity and drug release[END_REF] and freeze-drying (Škalko et al., 1996) techniques (Figure 3.3). Generally, DCL could be classified according to drug loading. Single loaded DCLs encapsulate the drug in the inner aqueous core of liposomes only in the form of CD inclusion complexes, while double-loaded DCLs are vesicles loaded with lipophilic drug both in its free form (in the lipophilic bilayer) and in CDcomplexed form (in the aqueous phase) [START_REF] Gharib | Liposomes incorporating cyclodextrin-drug inclusion complexes: Current state of knowledge[END_REF]. Since many of the hydrophobic drugs are not lipophilic enough to be efficiently entrapped into the lipid bilayer without destabilization of the structural integrity of liposome, DCL was proposed as a strategy to increase encapsulation efficiency (EE) of hydrophobic drugs into liposomes [START_REF] Mccormack | Entrapment of cyclodextrin-drug complexes into liposomes: potential advantages in drug delivery[END_REF]. The amount of hydrophobic drug incorporated into lipid bilayer is often limited in terms of drug to lipid ratio for small unilamellar vesicles. On the other hand, EE in the aqueous phase is limited by water-solubility of drug/CDs, preparation technique of DLCs and type of the used CD. It is well known that EE values of soluble drugs incorporated into the inner aqueous liposomal core are strongly limited by drug solubilization and are much lower than that of lipophilic molecules encapsulated into the lipid bilayer. Thus, for DCL, EE also depended on both the liposome preparation method and complex concentration in the aqueous phase and was ranged in the order MLV > LUV > SUV [START_REF] Maestrelli | Effect of preparation technique on the properties of liposomes encapsulating ketoprofencyclodextrin complexes aimed for transdermal delivery[END_REF]. In the case of SUV, double-loaded DCL (DL-DCL) is supposed to be efficient for drug encapsulation, but drugs incorporated in the lipid bilayers rather than in the aqueous core of the liposomes are prone to more rapid release. It worth noting that EE could be also improved by the increase in CD concentration.

However, one should be careful, because CDs exhibit the destabilizing effect towards the liposomal membrane at high concentrations [START_REF] Fatouros | Liposomes encapsulating prednisolone and prednisolone-cyclodextrin complexes: comparison of membrane integrity and drug release[END_REF].

The presence of CDs affects liposome characteristics and membrane integrity depending on CD functionalization and liposome composition [START_REF] Angelini | Liposomes entrapping β-cyclodextrin/ibuprofen inclusion complex: Role of the host and the guest on the bilayer integrity and microviscosity[END_REF][START_REF] Hatzi | Integrity of liposomes in presence of cyclodextrins: Effect of liposome type and lipid composition[END_REF][START_REF] Maestrelli | New "drug-in cyclodextrin-in deformable liposomes" formulations to improve the therapeutic efficacy of local anaesthetics[END_REF][START_REF] Zappacosta | Effect of the Incorporation of Functionalized Cyclodextrins in the Liposomal Bilayer[END_REF]. Generally, the addition of inclusion complexes increases the size of large liposomal vesicles [START_REF] Jain | Multivesicular liposomes bearing celecoxib-beta-cyclodextrin complex for transdermal delivery[END_REF][START_REF] Maestrelli | Effect of preparation technique on the properties of liposomes encapsulating ketoprofencyclodextrin complexes aimed for transdermal delivery[END_REF], while for small unilamellar vesicles the difference in size is negligible [START_REF] Dhule | Curcumin-loaded γ-cyclodextrin liposomal nanoparticles as delivery vehicles for osteosarcoma[END_REF][START_REF] Maestrelli | Effect of preparation technique on the properties of liposomes encapsulating ketoprofencyclodextrin complexes aimed for transdermal delivery[END_REF][START_REF] Mendonça | Enhanced antiproliferative activity of the new anticancer candidate LPSF/AC04 in cyclodextrin inclusion complexes encapsulated into liposomes[END_REF][START_REF] Zappacosta | Effect of the Incorporation of Functionalized Cyclodextrins in the Liposomal Bilayer[END_REF]. However, sometimes, the enhancement of the vesicle size during storage was observed [START_REF] Lapenda | Encapsulation of trans-References dehydrocrotonin in liposomes: an enhancement of the antitumor activity[END_REF][START_REF] Rahman | Native and βcyclodextrin-enclosed curcumin: entrapment within liposomes and their in vitro cytotoxicity in lung and colon cancer[END_REF]. A hypothesis could be that the high local CD concentration in the aqueous compartment of liposomes makes probable the replacing of drug molecule in CD cavity by lipids as a result of this reducing the stability of vesicles. In turn, the destabilization of the bilayer causes the changes in vesicles size and enables partial or complete leakage of drug content from DCL. The destructive effect of drug CD complexes on liposomes might be significantly different, depending on the affinity between CDs and drug molecules [START_REF] Zappacosta | Effect of the Incorporation of Functionalized Cyclodextrins in the Liposomal Bilayer[END_REF]. Thus, the extent of membrane destabilization due to CD is closely related to the amount of the free CD molecules which could interact with the membrane lipids [START_REF] Puskás | Influence of cyclodextrins on the physical stability of DPPC-liposomes[END_REF]. To obtain stable DCL, the CDs should have a higher affinity for drug molecules compared with liposomal membrane lipids.

Compared to drug CD complexes, administration of DCLs could serve as a better way to control the action of a wide range of therapeutic agents. DCLs presented more prolonged release profiles compared with both liposomes and CDs. The encapsulation of drug CD complexes into the inner aqueous core of liposomes could result in a modulation of the in vitro kinetics of release of drug molecules due to the additional barriers to the diffusion of a drug [START_REF] Jain | Multivesicular liposomes bearing celecoxib-beta-cyclodextrin complex for transdermal delivery[END_REF]. Since CD was found to be membrane impermeable [START_REF] Joguparthi | Effect of Cyclodextrin Complexation on the Liposome Permeability of a Model Hydrophobic Weak Acid[END_REF][START_REF] Zappacosta | Effect of the Incorporation of Functionalized Cyclodextrins in the Liposomal Bilayer[END_REF], the release of the drug should include the crossing of the lipid bilayer barrier [START_REF] Chen | Preparation, Characterization and Pharmacokinetics of Liposomes-Encapsulated Cyclodextrins Inclusion Complexes for Hydrophobic Drugs[END_REF]. Thus, the rate of drug release is depending on various factors such as CD type [START_REF] Fatouros | Liposomes encapsulating prednisolone and prednisolone-cyclodextrin complexes: comparison of membrane integrity and drug release[END_REF][START_REF] Piel | Betamethasone-in-cyclodextrin-in-liposome: The effect of cyclodextrins on encapsulation efficiency and release kinetics[END_REF], size and lammelarity of liposomes [START_REF] Maestrelli | Effect of preparation technique on the properties of liposomes encapsulating ketoprofencyclodextrin complexes aimed for transdermal delivery[END_REF] and lipid content (i.e. deformable liposomes) [START_REF] Gillet | Development of a new topical system: Drug-in-cyclodextrin-in-deformable liposome[END_REF][START_REF] Jain | Multivesicular liposomes bearing celecoxib-beta-cyclodextrin complex for transdermal delivery[END_REF].

At the same time, intravenously administrated DCLs will interact with plasma proteins and blood cells, resulting in the destabilization of liposomes and release of the drug in the form of CD inclusion complexes. In this case, the drug released not in a free form but in the CD inclusion complex which is ready to transport it to the target site. According to the literature data, encapsulation of drug CD complexes into liposomes results in a slow release profile in vitro, therefore, the systematic circulation time of the drug is prolonged (Arima et al.,

OBJECTIVES OBJECTIVES

The general objective of this study is an improvement of mTHPC delivery to the target tumor tissue with mTHPC-loaded cyclodextrin-based liposomal nanoparticles. The development of drug-in-cyclodextrin-in-liposome nanoparticles also includes the investigation of complexation processes of drug with CDs, development of detection methods for monitoring and control of these processes in complex systems and study of the inclusion complexes behavior in tumor tissues.

The objective of this work was to explore the cyclodextrin-based liposomal nanoparticles for effective delivery of mTHPC to target tumor tissue. For this purpose, the following studies were carried out:

• Study of inclusion complex formation between mTHPC and β-CD derivatives.

• CHAPTER II.

CD INCLUSION COMPLEXES

Characterization of inclusion complex formation

The first part of the results on the interaction of mTHPC with β-CDs is described in the article "Evaluation of temoporfin affinity to β-cyclodextrins assuming self-aggregation".

According to the literature data (Stella and He, 2008;[START_REF] Stella | Mechanisms of drug release from cyclodextrin complexes[END_REF], the biological behavior of drug/β-CDs complexes strongly depends on the binding affinity of the drug to β-CDs. Thus, we analyzed the processes of inclusion complex formation between mTHPC and three β-CD derivatives (hydroxypropyl-, methyl-and trimethyl-β-CD). Using several spectroscopic techniques and molecular dynamics simulations we experimentally assessed the binding affinity of β-CD derivatives to mTHPC and refined the value of the binding constant. Parameters of binding isotherms revealed that mTHPC strongly interacts with β-CD derivatives, forming 1:2 inclusion complexes in aqueous solution. We demonstrated that apparent binding constants strongly depend on mTHPC concentration due to the porphyrin self-aggregation. The accurate assumption of mTHPC self-aggregation effects in our model resulted in significantly higher values than those previously reported.

Development of spectral techniques for monitoring the distribution of inclusion

Development of spectral techniques for monitoring the distribution of inclusion complexes

The second part of the results on mTHPC/β-CDs complexes is described in the article "Soret band shape indicates mTHPC distribution between β-cyclodextrins and serum proteins".

In order to analyze PS microenvironment, the technique for recognition of molecules bound to mTHPC is required. With this aim, we analyzed fluorescence and absorbance characteristics of mTHPC in several organic solutions and complexes with various biological structures. Comparative data analysis showed that the shape of the Soret band was strongly dependent on the kind of biological structure bound with the PS. The well-detected difference in the ratio of two Soret band peaks is observed for mTHPC molecules bound to β-CD on the one hand and to serum proteins/lipid membranes on the other. Taking into account the possible mechanism of incorporation of mTHPC molecule into the β-CD cavities or lipid membranes, we suggested that the variability of Soret band shape is associated with the changes of mTHPC conformation, caused by the forced rotation of the aryl-substitutes. Using this spectral feature, we could control mTHPC distribution processes between β-CDs and biomembranes and assessed the fraction of inclusion of mTHPC/CD complexes inside DCLs. Moreover, this technique could be useful in analyzing both equilibrium and kinetic processes of PS distribution in the blood (Yakavets et al., 2018c).

Nanoshuttle mechanism of mTHPC delivery

The third part of the results has published the article "The alteration of temoporfin distribution in multicellular tumor spheroids by β-cyclodextrins" and reveals the features of mTHPC/β-CDs complexes behavior in tumor tissue.

We

GENERAL DISCUSSION

Nowadays, we observe certain saturation in the development of nanomaterials for drug delivery. The assembly of more than one type of nanomaterial in one hybrid nanoparticle could be considered as an advanced strategy in nanomedicine development [START_REF] Deljoo | Curcumin-hybrid Nanoparticles in Drug Delivery System (Review)[END_REF][START_REF] Sailor | Hybrid nanoparticles for detection and treatment of cancer[END_REF]Yakavets et al., 2019). Hybrid nanoparticles that contain two or more distinct nanoparticles assembled in a functional nanostructure could provide synergistic medical effects when nanomaterials are carefully selected. In the case of mTHPC, we suggested that combination of liposomes and cyclodextrin in "drug-in-cyclodextrin-inliposome" nanoassembly could restrain the dissociation of drug-mTHPC complexes, avoid rapid drug release and favorably alter PSs penetration into tumor tissues. Thus, we supposed that mTHPC-DCL could overcome the individual nanostructure limitations providing passive targeting of the tumor by liposomes and deep penetration of mTHPC/CD complexes in tumor tissue.

Liposomes represent bilayered phospholipid nanocapsules, which could be loaded with hydrophobic compounds in the lipid bilayer or hydrophilic drugs in the inner aqueous core [START_REF] Torchilin | Recent advances with liposomes as pharmaceutical carriers[END_REF]. After almost two decades of investigation, mTHPC liposomal formulation (Foslip ® ) is one of the most successful and the most studied mTHPC-based NPs [START_REF] Reshetov | Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo[END_REF]. Conventional liposomes encapsulate almost 8% (w/w) of mTHPC in lipid bilayer providing complete solubilization of a highly hydrophobic mTHPC molecule during its administration. Foslip ® exhibits lower dark toxicity and potentially could passively target tumor by EPR effect, thus improving the mTHPC selectivity. Moreover, it has been demonstrated that Foslip ® -PDT was more efficient at shorter DLIs of 6-15h compared with 24h DLI for Foscan ® [START_REF] Reshetov | Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo[END_REF]. Nevertheless, liposomes have limited penetration in the tumor tissue [START_REF] Gaio | Uptake and photo-toxicity of Foscan®, Foslip® and Fospeg® in multicellular tumor spheroids[END_REF]Millard et al., 2018) and rapidly release mTHPC in blood upon the interaction with serum components [START_REF] Reshetov | Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo[END_REF]. In summary, liposomes were suggested as potent nanocages.

Cyclodextrins (CDs) have been widely investigated as a unique pharmaceutical excipient for the past few decades and are still explored for new applications. Recent reports demonstrated the potency of β-CDs as an individual nanocarrier of mTHPC in in vitro and in vivo preclinical tumor models (Yankovsky et al., 2016). Deep investigations of CDs as pharmaceutical agents demonstrated that CD effect strongly depends on CD affinity to the drug (Stella and He, 2008). When we refined the value of binding constants assuming selfaggregation of mTHPC molecules, we obtained much higher values that were already reported in the literature [START_REF] Bautista-Sanchez | Photophysical properties of glucoconjugated chlorins and porphyrins and their associations with cyclodextrins[END_REF][START_REF] Desroches | A direct sensitized fluorimetric determination of5,10,15,20-tetra(mhydroxyphenyl)chlorin [m-THPC(Foscan)®] in human plasma using a cyclodextrin inclusion complex[END_REF]. According to our calculations (Yakavets et al., 2018b), completely methylated TM-β-CD exhibits the highest affinity (K = 1.1×10 7 M -1 ) as compared to randomly methylated Me-β-CD (K = 7.1×10 5 M -1 ) and 2-hydroxypropyl substituted Hp-β-CD (K = 1.7×10 5 M -1 ). One could suggest that β-CDs methylation increases their affinity to mTHPC, providing the opportunity to modulate PS behavior in biological media. Also, the detailed study of the photophysical properties of mTHPC/CD inclusion complexes revealed the changes of mTHPC Soret band shape upon the inclusion complex formation et al., 2017a). We suggested and further confirmed using molecular dynamics simulations (Yakavets et al., 2018b) that spectral changes were induced by the interaction of CDs with phenyl groups causing the rotation of phenyl substitutes (Yakavets et al., 2017a). It should be noted that the intercalation of mTHPC molecule in the lipid microenvironment is possible only in planar conformation, resulting in the opposite changes of mTHPC Soret band shape. Thus, the spectral technique based on the Soret band shape could be used for the monitoring of mTHPC microenvironment and could be applied for detection of mTHPC/CD complexes in the aqueous core of hybrid liposomes.

As was mentioned above, methylation of β-CDs increases their affinity to mTHPC, providing the opportunity to modulate PS behavior in biological media. To describe the mechanism of alteration, we studied mTHPC distribution in 3D multicellular tumor spheroids in the presence of different types of β-CDs [START_REF] Yakavets | The alteration of temoporfin distribution in multicellular tumor spheroids by β-cyclodextrins[END_REF]. We found that mTHPC accumulation in spheroids is strongly depended on the type and concentration of CDs, as was already reported for monolayer cells (Yankovsky et al., 2016). Varying the concentrations of β-CDs, we could increase PS uptake in 3D spheroids as well as inhibit it.

We showed that β-CD with lower affinity to mTHPC (Hp-β-CD) provided a threefold increase in total PS concentration, while Me-β-CD could enhance mTHPC uptake maximum 2-times.

However, higher affinity constant of Me-β-CD binding to mTHPC allows transporting the PS in the deeper cell layers than that of Me-β-CD. We suggested that the small size of inclusion complex (about 3 nm) along with its long life-time determine the transportation of PS in the tumor tissue depth via nanoshuttle mechanism. By this mechanism, mTHPC rapidly redistributes from serum proteins to cellular membranes (increased uptake) and easily diffuses into the interstitial medium in the host-guest complexes with β-CD (increased penetration). Thus, it was suggested that mTHPC/β-CDs could considerably improve the properties of mTHPC-loaded liposomes overcoming the limited penetration of liposomes in tumor tissue.

After all preliminary studies on the inclusion complex formation, we started to construct hybrid liposomes. In order to find the most optimal DCL composition for mTHPC delivery, we synthetized 9 various compositions based on three types of β-CD (Hp-, Me-and TMβ-CD)

and three PS loading concentrations (Yakavets et al., 2018a). Lipid composition of DCL vesicles was similar to Foslip ® , thus physical-chemical characteristics (size and surface charge) of DCLs were that of Foslip ® . Using developed spectral techniques we confirmed the possibility to encapsulate β-CD/mTHPC in the inner aqueous core of liposomes without destruction of liposomes and unexpected redistribution of mTHPC to the lipid bilayer.

According to our data, more than 85% of mTHPC was bound with CDs inside of liposomes.

We also addressed the issue of colloidal stability of mTHPC-DCL due to the reported destabilization of liposomes in the presence of β-CDs [START_REF] Hammoud | Cyclodextrin-membrane interaction in drug delivery and membrane structure maintenance[END_REF][START_REF] Piel | Betamethasone-in-cyclodextrin-in-liposome: The effect of cyclodextrins on encapsulation efficiency and release kinetics[END_REF][START_REF] Puskás | Features of the Interaction between Cyclodextrins and Colloidal Liposomes[END_REF]. According to the dynamic light scattering data, the colloidal stability of DCLs depended on the mTHPC:β-CD loading ratio and the type of β-CD. So, DCLs with the highest mTHPC loading based on the methylated β-CD exhibit the colloidal stability during more than 3 months, similar to Foslip ® . We supposed that the key factor of DCLs destabilization was the concentration of free β-CD molecules in the aqueous core, which remove lipid components forming inclusion complexes [START_REF] Fatouros | Liposomes encapsulating prednisolone and prednisolone-cyclodextrin complexes: comparison of membrane integrity and drug release[END_REF][START_REF] Szente | Cyclodextrin-Lipid Complexes: Cavity Size Matters[END_REF][START_REF] Uekama | Cyclodextrins in drug carrier systems[END_REF]. Nevertheless, the concept of synergistic mTHPC delivery by DL-DCLs which was clearly proved in 3D multicellular tumor spheroids should be also confirmed in vivo as potent nanoconstruct for mTHPC-PDT. Overall, DL-DCL is a perspective prototypical hybrid nanosystem which could be a good example of combined drug delivery.

CONCLUSION AND

PERSPECTIVES

Conclusion

CONCLUSION

The present thesis covers the study of hybrid liposomes that encapsulate inclusion complexes of CDs and second-generation photosensitizer mTHPC. The main research results are summarized as follows:

• Altogether, the proposed matryoshka-type lipid vesicles releasing mTHPC/CD complexes illustrate an optimal balance between plasma stability and efficient PS release.

PERSPECTIVES

Our ongoing studies are focused on the proof of the DCL concept on different cell cancer lines and 3D co-culture spheroid models. In fact, we investigate the influence of tumor microenvironment on distribution of mTHPC-DCLs in tumor tissue. With this aim, we analyzed the distribution profiles of mTHPC delivered by DL-DCL in co-culture stroma-rich spheroids.

Further continuation of research is related to the optimization of mTHPC-DCLs composition for in vivo application and PDT protocol. Optimization of PDT regimens necessarily requires knowledge of mTHPC/CD complexes pharmacokinetics and biodistribution, including the intratumoral spatial distribution of the PS. Also, the seruminduced destabilization of DL-DCLs in blood flow will be studied in detail. Based on these results, the optimal DLI for PDT treatment will be selected and PDT efficiency (necrotic/apoptotic response and tumor growth kinetics) will be analyzed. In addition, skin photosensitivity after DL-DCL administration studies should be performed.

Also, we suppose that the application of stimuli-response liposomes (e.g. lightresponsive) could provide external control of mTHPC/CD complexes release at the target site. Thus, additional optimization of mTHPC-DCL composition is required including screening in the preclinical in vitro and in vivo tumor models.

The overall results of such work would be beneficial for understanding the synergistic behavior of hybrid liposomes. Les liposomes représentent des nanocapsules phospholipidiques qui pourraient être chargées de composés hydrophobes dans la bicouche lipidique ou de médicaments hydrophiles dans le noyau aqueux interne [START_REF] Torchilin | Recent advances with liposomes as pharmaceutical carriers[END_REF]. Après environ deux décennies d'études, la formulation liposomale de mTHPC (Foslip ® ) est l'une des NP à base de mTHPC les plus efficaces et les plus étudiées [START_REF] Reshetov | Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo[END_REF] [START_REF] Reshetov | Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo[END_REF]. Toutefois, les liposomes ont une pénétration limitée dans les tissus tumoraux [START_REF] Gaio | Uptake and photo-toxicity of Foscan®, Foslip® and Fospeg® in multicellular tumor spheroids[END_REF]Millard et al., 2018), et libèrent rapidement la mTHPC dans le sang lors de l'interaction avec les composants sériques [START_REF] Reshetov | Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo[END_REF]. En résumé, les liposomes apparaissent comme des nanocages puissantes, qui pourraient être améliorées par l'encapsulation de complexes supramoléculaires nanométriques mTHPC/CD. Les cyclodextrines (CD) ont été largement étudiées comme excipient pharmaceutique unique au cours des dernières décennies et sont encore à l'étude pour de nouvelles applications. Des rapports récents ont démontré la puissance des β-CDs sur des modèles de tumeurs précliniques in vitro et in vivo comme nanovéhicule de la mTHPC (Yankovsky et al., 2016). Des études approfondies sur les CD en tant qu'agents pharmaceutiques ont démontré que l'effet de la CD dépend fortement de l'affinité de la CD avec le médicament (Stella and He, 2008). Lorsque nous avons affiné la valeur des constantes de liaison en supposant l'auto-agrégation des molécules de mTHPC, nous avons obtenu des valeurs beaucoup plus élevées que celles déjà rapportées dans la littérature [START_REF] Bautista-Sanchez | Photophysical properties of glucoconjugated chlorins and porphyrins and their associations with cyclodextrins[END_REF][START_REF] Desroches | A direct sensitized fluorimetric determination of5,10,15,20-tetra(mhydroxyphenyl)chlorin [m-THPC(Foscan)®] in human plasma using a cyclodextrin inclusion complex[END_REF]. Selon nos calculs (Yakavets et al., 2018b), le TM-β-CD complètement méthylé montre la plus forte affinité (K = 1,1×10 7 M -1 ) comparé au Me-β-CD (K = 7,1×10 5 M -1 ) et au Hp-β-CD (K = 1,7×10 5 M -1 ). On pourrait suggérer que la méthylation de β-CDs augmente leur affinité pour la mTHPC, permettant de moduler le comportement des PS dans les milieux biologiques. De plus, l'étude détaillée des propriétés photophysiques des complexes d'inclusion mTHPC/CD a révélé des changements de la forme de la bande de Soret de la mTHPC en fonction du complexe d'inclusion (Yakavets et al., 2017a). Nous avons confirmé, à l'aide de simulations de dynamique moléculaire (Yakavets et al., 2018), que les changements spectraux étaient induits par l'interaction des CD avec les groupes phényle, ce qui entraînait la rotation des substituts phényliques (Yakavets et al., 2017a). Il est à noter que l'intercalation de la molécule de mTHPC dans le microenvironnement lipidique n'est possible qu'en conformation planaire, entraînant des changements opposés de la forme de la bande de Soret de la mTHPC. Ainsi, la technique spectrale basée sur la forme de la bande de Soret pourrait être utilisée pour la surveillance du microenvironnement de la mTHPC et pourrait être appliquée pour la détection des complexes mTHPC/CD dans le noyau aqueux des liposomes hybrides.

SYNTHESE DES TRAVAUX DE THESE

Comme il a été mentionné plus haut, la méthylation des β-CDs augmente leur affinité avec la mTHPC, ce qui permet de moduler le comportement des PS dans les milieux biologiques. Pour décrire le mécanisme d'altération, nous avons étudié la distribution de de la mTHPC dans des sphéroïdes tumoraux multicellulaires 3D en présence de différents types de β-CDs (Yakavets et al., 2017). Nous avons constaté que l'accumulation de mTHPC dans les sphéroïdes dépend fortement du type et de la concentration des CD, comme cela a déjà été signalé pour les cellules monocouches (Yankovsky et al., 2016). En variant les concentrations de β-CDs, nous pourrions augmenter l'absorption de PS dans les sphéroïdes La présente étude a confirmé l'avantage des mTHPC-DCL à double charge par rapport aux formulations liposomales de mTHPC classiques (Foslip ® ) en termes de pénétration dans les tissus tumoraux. Dans l'ensemble, les vésicules lipidiques de type matryoshka proposées qui libèrent des complexes mTHPC/CD illustrent un équilibre optimal entre la stabilité plasmatique et la libération efficace de PS.

ABSTRACT

Photodynamic therapy (PDT) is an alternative cancer treatment which offers a more targeted and less invasive treatment regimen compared to traditional modalities. Temoporfin (mTHPC, medicinal product name: Foscan ® ), is one of the most potent clinically approved PS. However, its poor solubility in aqueous medium caused several complications of its administration. The present study is aimed at the development of drug-in-cyclodextrin-inliposome (DCL) nanoparticles by coupling two independent delivery systems: cyclodextrin/mTHPC inclusion complexes and liposomal vesicles to improve the transport and penetration of mTHPC to the target tissue. The formation of inclusion complexes between cyclodextrins and mTHPC was studied in detail. Based on these data, single and double loaded mTHPC-DCLs have been prepared, optimized and characterized. It was demonstrated that mTHPC-DCLs are stable and almost all mTHPC is bound to β-CDs in the inner aqueous liposome lumen. The influence of DCLs on mTHPC accumulation, distribution and photodynamic efficiency was studied in human adenocarcinoma HT29 cellular monolayer and spheroid models. Using 3D multicellular HT29 tumor spheroids we demonstrated that trimethyl-β-CD-based DCL provides homogenous accumulation of mTHPC across tumor spheroid volume thus supposing optimal mTHPC distribution.

Key-Words : Photodynamic therapy, mTHPC, cyclodextrins, binding constants, drugin-cyclodextin-in-liposomes, multicellular tumor spheroids.

RESUME

La thérapie photodynamique (PDT) est un traitement alternatif du cancer plus ciblé et moins invasif que les modalités traditionnelles. La Temoporfine (mTHPC, nom sous forme médicamenteuse : Foscan ® ), est l'un des PS les plus puissants cliniquement approuvés. Cependant, sa faible solubilité en milieu aqueux a provoqué plusieurs complications lors de son administration. La présente étude vise à mettre au point des nanoparticules constituées d'une molécule anticancéreuse couplée à la cyclodextrine intégré dans un liposome (drug-incyclodextrin-in-liposome, DCL) en couplant deux systèmes d'administration indépendants : les complexes d'inclusion cyclodextrine-mTHPC et les vésicules liposomales pour améliorer le transport et la pénétration de la mTHPC dans le tissu cible. La formation de complexes d'inclusion entre les cyclodextrines et la mTHPC a été étudiée en détail. Sur la base de ces données, des mTHPC-DCL à simple et double charge ont été préparées, optimisées et caractérisées. Il a été démontré que les mTHPC-DCL sont stables et que presque tous les mTHPC-DCL sont liés à β-CDs dans la lumière aqueuse interne des liposomes. L'influence des DCLs sur l'accumulation, la distribution et l'efficacité photodynamique de la mTHPC a été étudiée dans des modèles cellulaire en monocouche et sphéroïde multicellulaires 3D d'adénocarcinome de pharynx humain (HT29). En utilisant des sphéroïdes, nous avons démontré que le DCL à base de triméthyl-β-CD fournissait une accumulation homogène de la mTHPC dans tout le volume des sphéroïdes tumoraux, suggérant ainsi une distribution optimale de la mTHPC.

Figure 1 . 1 Figure 1 . 2 Figure 1 . 3 Figure 1 . 4 Figure 1 . 5

 1112131415 ABBREVIATIONS

Figure 1 . 6 Figure 3 . 1 Figure 3 . 2 Figure 3 . 3

 16313233 Figure 1.6 Structural skeletons of porphyrinoid-based PSs (Ben Mihoub et al., 2018) .........20 Figure 2.1 (A) Chemical structure of mTHPC. (B) Steric mTHPC conformation ...................21 Figure 3.1 Schematic structure of DCL ................................................................................47 Figure 3.2 Typical supramolecular equilibria of inclusion complexes with 1:1, 1:2, 2:1 and 2:2 stoichiometries. ....................................................................................................................54 Figure 3.3 Preparation of DCL by (A) thin-film hydration, (B) ethanol injection, (C) reverse

Figure 1 . 1

 11 Figure 1.1 Schematic illustration of PDT treatment process

Figure 1 . 2

 12 Figure 1.2 Photosensitization Processes Illustrated by a Modified Jablonski Diagram.

  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

 13 Figure 1.3 The main cell death subroutines induced by PDT. Reprinted from[START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF]

Figure 1 . 4

 14 Figure 1.4 Mechanisms of PDT-mediated photoeradication of tumor mass (from Agostinis et al. 2011)

Figure 1 . 5

 15 Figure 1.5 Light propagation through the tissues.

Figure 1 . 6

 16 Figure 1.6 Structural skeletons of porphyrinoid-based PSs (Ben Mihoub et al., 2018)

Figure 2 . 1

 21 Figure 2.1 (A) Chemical structure of mTHPC. (B) Steric mTHPC conformation

Figure 3 . 1

 31 Figure 3.1 Schematic structure of DCL

Table 3 . 1 3 *

 313 General CD structures (Ben Mihoub et al., 2018;Stella and He, 2008) Degree of substitution; ** n+2, number of glucopyranoside units CDs exhibit the shape of a truncated cone due to the chair conformation of the Dglucopyranose units. Free hydroxyls are on the outside of the CDs, while apolar C3 and C5

Figure 3 . 2

 32 Figure 3.2 Typical supramolecular equilibria of inclusion complexes with 1:1, 1:2, 2:1 and 2:2 stoichiometries. Km,n -association constant (K) of the guest-host complex with m:n stoichiometry, [G] -concentration of free guest molecules, [H] -concentration of free host molecules, [GmHn] -concentration of m:n complexes.

Figure 3 . 3

 33 Figure 3.3 Preparation of DCL by (A) thin-film hydration, (B) ethanol injection, (C) reversephase evaporation and (D) dehydration-rehydration methods[START_REF] Gharib | Liposomes incorporating cyclodextrin-drug inclusion complexes: Current state of knowledge[END_REF]

  Development of experimental techniques to control the distribution of mTHPC between β-CDs and lipid membranes • Investigation of the behavior of mTHPC loaded inclusion complexes in 3D in vitro tumor tissue model These tasks are addressed in the first research section (Chapter II). • Optimization of cyclodextrin-based liposomal nanoparticles loaded with mTHPC • Physical-chemical and photophysical characterization of drug-in-cyclodextrin-in-liposome formulation of mTHPC • Study of the behavior of drug-in-cyclodextrin-in-liposome formulation of mTHPC in 2D and 3D in vitro tumor cell cultures • Study of the mechanisms of mTHPC-CL photoinduced cell death in cultured cells in vitro • Study of PDT efficiency of mTHPC-CL in pre-clinical xenografted mice in vivo model. These points are addressed in the second research section (Chapter III).

  studied the ability of Me-β-CD and Hp-β-CD to alter the penetration and diffusion of mTHPC in HT29 multicellular tumor spheroid model. It was demonstrated that the presence of β-CDs significantly changed the distribution of mTHPC consisting in the increase of both the depth of PS penetration and accumulation in HT29 spheroids. Depending on the type and concentration of β-CD, mTHPC accumulation in spheroids could be increased up to 3 times for Hp-β-CD, while deeper penetration in the tumor tissue was observed for Me-β-CD-based complexes. Based on the obtained data, we suggested that β-CDs facilitate mTHPC transportation to the cells in the inner layers of spheroids via nanoshuttle mechanism. This study demonstrated the added value of β-CD to liposomes inducing improved accumulation, diffusion and penetration of the photosensitizer mTHPC in tumor tissue. and optimization of mTHPC-DCL This chapter will be devoted to the development, optimization and screening aspects of hybrid liposomes loaded with mTHPC/β-CD inclusion complexes. Firstly, we optimized and characterized liposomes encapsulated mTHPC/β-CD complexes. This part of the results is described in the article "Temoporfin-in-Cyclodextrin-in-Liposome -A New Approach for Anticancer Drug Delivery: The Optimization of Composition". We have synthesized 9 various DCL compositions encapsulated inclusion complexes of mTHPC with three different β-CD derivatives (HP-, Me-and TM-β-CD) containing three various mTHPC concentrations. The obtained DCLs were characterized using dynamic light scattering, atomic force microscopy and spectroscopic techniques. It was demonstrated that > 85% of mTHPC is bound to β-CD inclusion complexes in the inner aqueous core of liposomes. The study of colloidal stability showed that DCLs with methylated β-CDs and the highest mTHPC loading remained stable more than 3 months. Intracellular uptake, penetration and distribution of mTHPC delivered by DCLs were studied in HT29 monolayer cells and multicellular tumor spheroids in vitro. Among all tested DCLs, TM-β-CD-based mTHPC-DCL with the demonstrated a homogenous accumulation of mTHPC across tumor spheroid volume supposing better PS distribution in tumor tissue. Double loaded mTHPC-DCL 8. Double loaded mTHPC-DCL In the second part, we upgraded mTHPC-DCLs by additional loading of mTHPC in the lipid bilayer of liposomes. These results were reported in the article "Matryoshka-type liposomes offer the synergistic delivery of temoporfin to tumor spheroids". Double-loaded mTHPC-DCLs encapsulated inclusion complexes of mTHPC with Me-β-CD (MD) and mTHPC-TM-β-CD (TD) were prepared, characterized and tested in 2D and 3D in vitro models of HT29 human adenocarcinoma cells and tumor xenografted mice in vivo. The physico-chemical properties of double-loaded mTHPC-DCLs were comparable with single-loaded ones (Yakavets et al., 2018a). Based on the spectral characteristics, we assessed that almost 30% of mTHPC in DL-DCLs is bound to β-CDs in the inner aqueous core, while 70% of PS is localized in the lipid bilayer. In 2D monolayer cell culture, DL-DCLs behavior was similar to Foslip ® . At the same time, the distribution of mTHPC in 3D multicellular tumor spheroids was significantly changed. The kinetics of mTHPC distribution were studied by flow cytometry and confirmed using laser scanning confocal microscopy of spheroids' cryosections at 24h. Moreover, we analyzed the content of mTHPC in the medium supplemented with different serum concentrations by means of gel-chromatography. Our studies indicated that TM-β-CD-based DL-DCL provides deep penetration of mTHPC into the multicellular tumor spheroids via TM-β-CD nanoshuttles upon the liposomes destabilization by serum proteins. Finally, we assessed PDT efficiency of DL-DCLs in multicellular spheroids in vitro and xenografted mice in vivo demonstrating similar photokilling ability of hybrid liposomes and Foslip ® in both preclinical models.

  Concerning the screening of mTHPC-DCLs in HT29 colon adenocarcinoma monolayer cell culture and 3D multicellular tumor spheroids, we revealed the crucial role of mTHPC loading concentration on the intracellular uptake. Single loaded mTHPC-DCLs demonstrated improved penetration and distribution in the tumor spheroids, however, the total accumulation was significantly lower than Foslip ® due to the highly competitive mTHPC binding among CDs, liposomal membranes serum proteins and cells. Thus, we upgraded mTHPC-DCLs by mTHPC loading in the lipid bilayer wholly replicating Foslip ® . The physicochemical characteristics as well as colloidal stability of DL-DCLs were not changed compared to mTHPC-DCLs. The loading of high mTHPC concentrations in lipid bilayer resulted in the fluorescence quenching, confirmed spectroscopically. Overall, 70% of mTHPC was bound in the lipid bilayer and 30% of PS was encapsulated in the inner aqueous core with β-CDs that was very close for theoretical predictions. By this way, DL-DCLs provide an appropriate cellular accumulation of PS in both 2D and 3D cell cultures. However, since the main aim was to increase the penetration ability of mTHPC in tumor tissues, we mainly focused our studies on 3D multicellular spheroid model. We demonstrated significant improvement of mTHPC distribution upon utilization of TD nanoconstruct. TD-treated spheroids almost homogeneously accumulated mTHPC that was confirmed by flow cytometry and fluorescence microscopy, while MD provided mTHPC distribution mainly at the periphery of spheroids similar to Foslip ® . We supposed that the extremely high mTHPC affinity to TM-β-CD leads to the formation of long-living inclusion complexes capable to transfer mTHPC to the depth of tumor tissue. Thus, we could detect mTHPC/TM-β-CDs inclusion complexes in the culture medium after 24h incubation with spheroids, while Me-β-CD complexes possess a shorter complex lifetime resulting in partial penetration of mTHPC in spheroids.However, the benefits of DL-DCLs as mTHPC delivery system were not enough to overcome PDT efficiency of Foslip ® in multicellular tumor spheroid in vitro as well as tumorxenografted mice in vivo. Despite the improved mTHPC distribution in spheroids, PDTinduced cell death in spheroids treated with TD and MD was similar to Foslip ® . Similarly, PDT in tumor xenografted mice induced the tumor growth delay of 30 days for TD as well as Foslip ® . Probably, the intracellular concentration of mTHPC delivered by DL-DCL is below the limit of necrosis induction and mTHPC dose could be increased. It should be noted, that the improved PDT response in the case of homogeneous PS distribution is obvious however should be additionally investigated.

  The formation of mTHPC/CD inclusion complexes. The complexes between mTHPC and β-CD derivatives possess mainly 1:2 stoichiometry and very high affinity constants (up to 10 7 M -1 ) compared to other anticancer drugs. Strong binding of mTHPC between β-CD molecules results in the changes of PS molecule conformation, causing the changes of Soret band spectral band. Also, such high mTHPC affinity to β-CD provides the unique opportunity to alter PS biodistribution via CD nanoshuttle mechanism. • The drug-in-cyclodextrin-in-liposome formulations (DCL). DCLs were successfully prepared and characterized. The prepared hybrid liposomes replicated lipid composition of Foslip ® . mTHPC was localized in the aqueous core in the complexes with β-CDs. All prepared DCLs were colloidally stable for > 3weeks. The particular DCLs encapsulated methylated β-CD derivatives were stable more than 3 months. The possible mechanisms of DCLs destabilization were proposed. • Double loaded drug-in-cyclodextrin-in-liposomes (DL-DCL): Proof of the concept. DL-DCL were prepared and characterized. Double loading of DCLs significantly increased cellular uptake of mTHPC. The localization of mTHPC in DL-DCLs was analyzed spectroscopically (70% in lipid bilayer and 30% in CD complexes). The kinetic processes of DL-DCLs interaction with tumor cells were studied in 2D and 3D tumor culture models. Spheroids treated with TD displayed almost homogeneous mTHPC distribution between the cells. Hypothetically, TM-β-CD nanoshuttles are able to transport mTHPC in the tumor depth due to the high affinity to mTHPC compared to Meβ-CD. No difference in PDT efficiency was observed in 3D multicellular tumor spheroids in vitro as well as tumor xenografted mice in vivo. The data obtained confirmed the advantage of double-loaded mTHPC-DCLs over conventional mTHPC liposomal formulation (Foslip ® ) in terms of tumor tissue penetration.

  Afin d'améliorer le transport de la mTHPC vers le tissu cible et de renforcer son accumulation intra-tissulaire, il a été proposé de coupler deux de ces systèmes de libération indépendants. Ainsi, l'objectif principal du présent travail a été de développer et d'optimiser la nanoconstruction de la molécule chargée de témporfine dans la cyclodextrine dans les liposomes (DCL) afin d'obtenir une combinaison synergique de deux systèmes de libération pour un relargageefficace de la mTHPC dans le tissu tumoral cible. Dans ce but, la complexation de la mTHPC avec des CDs et le comportement de ces complexes dans un modèle 3D de tissu tumoral in vitro ont été étudiés. De plus, de nouvelles méthodes d'analyse de la libération de la mTHPC à partir de CD ont été mises au point pour caractériser la structure de la mTHPC-DCL. In fine, la composition des mTHPC-DCL développés a été optimisée en fonction des études sur des modèles tumoraux 2D et 3D in vitro.

  3D et aussi l'inhiber. Nous avons montré que la β-CD avec une plus faible affinité pour la mTHPC (Hp-β-CD) a permis de tripler la concentration totale de PS, tandis que la Me-β-CD pourrait améliorer l'absorption de mTHPC au maximum 2 fois. Cependant, une constante d'affinité plus élevée de liaison des Me-β-CD à la mTHPC permet de transporter le PS dans les couches cellulaires plus profondes. Nous avons suggéré que la petite taille du complexe d'inclusion (environ 3 nm) ainsi que sa longue durée de vie déterminent le transport du PS dans la profondeur du tissu tumoral. Par ce mécanisme, la mTHPC se redistribue rapidement à partir des protéines sériques aux membranes cellulaires (absorption accrue) et diffuse facilement dans le milieu interstitiel (pénétration accrue). Par conséquent, il a été suggéré que la mTHPC/β-CDs pourrait améliorer considérablement les propriétés des cette façon, les DL-DCL fournissent une accumulation cellulaire appropriée de PS dans les cultures cellulaires 2D et 3D. Cependant, en ce qui concerne l'objectif principal qui était d'augmenter la capacité de pénétration de la mTHPC dans les tissus tumoraux, nous avons principalement concentré nos études sur le modèle de sphéroïdes multicellulaire. Nous avons démontré une amélioration significative de la distribution de la mTHPC grâce à l'utilisation de la nanoconstruction TD. Les cellules des sphéroïdes traités par TD ont accumulé de façon presque homogène la mTHPC, ce qui a été confirmé par cytométrie de flux et microscopie de fluorescence, tandis que le MD a montré une distribution de la mTHPC principalement à la périphérie des sphéroïdes ainsi que Foslip ® . Nous avons supposé que l'affinité extrêmement élevée de la mTHPC pour TM-β-CD conduit à la formation de complexes d'inclusion à vie longue capables de transférer la mTHPC en profondeur du tissu tumoral. Ainsi, nous avons pu détecter des complexes d'inclusion de la mTHPC/TM-β-CDs dans le milieu de culture après une incubation de 24 heures avec des sphéroïdes, alors que les complexes de Me-β-CD ont une durée de vie plus courte, entraînant une pénétration limité de la mTHPC dans les sphéroïdes. Néanmoins, les avantages des DL-DCL en tant que système d'administration de la mTHPC n'ont pas été suffisants pour améliorer l'efficacité PDT dans le sphéroïde tumoral multicellulaire in vitro ainsi que chez les souris ayant subi une xénogreffe tumorale in vivo. Malgré l'amélioration de la distribution de la mTHPC dans les sphéroïdes, la mort cellulaire induite par la TPD dans les sphéroïdes traités par TD et MD était similaire à celle de Foslip ® . De même, chez les souris xénogreffées de tumeurs, le délai de croissance tumorale moyen était de 30 jours pour la TD ainsi que pour le Foslip ® . Nos études en cours portent sur différents paramètres expérimentaux permettant l'optimisation de la DL-DCL dans des modèles précliniques. L'une des solutions possibles est l'incorporation dans les liposomes d'un composant favorisant la libération de nanoshuttles CD après stimulation.

Table 1 .1. Photosensitizers

 1 approved for clinics for PDT (from[START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF][START_REF] Santos | Photodynamic therapy in cancer treatment -an update review[END_REF][START_REF] Lucky | Nanoparticles in Photodynamic Therapy[END_REF][START_REF] Ormond | Dye Sensitizers for Photodynamic Therapy[END_REF]

PS Trademark Approved λex, (nm) Application

	Porphyrins
	Porfimer	Photofrin ® ,			Bladder, esophageal, lung, cervical, endobronchial,
	sodium	Photogem ® ,	WW	630	and gastric cancers
		Photosan-3 ®
	Porphyrin precursors
	5-ALA 1	Levulan ® ,	WW	400	Treatment of actinic keratosis, basal cell carcinoma,
		Ameluz ®			head and neck, and gynecological tumors;
					diagnosis of bladder, brain, head & neck cancers
	MAL 2	Metvix ® ,	USA, EU,	630	Actinic keratosis, Bowen's disease, and basal cell
		Metvixia ®	New		carcinoma
			Zealand &
			Australia)
	HAL 3	Hexvix ® ,	USA	635	Diagnosis of bladder and colon cancers
		Cysview ®
	Benzoporphyrin
	Verteporfin	Visudyne ®	USA, EU,	689	Treatment for wet age-related macular
			Canada		degeneration, pathologic myopia, histoplasmosis
	Chlorins
	Temoporfin	Foscan ®	EU	652	Head and neck cancers
	Talaporfin or	Aptocine ® ,	Japan	664	Lung cancer
	MACE	Laserphyrin ®
	Chl e6 4	Fotolon ® ,	Belarus,	665	Nasopharyngeal, sarcoma, brain, skin cancer
		Radachlorin ® ,	Russia,
		Photodithazine	Ukraine
		®
	Phthalocyanines
	AlPCS4 5	Photosens ®	Russia	675	Stomach, skin, oral cavity, tongue, and breast
					cancers
	Texaphyrins
	Lutetium	Lutex ®	USA	732	Breast cancer and malignant melanomas
	texaphyrin
	Pheophorbides
	Pd-Bpheid 6	Tookad ®	EU	538/762	Prostate cancer
	Porphycenes
	ATMPn 7	ATMPn	Germany	630	Psoriasis and non-melanoma skin cancer

Table 2 . 1

 21 Photophysical properties of mTHPC in methanol[START_REF] Bautista-Sanchez | Photophysical properties of glucoconjugated chlorins and porphyrins and their associations with cyclodextrins[END_REF]

	Bonnett et al., 1989)
	Property

  . Les liposomes conventionnels encapsulent près de 8 % (p/p) de mTHPC dans une bicouche lipidique, ce qui permet la solubilisation complète d'une molécule de mTHPC hautement hydrophobe lors son administration. Le Foslip ® présente une toxicité plus faible dans le noir et pourrait potentiellement cibler passivement la tumeur par effet EPR, améliorant ainsi la sélectivité de la distribution de la mTHPC. De plus, il a été démontré que le traitement Foslip ® -PDT était plus efficace à des DLI plus courtes de 6 à 15 heures que la DLI de 24 heures pour le Foscan ®

Photodynamic therapy

5-Aminolevulinic Acid;

Methyl aminolevulinate;

Hethyl aminolevulinate;

Chlorin e6 derivatives;

Sulfonated aluminum phthalocyanines;

Palladium-bacteriopheophorbide;

7,12,porphycene

Meta-tetra(hydroxyphenyl)chlorin29 Meta-tetra(hydroxyphenyl)chlorin

Meta-tetra(hydroxyphenyl)chlorin31 Meta-tetra(hydroxyphenyl)chlorin

Meta-tetra(hydroxyphenyl)chlorin33 Meta-tetra(hydroxyphenyl)chlorin

Meta-tetra(hydroxyphenyl)chlorin35 Meta-tetra(hydroxyphenyl)chlorin

Meta-tetra(hydroxyphenyl)chlorin37 Meta-tetra(hydroxyphenyl)chlorin

Meta-tetra(hydroxyphenyl)chlorin39 Meta-tetra(hydroxyphenyl)chlorin

Meta-tetra(hydroxyphenyl)chlorin

Development of spectral techniques for monitoring the distribution of inclusion

Development and optimization of mTHPC-DCL

Development and optimization of mTHPC-DCL101 Development and optimization of mTHPC-DCL

Development and optimization of mTHPC-DCL103 Development and optimization of mTHPC-DCL

Development and optimization of mTHPC-DCL105 Development and optimization of mTHPC-DCL

Double loaded mTHPC-DCL111 Double loaded mTHPC-DCL

Double loaded mTHPC-DCL113 Double loaded mTHPC-DCL

Double loaded mTHPC-DCL115 Double loaded mTHPC-DCL

Double loaded mTHPC-DCL117 Double loaded mTHPC-DCL

Double loaded mTHPC-DCL119 Double loaded mTHPC-DCL

Double loaded mTHPC-DCL121 Double loaded mTHPC-DCL

Double loaded mTHPC-DCL123 Double loaded mTHPC-DCL

Double loaded mTHPC-DCL

SCIENTIFIC OUTPUT

Scientific output

colloïdale de la mTHPC-DCL grâce aux données de déstabilisation des liposomes en présence de β-CDs[START_REF] Hammoud | Cyclodextrin-membrane interaction in drug delivery and membrane structure maintenance[END_REF][START_REF] Piel | Betamethasone-in-cyclodextrin-in-liposome: The effect of cyclodextrins on encapsulation efficiency and release kinetics[END_REF][START_REF] Puskás | Features of the Interaction between Cyclodextrins and Colloidal Liposomes[END_REF]. Selon les données de diffusion dynamique de la lumière, la stabilité colloïdale des DCL dépend du rapport de charge de la mTHPC:β-CD et du type de β-CD. Ainsi, les DCL ayant la charge de mTHPC la plus élevée basée sur le β-CD méthylé présentent une stabilité colloïdale pendant plus de 3 mois. Nous avons supposé que le facteur clé de la déstabilisation des DCLs était la concentration de molécules libres β-CD dans le noyau aqueux, qui éliminent les composants lipidiques formant des complexes d'inclusion[START_REF] Fatouros | Liposomes encapsulating prednisolone and prednisolone-cyclodextrin complexes: comparison of membrane integrity and drug release[END_REF][START_REF] Szente | Cyclodextrin-Lipid Complexes: Cavity Size Matters[END_REF][START_REF] Uekama | Cyclodextrins in drug carrier systems[END_REF].En ce qui concerne le criblage des mTHPC-DCLs dans la culture cellulaire en monocouche d'adénocarcinome du côlon HT29 et des sphéroïdes tumoraux multicellulaires 3D, nous avons révélé le rôle crucial de la concentration de charge de la mTHPC sur l'absorption intracellulaire. Les mTHPC-DCL à charge unique ont démontré une meilleure pénétration et distribution dans les sphéroïdes tumoraux, mais l'accumulation totale était significativement inférieure à celle du Foslip ® en raison de la liaison de la mTHPC hautement compétitive avec les CD, les protéines sériques des membranes liposomales et les cellules.Ainsi, nous avons amélioré les mTHPC-DCL en les chargeant dans la bicouche lipidique comme c'est le cas pour le Foslip ® . Les caractéristiques physico-chimiques ainsi que la stabilité colloïdale des DL-DCL n'ont pas été modifiées par rapport aux mTHPC-DCL. La charge de concentrations élevées de mTHPC dans la bicouche lipidique a entraîné un quenching de fluorescence et a été confirmée par spectroscopie. Dans l'ensemble, 70% de la mTHPC était liée dans la bicouche lipidique et 30% du PS était encapsulé dans le noyau aqueux interne avec des β-CDs, ce qui était très proche pour les prévisions théoriques. De

Acknowledgements

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Dr. Vladimir Zorin for intellectual encouragement, scientific advice and discussions and guidance, and for steering me in the right direction. We have worked together for 7 long years, -3 years at the university, my Master's degree, and PhD years, which in totality were the making years for

LIST OF TABLES

 [START_REF] Agostinis | Photodynamic therapy of cancer: An update[END_REF][START_REF] Santos | Photodynamic therapy in cancer treatment -an update review[END_REF][START_REF] Lucky | Nanoparticles in Photodynamic Therapy[END_REF][START_REF] Ormond | Dye Sensitizers for Photodynamic Therapy[END_REF] 3. 1 General CD structures (Ben Mihoub et al., 2018;Stella and He, 2008)